ENTEROBACTERIACEAE

Microbiology Lecture Series

Luz Gregoria Lazo-Velasco, MD
Enterobacteriaceae
Characteristics:
Gram-negative rods
Motile with peritrichous flagella OR nonmotile
(Shigella, Klebsiella)
Grow on peptone or meat extract media without the addition of sodium chloride or other
supplements
Grow well on MacConkeys agar
Grow aerobically and anaerobically (facultative anaerobes)
Ferment rather than oxidize glucose, often with gas production
Catalase-positive
Oxidase-negative
Reduce nitrate to nitrite
39-59% G + C DNA content
Natural habitat: intestinal tract of humans and animals

Escherichia

Shigella

Salmonella

Enterobacter

Klebsiella

Serratia

Proteus
Some are part of normal flora (E. coli)
Some are regularly pathogenic for humans (Shigella bacillary dysentery, Salmonella typhoid fever)
Facultative anaerobes or aerobes
Possess a complex antigenic structure
Produce a variety of toxins and other virulence factors
Morphology & Identification
Short gram-negative rods
Capsules: Klebsiella, Enterobacter
Culture
Circular, convex, smooth colonies with distinct edges
Enterobacter more mucoid
Klebsiella large, very mucoid, coalesce with prolonged incubation
E. coli some produce hemolysis on blood agar

Growth Characteristics
Biochemical differentiation carbohydrate fermentation patterns and the activity of amino acid
decarboxylases and other enzymes

Rapid identification tests production of indole to tryptophan

less used Voges-Proskauer reaction (production of acetylmethylcarbinol from dextrose)

Culture on "differential" media that contain special dyes and carbohydrates (EMB, MacConkey, or
deoxycholate medium) distinguishes lactose-fermenting (colored) from non-lactose-fermenting
colonies (nonpigmented)

ANTIGENIC STRUCTURE
O (lipopolysaccharide) Antigens (IgM)- resistant to heat and alcohol
-detected by bacterial agglutination

K (capsular) Antigens (Vi in Salmonella)- associated with virulence

- E. coli strains (K1 antigen) prominent in neonatal meningitis
- K antigens of E. coli cause attachment of the bacteria to
epithelial cells prior to
gastrointestinal or urinary tract invasion

Enterobacteriaceae are classified by more than 150 different heat-stable somatic O (lipopolysaccharide)
antigens,
more than 100 heat-labile K (capsular) antigens, more than 50 H (flagellar) antigens
O (lipopolysaccharide) antigens - the most external part of the cell wall lipopolysaccharide; consist of
repeating units of polysaccharide
K (capsular) antigens - external to O antigens on some but not all; some are polysaccharides (K
antigens of E. coli); others are proteins;
may interfere with agglutination by O antisera; may be
associated with virulence
K (capsular) Antigens (Vi in Salmonella)
associated with virulence
Klebsiellae form large capsules consisting of polysaccharides (K antigens) covering the somatic (O or H)
antigens and can be identified by capsular swelling tests with specific antisera
Respiratory tract infection capsular types 1 and 2
Urinary tract infection types 8, 9, 10 and 24 cells possessing K antigen are more pathogenic than
those that lack them inhibits phagocytosis and the effects of serum antibody

H (flagellar) Antigens
located on flagella
denatured or removed by heat or alcohol
preserved by treating motile bacterial variants with formalin (H antigens agglutinate with anti-H antibodies
-IgG)
determinants in H antigens are a function of the amino acid sequence in flagellar protein (flagellin)

COLICINS (BACTERIOCINS)
High molecular weight bactericidal protein produced by certain strains of bacteria active against
some other strains of the same or closely related species
Production controlled by plasmids
COLICINS Escherichia coli
MARCESCENS Serratia
PYOCINS Pseudomonas

TOXINS AND ENZYMES

Most gram-negative bacteria possess complex lipopolysaccharides in their cell walls (endotoxins)
Many gram-negative enteric bacteria also produce exotoxins of clinical importance
A. ESCHERICHIA COLI
Escherichia coli

positive tests for indole, lysine decarboxylase and mannitol fermentation

produces gas from glucose

(+) hemolysis on blood agar
typical colonial morphology with an iridescent "sheen" on differential media (EMB agar)
positive spot indole test
Over 90% of isolates are positive for -glucuronidase using the substrate 4methylumbelliferyl- -glucuronide (MUG)
Isolates from anatomic sites other than urine can be confirmed as E. coli with a positive MUG test
Normal intestinal flora; small numbers part of the normal flora of the upper respiratory and genital tracts
Generally do not cause disease, may even contribute to normal function and nutrition in the intestine
pathogenic only when they reach tissues outside of their normal intestinal or other less common normal
flora sites

