Enterobacteriaceae

Objectives
• Describe general structure, biochemical and diagnostic
criteria of Salmonella, Shigella, Vibrio and Brucella.
• List the antigenic structure of each one.
• Illustrate the pathogenesis of infections caused by
each one.
• Describe and analyze the applied laboratory tests.

Reference:
Jawetz, Melnick & Adelberg s MEDICAL
MICROBIOLOGY 25the Edition
 Genus Salmonella
• They are Gram negative rods, motile with peritrichous flagella
except Gallinarum-pullorum
• Ferment glucose and mannose  acid only but not ferment
lactose or sucrose.
• Survive freezing in water for long period
• Aerobic and facultative anaerobes. Grow on simple media but
some strains require enrichment media with one or more
amino acids.
• Urease not produced
• Produce H2S  black color on kligler Iron agar or Triple Sugar
Iron agar media.
 Antigenic structure
1-Somatic O-Ag: side chain of repeating sugar projecting from the cell wall.
They are hydrophilic, heat-stable Ags.
2-Flagellar H-Ag: represent determinant groups on the flagellar protein,
they are heat-labile and Biphasic (phase I & phase II) detached flagella 
remains antigenic
 non-motile salmonella

Others as
Fimbrial Ag (F-Ag) –just like flagellar detached by heating & cause confusing
cross reaction
Capsular Ag (Vi-Ag) –acidic polysaccharide layer covering cell wall, also heat-
labile.
M-Ag –lose extracellular polysaccharide
R-Ag –responsible for criteria (rough)  mutation to S(smooth)
 Pathogenesis
Most important pathogenic species
• S. typhi
• S. Paratyphi A
• S. Paratyphi B
• S. Cholerasuis

In salmonella we need large infective dose due to effect of gastric acidity (at least
100.000).

*Ingestion of food & water contaminated with human & animal wastes.
*Typhoid fever transmitted only by humans.
*Human sources either acutely infected persons
 carriers

*Animal sources through poultry and eggs.

 Types of salmonella infections
1-Enterocolitis: Invasion of epithelial & subepithelial tissues of S.I & L.I.
 organisms penetrate through the mucosal cells into lamina
propria  inflammation
(stool culture usually positive for few weeks)
2-Bacteremia: (5-10% of salmonella infection mainly caused by
S.choleraesuis). Spreading to blood stream following oral infection
causing focal lesions of many organs (lungs, bones, meninges … etc).
(Blood culture usually positive)
3-Enteric fever (Typhoid fever): Mainly caused by S.typhi.
Infection start in S.I.  multiply in peyers patches  liver, gallbladder
& spleen  bacteremia.
Carrier state may develop in about 5% of patients due to invasion of
gall bladder causing excretion of the bacteria in the feces.
 Laboratory Diagnosis
Specimens include:
• Blood for culture in bacteremia, and often positive from
the 1st week in enteric fever
• Bone marrow may be useful
• Urine cultures may be positive after a second week.
• Stool specimens  positive in 2nd -3rd weeks in typhoid
fever and usually positive in enterocolitis.
• Duodenal drainage  positive results in carrier state
• Biopsy from rose spots
• Serum (Widal test) (agglutinating Ab appear 2nd – 3rd
weeks)
 Genus Shigella
Shigella species are the causative agents of bacillary dysentery
(enterocolitis of humans)(Shigellosis)
• Gram negative rods, non–motile, non–sporing and non-capsulated
• Aerobic and facultative anaerobes and can not grow on simple
media
• All are non lactose fermenter except for Sh.sonnei (late lactose
fermenter), but ferment other sugars producing acid only.
• >40 serotypes classified according to their biochemical &
serological criteria:
Group A (Sh.dysenteriae)  12 serotypes
Group B (Sh.flexneri)  1-6 serotypes
Group C (Sh.boydii)  18 different serotypes
Group D (Sh.sonnei)  antigenically homogeneous but present in 2
forms
Antigenic structure
The somatic O Ag of Shigella (LPS) and their serologic
specificity depends on cell wall polysaccharide.

