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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae Author: Mnr J.J. Gouws Licensed under a Creative Commons Attribution license. TABLE OF CONTENTS INTRODUCTION ...........................................................................................................................................2 Pathogenicity ............................................................................................................................................2 Isolation ....................................................................................................................................................2 Enrichment broths and selective media for Salmonella .................................................................3 Isolation procedures for Yersinia species ......................................................................................3 Identification ...................................................................................................................................4 Table 2.1: Tests that can give a presumptive identification of the opportunistic enterobacteria ...5 Table 2.2: Reactions of members of the Enterobacteriaceae in triple sugar iron agar (TSI ) .......5 Table 2.3: Biochemical reactions of some clinically significant members of the Enterobacteriaceae ........................................................................................................................8 ESCHERICHIA COLI SEROTYPING .........................................................................................................10 SALMONELLA SEROLOGY ......................................................................................................................11 Table 2.4: Division of Salmonella in species and subspecies .....................................................12 Table 2.5: Differential characteristics of Salmonella species and subspecies1 ..........................12 Table 2.6: Complete names of a number of serovars ..................................................................13 Table 2.7: Biochemical identification of Salmonella serotypes ....................................................13 SEROLOGICAL IDENTIFICATION ............................................................................................................13 APPENDIX 1.1 ............................................................................................................................................16 APPENDIX 1.2 ............................................................................................................................................16 REFERENCES ............................................................................................................................................21 1|Page INTRODUCTION Most members of the family Enterobacteriaceae are typical intestinal parasites of humans and animals, though some species may occur in other parts of the body, on plants and in soil or water. They share the following characteristics: Gram-negative, facultative anaerobic, non-sporing rods, all ferment glucose with the formation of acid or acid and gas, oxidase-negative, catalase-positive, and many are motile by means of peritrichous flagella. Most species reduce nitrate and are able to grow on non-enriched media such as nutrient agar. There are however, a few exceptions such as Shigella dysenteriae which is catalase-negative and Klebsiella spp., Shigella spp., Yersinia pestis, Salmonella gallinarum and S. pullorum which are non-motile. Pathogenicity The Enterobacteriaceae can be divided into three groups based on their pathogenicity for animals: 1. Major pathogens of animals such as Salmonella species, Escherichia coli and three of the Yersinia species (Table 2.4). 2. Opportunistic pathogens that are known to occasionally cause infections in animals. These include species within the genera Klebsiella, Enterobacter, Proteus, Serratia, Edwardsiella, Citrobacter, Morganella and Shigella. 3. Uncertain significance for animals. These include species from 17 genera of the Enterobacteriaceae. It must be taken into account that some of them may be isolated from clinical material. Isolation All enterobacteria will grow on blood and MacConkey agars and these are used routinely for their isolation in diagnostic laboratories. Brilliant green and xylose-lysine-deoxycholate (XLD) agars are more selective, and although these media will support the growth of some other enterobacteria, they are more often used for the isolation of Salmonella . Colonies of the various enterobacteria on blood agar are usually not sufficiently distinctive to enable their identification, except for the propensity of Proteus vulgaris and P. mirabilis to swarm; Serratia marcescens and S. rubidae to produce an orange-red pigment, although this does not often occur at 37°C; Enterobacter sakazakii is known to produce a yellow pigment; and Klebsiella spp., Enterobacter spp., and some strains of Escherichia coli produce