Most frequent sites:

urinary tract
biliary tract
other sites in the abdominal cavity
any anatomic site (bloodstream, prostate gland, lung, bone, meninges)
PATHOGENESIS & CLINICAL FINDINGS
Urinary Tract Infection
-Most common cause of urinary tract infection; ~90% of first urinary tract infections in young
women
-Urinary frequency, dysuria, hematuria, pyuria
- Flank pain associated with upper tract infection (uropathogenic E. coli)
- Can result in bacteremia with clinical signs of sepsis
- Strains that cause pyelonephritis express K antigen and elaborate P fimbriae
- Uropathogenic E. coli produces hemolysin (cytotoxic and facilitates tissue invasion)

E. coli-associated diarrheal diseases

- E. coli that cause diarrhea are classified by the characteristics of their virulence properties
and each group causes disease by a different mechanism
- The small or large bowel epithelial cell adherence properties are encoded by genes on
plasmids
- Toxins often are plasmid- or phage-mediated

Enteropathogenic E. coli (EPEC)

Important cause of diarrhea in infants
Previously associated with outbreaks of diarrhea in nurseries in developed countries
EPEC adhere to the mucosal cells of the small bowel
loss of microvilli (effacement), formation of filamentous actin pedestals or cup-like structures,
entry into the mucosal cells
watery diarrhea (self-limited or chronic); antibiotic tx
Enterotoxigenic E. coli (ETEC)
Common cause of "traveler's diarrhea"
Very important cause of diarrhea in infants in developing countries
Colonization factors specific for humans promote adherence to epithelial cells of the small bowel
Some strains produce a heat-labile exotoxin (LT) under the genetic control of a plasmid
Subunit B of exotoxin attaches to the GM1 ganglioside at the brush border of epithelial cells of the
small intestine and facilitates the entry of subunit A into the cell activates adenylyl cyclase

cyclic adenosine monophosphate (cAMP) intense and prolonged hypersecretion of water and
chlorides and inhibits the reabsorption of sodium
Gut lumen distended with fluid hypermotility, diarrhea for several days
LT is antigenic; cross-reacts with the enterotoxin of Vibrio cholerae; stimulates the production of
neutralizing Abs in the serum of persons previously infected with ETEC
Persons residing in areas where such organisms are highly prevalent are likely to possess
antibodies (less prone to develop diarrhea on reexposure to the LT-producing E. coli)
Assays for LT include:
fluid accumulation in the intestine of laboratory animals
typical cytologic changes in cultured Chinese hamster ovary cells or other cell lines
stimulation of steroid production in cultured adrenal tumor cells
binding and immunologic assays with standardized antisera to LT

Some strains produce the heat-stable enterotoxin STa (under the genetic control of a
heterogeneous group of plasmids)
STa activates guanylyl cyclase in enteric epithelial cells and stimulates fluid secretion; ST a-positive
strains also produce LT
Plasmids carrying the genes for enterotoxins (LT, ST) also may carry genes for the colonization
factors that facilitate the attachment of E. coli strains to intestinal epithelium
Antimicrobial prophylaxis can be effective but may result in increased antibiotic resistance in the
bacteria and probably should not be uniformly recommended
Antibiotic treatment effectively shortens the duration of disease

Shiga toxin producing E. coli (STEC)

Produce cytotoxic toxins Shiga-like toxin 1, Shiga-like toxin 2 (similar to Shiga toxin of shigella
dysenteriae type 1 but antigenically and genetically distinct)
Associated with hemorrhagic colitis, hemolytic uremic syndrome (ARF, microangiopathic
hemolytic anemia, thrombocytopenia)
O157:H7 (most common serotype producing Shiga toxin)
STEC O157:H7 does not use sorbitol, (-) on sorbitol MacConkey agar, (-) MUG tests; specific
antisera used in identification; assays done using commercially available enzyme immunoassays
Other methods: cell culture cytotoxin testing (use Vero cells), PCR

Enteroinvasive E. coli (EIEC)

