AN

ASSIGNMENT
ON

CHARACTERISTICS FEATURES OF ENTERIC BACTERIA

Submitted To: Submitted By:

VPH-602
Dr. C. V. Savalia Alpesh P.Suthar
(Theory Assign.)
Professor & Head M.V.Sc Scholar
Dept. of Vet. Public Health & Dept. of Vet. Public Health &
Epidemiology Epidemiology

Department of Veterinary Public Health and Epidemiology

Vanbandhu College of Veterinary Science and Animal Husbandry
Navsari Agricultural University
Navsari – 396 450
 INTRODUCTION
• Enteric bacteria are bacteria in the family
Enterobacteriaceae.
• These bacteria reside normally in the guts of many
animals, including humans, and some are pathogenic,
causing disease in certain animal species.
• Many cases of food poisoning are caused by infection
with enteric bacteria, as are some more serious
conditions, such as the plague.
• One of the most famous members of the family
is Escherichia coli, a bacterium which has been studied
extensively in laboratories all over the world.
 ENTEROBACTERIACEAE

Habitat
• digestive tube(colon) of human and animals
• 150 Species
• Facultative anaerobes
• Diarrheal illnesses
• 3 million death/year
• 4 billion infectoions/ worldwide
• Classification – more than15 different genera

Escherichia
Shigella Proteus
Edwardsiella Providencia
Salmonella Morganella
Citrobacter Yersinia
Klebsiella Erwinia
Enterobacter Pectinobacterium
Hafnia
Serratia
Morphology and General Characteristics
Are facultative anaerobes
Gram-negative, non-sporing, rod shaped bacteria
If motile, motility is by peritrichous
flagella(Except Shigella & Klebsiella)
Many are normal inhabitants of the intestinal tract
of man and other animals
Some are enteric pathogens and others are urinary
or respiratory tract pathogens
Differentiation is based on biochemical reactions
and differences in antigenic structure
 Morphology and Physiology

• Short gram-negative rods.

• Facultative anaerobes.

• Grow readily and rapidly on simple media.

• Klebsiella spp. have large capsule (form large and very

mucoid colonies); those of Enterobacter have smaller
capsule; the others produce diffusible slime layers (form
circular, convex and smooth colonies).
ENTEROBACTERIACEAE PHYSIOLOGY

• Glucose is fermented with strong acid formation and

often gas
• Reduce nitrates to nitrite
• Do not liquify alginate
• Oxidase negative
• Basis for speciation within a family
- differences in carbohydrate they ferment
- variations in end-product production
- variation in substrate utilization
 ENTEROBACTERIACEAE

Opportunistic
pathogens
Escherichia coli
Klebsiella pneumoniae
Enterobacter aerogenes Obligate pathogens
Serratia marcescens Salmonella spp.
Proteus spp. Shigella spp.
Providencia spp. Yersinia spp.
Citrobacter spp. Some E. coli strains
SITES OF INFECTIONS WITH MEMBERS OF
THE ENTEROBACTERIACEAE
 CULTURAL CHARACTERISTICS

• On non differential or nonselective media

blood agar or infusion agar
- no species distinction
- appear as moist, smooth, gray colonies
• Selective media -To isolate Shigella and salmonella
from fecal matter
• Differential media – selectively inhibit gram-positive
organisms and to separate enterics in broad categories
 MODES OF INFECTION
• Contaminated food and water (Salmonella spp.,
Shigella spp., Yersinia enterocolitica, Escherichia
coli O157:H7)

• Endogenous (urinary tract infection, primary bacterial

peritonitis, abdominal abscess)

• Abnormal host colonization (nosocomial pneumonia)

• Transfer between debilitated patients

• Insect (flea) vector (unique for Yersinia pestis)

