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POLSKIE TOWARZYSTWO MIKROBIOLOGÓW POLISH SOCIETY OF MICROBIOLOGISTS Polish Journal of Microbiology I am pleased to inform you that Polish Journal of Microbiology has been selected for coverage in Thomson Scientific products and customers information services. Beginning with No 1, Vol. 57, 2008 information on the contents of the PJM is included in: Science Citation Index Expanded (ISI) and Journal Citation Reports (JCR)/Science Edition. Stanis³awa Tylewska-Wierzbanowska Editor in Chief 2010 Polish Journal of Microbiology formerly Acta Microbiologica Polonica 2010, Vol. 59, No 3 CONTENTS MINIREVIEW Bacteriophage receptors, mechanisms of phage adsorption and penetration into host cell RAKHUBA D.V., KOLOMIETS E.I., SZWAJCER DEY E., NOVIK G.I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 ORIGINAL PAPERS Interactions between Borrelia burgdorferi and mouse fibroblasts CHMIELEWSKI T., TYLEWSKA-WIERZBANOWSKA S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 Clonal analysis of Staphylococcus aureus strains isolated in obstetric-gynaecological hospital SZCZUKA E., SZUMA£A-K¥KOL A., SIUDA A., KAZNOWSKI A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay BEDIR O., KILIC A., ATABEK E., KUSKUCU A.M., TURHAN V., BASUSTAOGLU A.C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Methanogenic diversity studies within the rumen of Surti buffaloes based on methyl coenzyme M reductase A (mcrA) genes point to Methanobacteriales SINGH K.M., PANDYA P.R., PARNERKAR S., TRIPATHI A.K., RAMANI U., KORINGA P.G., RANK D.N., JOSHI C.G., KOTHARI R.K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Optimisation of synthetic medium composition for levorin biosynthesis by Streptomyces levoris 99/23 and investigation of its accumulation dynamics using mathematical modelling methods STANCHEV V.S., KOZHUHAROVA L.Y., ZHEKOVA B.Y., GOCHEV V.K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Chromate reduction by cell-free extract of Bacillus firmus KUCr1 SAU G.B., CHATTERJEE S., MUKHERJEE S.K. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Occurence and chracterization of Colletotrichum dematium (Fr.) grove MACHOWICZ-STEFANIAK Z. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Cytotoxic activity of Serratia marcescens clinical isolates KRZYMIÑSKA S., RACZKOWSKA M., KAZNOWSKI A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Antibiotic susceptibility and genotype patterns of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa isolated from urinary tract infected patients ABOU-DOBARA M.I., DEYAB M.A., ELSAWY E.M., MOHAMED H.H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 SHORT COMMUNICATIONS rDNA- based genotyping of clinical isolates of Candida albicans NAWROT U., PAJ¥CZKOWSKA M., W£ODARCZYK K., MECLER I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 INSTRUCTIONS TO AUTHORS AND FULL TEXT ARTICLES (IN PDF FORM) AVAILABLE AT: www.microbiology.pl/pjm Polish Journal of Microbiology 2010, Vol. 59, No 3, 145155 MINIREVIEW Bacteriophage Receptors, Mechanisms of Phage Adsorption and Penetration into Host Cell D.V. RAKHUBA1, E.I. KOLOMIETS1, E. SZWAJCER DEY2 and G.I. NOVIK1* 1 Institute of Microbiology, National Academy of Sciences of Belarus, Minsk, Belarus University, Pure and Applied Biochemistry, Lund, Sweden 2 Lund Received 4 April 2010, accepted 2 July 2010 Abstract Bacteriophages are an attractive tool for application in the therapy of bacterial infections, for biological control of bacterial contamination of foodstuffs in the alimentary industry, in plant protection, for control of water-borne pathogens, and control of environmental microflora. This review is mainly focused on structures governing phage recognition of host cell and mechanisms of phage adsorption and penetration into microbial cell. K e y w o r d s: bacteriophage receptors, phage penetration mechanism Introduction Currently, the bacteriophage phenomenon may be regarded from different viewpoints. On one hand, bacterial viruses pose a grave challenge to industries based on bacterial agents applied in dairy processing, production of enzymes, antibiotics, solvents, insecticides, lactic and acetic acid, and various bacterial preparations when massive concentrations of biomass at active growth phase create favorable conditions for propagation of phages often responsible for lysis of industrial cultures. On the other hand, bacteriophages are objects that are attractive for application in medicine and veterinary practice for therapy of bacterial infections in humans and domestic animals. Bacterial viruses may also be used for biological control of bacterial contamination of foodstuffs in alimentary industry, agriculture; for control of water-borne pathogens, clinical pathogens causing aerogenic infections; control of environmental microflora, etc. Solution of industrial bacteriophage problems and search for practical virus application require fundamental studies to analyze interactions between bacteriophage and host cell and to elucidate correlations of viral infection process in bacterial cell. Such interactions are rather complicated and do not always result in cell lysis. Now phage-cell relations are considered as process consisting of several sequential stages: phage adsorption on host cell surface and penetration of phage nucleic acid into cell, intracellular synthesis of virus components and assembly of virions, lysis of bacterial cell and phage release. Adsorption is a key stage in virus recognition of sensitive host cell, i.e. specificity of phage infection is defined at this moment. Since bacteriophages, like any other viruses are obligate intracellular parasites, successful penetration into bacterial cell is an essential condition for continuation of their life cycle. This review is mainly focused on structures governing phage recognition of host cell and mechanisms of phage adsorption and penetration into microbial cell. Bacteriophage receptors on cell surface A specific bacteriophage strain is known to be able to infect a narrow host range or a concrete microbial species or strain. Such specificity in interaction of phage with bacterial cell is determined by specificity of adsorption, which in turn is dependent on the nature and structural peculiarities of receptors on bacterial cell surface (Braun and Hantke, 1997). In addition, a vital role is attributed to receptor localization on cell surface, their amount and density at various cell wall sites. * Corresponding author: G. Novik, Institute of Microbiology, National Academy of Sciences of Belarus, Kuprevich 2, 220 141 Minsk, Belarus; phone (+375-17) 2678620; e-mail: [email protected] 146 Rakhuba D.V. et al. The nature of receptors contacting bacteriophages is different for representatives of diverse taxonomic groups and is largely defined by composition of host cell wall and surface structures. Receptors localized in cell wall of gram-negative bacteria. The outer membrane of gram-negative bacteria differs in structure from the inner membrane and from the plasma membrane of gram-positive microorganisms. One of the distinctive features is its high permeability caused by increased levels of integral proteins forming transport channels up to 20 000 per cell (Nikaido, 2003). Another distinction is the presence in external lipid layer of a unique glycolipid lipopolysaccharide (LPS) typically exclusive for gram-negative bacteria. Proteins localized in membrane and various LPS sites may serve as bacteriophage receptors. In many cases phages require molecules of both types for adsorption (Lindberg, 1973). Protein receptors. Proteins of outer membrane may be subdivided into 5 classes: 1) structural proteins interacting with peptidoglycan layer; 2) specific and non-specific porins forming membrane channels; 3) enzymes; 4) substrate receptors with high affinity; 5) transport proteins responsible for secretion. Among structural proteins serving as receptors for virus adsorption, transmembrane protein OmpA was characterized. This protein comprises 8 antiparallel $-structures fixed inside membrane by non-covalent link to peptidoglycan layer with the free C-terminal vertex (Koebnik, 1999a; 1999b; Vogel and Jahnig, 1986). Mutants lacking the protein are distinguished by spherical shape and labile outer membrane. It was also shown that OmpA is involved in process of bacterial conjugation (Schweizer and Henning, 1977). OmpA protein-LPS complex is capable to inhibit phage Tulb (coliphage isolated from effluents). Binding of bacteriophage with protein-LPS complex occurs reversibly, and precipitation of this complex with Mg+2 leads to irreversible phage attachment. Apart from protein, the LPS molecule is not able to inhibit phage particles (Datta et al., 1977). OmpA protein inhibits bacteriophage K3 in the solution, while mutants defective in this protein are resistant to phage infection (Van Alpen et al., 1977; Scurray et al., 1974). Such findings testify to the receptor role of the protein with respect to Tulb and K3, yet phage-recognising sites are located at different areas of the molecule. This assumption is supported by the existence of mutants producing sufficient amount of OmpA protein and sensitive to phage K3 but showing resistance to phage Tulb (Henning et al., 1978). Porins were one of the first outer membrane proteins of gram-negative microorganisms to be characterized in detail (Nakae, 1976). These protein complexes are composed of 3 subunits forming the channel in bacterial membrane. Major proteins of this type in E. coli cells are OmpC and OmpF. 3 OmpC serves as a receptor for phages Hy2, ss4, Tulb and T4 (Scurray et al., 1974; Yu and Mizushima, 1982). Phage T4 utilized the protein as a receptor in combination with cell wall LPS. It was shown in experiments with LPS and OmpC mutants that absence of at least one receptor resulted in reduced efficiency of infection, whereas loss of both receptors induced bacteriophage resistance. Protein gp37 shaping tail fibers governs receptor recognition in phage T4 (Montag et al., 1990; Heller, 1990). The region is made up by approximately 14 amino acid residues and contains a large amount of histidine residues, responsible for OmpC recognition. OmpF is a receptor for phage T2 (Riede et al., 1985; Hantke, 1978). In contrast to T4 phage, the receptor recognizing site is located within the hypervariable region of protein gp38 attached to terminal part of protein gp37. Instead of hystidine bases, gp38 includes glycine sequences (up to 9 residues) at terminal and internal loci. Protein receptors in the cell wall of Shigella and E. coli were revealed for bacteriophage T6 (Jesaitis and Goebel, 1952; Michael, 1968). Manning and Reeves demonstrated that E. coli with tsx gene mutation displayed resistance to bacteriophage T6 infection and in subsequent papers they isolated and purified protein Tsx (product of tsx gene) controlling the transport of nucleotides and proved its receptor function (Manning and Reeves, 1976; 1978). Selective transport protein LamB is the receptor for phage 8 (Randall-Hazelbauer and Schwartz, 1973). Unlike non-selective porins OmpC and OmpF, this protein forms a narrow channel specific for transport of maltose and derived polymers, using aromatic positively charged aminoacid residues (Charbit et al., 1998). Bacteriophage 8 recognizes LamB via protein gpJ the factor defining host range of this phage. Among enzymes localized in the outer membrane are proteases OmpT and OmpX which may serve as receptors for T-like phages with host range mutations M1 and Ox2, respectively (Hashemolhosseini et al., 1994a; 1994 b). Proteins TonA (later renamed FhuA) and TonB serve as receptors for phages T7, T5 and n80. Receptors with high substrate affinity are components of active transport system. Their function is to carry out solid binding of substances below demand by passive transport system, namely vitamin B12 and iron ions as chelating agents (Frost and Rosenberg, 1975; Hancock et al., 1976; Bassford et al., 1976). Secretory transport proteins accomplish the function similar to substrate receptors, but in opposite direction, i.e. they transport diverse compounds out of the cell. So far phages using this protein type as receptors have not been detected. Lipopolysaccharide receptors. In addition to proteins, LPS is another constituent of the outer membrane in gram-negative bacteria serving as a receptor for 3 Receptors, adsorption and penetration of bacteriophage bacteriophage adsorption. LPS is a complex polymer made up of monosaccharides and fatty acids. Structurally, it incorporates 3 parts lipid A, core and O-chain (side chain, O-antigen). Lipid A usually is a disaccharide composed of two D-glycosamine moieties linked by $-1,6-bond with attached fatty acids (up to 8 residues) mediated by ester or amide group. Lipid A performs the role of hydrophobic anchor fixing in plasmatic membrane the whole construction bound via short oligosaccharide core to O-chain consisting of polymeric carbohydrate links (Wilkinson, 1996). There are two types of LPS: Smooth (S) type is characterized by typical LPS structure, i.e. comprising lipid A, core and side chain. Rough (R) type lacks O-chain but contains lipid A and the core. Some bacteriophages might adsorb to both LPS types. Phages specific to S-type LPS display an extremely narrow host range specificity determined by large variability of O-antigen structure in bacteria of different taxonomic groups. Bacteriophages recognizing R-type and vice versa show a broader host range since the structure of LPS core is rather conservative in various species and genera of gram-negative bacteria. A common feature of bacteriophages fixing to LPS O-chain is that their adsorption results in specific enzymatic cleavage of polysaccharide chain. g15 and P22 may be referred to such phages possesing endorhamnosidase activity and ability to lyse the bond Rha-1 → 3-Gal in O-antigen of Salmonella anatum and Salmonella typhimirium, respectively (Takeda and Uetake, 1973; Kanegasaki and Wright, 1973; Iwashita and Kanegasaki, 1973; 1976; Eriksson and Lindberg, 1977; Eriksson et al., 1979). Bacteriophage n1 (Reske et al., 1973) infecting Salmonella johannesbury is characterized by endo-1,3-N-galactoseaminidase activity (Chaby and Girard, 1980; Girard and Chaby, 1981). Bacteriophage S8 adsorbed on the surface of E. coli O8 shows endomannosidase activity, breaking down Man-1 → 3-Man link between repeating oligosaccharides and releases prevailing levels of hexa- and nonasaccharides (Reske et al., 1973; Prehm and Jann, 1976; Wallenfels and Jann, 1974). It was demonstrated for bacteriophage Sf6 isolated from strain Salmonella flexneri serotype 3a that its adsorption is associated with hydrolysis of Rha-1 → 3-Rha bond in O-chain of LPS (Lindberg et al., 1978). Virus H-F6S is able to bind to S. flexneri strains containing O-chain in LPS. Mutant strains lacking O-antigen are resistant to phage H-F6S but they are sensitive to other phages, like T3, T4, T7, with the respective receptors lying in the area of LPS core. It seems in wild-type strains these areas are hidden by O-chain complicating access for bacteriophages. In common, bacterial viruses adsorbing to O-antigen chain of LPS in gram negative bacteria recognize it 147 via enzyme localized at the tail end, which upon recognition and attachment hydrolyzes one of the bonds in polysaccharide chain of O-antigen. Besides, described bacteriophages have a similar morphology hexagonal head, short tail with base plate where spikes are localized. According to Bradley classification they are referred to group C (Bradley, 1967), and to podoviridae family according to modern classification. Position of a receptor in O-chain of S-type of LPS is described for phage 2. This bacteriophage infecting wild type strain of Pseudomonas aeruginosa B1 is affiliated to group B according to Bradley classification and to siphoviridae family according to modern classification because it has a long expanded tail (Bartell et al., 1971). This phage displays depolymerase activity owing to the constituent enzyme. After treatment of bacterial cells with purified enzyme isolated from virion, bacteria P. aerurinosa lose phage sensitivity (Castillo and Bartell, 1976). As mentioned above, structure of R-type LPS is limited by lipid A and the core region. In some mutant strains the structure of LPS core could be incomplete which according to a series of reports may result from disruption in core biosynthesis process occurring at different stages. Such structural aberrations could severely affect bacteriophage adsorption. For instance, phage F0 lysing wild-type Salmonella strains containing LPS with complete core. N-acetylglucosamine residue linked to the rest of the chain with "-1,2-bond is located at its terminal position. Mutants lacking this terminal glucosamine moiety are resistant to viral infection, and LPS isolated from such strains would not inactivate bacteriophage F0 (Lindberg, 1967; Lindberg and Hellerovist, 1971). Phages MX174, S13 and 6SR also require full LPS core for Salmonella and Shigella adsorption, with certain distinctions. Phage MX174 infects S. typhimirium strains showing on outer membrane surface LPS with complete core not protected by O-antigen. Mutants lacking terminal N-acetylglucosamine are still able to bind virus, although at lesser degree. On the other hand, absence of terminal glucosamine residue does not influence adsorption of phage S13 (Jazwinski et al., 1975). Whole core terminating in glucosamine is essential for optimal adsorption of phage 6SR to cells of S. typhimirium and S. flexneri. Yet, mutants of S. typhimirium defective in core biosynthesis or mutants of S. flexneri containing LPS with disaccharide composed of glucose and heptose moieties at its terminal position are also sensitive to phage infection, but at a lower rate (Lindberg and Hellerovist, 1971). The afore-mentioned phages behave in different way with respect to E. coli. Bacteriophage MX174 lysing E. coli C loses ability to adsorb to LPS lacking in its core terminal galactose residue (Feige and Stirm, 1976). Similarly, strain E. coli K12 sensitive to phage 148 Rakhuba D.V. et al. 6SR contains glucose moiety in the terminal position of LPS core (Picken and Beacham, 1977). It is well known that receptors for T-phages, specifically T3, T4 and T7 are components of R-type LPS of Shigella and Esherichia (Jesaitis and Goebel, 1952; Michael, 1968; Weidel, 1958). Phage T3 is adsorbed on the surface of S. flexneri mutants harboring core terminated with glucose linked to heptose by glycoside bond. The LPS isolated from these strains possesses the highest inactivating capacity towards this virus. Phage T7 adsorbs best on S. flexneri mutants with core terminated with galactose residue bound to glucose. Mutant strains E. coli K12 with core ending up in heptose and glucose are able to adsorb phages T3 and T7 (Picken and Beacham, 1977). The highest inactivating potential for phage T4 was displayed by LPS isolated from S. flexneri possesing in the core terminal disaccharide glucose-heptose. Mutants with complete core are also sensitive to phage T4 infection but inactivating ability of their LPS is lower. In E. coli B cells the optimal receptor proved to be LPS containing Glu-1→3-Glu-1→3-Hep in core terminal position (Prehm et al., 1976). Summing up, structure of LPS core responsible for recognition of the same bacteriophage may differ in bacteria of various microbial species and genera as demonstrated above by phages MX174, 6SR, T3, T4 and T7. It appears that major role in the receptor formation is played by spatial configuration around terminal glycosidic bond rather than terminal residue in polysaccharide chain of the core (Feige and Stirm, 1976). Receptors localized in cell wall of gram-positive bacteria. Cell wall of gram-positive bacteria significantly differs from the gram-negative species both in structure and chemical composition. The main component is peptidoglycan making up from 40 to 90% of the cell dry weight. Peptidoglycan is a heteropolymer composed of disaccharide monomer formed by N-acetylglucosamine and N-acetylmuramic acid. A tetrapeptide most often having the following structure: L-alanine D-glutamic acid L-diaminopimelic acid D-alanine is attached to a hydroxy group of N-acetylmuramic acid. This tetrapeptide mediates covalent links between peptidoglycan fibers so that cell wall represents a solid cover adjacent to the cell plasma membrane. Teichoic acids are the other vital constituents of gram-positive microorganisms. They are water-soluble polymers comprising glycerol or ribitol moieties linked together by phosphodiester bond and traversing peptidoglycan layer in direction perpendicular to the surface of plasmatic membrane. Most teichoic acids contain large ratio of D-alanine bound to free hydroxy groups, but other substitutes, like N-acetyl-D-glucosamine or D-glucose are found more often. Teichoic acids constitute the bulk of bacterial surface antigens. 3 Examined bacteriophages specific to Staphylococcus aureus, namely phages 3C, 52A, 71, 77, 79 and 80 are irreversibly inactivated by a complex of peptidoglycan and teichoic acids supplemented in addition by tetrapeptide attached to muramic acid. Reversible adsorption may be achieved during phage binding with teichoic acids connected with glycan fibers but irreversible procedure requires presence of tetrapeptide in the complex. Presence of N-acetylglucosamine in teichoic acid formula and O-acetyl groups in muramic acid residue is also essential for phage adsorption (Lindberg, 1973; Coyettl and Gheysen, 1968; Chatterjee, 1969; Gheysen et al., 1968; Murayama et al., 1968; Shaw and Chatterjee, 1971). Microorganisms of the genus Bacillus have the structure of peptidoglycan and teichoic acids similar to that of S. aureus. The only distinction is that N-acetylglucosamine as component of teichoic acids is substituted for D-glucose (Jazwinski et al., 1975). Due to this structural resemblance phages specific for S. aureus may adsorb on the surface of B. subtilis (Rakieten and Rakieten, 1937). D-glucose moiety plays a key role for adsorption of bacteriophages specific for B. subtilis. Phages M1, M25, M29, SP3, SP10, SP02 and µ were not able to adsorb on the surface of B. subtilis mutants lacking D-glucose in teichoic acid composition (Glacer et al., 1966; Hemphill and Whiteley, 1975; Young, 1968; Lindberg, 1973). Yet, some phages could infect bacterial cell without glycosylated teichoic acids in case growth occurred on the surface of solid nutrient media rather than in submerged culture (Yasbin et al., 1976). Protein GamR involved in adsorption of phage ( was identified in cell wall of B. anthracis. This protein is probably the component of cobalt transport system. B. cereus and B. thuringiensis also display on the surface GamR-like proteins. Only B. cereus is sensitive to phage ( infection although electron microscope studies have shown adsorption of phage particles to cells of both microbial species. It appears, missing additional surface structures in B. thuringiensis cells are indispensable for cell penetration and further phage propagation (Davison et al., 2005). Bacteriophages specific for Lactobacillus delbrueskii are inactivated by lipoteichoic acids isolated from cell wall of this microbial species. Inactivation degree depends on available D-alanine and L-glucose residues bound to fee hydroxyl groups of teichoic acids. An increase in D-alanine level results in reduced inactivating ability of lipoteichoic acids and their preliminary incubation with glucose-specific lectin ConA leads to complete inhibition of phage adsorption (Raisanen et al., 2007). Bacteriophages infecting Lactococcus lactis initially adsorb to polysaccharide cell wall. For some phages this step is irreversible (Monteville et al., 1994; 3 Receptors, adsorption and penetration of bacteriophage Schafer et al., 1991; Valyasevi et al., 1990; Valyasevi et al., 1994). Rhamnose, glucose and galactose moieties, as a part of extracellular polysaccharides are responsible for primary recognition and attachment of phage virions. Phage eb7 is characterized by adsorption to glucosamine or galactosamine residues (Keogh and Pettingill, 1983). Viruses of lactic acid streptococci belonging to group 2c and phage kh require specific protein (a phage infection protein) for irreversible secondary binding with bacterial cell wall (Monteville et al., 1994; Babu et al., 1995; Geller et al., 1993; Valyasevi et al., 1991). Receptors localized in capsular polysaccharides, pili and flagella. Many bacteriophages are attracted to bacterial pili, flagella, capsular and slime polysaccharides as receptors. Among viruses adsorbing to flagella several agents have been reported including phage P infecting representatives of Enterobacteriacae family Salmonella, Serratia, and E. coli, phage PBS7 attached to B. subtilis, B. pumilus, B. licheniformis, phage PBP7 specific to B. pumilis, phage 771 infects R. lupine (Shade et al., 1967; Lovett, 1972; Lotz et al., 1977). The phages have the same mechanism of adsorption, where the virion is fixed to the distal part of flagella via tail fibers. This adsorption stage is reversible; electron microscopic photos show that phage attachment does not result in release of nucleic acid from capside. Further on the virion moves closer to cell surface ultimately binding irreversibly to the baseplate of flagella. Phages MAcM4 and MAcS2 infecting Asticcacaulis biprosthecum also specifically adsorb to flagella via site connecting head and tail of the phage whereas distal part of the tail remains free for adsorption to the surface of bacterial cell (Pate et al., 1973). The attached virion is able to move along flagella towards cell and also may adsorb to the surface of neighbor cell. Many bacteria have external protective layers in the form of capsules or slime. Such layer may block access of bacteriophage to receptor localized in the cell wall or may be used for adsorption of phages, particularly those which fail to attach to bacteria devoid of capsules (Chakrabarty et al., 1967; Park, 1956). One of the bacteriophage receptors located in capsules of gram-negative bacteria is Vi-antigen typical for representatives of Salmonella, Citrobacter and E. coli. The polymer consists of residues of N-acetyl-Dgalactosaminuronic acid linked by "1,4-bond and partially O-acetylated (Luderitz et al., 1968). Studies on interaction of phage II with isolated Vi-antigen demonstrated that virion adsorption was accompanied by enzymatic cleavage of side acetyl groups while total chain depolymerisation did not occur (Taylor, 1965; 1966). The enzyme catalytically governing this reaction is localized in phage tail. Deacetylated Vi-polysaccharide loses capacity of further phage binding, but 149 this property is recovered after reverse acetylation. It should be noted that such interaction is reversible, while components of bacterial cell wall are essential for irreversible binding. Adsorption of other phages to capsular polysaccharides is also associated with enzymatic activity but in this case it is aimed at depolymerisation of main chain. Enzymes displaying endoglucosidase activity were characterized for phages of E. coli K29 and phage of Klebsiella K11 (Stirm et al., 1971a; 1971b). Virus K2 hydrolyzes capsule of Aerobacter aerogenesis using glucane hydrolase (Yurewicz et al., 1971) splitting "1, 3-bond between galactose residues. A common feature of these phages is their similar virion morphology and the interaction of phages with capsular polysaccharides is a reversible process. The capsule acts as a receptor for initial phage attachment whereas cell wall components are essential for irreversible binding (Taylor, 1966; Stirm et al. 1971b). Viruses of 2 types are present among bacteriophages that are able to adsorb to pili of RNA-containing viruses with isometric capsid and DNA-containing viruses in