Disease very similar to shigellosis
Most common in children in developing countries and traveler to these countries
Non-lactose or late-lactose fermenters; nonmotile
Produce disease by invading intestinal mucosal epithelial cells
Escherichia coli
Enteroaggregative E. coli (EAEC)
Causes acute and chronic diarhea (>14 days)
Produce ST-like toxin and a hemolysin
Sepsis
Meningitis - Leading cause of meningitis in infant with group B streptococci (+) K1 antigen
Newborns are susceptible to E. coli sepsis lack IgM antibodies
B. KLEBSIELLA-ENTEROBACTER-SERRATIA

Klebsiella species:
mucoid growth
large polysaccharide capsules
lack of motility
(+) tests for lysine decarboxylase and citrate
Enterobacter species:
(+) tests for motility, citrate, and ornithine decarboxylase
produce gas from glucose
positive Voges-Proskauer reactions
Serratia :
produces DNase, lipase and gelatinase

DISEASES:

Klebsiella pneumonia
- Present in the respiratory tract and feces of about 5% of normal individuals
- Causes a small proportion (about 1%) of bacterial pneumonias
- K. pneumoniae can produce extensive hemorrhagic necrotizing consolidation of the lung
- Urinary tract infection, bacteremia with focal lesions in debilitated patients
- Top 10 bacterial pathogens responsible for hospital-acquired
infections

Inflammatory conditions of the URT:

Klebsiella pneumoniae subsp. ozaenae (ozena fetid, progressive atrophy of mucous membranes)
Klebsiella pneumoniae subsp. rhinoscleromatis (rhinoscleroma) - destructive granuloma of the nose and
pharynx)
Klebsiella granulomatis (chronic genital ulcerative disease)
Enterobacter

E. cloacae, E. aerogenes, E. sakazakii

Ferment lactose, may contain capsules that produce mucoid colonies, motile
Broad range of hospital-acquired infections: pneumonias, UTI, wound and device infections
Most strains possess chromosomal b-lactamase (ampC) rendering them intrinsically resistant to
ampicillin and 1st and 2nd generation cephalosporins

Serratia marcescens
- pneumonia, bacteremia, and endocarditis esp in narcotics addicts and hospitalized patients
- only 10% of isolates form the red pigment (prodigiosin)
- resistant to aminoglycosides, penicillins
- treated with 3rd generation cephalosporins
C. PROTEUS-MORGANELLA-PROVIDENCIA
Characteristics:
deaminate phenylalanine

motile
grow on potassium cyanide medium (KCN)
ferment xylose

Proteus species
move very actively by means of peritrichous flagella; "swarming" motion on solid media
Proteus species and Morganella morganii urease-positive
Providencia species urease-negative
The proteus-providencia group ferments lactose very slowly or not at all
Proteus mirabilis more susceptible to antimicrobial drugs, including penicillins, than other members of
the group

DISEASES:
Proteus
- Produce infections in humans only when the bacteria leave the intestinal tract
- UTI, bacteremia, pneumonia, and focal lesions in debilitated patients or those receiving
intravenous infusions
P. mirabilis- UTI and other infections
P. vulgaris and Morganella morganii nosocomial pathogens
- Produce urease (rapid hydrolysis of urea with liberation of ammonia)
- UTI with proteus: urine becomes alkaline, promoting stone formation and making acidification
virtually impossible
- rapid motility may contribute to its invasion of the urinary tract
- P. mirabilis is often inhibited by penicillins
DIAGNOSTIC LABORATORY TESTS:
Specimens:
Urine, blood, pus, spinal fluid, sputum
Smears:
large capsules Klebsiella
Culture:
blood agar, differential media
IMMUNITY:
Specific antibodies develop in systemic infections
TREATMENT:
NO specific therapy available
Sulfonamides, cephalosporins, ampicillin, fluoroquinolones, aminoglycosides
Surgical correction (urinary tract obstruction, closure of a perforation in an abdominal organ,
resection of bronchiectatic lung)
Prevention of traveler's diarrhea:
o daily ingestion of bismuth subsalicylate suspension
o regular doses of tetracyclines or other antimicrobial drugs for limited periods
o caution in regard to food and drink in areas where environmental sanitation is poor
o early and brief treatment (ciprofloxacin or trimethoprim-sulfamethoxazole) substituted for
prophylaxis
D. CITROBACTER
Citrobacter
citrate-positive

differ from the Salmonellae - do not decarboxylate lysine

ferment lactose very slowly if at all
Epidemiology, Prevention & Control
The enteric bacteria establish themselves in the normal intestinal tract within a few days after birth and
from then on constitute a main portion of the normal aerobic (facultative anaerobic) microbial flora
Enterics found in water or milk are accepted as proof of fecal contamination from sewage or other
sources
Control measures are not feasible as far as the normal endogenous flora is concerned
Control depends on hand washing, rigorous asepsis, sterilization of equipment, disinfection, restraint in IV
therapy, and strict precautions in keeping the urinary tract sterile
Present
Designation
Shigella dysenteriae
Shigella flexneri
Shigella boydii
Shigella sonnei

