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VYTAUTAS MAGNUS UNIVERSITY FACULTY OF SCIENCE BIOLOGY DEPARTMENT Greta Mikučionytė (MGM09017) ANTIMICROBIAL ACTIVITY OF PROBIOTICS AND EFFECT OF PREBIOTICS AGAINST ENTERIC PATHOGENS Master thesis Biology program, state code 61201B103 Biology study area Supervisors : Assoc. Prof. Åsa Ljungh, Lund University ______________ (signature) Prof. Torkel Wadström, Lund University ______________ (signature) _______ (date) _______ (date) Prof. Limas Kupčinskas, Kaunas Medical University ______________ _______ (signature) Adviser: Dr. Kanthi Kiran Kondepudi., M.Sc., Ph.D, Lund University _______ (date) _______ (signature) (date) Opponent: Prof. Algimantas Paulauskas, Vytautas Magnus University _________ _______ (signature) KAUNAS, 2011 (date) Work has been carried out at Laboratory Medicine, Division of Bacteriology, Lund University, Sölvegatan, SE 22362, Lund, Sweden. Reviewer: Dr. B. Tamutė Work to be presented: Vytautas Magnus University, Department of Biology, for public critisim in auditorium 801, Vileikos 8, LT-44404 Kaunas, Lietuva, on June 2 at 9 o‘clock. Number of Protocol: Author of term paper: Greta Mikučionytė Supervisors: Assoc. Prof. Åsa Ljungh; Prof. Torkel Wadström; Prof. Limas Kupčinskas. Vytautas Magnus University, Biology Department. Head of the department A. Sruoga _________________ 2 TABLE OF CONTENTS ABBREVIATIONS………………………………………………………………………………….4 SUMMARY………………………………………………………………………………………….5 ABSTRACT…………………………………………………………………………………………6 INTRODUCTION……………………………………………………………………………………8 1. LITERATURE REVIEW………………………...……………………………………………...9 1.1 History and definition………………………………………………………………………….9 1.2 Microorganisms used as probiotics……………………………………………………………9 1.2.1. Genus Lactobacillus…………………………………………………………………….10 1.2.2. Genus Bifidobacterium………………………………………………………………….10 1.3. Bacteriocins and acid production by lactic acid and bifidobacteria…………………………11 1.4. Mechanism of action of Probiotics…………………………………………………………..14 1.4.1. Modification of the microbial flora……………………………………………………..15 1.4.2. Enhancement of intestinal barrier function……………………………………………..16 1.4.3. Immunomodulation……………………………………………………………………..17 1.5. Antimicrobial activity by Lactobacilli and bifidobacteria against enteric pathogens……….18 1.6. Prebiotics…………………………………………………………………………………….22 2. MATERIALS AND METHODS……………………………………………………………….22 2.1. Growth Substrates…………………………………………………………………………...22 2.2. Bacterial strains……………………………………………………………………………...22 2.3. Bacterial growth conditions…………………………………………………………………23 2.4. Antibacterial activity of selected bacteria…………………………………………………...23 2.4.1. Effect of cell free filtrates of probiotic strains against E.coli, S. typhimurium, S.aureus and B.cereus time course experiment…………………………………………………...23 2.4.2. Effect of prebiotics on the AMA of probiotic strains against E.coli, S. typhimurium, S.aureus and B.cereus…………………………………………………………………..24 2.4.3. Effect of bile stress on AMA of probiotics strains against E.coli, S. typhimurium, S.aureus and B.cereus…………………………………………………………………..24 2.4.4. Effect of probiotic strain combinations on AMA against E.coli, S. typhimurium, S.aureus and B.cereus…………………………………………………………………..25 2.4.5. Partial purification of proteinecious antimicrobial substance…………………………..26 3. RESULTS………………………………………………………………………………………..26 3.1. Antimicrobial activity of LAB and bifidobacteria of growth of enteropathogens (screening)…………………………………………………………………………………..26 3.2. A time course of antimicrobial activity of cell- free supernatant of probiotic bacteria against enteric pathogens……………………………………………………………………………27 3.3. AMA studies of LAB and Bifidobacteria in presence of prebiotics………………………..30 3.4. Effect of Bile on AMA of probiotic strains…………………………………………………34 3.5. Effect of probiotic strain combinations on AMA against E.coli, S. typhimurium, S.aureus and B.cereus……………………………………………………………………….36 3.6. AMA of partially purified bacteriocins from LAB and Bifidobacteria strains against enteric pathogens……………………………………………………………………………………44 CONCLUSIONS…………………………………………………………………………………...45 ACKNOWLEDGEMENT…………………….…………………………………………………..46 REFERENCES…..………………………………………………………………………………...47 3 ABREVIATIONS AMA – antimicrobial activity CFS – cell free supernatant EMP - Embden–Meyerhof–Parnas pathway ESBL – beta spectrum lactamase– producing Escherichia coli FOS – fructooligosaccharides GI /GIT– gastrointestinal tract GOS – galactooligosaccharides IBD – bowel disease IL – interleukin IMOS – Isomaltooligosaccharides LDH – lactate dehydrogenase MHB – Muller-Hinton broth MRSA - methicilin-resist Staphyloccocus aureus MRS - De Man, Rogosa, Sharpe broth MUC – mucin NEC – necrotizing enterocolits NF- κB - nuclear transcription factor SOS - soybean-oligosaccharides TLR – Toll – like receptors TSS - toxic shock syndrome XOS – xylooligosaccharides 4 SUMMARY Author of term paper: Greta Mikučionytė Full title of term paper: Antimicrobial activity of probiotics and effect of prebiotics against enteric pathogens. Supervisors: Assoc.Prof. Åsa Ljungh, Prof.Torkel Wadström, Prof. Limas Kupcinskas. Adviser: Dr. Kanthi Kiran. Kondepudi. M.Sc., Ph.D Work has been carried out at Laboratory Medicine, Department of Medical Microbiology, Division of Bacteriology, Lund University, Sölvegatan, SE 22362, Lund, Sweden. Number of pages: 53 Number of tables: 8 Number of figures: 32 Number of references: 76 5 ABSTRACT In this study we have focused on 9 human probiotic strains, which were tested using microtiter plate assays for their ability to inhibit the growth of some common enteropathogens: Salmonella typhimurium, ESBL (see list of abbreviations), Staphylococcus aureus and Bacillus cereus. Survivals at low pH, growth in the presence of bile and levels of inhibition of growth of enteropathogens at specific time intervals have been also studied. Several tested probiotic strains were combined with a range of prebiotic food supplements in an attempt to identify synbiotic combinations. Subsequently all tested Lactobacillus and Bifidobacterium strains were examined in co-culture experiment for their AMA to inhibit the growth of harmful bacteria. AMA of crude proteins precipitated by ammonium sulphate from the cell free supernatants of 4 candidate probiotic strains was also determined to define the possible role of proteinecious antimicrobial substances (bacteriocins). The results showed that four (2 Lactobacilli: L. paracasei F8, L. plantarum F44 and 2 Bifidobacteria: B. breve 46, B. lactis 88 of a total 9 Lactic acid and Bifidobacteria showed strong antimicrobial activity when tested against some Gram-negative and Gram-positive pathogens. In this study it was observed that AMA of collective candidate probiotic strains in different time course against 4 enteric pathogens inhibited was highest with acid supernatants. When the supernatants were neutralized to pH 6.0 there was a decrease of inhibition suggesting the possible synergistic action of secreted acids and other proteinecious or non-proteinacious metabolites by the probiotic strains (that inhibit the growth of pathogens). Probiotics grown with different carbohydrate sources were tested in a microtiter plate assay offered some inhibition of each of S. typhimurium, ESBL, S. aureus and B. cereus strains. The extent of inhibition was dependent on the probiotic strain and on the carbohydrate source used. AMA of top probiotics strains cell-free supernatants grown in presence of 5% porcine bile was also determined since it has been shown that bile stress often enhance AMA of probiotic strains. It was observed that bile stress did not abolish the AMA of LAB supernatants meanwhile AMA against Gram-negative pathogens (ESBL and S .typhimurium) by Bifidobacteria cell free supernatants was abolished when grown in presence of 5% porcine bile. Laboratory studies have shown that at acid cell free supernatants of probiotic strain combinations at 1:1 dilution inhibited the growth of S. typhimurium ESBL, S. aureus and B. cereus more than 80 % meanwhile neutralized cell free supernatant did not show antimicrobial activity or much less. 6 Crude proteins from two out of four candidate strains namely L. plantarum F44 and B. lactis 8:8 showed maximum activity against enteric pathogens at lower dilutions indicating the role of bacteriocins in AMA against 4 target enteric pathogens used in the present investigation. In conclusion, this study identified 4 potential probiotic active strains (2 lactobacilli and 2 bifidobacteria) that can inhibit some important pathogenic gram-positive or gram-negative enteropathogenic bacteria and could be potential candidate strains for in vivo studies in animal models. AIM OF THE PRESENT STUDY 1. To screen for AMA against human enteric pathogens by LAB and Bif strains and to select the top strains for further in vitro studies. 2. To establish the time course of AMA against the target pathogens. 3. To investigate the effect of selected probiotic strains in combination with prebiotics on human enteric pathogens. 4. To screen four top bile salt-resistatnt lactic acid bacteria for inhibitory activity against enteropathogenic bacteria. 5. To study the effect of a co-culture of LAB and Bif strains on AMA to suppress the growth of pathogens. 6. To fractionate proteinecious antimicrobial substances from probiotic strains using ammonium sulphate precipitation (salting out) and test these against the enteropathogens. 