the form of filaments. A peculiarity of such phages is that they use as receptors only sex pili of bacteria, able to adsorb several hundred phage virions. Phages P17, M12, fr, Q$, f2, f4 infecting E. coli are most thoroughly studied among RNA-containing viruses. All above-mentioned phages composed of vast amount of identical subunits are about 27 nm in diameter (Hohn and Hohn, 1970). The second capside component is protein A responsible for recognition and adsorption of virion to pili (Roberts and Steitz, 1967). This protein is available in virion as one copy and upon RNA injection it penetrates into host cell with nucleic acid (Steitz, 1968a; 1968b; Krahn et al., 1972). DNA-containing filamentous phages recognizing pili as receptors may be subdivided into 2 groups: Ff and If phages adsorbing to terminal parts of F and I pili, respectively (Meynell and Lawn, 1968; Schlesinger, 1932). Unlike RNA-containing phages, only few virions may adsorb to one pilus. Binding is also mediated by protein A, similar to RNA phage case (Meynell and Lawn, 1968). Mechanisms of phage adsorption and penetration Rate of adsorption is the value characteristic of each phage-host pair and it may vary depending on concentration of phage/host. Since bacteriophages do not have specific structures responsible for virion motion and, consequently, they cannot move independently, the adsorption process is the result of random phage-cell collision described by active mass law (Schlesinger, 1932). It appears therefore that as concentration of virions and bacterial cells grows, the number of random 150 Rakhuba D.V. et al. collisions tends to rise which, in turn, leads to higher adsorption rate. Rate of adsorption is also determined by a series of diverse non-specific physical-chemical factors (pH, temperature, presence in the media of certain substances and ions) and depends on host physiological state and cultural conditions (Hershey et al., 1994; Delbruck, 1940; Quiberoni and Reinheimer, 1998; Sillankorva et al., 2004). Virion adsorption on host cell surface is usually illustrated as the process consisting of 2 stages: reversible and irreversible binding. It should be noted that molecular mechanisms of interaction at both stages of adsorption are specific for different phagehost systems and they may vary significantly in representatives of diverse taxonomic groups. As a rule, penetration of nucleic acid takes place after irreversible adsorption phase. Mechanisms of this process are specific for each phage, or phage group. Electrochemical membrane potential, ATP molecules, enzymatic splitting of peptidoglycan layer or all three factors may be vital for penetration of genetic material inside the bacterial cell. Processes of adsorption and phage penetration into cells are investigated in most detail for viruses of E. coli, namely T4, T5, T7 (Letellier et al., 2004). Some findings are available on processes of adsorption and penetration of viruses incorporating plasmatic membrane. Below these mechanisms will be considered separately for each virus. T4-like phages. Initial stage of adsorption for T4-like bacteriophages consists in reversible attachment of long tail fibers to specific receptors on the surface of outer membrane. It is necessary for successful infection that 3 or more of tail fibers could adsorb to cell surface since they play a critical role in triggering conformational changes of phage tail essential for DNA penetration into the cell (Crawford and Goldberg, 1977; 1980; Arscott and Goldberg, 1976). After phage gets attached to the cell via long fibers, baseplate changes its shape and as a result it takes stellar conformation. Finally 6 short fibers are generated and they irreversibly adsorb to heptose moiety in LPS core (Riede et al., 1985, Montag et al., 1987). Conformational alteration of baseplate simultaneously launches contraction of tail sheaths so that inner hollow tube punctures bacterial outer membrane (Moody, 1973). To facilitate penetration through peptidoglycan layer, enzyme lysozyme an integral part of baseplate protein gp5 is localized at the end of the tube. X-ray spatial analysis of the complex has revealed that domain responsible for peptidoglycan degradation is located at C-terminal part of spike-form structure (Kanamaru et al., 2002). Contact of this site with phosphatidylglycerol of the inner membrane is a signal for DNA transport along tail tube and its introduction 3 into the cell. Specific mechanism of DNA penetration via inner membrane remains to be elucidated, yet it is clear that phage tail does not penetrate through inner membrane and the process requires electrochemical potential on the inner membrane (Labedan and Goldberg, 1979). Bacteriophage T7. Phage T7 infection results in restructuring of tail proteins making up a cylinder shape inside phage head. This structure consists of 3 protein gp16 copies, 12 protein gp15 copies and 18 protein gp 14 copies. In addition, phage head contains 2 other proteins playing a key role in DNA transport into the cell gp13 and gp7.3. DNA molecule is spiraled onto the cylinder formed within the capsid. Initially, phage T7 interacts with bacterial LPSs via tail fibers. As soon as such contact occurs, the signal triggering irreversible virion binding is transmitted into phage capsid. Phage tail, tail fibers and protein gp13 are involved in signal transfer. Irreversible binding is associated with degradation of proteins gp13 and gp7.3 while proteins gp1416 pass through phage tail channel and shape the pathway across bacterial cell wall (Molineux, 2001; 2005). N-terminal part of gp16 is homologous to bacterial lytic enzyme transglycosylase making a major contribution in the restructuring of the peptidoglycan layer. Perhaps this domain is responsible for penetration of formed tubular structure through peptidoglycan to the inner membrane. DNA transport via the channel slows down when the first 850 base pairs get into the cell. The reason is protein gp16 serves as a special clip retarding rate of DNA penetration. The essence of this mechanism is that partial DNA uptake initiates transcription process and produces inhibitor of cell restrictases. Slow penetration of nucleic acid allows to synthesize this inhibiting factor earlier than DNA sites sensitive to restrictases appear inside the cell (Molineux, 2001; 2005). T5 and similar phages. Bacteriophage T5 includes hexagonal head 90 nm in size and long flexible tail around 200 nm. Protein FhuA localized in cellular outer membrane and engaged in transport of iron into the cell acts as a receptor for this phage. Adsorption to such protein is energy-sparing and irreversible, leading to DNA release in absence of other factors (Letellier et al., 2004). It was shown that besides irreversible adsorption, T5 is able to bind reversibly to O-antigen of bacterial LPS (Heller and Braun, 1979; 1982). Irreversible binding accelerates DNA introduction into host cell but it is not a crucial factor in adsorption process. This conclusion was made after it was proven that loss of tail fibers by the phage and lack of LPS O-chain in bacteria would not affect plating efficiency. The precise mechanism of DNA cellular uptake has not been established so far. It is known that injection of genetic material proceeds in 2 stages. Introduc- 3 Receptors, adsorption and penetration of bacteriophage tion of 8% DNA into cytoplasm causes a pause lasting 4 minutes (Lanni, 1965; 1968). Entered viral DNA controls synthesis of proteins responsible for degradation of bacterial DNA and switching off its transcription. Later DNA transport is resumed and the rest of nucleic acid is transferred inside bacterial cell. Phages T1 and n80 use the same transport protein FhuA as a receptor, although adsorption to it requires energy. Electrochemical proton gradient generated on inner membrane of bacterial cell by electron transport chain is applied as energy source. Electrochemical potential is transmitted to outer membrane via mediation of protein TonB. Its N-terminal part is anchored to cell inner membrane, while C-terminal interacts with FhuA receptor. Specific mechanism of energy transfer and mechanism of DNA transport into bacterial cell remains to be decoded. Bacteriophages incorporating plasmatic membrane. Bacterial viruses structurally comprising lipid bilayer attract vivid interest due to large diversity of mechanisms engaged in viral infection of host cell. For instance, morphologically identical DNA-containing phages PRD1 and PM2 use different receptors for adsorption, have distinct cell penetration mechanisms and infect different hosts. Bacteriophage PRD1 is characterized by a relatively broad host range, including E. coli, P. aeruginosa, S. enteric, but it can infect only strains carrying conjugative plasmids of N, P or W type (Olsen et al., 1974). These plasmids encode bacteriophage receptor (Lyra et al., 1991). Capsid in the form of an icosahedron is constituted from 24 copies of protein P3, and each vertex is crowned with spikes consisting of proteins P2, P5 and P31 (Benson et al., 1999; Butcher et al., 1995; Mindich et al., 1982; Grahn et al., 1999; Rydman et al., 1999). Membrane vesicle surrounding double-stranded DNA is inside the capsid. Protein content in the membrane is approximately 50%. At the first stage of adsorption the phage is reversibly bound to cell receptor via protein P2 and as a result spiky protein complex (P2, P5, P31) and a part of capsid proteins (P3) are released. Such modifications produce a hole in capsid envelope (Rydman et al., 1999). Further on phage membrane within the capsid is subjected to structural regrouping, yielding tubular tail penetrating into the bacterial cell via outer membrane and peptidoglycan layer (Lundstrom et al., 1979; Bamford and Mindich, 1982). Two proteins possessing lytic activity P7 and P15 are localized in the newly generated membrane tube. These proteins acting concertedly break down peptidoglycan at the penetration site, generating small holes (Rydman and Bamford, 2000). Reaching internal membrane, bacteriophagederived tube fuses with it, releasing DNA into cytoplasm. The process is accompanied by massive extracellular secretion of potassium ions and ATP molecules (Daugelavicius et al., 1997). 151 Phage PM2 also includes intracapsid membrane vesicle surrounding double-stranded DNA molecule (Espejo and Canelo, 1968). Penetration mechanism of genetic material in host cell is not thoroughly investigated but available data indicate that it differs significantly from that in phage PRD1. Adsorption to host cell surface is followed by capsid dissociation into protein constituents. Increased membrane permeability for lipophylic molecules of gramicidine B is also observed, pointing to potential fusion with bacteriophage membrane (Kivela et al., 2004). Protein P7 possessing lytic activity and probably playing an important role in the process of penetration through peptidoglycan layer was identified in membrane vesicle (Kivela et al., 2004). Penetration of genetic material inside the cell is associated with depolymerisation of the microbial inner membrane. RNA-containing bacteriophage n6 entering P. syringae cell, apart from nucleic acid, should inject RNA-dependent-RNA-polymerase because the host cell does not contain enzymes able to transcribe viral RNA. Structural peculiarity of this phage is the presence of outer lipid-protein envelope surrounding a capsid with confined complex RNA plus RNApolymerase (Bamford et al., 1976; Butcher et al., 1997; Kenney et al., 1992; Vidaver et al., 1973; Daugelavicius et al., 2005). Main receptors for phage n6 are type IV pili (Bamford et al., 1976; Vidaver et al., 1973) where phage is attached via protein P3 (Daugelavicius et al., 2005; Romantschuk and Bamford, 1985). Integral protein P6 localized in plasmatic membrane of the virion initiates fusion of host outer membrane and phage lipid envelope (Daugelavicius et al., 2005; Bamford et al., 1987). As a result of such membrane integration the virus capsid with contained nucleic acid floods into the periplasmic space. Endopeptidase P5 localized in the capsid envelope splits the peptidoglycan layer at the point of attack and the virus nucleocapsid reaches the internal membrane of the bacteria, (Daugelavicius et al., 2005., Caldentey and Bamford, 1992; Mindich and Lehman, 1979). According to electron microscopy observations, the final stage of virus penetration into bacterial cell envisages generation of membrane vesicle incorporating phage nucleocapsid (Peisajovich and Shai, 2002). The process is similar to viral endocytosis in humans and animals (Smith and Helenius. 2004). Mechanisms of disclosing vesicle in host cytoplasm and virion decoating are not well established to date. DNA-containing phage Bam35 infects cells of gram-positive bacteria B. thuringiensis and contains plasmatic membrane located within the capsid. The mechanism of phage penetration into the cell is not fully investigated. It is known that N-acetylmuramic acid residue a cell wall component serves as a receptor. Penetration through the peptidoglycan layer 152 Rakhuba D.V. et al. is related to enzymes gp26 and gp30 localized in the capsid envelope. The transport of genetic material across the plasmatic membrane depends on presence of bivalent cations in the media, whereas phage adsorption and degradation of peptidoglycan are not dependent (Gaidelyte et al., 2006). Conclusions The process of bacteriophage adsorption to a receptor on cell surface is the first stage in virus-host interaction. Adsorption phase defines phage-host specificity and mechanisms governing resistance of bacteria to virus infection. The nature of receptor, aspects of its chemical composition and spatial configuration, structure of viral receptor-binding protein and specific interaction mechanisms all these factors play a key role in shaping stable bacteriophage-host population. In early studies on phage-host interactions it was assumed to regard processes of adsorption and penetration of nucleic acid into bacterial cell separately from each other as different stages of a virus life cycle. The massive amount of data collected so far evidences a deep correlation between virus adsorption and penetration into bacterial cell. Irreversible adsorption stage virtually always initiates penetration of genetic material inside host cell. It appears desirable therefore to consider the first phase of interaction between virus and bacterial cell as a complex process comprising adsorption, structural alterations of virus and host cell wall and transport of nucleic acid into the cell. It should be stated that analysis of literature findings indicates a large diversity of bacteriophage-host populations with respect to nature and structure of the receptor, virus antireceptor and molecular mechanisms of virion-cell interactions. 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Chem. 246: 56075616. 156 Rakhuba D.V. et al. 3 Polish Journal of Microbiology 2010, Vol. 59, No 3, 157160 ORIGINAL PAPER Interactions between Borrelia burgdorferi and Mouse Fibroblasts TOMASZ CHMIELEWSKI* and STANIS£AWA TYLEWSKA-WIERZBANOWSKA Laboratory of Rickettsiae, Chlamydiae and Spirochetes, National Institute of Public Health National Institute of Hygiene, Warsaw, Poland Received 18 June 2010, revised 15 July 2010, accepted 17 July 2010 Abstract Borrelia burgdorferi spirochetes are an infectious agent of Lyme borreliosis. The aim of our studies was to investigate the fate of engulfed B. burgdorferi cells in L-929 mouse fibroblasts and to observe development of intracellular infection in vitro after 2 and 48 h. Electron microscopic studies reveal consecutive stages of B. burgdorferi spirochetes penetration to mouse fibroblasts in vitro. It has been observed, as a first step attachment and engulfment of spirochetes followed by formation of vacuoles. After 48 hours of infection, vacuoles of fibroblastic cells have been seen full of B. burgdorferi bacteria and latter they have been released from infected cells to extracellular space. It can be the evidence that B. burgdorferi multiply intracellulary. K e y w o r d s: Borrelia burgdorferi, fibroblasts, interaction Introduction Borrelia burgdorferi spirochetes are an infectious agent of Lyme disease, also known as borreliosis, which is the most common tick-borne disease in the northern hemisphere. Early manifestations of infection include fever, headache, fatigue, and a characteristic skin rash called erythema migrans. Untreated, it can cause late symptoms involving tissue of the joints, heart, and nervous system (Stanek et al., 1996). Microscopic studies indicate that the bacteria can bind to the cell surface and enter the cytoplasm directly after inducing local engulfment and fragmentation of the plasma membrane. Several reports have described interactions between B. burgdorferi bacteria and different host cells. It has been shown that the spirochetes can enter mammalian immune cells and other cells as well as tick tissue. This probably allows the pathogen to survive in host tissues, to infect them and to escape the host defense (Hu et al., Linder et al., 2001, Sigal, 1997, Szczepanski et al., 1990, Thomas et al., 1989). However, there is no information on the fate of B. burgdorferi spirochetes inside eukaryotic cells and the way they leave the host cells. Various modes of bacterial entry into the host cell have been described as an essential pathogenic factors. Legionella pneumophila uptake is completed by a process termed bacteriopexis, followed by engulfment of the organisms with microvilli in association with intracellular cytoplasmic filaments (Oldcham et al., 1985). Another pathogen, Rickettsia prowazekii, can enter endothelial cells via induced phagocytosis and it is then released into the cell cytoplasm by disruption of the phagosomal membrane (Walker, 1984). Transmission electron microscopy has shown that viable or killed Candida organisms were attached to endothelial cells, then enveloped by cell membrane and incorporated into the endothelial cells within phagosomes (Rotrosen et al., 1985). The ability of Borrelia burgdorferi to attach to and invade human fibroblasts was investigated by confocal and scanning electron microscopy. Scanning electron microscopy has revealed that B. burgdorferi are tightly attached to fibroblast monolayers after 2448 h. Spirochetes were observed in the perinuclear region within human fibroblasts by laser scanning confocal microscopy (Klempner et al., 1993). The aim of our studies was to investigate the fate of engulfed B. burgdorferi cells in mouse fibroblasts and to observe the development of intracellular infection in vitro in electron and fluorescence microscopy. * Corresponding author: T. Chmielewski, Laboratory of Rickettsiae, Chlamydiae and Spirochetes, National Institute of Public Health National Institute of Hygiene; 24 Chocimska Street, 00-791 Warsaw;, Poland phone/fax (+48) 22 4521250; e-mail: [email protected] 158 3 Chmielewski T. and Tylewska-Wierzbanowska S. Experimental Material and Methods Borrelia afzelii VS461 strain (ATCC 51567) was grown in BSK-H medium Complete (Sigma Aldrich, St. Louis, USA) supplemented with 6% of rabbit serum for 7 days at 35°C in 5% CO2 atmosphere. The number of bacteria per 1 ml was counted in a Thoma counting chamber. Line L-929 (ATCC CCL-1, USA) were propagated in Eagles minimum essential medium (MEM) with L-glutamine and NaHCO3 (Biomed, Lublin, Poland), supplemented with 4% of fetal calf serum (ATCC, USA) at 37°C in 5% CO2 for 2 days. Cells were grown in shell-vials on glass coverslips inside tubes with screw caps (Sterilin, United Kingdom) until a confluent monolayer was obtained. Bottles containing cell line monolayer were inoculated with spirochetes culture containing 108 bacteria per 1 ml. L929 cells infected with B. burgdorferi (initial density 106 organisms/ml medium) were incubated for 2 and 48 hours at 35°C in 5% CO2 atmosphere. Infected mouse fibroblasts were fixed with acetone, washed three times with PBS and incubated 30 minutes with anti-B. burgdorferi human immune serum at 37°C. Next they were washed three times with PBS followed by incubation with rabbit anti-human immunoglobulins conjugated with FITC (DAKO, Denmark). Immunofluorescence was observed in the fluorescence microscope Eclipse E 400 (NIKON, Japan) at 500X magnification. All specimens were prepared according to standard technics (Glauert, 1975). The cultures were washed twice with PBS, fixed overnight with 2.5% glutaraldehyde. On the next day the cells were centrifuged at 750 g at 4°C and the pellet was washed with 2 ml 0.2 M sodium cacodylate buffer (pH 7.4) and re-centrifuged as above. After three washes, the cells were Fig. 1. Attachment of spirochetes to the cytoplasmic membrane of L-929 cell (X 30 000). fixed for 2 hours in 1% OsO4 in 0.1 M sodium cacodylate buffer. The cells were then washed three times in 0.1 M sodium cacodylate buffer and stained for 30 minutes by being resuspending in 1% aqeous uranyl acetate (Roth, Karlsruhe, Germany) solution. For embedding, fibroblasts were centrifuged and then dehydrated through a wash series in methyl alcohol solutions (from 25% to 100%) and embedded in epoxy resin and incubated overnight at 65°C. Transverse thin sections were cut and transferred to copper mesh 300 grids (Polysciences, St. Goar, Germany), stained with lead citrate and uranyl acetate and dried. The cells were observed in a JOEL 100C electron microscope (Japan) at magnifications from ×6000 to ×35000. Results Various stages of B. burgdorferi spirochetes infection in L-929 fibroblasts were observed. After two hours of incubation spirochetes were seen outside of the host cells. Their position suggested that most of them were motile. The first observed interaction step was attachment of the bacteria to the surface of the fibroblasts. In fluorescence microscopy it has been seen as adhesion to the cell surface. After two hours of incubation the spirochetes were bound and entered the mammalian cells. Most of them were bound apically. This contact triggered the engulfment of the bacteria in the cytoplasmatic host membrane (Fig. 1, Fig. 2). This process initiated the cellular uptake of single bacteria into phagocytic-like vacuoles. Within two hours of incubation such bacterial cell, surrounded by host cell membrane, were seen inside the fibroblast (Fig. 3). Some bacteria entered fibroblast cells in a different way. Fibroblast pseudopods bent around single spirochetes in the intercellular spaces. The pseudopods had a characteristic hook-like form, turned back to the fibroblasts membrane and slided along the Fig. 2. Cell membrane penetration (X 30 000). 3 Interactions between Borrelia burgdorferi and mouse fibroblasts 159 Fig. 6. Translocation of spirochetes to the intracellular space (X 6 700). Fig. 3. Single B. burgdorferi bacteria in vacuole in L-929 cytoplasm (X 33 000). were located in the peripheral part of the host cell adjacent to the cell membrane. Strong immunofluorescence of vacuoles and bacteria was observed in fluorescence microscopy. The vacuoles contained 15 to 20 bacterial cells as it has been seen on a cross sections. The number of spirochetes in the host vacuoles observed after two days of infection indicated that B. burgdorferi multiplied inside the fibroblasts (Fig. 5). Some of the vacuoles were disrupted and B. burgdorferi spirochetes were released to the extracellular space (Fig. 6). Discussion Fig. 4. Endocytosis of B. bugdorferi spirochetes (X 27 000). cell membrane. The observed manner of penetration resembled endocytosis however characteristic for coiling phagocytosis long appendages wrapping the spirochetes were also observed (Fig. 4). On the second day of infection the fibroblasts were seen to be vacuolized. Many vacuoles with bacteria Fig. 5. Multiplication of spirochetes within vacuoles (X4 000). Electron microscopic studies revealed the consecutive stages of B. burgdorferi spirochetes penetration into mouse fibroblasts in vitro. The first observed step was the attachment and engulfment of the spirochetes, followed by the formation of vacuoles and multiplication of bacteria inside vacuoles followed by release from the infected cells to the extracellular space. After 48 hours of infection, vacuoles of fibroblastic cells containing dozens of B. burgdorferi bacteria were seen. This can be taken as evidence that B. burgdorferi multiply intracellularly similarly to Legionella pneumophila, Coxiella burnetii and other obligatory intracellular parasites (Baca et al., 1983, Oldcham et al., 1985, Walker et al., 1984, Rotrosen et al., 1985). Studies by Comstock et al., (1989) with electron microscopy revealed that borreliae entered the endothelial cells and suggested that the organisms penetrated the host monolayers primarily by passing through them. Attachment of spirochetes is time and temperature dependent and pretreatment with heat, immune human serum or monoclonal antibodies to OspB reduce the binding to the endothelial cells (Thomas et al., 1989). Examination of spirochete-endothelial interactions demonstrated the presence of spirochetes in the intercellular junctions between endothelial cells as well as beneath the monolayers. Scanning electron microscopy identified a mechanism of transendothelial 160 Chmielewski T. and Tylewska-Wierzbanowska S. migration whereby spirochetes pass between cells into the amniotic membrane at areas where subendothelium is exposed (Szczepanski et al., 1990). Other study with Vero cells revealed that essential for the attachment process is metabolic activities of the spirochaete, not viability (ability to grow) (Hechemy et al., 1992). After entry of untreated B. burgdorferi, most of the spirochaetes were either free in the cytoplasm or tightly bound to the host membrane. In contrast, heat treated spirochaetes remained bound to host membrane in large phagosome-like vesicles (Comstock et al., 1989). It seems that several eukaryotic cells provide B. burgdorferi spirochetes with a protective environment contributing to their long-term survival (Peters et al., 1997, Rittig et al., 1998, Rittig et al., 1992). B. burgdorferi have been protected in fibroblasts for at least 14 days of exposure to ceftriaxone In the absence of fibroblasts, organisms did not survive. They were not protected from ceftriaxone by glutaraldehyde-fixed fibroblasts or fibroblast lysate, suggesting that a living cell was required. The ability of the organism to survive in the presence of fibroblasts was not related to its infectivity (Georgilis et al., 1992). Mouse keratinocytes, HEp-2 cells, and Vero cells showed a similar protective effect. Doxycycline or erythromycin were more effective in killing B. burgdorferi when they were grown in the presence of eukaryotic cells (Brouqui et al., 1996). Our findings show that in fibroblasts could occurs process of spirochetes multiplication. Difficulties with the isolation of B. burgdorferi from clinical material when cultured on artificial media also indicate that spirochetes are very fastidious bacteria, which require the presence of certain substances in the host cells for their growth. These observations allowed us to isolate several strains by inoculating cerebrospinal fluids, synovial fluids and blood of Lyme borreliosis patients into cell line culture (Chmielewski et al., 2003, Tylewska-Wierzbanowska et al., 1997). Literature Baca O.G. and D. Paretsky. 1983. Q fever and Coxiella burnetii: a model for host-parasite interactions. Microbiol. Rev. 47: 12749. Brouqui P., S. Badiaga and D. Raoult. 1996. Eucaryotic cells protect Borrelia burgdorferi from the action of penicillin and ceftriaxone but not from the action of doxycycline and erythromycin. Antimicrob. Agents. Chemother. 40: 15524. Chmielewski T., J. Fiett, M. Gniadkowski and S. TylewskaWierzbanowska. 2003. Improvement to laboratory recognition 3 of Lyme borreliosis with the combination of culture and PCR methods. Mol. Diagn. 