 Urinary Tract Infection
Pneumonia: Enterobacter spp., Klebsiella pneumoniae,
Escherichia coli, and Proteus mirabilis
• Wound Infection: Escherichia coli, Enterobacter spp.,
Klebsiella pneumoniae, and Proteus mirabilis
• Bacteremia: Escherichia coli, Enterobacter spp.,
Klebsiella pneumoniae, and Proteus mirabilis
Intestinal Infection
• Shigella sonnei (serogroup D)
• Salmonella serotype enteritidis
• Salmonella serotype typhimurium
• Shigella flexneri (serogroup B)
• Escherichia coli O157:H7
• Yersinia enterocolitica
 Pathogenesis and Immunity
• Common virulence factors
• Endotoxin (Lipid A of LPS)
• Capsule
• Antigenic phase variation
• Acquisition of growth factors (e.g. Fe)
• Resistance to serum killing
• Antimicrobial resistance

Type III secretion systems: possessed by some

Enterobacteriaceae pathogens, e.g., E. coli, Yersinia,
Salmonella, and Shigella; facilitate transport of
bacterial virulence factors directly into host cells.
 Escherichia coli
• Toxins: two types of enterotoxin; Shiga-type toxin;
Enteroaggregative ST-like toxin; Hemolysins;
Endotoxin

• Virulence factors that protect the bacteria from host

defenses: Capsule/Iron capturing ability
(enterochelin)
• Morphology:
• Gram negetive
• 1.1-1.5 x 2.0-5.0 m.
• Straight bacillli or coccobacilli
• Non spore-forming
• Motile by peritrichous flagella or non motile.
• Cultural characteristics:
• Colonies on nutrient agar may be smooth(S type), low
convex, shiny surface, entire edge, gray, Rype or mucoids
forms may occur.
• Colonies of E. hememanii are yellow pigmented , aerobic
facultative anaerobic
• Optimum growth temperature 37C .
• Grow readily on simple nutrient media like Nutrient agar or
nutrient broth
• For haemolysis use Trypticase Soy Agar + 5% sheep blood.
• Selective media: GEAM/ Levine-black colonies with metallic
surface, MacConkey agar- red colonies, Rambach agar- blue-
green colonies, leif son-red colonies.
• Biochemical character:
• Positive result for :
• Catalase
• Nitrate reduction
• Acid production from:
• L-arabinose
• D-mannitol
• D-mannose & trehalose
• Ngative result for:
• Oxidase
• H2S production
• Urease
• Voges-proskauer
• Citrate utilization
• Ecology:
• Widely distributed in nature, isolated from water, soil,
foods and clinical samples(feces, blood, urine, spinal
fluid, milk, wound infection)from human and animal.
 Enterobacter
• Morphology:
• Gram negative
• Straight bacilli, usually motile by 4-6 peritrichous flagella.
• Some encapsulated
• Measures 0.6-1.0 x 1.2-3.0
• Cultural characteristics
• Colonies on nutrient agar are round , 2-3 mm in diameter,
and slightly iridescent or flat with irregular edges, Non
pigmented.
• Exceptons : E. sakazakii is abale to grow at 44C.
• Grow on simple media and on selective media for
Enterobacteriaceae.
• Biochemical character:
• Positive result for:
• Catalase
• ONPG
• nitrate reduction
• Acid production from:
• L-arabinose
• Cellobiose
• Glucose
• Mannitol
• Maltose
• mannose
• Negative result for:
• Oxidase
• Indole production
• H2S production
• Gelatin hydrolysis
• Ecology:
• Can be found on human skin and plants as well as in
soil, water, sewage, intestinal tract of human and
animan, clinical specimens (feces, blood, urine, wound,
exudates, sputum) and some dairy products.
 Shigella
• Morphology:
• Gram negative rods
• Non motile
• Non-encapsulated
• Cultural characteristics:
• S-type colonies, non-pigmented, facultatively
anaerobic
• growth temperature 37C
• Media: Nutrient agar or Nutrient broth, Trypticase
Soy Agar + 5% sheep blood, Mac Conkey Agar.
 Shiga toxin

1. Chromosomally encoded
2. Neurotoxic
3. Enterotoxic
4. Cytotoxic
• Enterotoxicity can make the disease clinically
• appear as a diarrhea.
• The toxin inhibits protein synthesis (acting on the
• 60S ribosome and lysing 28S rRNA).
 Clinical significance
• man only "reservoir"
• mostly young children
– fecal to oral contact
– children to adults
• transmitted by adult food handlers
– unwashed hands
• Diagnosis of Shigella infection
• Specimen: stool.
• Culture and Identification
• Quick immunological methods:
• 1.Immunofluorescent “ball” test;
• 2.Coagglutination.
• Biochemical character:
• Positive result for:
• Nitrate reduction
• Methyl red
• Acid production from:
• D-mannose
• Negative result for:
• H2S production
• Oxidase
• Citrate utilization
• Voges-proskauer
• Urea hydrolysis
• Ecology
• Widely distributed in nature
• Isolated from feces, water, and soil.
• The normal habitat is intestinal tract of man and apes.