mucoid colonies. MacConkey agar is a differential medium and although it is also a selective medium, this is not the case for enterobacteria as all species tolerate bile salts as well as crystal violet and grow on this medium. Lactose fermenting species produce acidic metabolic products and the medium and colonies are pink. If the organism is unable to utilize the lactose, then it attacks the peptone in the medium with resulting alkaline metabolic products and the medium and colonies are pale straw-coloured (nonlactose-fermenter). Lactose fermenting species are also known as coliforms. Brilliant green agar incorporates lactose, sucrose, phenol red as pH indicator (red at pH 8,2 and yellow at pH 6,4) and brilliant green dye as an inhibitor that to some extent inhibits the growth of most enterobacteria, except Salmonella spp. Reactions are similar to those occurring on MacConkey agar 2|Page except that the bacteria may ferment one or both of the sugars with an acid reaction (yellowish-green colonies) or be unable to ferment either sugar and instead attack the peptone, with an alkaline reaction (red colonies and medium). XLD medium incorporates the fermentable sugars lactose, sucrose and xylose; lysine and chemicals for detecting H2S production; phenol red as pH indicator and bile salts (sodium deoxycolate) as inhibitor. Salmonella will first ferment the xylose creating a temporary acid reaction but this is reversed by the subsequent decarboxylation of lysine with alkaline metabolic products. Superimposed on the red (alkaline) colonies is the production of H2S, so most salmonellas have red colonies with a black centre. Edwardsiella tarda also gives this reaction although the H2S production is less marked and the periphery of the colonies tends to be a yellowish-red colour. The large amount of acid produced by enterobacteria that can ferment either lactose or sucrose, or both, prevents the reversion to alkaline conditions even if the bacterium is able to decarboxylate the lysine. Enrichment broths and selective media for Salmonella There are many enrichment broths for the isolation of salmonellas, but some however, can be toxic for some serovars. Strains of Salmonella Typhisuis, S. Choleraesuis, S. Pullorum and S. Gallinarum are inhibited by selenite and tetrathionate broths. Rappaport enrichment broth supports the growth of these serovars. The efficiency of selective enrichment broths is influenced by the type of specimen, the proportion of inoculum to broth, and the length and temperature of incubation used. The effectiveness of selenite and tetrathionate is considerably reduced by the addition of certain products such as egg albumen. Food products containing in excess of 1% dextrose should be diluted so that the final concentration of dextrose in the broth is less than 1%. Excessive amounts of inoculum inhibit selectivity of the enrichment broth. Faecal samples should be added in 5 - 10% quantities (1:9 ratio), and food products should not exceed 15% in the broth. All enrichment broths are incubated at 37°C. Subcultures from enrichment broths onto selective media can be made after 16 - 24 hours of incubation. It has been found that one subculture is sufficient to isolate the great majority of salmonellae. XLD agar, Brilliant green agar, MacConkey agar, Hektoen enteric agar, and Salmonella-Shigella (SS) agar are among the most generally used selective media. Brilliant green agar can be inhibitory to Salmonella Pullorum, S. Gallinarum, S. Typhi, S. Choleraesuis and S. Typhisuis. Modified brilliant green agar (Oxoid) was found to support growth of S. Typhisuis. S. Choleraesuis strains will grow on MacConkey and XLD agars, while some strains of S. Pullorum are inhibited by all the common selective media except for MacConkey agar. Isolation procedures for Yersinia species Yersinia enterocolitica is frequently recovered from animal faeces. Organisms of this genus can be cultivated on nutrient, tryptose, trypticase and blood agar. They also grow on selective and enrichment media such as MacConkey, SS, and desoxycholate agar, and also in selenite and Rappaport broth. 3|Page Isolation methods are the same for both Y. enterocolitica and Y. pseudotuberculosis. Although direct cultures are used as they are faster, a more sensitive technique is cold enrichment, which is similar to that used for Listeria. It is done as follows: Place faeces, approximately 5% by volume, in 0,67M phosphate buffered saline and hold at 4°C for 3 weeks. Inoculate MacConkey and SS agars after 7, 14 and 21 days of enrichment. For both direct culture and after enrichment, plates should be incubated at 22 - 25°C and at 37°C for not less than 48 hours. Although the bacteria will grow faster at 37°C, growth is better at lower temperatures. Longer incubation periods may be required for enrichment media targeting the recovery of Y. enterocolitica. Specimens for isolation of Y. pestis include oedematous tissue, lymph nodes, nasopharyngeal swabs, transtracheal aspirates and cerebrospinal fluid. Y. pestis grows poorly on agars