7: 155162. Comstock L.E. and D.D. Thomas. 1989. Penetration of endothelial cell monolayers by Borrelia burgdorferi. Infect. Immun. 57: 16261628. Georgilis K., M. Peacocke and M.S. Klempner. 1992. Fibroblasts protect the Lyme disease spirochete, Borrelia burgdorferi, from Ceftriaxone in vitro. J. Infect. Dis. 166: 440444. Glauert A.M. 1975. Practical methods in electron microscopy. In: Fixation, dehydration and embedding of biological specimens. Vol. III, part 1. North-Holland Publishing Co., Amsterdam. Hechemy K.E., W.A. Samsonoff, H.L. Harris and M. McKee. 1992. Adherence and entry of Borrelia burgdorferi in Vero cells. J. Med. Microbiol. 36: 229238. Hu L.T. and M.S. Klempner. 1997. Host-pathogen interactions in the immunopathogenesis of Lyme disease. J. Clin. Immunol. 17: 35465. Klempner M.S., R. Noring and R.A. Rogers. 1993. Invasion of human skin fibroblasts by the Lyme disease spirochete, Borrelia burgdorferi. J. Infect. Dis. 167: 10741081. Linder S., C. Heimerl, V. Fingerle, M. Aepfelbacher and B. Wilske. 2001. Coiling phagocytosis of Borrelia burgdorferi by primary human macrophages is controlled by CDC42Hs and Rac 1 and involves recruitment of Wiskott-Aldrich syndrome protein and Arp2/3 complex. Infect. Immun. 69: 173946. Oldcham L.J. and F.G. Rodgers. 1985. Adhesion, penetration and intracellular replication of Legionella pneumophila and in vitro model of pathogenesis. J. Gen. Microbiol. 131: 697706. Peters J.D. and J.L. Benach. 1997. Borrelia burgdorferi adherence and injury to undifferentiated and differentiated neural cells in vitro. J. Infect. Dis. 176: 470477. Rittig M.G., J.C. Jagoda, B. Wilske, R. Murgia, M. Cinco, R. Repp, G.R. Burmester and A. Krause. 1998. Coiling phagocytosis discriminates between different spirochetes and is enhanced by phorbol myristate acetate and granulocyte-macrophage colonystimulating factor. Infect. Immun. 66: 62735. Rittig M.G., A. Krause, T. Häupl, U.E. Schaible, M. Modolell, M.D. Kramer and E. Lütjen-Drecoll. 1992. Coiling phagocytosis is the preferential phagocytic mechanism for Borrelia burgdorferi. Infect Immun. 60: 420512. Rotrosen D., J.E. Edwards, T.R. Gibson, J.C. Moore, A.H. Cohen and I. Green. 1985. Adherence of Candida to cultered vascular endothelial cells: mechanism of attachment and endothelial cell penetration. J. Infect. Dis. 152: 12641273 Sigal L.H. 1997. Lyme disease: a review of aspects of its immunology and immunopathogenesis. Ann. Rev. Immunol. 15: 6392. 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Immun. 44: 20510 Polish Journal of Microbiology 2010, Vol. 59, No 3, 161165 ORIGINAL PAPER Clonal Analysis of Staphylococcus aureus Strains Isolated in Obstetric-Gynaecological Hospital EWA SZCZUKA1, ANNA SZUMA£A-K¥KOL2, ANNA SIUDA1 and ADAM KAZNOWSKI1* 1 Department of Microbiology, Institute of Experimental Biology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznañ, Poland 2 Hospital Laboratories, Obstetric-Gynaecological Clinical Hospital, Polna 33, 60-535 Poznañ, Poland Received 1 December 2009, revised 16 June 2010, accepted 17 June 2010 Abstract Epidemiological studies were carried out on 135 isolates of Staphylococcus aureus strains originating from medical staff, patients, and hospital environment. Restriction fragment length polymorphism (RFLP) analysis revealed genetic diversity of S. aureus isolates. Some clones were transmitted among nurses, doctors and patients. Our studies also demonstrate contamination of the hospital environment with S. aureus strains and there is a possibility that the patients acquire staphylococci from the environment. Moreover, we found that many medical staff workers were colonized with S. aureus and the transmission of these strains to patients is possible. K e y w o r d s: Staphylococcus aureus, epidemiological studies, RFLP analysis Introduction Staphylococcus aureus has been recognised as an important pathogen causing many infections such as septicemia, pneumonia, wound infections, septic arthritis, osteomyelitis and postsurgical toxic shock syndrome (Kloos and Bannerman, 1999). On the other hand, many healthy people are persistently or intermittently colonized with S. aureus at their anterior nares. Approximately 20% of individuals almost always carry one type of strain, 60% harbor S. aureus intermittently. About 20% of people almost never carry S. aureus. Colonization of human noses by S. aureus appears to play a role in the epidemiology and pathogenesis of infection (Kluytmans et al., 1997). S. aureus is one of leading agents of nosocomial infections therefore for public health epidemiologists and clinicians involved in patient management of prime importance is understanding the dynamics of the spread and transmission the of bacteria within a hospital, this being crucial for their control and eradication. Molecular typing approaches have been used to a great advantage in identifying and monitoring the local and international spread of S. aureus strains (tìpán et al., 2004). The aim of the present study was to evaluate the clonal composition of Staphylococcus aureus strains isolated from specimens taken from patients, medical staff and hospital environment at the ObstetricGynaecological Clinic Hospital in Poznañ, Poland. Moreover, we wanted to elucidate the spread of clones in different departments and the possible transmission routes of these clones. Experimental Material and Methods Bacterial strains. S. aureus strains were isolated from clinical specimens obtained from neonates and adult patients treated in different medical units in a hospital in Poznañ. Several members of the healthcare staff were screened for S. aureus nasal, throat and hand carriage. Also specimens from medical equipment and hospital environment were taken. All strains were identified as S. aureus by analysis of cell morphology, Gram stain, and catalase production, using the latex coagulase test and the ID 32 Staph Kit (bioMérieux, France). Methicillin susceptibility test * Corresponding author: A. Kaznowski, Department of Microbiology, Faculty of Biology, A. Mickiewicz University, ul. Umultowska 89, 61-614 Poznañ, Poland; phone (+48) 61 529 5937; fax (+48) 61 829 5590; e-mail: [email protected] 162 Szczuka E. et al. 3 Fig. 1. Dendrogram showing genetic relatedness of 135 strains of S. aureus determined by analysis of RFLP fingerprint patterns using Dice similarity coefficient and UPGMA cluster method. 3 163 Clonal analysis of S. aureus was determined by disc diffusion method and the results were interpreted in accordance to the criteria of the Clinical Laboratory Standards. The presence of the mecA gene was determined by PCR as described previously (Geha et al., 1994). RFLP analysis. Chromosomal DNA from the S. aureus strains were extracted according to methods described by Pitcher et al. (1989). A PCR was applied to simultaneously amplified part of the hypervariable region (HVR), a part of spa gene and a part of the coa gene based on primers established by Wichelhaus et al., 2001. The PCR product was incubated overnight with 10 units of HaeII restriction enzyme (MBI Fermentas) at 37°C. The resulting fragments were separated in 1.5% agarose gel. The DNA in gels were documented with V.99 Bio-Print system (Vilber Lourmat, Torcy, France). A computer analysis was carried out using GelCompar II (version 3.0; Applied Maths, Kortrijk, Belgium) software. Similarity between fingerprints was calculated with the Dice coefficient. Cluster analysis was performed using the unweighted pair-group method with average linkages (UPGMA). Results and Discussion All strains included in this study were methicillin/ oxacillin sensitive phenotypically and did not harbour the mecA gene. Our results demonstrated that many clones of S. aureus were coexisting in one hospital. This conclusion is based upon the results generated by clonal analysis of 135 strains of S. aureus (Fig. 1). We revealed 25 clusters at the 90% similarity level (Table I). Strains within these clusters were considered to be genetically related. We identified three major clones (cluster 8, 10, and 12), which included 28% strains of S. aureus. As shown in the dendrogram, there are five smaller clusters consisting from six to four strains. In addition, we found a considerable number of minor clusters, each harboring three or two strains. Minor clusters reached 36 strains (29%). Moreover, we identified 32 single strains with unique genotypes. The fact, that 25 clusters were identified next to 32 unique genotypes indicating a large genetic diversity among isolates of S. aureus obtained from the hospital. Similar result was obtained previously by Van Dijk et al., 2002, who described genetic diversity among S. aureus isolates from a Dutch Teaching Hospital. Although S. aureus has been described as the normal flora of nasal carriage, several epidemiological studies indicated that nasal carriage have increased risk for staphylococcal infections especially in specific group of patients (Archer and Climo 2001; KooistraSmid et al., 2004; Melless et al., 2004). Staphylococcus sp. infections are most commonly acquired from Table I Results of S. aureus clinical isolate typing by using RFLP analysis CluStrain No ster 1 MPU S 68, 69 2 MPU S 43 MPU S 76 3 MPU S 62, 136 4 MPU S 8, 9 MPU S 52, 58, 56 MPU S 158 5 MPU S 25 MPU S 26 6 MPU S 55, 61 7 MPU S 73, 128 MPU S 114, 151 MPU S 129 8 MPU S 10, 126, 133, 152, 155 MPU S 27, 41 MPU S 118, 156, 119, 145, 148 MPU S 153, 160 MPU S 154 MPU S 157 MPU S 159 9 MPU S 13, 54 MPU S 131 10 MPU S 33, 39, 42, 83, 99 MPU S 35, 84 MPU S 37, 38 MPU S 40 MPU S 123 11 MPU S 106 MPU S 107 MPU S 110 12 MPU S 18, 48, 108 MPU S 34, 53 MPU S 60 MPU S 109 MPU S 111 13 MPU S 67, 91 14 MPU S 29 MPU S 121 MPU S 122, 124 15 MPU S 36 MPU S 46, 49 16 MPU S 77, 142 MPU S 132, 135 MPU S 134 MPU S 137 17 MPU S 31 MPU S 32 18 MPU S 94 MPU S 98 MPU S 113 19 MPU S 70, 72, 130 MPU S 79 MPU S 93 MPU S 120 Source of isolation nasal swab of medical staff clothes of medical staff throat swab of medical staff throat swab of medical staff abscess of neonates nasal swab of medical staff hand basin throat swab of medical staff hands of medical staff nasal swab of medical staff throat swab of medical staff wound of patient patients bed throat swab of medical staff clothes of medical staff nasal swab of medical staff patients bed hand basin vagina of patient throat swab of neonate throat swab of medical staff skin of neonate clothes of medical staff nasal swab of medical staff hands of medical staff skin of neonate scale in delivery room hand basin abscess of patient clothes of medical staff nasal swab of medical staff throat swab of medical staff abscess of neonate vagina of patient hands of medical staff patients bed throat swab of medical staff hand basin patients bed clothes of medical staff skin of neonate hand basin patients bed catheter of neonate nasal swab of medical staff nasal swab of medical staff hands of medical staff hand basin patients bed vagina of patient nasal swabs of medical staff hands of medical staff patients bed scale 164 3 Szczuka E. et al. Table I continued CluStrain No ster 20 MPU S 86, 87 MPU S 89 21 MPU S 85, 92 22 MPU S 14 MPU S 19 23 MPU S 146 MPU S 150 24 MPU S 22, 80 25 MPU S 20 MPU S 23 Source of isolation medical equipment nasal swab of medical staff nasal swabs of medical staff skin of neonate hands of medical staff throat swab of medical staff wound of neonate nasal swab of medical staff nasal swab of medical staff conjunctive of neonate patients own flora, however patients may become infected from other healthy carriers. It is worthy to note that S. aureus carriers can contaminate their clothes and their surroundings through air, dust etc. It is well known that decolonization may reduce the risk of S. aureus infections in carries and prevent transmission to other patients. Recently, Gilpin et al. (2010) indicated that the standard decolonization protocol did not result in long-term clearance of MRSA carriage for most patients. In this study we found that many nurses and doctors harbored and/or were colonized with S. aureus strains. We also found strains of S. aureus on nurses hands and clothes. We found six clusters (4, 7, 8, 10, 11, 16) comprising strains isolated from patients, nurses and hospital environment. It is difficult to determine exactly where and to whom transmission of S. aureus occurred. It is probably, that the cross-transmission of S. aureus occurred via hands, which may be contaminated by contact with colonized or infected body sites of medical staff or colonised or infected patient or with devises. Nurses and doctors hands could be contaminated by strains existing in the hospital environment. For example, cluster 4 included strains of S. aureus isolated from two neonates, three nurses and one strain from environment. It is important to note that these neonates suffered from skin infection. We also identified clusters (9, 12, 22, 23) that included only strains isolated from patients and medical staff. For example cluster 22 included one strain isolated from skin of neonate and one isolate originated from nurses hands working in delivery room. Therefore, we think that the medical staff could be considered an important vector in the chain of S. aureus transmission. Previously, it has been reported that strains isolated from nurses hands could be regarded as the source of staphylococcal scaled skin syndrome (SSSS) of neonates in a maternity unit in Paris (Helali et al., 2005). Similary, Bertin et al., 2006, indicated that strains isolated from a healthcare worker suffering from otitis externa and carrries Staphylococcus aureus could be responsible for the outbreak of bloodstream infections in a neonatal intensive care unit. Hand hygiene has been recognized as the key to prevent transmission of S. aureus strains and to reduce the nosocomial infections. Sroka et al. (2010) indicated that the increasing consumption of hand antiseptics was associated with a significant reduction of S. aureus rate. We isolated strains from medical equipment, patients beds or hand wash basins. Previously, several authors also demonstrated that hospital equipment and environment could be the reservoirs of S. aureus (Ohara et al., 1998; Embil et al., 2001; Hardly et al., 2006; Sexton et al., 2006). In addition, it is noteworthy that staphylococci can persist in clinical areas for a long period of time (Sexton et al., 2006). Recently, Aldeyab et al. (2009) indicated that environmental decontamination using detergents and hypochlorite was effective in eliminating MRSA strains from hospital environment. We identified many clusters, which grouped strains isolated from patients and hospital environment. This suggests that patients acquired S. aureus from the hospital environment. However, we can not exclude the possibility that patients contaminate their surrounding such as hospital beds, hand wash basins etc. We found cluster 10, which included strains isolated from neonate, from scales used to weigh neonates after birth in the delivery room. In this cluster we also found strains, which were derived from cultures originated from healthcare workers. It might be speculate that strains obtained from scales were via contaminated nurses hands transformed on childish skin. It is well known that shared equipment in common places is an additional source of dissemination of S. aureus. Our results illustrate the great genetic diversity among S. aureus strains in a hospital. This study also reveals contamination of hospital environment with S. aureus strains and the need for more effective cleaning of the hospital environment in order to eliminate reservoirs of these strains. 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Simouns, H.A Verbrugh and A. van Belkum. 2004. Natural population dynamics and expansion of patogonic clones of Staphylococcus aureus. J. Clin. Investig. 114: 17321740. Ohara T., Y. Itoh and K. Itoh. 1998. Ultrasound instruments as possible vectors of staphylococcal infections. J. Hosp. Infect. 40: 7377. Pitcher D.G., N.A. Saunders and R.J. Owen. 1989. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol. 8: 151156. Sexton T., P. Clarke, E. ONeill, T. Dillane and H. Humphreys. 2006. Environmental reservoirs of methicillin-resistant Staphylococcus aureus in isolation rooms: correlation with patient isolates and implications for hospital hygiene. J. Hospit. Infect. 62: 187194. Sroka S., Gastmeier P. and E. Meyer. 2010. Impact of alcohol hand-rub use on methicillin-resistant Staphylococcus aureus: an analysis of the literature. J. Hosp. Infect. 74: 204211. tepán J. R., J. Pantuèek and J. Dokaø. 2004. Molecular diagnostics of clinical important staphylococci. Folia Microbiol. 49: 355386. Van Dijk Y., C.L.C. Wielders, A.C. Fluit, A. Paauw, R.J.A. Diepersloot and E.M. Mascini. 2002. Genotyping of clinical methicillin-susceptible Staphylococcus aureus isolates in Duch Teaching Hospital. J. Clin. Microbiol. 40: 663665. Wichelhaus T., K-P. Hunfeld., B. Böddinghaus, P. Kraiczy, V. Schäfer and V. Brade. 2001. Rapid molecular typing of methicillin-resistant Staphylococcus aureus by PCR-RFLP. Infect. Control. Hosp. Epidemiol. 22: 294298. 166 Szczuka E. et al. 3 Polish Journal of Microbiology 2010, Vol. 59, No 3, 167173 ORIGINAL PAPER Simultaneous Detection and Differentiation of Pathogenic and Nonpathogenic Leptospira spp. by Multiplex Real-Time PCR (TaqMan) assay ORHAN BEDIR1, ABDULLAH KILIC1*, ERDINC ATABEK2, AHMET MERT KUSKUCU 1, VEDAT TURHAN3 and A. CELAL BASUSTAOGLU1 1 Department of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy and School of Medicine 06018, Ankara, Turkey 2 Central Veterinary Control and Research Institute, Etlik, Ankara, Turkey 3 Department of Infectious Diseases, Gulhane Military Medical Academy and School of Medicine, Haydarpasa Training Hospital, Istanbul, Turkey Received 3 August 2009, revised 1 June 2010, accepted 15 June 2010 Abstract Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1× 102 cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity. K e y w o r d s: Leptospira genus, leptospirosis, multiplex real-time PCR (TaqMan) assay, pathogenic Leptospira, saprophytic Leptospira Introduction Leptospirosis is caused by spirochetes of the genus Leptospira (Guerra, 2009). The Leptospira genus has been classified into 17 species by DNA-DNA hybridization. This genus is further divided into three groups as pathogenic, nonpathogenic, and opportunistic/possibly pathogenic. The pathogenic leptospires include eight species: Leptospira interrogans, Leptospira kischneri, Leptospira borgpetersenii, Leptospira santarosai, Leptospira weilii, Leptospira alexanderi, Leptospira genomospecies 1 and Leptospira noguchii (Brenner et al., 1999). The Leptospira genus has also been classified on the basis of surface antigen patterns. They are divided into at least 250 serotypes that have major antigens in common and are combined into 24 serogroups (Dutta and Christopher, 2005). Leptospirosis is identified as one of the emerging infectious diseases and a major public health concern worldwide. The organism affects virtually any mammal, including humans. Humans acquire the organisms through contact with contaminated soil, water, vegetation or with the body fluids of animals harboring leptospires (Palaniappan et al., 2005). More than 500 000 severe leptospirosis cases have been reported each year around the world (WHO, 1999). The rate of leptospirosis cases varies depending on the climate and animal reservoirs. The incidence of leptospirosis is especially highest during the summer season with heavy rains and floods (Xue et al., 2008). Since the currently available microscopic agglutination test (MAT) that is a known gold standard does not permit early diagnosis and other serologic methods have low sensitivity, more rapid and sensitive methods * Corresponding author: A. Kilic, Department of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy, School of Medicine, 06018, Ankara, Turkey; phone: (+90) 312-3043412; e-mail: [email protected] 168 Bedir O. et al. are needed for detection of Leptospira as well as the ability to distinguish pathogenic and nonpathogenic Leptospira. Therefore, a number of molecular methods such as conventional multiplex PCR (Kositanont et al., 2007), real-time PCR (Slack et al., 2007; Levett et al., 2005), nested-PCR (Bomfim et al., 2008), loop mediated isothermal amplification (LAMP) (Lin et al., 2009), nested PCR-restriction fragment length polymorphism (RFLP) (Djadid et al., 2009) have been developed for specific detection of pathogenic Leptospira spp. in diagnostic laboratories. The TaqMan real-time PCR method has also been used for detection of pathogenic Leptospira spp. based on specific target sequences including the ribosomal 16S ribosomal RNA gene, and the Leptospira immunoglobulin-like protein A and B gene (lig A and ligB) (Palaniappan 2005). The lig A and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 big domain repeats. The lig genes are only detected in pathogenic Leptospira spp. as a unique virulence factor. Conversely, these genes are not found in nonpathogenic Leptospira spp. (Matsunaga et al., 2003). The aim of this study was to develop a rapid and simple assay for the specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR TaqMan method using primers and probes specific for Leptospira genus 16S ribosomal RNA gene, pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Experimental Materials and Methods Leptospira reference strains and control bacterial strains. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic were obtained from the Etlik Central Veterinary Control and Research Institute (ECVCRI), WHO Collaborating Center, Ankara, Turkey (Table I). All strains were stored in Fletcher media (Difco, Detroit, MI, USA) and then cultured in liquid Ellinghausen McCullough Johnson Harris (EMJH) (Difco) media supplemented with 10% serum of hemolysed rabbit blood at 30°C for 7 days. Other control bacterial strains including Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 13883, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 27270, Salmonella typhimurium NCTC 12023, Legionella pneumophila, Neisseria gonorrhoeae NCTC 8375, Borrelia burgdorferi strain B31, and Streptococcus pyogenes NCTC 12696 were selected from stock reference culture collection in our laboratory. The isolates were stored at 70°C 3 in trypticase soy broth (Merck, Darmstadt, Germany) supplemented with 15% glycerol before being tested. Design of primers and probes. Oligonucleotide primers and probes for multiplex real-time PCR (TaqMan) assay were designed based on a particular region of the ligA/B gene sequence (ligA/B-P1-5cggttc tcacttctattcaa-3, ligA/B-P2-5-attgaagaatcggat gagaa-3, and ligA/B-Probe-Texas red-5-atcctgtaaa tcctt ctcttgcaaa-3-Bhq-2) for pathogenic Leptospira spp. (Genbank accession nos FJ030916, EF517920, AF534640, AF368236, and AY221109), the 16S ribosomal RNA gene sequence (16S-P1-5-tagtgaacgg gatagatac-3, 16S-P2-5-ggtctacttaatccgttagg-3, and 16S-Prob-Fam-5-aatccacgccctaaacgttgtctac-3-Bhq-1) for Leptospira spp. genus (Genbank accession nos FJ154560, FJ154600, FJ154577, FJ154571, FJ154569, FJ154568, FJ154564, FJ154563, FJ154556, FJ154555, and FJ154553), and the 23S ribosomal RNA gene sequence (23S-P1-5-acaatcttaccaaaccctatc-3, 23SP2-5-ttaccacttagcgtagattt-3, and 23S-Prob-Joe-5tccgaatactgtaacttgaagtactgca-3-Bhq-1) for non-pathogenic L. biflexa (Genbank accession no CP000786) by using oligo analysis and design program Oligaware 3.0 developed in our institution (Table II). BLAST program was used to initially asses the ability of the primers and probes to identify target sequences (Smythe et al., 2002; Altschul et al., 1997). The primers and probes were synthesized by Metabion International, Germany. DNA extraction. DNA was extracted from the samples by treatment with 1% SDS and 100 mg proteinase K (Sigma Chemicals, St. Louis, Missouri, USA) in a buffer containing 50 mM Tris (pH 8.0), 50 mM EDTA, 100 µM NaCl. After 2 hours of incubation at 55°C in a waterbath, the DNA was purified by repeated extraction with phenol/chloroform/isoamyl alcohol (25:24:1). The DNA was concentrated by precipitation with 99% ethanol. The precipitate was collected by centrifugation, then dried and resuspended in deioinized sterile water (Veloso et al., 2000). Multiplex real-time PCR (TaqMan). The multiplex real-time PCR (TaqMan) method was performed by using a 7500 ABI Prism Sequence Detector (Applied Biosystems, Foster City, Calif., USA). In brief, 2 ml of the extracted nucleic acid solution was added to 23 µl of reaction mixture containing 0.8 µM of each primer and 0.4 µM each fluorophore probe (final concentration), and mixed with 12,5 µl of TaqMan Universal PCR Master Mix (Applied Biosystems). The TaqMan cycling conditions included a 10 min degradation of the preamplified templates at 95°C and then 40 cycles of denaturation at 95°C for 15 s and annealing and extension at 60°C for 60 s. All experiments were repeated at least twice for testing the reproducibility of the assay. Detection limits determination and assay validation. Pathogenic L. interrogans strain Wijnberg and 3 Detection and differentiation of pathogenic and nonpathogenic Leptospira spp. 169 Table I Leptospira strains used in this study Serovar Genomospecies Leptospira interrogans Leptospira interrogans Leptospira interrogans Leptospira interrogans Leptospira interrogans Leptospira interrogans Leptospira interrogans Leptospira. interrogans Leptospira interrogans Leptospira interrogans Leptospira interrogans Leptospira borgpetersenii Leptospira borgpetersenii Leptospira borgpetersenii Leptospira kirschneri Leptospira biflexa australis autumnalis bataviae bratislava hebdomadis icterohaemorrhagiae hardjo canicola icterohaemorrhagiae pomona pyrogenes ballum javanica tarassovi grippotyphosa patoc Strain Ballico Akiyami A Swart Jes Bratislava Hebdomadis Wijnberg Hardjoprajitno Hond Utrecht IV RGA Pomona Salinem Mus 127 Veldrat Batavia 46 Perepelitsin Moskova V Patoc I Table II Oligonucleotide sequence of the primers and probes used in this study Target region Oligo Name Sequence (5'3') ligA/B gene for pathogenic ligA/B-P1 (Forward) 5-cggttctcacttctattcaa-3 Leptospira spp. ligA/B-P2 (Reverse) 5-attgaagaatcggatgagaa-3 ligA/B-Prob Texas Red-5 -atcctgtaaatccttctcttgcaaa-3-Bhq-2 23S rRNA gene for non23S-P1 (Forward) 5-acaatcttaccaaaccctatc-3 pathogenic Leptospira spp. 23S-P2(Reverse) 5-ttaccacttagcgtagattt-3 23S-Prob Joe-5-tccgaatactgtaacttgaagtactgca-3-Bhq-1 16S rRNA gene 16S-P1(Forward) 5-tagtgaacgggattagatac-3, for Leptospira genus 16S-P2 (Reverse) 5-ggtctacttaatccgttagg-3, 16S-Prob Fam-5-aatccacgccctaaacgttgtctac-3-Bhq-1 L. biflexa strain Patoc I were cultured in EMJH (Difco) media to obtain a cell density of 1×108 cells/ml for each of the target species, and then total DNA was extracted. Serial 10-fold dilutions of extracted DNA were prepared ranging from 1× 106 cells/ml to 1× 100 cells/ml by using sterile ddH2O. The threshold cycle (Ct) value of each dilution was recorded. The lowest number of leptospires detected visually was defined as the detection limit of the assay. Results Multiplex real-time (TaqMan) PCR assay using LigA/B, 16S, and 23S primers and probes. The sixteen standard strains of Lepotospira spp. were tested for all genes. The multiplex real-time PCR (TaqMan) assay was used to amplify specific leptosiral sequences including ligA/B genes, 16S ribosomal RNA gene and Genbank accession no FJ030916, EF517920, AF534640, AF368236, and AY221109 CP000786 FJ154560, FJ154600, FJ154577, FJ154571, FJ154569, FJ154568, FJ154564, FJ154563, FJ154556, FJ154555, and FJ154553 23S ribosomal RNA gene simultaneously in a single tube. The ligA/B primers and probe designed based on particular region of ligA/B of pathogenic leptospires gave a detectable product, Ct values ranged from 17 to 39 and averaged 24 for only all pathogenic leptospires, whereas the 23S primers and probe designed based on the particular region of 23S ribosomal RNA non-pathogenic leptospires gave a detectable product Ct values ranged from 18 to 36 and averaged 23 for only nonpathogenic leptospires. The 16S primers and probe designed from the conserved region of 16S ribosomal RNA of Leptospira genus amplified genomic DNA from both pathogenic and non-pathogenic leptospires. Sensitivity and specificity of multiplex real-time PCR (TaqMan) method. The reaction efficiency of each assay was counted from the slopes of standard curves. The reaction efficiencies were found to be 83105% with decision coefficients of more than 0.99 in all multiplex assays. To determine assay sensitivity, 170 3 Bedir O. et al. Fig. 1. Amplification curves and dilution end point standard curves of log10 genome equivalents versus CT cycle number. The analytical sensitivity of this assay for ligA/B (A1-A2) and 23S ribosomal RNA (B1-B2) assays were approximately 100 genome per ml and 16S rRNA (C1-C2) assay was approximately 10 genome equivalents per ml. Leptospiral DNA was adjusted by 10-fold serial dilution ranging from 1× 106 cell/ml to 1× 100 cells/ml from both pathogenic Leptospira interrogans strain Wijnberg and Leptospira biflexa strain Patoc I. Linear dynamic range of multiplex real-time PCR (TaqMan) assay was performed with triplicates of 10-fold dilutions of the purified genomic DNA extracted from pathogenic and nonpathogenic leptospires, and was observed constantly (Fig. 1). The standard curves obtained with 10-fold serially diluted genomic DNA preparations were linear over seven orders of magnitude for targeting sequence. The assay detection limit, that is the sensitivity, was found to be approximately 1× 102 cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. To determine the assay specificity of multiplex real-time PCR (TaqMan) assay, other control bacteria were tested with ligA/B, 23S and 16S primer and probes. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. The ligA/B, the 16S, and 23S primers and probes were specific for pathogenic leptospires, Leptspira genus, and nonpathogenic leptospira, respectively (Fig. 2). Discussion Leptospirosis is known to be an emerging zoonotic disease caused by the genus Leptospira worldwide (Guerra, 2009). Since there is no national Leptospira 3 Detection and differentiation of pathogenic and nonpathogenic Leptospira spp. 171 Fig. 2. Multiplex amplification results with Leptospira interrogans strain Wijnberg, Leptospira biflexa strain Patoc I and mix DNA samples. In 10 fold dilutions 4 times. reference center in Turkey, reliable data about the incidence of leptospirosis are not available. Twelve Turkish cases were reported from south region of Turkey in 1997 (Saltoglu et al., 1997). In 2005, three cases of anicteric leptospirosis with mild and sever complications, a case of icteric leptospirosis with resistant immune hemolytic anemia, and four cases of anicteric leptospirosis with mania and psychosis were reported from the central Anatolia and northwest region of Turkey (Erdinc et al., 2006; Solmazgul et al., 2005; Semiz et al., 2005). Recently a Turkish leptospirosis case complicated with cerebral venous thrombosis was reported from the northwest region of Turkey (Turhan et al., 2006). A spectrum of human leptospirosis is recognized ranging from subclinical infection to a severe syndrome of multiorgan infection such as severe vasculitis and intravascular coagulation (Guerra, 2009). However, the symptoms of leptospirosis at the prodromal stage are almost indistinguishable from various other bacterial and viral febrile infections such as salmonellosis and dengue virus infection (Srimanote et al., 2008). In addition, the lack of reliable techniques for rapid diagnosis of leptospirosis may cause delay in treatment of patients and lethal sequela (Djadid et al., 2009). In diagnosis of leptospirosis, serological methods such as MAT and enzyme-linked immunosorbent assay (ELISA) based on the immunogenic response of the host and culture method to observe the organisms from clinical samples are usually per- formed in many laboratories. MAT is used to detect leptospiral antibody from the patient sera using live organisms as sources of antigens. Since antileptospiral antibody becomes detectable only after 810 days from the onset of illness, MAT cannot provide early diagnosis of leptospirosis (Smythe et al., 2002). In addition to MAT, other serologic tests detecting leptospires from clinical samples are not appreciable, especially during the early phase of the infection (Palaniappan et al., 2005; Srimanote et al., 2008). Leptospira is a fastidious microorganism and difficult to grow in culture medium. Moreover, culture which requires a special medium and at least two weeks to yield the organisms, cannot provide an early diagnosis. Thus, these techniques are used mainly for epidemiological purposes (Palaniappan et al., 2005; Srimanote et al., 2008). Therefore, there is a need for a reliable and accurate method for detection and differentiation of leptospires from clinical samples. The multiplex realtime PCR (TaqMan) assay is a rapid, sensitive and specific assay for detection of leptospires during early stage of infection (Palaniappan et al., 2005; Smythe et al., 2002; Slack et al., 2006). This assay has also been used as a diagnostic tool for the discrimination of pathogenic and non-pathogenic leptospires using particular gene sequence (Smythe et al., 2002). In this study, we developed a multiplex real-time PCR (TaqMan) assay to detect infection and the same time distinguish pathogenic leptospires from nonpathogenic leptospires in same reaction tube. The 172 3 Bedir O. et al. ligA/B, 23S, 16S primers and probes were used for specific detection of pathogenic, non-pathogenic leptospires and leptospira genus. This assay was able to detect rapidly leptospira genus and distinguish pathogenic leptospires from non-pathogenic ones. The detection limit of the assay was 100 leptospira cells/ml for pathogenic, non-pathogenic leptospires, and 10 cells/ml for leptospira genus. While the 16S primers amplified the target from both pathogenic and non-pathogenic leptospires, ligA/B amplified target DNA only from pathogenic leptospires and the 23S primers were specific for non-pathogenic ones. Thus, this assay may enable the recognition and discrimination between pathogenic and non-pathogenic, environmental contaminant leptospires in the same reaction tube. A number of gene sequences have been used to detect and distinguish pathogenic leptospires from non-pathogenic ones. Woo et al. developed a realtime PCR (TaqMan) method using 23S rRNA gene sequence for identification of pathogenic Leptospira (Woo et al., 1996). Levett et al. evaluated a highly sensitive and specific real-time PCR (TaqMan) method for lipL32 gene, which is a probable virulence gene, for detection of pathogenic leptospires (Levett et al., 2005). Slack et al. used DNA gyrase subunit B gene for identification of pathogenic Leptospira spp. When compared to the 16S rRNA gene, the gyrB gene showed greater nucleotide/evolutionary divergence allowing superior identification (Slack et al., 2006). Kawabata et al. used flab gene and suggested that PCR-RFLP was an efficient tool for rapid detection and identification of species of Leptospira from clinical specimens (Kawabata et al., 2001). Djadid et al. developed a nested PCR-RFLP assay using 16S rRNA as a rapid and specific available technique for differentiate pathogenic and non-pathogenic Leptospira spp. in the early stage of infection (Djadid et al., 2009). In this study, the ligA/B primers were used in a multiplex real-time (TaqMan) PCR assay to detect and distinguish pathogenic Leptospira serovar. The lig genes encode surface proteins containing immunoglobulin-like repeat predicted to play a role in adhesion to host tissues. Previous studies have demonstrated that pathogenic leptospires contain these genes, while they are absent from the non-pathogenic saprophyte (Xue et al., 2008). Diagnostic methods such as PCR and ELISA targeting lig genes have been recently developed to discrimination pathogenic and non-pathogenic leptospires (Palaniappan et al., 2005; Srimanote et al., 2008). Palaniappan et al. developed a real-time PCR (TaqMan) assay using lig1/lig2 primers targeting the conserved region of ligA and B as a sensitive and rapid tool for early diagnosis of leptospirosis (Palaniappan et al., 2005). One of the most significant advantages of our multiplex real-time PCR (TaqMan) assay is that both pathogenic and non-patho- genic leptospires were detected at the same cycling conditions allowing all three reactions to be performed in a single PCR tube simultaneously. We also analyzed leptospires based on the genus in the same reaction tube as an internal control. To our knowledge, this is the first study detecting three leptospire gene sequences in the same reaction tube. The efficiency of each assay ranged between 83 and 105%; the accepted ranges for PCR efficiency are generally between 80.0 and 110.0%. In conclusion, the developed multiplex real-time PCR (TaqMan) assay targeting ligA/B gene, 16S ribosomal RNA and 23S ribosomal RNA sequences is highly useful for early diagnosis of leptospirosis and differentiation between pathogenic and non-pathogenic leptospires in the same reaction tube with high sensitivity and specificity. Literature Altschul S.F., T.L. Madden, A.A. Schäffer, J. Zhang, Z. Zhang, W. Miller and D.J. Lipman. 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 33893402. Bomfim M.R., E.F. Barbosa-Stancioli and M.C. Koury. 2008. Detection of pathogenic leptospires in urine from naturally infected cattle by nested PCR. Vet. J. 178: 251256. Brenner D.J., A.F. 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Infect. 11: 328333. 174 Bedir O. et al. 3 Polish Journal of Microbiology 2010, Vol. 59, No 3, 175178 ORIGINAL PAPER Methanogenic Diversity Studies within the Rumen of Surti Buffaloes Based on Methyl Coenzyme M Reductase A (mcrA) Genes Point to Methanobacteriales KRISHNA M. SINGH1, PARESH R. PANDYA2, SUBHASH PARNERKAR2, AJAI K. TRIPATHI1, UMED RAMANI1, PRAKASH G. KORINGA1, DHARAMSHI N. RANK3, CHAITANYA G. JOSHI1 and RAMESH K. KOTHARI4 1 Department of Animal Biotechnology, College of Veterinary Science and A.H., Anand Agricultural University, Anand (388 001), Gujarat, India 2 Animal Nutrition Research Station, College of Veterinary Science and A.H., Anand Agricultural University, Anand (388 001), Gujarat, India 3 Department of Animal Genetics & Breeding, College of Veterinary Science and A.H., Anand Agricultural University, Anand (388 001), Gujarat, India 4 Department of Microbiology, Christ College, Rajkot, Gujarat, India Received 3 April 2010, revised 21 June 2010, accepted 12 July 2010 Abstract Methane emissions from ruminant livestock are considered to be one of the more potent forms of greenhouse gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, basic knowledge of the diversity of these microbes in breeds of buffalo is required. Therefore, the methanogenic community in the rumen of Surti buffaloes was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) gene. A total of 76 clones were identified, revealing 14 different sequences (phylotypes). All 14 sequences were similar to methanogens belonging to the order Methanobacteriales. Within Methanobacteriales, 12 clones (6 OTUs) were similar to Methanosphaera stadtmanae and the remaining 8 phylotypes (64 clones) were similar to unclassified Methanobacteriales. Overall, members of the Methanobacteriales dominated the mcrA clone library in the rumen of Surti buffalo. Further studies and effective strategies can be made to inhibit the growth of Methanobacteriales to reduce methane emission from the rumen which would help in preventing global warming. K e y w o r d s: methyl coenzyme-M reductase, PCR, ruminant methanogens Introduction The rumen is characterized by high microbial population density and complexity of micro-ecological interactions. Methane is biologically produced by the metabolism of a diverse group of methanogenic microorganisms, methanogens, which are phylogenetically placed exclusively as members of the domain archaea. They inhabit typical anaerobic environments, such as wetlands, sediments, geothermal springs and the digestive tracts of mammals (Garcia et al., 2000). Methane is an important greenhouse gas which significantly contributes to global warming. Livestock is a major anthropogenic source of methane emission from agriculture and contributes about 18% of global greenhouse gas (GHG) emissions, and as much as 37% of anthropogenic methane, mostly from enteric fermenta- tion by ruminants (FAO, 2006). Livestock rearing has been an integral part of the agricultural system in India. Currently, India possesses the worlds largest livestock population of 485 million, which accounts for 13% of the global livestock population (MOA, 2003). It has 57% of the worlds buffalo and 16% of the cattle population. The contribution of methane emission in India by buffalo is 42% (Chhabra et al., 2009). Several groups have reported the monitoring of methanogen populations from environmental samples through targeting of the 16S ribosomal gene (Yu et al., 2005; Stewart et al., 2006; Wright et al., 2007). While researchers have traditionally used the 16S rRNA gene for phylogenetic diversity, many researches are now addressing the diversity of the methanogenic archaea by studying sequence divergence within the methyl coenzyme-M reductase subunit A (mcrA) gene (Lueders * Corresponding author: Ramesh K. Kothari /Krishna M. Singh, Department of Microbiology, Christ College, Rajkot, Gujarat, India; phone 02692 261201; fax 02692 261201; e-mail: [email protected], e-mail: [email protected] 176 Singh K.M. et al. et al., 2001; Luton et al., 2002; Hallam et al., 2003; Tatsuoka et al., 2004; Rastogi et al., 2008). Methyl coenzyme-M reductase is ubiquitous to methanogens and is crucial to the terminal step of methanogenesis where it is involved in the reduction of the methyl group bound to coenzyme-M. There is no report concerning mcrA genes from buffalo rumen, therefore, we examined the community of methanogens using comparative sequence analysis of the mcrA amplified from total DNA extracted from rumen fluid of Surti buffaloes. Experimental Materials and Methods Sampling and DNA extraction. The experiment was carried out on 3 adult Surti buffaloes reared at the Department of Animal Nutrition, College of Veterinary Science and A.H., Anand. All the animals were maintained under uniform feeding regime (I.C.A.R., 1998) for minimum 30 days. The diet consisted of green fodder (Sorghum and NB21), dry mature pasture grass (Dicanthium annulatum) and compound concentrate mixture (20% CP, 65% TDN). The animals were offered 10 kg green, ad-lib dry grass and 2.5 kg of concentrate mixture daily. Approximately 500 ml of liquor was collected via a stomach tube located in the mid part of the rumen and connected to a vacuum pump at 0, 2, 4 and 6 hrs post feeding (Khampa et al., 2006). About 100 ml liquor was passed through four layers of cheese cloth to remove particulate matter. Remaining liquor was stored at 80°C for further study. Total DNA (each hrs) was extracted separately by using a commercially available kit according to the manufacturers instructions (QIAGEN Stool kit; QIAGEN, CA). The individual DNA was used as a template in PCR to amplify mcrA gene. PCR primers and amplification. The mcrA primers used were ME1 (5-AGCMATGCARATHGGWA TGTC-3-) and ME2 (5-ATCATKGCRTAGTTDGG RTAGT-3) (Hales et al., 1996), subsequently mcrA gene were amplified (760 bp) by PCR using metagenomic DNA and Master mix (Fermentas, USA). A total of 25 µl of reaction mixture consisted of 10 pmol of each primer, 30 ng of template DNA, 12.5 µl of Master mix (Fermentas, USA). The PCR amplification was carried out as follows: 1 cycle at 95°C for 3 min, 35 cycles of 95°C for 30 s, 60°C for 1 min, 72°C for 1 min and a final elongation at 72° for 10 min by using thermal Cycler (ABI, USA). The anticipated product of approximately 760 bp was cleaned separately using a Qiagen DNA Gel Extraction Kits (QIAGEN, CA) in accordance with the directions of the manufacturer and pooled the purified PCR products in equimolar concentration. 3 Cloning and sequencing. The purified PCR products were cloned in InstaTA cloning kit (Fermentas, USA) as per the instructions of the manufacturer. The recombinant plasmids then were extracted by the Qiagen mini-prep plasmids extraction kit (QIAGEN, CA). Sequencing was performed for all the clones in the library with an ABI Prism 310 Genetic analyser (Applied Biosystems Inc., CA) using BigDye Terminator (version 3.1) at Animal Biotechnology laboratory, AAU, Anand, Gujarat, India. Sequence analyses and phylogenetic tree construction. All reference sequences were obtained from the Genbank/EMBL/DDBJ (Benson et al., 2007). Sequences (~600 bp) from the current study were analysed by the CHECK_CHIMERA program (Maidak et al., 2001) to remove any chimeric clone. The similarity searches for sequences were carried out by BLAST (http://www.ncbi.nlm.nih.gov/ BLAST/ Blast.cgi (Madden et al., 1996) and alignment was done using CLUSTAL W (http://www.ebi.ac.uk/ Tools/clustalw2/index.html (Thompson et al., 1994). Ambiguously and incorrectly aligned positions were aligned manually. The distance matrix was calculated using the PRODIST program included in PHYLIP (Felsenstein, 1985) and used to assign sequences in various operational taxonomic units (OTUs) or phylotypes by DOTUR (Schloss and Handelsman, 2005) and total of 14 OTUs were generated. The percentage of good coverage was calculated as [1 (n/N)] X 100, where n is the number of single clone OTUs and N is the library size. Phylogenetic tree was constructed by the neighbour joining method using MEGA 4.0 (Tamura et al., 2007). Bootstrap re-sampling analysis for 1000 replicates was performed to estimate the confidence of tree topologies. The prefix mcrA was used to denote OTU identified and nucleotide sequences have been deposited in the Genbank database under the accession numbers GQ120890-GQ120965. Fig. 1. Ethidium bromide-stained agarose gel showing PCR products (670 bp) amplified from DNAs extracted from the three rumen samples. Template DNAs are as follows: lane 1, sample1; lane 2, sample 2; lane 3; sample 3 and lane 4, shows DNA size marker. 3 Sequencing of mcrA genes of ruminant methanogens Results Methanogen-specific DNA fragments were amplified from DNA extracted from the Surti rumen fluid by PCR with primers targeting mcrA genes. The amplified fragments from three rumen fluids, which were approx. 760 bp, are shown in Fig. 1. All the clones were subjected to sequence analysis followed by online homology search, Genbank which implements the BLAST algorithm (Madden et al., 1996). None of clone (76) from our library was assigned any genera/species. Because the similarity values of our sequences were too low to assign them to particular taxa with a reasonable degree of confidence. In our library 14 sequences (phylotypes) were generated. Phylogenetic analysis was performed to clarify their taxonomic position. The phylogenetic placements of the deduced DNA sequences are shown in Fig. 2. The good coverage of mcrA library was 94.73% in present study. This level of coverage showed that the mcrA sequences identified in library represent the majority of methanogen diversity. The sequences obtained in the present study were placed in the single cluster Methanobacteriales. 177 Table I Analysis of mcrA gene phylotypes diversity retrieved from the rumen fluid of Surti buffaloes Items Library size (N) OTUs b Single clone OTU c (n) Goods coverage d (%) Clone distribution (i) Methanobacteriales a Methanosphaera stadtmanae b. Unidentified Methanobactriales c. Unknown methanogen a 16S r DNA library 76 14 4 94.73 14 OTUs (76 clones) 6 OTUs (12 clones) 7 OTUs (63 clones) 1 OTU (01 clone) a Number of clones analyzed from library; b OTUs based on mcrA gene sequences; c OTUs containing only single clone; d The higher percentage coverage means more diversity is captured Within the Methanobacteriales, 63 clones (7 OTUs) belonged to the unidentified Methanobacteriales and 12 clones (6 OTUs) sequences were found to be the closest to Methanosphaera stadtmanae. Of the clones isolated from rumen sample, only one clone represented unknown methanogens (Table I). Fig. 2. The evolutionary history was inferred using the Maximum Parsimony method. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Phylogenetic analyses were conducted in MEGA4. (UNI = u nidentified and MSS = Methanosphaera stadtmanae). 178 Singh K.M. et al. Discussion Of 14 phylotypes, 13 sequences (Phylotypes) of the DNA were obtained from the 76 clones in the present study; these sequences were placed in the same cluster, which was relatively close to unidentified Methanobacteriales (63 clone) and M. stadtmanae (12 clones), in phylogenetic placements (Fig. 2). Whitford et al. (2001) also found several sequences that were related to Methanosphaera stadtmanae. Wright et al. (2007) also found a sequence (ON-CAN.13) in cattle from Ontario that was 95.8% similar to that of Methanosphaera stadtmanae and 99.8% similar to that of their clone ARC29. Methanogens similar to Methanosphaera stadtmanae have also been reported in pasture-fed dairy cattle (Skillman et al., 2006) The DNA sequences of mcrA genes, isolated in this study, showed similarities with unidentified methanobacteriales and to M. stadtmanae. It has been known that mcrA genes could be used as phylogenetic tool for the specific detection and the identification of methanogenes, because the phylogeny of the mcrA genes and 16S rDNA from the recognized orders of methanogens clearly had strong similarity (Luton et al., 2002). Our results show that rumen of Surti buffaloes contains one of the essential and diagnostic genes of the methanogenic pathway. The identification of these genes provides a means to identify cluster/group on the basis of mcrA sequence. Moreover, identification of rumen associated mcrA groups defines a functional genomic link between methanogenic and putative reverse methanogenic archaea. Specific questions relating to methanogenic protein function in buffalo rumen await further genomic, biochemical, structural, and proteomic analysis. Acknowledgements Financial support provided by the Department of Biotechnology Govt. of India, New Delhi to conduct the study reported here is acknowledged with respect and gratitude. Literature Benson D.A., I .Karsch-Mizrachi, D.J. Lipman and J. Ostelland. 2007. GenBank. Nucleic Acids Res. 35, D1D25. Chhabra A., K.R .Manjunath, S. Panigrahy and J.S. Parihar. 2009. Spatial pattern of methane emissions from Indian livestock. Current Science 96: 510. Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783791. FAO, FAOSTAT. 2006. Online Statistical Service, Food and Agriculture Organization of the United Nations, Rome. Garcia J.L., B.K.C. Patel and B. Olliver. 2000. Taxaonomic, phylogenic, and ecological diversity of methanogenic Archaea. Anaerobe 6: 205226. Hales B.A., C. Edwards, D.A. Ritchie, G. Hall, R.W. Pickup and J.R. Saunders. 1996. 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KOZHUHAROVA2, BORIANA Y. ZHEKOVA3 and VELIZAR K. GOCHEV 4 1 Department of Automatics, Information and Control Systems, University of Food Technologies, Plovdiv, Bulgaria 2 Department of Biotechnology, University of Food Technologies, Plovdiv, Bulgaria 3 Department of Biochemistry and Molecular Biology, University of Food Technologies, Plovdiv, Bulgaria 4 Department of Biochemistry and Microbiology, P. Hilendarski University, Plovdiv, Bulgaria Received 3 April 2010, accepted 25 May 2010 Abstract The composition of a synthetic culture medium for levorin biosynthesis by Streptomyces levoris 99/23 was optimised using mathematical modelling methods. The optimal concentrations of the medium components were established by means of an optimum composition design at three factor variation levels. An adequate regression model was obtained. Levorin biosynthesis by Streptomyces levoris 99/23 in the optimised synthetic medium was over 38% higher than in the initial medium. The antibiotic biosynthesis dynamics in the optimised culture medium was studied by means of a non-linear differential equation system. The resultant model was valid. K e y w o r d s: Streptomyces levoris 99/23, biosynthesis dynamics, mathematical modelling, optimisation of levorin biosynthesis Introduction Levorin is an antifungal preparation widely used in medicine. The antibiotic biosynthesis mechanism and the characteristics of its producers in a physiological aspect are studied mainly by means of synthetic culture media (Belousova et al., 1970; Jakovleva, 1980). These media have strain-specific compositions which are determined experimentally. Culture medium optimisation in a quantitative and qualitative aspect using mathematical modelling methods is insufficiently studied. Orthogonal Latin rectangles were used by Jakovleva (1980), and linear models were applied by Gotchev et al. (2002). There are individual reports on biosynthesis description using neural networks (XianFa et al., 2000). From a practical point of view, however, they are more suitable for process control rather than investigation since kinetic constant values are most often hidden either in the architecture or in the weight coefficients of the neural network. This paper aimed to determine the optimal concentrations of the synthetic culture medium components for levorin biosynthesis by Streptomyces levoris 99/23 and study levorin accumulation dynamics using mathematical modelling methods. Experimental Materials and Methods Microorganism. A Streptomyces levoris 99/23 strain stored in a lyophilised form in the Biotechnology Departments collection at UFT was used as a levorin producer (Kozhuharova et al., 2002). The culture was maintained on a medium described by Kozhuharova et al. (2008). Media and cultivation conditions. The initial nutrient medium for S. levoris 99/23 cultivation, which was subject to optimization, had the following composition (%): glucose 1.5; starch 2; (NH4)2SO4 0.6; KH2PO4 0.005; KCl 0.1; MgSO4 0.25; CaCO3 0.3. After pH adjustment to 7.2, and sterilization at 121°C for 30 min, the nutrient medium was inoculated with * Corresponding author: V.S. Stanchev, 26 Maritza Boulevard, 4002 Plovdiv, Bulgaria; phone: (+359) 32603898; fax: (+359) 32644102; e-mail: [email protected] 180 2% (v/v) spore inoculum containing 2.109 cfu/ml. Strain cultivation and levorin biosynthesis were carried out in 500 ml Erlenmayer flasks containing 50 ml of each nutrient medium at a temperature of 28°C, on a rotary shaker (220 min1) for 96 h. Mathematical modelling. The optimal concentrations of medium components were determined using optimal composition design with three variation levels of the factors (Koleva et al., 2005; Mason et al., 2003). Such an approach enables generation of nonlinear regression models with a minimum number of experiments: Ymod = b0 + 3 Stanchev V.S. et al. k Σ i=1 k k1 k Σ bii.xi2 + iΣ= 1 j=Σ2 bij.xj.xj i=1 bi.xi + (1) where: Ymod is the predicted response, bi, bij and bii are coefficients accounting for the effect of each factor (xi), of their interrelations (xi.xj), and those to the square of two (xi2) respectively, and k is the number of factors. The experimental data statistical processing and the results analysis were performed using Anova (Microsoft Excel 2003). The process dynamics was studied by submerged cultivation of the strain in the optimised culture medium. The input parameter values were approximated by third order spline functions (Mathews and Fink, 2001). The calculation and optimisation procedures were performed within the Eureka software environment (The Software Eureka 2000). The graphic presentation was based on Microsoft Excel 2003 and Sigma Plot 9.0. Assays. Levorin concentration in the culture medium was analysed according to the spectrophotometric method suggested by Bob et al. (1978) and expressed in mg/ml. One antibiotic activity unit (IU) corresponds to 0.04 µg of levorin (Bolshakova et al., 1989). Reducing sugars (substrate) concentration was determined by the dinitrosalicylic acid method (Miller, 1959). The biomass quantity was determined after drying at 105°C to constant weight. pH was measured potentiometrically. Results and Discussion Following a series of single-factor experiments, the variation interval for the basic culture medium components was determined. The real and coded values of the independent variables are shown in Table I. The remaining constituents were fixed at the following levels (%): KCl 0.1; MgSO4 0.25; CaCO3 0.3. The experimental data was formed as the mean value of the results of six parallel experiments. The design matrix, the experimental results (Yexp) and model values (Ymod) obtained by equation (2) are presented in Table II. Table I Real and coded values of independent variables Coded value Factor (%) 1 0 +1 X1: glucose 1.0 1.5 2.0 X2: starch 1.0 2.0 3.0 X3: (NH4)2SO4 0.4 0.6 0.8 X4: KH2PO4 0.001 0.0055 0.01 Table II Optimum composition design for 4 factors and three levels of their variation No X1 X2 X3 X4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 1 1 Yexp (mg/ml) Ymod (mg/ml) 0.667 0.724 0.785 0.576 1.242 0.581 0.860 0.529 0.718 0.726 0.851 0.573 0.894 0.572 0.588 0.527 0.592 0.691 0.903 0.812 0.714 0.700 0.635 0.620 0.597 0.748 0.737 0.603 1.135 0.581 0.806 0.537 0.781 0.751 0.921 0.605 0.950 0.578 0.622 0.535 0.693 0.693 0.820 0.820 0.739 0.646 0.691 0.590 The analytical expression of the regression equation obtained is: Ymod = 0.6928 +0.0472.X3+ 0.1007.X4 0.0467.X1.X2 0.0459.X2.X3 0.0595.X2.X4 0.0454.X1.X2.X4 0.0714.X2.X3.X4+ 0.1276.X22 0.1024.X42 (2) The model was adequate at confidence level " = 0.05 and degrees of freedom < = 9 (Table III). The analysis of (2) revealed several considerations. The two carbon sources (X1, X2) were not present on their own in the model but the coefficient before X22 in (2) had higher positive value. Its effect in Ymod in relation to b0 was 18.4%. This showed that S. levoris 99/23 preferred a carbon source with a relatively high molecular mass. On the other hand, X1 3 Mathematical modeling for levorin biosynthesis optimisation 181 Table III Statistical analysis results according to Anova Parameter Df SS MS F Significance F Regression Residual Total 9 14 23 0.5176539 0.0788534 0.5965073 0.057517098 0.005632389 10.21185 9.32311E-05 Df degree of freedom; SS sum square; MS mean square; F Fisher coefficient participated in two terms of (2) with total weight of 13.3% in relation to b0. Since the effect of both factors in Ymod is commensurate, there is sufficient ground to believe that they are in optimum correlation ensuring the absolute extremum of (2). X4 had a pronounced individual influence in the model (15%, which is comparable to that of X22). KH2PO4 was a source of phosphorus, an important element for the S. levoris growth and levorin biosynthesis regulator. In this respect, the results obtained are in agreement with the theoretical formulations and data reported by other authors (Belousova et al., 1970). The nitrogen source can be seen as having no significant effect on levorin biosynthesis. The conditions maximising (2) were found by means of a gradient optimisation method (The Software Eureka 2000): max Y mod = 1.135 mg/ml, at: X1 = + 1; X2 = 1; (3) X3 = + 1; X4 = + 1 The response function (2), in graphic form, with variation of X1 and X2 within the limits set in Table I and optimum values of X3 and X4 (+1), is presented on Fig. 1. The hypothesis of equality of the mathematical exmax pectation of the experiment results Yexp under the optimal conditions with that of the predicted max Ymod = 1.135 was checked (Mason et al., 2003). The max values (mg/ml) were as follows: Yexp, 1 = 1.20; max max max max Y exp, 2 = 1.22; Yexp, 3 = 1.16; Yexp, 4 = 1.15; Yexp, 5 = 1.10; max max max Yexp, 6 = 1.12; Yexp, 7 = 1.06; Yexp, 8 = 1.00. For degrees of freedom< = 7 and confidence level "= 0.05, tcrit.