XLD Agar: Growth of Shigella and Proteus

 Salmonella
• Morpholgy:
• Gram negative rods
• 2-5/0.7-1.5 m
• Non-capsulated
• Non-sporulated
• Cultural characteristics:
• Colonies are 1-3 mm diameter, S-type, but sometimes
R-type colonies may appear.
• Motile, with the exception of Gallinarum and
Pullorum serovars
• Facultative anaerobic , growth temperature 37C
• Grow easily on various media:Nutrient Agar or
Nutrient broth, trypticase Soy Agar+ 5% sheep blood
• Mac Conkey Agar- red colonies, SS Agar-colourless
colonies, Rambach Agar- red colonies, XLD Agar- red
colonies, some with black center, Muller- Hinton Agar.
• Only a few types that are commonly associated
• with characteristic human diseases -
• 1. S. enteritidis
• 2. S. cholerae-suis
• 3. S. typhi
• several syndromes including gastroenteritis, enteric
(typhoid) fever or septicemia
• Biochemical character:
• Positive result for:
• Catalase
• Nitrate reduction
• Methyl red
• Acid production from:
• Glucose
• Mannitol
• D-mannose
• Negative result for:
• Indole production
• Urea hydrolysis
• Voges-proskauer-oxidase
• Acid production from:
• Sucrose,Cellobiose
 Diagnosis

• A. Specimens
a) Enteric fever: blood, bone marrow, stool, urine.
b) Food poisoning: stool, vomitus, suspected food.
c) Septicemia: blood.
• B. Culture and identification
• C. Widal test
XLD Agar: Appearance of Salmonella
Ecology:
Widely distributed in nature, isolated from water,
foods( raw meat, poultry, eggs, milk and dairy products,
fish, shrimp, frog legs, yeast, coconut etc.), soil, plant,
feces, animal organ and carcases( which may be
contaminated during slaughtering and processing).
Also have been isolated from the outside of egg shells.
 Klebsiella
• Morphology :
• Gram negative
• Straight, non-motile, capsulated
• 0.3-1.0 m x 0.6-6 m bacilli.
• K. oxytoca strains produce a dark brown pigment when
growth on media containing gluconate and ferric citrate.
• Cultural characteristics:
• Glistening moist colonies of varying degrees of
stikiness(M-type colonies).
• K. granulomatis grow intra cellulary only; it has not
been cultured.
• No special requirement.
• Facultatively anaerobic, optimum growth temperature
35-37C .
• Media : Nutrient Agar/broth, Trypticase Soy Agar + 5%
sheep blood , Mac Conkey , Muller-Hinton Agar.
• K.granulomatis grow in the yolk sac of developing
embryo or in cell cultures(fresh mononuclear cell or
Hep-2 cell line.