containing desoxycholate, whereas Y. enterocolitica and Y. pseudotuberculosis grows well on these media. Great care must be exercised if a live or dead animal is presented with suspected Y. pestis infection. The public health authorities should be notified immediately. The animal, whether alive or dead, should be treated promptly to kill any fleas. It is advisable to wear a gown, mask and gloves when handling the animal. All bacteriological culture work should be carried out in a biosafety cabinet. Identification Reactions on MacConkey agar indicate whether the bacterium ferments the lactose in the medium or not. The colonies of most of the members of the Enterobacteriaceae are similar on blood agar. They are usually relatively large, 2 - 3 mm after 24 hours incubation, usually non-haemolytic except for some strains of E. coli especially pathogenic porcine and canine strains, shiny, round and greyish. However, a few enterobacteria have distinctive colonial characteristics. Most Proteus mirabilis and P. vulgaris will swarm on blood agar. Normally the bile salts in MacConkey agar prevent the swarming of Proteus species. On blood agar, particularly, the powerful and foul odour of Proteus spp. will be noticed and the bacteria tend to turn blood agar a chocolate-brown colour. Most P. vulgaris and P. mirabilis strains produce H2S in triple sugar iron (TSI) and XLD media. As they are also lactose-negative, they can give a reaction similar to most of the salmonellae in TSI. However, Proteus spp. are almost always lysine-decarboxylase negative and urease positive. Similarly, on XLD medium some strains of Proteus can mimic Salmonella colonies by having a black centre but the periphery of the colony tends to have a yellowish tinge. Klebsiella pneumoniae and Enterobacter aerogenes have very mucoid colonies on primary isolation indicative of the presence of a large capsule around individual cells. Both are lactose fermenters but the colonies are pale pink on MacConkey agar. The rare strains of E.coli that are mucoid are usually a more vivid pink. Both Serratia marcescens and S. rubidae produce a red pigment called prodigliosin. A number of the enterobacteria produce a yellow pigment as demonstrated by Enterobacter agglomerans and E.sakazakii. 4|Page Serratia rubidae Enterobacter cloacae Citrobacter diversus Proteus mirabilis Proteus vulgaris Serratia marcescens Edwardsiella tarda Morganella morganii Shigella spp Yersinia enterocolitica Salmonella spp. Yersinia pestis TEST Lactose McConkey Swarm. ( BA ) Mucoid colony Red pigment Spot indole Citrate Urease H2S ( TSI ) Motility Xylose Sucrose Lysine decarboxyla se + + + + v - - - - - - - - - - - - - - - - + + - - - - - - - - + + v - - - - - - - - - - - - - - - - + - - - - + - - - - - - - - - + - + + - + - + + v v - - - + - + + - + - + v + v v + v + + v - + - v + - - + - - - - - - + + - + - - - + - - + + + + + + + v v + + + + + + + v + + v + + + + + + - + - - v + + + - + - + - + + (+) - + - - - + + - - - + - - + = positive - = negative v = variable This combination of tests, plus a TSI agar slope, while actually designed for the presumptive identification of Salmonella, can also be useful for identification of other enterobacteriaceae (Table 2.3). Table 2.2: Reactions of members of the Enterobacteriaceae in triple sugar iron agar (TSI ) Alkaline slant (red) Salmonella (most) Acid butt (yellow ) Edwardsiella tarda H2S (black) Citrobacter freundii (some) R/Y/H2S+ Proteus mirabilis (most) Alkaline slant (red) Salmonella Choleraesuis Acid butt (yellow) Hafnia alvei No H2S Yersinia ruckeri (some) R/Y/H2S- Salmonella Typhisuis (some) 5|Page Yersinia pseudotuberculosis Escherichia coli + Enterobacterer aerogenes Klebsiella pneumoniae Table 2.1: Tests that can give a presumptive identification of the opportunistic enterobacteria Yersinia pestis Y. pseudotuberculosis Y. ruckeri Morganella morganii Shigella spp. Providencia spp. (some) Citrobacter spp. (some) Acid slant (yellow) Salmonella Arizonae (some) Acid butt (yellow) Proteus vulgaris (most) H2S (black) P. mirabilis (some) Y/Y/H2S+ Citrobacter freundii (some) Acid slant (yellow) Edwardsiella spp. (most) Acid butt (yellow) E. coli No H2S Klebsiella pneumoniae Y/Y/H2S- Klebsiella spp. (most) Enterobacter aerogenes E. gergoviae Serratia spp. (most) Kluyvera spp. Enterobacter spp. (most) Yersinia enterocolitica Citrobacter spp. (most) Klebsiella spp. (some) Providencia spp. (some) Serratia spp. (some) 6|Page Cedecea spp. Tatumella spp. R = red (alkaline); Y = yellow (acid); H2S+ = hydrogen sulphide produced; H2S- = hydrogen sulphide not produced. The general interpretation of the reactions in TSI medium is as follows: Alkaline (red) slant and acid (yellow) butt: glucose fermentation only. Acid (yellow) slant and acid (yellow) butt: lactose and/or sucrose attacked as well as glucose. Blackening of the medium: hydrogen sulphide production. If the given reactions are equivocal and the isolate cannot be identified, further biochemical tests should be carried out (Table 2.2) or an identification system