Toxins:
• Endotoxin: Toxic polysaccharide released during
autolysis causing irritation of bowel wall.
• Exotoxin: An antigenic protein released by
Sh.dysenteriae type 1 (Shiga bacillus). Like E.coli
verotoxin, act on gut  diarrhea and on CNS
(Neurotoxin)  meningism & coma.
The pathologic process of bloody diarrhea
(dysentery)
After feco-oral transmission  it reach LI 
Invade mucosal epithelial cells of large intestine
and terminal ileum by induce phagocytosis 
micro-organisms multiply within the cytoplasm
and pass to adjacent cells  microabscesses of
the wall  necrosis, superficial ulceration,
bleeding and pseudomembrane formation (fibrin,
leukocytes, cell depris, necrotic tissues and
bacteria)  then subsides by granulation and
formation of scar tissues.
Laboratory Diagnosis
1-Specimens, stool or rectal swab  culture.
2-Cultured on same media used for isolation of
Salmonella  non lactose fermentor (NLF) but no
H2S production and then identified by biochemical
tests and agglutination produced by mixing with
specific antisera.
3-Serum specimen may be done for detection of
agglutinins, but not diagnostic.(Anti bodies appear
after recovery but not protective).
4- Although it is very rare but some remains as carriers
shedding the bacilli with their stool .
 Genus Vibrio

1-Halophilic, require 8.5% and the isolated human pathogens

included are:-
-V.parahaemolyticum  self limited enteritis from contaminated
seafood.
-V.vulnificus wound infection
2-Non - halophilic , the most important pathogens are:-
-V. cholera  cholera
-Non – cholera Vibrio (Non – agglutinable vibrio)  sever
enteritis
General characteristics of V. cholera
-actively motile Gram negative, curved rods ferment
glucose, sucrose & mannose but require few days to
ferment manitol(N L F)
-All are oxidase positive
-optimum PH required (8.5)
( T . C . B . S .) alkaline media

Indicator bromothymol
Blue
Green  yellow


Acid production
PH
-V.cholera killed by acidity and destroyed by 55C15
min. & 0.5%phanol.
-It reduce nitrates nitrite with production of
Indole← used for diagnosis of v.cholera
by (Nitrose indol reaction)
or (cholera red reaction)
This is done by isolating suspected micro – organism on
alkaline peptone water with nitrate, then after
incubation  add few drops of concentrated H2SO4
 if red color appear means the presence of indole.
Antigenic structure of V.cholera
-The O, lipopolysaccharide, there are 139 O Ag.
The most important (O1), but O139 also cause classical
cholera, and this strain shares the other non- O1
cholera strains the presence of a polysaccharide
capsule.
The O1 organisms can be subdivided into:
-Two biological types Eltor and Classical biotype.
-Three serological sub types Inaba, Ogawa and every
rare Hikojima.
* Vibrio which lack O Ag called NAG vibrio which cause
cholera like disease.
V. cholerae Enterotoxin
Called (choleragen) composed of 2 subunits (A&B), Epithelial
ganglioside Gm1 serves as a receptor for B subunit ,and this
will help entery of A subunit, then activation of A subunit 
increase level of intracellular CAMP  Prolong
hypersecretion of water & electrolyte  causing watery
diarrhea without inflammatory cells (non– invasive)
Pathogenesis of V. cholera
Cholera is transmitted by fecal contamination of water and
food from human sources. large infective dose must be
ingested because they are sensitive to stomach acid.
The micro – organism adhere to epithelial cells of brush
border of the gut, multiply and secret enterotoxin and
mucinase enzyme which dissolves the protective
glycoprotein coating
Laboratory diagnosis:-
Specimen including Stool or mucus flecks, Rectal swab or
catheter or Vomitus (unusual).
1-Microscopical examination
2-Motility test.
a-by stabbing the micro-organism with needle into semisolid
media (incubation 24hr at 37c ̊ )
b-Hanging drop preparation
3-Cholera immobilization test
4-culture
5-Biochmical tests
*Cholera red test.
*Oxidase test.
6-Typing of the isolated micro-organism with specific antisera
7-Serological test
Antitoxin can be detected by ELISA.
Brucella
Obligate intracellular parasite of animal and
human Brucella spp. are causative agent of
brucellosis, malta fever or undulant fever
4 major human pathogens and their animal
reservoirs:-
-B.melitensis (goats & sheep).
-B.abortus (cattle).
-B.suis(pigs).
-B.canis(dogs).
General characters:
-Gram negative rods, non-motile, non spore–forming & some are
capsulated.
-Obligate aerobes, grow on blood agar and enriched media
-Serum of susceptible animals contain globulin and a lipoprotein that
suppress growth of a virulent type and favor the growth of virulent
one.
-Brucella utilize CHO with no acid or gas, reduce nitrate, catalase &
oxidase positive and H2S is produced by many strains. They are
sensitive to heat and killed by milk pasteurization for 10 min at 60oC
and by acidity of sour milk, also killed by exposure to 1% phenol for
15 min.
Antigenic structure:
-The 2 lipopolysaccharide antigens A&M are present in different
proportions in the 4 spp.
-Superficial L Ag also present that resembles the Vi Ag of Salmonellae.
Pathogenesis:
The organisms → localize in RES (L.N, liver, spleen and bone
marrow) many killed by macrophages, but some survive
within the cells (intracellular) protected from Abs.
Granulomas may appear which can progress to focal
abscesses and caseation. Osteomylitis or cholecystitis also
occasionally occur.
- The mechanism of pathogenesis in not well defined except
the role of endotoxin in active brucellosis.
*B.abortus →mild disease without suppurative complications
and non caseating granuloma.
*B.canis →mild disease.
*B.suis →chronic disease with suppurative and caseating
granulomas.
*B.melitensis → more acute and sever disease.
Laboratory diagnosis:
-Blood for blood culture during acute phase
-Biopsy material for culture
-Serum for Antibody detection