7 INTRODUCTION Gastrointestinal microflora consists of hundreds of different types of microorganisms and is biologically important component of the gut to inhibit gut colonization by incoming pathogens. In the 19th century, microbiologists identified the gastrointestinal (GI) microflora in the GI tract of healthy individuals. The beneficial microflora found in the GI was termed probiotic microbes. The term probiotic was defined more than 20 years ago and is usually defined as live microorganisms or microbial food supplements that confer health benefits for the host, when administered in adequate amounts (Reid et al., 2003). Most probiotics fall into the group of organisms known as lactic acid producing bacteria such as Lactobacillus and Bifidobacterium which have a long safe history in the manufacture of dairy products normally consumed in the form of yogurt, fermented milks or other fermented foods. The efficiency of these probiotic products on animal performance has been discussed extensively, but the mode of action of probiotics still remains unclear. Number of mechanisms have been proposed to explain their mode of action viz., decreasing luminal pH via the production of volatile short chain fatty acids (SCFAs), competing for specific nutrients with pathogens, modulation of the host immune system and producing specific inhibitory compounds such as organic acids (lactic acid and acetic acid etc), oxygen catabolites (e.g. hydrogen peroxide) and antibacterial proteinaceous compounds (e.g. bacteriocins) (Sanders, 1993; Helender et al., 1997; Magnusson et all., 2003; Servin. 2004; Valerio et al., 2004). In the normal intestinal flora these mechanisms are essential components of bacterial populations, with many advantages over competing bacterial pathogens. However factors such as unhealthy diet, stress, microbial infections and other diseases disturb the balance, which often lead to intestinal dysfunction by decreasing number of viable lactobacilli and bifidobacteria (Fuller and Gibson, 1997). For the last three decades the most extensively studied probiotics are Lactobacillus and Bifidobacterium strains of many species, which contribute to inhibit a wide range of infections, such as antibiotic-associated diarrhea (AAD), Helicobacter pylori gastroenteritis and urovaginal infections have been demonstrated in both in vitro and in vivo experimental studies as well as clinical trials (Sgouras et al., 2004; Wult et al., 2003). 8 1. LITERATURE REVIEW 1.1 History and definition Although the term of probiotics was not established until 1965 the history of probiotics is as old as the consumption of fermented milk which first documented in The Old Testament and exists for over 2000 years (JoMay, 2002). Before the term probiotics was even coined the Russian scientist Ellie Metchnikoff in 1908 at the Pasteur Institute in Paris was one of the first developed definable notion that fermented milk product might have beneficial effect on the intestine. He knew that Bulgarian peasants who lived certain part of Bulgaria and consumed a large amount fermented milk products were known for their extraordinary longevity. Metchnikoff was using a pure Grampositive rod shaped bacterium in those days called Bulgarian bacillus and latter Bacillus bulgaricus of what is now called Lactobacills delbrueckii subsp. Bulgaricus which together with Steptococcus thermophilus produce fermented milk products such as yoghurt, to modify the colonic microflora through ingestion of soured milk. He established that normal microflora of the lower gut of humans was having an adverse effect on the host and that consumption of soured milks reserved this effect (Stiles and Holzaphel, 1996). Probiotics, derived from the Greek and meaning “for life” , are defined as cultures of viable microorganisms or microbial food ingredient when ingested in adequate amounts embrace the antagonistic activity against gastrointestinal pathogens, stimulate the immune system and beneficially effect the host by improving its intestinal microbial balance (Quigley and Quera, 2006; Walker and Duffy, 1998). It was first used by Lilly and Stillwell in 1965 to describe as “microbial substances secreted by one microorganism which stimulates the growth of another" and thus was conversed with the term antibiotic (Lilly, 1965). Meanwhile Sperti (1971) designated it as tissue extract which can improve the colonic bacterial growth. These conceptions did not establish. The classical definition which is used until now was R.B Parker (1974) who defined probiotics as “organisms and substances which contribute to intestinal microbial balance” (Parker, 1974). Subsequently in 1989, Fuller redefined probiotics by removing the “substance” which could include antibiotics and microbial stimulant and revised it as "A live microbial feed supplement which beneficially affects the host by improving its intestinal microbial balance (Chen and Walkerl, 2005; Fuller, 1989). 1.2 Microorganisms used as Probiotics The group of microorganism most frequently regarded as probiotics consist of either yeast, especially Saccharomyces, or bacteria, especially lactic acid bacteria which is the most commonly 9 used as nonpathogenic “viable cells”. An overall, traditional dairy strain of probiotics bacteria belongs to the Lactobacillus and Bifidobacterium genera (in complete), but the yeast such as Saccharomyces cerevisiae and some E. coli and Bacillus species are also used as probiotics (Francisco et al., 2008). 1.2.1 Genus Lactobacillus According to second edition of Bergey’s Manual of Systematic Bacteriology (2004) published in a third volume (2009), the genus Lactobacillus belongs to the phylum Firmicutes, class Bacilli, order Lactobacillales, family Lactobacillaceae and its closest relatives, being grouped within the same family, are the genera Paralactobacillus and Pediococcus (Hammes and Hertel, 2009). Lactobacilli are almost ubiquitous: they grow and found in environments where carbohydrates are available, such as food: beer, fruit, grain mashes, marinated fish, sugar processing, sour dough’s, milk, meat and meat products, fermented beverage, plants and plant materials: soil, water, sewage and manure and human or animal habits: respiratory, oral cavity, GI and genital tracts. Lactobacilli are Gram-positive, non-sporeforming, non-motile facultative anaerobic or microaerophilic organisms (Orla-Jensen, 1919) and can vary from long and slender, sometimes bent rods to short, often coryneform coccobacilli (Hammes and Hertel, 2009). With a DNA base composition lower than 54 mol% G+C content and not more than 10% range in G+C content exist in a well-defined genus (Stiles and Holzapfel, 1996). 1.2.2 Genus Bifidobacterium Before the 1960s, Bifidobacteria strains were first discovered in infant faces by Tisser in 1900 and named as "Bacillus bifidus" which later in 1920 was referred as "Lactobacillus bifidus" by Holland. In 1924 Orla-Jensen recognized the existence of genus Bifidobacterium a separate taxon but given their similarities of bifidobacteria with the genus Lactobacillus and bifidobacteria were included in this genus as listed in the 7th edition of Bergey Manual of Determinative Bacteriology (Biavati et al., 2000). In the eight edition of this manual that the genus Bifidobacterium was included in the phylum Actinobacteria, class Actinobacteria, order Bifidobacteriales, family Bifidobacteriaceae (Biavati and Mattarelli, 2006) Bifidobacterium are Gram positive polymorphic branched anaerobic (although some species can tolerate oxygen) rods that occur singly, in chains or clumps (Felis and Delaglio, 2007). They are, non-motile, non-sporulating, non-filamentous and non-gas producing (produce only acid 10 from a variety of carbohydrates), and chemoorganotrophic, having a fermentative type of metabolism (Ventura et al., 2004). The G+C content is quite high compared to Lactobacillus with a value ranging from 42 to 67 mol% (Lee and O’Sullivan, 2010). In the studies on the ecology of bifidobacteria described so far are grouped in six different niches: the human intestine and vagina, dental carries and oral cavity, food (fermented milk), the animal gastrointestinal tract, the insect intestine and sewage (Ventura et al., 2004). 