such as API 20E should be used. 7|Page Indole Methyl Red Voges-Proskauer Citrate Urease Phenylalanine deaminase H2S Lysine decarboxylase Ornithine decarboxylase Motility (36°C) Gelatin liquefaction Growth in KCN broth ONPG Acid from: Dulcitol Inositol Lactose Maltose Mannitol Mannose Rhamnose Sorbitol Sucrose Xylose Red pigment Swarming (BA) Mucoid colonies (MAC) 8|Page Yersinia pseudotuberculosis Yersinia pestis Yersinia enterocolitica Shigella species Serratia rubidaea Serratia marcescens Salmonella II Salmonella I (arizonae) Providencia species Proteus vulgaris Proteus mirabilis Morganella morganii Klebsiella pneumoniae Hafnia alvei Escherichia coli Enterobacter cloacae Enterbacter aerogenes Test Edwardsiella tarda Citrobacter diversus Table 2.3: Biochemical reactions of some clinically significant members of the Enterobacteriaceae + + + (+) + + + + + + + + + - + + + + + + + + + D + + + + + + (+) D (+) + D (+) + + (+) + + (-) + + + + + + + + + + + + + - + (-) D + + + + + + + - + + (-) + + + + + + - + + + V + + + - + + + + + + - + + + + + + + (-) + + (-) + + + + + + (-) + + D (+) + (-) + V + V V D + (+) + + (+) (+) + + D D d + + + + + (-) + - + + - + + + + + + + + + + (-) (-) + + + + + + + + - D + + + + (+) + D + (-) + + + + + - D + + + + + + + + + + + - (-) + + - + + + + - V (-) V + V V + - + D + + + + + + - + D + + + + + + - (+) + + + + + + - (-) + + + + + + + - V V + V V - D DD + + + + D - (+) + + + - + + + + + - Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae Spot Indole Test Principle The indole end product of the action of tryptophanase on tryptophan can be detected by its ability to combine with certain aldehydes to form a coloured compound. The pink compound formed by indole and cinnamaldehyde is visualized by rubbing bacteria that produce tryptophanase on filter paper impregnated with the substrate. Method 1. Prepare indole reagent (1% para-dimethyl-aminocinnamaldehyde, available from Sigma Chemical Co and other chemical suppliers, dissolved in 10 % [vol/vol] hydrochloric acid). Store in a dark bottle in the refrigerator. NB Kovacs and not James reagent should be used. 2. Saturate a qualitative filter paper (Whatman No. 1 is fine) in a Petri dish or on a slide with the reagent. 3. Using a wooden stick or loop, rub a portion of the colony on the filter paper. Rapid development of a pink colour indicates a positive test. Most indole positive organisms will turn pink within 30 seconds. Quality control Test a fresh subculture of E. coli ATCC25922 and Enterobacter cloacae ATCC 23355. Expected results Positive organisms such as E. coli will display a pink colour on the filter paper; negative organisms such as E. cloacae will remain colourless. Performance schedule Perform when a new lot number of reagent is received and each day that tests are performed. 9|Page Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae ESCHERICHIA COLI SEROTYPING Author: Dr. Marijke Henton As E. coli is a very common contaminant, it is often isolated in veterinary laboratories. There are various ways of establishing whether an isolate is significant or not, and these are: A. Toxin production Toxins produced by E. coli include enterotoxigenic heat labile (LT),and heat stable toxins (ST) and Verotoxin (VT) or Shiga toxin-producing E. Coli (STEC). These are tested in infant mice, ligated bowel loops, on tissue culture, ELISA tests, immunodiffusion or PCR assays. B. Attaching and effacing E. coli (AEEC) and enteroaggregative E. coli (EAggEC). These are tested using cell culture techniques and PCR. C. Serotyping E. coli strains produce O or somatic antigens, K or capsular antigens if they are encapsulated, and H or flagellar antigens if they are mobile. There are more than 150 O antigens, more than 100 K antigens, and more than 70 H antigens. . Selection for serotyping is based on the following criteria: Selection of potentially typeable/pathogenic E. coli E. coli is very commonly isolated and may be significant or just a contaminant. The challenge for the laboratory is to determine whether an isolate is normal flora or an agent of disease. To help you decide: 1. Freshness of samples – E. coli multiplies in carcasses after death and spreads from the intestine to the organs closest to the gut. If an animal is moribund for a long time, E. coli can leave the intestine about 4 hours before death and already be circulating to all the organs via the blood. Other bacteria from the intestines are also spread in this way, and mixed cultures are seen if this 2. has occurred. The same thing is seen if the intestine is damaged by e.g. viruses, arsenic, etc. as the intestinal 3. barrier cannot prevent bacteria from getting into the bloodstream. Purity of the culture – a pure growth is more significant than a mixed one. The different E. coli strains may also look different and so it is important to see whether the colonies all look the same or whether there are many different shapes and sizes of E. coli on one plate which would indicate a mixed growth of different E. coli strains. 