= 2.365 (Students table). Since tcalc.= 0.535<tcrit., there was no max and Y max statistically significant difference betweenY exp mod A significant increase in levorin biosynthesis was detected with the optimised medium. The yield achieved was with 38% higher in comparison to the yield with the initial medium. The dynamics of levorin biosynthesis by S. levoris 99/23 with the optimised medium was modelled by means of a system of the following non-linear differential equations: dX(t) = µ(t) X(t) dt (4) 1 dX(t) dS(t) = Y2X(t) Y1 dt dt dP(t) dX(t) = "1 + "2 X(t) dt dt S(t) $X(t) µ(t) = µm ks + S(t) (5) (6) (7) X(t), S(t), and P(t) are the biomass concentrations, substrate concentration, and levorin concentration respectively when the process operates in the periodic mode, Y1 is an economic coefficient, Y2 is related to the rate of substrate assimilation by the cells in a stationary phase of the process, ks is a saturation constant, µm is the maximum specific growth rate, $ is the decay coefficient, "1 is the coefficient of substrate transformation into a metabolism product, and "2 is levorin accumulation rate in the stationary phase of the process. Using (7), the microbial population growth was modelled in the presence of substrate limitation modified Verhulst law. The experimental data were formed by using the mean value of the results from six parallel experiments on the process dynamics. The lag phase time (24 h) was excluded from the data set. The kinetic constant values in the model were determined using an optimisation procedure minimising the following criterion: Fig. 1. Ymod = f (X1, X2) at an optimum value of X3 and X4 + 1. 182 J= Stanchev V.S. et al. n [(Xexp, i Xmod, i)2 + (Sexp, i Smod, i)2 Σ i=1 3 + + (Pexp, i Pmod, i)2] → min (8) where exp, i and mod, i denote the process parameters according to the experimental data and models (47), and n is the number of observations. With this setup, the numerical values of the kinetic constants for the model were determined to be as follows: µm = 0.05583 h1; ks = 0.97 mg/ml; $ = 0.01054 ml/mg.h; Y1 = 0.233; Y2 = 0.0077 h1; "1 = 0.169; "2 = 0.00117 h1 The process dynamics is presented graphically in Fig. 2, Fig. 3 and Fig. 4. There was a good coincidence between the experimental and model results. P(t) reached a maximum equal to 1.08 mg/ml at t =131.5 h (Fig. 4). This value was obtained after approximation of the analytically calculated P(t) data according to (6), with a second-order spline function and maximisation of P(t), within the 108144 h time interval and degree of freedom t. No check of the experiment reproduci- Fig. 2. Growth dynamics of S. levoris 99/23: (◆) experimental data; (¨) model data. Fig. 3. Dynamics of substrate assimilation by S. levoris 99/23: (◆) experimental data; (¨) model data Fig. 4. Dynamics of levorin biosynthesis by S. levoris 99/23: (◆) experimental data; (¨) model data. bility was run since it generally coincided with that of the studies on the process statics. The kinetic constants can be interpreted in the following manner in a biotechnological aspect. Under the experimental conditions, the value of decay coefficient $, was 5.3 fold lower than the value of µm. At time point t = 131.5 h, the available biomass exceeded the initial biomass by 10.7 folds. Over 23% of the substrate was assimilated for biomass accumulation (Y1), and around 18.5% was used for maintaining the life activity in the stationary phase of the process. Since the values mentioned were close, it could be considered that approximately the same substrate amount was spent for both purposes. The main antibiotic quantity was synthesised in the process stationary phase, and it was 6 fold higher than the value in the exponential phase, at a priori specified stationary phase duration of 36 h. The ratio "1/("2.)t) for )t = 36 h, in the stationary phase was 1:6. This came as another proof that levorin biosynthesis took place mainly at this stage of the microbial population growth. At the end of the process, over 15.6% of the substrate remained unassimilated (Fig. 3). This was an indication for the presence of a critical value in respect to S0 at which, the function would reach its maximum along with the complete substrate utilisation. Such an effect was not registered under the conditions of the experiment. The material balance of the process came to the following considerations. The substrate consumption for biomass accumulation in the exponential growth phase of S. levoris 99/23 was about 23.3%, and the value for life activity during the process stationary phase was about 18.5%. About 15.6% of the substrate was unassimilated at the end of the biotechnological process. For antibiotic synthesis in the exponential growth phase about 16.9% of the substrate was used, and the corresponding value for the stationary phase 3 Mathematical modeling for levorin biosynthesis optimisation was 27.7%. Total carbon source consumption was determined to be 102%. We consider that the results obtained provide an objective idea of the material balance in the system taking into account the subjective, methodological and instrumental error in the experimental data analysis. The dynamics model of the levorin biosynthesis by S. levoris 99/23 was valid. It described in detail even the lysis processes at the end of the stationary phase. In proof of this statement, the experimentmodel error dispersion values are presented for all observation points as follows: F2x=0.0025; F2s=0.26; F2p=0.0005. The main share in criterion (8) is mainly attributed to S since its natural values are 100 and more times higher than those of X and P. Conclusion. As a result of the optimisation of the nutrient medium for levorin biosynthesis by S. levoris 99/23 using mathematical modelling methods, the optimal composition of the medium was determined. The yield achieved with this medium was 38% higher in comparison to the initial one. The dynamics of the antibiotic biosynthesis by S. levoris 99/23 was studied by means of a non-linear differential equation system. The kinetic constant values were calculated for Verhulst model, describing the presence of substrate limitation. A valid model of the dynamics of levorin biosynthesis by S. levoris 99/23 was obtained and the material balance of the process was assayed. Literature Belousova I.I., E.B. Lishnevskaya and R.E. Elgat and I.M. Tereshin. 1970. Effect of mineral phosphorus on the formation of 183 levorin and fatty acids by Actinomyces levoris Krass, Antibiotics 15: 224228. Bob T.G., G.B. Barabanshchikova, V.Y. Raigorodskaya, E.D. Etingov, T.A. Fradkova, N.B. Kishkurno, V.M. Orekhova and A.F. Aleshkova. 1978. Differential spectrophotometric method of levorin analysis in culture broth and mycelium. Antibiotics 23: 882885. Bolshakova L.O., Y.D. Shenin, T.A. Fradkova, O.B. Ermolova, L.N. Astanina and V.M. Grigoryeva. 1989. Determination the biological activity of levorin by the international standard of candicidin. Antibiot. Chemother. 34: 732736. Gochev V., L. Kozuharova and M. Diltcheva. 2002. Optimisation of synthetic culture medium composition for levorin biosynthesis by Streptomyces levoris 99/23, Proceedings of the Tenth Congress of the Bulgarian Microbiologists 2002. Plovdiv, 2:105108. Jakovleva E.P. 1980. Synthetic medium for biosynthesis of polyenic antibiotics levorin and amphotericin B. Antibiotics 25: 817821. Koleva B., V. Stanchev, D. Spasova and S. Bahchevanska. 2005. Investigation of the maceration process of Sofora japonica flowers. Sci. Works UFT, LII: 380385. Kozhuharova L. and V. Gochev. 2002. Selection of a highly active levorin producer strain. Science Conference with International Participation for Food, Health, Longevity 2002. Smolyan 2002, 159164. Kozhuharova L., V. Gochev and L. Koleva. 2008. Isolation, purification and characterization of levorin produced by Streptomyces levoris 99/23. World J. Microbiol. Biotechnol. 24: 15. Mason R., R. Gunst and J. Hess. 2003. Statistical design and analysis of experiments with applications to engineering and science. John Wiley & Sons. Mathews J. and K. Fink. 2001. Numerical methods using Matlab. Prentice Hall, Upper Saddle River, NJ. Miller G.L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426428. The Software Eureka. 2000. Manual for Users. Xian-Fa J., X. Jun-Ja, Zhu-Qiang and L. Guo-Yong. 2000. Model construction of the lycomycin fermentation process based on neural networks for identification. J. Luoyang Inst. Technol. 21: 8588. 184 Stanchev V.S. et al. 3 Polish Journal of Microbiology 2010, Vol. 59, No 3, 185190 ORIGINAL PAPER Chromate Reduction by Cell-Free Extract of Bacillus firmus KUCr1 GOPI BALLAV SAU, SWAGATA CHATTERJEE and SAMIR KUMAR MUKHERJEE* Department of Microbiology, University of Kalyani, Kalyani 741235, India Received 9 February 2010, revised 10 June 2010, accepted 20 June 2010 Abstract Microbial enzymatic reduction of a toxic form of chromium [Cr(VI)] has been considered as an effective method for bioremediation of this metal. This study reports on the in vitro reduction of Cr(VI) using cell-free extracts from a Cr(VI) reducing Bacillus firmus KUCr1 strain. Chromium reductase was found to be constitutive and its activity was observed both in soluble cell fractions (S12 and S150) and membrane cell fraction (P150). The reductase activity of S12 fraction was found to be optimal at 40 µM Cr(VI) with enzyme concentration equivalent to 0.493 mg protein/ml. Enzyme activity was dependent on NADH or NADPH as electron donor; optimal temperature and pH for better enzyme activity were 70°C and 5.6, respectively. The Km value of the reductase was 58.33 µM chromate having a Vmax of 11.42 µM/min/mg protein. The metabolic inhibitor like sodium azide inhibited reductase activity of membrane fraction of the cell-free extract. Metal ions like Cu2+, Co2+, Ni2+ and As3+ stimulated the enzyme but others, such as Ag+, Hg2+, Zn2+, Mn2+, Cd2+ and Pb2+, inhibited Cr(VI) reductase activity. K e y w o r d s: Bacillus firmus, cell-free extract, chromium reductase Introduction Application of Cr-resistant bacteria to detoxify Cr(VI) has been considered as an effective bioremediation method (Ohtake and Silver, 1994; Shakoori et al., 1999; Ganguli and Tripathi, 2002; Cheung and Gu, 2007; Sau et al., 2008). Bioremediation of Cr(VI) can occur by enzymatic reduction of Cr(VI) to Cr(III) via Cr(V) and Cr(IV) intermediates (Camargo et al., 2003; Xu et al., 2004; Xu et al., 2005; Pal et al., 2005; Cheung et al., 2006) or through immobilization (Losi et al., 1994). In the course of aerobic reduction, the cytosolic fractions of most chromium resistant bacteria exhibit Cr(VI) reductase activity (Campos et al., 1995; Cervantes et al., 2001), while under anaerobic conditions, CrO42 is used as a terminal electron acceptor and is reduced in the cell membrane (McLean and Beveridge, 2001). Requirement of electron supply is obvious during the reaction process, considering the process a redox reaction. The enzyme responsible for Cr(VI) reduction has not yet been well characterized, though there are reports on reduction kinetics of Cr(VI) reduction by cell-free extracts (CFE) with varied results (Ishibashi et al., 1990; Suzuki et al., 1992; McLean and Beveridge, 2001; Park et al., 2000; Camargo et al., 2003; Pal et al., 2005). Studies using partially purified Cr(VI) reductase from Pseudomonas ambigua G-1 (Suzuki et al., 1992) and P. putida MK1 (Park et al., 2000) have indicated Cr(VI) reduction using NADH or NADPH as a source of electrons. In Bacillus sp. ES29, Cr(VI) reductase was found in the soluble fraction of CFE, which utilizes NADH as electron donor (Camargo et al., 2003). Cr(VI) reducing Bacillus firmus KUCr1 has been reported and its potential to reduce Cr(VI) using whole cells under culture condition was documented earlier (Sau et al., 2008). This study reports on the reduction of Cr(VI) by CFE of this strain, effects of different electron donors, metal ions, inhibitors, pH and temperature on its Cr(VI) reducing activity, and cellular localization of Cr(VI) reductase. Experimental Materials and Methods Microorganism and growth condition. A Cr(VI) resistant Bacillus firmus KUCr1 (NCBI GenBank 16S rDNA sequence Accession No EU784699) was earlier reported (Sau et al., 2008) and used in this study. * Corresponding author: S.K. Mukherjee, Department of Microbiology, University of Kalyani, Kalyani 741235, India; phone: (+91) 3325827315; fax: (+91) 3325828282; e-mail: [email protected] 186 3 Sau G.B. et al. Cells were grown aerobically in PYG medium (peptone 10 g/l; yeast extract 5 g/l; glucose 3 g/l; pH 7.2) supplemented with or without (control) 0.1 mM Cr(VI) as K2CrO 4 under continuous shaking on a rotary shaker at 35°C for 24 h. Preparation of cell-free extract (CFE) and cellular fractions. Cell-free extract (CFE) and cellular fractions were prepared following McLean and Beveridge (2001) with little modification. Cells grown in PYG medium were harvested at the exponential phase, washed three times in 0.2 M phosphate buffer (pH 7.2) by centrifugation (4000× g at 4°C), resuspended in the same buffer to have a 10 ml suspension and kept in an ice bath. Cells were disrupted with an ultrasonicator (Hielscher Ultrasound Technology, UP50H). Power was applied five times in 1min pulses with 100% amplitude. After sonication the suspension was centrifuged at 12 000 × g for 10 min at 4°C to obtain a soluble fraction (S12). Five ml of S12 fraction was centrifuged at 150 000× g for 90 min at 4°C to obtain S150 fraction. The pellet fraction was washed twice with 0.2 M phosphate buffer (pH 7.2) to remove soluble chromium reductase, if any and was resuspended in same buffer (5 ml) to obtain membrane fraction (P150). Equivalent volume of S12, S150 and P150 fraction were assayed for studying the cellular localization of Cr(VI) reductase enzyme. Cr(VI) reductase assay. Cr(VI) reductase activity of CFE was assayed following the procedure of Park et al. (2000). The reaction mixture (1 ml) for the enzyme assay contained 0.2 mM K2CrO4, 0.2 mM NADH and 400 µl of CFE in 0.2 M phosphate buffer (pH 7.2). The reaction was stopped by adding 0.5 ml of 20% trichloroacetic acid (Horitsu et al., 1987). Reduction of Cr(VI) was measured by estimating the decrease in Cr(VI) in the reaction mixture after 30 min of incubation at 35°C and quantified colorimetrically using 1,5-diphenylcarbazide as the complexing reagent (Urone, 1955). To eliminate the abiotic reduction of Cr(VI), if any, a control set was prepared through out the course of the study without putting any biotic component including bacterial cell. Whenever required, abiotic reduction of Cr(VI) has been subtracted from the total reduction to get the exact influence on chromium reduction by the biotic factors. Amount of protein in the CFE was estimated by the folin-phenol method (Lowry et al., 1951) using bovine serum albumin as the standard. One unit of Cr(VI) reductase activity was defined as the amount of enzyme which decreased l.0 µM Cr(VI) per min at 35°C. The effect of pH and temperature on Cr(VI) reductase were measured at different pH values (4 to 10.6) of the reaction mixture at 35°C and at different reaction temperature (20 to 100°C) at pH 7.2 respectively. Results and Discussions The reductase activities of the soluble fraction (S12) from both the induced and uninduced cells of B. firmus KUCr1 were almost similar with regard to time course (Fig. 1). This study revealed that the chromate reductase in this strain is constitutive, which supports earlier reports on the enzymatic reduction of Cr(VI) under aerobic conditions (Bopp and Ehrlich, 1988; Campos et al., 1995; Wang and Xiao, 1995; McLean and Beveridge, 2001; Pal et al., 2005), though an inducible reductase in the soluble fraction of CFE of Ochrobactrum sp. was reported (Thacker and Datta, 2005). As the reductase activity was found to be constitutive, thus for further experiments CFE (S12) was prepared from cells grown in Cr(VI)-free medium for characterization of chromate reductase activity. The effect of initial concentration of Cr(VI) on reductase activity of S12 fraction was determined at a concentration range of 0 to 80 µM of Cr(VI). Specific activity increased with increasing initial concentration of chromate up to 40 µM, after that it slowed down but reduction continued (Fig. 2a) with enzyme equivalent to 0.493 mg protein/ml. The saturation kinetics of Cr(VI) reduction of S12 fraction fit with the linearized Lineweaver-Burk plot, and the apparent Michaelis-Menten constant (Km) was found at 58.33 µM chromate and Vmax was 11.42 µM per min/mg protein (Fig. 2b). The Km and Vmax values differed from the enzyme activity of the CFE of Bacillus sp. ES 29 (Camargo et al., 2003), B. subtilis (Garbisu et al., 1998), B. Sphaericus AND 303 (Pal et al., 2005), P. putida (Ishibashi et al., 1990; McLean and Beveridge Fig. 1. Cr(VI) reduction by the extracts (S12) from both induced [0.1 mM Cr(VI) in the medium] and non-induced cells of B. firmus KUCr1. The reaction mixture contained 0.2 mM Cr(VI) and 0.2 mM NADH as electron donor in 0.2 M phosphate buffer (pH 7.2) and the reaction temperature was 35°C. 3 187 Chromate reduction by B. firmus cell-free extract Fig. 3. Effect of pH on chromate reductase activity (S 12) of B. firmus KUCr1 at 35°C for 30 min of incubation. Fig. 2. Kinetics of Cr(VI) reduction of cell-free extract (S12) of B. firmus KUCr1 at different Cr(VI) concentrations. Reaction time was 30 min at 35°C (a). Linearized Lineweaver-Burk plot for Cr(VI) reduction of cell-free extract (S12) (b). 2001; Park et al., 2000) and in P. ambigua G-1 (Suzuki et al., 1992). Lower K m value of Cr(VI) reductase suggests higher affinity for the substrate, at least with what was found with the cell-free extract in this strain. The effect of pH on reductase activity was assessed at a pH range of 4.0 to 10.6 using 0.2 M of citrate buffer (pH 4.0 to 5.6), 0.2 M of phosphate buffer (pH 6.0 to 7.6), 0.2 M of tris-HCl buffer (pH 8.4 to 8.8) and 0.2 M of glycine-NaOH buffer (pH 9.2 to 10.6) separately. The reductase activity achieved a maximum at pH 5.6 (Fig. 3). Similarly, the optimum temperature for highest Cr(VI) reduction was found at 70°C (Fig. 4). These results varied from other earlier reports with B. sphaericus AND 303, where they were 30°C and 6.0 respectively (Pal et al., 2005) and with P. putida MK1 (Park et al., 2000) or P. ambigua G-1 (Suzuki et al., 1992), the optimal temperature and pH being 80°C, 50°C, and pH 5.0, 8.6 respectively. The effect of electron donors, inhibitors and metal ions on chromate reduction by CFE (S12) of B. firmus KUCr1 was determined. 0.2 mM each of NADH, NADPH, glutathion, D-glucose, and D-fructose were Fig. 4. Effect of temperature on chromate reductase activity (S12) of B. firmus KUCr1. The assay was conducted in 0.2 M phosphate buffer (pH 7.2) for 30 min. used as electron donors. Among the electron donors used, only NADH showed a significant effect on chromate reductase activity (72% activity over control). NADPH gave 32% less activity than NADH (Table I). Table I Effect of electron donor on Cr(VI) reductase activity in cell-free extracts (S12) of B. firmus KUCr1 Electron donor (0.2 mM) None (control) NADH NADPH Glutathione D-glucose D-fructose a b Specific activitya (U/mg protein) 2.40 (± 0.01) 8.65 (± 0.06) 5.87 (± 0.08) 2.53 (± 0.02) 2.89 (± 0.01) 2.70 (± 0.07) Relative specific activityb (%) 27.74 100.00 67.86 29.24 33.41 31.21 Data are the mean of three replications plus standard error. The reaction mixture contained 0.2 mM Cr(VI) in 0.2 M phosphate buffer (pH 7.2) and was incubated for 30 min at 35°C. {(Specific activity) / (specific activity)NADH } X 100 188 3 Sau G.B. et al. Fig. 5. Effect of azide (NaN3, 0.2 mM)) and some selected metals (0.2 mM) on Cr(VI) reductase activity in the cell-free extracts (S12) of B. firmus KUCr1. Data are the mean of three replications with error bars. In the presence of glutathion, D-glucose, and D-fructose, the non-enzymatic reductants of Cr(VI), the activity of the reductase was almost equal to that of the control. The cell-free enzyme of B. firmus KUCr1 required NADH or NADPH as an electron donor for better enzymatic Cr(VI) reduction. The reductase became sharply more active in the presence of NADH than NADPH, suggesting the requirement of a cofactor for catalytic activity. NADH dependent Cr(VI) reduction was also advocated by several researchers earlier in Bacillus (Garbisu et al., 1998; Camargo et al., 2003) and in Pseudomonas (Suzuki et al., 1992; Park et al., 2000). Our study also supports the earlier observations on the role of nonenzymatic reductants like glutathione, D-glucose and D-fructose on Cr(VI) reduction by CFE (Branca et al., 1990; Shi and Dalal, 1990; Suzuki et al., 1992). Cr(VI) reduction by CFE was significantly inhibited in the presence of 0.2 mM sodium azide (Fig. 5). The metal cations, Ag+, Zn2+, Cd2+, Pb2+, Mn2+, and Hg2+ inhibited reductase activity by more than 50% over the control in the reaction mixture (Fig. 5). However, Co2+, Ni2+, As3+ and Cu2+ stimulated the activity of CFE. The order of stimulation by these metal cations in reductase activity was found to be As3+ > Ni2+ > Co2+ > Cu2+. In this study, Cr(VI) reductase activity was found to be inhibited by a respiratory inhibitor, sodium azide (0.2 mM). Though chromate reductase in CFE was reported to be unaffected by azide in Escherichia coli ATCC 33456 (Shen and Wang, 1993), Bacillus sp. ES29 (Camargo et al., 2003) and in B. megaterium TKW3 (Cheung et al., 2006) by 1.0 mM NaN3. However, inhibition of Cr(VI) reduction due to NaN 3 in live cells of a Bacillus subtilis strain was reported (Garbisu et al., 1998). Inhibition of a cytoplasmic membrane associated Cr(VI) reductase by azide was also reported in Shewanella putrefaciens MR-1 (Myers et al., 2000). The interference of azide in Cr(VI) reduction in this study and views from earlier reports suggest the possible coexistence of Cr(VI) reductase in the cytosol and membrane as well. However, further investigations are required to elucidate this particular feature. The cations Hg2+ and Ag + (0.2 mM) inhibited reductase activity. The noncompetitive inhibitory effect of Hg2+ and Ag+ on Cr(VI) reduction in P. putida was reported earlier (Ishibashi et al., 1990). The inhibitory effect of Hg2+ is expected, because of its affinity for ligands containing thiol (-SH) group of a variety of enzymatic proteins. Enzymatic reduction of Cr(VI) by CFE of B. firmus KUCr1 was stimulated by Cu2+, As3+, Ni2+ and Co2+ at 0.2 mM concentration separately, though Pal et al. (2005) reported the inhibition of Cr(VI) reductase activity of B. sphaericus AND 303 by Ni2+ and Co2+ at 100 µM concentration. The reductase activity was found to be unaffected by As3+ in P. putida MK1 (Park et al., 2000). Stimulation of enzyme activity by Cu2+ might be due to its nature as a prosthetic group of many reductase enzymes and also indirectly involved in the protection of chromate reductase from O2, for oxygen-sensitive enzyme (Ettinger 1984; Camargo et al., 2003). Abe et al. (2001) reported that Cu2+ acting as electron-transport protection or acting as a single electron redox center and as a shuttle for electron between protein subunits. Zn2+, Cd2+, Pb2+ and Mn2+ inhibited the reductase activity at 0.2 mM. Inhibitory effects by Cd2+ and Zn2+ support the earlier views (Park et al., 2000; Pal et al., 2005; Desai et al., 2008). On the contrary, Camargo et al. (2003) showed slight stimulatory effect of Mn2+ on Cr(VI) reductase in Bacillus sp. ES 29. These variations seem to be due to the different functional nature of the Cr(VI) reductase in B. firmus KUCr1, which warrants further investigation. Cr(VI) reductase activities by different cellular fractions are presented in Table II. In the presence of electron donor (0.2 mM NADH) S150 fraction showed higher activity than fractions S12 and P150. Table II Localization of Cr(VI) reductase in cell fractions of B. firmus KUCr1 and its catalytic activity Cellular fractiona a b c % Cr(VI) reductionb Specific activityc (U/mg protein) S12 14.21 9.12 (± 0.03) S150 15.66 14.68 (± 0.04) P150 6.98 7.34 (± 0.04) S stands for soluble cellular fractions and P stands for membrane fractions (see Materials and Methods). Reaction mixture contained 0.2 mM Cr(VI) in phosphate buffer (pH 7.2) and was incubated for 30 min at 35°C. Data are the mean of three repetitions plus standard error. 