1.3 Bacteriocins and acid production by lactic acid bacteria and bifidobacteria The earliest classifications of lactic acid bacteria are based on the production of speciesspecific stereoisomers of lactate from glucose fermentation as a means of identifying and categorizing the various species in this group. Swedish scientist Schele in 1780 studied sour milk and found that lactic acid is one of the most important organic acids produced by lactic acid bacteria (LAB). Lactic acid exists in two optically active-stereo isomers, the L (+) and the D (-). Since explored of D (-) lactic acid is harmful to humans, L (+) lactic acid is preferred isomer in food and pharmaceutical industries as human have only L – lactate dehydrogenase (LDHs) that metabolizes L ( +) lactic acid isomer normally found in the blood and irune (Reddy et al., 2008). Lactic acid bacteria can be divided into groups on their type of fermentation, that bacterial species are able to produce lactic acid as main half end-product of the fermentation of carbohydrates which has beneficial effects. Three groups of lactobacilli fermentation have been established: 1. Obligately homofermantive lactobacilli are able to ferment hexoses into 85 % lactic acid by the Embden–Meyerhof–Parnas (EMP) pathway while pentoses and gluconate are unable to ferment; 2. Facultatively heterofermentative lactobacilli degrade hexoses only to 50 % lactic acid by the EMP pathway and may produce gas from gluconate but not from glucose. They are also able to degrade pentoses and often gluconate as they possess both aldolase and phosphoketolase; 3. Obligately heterofermentative lactobacilli degrade hexoses by the phosphogluconate pathway producing lactic acid, ethanol or acetic acid and carbon dioxide (Connoly and Lonnerdal., 2004; Mayo and Sinderen., 2010). It is currently been discussed that Lactobacilli has been attributed to the production of metabolites such as organic acid (lactic and acetic acid), hydrogen peroxide, ethanol, diacetyl, acetaldehyde which makes their environments acidic, and it may help to inhibit the growth of some 11 harmful pathogens or stabilize of the intestinal microflora. These microbes generate small molecular metabolic products that exert beneficial regulatory influence on host biological functions.These metabolic products are sometimes referred to as “postbiotics” and may function biologically as immune modulators (Thomas et al., 2010). It has been also observed that some of LAB produce low molecular mass (< 1000 Da) peptides – bacteriocins (antimicrobial substances) or ribosomally synthesized peptides/ proteins with bactericidal activity against related species or across genera. Bacteriocins serve as an important mechanism of pathogen exclusion in fermented foods as well as in the gastrointestinal environment. They are sub-divided into 3 or 4 classes. The most famous bacteriocins produces by lactic acid bacteria which has beneficial effect in food industry is I class type A lantibiotic – nisin produced by Lactococcus lactis. It is used to extend the shelf life of food products by inhibition of Gram-positive bacteria, such as Actinomycetes, Bacillus, Clostridium, Corynebacterium, Enterococcus, Gardnerella, Lactococcus, Listeria, Micrococcus, Mycobacterium, Proprionbacterium, Streptococcus and Staphylococcus (Suskovic et al., 2010; Mayo and Sinderen., 2010). Table 1 shows the different species of Lactobacillus from animals and humans which has most beneficial properties and predominantly used in probiotics based upon these criteria: fermentation group, genome GC content, lactic acid isomer and antimicroial metabolites. Many of these isolates were found in healthy human and animal gut and licensed as safe considering that species differ between human and various animals. Table 1. Subdivision and characteristics of Lactobacillus genus Obligative homofermentative Facultative heterofermentative L. acidophilus NCFM 34.7 DL B, C, E, H 1,2,3 L. johnsonii NCC533 L .salivarius subs. salivarius UCC118 L. delbrueckii spp. bulgaricus ATTCC 11842 L. casei ATTCC334 L. curvatus ATCC 33820 L. lactis IL1403 L. plantarum WCFS1 34.9 33.04 DL L B, C, E, B, C, E, 4,5, 3 6, 5,3 49.7 D B, C, E, H 6, 5,3 46.6 36.5 L DL A,B, C, E, G A,B, C, E,G 7, 3 8, 9,3 35.4 45,6 L DL A,B, C, E,G A,B, C, E,G,H,K,L,M 10,5, 3 4,9, 5, 3 L. casei Shirota L. rhamnosus ATCC 53103 46.3 45-47 L L A,B, C, E, G A,B, C, E, G,H 7, 3,11 5, 3 12 Table 1. – continued Obligately heterofermentative L. fermentum CECT 5716 L. reuteri JCM1112 51.9 38.9 DL DL A,B, C, E, G A, B, C, E, G,N,P 12,9, 3 13, 3 46.2 DL A, B, C, E, G ,H 5, 3 L. brevis ATCC367 *A – acetic acid; B – lactic acid; C – diacetyl acetaldehyde acetoin; E – hydrogen peroxide, G - carbon dioxide, H – 3phenyllactic acid, 4-hydroxyphenyllactic acid; K – 3-hydroxy fatty acids; L – benzoic acid methylhydantoin mevalonolactone; M - cyclic dipeptides; N – reuteryciclin; P – reuterin. **1- Altermann et al., 2005; 2- Connolly and Leonnerdal, 2004; 3- Suskovic et al., 2010; 4- Boekhorst et al., 2004; 5Ljungh and Wadström, 2009; 6- Claesson et al., 2004; 7- Cai et al., 2009; 8- Felis et al., 2009; 9 - Felis and Dellaglio, 2007; 10- Bolotin et al., 2001; 11- Watanabe et al., 2009; 12 - Jime´nez et al., 2010; 13- Morita et al., 2008; Although genus Bifidobacterium phylogenetically not related to LAB it is often grouped part of the LAB for its beneficial health effects, including the regulation of intestinal microbial homeostasis, the inhibition of pathogens and harmful bacteria, the modulation of local and systemic immune responses, the repression of procarcinogenic enzymatic activities within the microbiota, the production of vitamins, and the bioconversion of a number of dietary compounds into bioactive molecules (Mayo and Sinderen., 2010; Rastall et al., 2005). However much metabolic research of bifidobacteria has focused on carbohydrate fermentation and they play an important role in been observed that genus Bifidobacterium oligosaccharide metabolism in the colon. It has possesses a unique fructose-6-phosphate phosphoketolase pathway employed to ferment carbohydrates which produce lactic acid and additional amount acetic acid, larger than the amounts secreted by lactobacilli, as the main product of carbohydrate metabolism that inhibit various pathogenic bacteria (Biavati et al., 2000). L-isomer produced by bifidobacteria is more potent than the D-isomer (Mayo and Sinderen, 2010). Unfortunately it is unclear which active stereo-isomers are produced by different Bifidobacterium species. Unlike lactobacilli, very little is known about the production of bacteriocins by bifidobacteria. They produce at least six categories of inhibitory substances other than small terminal metabolites or other non – peptide compounds. It is only clear that some types of bacteriocins produced by bifidobacteria have the physiochemical stability making them suitable as potential antimicrobial agents in food for inhibiting some gastrointestinal pathogens and possibly provide an adjunct or alternative to antibiotic therapy. The most promising bacteriocins produced by Bifidobacterium bifidum NCDO 1452 is bifidin and the second one called as bifidocin B is produced by B. bifidum NCFM 1454 (Mayo and Sinderen, 2010). Table 2 shows the different species of genus Bifidobacterium for human consumption which has most beneficial properties and extensively used in commercial probiotic preparations. Many of these isolates were found in healthy 13 human gut and licensed as a safe considering that different species effects differ between species in human or animal studies. Table 2. Subdivision and characteristics of Bifidobacterium genus Species B.adolescentis: ATCC15703; L2-32; B. angulatum DSM 20098 B.animalis subsp. lactic: AD011 B1-04 DSM 10140 HMN019 B.bifidum: S17; PRL 2010; NCIMB 41171 B.breve DSM 20213 B. catenulatum DSM 16992 B.dentium: Bd1; ATCC 27678; B.gallicum DSM 20093 B.longum subsp. infantis: ATCC15697; ATCC 55813; CCUG 52486; B.longum subsp. longum: DJO10A; NCC 2705; G+C (mol %) *Refernces 59.18 59 59 Ventura e al., 2007 Turoni et al., 2011 60.49 60.49 60.48 60.48 60.48 Kim et al., 2009 Lee and O’Sullivan 2010 Mayo and Sinderen, 2010 62 63 59 56 Turnoei et al., 2011 58.94 59 58 Lee et al., 2010 59.86 60.15 60.13 Lee and O’Sullivan 2010 Lee et al., 2010 Mayo and Sinderen, 2010 1.4 Mechanism of action of probiotics Critera for Lactobacilli and Bifidobacteria strains to be called as probiotic are: bacterium must survive the acidic conditions of the upper GIT and colonize the intestine microbiota. Fermentation products and cell components must not contain pathogenic toxins, mutagens or carcinogens and must be genetically stable. Finally it must be easily reproducible and remain viable during processing and storage (Ouigley, 2010). One of the most exciting areas of research is the mechanism of action of probiotics. Mechanism by which probiotics strains affect the micro ecology of the intestinal tract are not well understood with at least three modes of action: modification of the microbial flora, enhancement of intestinal barrier function and stimulation of immune response have been observed (Table 3). 14 Table 3. Probiotic effects on the development of host defense Mechanism of action of probiotics Modification of the Enhancement of microbial floraintestinal barrier antimicrobial activity function Decrease luminal pH Increase mucus production (Trefoil Produce inhibitory factors) compounds Enhance barrier Inhibit bacterial invasion integrity (tight (gene expression) junctions) (epithelial Block bacterial adhesion barrier function) to epithelial cells. Immunomodulation Effect on epithelial cells Effects on dendritic cells Stimulation and effect on macrophages Stimulation and effects on lymphocytes: - B lymphocytes - NK cells - T cells - T cells redistribution - Interferon Secrete polymeric IgA secretion 1.4.1 Modification of the microbial flora Probiotics possess the ability to colonize the GI tract by increasing the numbers of beneficial microbes and decreasing the population of potentially pathogenic microorganisms and creates a balance in the gut microbiota of the host. A front line of defense against pathogens is antimicrobial effect: By reducing luminal pH. Lactic acid bacteria produce organic acid the most lactate and acetate, which create acidic microenvironment in the gut lumen and that is inhibitory to virulent organism growth (Sherman et al., 2009). By producing inhibitory (antimicrobial) compounds (substances). Probiotics secrete antimicrobial compounds – inhibitory peptides such as : lantibiotics (class I), peptide bacteriocins (class II), and bacteriolysins (class III) and may become active participants in the fight against certain infections while others are potent anti-inflammatory agents and produce (L.reuteri) antimicrobial-multi compound such as reuterins, which also has broad spectrum activity against of some harmful Gram-negative and Gram-positive bacteria (Suskovic et al. 2010). The general functions of effect are depicted in Figure 1. By inhibiting bacterial invasion. It has been observed that some strains of probiotics may influence virulence gene expression of microbial pathogens ( MedellinPena et al., 2007). 