4. Site of isolation – E. coli lives in the intestinal tract normally, and also in small numbers on mucous membranes. Young animals normally have more E. coli in their intestines than older ones. Pure cultures of E. coli from organs such as the liver or heart, which are usually sterile, are more significant than from the intestine. 10 | P a g e Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae 5. Correlation with disease – E. coli strains causing enteritis are isolated from the intestine and mesenteric lymph nodes, and the other organs remain sterile; E. coli strains causing septicaemia are isolated from all organs; E. coli strains acting as opportunists causing e.g. mastitis, cystitis, etc. would only be isolated from that organ. The veterinarian must give this type of information to the laboratory if he wants a meaningful result. 6. Roughness of the colonies – smooth colonies can be typed to see whether they belong to pathogenic strains or not. Rough colonies are not typable because they cross-agglutinate and are usually non-pathogenic. It is difficult to see whether colonies are rough or smooth on blood agar or if the culture is only 1 day old. MacConkey agar 2-3 days old is best for assessing roughness. Smooth colonies are round, shiny and domed and rough colonies are irregular, have a pitted surface and are flat. 7. If a culture is made up of a mixture of rough and smooth colonies and there are more rough colonies than smooth colonies, they are less significant than if there are more or mostly smooth 8. colonies. Haemolysis – pathogenic E. coli from pigs are usually haemolytic, but not from other animals. If a haemolytic suspected E. coli is isolated from an animal which is not a pig, make sure that it isn’t Citrobacter, as the two are easily confused. 9. E. coli that act as opportunists and are isolated from unusual sites such as from the udder causing mastitis, may be rough or smooth, and would be acting as opportunists and not as primary pathogens, and so the selection criteria as above do not apply. 10. It is then only important whether E. coli is isolated in heavy pure or almost pure growth. 11. Capsule – E. coli strains that have large capsules usually grow as mucoid pink colonies on MacConkey agar. These often become yellow on aging. 12. Small dark pink E. coli colonies on MacConkey often indicate that the strain has no capsule. 13. If a bacteriophage infects E. coli colonies, they become wrinkled and appear dry after 2-3 days. SALMONELLA SEROLOGY Author: Dr. Martie van der Walt Introduction The genus Salmonella consists of more than 2 500 different serological types, called serovars or serotypes. These different serovars can be grouped under 2 species, viz. S. enterica and S. bongori. Salmonella enterica has been divided into 6 subspecies, based on biochemical differences (Tables 2.5 and 2.6). The majority of salmonellae of veterinary importance belong to Salmonella enterica subspecies enterica. Serotypes are designated S. enterica subspecies enterica serotype Typhimurium. In general, the shortened version is written, namely Salmonella Typhimurium. 11 | P a g e Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae Table 2.4: Division of Salmonella in species and subspecies Species enterica Subspecies enterica salamae arizonae diarizonae houtenae indica Older names Salmonella I II IIIa; Arizona IIIb; Arizona IV V VI bongori Table 2.5: Differential characteristics of Salmonella species and subspecies1 Species Subspecies Dulcitol ONPG Malonate Gelatinase Sorbitol Growth on KCN L(+)-tartrate (a) Galacturonate -glutamyltransferase -glucuronidase (ONPG) Mucate Salicin Lactose Lyses by phage O1 Inositol (a) enterica I# salamae II + + + +(*) d + + + + + + + + + d + + - S. enterica arizonae diarizonae IIIa IIIb + + + + + -(75%) - + + + + + + + -(70%) +(75%) + - S. bongori houtenae IV indica V + + + + + + - d d + + + d + d + - + + + + + + + d - = d-tartrate (*) = Typhimurium d, Dublin -, + = 90% or more positive reactors - = 90% or more negative reactors d =different reactions given by different serovars # = warm blooded animals, the rest cold blooded animals and environment (1) L. Le Minor, M. Véron, M Popoff. Ann. Microbiol. (Inst. Pasteur), 1982, 133, 223-243 & 245-254. (2) L. Le Minor, M. Y. Popoff, B Laurent, D Hermant., Ann. Inst. Pasteur/Microbiol., 1986, 137 B, 211-217 (3) M.W. Reeves, G.M. Evins, A.A. Heiba, B.D. Plikaytis, J.J. Farmer. Journal of Clinical Microbiology, 1989, 27: 313-320 The names by which salmonellas are generally known, e.g., Salmonella Typhimurium, Dublin, or Gallinarum, do not have species status and are therefore not italicized. These names have taxonomically the same value as the antigenic formula of a serovar. See Table 2.7 for the complete name of a serovar. For practical everyday use, the name of S. Typhimurium is still used, but not underlined. 