A-Culture : on Trypticase – soy broth and sub culture

every 3-5 days on solid media (under aerobic and 10%
CO2), keep blood for 4wks before being discard as
negative. The colonies appear as small, transparent and
non hemolytic, Gram’s stain showing the typical
coccobacilli. Biochemically they are (oxidase positive &
urease positive).
The isolated brucellae typed by H2S production and
agglutination by specific antisera.
B-Serology
1-Agglutination test (Rose Bengal) For detection of IgG Ab, serial
tube dilution used, IgG titers >1:80 indicate active infection
This test can give:
false positive because of cross reaction with other infection and
this can be overcomed by application of 2- Mercaptoethanol
test – the addition of 2me will destroy IgM and leaves IgG.
false negative because of the presence of blocking Abs (IgA)
which appear during subacute stage and remain for years
causing a negative test in low serum dilution ,so increase
dilution and using coombs antiglobulin method(patient serum
+ Brucella antigen +Anti human globulin)
2-ELISA & EFAT
C. Brucella skin test
 Summary
- Salmonella are Gram’s negative rods producing H2S giving black
colour on KIA and having somatic O antigen and flagellar H
antigen.
- Lab diagnosis involve microscopical, cultural, biochemical and
interpretation of Widal test results is the most important step
in the diagnosis
• Shigella species are Gram negative rods, non–motile, non–
sporing, non-capsulated and H2S not produced causing
bacillary dysentery.
• It has somatic O Ag (LPS) and produce Endotoxin and
Exotoxin(Shiga bacillus).
• Symptoms are related to effect of exotoxin and localized
invasion of the intestinal mucosa
 Summary
- V.Cholera actively motile, Gram negative, curved rods isolated on alkaline
media (pH 8.5)(TCBS)
-V.Cholera reduce nitrates nitrite with production of Indole← (cholera red
reaction)
-The O, lipopolysaccharide, there are 139 O Ag
-Enterotoxin (choleragen) composed of 2 subunits (A&B)causing watery
diarrhea without inflammatory cells

- Brucella spp. are Obligate intracellular parasite of animal and human, they are
causative agent of malta fever.
- Gram negative rods, non-motile, non spore–forming & some are capsulated,
grow aerobically. and killed by milk pasteurization for 10 min at 60 oC and by
acidity of sour milk.
- Lab diagnosis involve culture of blood sample or biopsy material on selective
media and detection of IgG Ab by Rose Bengal test(serology).