K. Pneumoniae On BA
• Biochemical character:
• Positive result for:
• Catalase
• Nitrate reduction(except K. singaporensis)
• Acid production from:
• L-arabinose
• Cellobiose
• Glucose
• Mannitol
• D-mannose
• Negative result for:
• Oxidase
• H2s production
• Arginine dihydrolase
• Gelatin hydrolysis
• Ecology:
• Widely distributed in nature
• Isolated from soil and plants they seem to be responsible
for nitrogen fixation, also isolated from water, and some
from clinical specimens(human and animal).
 Serratia
• Morphology:
• Gram negative, straight rods
• 0.5-0.8 x 0.9-2.0 m
• Non-capsulated
• Some strains are capsulated
• Motile by peritrichous flagella
• Some species produce a red, water insoluble
pigment(prodigiosin).Pigment production is variable
and not all strains produce it.
• Production is influenced by cultural conditions(30C)
and medium composition.
• Cultural characteristic:
• Colonies are 1 to3 mm in diameter, circular, shiny,
opaque, cream-white and smooth with entire margin.
• Some strains may have a distinctive musty odour.
• Facultatively anaerobic
• Growth temperature 20-35C (range may vary from 10-
40C).
• For S.plymuthica many test that are positive at 28-35C,
give negative result at 37C.
• Don’t require growth factors.
• Media: Nutrient Agar or Nutrient Broth, Trypticase Soy
Agar  5 % shee blood, Mac Conkey Agar, Milk Agar.
• Biochemical character:
• Positive result for:
• Catalase
• ONPG
• Citrate utilization
• Acid production from:
• Glucose
• Mannitol
• Maltose
• Glycerol
• D-mannose
• S.marcescens subsp. Sakuensis differes from
S.marcescens subsp. Marcescens in negative L-
arabitol forming(positive for S.marcescens subsp.
Marcescens ).
• S.quinivorans can be differentiated by negative
aesculin hydrolysis and acid production from salicin.
• Ecology:
• Widely distributed in nature
• Isolated from water, soil, foods, human and animal
clinical specimens, mainly from wounds and the
respiratory tract (more frequently S. marcescens) also
from moluscs and insects.
• S.fonticola and S. ureilytica have been isolated from
water.
• S.plymuthica has been isolates from various parts and
S.rubidaea also from plants, mostly coconuts.
• S. ficaria has been isolates from male figs and from
Blastophaga psenes (a fig tree-specific polinator that
breeds in male figs)
• S. glossinae has been isolated from the midgut of the
tsetse fly Glossina palpalis gambiensi.
• S. nematodiphila has been isolated from the intestine of
the nematode Heterorhabditidoides chongmingensis.
 Proteus
• Morphology:
• Gram negetive , straight rods, .4-0.6 x 1.0-3.0 m.
• Cultural characteristic:
• Facultatively anaerobic , growth temperature 37C .
• Highly motile by peririchous flagella, resulting a thin
film of bacteria on the agar surface (swarming).
• S-type, non-pigmented colonies on swarming
ihibitory media, majority are haemolytic on blood
agar.
• Isolation and growth media:
• Nutrient Agar or Nutrient broth, Muller-Hinton agar,
Trypticase Soy Agar  5% sheep blood : swarming ,
transparent,non-hemolytic colonies (with few
exceptions );
• Mac Conkey Agar: white transparent colonies (lactose
negative)-swarming inhibited;
• Rambach agar : white transparent colonies(lactose
negetive)-swarming inhibited.
• Biochemical character:
• Positive result for:
• Catalase
• Methyl red
• Urease
• Nitrate reduction
• Acid production from:
• Glucose
• Glycerol
Negative result for:
Oxidase
ONPG
Acid production from:
L-arabinose
D-arabiol
D-mannitol
D-mannose
Lactose
Ecology:
Isolate from urine, faeces,(human and animal sources),
soil and sewage.
Commonly found in the intestinal tract as part of normal
flora.P.myxofaciens has been isolated only from living
and dead gypsy moth larvae.
 Yersinia
• Morphology:
• Gram negative, bipolar, 1.0-3.0 x 0.5-0.8 m rods.
• Motile in vitro, but non motile in vivo.
• Non motile at 37C , but motile with 2-15
peritrichous flagella when grown below 30C, except
for Yersinia pestis, which is always nonmotile .
• Non capsule and no spores are produced.
• Cultural characteristic:
• Small, gray white, opaque, irregular colonies,0.1 to
1.0 mm in diameter after 24 hours incubation.
• Y. pestis strains do not produce pigment but adsorb
large amounts of exogenous hemin that cause
formation of apparently pigmented colonies at 26C.
• Facultatively anaerobic, optimum growth temperature
28C.
• Grow very slow at 4C (1-3 weeks).
• Media: Nutrient Agar or Nutrient broth, Trypticase Soy
Agar  5% sheep blood , MacConkey-lactose negative
colonies
• Biochemical character:
• Positive result for:
• Catalase+
• Methyl red+
• Ntrate reduction+
• acid production from
• Glucose+
• D-mannitol+
• D-mannose+
• Trehalose+
Negative result for:
Oxidase-
H2s production-
Voges-Proskauer-
Citrate utilization-
Acid production from
Lactose-
Meso-inositol-
Ecology:
Isolated mostly from some fron fish, snails, oysters,
frogs.
Also isolated from humans (feces, sputum, blood, urine,
absceses, lymph nodes), animals(rodents, swine, cattle,
birds), soil and foods(milk, meat).
 Oxidase Test