12 | P a g e Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae Table 2.6: Complete names of a number of serovars Serovar Complete name S. enterica ssp. enterica serovar Typhimurium S. enterica ssp. enterica serovar Dublin Typhimurium Dublin Antigenic formula 1,4,5, 12; i; 1,2 1,9,12; gp; - - + + V (58%) + - + + + - + + - (+) - (+) - Salmonella (most serotypes) S. Gallinarum S. Pullorum S. Choleraesuis S. Tphisuis TEST Glucose (gas) Lysine decarboxylase Ornithine decarboxylase Citrate (Simmons) H2S Dulcitol Inositol Maltose Rhamnose Sorbitol Trehalose S. Choleraesuis biotype Kunzendorf Table 2.7: Biochemical identification of Salmonella serotypes (+) - + - - + + + - + + + + + + V + + + + SEROLOGICAL IDENTIFICATION The Kauffmann-White Scheme Only serovars belonging to subspecies I have names, while those belonging to the other subspecies are only known by the subspecies number and its antigenic formula, e.g. S. II 6, 7; Z4 Z24; z42 and S. IIIb; iv; z. Each Salmonella serovar that exists is identified according to the antigens it possesses and classified into a subspecies based on its biochemical reactions. All the Salmonella serovars are contained in a scheme, the Kauffmann-White Scheme which is based on the antigenic formulae of all the serovars. As new Salmonella serovars constantly emerge, based on the identification of new antigens, or mixing of existing antigens, the Kauffmann-White Scheme is not a fixed document but is updated every 5 years by the WHO Collaborating Centre for Reference and Research on Salmonella. (Institute. Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, FRANCE). In the Kauffmann-White Scheme the serovars are grouped together based on the O-antigens (somatic antigens, or LPS antigens), and further divided based on H-antigens (flagellar antigens). The O-antigens have numerical values, from 1-67. Some O-antigens occur on their own, i.e. 0, 11, while others are in groups, i.e. 1, 4, 5, 12. 13 | P a g e Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae In some rare instances, Salmonella can lose its O-antigens, and such a strain is then called “rough”. When it has lost its O-antigens, it is very difficult to determine to what serovar it belongs, as the Oantigen is the primary antigen used for serovar identification. However, when a strain has lost its Oantigen, it has also lost a very important virulence factor, and such an isolate is avirulent, and therefore not of clinical significance. If an organism has turned rough, it agglutinates in saline (autoagglutination). All the antisera used for the serotyping of Salmonella are diluted in saline. Therefore rough Salmonella will agglutinate in most diluted typing sera as well, and can be misdiagnosed as possessing that O-antigen, especially if only a few sera are tested. All salmonellae are pre-tested for auto-agglutination in a saline solution. The H-antigens are known by alphabetical numbers a to z, and by z1 – z81, by numerical from 1 – 7. Most serovars possess 2 types of H-antigens, and these are known as the 2 phases of the H-antigens. To be able to optimally determine the presence of the flagellar antigens, the organisms are grown on a semi-solid medium, called “Swarm agar”. This agar medium optimally stimulates the formation of Hantigens. Therefore, for the complete identification of a Salmonella isolate, the following has to be determined: 1. To which biochemical subspecies it belongs (serovars of a different subspecies may have the same serological formulae); i.e. S. Limete 4, 12; b; 1, 5 and S II 4, 12; b; 1, 5 2. Determination of rough characteristic 3. 4. The O-antigens have to be determined. Both phases of the H-antigens. Method of Serotyping Subspecies identification Biochemical subdivision is done based on the tests in Table 2.9. For the determination of the rough characteristic as well as the O-antigens, the organisms are grown for 18 hours at 37°C on blood agar or nutrient agar plates. Selective media e.g. MacConkey should not be used, as it may inhibit the formation of antigens. A small amount of growth is mixed with acriflavine/saline (20l) on a glass plate. It is mixed by gentle rotation for 2 minutes, and the agglutination is observed against a black background. The presence of agglutination is indicative of roughness. O-antigen typing Most laboratories use commercially produced sera. These antisera are available as monovalent sera, i.e. specific for only antigen O, 4. Although O, 4 is always associated with 1, 4, 5, 12 not all the antigens in an antigenic formula are determined, only the key antigens. For examples for S. Typhimurium 1, 4, 5, 12; i; 1, 2; only O-antigen 4, and sometimes O, 12 is determined. Only i and 2 Hantigens are determined. 