3 Chromate reduction by B. firmus cell-free extract The percentage of Cr(VI) reduction by fraction P150 is significantly less compared to other fractions, but the specific activity signifies its catalytic function, suggesting the occurrence of some membrane-bound protein responsible for Cr(VI) reduction. In Bacillus QC1-2 (Campos et al., 1995), B. sphaericus AND 303 (Pal et al., 2005) and P. putida (Ishibashi et al., 1990; Park et al., 2000) the chromium reductase activities were reported to be associated with the cytosolic and soluble fractions. In Enterobactor cloacae (Wang et al., 1990), S. putrefaciens MR-1 (Myers et al., 2000), B. megaterium TKW3 (Cheung et al., 2006) and P. fluorescences (Bopp and Ehrlich, 1988) Cr(VI) reductase appears to be membrane associated. Lovley and Phillips (1994) reported that both the soluble and membrane fractions reduced chromate in Desulfovibrio vulgaris but soluble protein fraction reduced Cr(VI) faster than the membrane fraction did. In Bacillus cereus S-6 (Iftikhar et al., 2007) Cr(VI) reduction occur both in cytosolic and membrane fraction of this strain but percentage of reduction in cytosolic fraction is higher than membrane fraction. It seems that KUCr1 harbors Cr(VI) reductase constitutively both in membrane and cytosol. 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Chemosphere 57: 609613. Xu X.R., H.B. Li, J.D. Gu and X.Y. Li. 2005. Kinetics of the reduction of chromium (VI) by vitamin C. Environ. Toxicol. Chem. 24: 13101314. Polish Journal of Microbiology 2010, Vol. 59, No 3, 191200 ORIGINAL PAPER Occurrence and Characterization of Colletotrichum dematium (Fr.) Grove ZOFIA MACHOWICZ-STEFANIAK Department of Phytopathology, University of Life Sciences Lublin, Poland Received 9 November 2009, revised 20 January 2009, accepted 1 March 2010 Abstract Colletotrichum dematium was isolated from caraway for the first time in Poland in 2005. Isolations of this fungus were repeated in 2006 and 2007. The cultures of fungus were obtained from superficially disinfected leaves, root necks, roots, stems and umbels. The isolates were identified on culture media: PDA and malt agar with addition of pieces of caraway stems and on the base of macro and microscopic structures. Studies on the biotic effect between C. dematium and other species of phyllosphere fungi of caraway showed that the majority of the studied species limited the growth and development of C. dematium, but the size of the limiting effect was different. The species from Trichoderma and Gliocladium genera were the most effective against C. dematium, causing degeneration and lysis of hyphae and precluded the formation of the pathogens acervuli and conidia. C. dematium in dual culture with E. purpurascens, A. radicina, S. sclerotiorum, B. cinerea and R. solani produced an inhibition zone which indicated its capacity for antibiosis. K e y w o r d s: Colletotrichum dematium, biotic activity, caraway isolates, phylosphere fungi Introduction Fungi from the genus Colletotrichum occur in all climatic zones and cause diseases of various plant species, especially in hot and moderate climates. They can be polyphagic but some of them are pathogens of only one species of host plant (Frencel et al., 1997; Sutton, 1980). Till the middle of the 20th century, over 1000 species of these fungi were described basing on the morphological structures of conidia, setoses and host plant (Von Arx, 1957). As a result of studying these fungi on artificial culture media in the laboratory, the number of species was reduced to 40 (Sutton, 1980). At present, the numerous existing species of the genus Colletotrichum are gathered in one common species on the basis of genetic diversity (Frencel et al., 1997). The following species belong to the commonly occurring pathogenic species: C. gloeosporioides (Penz.) Sacc. (teleomorph: Glomerella cingulata (Stonem.) Spauld. et Schrenk), C. lindemuthianum (Sacc. et Magn.) Br. et Cav., C. atramentarium (Berk. et Br.) Taubenh., C. acutatum Simmonds, C. lini (Manns) Bolley, C. orbiculare (Berk. et Mont.) Arx and others (Sutton, 1980; Farr et al., 1995; Frencel et al., 1997; Gärber and Schenk, 2001). C. dematium, the genus typical species, is a saprotroph and colonizes various plant species as a second- ary pathogen and pathogenic strains of this fungus cause plant diseases (Von Arx, 1957; Sutton, 1980; Farr et al., 1995). C. dematium f. circinans cause anthracnose of onion cultivated in various climatic regions in the world (Sutton, 1980). The occurrence of C. dematium was ascertained in India on various pea-plant cultivars and in South Africa on stems and pods of cowpea (Smith et al., 1999; Shinde et al., 2003). The species was isolated from dying plants of Catharanthus roseus (L.) G. Don in Florida in 1991 and positive results of artificial infection of plants were obtained (McMillan and Graves, 1996). C. dematium caused anthracnose of spinach on some farms in Australia and the harmfulness of this fungus for various spinach cultivars was confirmed by pathogenicity tests (Washington et al., 2006). In Japan, the harmfulness of C. dematium for Japanese radish was confirmed by pathogenicity tests (Smith et al., 1999). Anthracnose caused by C. dematium was discovered on various cultivars of strawberry in India, which fact was confirmed by pathogenicity tests (Singh et al., 2003). C. dematium occurred on mulberry in Japan and on various species of plants from the Amaryllidaceae family (Yoshida and Shirata, 1999; BonillaBernal et al., 2003). In post-culture liquids of C. dematium f. sp. epilobii, the pathogenic species for willow herb (Epilobium angustifolium L.), the presence of * Corresponding author: Z. Machowicz-Stefaniak, Department of Phytopathology, University of Life Sciences Lublin, Poland; e-mail: [email protected] 192 3 Machowicz-Stefaniak Z. secondary metabolites with phytotoxic and zootoxic abilities was found (Abou-Zaid et al., 1997; Mendiratta et al., 2005). As a result of many-years studies conducted on diseases of herb plants C. dematium was isolated for the first time on caraway in Poland in 2005. Therefore, attention to the occurrence of this fungus in the next years of studies was directed and macroscopic and microscopic features of isolates of the pathogen and the biotic effects between C. dematium and other species of phyllosphere fungi of caraway were studied. Material and Methods The studied material consisted of isolates of Colletotrichum dematium (Fr.) Grove obtained from the organs of one-year-old and two-years-old plants of caraway cultivated in the Lublin region in the years 20052007 (Table I). The artificial culture method and malt agar medium were used for the isolation of this fungus (Machowicz-Stefaniak and Zalewska, 2008). One- spore cultures of 15 isolates of fungus: K117, K123, K425, K426, K510, K514, K612, K625, K626, K 628, K630, K631, K633, K651, K657 were chosen randomly from our professional collection. For identification, the isolates were cultured on malt agar medium with an addition 50 g/dm 3 of 23 mm pieces of caraway stems and on PDA medium (Difco), in a thermostat, at the temperature 24°C, in dark conditions for 14 days (Machowicz-Stefaniak, 2009). The character of cultures, the color of the averse and the reverse, the formation and morphology of fungus acevuli and conidia were studied at this time. To determine the structures mentioned above, the measurements of 150 acervuli (15 isolates per 10 acervuli) and 600 conidia (15 isolates per 40 spores) were made. Moreover, the size of setoses and appressoria of the studied isolates was determined. The photos of the above mentioned morphological elements were taken using light and scanning SEM microscope. To identify the studied isolates, the descriptions of Von Arx (1957), Pidoplièko (1977) and Sutton (1980) were used. To study the biotic effects of fungi, 3 isolates of C. dematium: K425, K426, K625 and 22 isolates of fungi species mentioned in Table III were taken. Those isolates were chosen randomly from our professional collection of fungi gathered in the years 20012008, as a result of a study on diseases of caraway (Machowicz-Stefaniak and Zalewska, 2004; 2008; Machowicz-Stefaniak, 2009). Because of the lack of information concerning the biotic relation between C. dematium and other fungi, the maximum number of fungal species was taken for this study, irrespective of the frequency of their isolation from caraway (Machowicz-Stefaniak and Zalew- Table I Occurrence of C. dematium on aboveground organs of caraway (Carum carvi L.) in 20052007 Organs Years leavs stems base of stems roots umbels 2005 ++ ++ +++ +++ +++ Participation of izolates 2006 2007 ++ ++ + +++ + ++ ++ ++ +++ + frequency of occurrence < 5% ++ frequency of occurrence from 5 to 10% +++ frequency of occurrence > 10% ska, 2004; 2008; Machowicz-Stefaniak, 2009). The species Gliocladium catenulatum, G. fimbriatum, G. roseum and Trichoderma viride were taken from other cultivated plants, because they were not isolated from caraway. The study of biotic effects was carried out using the biotic series method on PDA (Difco) medium, which was elaborated for soil fungi community firstly (Mañka, 1974; Mañka, 1995). This method was adapted for fungi colonizing the phyllosphere of plants (Mañka, 1995; Machowicz-Stefaniak, 1998; Król and Machowicz-Stefaniak, 2008). The two-organism cultures consisting of C. dematium and one of the fungi representing the community component were studied on PDA medium in Petri dishes, according to the method by Mañka (1974) and Mañka (1995). The dishes with medium on which the mycelium of a single fungal species was placed constituted the control. For each combination, i.e. for C. dematium with a species of fungus representing the community component and control, 4 replications were made. The biotic effect of the fungi in dual cultures was evaluated after 12 days of common growth, but in the case of Gliocladium spp. after 24 days, at 23°C, in dispersion light, based on an eight-degree scale. One colony being surrounded by other species, the occurrence of the inhibition zone between them and the reduction of the colony size were taken into account while the IBEs were evaluated (Mañka, 1995). If the colony of C. dematium was overgrown by other species of fungi, the appearance of mycelium and conidia of the studied fungus were evaluated. The biotic effect of fungi representing the phyllosphere of caraway on C. dematium was estimated as an individual biotic effect (IBE). The size of IBE consisted of the arithmetic sum of values for surrounding of the colony, the inhibition zone and the reduction of colony size. The size of IBE indicates the effect of one isolate of the community species on pathogen growth (Mañka, 1974). Positive IBE indicates suppressive effect on pathogen growth, negative indi- 3 193 Grove of Colletotrichum dematium (Fr.) from caraway Fig. 2. Acervuli and setose of C. dematium in light photo microscope (Photo E. Zalewska). Fig. 1. 14-day-old colony of C. dematium isolate K 425 on the PDA medium, at 25°C (Photo E. Zalewska). cate slack of suppressive effect on pathogen growth, while the value of the effect may be 0 indicating neutral influence (Mañka, 1974; Mañka, 1995). Results The isolates of C. dematium were obtained for the first time in 2005 from the leaves, the neck of roots and the roots of 0.66% of caraway seedlings. The isolations of fungi repeated in 2006 and 2007, respectively from 1.2% and 2.16% of plants in the second year of cultivation. The cultures of fungus were obtained most often from the stems, the umbels and the roots with different frequency in different years (Table I). C. dematium was isolated from the parts of plants with nonspecific etiological and disease lesions, which can indicate the presence of the pathogen in the plant tissues. On the other hand, among the fungi isolated from plants on malt agar medium there were isolates coloring the medium violet and forming dark gray, velvet mycelium with numerous brown appressoria an the top and crossing hyphae, the organs characteristic of Colletotrichum genera. However, acervuli and spores of fungi were obtained scarcely on malt agar medium with an addition of pieces of caraway stems and on PDA medium. The one-spore-cultures of this fungus chosen for the subsequent studies were cultivated on the above mentioned two artificial media for 14 days with their size ranging from 3.5 to 5.5 cm. The colonies were loose and slightly fluffy in the beginning but later the hyphae became more and more thickened and formed a compact surface. The colour of the mycelium was gray or dark gray. The reverse of the colonies was pink to violet on PDA medium with a visible diffusion of the dye to the culture medium (Fig. 1). On the malt agar medium the reverse was beige-yellow. At the beginning of day 6 of cultivation, acervuli were formed on PDA medium and after 10 days on malt agar medium with an addition of caraway stems pieces. Numerous acervuli were observed after 14 days of the cultivation, and covered the entire surface of the colony but sometimes they were formed in sectors. Acervuli were slightly immersed in the medium, almost black, lenticular, flat or pulvinate with sufficiently visible and high rends. The diameter of acervuli was 495.44× 371.48 µm (Table II). All around the ostiole and on the surface of acervuli numerous setoses were formed. (Fig. 2, 3). The setoses were dark brown or almost black, generally septate (Fig. 2), unpliant, smooth, tapered to the apices, their size ranging from 36.05 to 202.99 µm in length and at the Table II Size (µm) of morphological structures of C. dematium on PDA medium (mean for 15 isolates) Author acervuli (µm) setose (µm) conidia (µm) appressoria Own measurements Sutton 1980 Pidoplièko 1977 Von Arx 1957 495.44 × 371.48 36.05202.99 × 3.835.74 7.6615.28 × 5.7413.37 814.5 × 6.58 250 150 × 4 100600 17.1924.83 × 3.825.72 19.524 × 22.5 (3.5) 25 × 5 1830 × 34.5 194 3 Machowicz-Stefaniak Z. (a) (b) Fig. 3. Acervuli and setose of C. dematium (a), setose (b) in SEM (Photo M. Wróbel). base from 3.83 to 5.74 µm in width (Table II). Thick, almost black, shining drops resulting from the large number of conidia were emerged from the mature acervuli. The conidia were hyaline, aseptate, smooth, falcate or fusiform, and they had acute apices (Fig. 4). A visible gutate breaking the light was observed in the middle of the conidia. The size of the studied isolates conidia was 17.1924.83× 3.25.72 µm on PDA, and 18.526.74× 2.883.7 µm on malt agar with an addition of caraway stem pieces (Table II). At the end of hyphae or in the middle of them abundant appressoria occurred. Unlike the vegetative hyphae, they were brown or almost black, irregular in shape, single or gathered and their size was 7.6615.28×5.7413.37 µm (Fig. 5). The formation of sclerotia was not observed in the studied isolates. Among the 22 tested species of phyllosphere fungi, the majority, i.e. 18 species limited the growth and development of two isolates of Colletotrichum dematium K425 and K426, and 17 species of isolate K625, Table III Biotic effect of fungi isolated from caraway (Carum carvi L.) on Colletotrichum dematium Fungal isolates Alternaria alternata (Fr.) Keissler (K 461) Alternaria radicina Meier, Drechsler et Eddy (K1723) Botrytis cinerea Pers. (K 1777) Cladosporium cladosporioides (Fres.) de Vries (K 518) Epicoccum purpurascens Ehrenberg (K 1696) Fusarium avenaceum (Fr.) Sacc. (K 56) Fusarium culmorum (W.G.Smith) Sacc. (K 284) Fusarium equiseti (Corda) Sacc. (K304) Fusarium oxysporum Schlecht (K 271) Fusarium sporotrichioides Sherb (K 465) Phoma exigua Desm. var exigua (K 1503) Phomopsis diachenii Sacc. (K 255) Rhizoctonia solani Kühn (K 1561) Septoria carvi Syd. (K 1833) Sclerotinia sclerotiorum (Lib.) de Barry (K 2313) Stemphylium botryosum Wallv. (K 296) Gliocladium catenulatum Gilman et Abbott (L 4940) Gliocladium fimbriatum Gilman et Abbott (W 76) Gliocladium roseum Bainier (L 830) Trichoderma harzianum Rifai (K 428) Trichioderma koningii Oud. (K 437) Trichoderma viride Pers. et Gray (W 1222) Individual biotic effect IBE after 12 days C. dematium isolates K 425 1 +1 +4 2 +1 +4 +5 +5 +5 +7 +3 +6 +5 6 +5 4 +2 +2 +2 +8 +7 +7 K 426 1 +1 +3 2 +1 +4 +5 +4 +5 +6 +3 +6 +5 6 +5 3 +2 +2 +3 +8 +7 +7 K 625 0 +1 +4 2 2 +4 +4 +4 +5 +7 +3 +5 +5 6 +5 3 +2 +2 +3 +8 +8 +7 3 Grove of Colletotrichum dematium (Fr.) from caraway (a) 195 (b) Fig. 4. Conidia of C. dematium in light photo microscope (magnification × 500) (a) (Photo E. Zalewska), SEM (b) (Photo M. Wróbel). Fig. 5. Appressoria of C. dematium on PDA medium (magnification × 500) (Photo E. Zalewska). which indicates the positive individual biotic effects observed after 12 days of dual growth (Table III). The species of fungi from Trichoderma genus limited the growth of the studied isolates of C. dematium to the highest degree because their individual biotic effect was + 8 for T. harzianum and + 7 for T. viride and T. koningii (Table III, Fig. 6). The studied species from Trichoderma genus caused lyses and degeneration of C. dematium hyphae (Fig. 7). The observation showed that the colonies of each studied Trichoderma species totally overgrew the inoculums of C. dematium and made the growth and sporulation of the pathogen impossible. In the slides of 12-daysold colonies of C. dematium and T. harzianum and of C. dematium with T. viride the concentration and disintegration of cytoplasm in C. dematium hyphae were observed. Moreover, after 12 days of dual growth of the pathogen with T. koningii deformed hyphae of C. dematium with a big concentration of melanine were observed (Fig. 7). The fungi from the genera Gliocladium slightly limited the growth of C. dematium in the first day of common growth. G. catenulatum and G. fimbriatum grew on 1/3 and G. roseum on 1 of the surface of the pathogens colony after 12 days of dual growth (Fig. 8). In the slides chains of miss-shapen, dark hyphae of C. dematium and numerous hyphae of the pathogen being subject to lysis were observed (Fig. 9). Moreover, it was noticed that after 18 days of dual growth micoparasitic fungi practically overgrew the whole surface of the pathogen, i.e. 7/8 of the pathogens colony and after 24 days the whole surface of C. dematium colony was overgrown by them causing total lyses of the pathogen»s hyphae. The growth of C. dematium colony was strongly limited by P. diachenii as its IBE was + 6 and in the case of isolate K625 IBE was + 5 (Table III). Among fungi of the Fusarium genus the growth of the pathogen was limited more strongly by F. sporotrichioides species and its IBE was + 7 (Table III). On the other hand, F. avenaceum, F. culmorum, F. equiseti, F. oxysporum, Rhizoctonia solanii, Sclerotinia sclerotiorum and Botrytis cinerea slightly limited the growth of C. dematium colony in comparison to F. sporotrichioides (Table III). Moreover, it was observed that C. dematium in dual growth with S. sclerotiorum, B. cinerea and R. solani formed an inhibition zone (Fig. 10). The fungi Alternaria radicina and Epicoccum purpurascens inhibited the growth of C. dematium colonies in a small degree and their IBEs were +1. However, isolate K625 C. dematium limited the growth of Epicoccum purpurascens, which was shown by the negative IBE value (Table III). Moreover, the fungus C. dematium limited the growth of Septoria carvi giving IBE-6, Cladosporium cladosporioides-2, Alternaria alternata-1, Epicoccum purpurascens and Stemphylium botryosum from 3 to 4 (Table III). 196 (a) Machowicz-Stefaniak Z. 3 (b) (c) Fig. 6. C. dematium isolate K 425 (left) and T. harzianum (right) after twelve days of dual growth (a), individual growth of C. dematium (b) and T. harzianum (c) (Photo E. Zalewska). (a) (b) Fig. 7. Degeneration of C. dematium hyphae isolate K 425 caused by T. harzianum (magnification x 500) (Photo E. Zalewska). 3 Grove of Colletotrichum dematium (Fr.) from caraway (a) 197 (b) (c) Fig. 8. C. dematium isolate K 425 (left) and Gliocladium catenulatum (right) after twelve days of dual growth (a), individual growth of C. dematium (b) and G. catenulatum (c) (Photo E. Zalewska). Fig. 9. Degeneration of C. dematium isolate K 425 caused by G. catenulatum, condense of cytoplasma (a), degeneration of the hyphae (b) (magnification × 750) (Photo E. Zalewska). 198 Machowicz-Stefaniak Z. (a) 3 (b) (c) Fig. 10. C. dematium isolate K 425 (left) and Sclerotinia sclerotiorum (right) after twelve days of dual growth (a), individual growth of C. dematium (b) and S. sclerotiorum (c) (Photo E. Zalewska). Discussion Including the studied isolates in the species Colletotrichum dematium was possible on the basis of macroscopic and microscopic features of their colony and on the basis of morphology and size of acervuli and conidia. The above-mentioned features were compared to those shown by Von Arx (1957), Pidoplièko (1977) and Sutton (1980). Small differences, especially in the size (length and weight) of conidia are a consequence of differentiation of culture medium on which the fungus grows. The dependence of the growth and development of numerous fungi species from the culture conditions and from the composition of culture medium was indicated by a lot of authors (Uecker 1988, Sutton 1980). The straight growth of studied isolates as well as the ability to produce the acervuli and conidia just after a few days old colonies on PDA medium suggested that medium is favorable to the culture and identification of C. dematium. The production of a numerous number of acervuli by C. dematium on PDA was indicated earlier by Azad et al. (2005). The presence of a numerous and high setoses occurring all around the ostiole of acervuli and many brown appressoria should be recognized as a favorable and characteristic micromorphological feature causing the identification of the studied fungus easier, which was indicated earlier by Von Arx (1957). The latter organs, i.e. appressoria could be significant in pathogenesis because they could attach hyphae of the pathogen to the surface of plants (Von Arx, 1957). The 3 Grove of Colletotrichum dematium (Fr.) from caraway high tinctorial power of the culture medium characteristic of all the studied isolated of C. dematium points to the possibility of this fungus producing secondary metabolites. The detection of C. dematium in Poland for the first time on caraway plants increased the number of host plant to this fungus. The present studies showed that a lot of caraway phyllosphere fungi limited the growth of C. dematium and the positive values of IBE indicate it, but the size of limiting possibilities was different. Among the fungi inhibiting the growth of C. dematium there were the species from the genera Trichoderma and Gliocladium. These fungi are known for their antagonistic influence against pathogenic fungi (Fokkema, 1993; Machowicz-Stefaniak, 1998; Król and Machowicz-Stefaniak, 2008). T. harzianum, T. koningii and T. viride should be recognized as the most effective antagonists for C. dematium, which was indicated by complete overgrowth and destruction of the pathogen colonies by these antagonists just after a few days of dual growth. Similarly, these special high antagonistic abilities of Trichoderma spp., were indicated earlier for other pathogens of caraway like Septoria carvi and Phomopsis diachenii (Machowicz-Stefaniak et al., 2008; MachowiczStefaniak, 2009). It is probably possible thanks to the emission of constitutional enzymes and those produced as an effect of contact with the pathogen, as well as the ability to form toxic metabolites and the ability to mycoparasite (Fokkema, 1993). Thanks to these abilities, Trichoderma spp. are used in the production of biopreparates (Cohen et al., 1996). A significantly slower antagonistic effect of Gliocladium spp., (unlike that of Trichderma spp.) towards C. dematium and other pathogenic fungi results from antibiosis and the ability to mycoparasite in the lack of competitive abilities (Fokkema, 1993; MachowiczStefaniak et al., 2008; Machowicz-Stefaniak, 2009). Therefore, full antagonistic activity of Gliocladium spp. to C. dematium was not shown until 20 days in vitro. On the other hand, recently Epicoccum purpurascens have been recognized as a fungus strongly inhibiting the growth of different microorganisms thanks to the possibilities to produce siderophores, flavipine and Epicorazine B (Frederick et al., 1981; Mallea et al., 1991; Fokkema, 1993) In the present studies the fungus showed only slight possibilities of inhibiting the growth of C. dematium. On the other hand, the production of inhibition zone by one of the pathogens studied isolates in a dual culture with E. purpurascens confirmed the ability of C. dematium to produce secondary metabolites, which were detected in ethyl-acetyl extract of culture liquids pathogenically forms of C. dematium (Abou-Zaid et al., 1997, Mendirata et al., 2005). The inhibiting effect of C. dematium isolates to A. alternata, C. cladosporioides, S. carvi and S. botry- 199 osum also seems to confirm the ability of C. dematium for antibiose. Fast-growing phytopathogenical fungi possessing a big enzymatic ability and used in the present studies, i.e. S. sclerotiorum, B. cinerea and R. solani only partly limited the growth of C. dematium because the last mentioned species produced an inhibition zone during the common growth with each of the above mentioned species. The inhibition zone formed by C. dematium was not observed in dual growth with the species of genera Fusarium. The last mentioned species and especially F. sporotrichioides suppressed the colonies of C. dematium from the culture medium. It was probably possible thanks to the big power of their growth and the ability to produce secondary metabolites (Kiecana and Perkowski, 1998). Taking into account the inhibiting effect of the studied phyllosphere fungi towards C. dematium, it seems that the species of Trichoderma and Gliocladium genera could be recognized as positive antagonistic fungi. In perspective, these species may be used in biological control of C. dematium. The other studied species of fungi, despite only partly limiting C. dematium growth, are dangerous pathogens of many cultivated plant and their occurrence in culture plants phyllosphere is undesirable. Comparing the results of the present studies with the results of similar studies conducted for other pathogens of caraway like Septoria carvi (MachowiczStefaniak et al., 2008) and Phomopsis diachenii (Machowicz-Stefaniak, 2009) it is possible to notice that they have numerous antagonists among phyllosphere fungi. That is why their isolation from plant tissues on artificial media may be difficult. Among the three above-mentioned pathogenic fungi S. carvi showed the worst antagonistic activity (MachowiczStefaniak et al., 2008). It seems that isolates of P. diachenii have better competitive abilities than C. dematium (Machowicz-Stefaniak, 2009). On the other hand, C. dematium have high abilities for antibiosis. On the basis of the present results and the data in literature we can suggest the need to study the secondary metabolites of fungi not only in the aspect of their phytotoxicy and zootoxicy but also in the direction of knowing about their mechanisms of antibiosis. It is interesting if it results from secretion of antibiotic, lytic enzymes or from excessive acidification or alkalization of the medium (Fokkema, 1993). Literature Abou-Zaid M., M. Dumas, D. Chauret, A. Watson and D. Thompson. 