Group &
type
A
B
C
D

Mannitol

Ornithine decarboxylase

+
+
+

E. SHIGELLAE
Shigella
Non-motile
Do not ferment lactose
Ferment other carbohydrates producing acid but not gas
Do not produce H2S
The 4 Shigella species are closely related to E. coli
Shigellae Bacillary Dysentery
Limited to the intestinal tracts of humans and other primates
MORPHOLOGY & IDENTIFICATION
Typical Organisms slender, G(-) rods
Culture
- Facultative anaerobes but grow best aerobically
- Convex, circular, transparent colonies w/ intact edges
- Reach a diameter of about 2mm in 24 hours
Growth Characteristics
- Ferment glucose
- Do not ferment lactose (except S. sonnei) distinguishes them on differential media
Shigellae Bacillary Dysentery
MORPHOLOGY & IDENTIFICATION

Growth Characteristics
- Form acid from carbohydrates but rarely produce gas
- Divided into those that ferment mannitol and those do not
ANTIGENIC STRUCTURE

Somatic O antigens (LPS)

PATHOGENESIS & PATHOLOGY
Shigella infections are almost always limited to the GIT; bloodstream invasion is quite rare
highly communicable; Infective dose 103 organisms
essential pathologic process: invasion of the mucosal epithelial cells by induced phagocytosis, escape
from the phagocytic vacuole, multiplication and spread within the epithelial cell cytoplasm, and passage to
adjacent cells
Microabscesses in the wall of the large intestine and terminal ileum necrosis of the mucous membrane,
superficial ulceration, bleeding, formation of a "pseudomembrane" on the ulcerated area
consists of fibrin, leukocytes, cell debris, a necrotic mucous membrane, and bacteria
As the process subsides, granulation tissue fills the ulcers and scar tissue forms
TOXINS
Endotoxins:

Upon autolysis, all shigellae release their toxic LPS (irritation of the bowel wall)
Shigella dysenteriae Exotoxin:
S. dysenteriae type 1 (Shiga bacillus) produces a heat-labile exotoxin that affects both the gut and the
CNS protein that is antigenic (stimulating production of antitoxin) and lethal for experimental animals
o acts as an enterotoxin (diarrhea )
o acts as an neurotoxin (meningismus, coma)
CLINICAL FINDINGS
short incubation period (12 days)
sudden onset of abdominal pain, fever, watery diarrhea
Diarrhea attributed to an exotoxin acting in the small intestine
in a day or two, infection involves the ileum and colon (number of stools increases, less liquid but often
contain mucus and blood)
bowel movement accompanied by straining and tenesmus (rectal spasms), with resulting lower abdominal
pain
fever and diarrhea subside spontaneously in 25 days
in children and the elderly, loss of water and electrolytes may lead to dehydration, acidosis, and even
death
illness due to S. dysenteriae may be particularly severe
On recovery, most persons shed dysentery bacilli for only a short period, but a few remain chronic
intestinal carriers and may have recurrent bouts of the disease
Upon recovery from the infection, most persons develop circulating antibodies to shigellae but do not
protect from reinfection
DIAGNOSTIC LABORATORY TESTS
Specimens: Fresh stool, Mucus flecks, Rectal swabs
Large numbers of fecal leukocytes and some red blood cells often are seen microscopically
Serum specimens must be taken 10 days apart to demonstrate a rise in titer of agglutinating
antibodies
Culture:
differential media (MacConkeys or EMB agar)
selective media (Hektoen enteric agar or salmonella-shigella agar), which suppress other
Enterobacteriaceae and gram-positive organisms
Culture:
Colorless (lactose-negative) colonies are inoculated into triple sugar iron agar
Organisms that fail to produce H2S, that produce acid but not gas in the butt and an alkaline slant
in triple sugar iron agar medium, and that are nonmotile should be subjected to slide agglutination
by specific shigella antisera
Serology: not used to diagnose Shigella infections
Serial determinations of antibody titers may show a rise in specific antibody
IMMUNITY
Infection is followed by a type-specific antibody response
Injection of killed Shigellae stimulates production of antibodies in serum but fails to protect humans
against infection
IgA antibodies in the gut may be important in limiting reinfection; these may be stimulated by live
attenuated strains given orally as experimental vaccines
Serum antibodies to somatic shigella antigens are IgM
TREATMENT
Ciprofloxacin, ampicillin, doxycycline, and trimethoprim-sulfamethoxazole most commonly inhibitory for
shigella isolates and can suppress acute clinical attacks of dysentery and shorten the duration of
symptoms; may fail to eradicate the organisms from the intestinal tract
Multiple drug resistance can be transmitted by plasmids, and resistant infections are widespread
Many cases are self-limited
Opioids should be avoided in Shigella dysentery
EPIDEMIOLOGY, PREVENTION & CONTROL
Transmission: Food, Fingers, Feces, Flies from person to person
Most cases of shigella infection occur in children under 10years of age
control efforts must be directed at eliminating the organisms from humans by:

o
o
o

sanitary control of water, food, and milk; sewage disposal; fly control, isolation of patients and
disinfection of excreta
detection of subclinical cases and carriers, particularly food handlers
antibiotic treatment of infected individuals

F. SALMONELLAE
Salmonella
Motile rods
Ferment glucose and mannose without producing gas but do not ferment lactose or sucrose
Most produce H2S
Often pathogenic for humans or animals when ingested
Salmonella
Salmonellae are often pathogenic for humans or animals when acquired by the oral route
Transmitted from animals and animal products to humans,
Enteritis, systemic infection, and enteric fever
MORPHOLOGY & IDENTIFICATION
Most are motile with peritrichous flagella
Grow readily on simple media, but they almost never ferment lactose or sucrose
They form acid and sometimes gas from glucose and mannose
Produce H2S
Survive freezing in water for long periods; resistant to certain chemicals (brilliant green, sodium
tetrathionate/deoxycholate)

CLASSIFICATION
The members of the genus Salmonella were originally classified on the basis of epidemiology,
host range, biochemical reactions, and structures of the O, H, and Vi (when present) antigens
Divided into 2 species, each with multiple subspecies and serotypes
Salmonella enterica
Supspecies: I enterica*
IIIb - diarizonae
II- salamae
IV - houtenae
IIIa - arizonae VI - indicae
Salmonella bongori
PATHOGENESIS & CLINICAL FINDINGS
Salmonella Typhi, Salmonella Choleraesuis, and perhaps Salmonella Paratyphi A and Salmonella
Paratyphi B are primarily infective for humans, and infection with these organisms implies
acquisition from a human source
The vast majority of salmonellae are chiefly pathogenic in animals that constitute the reservoir for
human infection: poultry, pigs, rodents, cattle, pets (from turtles to parrots), and many others
The organisms almost always enter via the oral route, usually with contaminated food or drink
Mean infective dose 105108 (Salmonella typhi 103)
Salmonella Typhi, Salmonella Choleraesuis, and perhaps Salmonella Paratyphi A and Salmonella
Paratyphi B are primarily infective for humans, and infection with these organisms implies
acquisition from a human source
The vast majority of salmonellae are chiefly pathogenic in animals that constitute the reservoir for
human infection: poultry, pigs, rodents, cattle, pets (from turtles to parrots), and many others
The organisms almost always enter via the oral route, usually with contaminated food or drink
Mean infective dose 105108 (Salmonella typhi 103)
Among the host factors that contribute to resistance to salmonella infection are:
o gastric acidity
o normal intestinal microbial flora
o local intestinal immunity
Enteric Fever (Typhoid Fever):
o produced by only a few of the salmonellae, of which Salmonella Typhi (typhoid fever) is the most
important

o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o

The ingested salmonellae reach the small intestine, from which they enter the lymphatics and then the
bloodstream
They are carried by the blood to many organs, including the intestine
The organisms multiply in intestinal lymphoid tissue and are excreted in stools
After an incubation period of 1014 days, fever, malaise, headache, constipation, bradycardia, and
myalgia occur
The fever rises to a high plateau, and the spleen and liver become enlarged
Rose spots, on the skin of the abdomen or chest seen briefly
The white blood cell count is normal or low
In the preantibiotic era, the chief complications of enteric fever were intestinal hemorrhage and
perforation, and the mortality rate was 1015%
Treatment with antibiotics has reduced the mortality rate to less than 1%
Principal lesions : hyperplasia and necrosis of lymphoid tissue (eg, Peyer's patches), hepatitis, focal
necrosis of the liver, and inflammation
Fever rises to a high plateau, spleen and liver enlarge
Rose spots, on the skin of the abdomen or chest seen briefly
The white blood cell count is normal or low
In the pre-antibiotic era, the chief complications of enteric fever were intestinal hemorrhage and
perforation, and the mortality rate was 1015%
Treatment with antibiotics has reduced the mortality rate to <1%
Principal lesions : hyperplasia and necrosis of lymphoid tissue (eg, Peyer's patches), hepatitis, focal
necrosis of the liver, and inflammationof the gallbladder, periosteum, lungs, and other organs