14 | P a g e Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae For ease of use, the monovalent typing antisera are grouped together in polyvalent groups, e.g. OA, OB, etc., or HA, HB etc. When an isolate is examined, it is first tested with the polyvalent antisera to determine under which polyvalent group its antigens are grouped. Refer to Appendix 1.2. H-antigen typing The organism is grown for 18 hours at 37°C on swarm agar, and the presence of the H-antigens are determined by the same procedure as for the O-antigens, but using H-polyvalent and monovalent sera. (Refer to Appendix 1.2). After completion of all these steps it should be possible to completely identify a Salmonella isolate. (Refer to Appendix 1.1). The level of identification of a Salmonella isolate by a smaller laboratory will be determined by the amount of information required by the sender of the sample. It is impractical for a small laboratory to keep the wide range of typing sera or biochemical tests. Therefore, most laboratories only isolate Salmonella species and send the culture for complete serotyping to a reference laboratory. Otherwise, if a laboratory wishes to be able to identify only a small number of clinically important serovars, it may wish to acquire only those antisera. However, the chance of misdiagnosis is great if only a limited number of sera are used. Identification of salmonellae of clinical importance For veterinary medicine, a number of serovars which have atypical biochemical reactions are important. They are often adapted to a specific host. They are often misidentified as they do not appear biochemically like true Salmonella. 15 | P a g e Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae APPENDIX 1.1 ANTI-SALMONELLA AGGLUTINATING SERA Sera for O-antigen identification Polyvalent O-sera Polyvalent group OA OB OC OD OE OF OG Contains agglutinins for K-W Groups A, B, D, E, L C, F, G, H I, J, K, M, N, O, P Q, R, S, T, U, V, W X, Y, Z, 51 - 53 54, 55, 56, 57, 58, 59 60, 61, 62, 63, 65, 66, 67 Corresponding O-somatic antigen 1, 2, 4, 5, 9, 12, 46, 27, 3, 10, 15, 19, 21 6, 7, 8, 14, 11, 13, 22, 23, 24, 25, 8, 20 16, 17, 18, 28, 30, 35, 38 39, 40, 41, 42, 43, 44, 45 47, 48, 50, 51, 52, 53 Monovalent O-sera The following monovalent sera are available: 1, 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12, 13, 14, 19 – 25, 28, 30, 35, 38 – 67 Sera for H-antigen identification Polyvalent H-antisera Polyvalent sera HA Corresponding flagellar antigens a, b, c, d, I, z10, z29 e, h, n, x, z15, g, m, p, q, r, s, t, u k, y, z, l, z4, z23, z13, z24, z28, z32, r, v, w z35, z36, z38, z39, z41, z42, z44, z60 1, 2, 3, 6, 7 Z52, z53, z54, z55, z57, z61 HB HC HD HE H III (H factors for IIIa and IIIb) Monovalent H-antisera The following monovalent antisera are available: A, b, c, d, g, m, p, q, s, t, u, h, I, k, r, v, w, x, y, z, z10, z15, z29, 2, 5, 6, 7 APPENDIX 1.2 KAUFFMANN-WHITE SCHEME: The antigenic formulae of the Salmonella serovars Type Group O:2 (A) Paratyphi A Nitra 16 | P a g e Somatic (O) antigen 1, 2, 12 2, 12 Flagellar (H) antigen Phase 1 Phase 2 a g, m [1, 5] - Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae Kiel Koessen Group O:4 Kisangani Hessarek1 Fulica1 Arechavaleta Bispebjerg Tinda II Huettwillen Nakuru II Paratyphi B2(a,b) Limete II Derby Agona2(b) II Essen Hato II Group O:4 II II California Kingston1 Budapest Travis Tennyson II Banana Madras Typhimuriium Lagos Agama Farsta Tsevie Gloucester Tumodi II Massenya Neumuenster II Ljubljana Texas Fyris Azteca Clackamas Bredeney2 Kimuenza 1. 1, 2, 12 2, 12 g,p l, v 1, 5 1, 4, [5], 12 4, 12, 27 4, [5], 12 4, [5], 12 1, 4, [5], 12 1, 4, 12, 27 1, 4, [5], 12, 27 1, 4, 12 1, 4, 12, 27 1, 4, 12, 27 1, 4, [5], 12 1, 4, 12, 27 4, 12 1, 4, [5], 12 1, 4, 12 1, 4, [5], 12 4, 12 4, [5], 12 1, 4, 12, 27 a a a a a a a a a a b b b f, g f, g, s f, g, t g, m g, m, s g, [m], [s], t 1, 2 1, 5 1, 7 e, n, x e, n,z15 e, n, x l, w z6 z39 1, 2 1, 5 1, 5 [1, 2] [1, 2] z6, z42 e, n, x 1, 4, 12, 27 4, 12 4, 12 1, 4, [5], 12, 27 1, 4, 12, 27 4, [5], 12 4, 5, 12 4, 12 4, [5], 12 4, [5], 12 1, 4, [5], 12 1, 4, [5], 12 4, 12 4, 12 4, 12 1, 4, 12, 27 1, 4, 12 4, 12, 27 1, 4, 12, 27 1, 4, 12, 27 1, 4, 12, 27 4, 12, 27 4, [5], 12 4, [5], 12 4, [5], 12, 27 4, 12 1, 4, 12, 27 1, 4, 12, 27 g, [m], t g, m, t g, m, t g, s, t g, t g, z51 g, z51 g, z62 m, t m, t i i i i i i i i k k k k k l, v l, v l, v l, v l, v [1, 5] z39 [1, 2] 1, 7 e, n, z15 1, 5 e, n, z15 1, 2 1, 5 1, 6 e, n, x e, n, z15 l, w z6 z35 1, 5 1, 6 1, 6 e, n, x e, n, z15 1, 2 1, 5 1, 6 1, 7 e, n, x Serovar Hessarek and Fulica with same global antigenic formula are not combined (as Miami/Senda) because their biochemical characters are very different: rhamnose, gaz/glucose, dulcitol, trehalose, Simmons citrate agar, L(+) tartrate (=d-tartrate), mucate, H2S, tetrathionate-reductase: + for Hessarek, - for Fulica. This last serovar is very rare. 2 (a) Variety L(+) tartrate (=d-tartrate) positive is often called variety Java. 2 (b) May possess a R-phase H antigen 1 2 May possess a R-phase H antigen: z43 May possess a R-phase antigen: z40 17 | P a g e Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae Type Flagellar (H) antigen Somatic (O) antigen Phase 2 Group O:7 (C1) Strains of this group may be lysogenized by phage 14 (O: 6, 7, to O: 6, 7, 14). The strains possessing O: 6,7,14 had been classified in a special group, C4. They are now classified into group C1. Names formerly given to serovars of group C4 are deleted. Sanjuan 6, 7 a 1, 5 II 6, 7, 14 a 1, 5 Umhali 6, 7 a 1, 6 Austin 6, 7 a 1, 7 Oslo 6, 7, 14 a e, n, x Denver 6, 7 a e, n, z15 