1997. C-methyl flavonols from the fungus Colletotrichum dematium f. sp. epilobii. Phytochemistry 45, 5: 957961. Azad A.K.M., M.M. Kamal, S.H. Howlader, M. Hossain and A.M. Akanda. 2005. Morphology of six isolates of Colletotrichum 200 Machowicz-Stefaniak Z. species and their host range. Bangladesh Journal Plant Pathology 21 (1/2): 7176. Bonilla-Bernal T., I., Sandoval-Ramirez, G. Estrada-Villardel and M.O. Lopez. 2003. Species of fungi found on Amaryllidaceae. Fitosanidad 7 (3): 1316. Cohen A., Y. 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A Word list of Phomopsis names with notes on nomenclature. Morphology and biology. Mycol. Mem. 13: 231. Washington W.S., G. Irvine, R. Aldaoud, S. DeAlwis, J. Edwards and I.G. Pascoe. 2006. First record of anthracnose of spinach caused by Colletotrichum dematium in Australia. Australasian Plant Pathology 35 (1): 8991. Von Arx J.A. 1957. Die Arten der Gattung Colletotrichum Cda. Phytopathol. 29: 413468 [in Germany]. Yoshida S. and A. Shirata. 1999. Survival of Colletotrichum dematium in soil and infected mulberry leaves. Plant Disease 83, 5: 465468. Polish Journal of Microbiology 2010, Vol. 59, No 3, 201205 ORIGINAL PAPER Cytotoxic Activity of Serratia marcescens Clinical Isolates SYLWIA KRZYMIÑSKA, MARTA RACZKOWSKA and ADAM KAZNOWSKI* Department of Microbiology, Faculty of Biology, A. Mickiewicz University, Poznañ, Poland Received 3 March 2010, revised 27 May 2010, accepted 1 June 2010 Abstract Twenty Serratia marcescens isolates from clinical specimens were examined for their cytotoxic activity on four cell lines (HEp-2, Vero, CHO, J774). Most of the isolates were found to be cytotoxic to CHO (70%), Vero (75%) and HEp-2 cells (90%). CHO cells were the most sensitive to cell-free supernatants, followed by HEp-2 and Vero cells. Two strains produced cytotonic toxins which caused elongation of CHO cells. Moreover, twelve isolates (60%) revealed cytotoxic potential to macrophage cell line J774. The results indicate that these bacteria may destroy phagocytes and epithelial cells, which may lead to spread within the host. K e y w o r d s: Serratia marcescens, cytotoxicity Introduction Strains of Serratia marcescens have been recognized as an important nosocomial pathogen causing a variety of diseases, including respiratory and urinary tract infections, meningitis, peritonitis and bacteremia. The bacteria are becoming increasingly important cause of many outbreaks and endemic nosocomial infections, particularly among newborns and patients submitted to invasive procedures (Buffet-Batoillon et al., 2009; Friedman et al., 2008; Grimont and Grimont, 2006). Although S. marcescens is the common Serratia species causing nosocomial infections, little is known about the factors impacting their pathogenicity and virulence. The possible mechanism of the pathogenesis is complex and multifactorial, with the involvement of a number of putative virulence factors whose role in development of disease is not clear. The first step of pathogenesis is colonization of epithelial cells. After adhesion to the cells strains produce many potential virulence factors, including extracellular toxins which are probably the most common mechanism of pathogenicity. Some S. marcescens isolates produce hemolysin, which represents the prototype of a family of pore forming toxins with hemolytic and cytotoxic activity (Hertle, 2005). Strains of S. marcescens have been reported to produce many extracellular proteins, including proteinases, lipases, nucleases, chitinases, lecithinases, which may directly contribute to cellular cytotoxicity by exerting their damaging effects upon host cells (Grimont and Grimont, 2006). The major defense mechanism of host nonspecific immunity represents cell-mediated killing. Phagocytic cells such as macrophages and neutrophils contribute to the primary line of innate defense against bacterial pathogens by providing their removal and destruction at the level of the epithelial barrier. Therefore, many bacterial pathogens have developed specific strategies to suppress the effective antimicrobial immune response of macrophages to avoid the innate immune defense of the host (Navarre and Zychlinsky, 2000). S. marcescens may infect numerous sites of the host body, including lungs and respiratory epithelia, muscle and soft tissues, therefore in the current study, we have examined the cytotoxic activity of human isolates to different mammalian epithelial cell lines (Vero, CHO, HEp-2). Moreover, we investigated the effect of S. marcescens cell-free filtrates on macrophage cell line J774. Experimental Materials and Methods Bacterial strains. A total of 20 isolates (listed in Table I) identified as Serratia marcescens by biochemical test kit (API20E, bioMerieux) were analyzed. They were originated from specimens of hospitalized * Corresponding author: A. Kaznowski, Department of Microbiology, Faculty of Biology, A. Mickiewicz University, ul. Umultowska 89, 61-614 Poznañ, Poland; phone (+48) 61 529 5937; fax (+48) 61 829 5590; e-mail: [email protected] 202 3 Krzymiñska S. et al. Table I Serratia marcescens strains used in the study Source of origin (number of strains) Ulceration (4) Urine (4) Postoperative wound (3) Catheter (1) Feces (1) Blood (1) Aspirate (2) Pus: pharynx, ear, abscess, drain (4) Isolates No MPU S1, MPU S4, MPU S7, MPU S23 MPU S3, MPU S12, MPU S18, MPU S21 MPU S6, MPU S9, MPU S11 MPU S20 MPU S15 MPU S22 MPU S2, MPU S10, MPU S5, MPU S14, MPU S19, MPU S13 patients and belonged to Bacterial Culture Collection of Department of Microbiology Poznañ University (MPU). Seven isolates originated from wounds (postoperative and ulcerations), 4 from secretions (2 from aspirates and 1 from ear and pharynx), 4 from urine, 1 from blood, 2 from catheter and drain, 1 from fecal specimen. The isolates were maintained at 75°C in trypticase soy broth (TSB, Difco) containing 50% (vol/vol) glycerol. Cell cultures. Human epidermoid carcinoma cells from the larynx (HEp-2), Chinese hamster ovary cells (CHO) and African monkey kidney (Vero) were cultured in Eagle Minimum Essential Medium (EMEM, Sigma) with 5% fetal calf serum (FCS, Sigma) containing 2 mM glutamine, 50 IU of penicillin per milliliter, streptomycin (100 µg/ml) and nystatin (1 mg/ml). The murine macrophage cell line, J774 was maintained in growth medium (GM), containing RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, gentamicin (5 µg/ml) and 2 mM L-glutamine (Krzymiñska et al., 2009) The cells in number 1× 104 cells per well seeded with 100 ml of suspension and incubated at 37°C in an atmosphere with 5% CO2. Preparation of bacterial cell-free supernatants. For preparing bacterial filtrates, the strains were cultured in brain heart infusion broth (BHI, Difco) at 37°C. The cultures were incubated on Luria-Bertani medium (LB, Difco) in a shaking incubator with agitation at 300 rpm at 37°C for 24 h. After centrifugation at 3000 rpm for 20 min, the supernatants were sterilized through 0.22 mm-pore size membrane filters Millex-GV (Millipore). Sterile culture supernatants were heated at 56°C for 20 min to destroy activity of heat-labile toxins (Carbonell et al., 1997). Cytotoxic and cytotonic activity to epithelial cells. The assay was performed according to Krzymiñska et al. (2009). Twofold serial dilutions (from 1:2 to 1:512) of culture filtrates in phosphate buffered saline (PBS, Biomed) added to the wells of tissue culture plate containing confluent Vero and CHO mono- layers and incubated for 24 hours at 37°C. As negative controls the wells received non-pathogenic E. coli K12C600 filtrates. Cytotonic activity revealed as elongation of CHO cells. The cytotoxic titer of each isolate was calculated by determining the reciprocal of the highest dilution of culture filtrates which produced a cytopathic effect. The results were observed under an inverted microscope. The results were presented as mean titers from two experiments in triplicate. Cytotoxic activity to murine macrophage J774 cells by Neutral Red retention assay. Neutral Red (NR) is a biomarker of cellular stress and supravital dye taken up in the lysosomes of viable cells (Maleri et al., 2008). The assay was performed in microtitration plates with a method by Carbonell et al. (1997) with slightly modifications. Macrophage monolayer was incubated with bacterial culture filtrates at 37°C for 24 h. As a negative control, the cells were infected with non-pathogenic E. coli K12C600 filtrate. Next, the medium was removed and the cultures were washed with PBS and 200 µl of NR (50 µg/ml) was added to each well and incubated for 3 h at 37°C. After incubation the dye solution was aspirated, cells were rinsed in PBS before being fixed with formalin in calcium chloride solution (40% formaldehyde, 10% anhydrous calcium chloride) which was next removed, and incorporated dye was eluted from the cells by adding ethanol/acetic acid mixture (50% ethanol and 1% acetic acid). The absorbance at 540 nm was measured using a plate reader. All absorbance values were corrected against blank wells which contained growth medium alone which were processed as described above. Cell viability was determined by comparing the absorbance values obtained from the control wells (taken as 100% viability). Results Cytotoxic and cytotonic activity to epithelial cells. Cytotoxic potential of S. marcescens isolates was studied on three different cell lines, including Vero, CHO and HEp-2 cells. Microscopic examination of the cells following incubation with cell-free supernatants revealed a number of changes: rounding and shrinking of cells, followed by detachment, loss of cytoplasmic extension, disorganization of cell monolayer (Fig. 1B, E, G.). Cytotoxicity to CHO cells (Fig. 1B) was induced by 14 strains (70%) with cytotoxic titers ranging from 1 to 128. Eighteen of 20 (90%) S. marcescens strains were found to be cytotoxic to HEp-2 cells (Fig. 1E) with cytotoxic titers ranging from 1 to 32. The highest cytotoxic activity revealed 10 isolates (from MPU S1 to MPU S7, MPU S10, MPU S11, MPU S22). Fifteen strains (75%) were cytotoxic to Vero cells (Fig. 1G) with lower cytotoxic titer 3 203 Cytotoxicity of Serratia marcescens A C B D E F G Fig. 1. Cytotoxic effects of S. marcescens culture filtrates to CHO (B), HEp-2 (E) and Vero cells (G). Cytotonic activity of S. marcescens MPU S15 to CHO cells (C). A, D, F uninfected cells. Magnification, ×100. (132). Preheating (56°C for 20 min) of the supernatants caused a decrease in cytotoxic activity to CHO, Vero and HEp-2 cells (Table II). Mean cytotoxicity titers for individual cell lines taking all isolates into account were in the 3.5522.7 range (Table III). CHO cells expressed the highest sensitivity with mean titer 22.7 ± 0.11 whereas the least sensitive were Vero cells (mean titer = 3.6 ± 0.07). Cytotonic activity identified as elongation of CHO cells (Fig. 1C) was observed for 2 isolates (MPU S3, MPU S15). Non pathogenic strain E. coli K-12 C200 was not cytotoxic to epithelial cells. Cytotoxic activity of S. marcescens strains to murine macrophages. Twelve strains (60%) were cytotoxic to murine J774 cells after 24-hour infection, as measured by NR assay (Table IV). The maximum cytotoxicity (5268%) was observed in macrophages infected with two strains (MPU S21, MPU S22), the lowest cytotoxic activity was demonstrated by seven strains (35%). Cytotoxic activity to J774 cells was not inhibited after preheating of the supernatants. E. coli K-12 C200 strain, the negative control, was not cytotoxic to murine macrophages. Table II Cytotoxic activity of culture supernatants of S. marcescens strains Mean titers1 64 128 8 32 14 0 1 2 CHO Vero HEp-2 Non treated Heat inactivated Non treated Heat inactivated Non treated Heat inactivated supernatant supernatant supernatant supernatant supernatant supernatant 3 (15)2 6 (30) 5 (25) 6 (30) 0 1 (5) 7 (35) 12 (60) 0 2 (10) 13 (65) 5 (25) 0 1 (5) 8 (40) 11 (55) 0 9 (45) 9 (45) 2 (10) 0 2 (10) 10 (50) 8 (40) Mean of the reciprocal of the highest dilution yielding rounding, detachment and destruction of 50% of CHO, Vero or HEp-2 cells. Number (and percentage) of strains that revealed cytotoxic activity. 204 Krzymiñska S. et al. Table III Mean and standard error (SE) of cytotoxicity titers for different cell lines Mean ± SE Cell line 22.7 ± 0.11 10.8 ± 0.08 3.6 ± 0.07 CHO HEp-2 Vero Table IV Cytotoxic activity of S. marcescens culture supernatants to murine J774 macrophages Cytototoxicity range1 0 7.8 19.2 21.6 29.4 52.3 68.1 1 2 Number of cytotoxic isolates (%) Non treated Heat inactivated supernatant supernatant 8 (40)2 9 (45) 7 (35) 7 (35) 3 (15) 2 (10) 2 (10) 2 (10) The percentage of cytotoxicity was determined 24 h after infection by NR assay, Number and (percentage) of strains revealing cytotoxic activity. Discussion Serratia marcescens is increasingly recognized as a cause of morbidity in nosocomial units. It has been considered as an etiologic agent in all kinds of infection in humans. However, the exact mechanism of pathogenicity has not been sufficiently understood. The analysis of incidence of cytotoxic activity in S. marcescens isolates revealed that most strains produced cytotoxic toxins, which were noticed in the case of CHO (70%), Vero (75%) and HEp-2 cells (90%). The highest cytotoxic activity was detected in strains isolated from ulceration (3), postoperative wounds (2) and one each from urine, blood, aspirate and pus. Previously, Carbonell et al. (1997) demonstrated that culture filtrates from 22% S. marcescens isolates were cytotoxic to Vero and HeLa cells. Marty et al. (2002) confirmed earlier reports that the strains are cytotoxic to mammalian cells. Toxin production by S. marcescens strains is still not clearly defined. Marty et al. (2002) examined the 56-kDa metalloprotease of S. marcescens strains and found it to be the most potent cytotoxic factor. They suggested that the enzyme may possess a binding site for specific host proteins that are internalized by an endocytic mechanism into host cells. Carbonell et al. (2004) isolated a cytotoxic enterotoxin from a clinical isolate of S. marcescens which was highly cytotoxic to CHO cells but did not reveal hemolytic activity, suggesting that the cytotoxin is distinct from S. marcescens hemolysins. In consecutive reports, Carbonell et al. (2003) observed that the cytotoxic toxin was bound to the CHO cell surface, without being internalized and was 3 able to trigger changes in the intracellular metabolism of the cells and to induce cell death by apoptosis. In the past several years there have been reports about the family of Serratia-type pore forming toxins (Hertle, 2005). The hemolysin (ShlA) represents the prototype of a new type of cytolysins which are distinct from E. coli type "-hemolysins, staphylococcal "-toxins or other related toxins. ShlA pore formation in nucleated eukaryotic cells and erythrocytes results in cell lysis. The toxin additionally brings about cytoskeleton rearrangement and apoptosis. The S. marcescens culture filtrates incubated at 56°C for 20 min revealed a decrease in cytotoxic activity to CHO (86% filtrates), HEp-2 (90% filtrates) and to Vero cells (43%). The strains probably produce heatlabile cytotoxins. Carbonell et al. (2003) showed that a monolayer of Vero cells lost 30% cell viability when the filtrates were heated to 60°C, and no cytopathic effect was observed after incubation at 70°C. In order to choose a suitable target cell line, we compared the sensitivity of three epithelial cells to the cytotoxic activity of clinical isolates. In the present study CHO cells appeared to be the most sensitive to the toxic effect of S. marcescens culture filtrates, followed by HEp-2 and Vero cells. The results indicate that CHO cells could be used as a model to study the exact mechanism of action of cytotoxic factors. Carbonell et al. (2003) also found that CHO and HEp-2 cells are highly sensitive to S. marcescens cytotoxin. Interestingly, two isolates MPU S3 and MPU S15, appeared to be positive for cytotonic activity, which revealed as elongation of CHO cells. Singh et al. (1997) noticed that 4 of 6 S. marcescens strains isolated from food produced cytotonic toxins. There is no evidence of cytotonic toxins production by strains originating from human specimens. Cytotonic and heat-labile enterotoxins were produced, respectively, by Vibrio cholerae and E. coli strains. The toxins activated adenylate cyclase, which caused an increase in intracellular cAMP concentration, inducing morphological alterations in CHO cells and producing an imbalance in electrolyte movement in epithelial cells, resulting in abundant net fluid loss from the intestine (Sanchez et al., 2005). The high cAMP concentration impairs host cells functions, such as phagocytosis and oxidative ability (Moss et al., 2000). Moss et al. (2000) reported that Bordetella pertusis produced an adenylate cyclase-hemolysin (AcHly) toxin which caused increased cAMP level. An increase in the intracellular concentration of cAMP leads to apoptosis of mammalian cells. Phagocytes, either resident in tissues or circulating in blood contribute to the primary line of innate defense against bacterial pathogens by providing their removal and destruction at epithelial barrier level (Navarre and Zychlinsky, 2000). Some bacterial ente- 3 Cytotoxicity of Serratia marcescens ropathogens have developed strategies for avoiding antimicrobial effects of phagocytes and have evolved mechanisms which kill the immune cells. In this study we have demonstrated that 60% of isolates were cytotoxic to murine macrophages J774 cells. The highest activity was observed for strains isolated from blood and urine. The results suggest that the cytotoxic activity of these bacteria may be an important mechanism for evasion of host immune response during infection, which may induce inflammation and development of fatal bacteremia in weakened patients. The results of the study demonstrate that S. marcescens clinical isolates reveal cytotoxic activity that may modulate the properties of host epithelial cells. Moreover, we have observed the incidence of an antihost strategy based on the elimination of host immune cells. Literature Buffet-Batoillon S., V. Rabier, P. Betremieux, A. Beuchee, M. Bauer, P. Pladys, E. Le Gall E, A. Cormier and A. JolivetGougeon. 2009. Outbreak of Serratia marcescens in a neonatal intensive care unit: contaminated unmedicated liquid soap and risk factors. J. Hosp. Infect. 72: 1722. Carbonell G.V., A.F. Alfieri, A.A. Alfieri, M.C.Vidotto, C.E. Levy, A.L.C. Darini and R.M. Yanaguita. 1997. Detection of cytotoxic activity on Vero cells in clinical isolates of Serratia marcescens. Braz. J. Med. Biol. Res. 30: 12911298. Carbonell G.V., C.R.N. Amorim, M.T. Furumura, A.L.C. Darini, B.A.L. Fonseca and T. Yano. 2003. Biological of Serratia marcescens cytotoxin. Braz. J. Med. Biol. Res. 36: 351359. 205 Carbonell G.V., R. Falcon, A.T. Yamada, B.A.L. Fonseca and T. Yano. 2004. Morphological and intracellular alterations induced by Serratia marcescens cytotoxin. Res. Microbiol. 155: 2530. Friedman D., D. Kotsanas, J. Brett, B. Billah and T.M. Korman. 2008. Investigation of an outbreak of Serratia marcescens in a neonatal unit via a case-control study and molecular typing. Am. J. Infect. Control 36: 2228. Grimont F. and P.A.D. Grimont. 2006. The genus Serratia. pp. 219244. In: Dworkin M., S. Falkow, E. Rosenberg, K.H. Schleifer, and E. Stackebrandt (eds). The Prokaryotes. 3rd ed. Springer Verlag, Berlin. Hertle R. 2005. The family of Serratia type pore forming toxins. Curr. Protein and Peptide Sci. 6: 313325. Krzymiñska S., J. Mokracka, R. Koczura and A. Kaznowski. 2009. Cytotoxic activity of Enterobacter cloacae human isolates. FEMS Immunol. Med. Microbiol. 56: 248252. Maleri R.A., F. Fourie, A.J. Reinecke and S.A Reinecke. 2008. Photometric application of the MTT and NRR-assays as biomarkers for the evaluation of cytotoxicity ex vivo in Eisenia andrei. Soil Biol. Biochem. 40: 10401048. Marty K.B., C.L. Williams, L.J. Guynn, M.J. Benedic and S.R Blanke. 2002. Characterization of a cytotoxic factor in culture filtrates of Serratia marcescens. Infect Immun. 70: 11211128. Moss J.E., I. Idanpaan-Heikkila and A. Zychlinsky. 2000. Induction of apoptosis by microbial pathogen. pp. 275290. In: Cossard P., P. Bouquet, S. Normark, and R. Rappuoli (eds). Cellular Microbiology. Washington, D.C. Navarre W.W. and A. Zychlinsky. 2000. A Pathogen-induced apoptosis of macrophages: a common end for different pathogenic strategies. Cell Microbiol. 2: 265273. Sanchez J. and J. Holmgren. 2005. Virulence factors, pathogenesis and vaccine protection in cholera and ETEC diarrhea. Curr. Op. Immunol. 17: 388389. Singh B.R., Y. Singh and A.K. Tiwari. 1997. Characterisation of virulence factors of Serratia strains isolated from food. Int. J. Food Microbiol. 34: 259266. 206 Krzymiñska S. et al. 3 Polish Journal of Microbiology 2010, Vol. 59, No 3, 207212 ORIGINAL PAPER Antibiotic Susceptibility and Genotype Patterns of Escherichia coli, Klebsiella pneuomoniae and Pseudomonas aeruginosa Isolated from Urinary Tract Infected Patients M.I. ABOU-DOBARA1*, M.A. DEYAB1, E.M. ELSAWY2 and H.H. MOHAMED2 1 Faculty of Science (Damietta), Damietta Branch, Mansoura University and and Nephrology Center, Mansoura University, Egypt 2 Urology Received 13 October 2010, revised 1 May 2010, accepted 15 May 2010 Abstract Thirty nine isolates of Escherichia coli, twenty two isolates of Klebsiella pneumoniae and sixteen isolates of Pseudomonas aeruginosa isolated from urinary tract infected patients were analyzed by antimicrobial susceptibility typing and random amplified polymorphic DNA (RAPD)-PCR. Antibiotic susceptibility testing was carried out by microdilution and E Test methods. From the antibiotic susceptibility, ten patterns were recorded (four for E. coli, three for K. pneumoniae and three for P. aeruginosa respectively). Furthermore, genotyping showed seventeen RAPD patterns (seven for E. coli, five for K. pneumoniae and five for P. aeruginosa respectively). In this study, differentiation of strains of E. coli, K. pneumoniae and P. aeruginosa from nosocomial infection was possible with the use of RAPD. K e y w o r d s: Escherichia coli, Klebsiella pneumoniae, Pseudomonas areuginosa, antibiotic susceptibility, urinary tract infection Introduction Urinary tract infections (UTIs) are most prevalent among geriatric and critically ill patients and occur more commonly after urinary catheterization (Turck and Stamm, 1981; Centers for Disease Control, 1983). Many bacterial species are associated with this infection, including Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa (Gales et al., 2000; Gupta et al., 2007). Multi-drug resistance of antimicrobial classes is common among the uropathogenic bacteria such as Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa (Yuksel et al., 2006; Foda, 2008). Among whole genome fingerprinting PCR methods, random amplified polymorphic DNA (RAPD) (Williams et al., 1990) is used for demonstrating differences between bacteria. RAPD can be used for typing of organisms without previous knowledge of DNA sequences. The use of a single primer leads to amplification of several DNA fragments randomly distributed throughout the genome. The primers used in RAPD are short, usually 812 mers, with random sequence composition (Lübeck and Hoorfar, 2003). RAPD has received considerable attention in recent years as a molecular typing method due to its simplicity, sensitivity, flexibility and relatively low cost (Welsh and McClelland, 1990; Williams et al., 1990; Belkum, 1994). The ability of RAPD to type a wide variety of bacteria strains in a short time suggests that it will be a useful molecular epidemiological tool (Gori et al., 1996; Hilton and Penn, 1998; Bosi et al., 1999; Patton et al., 2001). Typing helps in the identification of environmental sources as well as indicating whether transmission of strains has occurred between patients (Kerr et al., 1995). It also reveals whether strains emerging after antibiotic therapy are variants of the original or newly acquired strains (Chit and Chew, 1993). Knowledge of the spread of specific strains is of epidemiological importance in order to monitor the broad spreading of both their pathogenicity and multiresistance (Lopes et al., 2005). The aim of this study was to determine antibiotic susceptibility and to apply RAPD typing method for characterization of the isolated bacterial pathogens. * Corresponding author: M.I. Abou-Dobara, Botany Dept., Faculty of Science, New Damietta, Damietta P.O. Box 34517, Egypt; e-mail: [email protected] or [email protected] 208 Abou-Dobara M.I. et al. Experimental Material and Methods Collection and transport of the samples. A total of 77 clinical samples were collected from urine samples in sterile tightly locked containers from different hospitalized patients in Urology and Nephrology Center, Mansoura University. The patients were not treated with any of the eight antibiotics tested (Table I). Table I Antibiotic types retrieved in this studya Antibiotic IP AK PT XL NI CT NX TS a b MICb S ≤4 ≤ 16 ≤ 16 ≤8 ≤ 32 ≤8 ≤4 ≤2 I 8 32 32 16 64 16 8 Conc. Range [µg/ml] R ≥ 16 ≥ 64 ≥ 128 ≥ 32 ≥ 128 ≥ 64 ≥ 16 ≥4 Broth 1 16 2 64 8 128 4 32 16 128 8 64 4 16 24 E test 0.002 32 0.016 256 0.016 256 0.016 256 0.032 512 0.016 256 0.016 256 0.002 32 Abbreviations: IP, Imipenem; AK, Amikacin; PT, Piperacillin/tazobactom; XL. Amoxicillin/clavulanate; NI, Nitrofurantoin; CT, Cefotaxime; NX, Norfloxacin; TS, Trimethoprim-sulfamethoxazole; Susceptibility was performed as described by National Committee for Clinical Laboratory Standards [NCCLS, 2006], which was used to categorize strains as susceptible or sensitive (S), intermediate (I) or resistant (R). Isolation of the bacterial isolates. The collected samples were inoculated into sterile Petri dishes containing ready prepared Cled agar and blood agar media and incubated at 35°C for 1824 hours. After the incubation period, the plates were examined for growing bacterial colonies. The isolated colonies were subcultured and purified for characterization. Identification of the different isolates. The isolated bacteria were identified according to Bergeys Manual of Determinative Bacteriology (Brenner, 1986; Ørskov, 1986a and b; Palleroni, 1986) and confirmation of species identification was carried out by using automated Microscan (DADE BEHRING, USA). Antibiotics sensitivity tests. The antimicrobial susceptibility tests of the isolated bacteria were carried out using the following methods: 1 Broth Microdilution MIC Method. A sterile plastic tray containing various concentrations of antimicrobial agents (Table I) was inoculated with a standardized number of test bacteria in Mueller Hinton broth. After overnight incubation at 35°C, the minimal inhibitory concentrations (MIC) were determined and interpreted as susceptible, intermediate, or resistant (Table I). 2 E Test. The system comprises a E test strip with predefined antimicrobic gradients (Table I), to deter- 3 mine the Minimum Inhibitory Concentration (MIC), in µg/ml of individual agent against microorganisms as tested on agar media (Mueller Hinton Agar). The inoculated media were incubated at 3537°C for 1824 hours. After the incubation period the MIC values were recorded at the point of intersection between the inhibition ellipse edge and the E test strip (Table I). Genotyping of the bacterial isolates Isolation of the bacterial DNA. Bacterial colonies were removed and suspended in 1 ml distilled water, then centrifuged for 10 min at 5000× g. DNA was extracted using High Pure PCR Template Purification Kit, Germany) as follow: The bacterial pellets were suspended in 200 µl phosphate buffered saline. 