15 Inhibiting bacterial adherence. Some probiotics strains such as L. crispatus and L. helveticus has a surface- protein layer ( S-layer) and are able to bind to host cell surfaces of epithelial cells thereby competing with pathogenic bacteria for the same glycoconjugate epithelial surface receptor as those used by pathogens as receptors for adherence (Saulnier et al., 2009). The general functions of effect are depicted in Figure 2. Fig. 1: Antibacterial substances (Chen. and Walker, 2005) Produce and secrete antimicrobial substances. Fig. 2: Inhibition of adherence (Chen. and Walker, 2005) Compete with pathogens for glycoconjugate receptors on the mucosal surface thereby limiting adherence and colonization. 1.4.2. Enhancement of intestinal barrier function The mucosal epithelial cell barrier is the first line of defense against pathogen attack on that ground probiotic can help: By enhancing the production and secretion of mucin (mucin gene expression- MUC2 or MUC3) or reduction of gut permeability. Promoting mucin production thereby enhanced mucus layer overlying the epithelial and reducing intestinal permeability it may serve as antimicrobial shield that prevents penetration of pathogenic organism and toxic substances ( (Lindenet al., 2008). Mucin producing cells can easily secrete antimicrobial peptides – treifol factors (human proteins) in response to microbial pathogens binding to the epithelial cell surface (Sherman et al., 2009). Direct effect on enhancing epithelial barrier function to modify mucus or chloride secretion or changes in tight junction protein expression by epithelial cells. The general functions of effect are depicted in Fig. 3. 16 Fig. 3: Strengthen tight junction Enhance tight junction proteins to strengthen against the mucosal barrier. Fig. 4: innate immunomodulation Stimulate specific mucosal host defenses pathogens. 1.4.3 Immunomodulation Probiotics are able to affect or suppress aspects of the immune response. Some of the strains affect the epithelial cells and may decrease chemokine interleukin (IL)-8 secretion by epithelial cells compared with some intestinal pathogens such as enteropathogenic E. coli and Salmonella dublin, Shigella dysenteriae and Listeria monocytogens and indicate that probiotic bacteria may override the effects of pathogenic bacteria. On the other effect of probiotic bacteria on epithelial cells is the ability to produce the innate immune system receptors – Toll-like (TLR) such as TLR-2 and TLR-4 on the surface of epithelial cells and can induce the production of cytokines that enhance epithelial cell regeneration and inhibit epithelial cell apoptosis. Probiotic bacteria can also reduce a proinflammatory response in intestinal epithelial cells by blocking phosphorylation and degradation by ubiquitination of IκB, meanwhile some pathogenic bacteria induce proinflammatory response by activating the nuclear transcription factor , NF-κB.( NF-κB is a protein complex that controls the transcription of DNA and plays a key role in regulating the immune response to infection (Ng et al., 2009). It has been also observed that probiotic bacteria can have effect on dendritic cells. Dendritic as antigen-presenting cell (APCs) are important in bacterial recognition and subsequent T – cell responses, but the most important aspect are their ability to recognize and responds bacterial components, to initiate primary immune response, and to direct developing T- and B-cells response (Walker, 2008). Some of probiotic strains can increase IL-10, IL-12, IL-18, IFN-γ, TNFα synthesis and secretion in macrophages (Ng et al., 2009). 17 Meanwhile others can elevate the B lymphocytes, NK cells, T cells and interferon activities. Probiotics may also promote the differentiation of B cells into plasma cells and thereby increase the production of secretory immunoglobulin A that plays a critical role in mucosal immunity. IgA, in turn, coats the muscusal surface to control microbial and antigen penetration (Walker, 2008; Sherman et al., 2009). The general functional affects are depicted in Fig. 4. 1.5 Antimicrobial activity by lactobacilli and bifidobacteria against enteric pathogens Lactic acid bacteria play a key role in maintaining the balance of the normal gastrointestinal microflora (Quigley, 2010). However factors such as diet, stress, an enfeebled immunity system, microbial infections, other diseases and a prolonged antibiotic usage disturbs the balance, which often leads to a decrease of viable lactobacilli and bifidobacteria. The subsequent uncontrolled proliferation of pathogens bacteria may often lead to viral or bacterial diarrhea, inflammatory bowel disease (IBD), necrotizing enterocolits (NEC), cancer and other clinical disorders. There are a number of studies that suggest that lactic acid bacteria can decrease the incidence, duration and severity of some pathogens which can cause gastric and intestinal illness. Protective effects of probiotics against gastric and intestinal infections have been demonstrated in well-designed in vitro and in vivo experimental studies (Table 4). These mechanisms may include lowering of the pH, production of lactic acid and antimicrobial compounds such as bacteriocins, hydrogen peroxide and competition for nutrients or adhesion receptors. In addition it is important to mention that the antimicrobial activity of lactobacilli and bifidobacteria is a strain specific property and cannot be extrapolated to other lactic acid bacteria that is why it is necessary to screen other members of Lactobacillus and Bifidobacterium and select the most effective strains to prevent and treat infectious bacterial and viral diarrhea, gastroenteritis or urovaginal infections. We focused on four enteric pathogens: (a) two Gram negative enteropathogens; Salmonella typhimurium which causes gastroenteritis in humans and other mammals; and extended-spectrum beta-lactamase ( ESBL) – producing Escherichia coli which can various forrms of diarrhea and (b) two Gram positive pathogens methicilin-resist Staphyloccocus aureus (MRSA) which is responsible for various diseases including: mild skin infections (impetigo, folliculitis, etc.), invasive diseases (wound infections, osteomyelitis, bacteremia with metastatic complications, etc.), toxin mediated diseases (food poisoning, toxic shock syndrome or TSS, scaled skin syndrome, etc.) and spore-forming Bacillus cereus which causes two types of food poisoning in humans including 18 diarrheal syndrome and emetic syndrome. A representative summary of recent scientist research of probiotics antimicrobial effects against S. typhimurium, ESBL, S. aureus and Bacillus cerues infection is listed in Table 4. Table 4. Inhibition effects of probiotics on S. typhimurium, ESBL, S. aureus and B.cerues Barrier function and Probiotics Tested L. acidophilus: LB Pathogens Epithelial adherence S.typhimurium: (SL 1344) E.coli: JPN15[pMAR7] and S.typhimurium : (CECT 1456), (SL 1344), (C5) Model References In vitro (Caco2/TC-7) Coconnrie et al., 1999;2000 + in vivo (gem-free mouse) Santos et al., 2003 Bernet-Camard et al., 1997 + HT-29, HT29 MTX Gopal et al., 2001 In vitro (Caco-2) Nesser et al., 2000 In vitro (intestine mucus) In vitro (Caco-2 or intestine mucus) In vitro (intestine mucus Tuomola et all., 1999 Lee et al., 2003, Lee and Puong., 2002 Tuomola et all., 1999 Lee et al., 2003, Lee and Puong., 2002 Tuomola et all., 1999 L.gasseri : (UO 002) L. kefiranofaciens: ( CYC 10058) L.delbruckii: ( CYC 10048) L.helveticus: ( R0052) Bifidobacteria strains : (CA1) and (F9) E.coli : (0157:H7) E.coli: (ETEC)-enterotoxigenic; (EPEC) – enteropathogenic; S.typhimurium: (ATCC 14028) E.coli: (ATCC 11775), (TG-1), (0157) S.typhimurium : (ATCC 14028), (E10), (E12) SfaIIfimbrated E.coli HB101 (paZZ50), E.coli: (ATCC 11775), (TG-1), (0157) S.typhimurium: ( ATCC 14028), (E10), (E12) SfaII- fimbrated E.coli HB101 (paZZ50), E.coli : ( 0157:H7) E.coli : (ETEC) (ATCC 31705)enterotoxigenic, (EIEC) IID 995enteroinvasive, (EPEC) IID 956 enteropathogenic E.coli : (0111) S.typhimurium : (CECT 1456), E.coli : (CECT 4076) E.coli: (0157:H7) S.typhimurium: (SL 1344) lactis (DR10) E.coli : ( 0157:H7) In vitro (Caco-2, HT-29, HT29 MTX (CYC 10051) (LA1-SCS ) (HN017) L.johnsonii : (La1) (LJ1) L.casei subsp: Shirota (Yakult;Singapore) rhamnosus : GG (ATCC 53103) GG (ATCC 53103) and (LC-705) (DR20) L.crispatus: (JCM 5810) In vitro (Caco-2) In vitro (intestine mucus) In vitro (Caco-2, HT-29, HT29 MTX) Gopal et al., 2001 In vitro (Matrigel) Horie et al., 2002 In vitro (Caco-2) In vitro (Caco-2) Fernanadez et al., 2003 Santos et al., 2003 In vitro (Hep-2, T84) In vitro (Caco-2) and in vivo (axenic mouse) Johnson-Henry et al., 2007 Lievin et al., 2000 Gopal et al., 2001 19 Table 4 - continued Probiotic antimicrobial factors P.acdilactici : (NCIM 2292) P.pentosaceous (NCIM 2296) P. cervisiae (NCIM 217) L.plantarum: (42) L.curvaus: ( DF 38) P.pentosaceus : (34) L.salivarius: (241) L.casei LHS L.acidopillus : (CRL 1259), (I 26) ,(I 16) ( YIT0070) L.crispatus: (I 12) L.brevis: (I 23), (I 211,) (I 218), (C10), (C1, C17) L.fermentum: (I 24),( I 25), (C 16) L.casei subsp: Shirota: (YT 9029) (DN -114001) rhamnosus: (GR1) (ATCC 4356) casei : (ATCC 393) E. faecium: (ATCC 19434) L.johnsonii: (La1) L.sakei: (CWB 030202) S.aureus : (MTCC 737) and B.cereus : (MTCC 1272) E.coli, S.typhimurium including strains isolated from patients diagnosed with HIV/AIDS E.coli.: (O1:K1), (O2:K1), (O78:K80), S.typhimurium (0157:H7) E.coli: O1:K1, O2:K1, O78:K80 and S.typhimurium E.coli : (0157:H7) S.typhimurium: (SL 1344) In vitro Jamuna and Jeevarantnam, 2004 In vitro Brink et al., 2006 In vitro Juarez et al., 2003 Jin et al., 1996 Ogawa et al., 2001 In vitro Jin et al., 1996 In vitro Ogawa et al., 2001 Fayol-Messaoudi et al., 2005 Enteroaggregative E.coli (EaggEC TN-2) In vitro Miyazaki et al., 2010 S.typhimurium: (SL 1344) In vitro Fayol-Messaoudi et al., 2005 20 1.6 Prebiotics The term prebiotic, first coined by Gibson and Reberfroid, refers to non-digestible but fermentable food ingredients that beneficially affect the host by selectively stimulating the growth of a limited number of health-promoting commensal flora. A number of food ingredients could act as prebiotics but the most commonly evaluated prebiotics are those stimulate probiotic microorganism already resident in the lower gut (Gibson et al., 2010). Certain carbohydrates, oligoand poly-saccharides, occur naturally and meet the criteria of prebiotics. These consist mainly of non-digestible fructooligosaccharades and inulin –type fructans, which are linked by β (2-1) bonds that limit their digestion by intestinal enzymes. Crittenden and Playne (1997) describe food-grade oligosaccharides in commercial production which include fructooligosachharides (FOS), galactooligosaccharides (GOS), gentiooligosaccharides, inulin, isomaltooligosaccharides (IMOS), maltooligosaccharides, palatinose-oligosaccharides lactulose, glucosyl sucrose, lactosucrose, soybean-oligosaccharides (SOS), xylooligosaccharides (XOS) and cyclodextrins ( Gibson et al., 2010). Most of them are present in significant amounts in many edible fruits and vegetables including wheat, onion, chicory, garlic, leeks, artichokes and bananas (Salvatore and Vandenpalas, 2010). Because of their chemical structure, prebiotics are not hydrolyzed and absorbed in the small intestine but are fermented in the colon by endogenous bacteria to act as energy and metabolic substrates, with lactic and short chain carboxylic acid as end products of fermentation (Salvatore and Vandenpalas, 2010). Some non-digestible carbohydrates have a number of functional effects on the GIT which have been used to validate functional and health claims. The carbohydrate group that has received the most attention and research is the oligosaccharides – FOS may selectively stimulate the growth of bifidobacteria in the large intestine, increase calcium absorption, and fecal weight, shortening of gastrointestinal transit time and possibly, lowering the blood lipid level (JoMay, 2002). When prebiotics are added in combination with probiotic strains, the combination referred to as “synbiotic”, can selectively stimulate the growth of probiotic bacteria, especially, but not exclusively lactobacilli and bifidobacteria. Prebiotics can serve as a selective growth substrate for the probiotic strain during fermentation. Combining probiotics and prebiotics could beneficially affect the host by improving the survival and of live microbial dietary supplements into the gastrointestinal flora and by improving the microbial balance of the gastrointestinal tract (Chen. and Walker, 2005). 21 2. MATERIAL AND METHODS 2.1 Growth substrates Prebiotics used in the present study are listed in Table 5. Table 5. Carbohydrate source list used in this study Carbohydrate source Purity Company Fructooligosaccharides (FOS) 93.7% Orafti (Tienen, Belgium) Galactooligosaccharides (GOS) 96.2% Friesland Campina Domo (The Netherlands) Lactulose 95% Sigma-Aldrish (Canada) Xylooligosaccharides (XOS) 95% Sweet Town Bio-tech (Taiwan) Isomaltooligosaccharides (IMOS) 60% Wako Pure chemical, Ltd (Germany) 2.2 Bacterial strains Probiotics and enteric pathogens strains used in this study are listed in a Table 6. Table 6. List of bacterial strains used in the present investigation No. 1 2 3 4 5 6. 7. 8 9. 1. 2. 3. 4. Name of strain Probiotic strains L. crispatus LMG 12003 L. paracasei F8 L. rhamnosus LС 35 L. plantarum F44 L. plantarum F17 B. breve 46 B. lactis 88 B. longum 618 B. pseudocatenulatum JCM 1200 Enteropathogens S. typhimurium (Gram Negative/resistant) ESBL (Gram Negative/resistant) MRSA-S. aureus (Gram Positive/resistant) B. cereus (Gram Positive /resistant) Source Infant, feaces Biobank, LUH Lyocentre, France Biobank, LUH Biobank, LUH Biobank, LUH Biobank, LUH Biobank, LUH Feces of infant Biobank, LUH Biobank, LUH Biobank, LUH Biobank, LUH 2.3 Bacterial growth conditions Bifidobacteria and lactobacilli strains were screened for antimicrobial properties against Gram-negative and Gram-positive bacteria. Lactic acid bacteria were grown microaerobically in 5ml of De Man, Rogosa, Sharpe (MRS) broth (Difco Laboratories, Detroit, MI) 24h at 37 oC and bifidobacteria strains were grown in 5 ml MRS broth supplemented with 0.05 % 22 L- cysteine hydrochloride (MP Biomedicals, LLC, France) 24h at 37 oC under anaerobic conditions, achieved using the Anoxomat system (Mart Microbiology, The Netherlands). Enteropathogens used were E. coli, S. typhimurium, S. aureus and B .cereus were grown aerobically in 5 ml Muller-Hinton broth (Becton Dickinson company Cockeysville, MD21010, USA) or on agar (OXOID AB LTD, Sollentuna, Sweden). 2.4 Antibacterial activity of selected bacteria 2.4.1 A time course of antimicrobial activity of cell-free supernatant of probiotic bacteria against enteric pathogens AMA of LAB and bifidobacteria was determined against E. coli, S. typhimurium, S. aureus and B. cereus using microtiter plate method. Preparation of cell free culture filtrates: Briefly, a cell free culture filtrate was obtained by harvesting 24 h old cultures of lactobacilli and bifidobacteria grown on MRS and MRSC broth by centrifugation (15.000 x g for 15 min at 4 oC in an eppendorfs centrifuge 5810, Hamburg). The supernatants were collected, pH determined and adjusted to pH 6.0 with 2 M. NaOH (MERCK, Germany) and filter sterilized by passing through a sterile syringe and 0.2- µm pore size with cellulose acetate membrane filter (VWR INTERNATIONAL AB, Stockholm, Sweden). To determine if the antimicrobial activity recorded was bacteriostatic or bactericidal, the cell free culture filtrates were added on pathogens in a sterile 96 well micro titer plates (TPP, 92696, Switzerland) (Catherine and Rolfe., 2000). Dilutions 1:1 and 1:10 (in separate experiment 1:100 and 1:500) were prepared in Muller-Hinton broth. One hundred of the culture filtrates of lactic acid bacteria and bifidobacteria was added to the microtiter plate wells and incubated overnight at 37oC. Preparation of enteric pathogens for antimicrobial activity: E. coli, S. typhimurium, S. aureus and B. cereus cells were grown in Muller-Hinton broth (MHB) for 24 h in aerobic conditions at 37 oC (cells were harvested by centrifugation and washed twice in sterile phosphate buffer saline 0.015 M. PBS, pH 7.2) and suspended in PBS. Ten micro liters of E. coli, S. typhimurium, S. aureus and B. cereus cells with 0.5 OD (optical density) at 620 nm was prepared in PBS and 10 µl of the suspension was added to 100 µl of test supernatants in the microtiter plates. After 24 h, the growth in microtiter plates was measured using microtiter plate reader ( iMarkTM Microplate Absorbance Reader catalog number 168-1130, BIO-RAD Laboratories AB, 23 Sundbyberg, Sweden). The antimicrobial activity was expressed as the percent inhibition of the growth of pathogen compared to control. 2.4.2 Effect of prebiotics on the AMA of probiotic strains against E. coli and S. typhimurium, S. aureus and B. cereus The lactic acid bacteria and bifidobacteria were grown in MRS and MRSC for 24 h. Cells were centrifuged and washed twice in PBS and the biomass density was determined by measuring the optical density at 620 nm. Pre-reduced MRS and MRSC broth supplemented with filter sterilized (0.2 µm) 1% each of fructooligosaccharides (FOS), galactooligosaccharides (GOS), lactulose, xylooligosaccharide (XOS), isomaltooligosaccharides (IMOS) was inoculated with probiotic strains to get an OD of 0.5 in 5 ml broth. After 24 h of growth at 37oC, the cells were washed with sterile phosphate buffer saline (PBS, pH 7.2, 0.015) and cell –free culture filtrates were adjusted to 6.0 pH and tested for antimicrobial activity against E. coli , S. typhimurium, S. aureus and B. cereus cells using 96 well sterile microtiter plates (Brink et al., 2006). 2.4.3 Effect of bile stress on AMA of probiotic strains against E. coli and S. typhimurium, S. aureus and B. cereus Effect of bile was determined as described by Brink et al., 2006 with a slight modification. Briefly, LAB and bifidobacteria were grown in MRS and MRSC broth supplemented with 5% porcine bile instead of oxbile (Brink et al., 2006). After 24 h of growth at 37oC, the cell free culture filtrates were obtained by centrifugation. The pH was adjusted to 6.0 and was used to determine antimicrobial activity against cells of the E. coli, S. typhimurium, S. aureus and B. cereus strains using microtiter plate. 