Coleypark 6, 7, 14 a l, w Damman 6, 7 a z6 II 6, 7 a z6 II 6, 7 a z42 Brazzaville 6, 7 b 1, 2 Edinburgh 6, 7, 14 b 1, 5 Adime 6, 7 b 1, 6 Koumra 6, 7 b 1, 7 Lockleaze 6, 7, 14 b e, n, x Georgia 6, 7 b e, n, z15 II 6, 7 b [e, n, x]: z42 Ohio1 6, 7, 14 b l, w Leopoldville 6, 7 b z6 Kotte 6, 7 b z35 II 6, 7 b z39 Hissar 6, 7, 14 c 1, 2 Paratyphi C 6, 7, [Vi] c 1, 5 Choleraesuis 6, 7 c 1, 5 Typhisuis 6, 7 c 1, 5 Birkenhead 6, 7 c 1, 6 Schwabach 6, 7 c 1, 7 Namibia 6, 7 c e, n, x Kaduna 6, 7, 14 c e, n, z15 Kisii 6, 7 Phase 1 d 1, 2 1: May possess a R-phase antigen : z59 Type Group O:7 (C1) Isangi Kivu Kambole Amersfort Gombe Livingstone Wil Nieukerk II Larochelle Lomita Norwich Nola Braenderup II Rissen Eingedi Afula Montevideo II II 18 | P a g e Somatic (O) antigen 6, 7, 14 6, 7 6, 7 6, 7, 14 6, 7, 14 6, 7, 14 6, 7 6, 7, 14 6, 7 6, 7 6, 7 6, 7 6, 7 6, 7, 14 6, 7 6, 7, 14 6, 7 6, 7 6, 7, 14 6, 7 6, 7 Flagellar (H) antigen Phase 1 d d d d d d d d d e, h e, h e, h e, h e, h e, n, x f, g f, g, t f, g, t g, m, [p], s g, m, [s], t (g), m, [s], t Phase 2 1, 5 1, 6 1, [2], 7 e, n, x e, n, z15 l, w l, z13, z28 z6 z42 1, 2 1, 5 1, 6 1, 7 e, n, z15 1, 6: z42 1, 2, 7 e, n, x [1, 2, 7] e, n, x 1, 5 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae II Othmarchen Menston II Riggil Alamo IV Haelsingborg II Oranienburg Augustenborg Oritamerin Garoli Group O:7 (C1) Phaliron Kalumburu Kuru Daula Bellevue Lesennes Breda Chailey Dabou Corvallis Albany1 Duesseldorf Tallaheassee Bazenheid Zerifin Paris Mapo Cleveland Istanbul Hadar Chomedy Glostrup Molade Wippra II II Tamale Uno II Kolda Yarm Angers Apeyeme Diogoye Aesch 6, 7 6, 7, 14 6, 7 6, 7 6, 7 6, 7 6, 7 6, 7 6, 7 6, 7, 14 6, 7, 14 6, 7 6, 7 g, [m], s, t g, m, [t] g, s, [t] g, t g, (t) g, z51 g, z51 m, p, t, [u] m, t m, t i i i [z42] [1, 6] e, n, x: z42 1, 5 [z57] 1, 2 1, 2 1, 6 8 6, 8 6, 8 8, 20 8 6, 8 6, 8 6, 8 8, 20 8, 20 8, 20 6, 8 6, 8 8, 20 6, 8 8, 20 6, 8 6, 8 8 6, 8 8, 20 6, 8 8, 20 6, 8 6, 8 8 8, 20 6, 8 6, 8 8, 20 6, 8 8, 20 8, 20 8, 20 6, 8 z z z z z4, z23 z4, z23 z4, z23 z4, z23 z4, z23 z4, z23 z4, z24 z4, z24 z4, z32 z10 z10 z10 z10 z10 z10 z10 z10 z10 z10 z10 z29 z29 z29 z29 z29 z35 z35 z35 z38 z41 z60 e, n, z15 e, n, z15 l, w z6 1, 6 1, 6 e, n, x e, n, z15 l, w [z6] 1, 2 1, 2 1, 5 1, 5 1, 7 e, n, x e, n, x e, n, z15 e, n, z15 z6 z6 1, 5 e, n, x: z42 [e, n, z15] [e, n, z15] e, n, x 1, 2 1, 2 z6 z6 1, 2 1: May possess a R-phase H antigen z45 Type Group O:7 (C1) Sendai1 Miami1 II Os Saarbruecken Lomalinda II Durban II II 19 | P a g e Flagellar (H) antigen Somatic (O) antigen 1, 9, 12 1, 9, 12 9, 12 9, 12 1, 9, 12 1, 9, 12 1, 9, 12 9, 12 9, 12 1, 9, 12 Phase 1 a a a a a a a a a a Phase 2 1, 5 1, 5 1, 5 1, 6 1, 7 e, n, x e, n, x e, n, z15 z39 z42 Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae Orarimon Frintrop II II II Goeteborg Ipeko Elokate Alabama Ridge Ndolo Tarshyne Eschberg II Bangui Zega Jaffna II Typhi2 Bournemouth Eastborne 1, 9, 12 1, 9, 12 1, 9, 12 1, 9, 12 1, 9, 12 9, 12 9, 12 9, 12 9, 12 9, 12 1, 9, 12 9, 12 9, 12 9, 12 9, 12 9, 12 1, 9, 12 9, 12 9, 12 [Vi] 9, 12 1, 9, 12 a b b b b c c c c c d d d d d d d d d e, h e, h 1, 2 1, 5 e, n, x z6 z39 1, 5 1, 6 1, 7 e, n, z15 z6 1, 5 1, 6 1, 7 e, n, x e, n, z15 z6 z35 z39 1, 2 1, 5 1: Sendai (adapted to man) is auxotroph, Miami is prototroph 2: Uncommon strains possess: a) the R-phase H:j (instead of H:d) as first phase of the H-antigen; b) Either the H:d antigen or the R-phase H:j as first phase, and the R-phase H: z66 as second phase of the H antigen. Type Group O:7 (C1) Westafrica Israel II II Berta Enteritidis1 Blegdam II II Dublin Naestved Rostock Moscow II Newmexico II Antartica II Pensacola Seremban Claibornei Goverdhan Mendoza Panama2 Kapemba Flagellar (H) antigen Somatic (O) antigen 9, 12 9, 12 9, 12 9, 12 1, 9, 12 1, 9, 12 9, 12 1, 9, 12 1, 9, 12 1, 9, 12[Vi] 1, 9, 12 1, 9, 12 9, 12 9, 12 9, 12 1, 9, 12 9, 12 9, 12 1, 9, 12 9, 12 1, 9, 12 9, 12 9, 12 1, 9, 12 9, 12 Phase 1 e, h e, h e, n, x e, n, x [f], g, t g, m g, m, q g, m, [s], t g, m, s, t g, p g, p, s g, p, u g, q g, s, t g, z51 g, z62 g, z63 m, t m, t i k k l, v l, v l, v Phase 2 1, 7 e, n, z15 1, [5], 7 1, 6 [1, 5, 7]:[ z42] e, n, x e, n, x 1, 5 e, n, x [1, 2] 1, 5 1, 5 1, 6 1, 2 1, 5 1, 7 1: In addition to H:g,m factors, some variants may possess the H:p or H:s or H:f or H:t factor. Very uncommon strains may possess the H:1,7 antigen as second phase of the H antigen. 2: May possess a R-phase antigen : z40 20 | P a g e Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 2: The Enterobacteriaceae REFERENCES 1. Carter, G.R., Cole, J.R. jr. Diagnostic Procedures in Veterinary Bacteriology and Mycology. Fifth Edition. Academic Press, 1990. ISBN 0-12-161775-0. 2. Quinn, P.J., Carter, M.E., Markey, B. and Carter, G.R. Clinical Veterinary Microbiology. Wolfe Publishers, 1994. ISBN 0 7234 1711 3. 3. Quin, P.J., Markey, B.K., Leonard, F.C., FitzPatrick, E.S., Fanning, S., Hartigan, P.J. Veterinary Microbiology and Microbial Disease. Second Edition, Wiley-Blackwell, 2011. ISBN 78-1-40515823-7. 21 | P a g e