Negative
Positive
Enterobacteriaceae
Pseudomonas
MacConkey’s agar & TSI
 O/F test: O+/F-
 Nitrate test: +ve further
Pink colonies on MacConkey colorless colonies on MacConkey reduction to N2
& acidic butt and slant on TSI & acidic butt alkaline slant onTSI
 Growth on cetrimide agar:
Lactose fermenter Lactose non-fermenter Pale colonies with green
pigmentation
IMViC test
No H2S production H2S production
& EMB (no blacking in TSI) (blacking in TSI)

IMViC IMViC Urease production

++ - - - - ++ Shigella
& black colonies Motility +ve -ve
with metalic
shines on EMB SS agar
Not motile
Motile Proteus colorless colonies with black centers
E.coli
Klebsiella Salmonella
 Biochemicalresult
Biochemical resultofofsome
some
Enterobacteriaceae
Enterobacteriaceae
 Negative test Positive test
e.g. Klebsiella e.g. E. coli
Citrate Utilization Test

Urease Test

Positive Negative test Indole test

test
Special Features:
Positive Used in the differentiation of
Klebsiella, Negative genera and species. e.g.
Enterobacter E. coli E. coli (+) from
Klebsiella (-).
 MR/VP test

Methyl Red test

Voges-Proskauer test
Red: Positive MR (E. coli)
Pink: Positive VP (Klebsiella)
Yellow or orange: Negative MR (Klebsiella)

No pink: Negative VP (E. coli)

 Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
EMB SS MacCo O/F Nitrate Oxidase Gram
nkey reductase stain

Metallic LF LF O+/F+ +ve -ve -ve rod E. coli

sheen
Dark LF LF O+/F+ +ve -ve -ve rods Citrobacter

Dark LF LF O+/F+ +ve -ve -ve rods Klebsiella

Dark LF LF O+/F+ +ve -ve -ve rods Enterobacte

r
Colorless NLF/ NLF O+/F+ +ve -ve -ve rods Salmonella
H2S
Colorless NLF NLF O+/F+ +ve -ve -ve rods Shigella

Colorless NLF/ NLF O+/F+ +ve -ve -ve rods Proteus

H2S
 Summary of morphology, cultural characteristics,
and biochemical reactions of Enterobacteriaceae
Motility Urease Citrate VP MR Indole TSI

Motile -ve -ve -ve +ve +ve A/A/- E. coli

Motile -ve +ve -ve +ve +ve A/A/- Citrobacter

freundii

Non +ve +ve +ve -ve -ve A/A/- Klebsiella

motile pneumoniae

Motile +ve +ve +ve -ve -ve A/A/- Enterobacter

cloacae

Motile -ve +ve -ve +ve -ve A/Alk/+ Salmonella

typhi

Non -ve -ve -ve +ve -ve A/Alk/- Shigella

motile boydii

Motile +ve +ve -ve +ve -ve A/Alk/+ Proteus

Swarwing mirabilis
 REFERENCES

• http://www.cram.com/flashcards/enteric-
bacteria-1918627
• Bergey, D. H., Harrison, F. C., Breed, R. S., Hammer, B.
W. and Huntoon, F. M. (1957). Bergey's manual of
determinative bacteriology. Bergey's Manual of
Determinative Bacteriology., (Edn 7).
• J.G.Holt et al. (1994). Facultatively anerobic gram –
negative rods.Subgroup1.Family Enterobacteriaceae. In
Bergey's manual of determinativebacteriology.Williams
and Wilkins, pp- 175-189.
Thank
you