2.4.4 Effect of probiotic strain combinations on AMA against E. coli, S. typhimurium, S. aureus and B. cereus LAB and bifidobacteria strains were checked for compatibility and various mixed cultures (Co-cultures) of different lactic acid bacteria and bifidobacteria were performed in 5 ml MRSC broth and incubated under anaerobic condition. After 24 h of growth at 37 oC, cell free culture filtrates were obtained by centrifugation and neutralized to pH 6.0 with 2 M NaOH, filter sterilized and was tested for antimicrobial activity against E. coli and S. typhimurium S. aureus and B. cereus cells using micro titer plate and listed in Table 7. 24 Table 7. List of co-culture mixes tested for antimicrobial activity Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Strain combinations F44+F8+88 F44+F8+46 F8+46+88 F17+F8+88 F17+46+88 F8+88 F44+46 F17+46 F17+88 F8+46 8:8+618 46+88 46+618 46+88+F8+F44 F44: L. plantarum; F8: L. paracasei; 46: B. breve; 618: B. longum & 88: B. lactis LUH strain Biobank collection (Åsa Ljungh; to be published; www.qualvivo.eu) A part of the work will be presented in “International conference on probiotics and probiotics ICP2011” Slovakia during June 2011. 2.4.5 Partial purification of proteinecious antimicrobial substance LAB and bifidobacteria strains were grown in MRS or MRSC broth for 24 h at 37 °C. After incubation, the supernatants were collected by centrifugation at 5000 × g for 10 min. and used for the precipitation of antimicrobial peptides with 80% saturation of ammonium sulphate (Joshi et al., 2006). After stirring on a magnetic stirrer, it was kept undisturbed at 4 °C overnight. Precipitates formed were collected by centrifugation at 10 000 × g for 10 min and redissolved in 10 mM sodium phosphate buffer, pH 6.0. Crude protein extract was dialyzed using 1000 Dalton cutoff dialysis membrane against 10 mM Phosphate buffer of pH 6.0 extensively. Similarly proteins precipitated from MRSC broth uninoculated with probiotic strains served as a control. Antimicrobial activity of the crude protein was determined against enteric pathogens at 1:1; 1:10; 1:100 and 1:500 dilutions. 25 3. RESULTS AND DISCUSSION 3.1 The antimicrobial activity of LAB and Bifidobacteria of growth of enteric pathogens (screening) The antimicrobial activity (AMA) of LAB and bifidobacteria and inhibition of growth of enteropathogens is shown in Table 8. From a total 5 lactic acid and 4 bifidobacteria, the culture supernatants of two Lactobacilli (L. paracasei F8, L. plantarum 44) and two bifidobacteria ( B. breve 46, B. lactis 88) has shown strong antimicrobial activity when tested against the two Gram negative and Gram positive pathogens. The inhibition of enteropathogens ranged from 70 to 83%. The strongest inhibition (84%) was obtained with culture supernatant of strains L. paracasei on ESBL. In contrast, B. cereus was the most resistant strain; only inhibited at pH 6 at 1:1 dilution. In the present study, the acid supernatants (pH 3.5-4.9) of the L. paracasei, L. plantarum, B. breve and B. lactis strains inhibited the growth of all tested enteropathogens. The most sensitive pathogen was the Gram positive S. aureus to inhibitory substances produced by the LAB and bifidobacteria. It was observed that four top probiotic cell-free supernatants at 1:1 and 1:10 dilutions suppressed the growth of S. aureus. We also found that the inhibitory effect of L. paracasei, L. plantarum, B. breve and B. lactis on growth also decreased after adjustment of the pH to 6.0. Table 8a. Antimicrobial activity (AMA) of acid cell-free supernatants of probiotic strains against enteropathoges by microtiter plate assay Pathogens Acidic cell free supernatants Strains S. typhimurium ESBL S. aureus 1:1 1:10 1:1 1:10 1:1 1:10 B. breve 46 3+ 3+ dW 2+ 2+ B. longum 6:18 2+ 3+ W+ 2+ 2+ B. lactis 8:8 3+ 3+ dW 2+ B.pseudocatenulatum JCM 1200 2+ 2+ dW 2+ + L.paracasei F8 3+ 3+ 3+ + L.plantarum 44 3+ 3+ W+ 2+ W+ L.plantarum F17 2+ dW 3+ + 2+ W+ L.crispatus LMG 12003 2+ 3+ dW 2+ + L.rhamnosus LC35 2+ 3+ + 2+ + B. cereus 1:1 1:10 2+ W+ 2+ 2+ + 2+ 2+ W+ 2+ + - ((3+) - 80% to 90% inhibition, (2+) –60% to 80% inhibition, (+) –30% to 60% inhibition, (W) –10% to 30% inhibition, (dW) –5% to 10 % inhibition, (-) – no inhibition 26 Table 8b. Antimicrobial activity (AMA) of neutral cell-free supernatants of probiotic strains against Enteropathoges by microtiter plate assay Pathogens Neutralized cell free supernatants Strains S. typhimurium ESBL S. aureus B. cereus 1:1 1:10 1:1 1:10 1:1 1:10 1:1 1:10 B. breve 46 2+ + 2+ dW B. longum 618 + + + B. lactis 88 + + + B. pseudocatenulatum JCM 1200 dW dW + L. paracasei F8 + dW 2+ L. plantarum F44 + W+ 2+ L. plantarum F17 + W+ + L. crispatus LMG 12003 + + 2+ L. rhamnosus LC35 + + 2+ ((3+) - 80% to 90% inhibition, (2+) –60% to 80% inhibition, (+) –30% to 60% inhibition, (W) –10% to 30% inhibition, (dW) –5% to 10 % inhibition, (-) – no inhibition 3.2 A time course of antimicrobial activity of cell - free supernatants of probiotic bacteria against enteric pathogens Time course of AMA of cell free supernatants (acid and neutralized) of two lactobacilli and two bifidobacteria strains against four enteric pathogens was evaluated by collecting the cell free supernatants of the probiotic strains at 4, 8, 12 and 24 h of growth. (Fig. 5, 6, 7 and 8). L. paracasei F 8 Acid supernatants of L. paracasei F8 obtained at 4 h of growth showed strong inhibition of the 4 enteric pathogens at 1:1 dilution (Fig. 5). AMA of supernatants obtained at 8, 12 and 24h showed a reduction against 4 enteric pathogens. Neutralized supernatants (pH 6.0) obtained at 8 h of growth at 1:1 dilution showed highest AMA against S. aureus. However neutralized supernatants showed a reduction in AMA against S. typhimurium, ESBL E. coli and B. cereus after 12 hours of incubation. 27 Fig. 5 Time course of AMA of L. paracasei F8 against 4 enteropathogens (a) acid supernatants and (b) neutralized supernatants (pH 6.0) L. plantarum F44 Time course of AMA of L. plantarum F44 against 4 enteric pathogens is shown in Fig. 6. Acid supernatants of L. plantarum obtained at 4h of growth at 1:1 dilution inhibited the growth of enteric pathogens by more than 80%. However a decrease in AMA was observed with the supernatants obtained at 8, 12 and 24 h of growth. Neutralized supernatants (pH 6.0) obtained at 4 h of growth showed a reduction in AMA against ESBL, B. cereus and S. typhimurium, meanwhile S. typhimurium was decreased by incubation with the supernatants of L. plantarum strain grown after 8 h of incubation. However AMA against S. aureus remained the same. Fig. 6 Time course of AMA of L. plantarumF44 against enteropathogens (a) acid supernatants and (b) neutralized supernatants (pH 6.0) 28 B. breve 46 Time course of AMA of cell free supernatants of B. breve 46 against 4 enteric pathogens is shown Fig. 7. Acid extracts obtained at 4 h of growth of B. breve 46 showed strong inhibition of S. aureus and S. typhimurium where as AMA against ESBL and B. cereus was highest with supernatants obtained at 8 and 12 h of growth. AMA of acid supernatants obtained after 24 h showed a slight reduction against the 4 enteric pathogens. Neutralized extracts obtained at 4 h of growth at 1:1 dilution showed strong AMA against S. aureus and stabilized after 12h and did not decrease after 24h of incubation, whereas AMA against B. cereus was about 50% with the neutralized supernatants obtained after 12h of growt at 1:1 dilution. AMA against ESBL and S. typhimurium was highest with neutralized extracts obtained after 24 h of growth at 1:1 dilution. Fig. 7 Time course of AMA of B. breve 46 against 4 enteropathogens (a) acid supernatants and (b) neutralized supernatants (pH 6.0) B. lactis 88 Time course of AMA of cell free supernatants of B. lactis 88 against 4 enteric pathogens is shown Fig. 8. Acid supernatants obtained at 12 h of growth at 1:1 dilution showed highest inhibiton of all the enteric pathogens. However the inhibition was decreased with the supernatants obtained at 24 h of growth. Neutralized supernatants (pH 6.0) of B. lactis 88 obtained at 4 h of growth showed strong inhibiton of S. aureus and the AMA was retained upto 12 h and was decreased with the supernatants obtained at 24 h of growth. 29 Neutralized supernatants of B. lactis 88 obtained at 8 and 12 h of growth at 1:1 dilution showed strong AMA against B. cereus and the AMA greatly reduced with the neutralized supernatants obtained at 24 h of growth. However neutralized supernatants of B.lactis 88 obtained after 24 h of growth at 1:1 dilution showed highest AMA against S. typhimurium and ESBL. It was observed that AMA at 1:10 dilution was completely lost or imperceptible. Fig. 8 Time course of AMA of B. lactis 8:8 against Enteropathogens (a) acid supernatants and (b) neutralized supernatants (pH 6.0) In conclusion, the results on time course of AMA of probiotic strains against the 4 enteric pathogens showed that the inhibition was highest with acid supernatants. When the supernatants were neutralized to pH 6.0 there was a decrease in AMA. This could be due to the synergistic action of acid metabolites like lactic, acetic, propionic and butyric acids and also the possible secretion of proteinecious bacteriocins (and bacteriocin like substances or non-proteinecious substances (H2O2) by probiotic strains in acid supernatants which further needs to be investigated). 