15 µl lysozyme was added and incubated for 15 min at 37°C. Subsequently 200 µl binding buffer and 40 µl proteinase K were added, mixed immediately and incubated for 10 min at 72°C, then 100 µl isopropanol was added to precipitate DNA. The filter tubes and the collection tubes were combined and the samples were pipetted, and then centrifuged for 1 min at 5000× g. The upper reservoir was washed twice with 500 µl washing buffer and centrifuged for 1 min at 5000× g. 200 µl of prewarmed (70°C) elution buffer was added and the tubes were centrifuged for 1 min at 500× g. Randomly Amplified Polymorphic DNA (RAPD) Fingerprinting (Williams et al., 1990). RAPD was carried out with some modification. The PCR mixture was composed of 10× PCR buffer: 100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl2, 0.01% (w/v) gelatin, pH 8.3.2 mM of each dGTP, dATP, dCTP, and dTTP was added. Taq DNA polymerases: Taq DNA polymerase (5 U/µL; Sigma). Template DNA: 10 to 25 ng/µl stock solution containing good-quality, protein-free, DNA can be resuspended in high-quality sterile, deionized water or TE (Tris-EDTA) pH 8.0. RNase (20 ng per 1 ng of DNA). The following primer (OPA-02 5'TG CCGAGCTG3') was used in this study at 25 pmol/µl. The amplifications were done in thermal cycler (PerkinElmer model 9700) programmed for the first five cycles to denature for 1 min at 94°C, anneal for 2 min at 27°C followed by primer extension for 2 min at 72°C. Then, a program for 45 cycles of 1 min denaturation at 94°C, 2 min of annealing at 32°C and 2 min primer extension at 72°C followed by a final extension period for 15 min at 72°C, was run. Gel preparation and sample loading (Maniatis et al., 1982). A 0.7% agarose was prepared in 1× TBE and mixed with 0.5 ug/ml of ethidium bromide. The gel was transferred to electrophoresis cell with 1× TBE buffer. Each sample (20 µl) was mixed with 4 ul loading dye and loaded into the gel and 1 µl DNA marker ØX 174 Hae III was loaded into one well of the gel. 3 E. coli, K. pneumoniae, P. aeruginosa isolates from urinary tract Electrophoresis and detection. 80 volts for 2 hours as 7.5 v/cm of the gel was applied. The gel was visualized using UV transilluminator and photographed by Polaroid film in Polaroid camera with 4 seconds exposure time. Results Characterization and identification of the isolated bacteria. Seventy-seven isolated bacterial samples were divided into three groups namely; group 1 (thirty-nine isolates were identified as Escherichia coli), group 2 (twenty-two isolates were identified as Klebsiella pneumoniae) and group 3 (sixteen isolates were identified as Pseudomonas aeruginosa) according to their colony morphology, colony smell, Gram stain response, shapes, pigmentation and biochemical properties. E. coli was the most frequent bacterium isolated (50%) followed by K. pneumoniae (29 %) and P. aeruginosa (21%). Imipenem, amikacin and piperacillin/ tazobactam were the most commonly used drugs for the treatment of E. coli with 100% effectivity. Also imipenem and amikacin were effective against K. pneumoniae and P. aeruginosa as the effectiveness of both agents on them was 100%. Amoxicillin/clavulanate, had a lower effect against E. coli (72%). Nitrofurantion, cefotaxime, norfloxacin and trimethoprim/sulfamethoxazole had a low effect against E. coli (46%, 26%, 26% and 26% respectively). Amoxicillin/clavulanate, nitrofurantion, cefotaxime and norfloxacin had a low and similar effect on K. pneumoniae (27%). Cefotaxime and norfloxacin also showed low-level effectiveness against P. aeruginosa (50%). In addition, P. aeruginosa were completely resistant to nitrofurantion. Finally, trimetho- 209 prim/sulfamethoxazole had the lowest effect among the tested antibiotics with K. pneumoniae and P. aeruginosa being resistant to them. From the antibiotic susceptibility, ten patterns were recorded (four for E. coli, three for K. pneumoniae and three for P. aeruginosa respectively). For isolates of E. coli, the first pattern was resistant to amoxicillin/clavulanat, nitrofurantion, cefotaxime, norofloxacin, and trimethoprim/sulfamethoxazole. The second pattern was resistant to nitrofurantion, cefotaxime, norofloxacin, and trimethoprim/sulfamethoxazole. Pattern two was resistant to cefotaxime, norfloxacin, and trimethoprim/sulfamethoxazole. Finally, pattern four was susceptible to all the tested antibiotics. The isolates of K. pneumoniae were distributed into three patterns. The first pattern was resistant to piperacillin /tazobactama, amoxicillin/clavulanat, nitrofurantion, cefotaxime, norfloxacin, and trimethoprim/ sulfamethoxazole. Pattern two was resistant to amoxicillin/clavulanate, nitrofurantion, cefotaxime, norfloxacin, and trimethoprim/sulfamethoxazole. Pattern three was resistant to trimethoprim/sulfamethoxazole. On the other hand, P. aeruginosa isolates were separated into three patterns. The first one was resistant to amikacin, piperacillin/tazobactam, nitrofurantion, cefotaxime, norofloxacin and trimethoprim/ sulfamethoxazole. The second was resistant to nitrofurantion, cefotaxime, norfloxacin and trimethoprim/ sulfamethoxazole. Finally pattern three was resistant to nitrofurantion and trimethoprim/ sulfamethoxazole. RAPD-PCR analysis revealed different genotypes for all the identified bacteria. E. coli had a different RAPD pattern (Fig. 1), as did K. pneumoniae and P. aeruginosa (Fig. 1 and 2). RAPD technique allowed the amplification of many bands in all the isolated bacteria. There was a difference in intensity of bands within the same pattern or between the different Fig. 1. Effect of different antibiotics on Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. 210 3 Abou-Dobara M.I. et al. P. aeruginosa. All isolates were found to have a band of 1078 bp. Three patterns of P. aeruginosa resulting from the antibiotic susceptibility testing were divided into five distinct patterns in RAPD analysis (Fig. 2). The RAPD patterns of different isolates of E. coli, K. pneumoniae and P. aeruginosa from Urology and Nephrology Center, Mansoura University may suggest that those isolates constitute a clonal lineage. Fig. 2. Polymerase Chain Reaction-Amplified DNA of a selection of isolates of E. coli and Klebsiella pneumoniae showing random amplification of polymorphic DNA assay fingerprints, separated by gel electrophoresis and detected by ethidium bromide staining. Lane 1: DNA molecular weight marker of Hae III digested phage øx174; Lanes 2 to 8 E. coli DNAs; Lane 9 and 10, K. pneumoniae DNAs; Lane 11 DNA ladders. patterns. However, many isolates were found to be identical in genotype-displayed variability in antibiotic susceptibility pattern. For E. coli isolates, the amplification of eight bands ranging in size from 280 to about 1078 bp occurred. All isolates were found to have a band of 975 bp. The four patterns resulting from the antibiotic susceptibility testing of E. coli were divided into seven patterns in RAPD analysis (Fig. 1). On the other hand, eleven bands ranging in size from 360 to about 1900 bp were amplified in the isolates of K. pneumoniae. There was no common band in all the isolates. Three patterns of K. pneumoniae resulting from the antibiotic susceptibility testing were divided into five distinct patterns in RAPD method (Fig. 1 and 2). Furthermore, nine bands ranging in size form 502 to about 1840 were amplified in the isolates of Fig. 3. Polymerase Chain Reaction-Amplified DNA of a selection of isolates of Klebsiella pneumoniae and Pseudomonas aeruginosa showing random amplification of polymorphic DNA assay fingerprints, separated by gel electrophoresis and detected by ethidium bromide staining. Lane 1: DNA molecular weight marker of Hae III digested phage øx174; Lanes 2 to 4 K. pneumoniae DNAs; Lane 5 and 9 P. aeruginosa DNAs; Lane 10 DNA ladders. Discussion Most of the clinical isolates of E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa in this study were susceptible to imipenem and amikacin with 100% susceptibility rate and the degree of resistance to the other tested multiple antibiotics varied according to the antibiotics. Foda (2008) recorded that meronem and amikacin were highly active towards E. coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis and Pseudomonas aeruginosa. He also reported that the susceptibility rate of urinary isolates was 76.19% for meronem followed by amikacin (70.27%). Das et al. (2006) showed that the susceptibility rate of urinary isolates was the highest for amikacin (87.2%). The simplicity and wide applicability of the RAPD method is dependent on the use of short nucleotide primers, which are not related to known DNA sequences of the target organism. They are designed within constraints including (i) a length of not less than nine nucleotide residues, (ii) a GC content of > 50% and (iii) a lack of palindromic sequences (William et al., 1990). These primers used in PCRs have been able to efficiently detect DNA polymorphisms and identify interstrain variations in an increasing number of species (Bingen et al., 1993a; 1993b). Genetic mapping and determination of the degree of relatedness between strains have been performed with validation by ribotyping (Williams et al., 1993). The banding pattern derived in this process allows the identification of similar strains by a method significantly less complicated and time consuming than ribotyping. When directly compared in the analysis of bacterial sample outbreak in a maternity unit, RAPD and ribotyping were equivalent in their abilities to discriminate between strains (Bingen et al., 1993a). It is of paramount importance that reaction conditions, including DNA template concentration, annealing temperature, and other PCR mixture concentration are strictly standardized to avoid artifactural variation in RAPD patterns (Ellsworth et al., 1993). While RAPD gives information regarding similarity between isolates, the application of PCR based techniques has a revolutionary impact on the diagnosis of infectious diseases. The most commonly used 3 E. coli, K. pneumoniae, P. aeruginosa isolates from urinary tract molecular genetic fingerprinting technique by RAPD revealed more genetic differences among avian bacterial strain than amplified fragment length polymorphism (AFLP) analysis (Gomes et al., 2005). In recent years with the advent of molecular DNA techniques, several arbitrary primer based RAPD-PCR techniques have been used for delineating the bacteria according to their genetic relatedness (Muzurier and Wernas, 1992; Eisen et al., 1995; Lin et al., 1996). Earlier researchers were of the opinion that RAPD was the best method for detecting genetic differences with respect to its speed and ability to type a wide variety of bacterial species and suggested it would be an increasingly useful molecular epidemiologic tool. In the past, dendrogram-based analysis of the RAPD profiles of various bacteria allowed understanding the genetic relationship between isolates grouped into several clusters. These phylogenetic studies successfully showed the predominance of a single epidemic strain that was transmitted between hosts and its persistence over a period of time (Gomes et al., 2005). In this study the four patterns resulting from the antibiotic susceptibility testing of E. coli were divided into seven patterns in the DNA method. There is a difference in intensity of bands within the same pattern or between the different patterns, i.e. DNA method identified additional heterogeneity among the related strains. Seven patterns of E. coli generated by DNA based method differed by the presence or absences of one or two single DNA fragment when compared one with another. Sometimes smearing is observed when multiple DNA fragments, which differ slightly in length, are visible. Eisen et al. (1995) studied multi-resistant Klebsiella pneumoniae strains by typing the isolates phenotypically and with random amplified polymorphic DNA analysis (RAPD) and plasmid analysis and they showed the predominance of a single epidemic strain that was transmitted between patients in the Newborn Services Unit. Lopes et al. (2005) found that 26 RAPD genotypes among studied 30 K. pneumoniae and they demonstrated the high discriminatory power of RAPD. RAPD analysis in their research indicated that pathogenic K. pneumoniae strains comprise a genetically high variable group of organisms. In this study, three patterns of K. pnuomoniae resulting from antibiotic susceptibility testing were divided into five patterns in the RAPD analysis. Lai et al. (2000) reported that a pathogenic K. pneumoniae strain was highly heterogeneous, based on the distribution of different nucleotide sequences. The high number of serotypes in this species (Orskov and Orskov, 1984) could ahlso explain the relevant degree of genetic diversity highlighted by RAPD. Kerr et al. (1995) applied RAPD to 10 cases of pneumoniae associated with sputum culture of Pseudo- 211 monas aeruginosa and they suggested that a single strain of P. aeruginosa, isolated from 10 ICU patients, was responsible for this outbreak of pneumonia. Nazik et al. (2007) studied the typing of the Pseudomonas aeruginosa isolates recovered from cystic fibrosis (CF) patients by random amplified polymorphic DNA (RAPD)-PCR and to determine the antibiotic susceptibility of these strains. Their study revealed that most the P. aeruginosa isolates with dissimilar colony morphology or antibiotic susceptibility isolated from these CF patients were of the same genotype, but colonization or infection with only one genotype is, however, not a rule. These results were also recorded in other earlier studies (Sener et al., 2001; Horrevorts et al., 1990). In this study, three patterns of P. aeruginosa resulting from antibiotic susceptibility testing were divided into five patterns in the DNA method, that is the DNA method identified additional heterogeneity among the related strains. Khalifa et al. (2010) reported that genotyping of Pseudomonas aeruginosa isolated from clinical samples showed 83 RAPD types and they also recorded that the isolates showing the same serotype could show different genotypes. In addition, by using RAPD, Trautmann et al. (2006) showed that isolates of Pseudomonas aeruginosa from patients showed a similar distribution of genotypes. 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Aysev, M., Ekim and F. Yalcinkaya. 2006. Antibiotic resistance or urinary tract pathogen and evaluation of empirical treatment in Turkish children with urinary tract infections. Int. J. Antimicro. Agents. 5: 413416. Polish Journal of Microbiology 2010, Vol. 59, No 3, 213216 SHORT COMMUNICATION rDNA-Based Genotyping of Clinical Isolates of Candida albicans URSZULA NAWROT, MAGDALENA PAJ¥CZKOWSKA, KATARZYNA W£ODARCZYK, IZABELA MECLER Department of Microbiology, Medical University of Wroclaw, Poland Received 25 January 2010, revised 2 July 2010, accepted 7 July 2010 Abstract The study presents an analysis of the restriction pattern of rDNA fragments of 95 C. albicans isolates previously classified on the basis of the presence of the intron in rDNA into genotypes A (62 isolates), B (28), and C (5). Most isolates (61) with genotype A were classified as subtype a and one as subtype d (Karahan and Akar; 2005). No differences were observed in the restriction patterns of the tested genotype B isolates. Similarly, most genotype C strains (4/5) showed the same restriction pattern. The results indicate low subtyping variations of the analyzed isolates, which is in contrast to published data obtained from a Turkish collection of yeasts. K e y w o r d s: Candida albicans, group I self-splicing intron, rDNA genotyping, RFLP Candida albicans is the most frequent fungal pathogen, being the causative agent of both superficial and deep-seated and life-threatening mycoses. Many molecular methods have been employed in investigating species evolution and epidemiology. One of them is the genotyping developed by Mercure et al. (1993) and McCullough et al. (1999a) based on the presence or absence of the self-splicing group I intron in the large subunit (CaLSU) of rRNA genes. Strains can be differentiated into three genotypes: genotype A without the intron, genotype B harboring the intron, and genotype C possessing LSU rDNA with and without the intron in a single genome. In the last decade, several authors reported the distribution of the A, B, and C genotypes in different groups of clinical C. albicans isolates (Tamura et al., 2001, Millar et al., 2002; Karahan, 2004; Karahan and Akar, 2005; Qi et al., 2005; Millar et al., 2005; Girish Kumar et al., 2006, Nawrot et al., 2004; Nawrot et al., 2008). Although the data obtained by the particular authors differ in detail, genotype A has been reported as prevalent in most groups of clinical isolates, including those regarded as invasive. It is supposed that intron-containing genotypes can be eliminated because of their high susceptibility to some drugs, for example 5-fluorocytosine, pentamidine, and bleomycin, interfering with the selfsplicing process (Mercure et al., 1993; Zhang et al., 2002; Jayaguru and Raghunathan, 2007). On the other hand, some authors observed increased occurrence of genotype C (McCullough et al., 1999b; Gurbuz and Kaleli, 2010). Recently, Karahan and Akar (2005) found significant differences in the LSU rDNA sequences of genotype A isolates and constructed an RFLP-based method for differentiating genotype A into eight subtypes. In this communication we present the results of the RFLP analysis of LSU rDNA of C. albicans isolates, which is complementary to our previous study on the distribution of CaLSU among C. albicans isolated from blood and the respiratory tract (Nawrot et al., 2008). The study was performed on 95 isolates of C. albicans, including 55 blood isolates obtained from BCCM/ IHEM (30 genotype A, 20 B, and 5 C) and 40 isolates (32 genotype A and 8 B) from the laboratory collection of Wroc³aw Medical University and originating from different clinical samples (22 from sputum or pharyngeal swabs, 7 from blood, and 8 from body fluids or pus). Genomic DNA of the tested yeasts was extracted using the CTAB method according to ODonnell et al. (1997). The PCR assay was performed with the primer pair CA-INT-L (5-ATA AGG GAA GTC GGC AAA ATA GAT CCG TAA-3) and CAINT-R (5CCT TGG CTG TGG TTT CGC TAG ATA GTA GAT-3), described previously by McCullough et al. (1999b). DNA samples were denatured at 94°C for 3 min before 30 cycles of 94°C for 1 min, 65°C * Corresponding author: U. Nawrot, Department of Microbiology, Medical University of Wroclaw, Cha³ubinskiego 4, 50-368 Wroc³aw, Poland; e-mail: [email protected] 214 Nawrot U. et al. 3 A) B) Fig. 1. Electrophoretic gel image of PCR products digested with Hae III (A) and MspI (B). M-QX DNA Size Marker FX 17. Lines 15 strains with genotype C (IHEM19482, 19076, 19265, 19491, 19608), line 6 genotype B (IHEM 19651), line 7 genotype A subtype d (1228, laboratory collection), lines 8 and 9 genotype A subtype a (IHEM 19069, 19144). Performed with the help of Biocalculator software (Qiagen). for 1 min, and 72°C for 4 min, with a final extension at 72°C for 4 min following the last cycle. The PCR products were digested, separately overnight with the enzymes Hae III (BsuRI) and MspI (HpaII, Fermentas), in accordance with the protocol of Karahan and Akar (2005). The undigested and digested PCR products were analyzed by electrophoresis in 4% agarose gel and visualized in UV after staining with EtBr. The selected DNA samples were additionally analyzed by capillary electrophoresis in the QIAxcel system (Qiagen) using a QIAxcel DNA High Resolution Kit, the QX Alignment Marker 15-bp/3-kb, and the QX DNA Size Marker FX 174. The tests were performed with the OM500 method and the results were analyzed by Biocalculator software and presented as both simulated bands on gel images and peaks in electrophoregrams. PCR with the primer pair CA-INT-L/CA-INT-R and DNA of genotype A resulted in a single PCR product of ~ 460 bp (McCullough et al., 1999b), 3 Short communication 215 Fig. 2. A diagram presenting the restriction sites of a fragment of sequence DQ465844 (mether line) and the hypothetical sequence in which the intron of DQ465844 was replaced by the X74272 sequence (performed with the help of SeqBilder, Lasergene software). which can be slightly different in particular subtypes (Karahan and Akar, 2005). The strains of genotype A tested in this study give PCR products of typical size, except for one isolate (no 1228, laboratory collection), which gave a larger product (~470 bp). Most isolates (61/62, 98%) of genotype A showed the same RFLP pattern, namely three fragments of 294, 92, and 72 bp, after digestion with Hae III and two fragments of 289 and 171 bp after digestion with MspI (Figs. 1 A and B). This result corresponds well with subtype a described by Karahan and Akar (2005), which was characterized by three bands of 296, 93, and 71 bp after digestion with Hae III. Isolate no 1228 showed a different restriction pattern, consisting of two bands of ~ 400 and 72 bp with enzyme Hae III and three bands (290, 93, and 84 bp) with MspI, which is in accordance with the subtype classified as subtype d by Karahan and Akar. In their study, Karahan and Akar tested 144 genotype A isolates obtained from different clinical samples from three Turkish hospitals and 52% of them were identified as subtype a, whereas the other strains were distributed among seven different subtypes. In a recently published paper, Gurbuz and Kaleli (2010) found 84 (84.8%) subtype a samples among 99 genotype A isolates. The genotype A isolates tested in this study were highly homogenous (98% with subtype a). Analysis of the results obtained by us and by other authors, indicates that the level of diversity of LSU rDNA varies in particular C. albicans populations and may be geographically related. It is interesting that subtype d identified in this study, as well as 9 isolates with subtype d described by Karahan and Akar (2005), were obtained from blood, which may suggest a high invasiveness of subtype d. A future study performed with a higher number of invasive and non-invasive isolates can be helpful in verifying such an hypothesis. In this study we also performed the RFLP analysis of the PCR products obtained for genotypes B and C. The public PubMed database (http://www.ncbi.nlm. nih.gov/sites/entrez?db=pubmed) contains only two sequences (accession nos. DQ465844 and DQ465845) of LSU of C. albicans genotype B which include the DNA fragment flanked by the CA-INT-L and CA-INT-R primers. The restriction analysis of these sequences performed with the use of SeqBuilder Lasergene software indicated the same pattern with MspI (four bands of 310, 225, 169, and 131 bp) and two different patterns with Hae III (398, 242, 93, 71, 31 bp for DQ465844, and 248, 242, 182, 93, and 71 bp for DQ465845). This finding suggests sequence diversity in the analyzed LSU fragment of genotype B and the usefulness of the Hae III enzyme in its testing. The results obtained experimentally differed from those predicted on the basis of an analysis of the reference sequences. In our study, all the investigated strains with genotype B showed the same PCR product size (~840 bp) and the same restriction pattern with enzymes Hae III (six bands of 251, 239, 101, 90, 68, and 35 bp) and MspI (540, 170, and 130 bp). It is worth noting that there was some discrepancy between the results obtained by capillary electrophoresis (the above pattern) and classical gel electrophoresis, which cannot easily distinguish the bands of ~239, 90, and 35 bp after digestion with Hae III (data not shown). What is interesting is that replacing the intron present in DQ465844 by the sequence of the intron from another strain (accession no X74272.1) resulted in changing the restriction pattern to one more similar to our finding for genotype B, namely seven bands of 247, 242, 103, 93, 72, 32, and 20 bp for Hae III and three bands of 539, 170, and 131 bp for MspI (Fig 1B). An analysis of the DQ465844 and DQ465845 sequences performed after excluding the intron indicated their high similarity (99.8%) to subtype a. This suggests that the strains with genotype B tested in this study can also be related to subtype a and their intron to the X74272.1 sequence. Genotype C generates two amplicons, one of ~460 bp and one of ~ 840 bp, so the PCR-RFLP result could reflect the sequence diversity of the two products. The five strains of genotype C tested in this study displayed the typical sizes of the PCR products and the same restriction pattern with MspI (four bands of 580, 290, 170, and 130 bp), whereas with Hae III, four strains showed the same pattern (five bands of 290, 251, 239, 90, and 70 bp), but the fifth differed from them by the presence of an additional band of ~400 bp (Fig. 1A). The evolutionary processes which 216 Nawrot U. et al. resulted in the formation of the heteroallelic genotype C were discussed by many authors. McCullough et al. (1999) proposed two hypotheses of genotype C development. The first assumes that genotype C arises after losing the intron by genotype B and the second is that genotype C is formed due to the acquisition of the intron by genotype A, most probably as a result of sexual recombination, which has not yet been detected in C. albicans. Recently, Miletti-Gonzalez and Leibowitz (2008) studied the genetic arrangement of the CaLSU intron in the rDNA of an isolate with genotype C and showed that intron-possessing rDNA and intron-less rDNA copies are arrayed in tandem and adjacent to each other, forming rDNA clusters present in two R chromosomes. The authors observed high variability in the number of rDNA complex copies among clones of genotype C. The simple RFLP experiment performed in this study showed that the diversity of genotype C described above can be wider due to variability in the rDNA sequence. In summary, the presented results indicate a high homogeneity of the analyzed fragment of LSU rDNA in the clinical isolates of C. albicans, in contrast to published data obtained with a Turkish collection of yeasts. The restriction analysis of the amplicons obtained after A, B, and C genotyping is a simple and reproducible method enabling broader strain characteristics and can be useful in epidemiological and evolutionary studies. Acknowledgments This study was supported by the Polish Ministry of Science and Higher Education (Nr 402 055 31/1808) Literature Girish Kumar C.P., A. M. Hanafy, M. Katsu, Y. Mikami and T. Menon. 2006. Molecular analysis and susceptibility profiling of Candida albicans isolates from immunocompromised patients in South India. Mycopathologia 161: 153159. Gurbuz M. and I. Kaleli. 2010. Molecular analysis of Candida albicans isolates from clinical specimens. Mycopathologia 169: 261267. Jayaguru P. and M. Raghunathan. 2007. Group I intron renders differential susceptibility of Candida albicans to bleomycin. Mol. Biol. Rep. 34: 1117. 3 Karahan Z.C. and N. Akar. 2005. Subtypes of genotype A Candida albicans isolates determined by restriction endonuclease and sequence analyses. Microbiol. Res. 160: 361366. Karahan Z.C., H. Guriz, H. Agirbasli, N. Balaban, J.S. Gocmen, D. Aysev and N. Akar. 2004. Genotype distribution of Candida albicans isolates by 25S intron analysis with regard to invasiveness. Mycoses. 47: 465469. McCullough M.J., K.V. Clemons and D.A. Stevens. 1999a. Molecular epidemiology of the global and temporal diversity of Candida albicans. Clin. Infect. Dis. 29: 12201225. McCullough M.J., K.V. Clemons and D.A. Stevens. 1999b. Molecular and phenotypic characterisation of genotypic Candida albicans subgroups and comparison with Candida dubliniensis and Candida stellatoidea. J. Clin. Microbiol. 37: 417421. Mercure S., S. Montplaisir and G. Lemay. 1993. Correlation between the presence of a self-splicing intron in the 25S rDNA of C. albicans and strains susceptibility to 5-fluorocytosine. Nucleic. Acids. Res. 21: 60206027. Miletti-González K.E. and M.J. Leibowitz. 2008. 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