Download Chapter 2: The Enterobacteriaceae

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.
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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.
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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.
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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.
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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.
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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 (20l) 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.
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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.
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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
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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
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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
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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.
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