3.3 AMA studies of LAB and Bifidobacteria in presence of prebiotics The ability of FOS, GOS, IMOS, LACT and XOS to enhance lactobacilli and bifidobacteria antimicrobial activities against enteric pathogens were analyzed (Fig. 9, 10, 11, 12, 13). We have observed that addition of prebiotics to MRS or MRSC resulted to increased antimicrobial activity, but differed from strain to strain. B. breve 46 and B. lactis 8:8 growth with FOS, GOS, IMOS, LACT and XOS (B. lactis 8:8), were effective in inhibiting growth of all tested enteric pathogens with acid supernatants. Cell free supernatants consistently conferred a significantly greater inhibitory effect than neutralized supernatant fractions. It was observed that B. 30 breve 46 cell free supernatants in presence of all tested prebiotic sources showed stronger antimicrobial activity at pH 4 compared with B. lactis strain. In contrast, B. breve was most effective with FOS, GOS and XOS imparting high inhibition levels against all four enteropathogens (Fig. 9, 10 and 13). No AMA or very slight was recorded with neutralized cell free supernatants of B. breve and B. lactis in presence of IMOS, LACT and XOS (Fig. 11, 12, 13). Highest inhibition of the pathogens by the neutralized cell free supernatants of B. breve strain against S .aureus was obtained with FOS and GOS (Fig. 9 & 10) meanwhile GOS was the only carbohydrate source where inhibition of B. cereus was observed with AMA of neutralized cell free supernatants of B. lactis strain at pH 6 (Fig. 10) AMA of cell-free supernatants obtained from L. plantarum and L. paracasei cultured in MRS broth with 1 % each of GOS, IMOS, LACT was determined (Fig.10, 11 & 12). L. plantarum grown with GOS was the most effective at inhibiting pathogen growth at no adjustment pH (Fig. 9) while no antimicrobial activity or very slight was recorded in the cell free supernatant of L.planatrum in present of lactulose (Fig. 12). It was observed that at neutralized pH of strain supernatants (1:1) in the presence of GOS and IMOS were ineffective against S. aureus and B. cereus) (Fig. 10 & 11). In contrast, L. paracasei in presence of IMOS has shown high AMA at acid pH only against Gram-positive pathogens (Fig. 16). Fig. 9 Inhibition of 4 enteropathogens by B. breve and B. lactis in presence of FOS 31 Fig. 10 Inhibition of 4 enteropathogens by B.breve, B. lactis and L. plantarum in presence of GOS Fig. 11 Inhibition of 4 enteropathogens by B. lactis, B. breve, L. plantarum and L. paracasei in presence of IMOS 32 Fig. 12 Inhibition of 4 enteropathogens by B. lactis, B. breve and L. plantarumin presence of LACT Fig. 13 Inhibition of 4 enteropathogens by B. lactis in presence of XOS 33 3.4 Effect of bile on AMA of Probiotic strains Bile is reported to enhance cell surface hydrophobicity of probiotic strains and certain pathogens for better adhesion to the mucus lining. Therefore the effect of 5% porcine bile on AMA of probiotic strains was evaluated and compared with AMA of supernatants obtained from unstressed cells. As data shown in Fig. 14 a & b, the acid supernatants of L. plantarum in presence of bile and without bile at 1:1 dilution are able to inhibit the growth of 4 enteric pathogens by more than 60%. Growth was much more vigorous at a neutralized pH in the presence of bile cell-free supernatant at 1:1dilution inhibited the growth of all target pathogens. Fig. 14 AMA of L. plantarum cell free supernatant against 4 enteropathogens (a) grown in presence of 5% (w/v) porcine bile and (b) grown without bile in the growth medium AMA of bile stressed cell-free supernatant of L.paracasei against 4 pathogens are represented in Figure 15. Acid supernatants of L. paracasei at 1:1 dilution showed slight or moderate inhibition of the growth of enteropathogens meanwhile inhibition of pathogens was much more inhibited in the presence of bile. It was observed that among the strains Gram-negative pathogens are able to survive bile conditions at neutral pH, whereas Gram-positive bacteria (S. aureus and B. cereus) were significantly suppressed with the neutralized supernatants of bile at 1:1 dilutions. 34 Fig. 15 AMA of L. paracasei cell free supernatants against 4 enteropathogens (a) grown in presence of 5% (w/v) porcine bile and (b) grown without bile in the growth medium The inhibition of growth of four target pathogens in presence of B.breve cell-free supernatant with 5% bile was also determined. Data obatined and sumarised in Figure 16. In this study it was observed that AMA of acid extract at 1:1 dilution showed strong AMA against all enteric pathogens meanwhile AMA of supernatant obtained from bile grown cells against S.typhimurium and ESBL was lost completely. It was determine that Gram-negative strains displayed good survival at pH 6 whereas larger differences were observed between Gram-positive strains such as S.aures and B.cereus where neutralized supernatants of B. breve caused maximum inhibition at 1:1 and 1:10 dilutions of S. aureus and B. cereus. Fig. 16 AMA of B. breve cell free supernatants against 4 enteropathogens (a) grown in presence of 5% (w/v) porcine bile and (b) grown without bile in the growth medium 35 Simmilar results were obtained in presence of B. lactis cell-free supernatant with 5% of bile (Fig. 17b). Results contrasted without bile showed that B.lactis cell-free supernatant at pH 4 at 1:1 dilution can completely supressed the growth of three target enteric pathogens, B. cereus was was the most resistant strain. AMA of B. lactis extract in presence of bile showed highest inhibition of B. cereus. Fig. 17 AMA of B. lactis cell free supernatants against 4 enteropathogens (a) grown in presence of 5% (w/v) porcine bile and (b) grown without bile in the growth medium. In conclusion, bile stress did not abolish the AMA of LAB supernatants meanwhile AMA against ESBL and S. typhimurium by bifidobacteria cell free supernatants was abolished when grown in presence of 5% porcine bile. However B. cereus was inhibited by supernatant of bifidobacteria strains grown in presence of 5% porcine bile. 3.5 Effect of probiotic strain combinations on AMA against E. coli, S. typhimurium, S. aureus and B. cereus AMA of cell-free culture supernatants collected from 14 different combinations of Lactic acid bacteria and bifidobacteria against E. coli and S. typhimurium S. aureus and B .cereus were examined. Acid cell free supernatant of probiotics strains combinations at 1:1 dilutions inhibited the growth of E. coli and S .typhimurium, S. aureus and B. cereus more than 80%. At higher dilutions (1:10, 1:100 &1:500), cell free supernatants did not show antimicrobial activity or it was much less on S. typhimurium, E. coli and B. cereus. Neutralized cell free supernatants of tested probiotics strain combinations did not show antimicrobial activity on the Gram-positive and Gram-negative bacteria tested except S. aureus. Generally, acid cell free supernatants of 14 probiotics strain combinations were more 36 effective in inhibiting the growth of S. aureus than S. typhimurium, B. cereus and E. coli. However B. cereus was found to be the most resistant strain Fig. 18 AMA of cell free supernatant of co-cultured probiotic strain ombination of F44+F8+88 (a) acid supernatants (b) neutralized supernatants As shown in Fig. 18a, the acid supernatants of F44+F8+88 combinations at 1:1 dilution inhibited the growth of E. coli and S. typhimurium, S. aureus and B. cereus by more than 80%. At higher dilutions (1:10, 1:100, 1:500), cell free supernatant did not show antimicrobial activity or it was very less except S. aureus which growth has been suppressed at all dilutions by more than 30%. When pH was adjusted to 6, inhibitory effect on S. typhimurium, E. coli and B. cereus pathogens was detected except S. aureus which growth was suppressed (Fig. 18b). Fig. 19 AMA of cell free supernatant of co-cultured probiotic strain combination of F44+F8+46 (a) acid supernatants (b) neutralized supernatants 37 As shown in Fig. 19a, the acid supernatants of F44+F8+46 probiotic strain combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium. S. aureus and B. cereus by more than 80%. At higher dilutions (1:10, 1:100, 1:500), cell free supernatants showed slight bactericidal effect on the growth of the E. coli and the most observable of the S. aureus, whereas no inhibitory effect of supernatant of B. cereus was noticeable . In contrast to the supernatant of the F44+F8+46, the bactericidal effect on tested pathogens was also detected on S. aureus (in all dilutions) even when the pH of the supernatants has been adjusted to pH 6.0 (Fig. 19b). Fig. 20 AMA of cell free supernatants of co-culture of probiotic strain combinationF8+46+88 (a) acid supernatants (b) neutralized As shown in Fig. 20a, the acid supernatants of F8+F88+46 strain combinations at 1:1 dilution inhibited the growth of E. coli, S typhimurium. S. aureus and B. cereus by more than 80%. At higher dilution (1:10, 1:100 & 1:500), the cell free supernatants showed slight bactericidal effect on the growth of all tested pathogens however the highest against S. aureus. In contrast when the supernatants of the F8+88+46 was adjusted to pH 6 the bactericidal effect on tested pathogens was also detected on 1:1 dilution, meanwhile on the growth of S. aureus it has been retained in all dilutions (Fig. 20b). 38 Fig. 21 AMA of cell free supernatants of co-cultured probiotic strain combination F17+F8+88 (a) acid supernatants (b) neutralized supernatants As shown in Fig. 21a, the acid cell free supernatant of F17+F8+88 strain combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium, S. aureus and B. cereus by more than79 %. At higher dilution (1:100 and 1:500), the cell free supernatants did not show (or it was very less) strong antimicrobial activity against Gram-positive and Gram-negative bacteria. It was observed that pH adjustment to 6.0 of F17+F8+88 supernatant enhanced the growth (except 1:1 unlisted S. aureus) of all tested pathogens. Fig. 22 AMA of cell free supernatants of co-cultured probiotic strain combination F17+46+88 (a) acid supernatants (b) neutralized supernatants As shown in Fig. 22a, the acid cell free supernatants of F17+46+88 probiotic strain combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium. S. aureus and B. cereus by more than 80 %. Three pathogens ( E. coli-39%, S. aureus 86% and B.cereus-35%) were significantly decreased by incubation with the supernatant of F17+46+88 at 1:10 dilution. When 39 pH was adjusted to 6 the inhibitory effect of cell free supernatants of probiotic strain combination on the 4 enteropathogens was disappeared except S. aureus (Fig. 22b). Fig. 23 AMA of cell free supernatant of F8+88 strain co-culture (a) acid supernatants (b) neutralized supernatants As shown in Fig. 23a, the acid cell free supernatants of F8+88 probiotic strain combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium, S. aureus and B. cereus by more than 80%. In this experiment it was observed that the acid supernatant of F8+88 first time suppressed growth of B. cereus in all serial dilutions and it has been also showed a quite strong bactericidal effect on other pathogens growth in spite that it was lost after adjustment of the pH to 6 (except S.aureus) (Fig. 23b). Fig. 24 AMA of cell free supernatants of 44+46 co-culture (a) acid supernatants (b) neutralized supernatants 40 As shown in Fig. 24a, the acid cell free supernatants of 44+46 probiotics strain combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium, S. aureus and B. cereus by more than 74%. In 1:100 and 1:500 dilutions the effect of non-pH adjusted supernatant did not show a bactericidal effect on the growth of tested enteropathogens. Neutralized CFS did not inhibit the pathogens (Fig. 24b). Fig. 25 AMA of cell free supernatants of F17+46 co-culture (a) acid supernatants (b) neutralized supernatants As shown in Fig. 25am, the acid cell free supernatants of F17+46 probiotic strain combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium, S. aureus and B. cereus by more than 76%. Tested strain combination supernatants at acid pH were able to inhibit the growth at original cell free extract meanwhile neutralized supernatant did not show antimicrobial activity or it was very less in all higher dilution cases (Fig. 25 b). Fig. 26 AMA of cell free supernatant of F17+88 co-cultured probiotic strain (a) acid supernatants (b) neutralized supernatants 41 As shown in Fig. 26a, the acid cell free supernatants of F17+88 probiotic strain combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium, S. aureus and B. cereus by more than 79%. At higher dilution (1:10, 1:100& 1:500), cell free supernatants showed slight bactericidal effect on the tested enteropahogens. We found that inhibitory effect of F17+88 was also disappeared after adjustment of the pH (Fig. 26b). Fig. 27 AMA of cell free supernatants of F8+46 co-cultured probiotic strains (a) acid supernatants (b) neutralized cell free supernatants As shown in Fig. 27a, acid cell free supernatants of F8+46 probiotics strain combination at 1:1 dilution inhibited the growth of E. coli, S. typhimurium. S. aureus and B. cereus by more than 77 %. At higher dilution (1:10, 1:100 & 1:500) cell free supernatants showed bactericidal effect on the growth of S. aureus. In contrast when pH value of the F8+46 supernatant was adjusted to pH 6.0, the bactericidal effect on tested pathogens was also detected on 1:1 dilution meanwhile the growth of S. aureus has been retained at all dilutions (Fig. 27b). Fig. 28 AMA of cell free supernatant of 88+618 co-culture (a) acid supernatants (b) neutralized cell free supernatants 42 As shown in Fig.28a, the acid cell free supernatants of 88+618 probiotic strain combination at 1:1 dilution inhibited the growth of E .coli, S. typhimurium. S. aureus and B. cereus by more than 80%. AMA of serially diluted supernatants from 88+618 showed antimicrobial spectrum just on S. aureus. In contrast to the supernatant of the 88+618, the bactericidal effect on tested pathogens was also detected on S. aureus growth (in all dilutions) even when the pH of the supernatants has been adjusted to pH 6.0 (Fig. 28b). Fig. 29 AMA of cell free supernatant of 46+88 co-culture (a) acid supernatants (b) neutralized supernatants As shown in Fig. 29a, the acid cell free supernatants of 46+88 probiotics strain combination at 1:1dilution inhibited the growth of E. coli, S typhimurium, S. aureus and B. cereus by more than 80 %. At higher dilution (1:10, 1:100 & 1:500), cell free supernatants did not show antimicrobial activity (or it was much less except for S. aureus growth suppressed in all serial dilution more than 35%). When pH was adjusted to 6, there was no inhibitory effect on S. typhimurium, E. coli and B. cereus pathogens was detected except S. aureus which growth was suppressed (Fig. 29b). Fig. 30 AMA of cell free supernatant of 46+618 co-culture (a) acid supernatants (b) neutralized supernatants 43 As shown in Fig. 30a, the acid cell free supernatants of 46+618 probiotic strain combination at 1:1dilution inhibited the growth of E .coli, S. typhimurium, S. aureus and B. cereus by more than 80 %. S. aureus (77% ) were significantly decreased by incubation with the supernatants of 46+618 at 1:10 dilution. When pH was adjusted to 6.0, the inhibitory effect of probiotic strain combination on S. aureus did not decrease significantly (Fig. 30b). 3.6 AMA of partially purified bacteriocins from LAB and Bifidobacteria strains against enteric pathogens Ammonium sulphate precipitated crude proteins from two of the 4 candidate probiotic strains showed strongest antimicrobial activity against target pathogens (Fig. 31 & 32). Partially purified proteins from L. plantarum and B. lactis at 1:10, 1:100 dilution showed maximum AMA of 92 % against E. coli, S. typhimurium, S. aureus and B. cereus. Fig. 31 AMA of partially purified proteins from L. plantarum against 4 enteric pathogens Fig. 32 AMA of partially purified proteins from L. plantarum against 4 enteric pathogens 44 CONCLUSIONS 1. In this study it was observed that AMA of a collection of candidate probiotic strains (different time course) against 4 enteric pathogens showed inhibition of all 4 target enteric pathogens was highest with acid supernatants. When the supernatants were neutralized to pH 6.0 there was a decrease suggesting the possible synergistic action of secreted acids and other proteinecious or non-proteinecious metabolites by the probiotic strains inhibited the growth of pathogens. 2. Probiotics grown with different prebiotics tested in a microtiter plate assay showed inhibition of each of the pathogenic strains, S. typhimurium, ESBL, S. aureus and B. cereus. The extent of inhibition was dependent on the probiotic strain and on the prebiotic source used. 3. AMA of top probiotics strains cell free supernatant in presence of 5% porcine bile was also determined since it has been shown that bile stress enhanced AMA of some probiotic strains. In the present investigation it was observed that bile stress did not abolish the AMA of LAB supernatants meanwhile AMA against Gram negative pathogens (ESBL and S. typhimurium) by bifidobacteria cell free supernatant was abolished when grown in presence of 5% porcine bile. 4. In conclusion acidic supernatants all of probiotic strain combinations at 1:1 dilution inhibited the growth of S. typhimurium ESBL, S. aureus and B. cereus by more than 80% meanwhile neutralized cell free supernatant did not show antimicrobial activity or it was much less. 5. This study has lead to the identification of 4 potential probiotic strains (2 lactobacilli and 2 bifidobacteria) that can inhibit some important Gram positive or Gram negative enteropathogenic bacteria and could be potential candidate strains for in vivo studies in animal models. 6. Ammonium sulphate precipitated crude proteins from L. plantarum and B. lactis showed highest inhibition against four target enteric pathogens at a lower dilution of 1:10 and 1:100. No AMA at a higher dilution of 1:1 could be due to the potential protein aggregation. 45 ACKNOWLEDGEMENT This Master’s thesis could not have been written without Assoc. Prof. Åsa Ljungh, Prof. Torkel Wadström and Dr. Kanthi Kiran Kondepudi, Prof. Limas Kupčinskas who encouraged and challenged me through my learning program. They never accepted less than my best efforts. Thank you. I would also like to warmly acknowledge Dr. Kanthi Kiran Kondepudi who went out of his way and invested time on me. Thank you for your help and support in guiding me. Without you, this work would have taken years of my life. I would also like to express my gratitude to Ingrid Nilsson, Padma Ambalam, Peren Karagin, Oksana Poltavska and Praveen Babu for their time and valuable feedback. 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