Bacteremia with focal lesions:

o This is associated commonly with S. choleraesuis but may be caused by any salmonella serotype
o Following oral infection, there is early invasion of the bloodstream (with possible focal lesions in lungs,
bones, meninges, etc) but intestinal manifestations are often absent
o Blood cultures are positive

Enterocolitis:
o Most common manifestation of salmonella infection
o 8-48 hours after ingestion of salmonellae nausea, headache, vomiting, and profuse diarrhea, with few
leukocytes in the stools
o Low-grade fever is common
o Usually resolves in 23 days
o Inflammatory lesions of the small and large intestine are present
o Bacteremia is rare (24%) except in immunodeficient persons
o Blood cultures are usually negative
o Stool cultures positive for salmonellae, may remain positive for several weeks after clinical recovery
DIAGNOSTICS AND LABORATORY TESTS
Specimens:
o Blood for culture taken repeatedly
o In enteric fevers and septicemias, blood cultures are often positive in the first week of the disease
o Bone marrow cultures may be useful
o Urine cultures may be positive after the second week
o Stool specimens taken repeatedly
o In enteric fevers, the stools yield positive results from the second or third week on; in enterocolitis, during
the first week
o A positive culture of duodenal drainage establishes the presence of salmonellae in the biliary tract in
carriers
Bacteriologic Measures for Isolation of Salmonellae:
Differential Medium Cultures:
o EMB, MacConkey, or deoxycholate medium permits rapid detection of lactose nonfermenters)
o Bismuth sulfite medium permits rapid detection of salmonellae which form black colonies because
of H2S production
o Many salmonellae produce H2S
Selective Medium Cultures:

Salmonella-Shigella (SS) agar, Hektoen enteric agar, XLD, or deoxycholate-citrate agar, which
favor growth of salmonellae and shigellae over other Enterobacteriaceae
Enrichment Cultures:
o Selenite F or tetrathionate broth (inhibit replication of normal intestinal bacteria, permit
multiplication of salmonellae )
o After incubation for 12 days, plated on differential and selective media
Final Identification:
o Suspect colonies from solid media are identified by biochemical reaction patterns and slide
agglutination tests with specific sera
Serologic Methods:
o Used to identify unknown cultures with known sera
o Used to determine antibody titers in patients with unknown illness
Agglutination Test:
o known sera and unknown culture are mixed on a slide
o Clumping, when it occurs, can be observed within a few minutes
o Useful for rapid preliminary identification of cultures
o Commercial kits available to agglutinate and serogroup salmonellae by their O antigens: A, B, C 1,
C2, D, and E
Tube Dilution Agglutination Test (Widal Test):
o Serum agglutinins rise sharply during the second and third weeks of Salmonella Typhi infection
o Detect these antibodies against the O and H antigens
o At least two serum specimens, obtained at intervals of 710 days needed to prove a rise in
antibody titer
o Serial dilutions of unknown sera are tested against antigens from representative salmonella
o a titer against the O antigen of > 1:320 and against the H antigen of > 1:640 is considered
positive
o test is not useful in diagnosis of enteric fevers caused by salmonella other than Salmonella Typhi
IMMUNITY
Infections with Salmonella Typhi or Salmonella Paratyphi usually confer a certain degree of
immunity
Reinfection may occur but is often milder than the first infection
Circulating antibodies to O and Vi are related to resistance to infection and disease
Relapses may occur in 23 weeks after recovery in spite of antibodies
Secretory IgA antibodies may prevent attachment of salmonellae to intestinal epithelium
Persons with S/S hemoglobin (sickle cell disease) are exceedingly susceptible to salmonella
infections, particularly osteomyelitis
Persons with A/S hemoglobin (sickle cell trait) may be more susceptible than normal individuals
(those with A/A hemoglobin)
TREATMENT
Antimicrobial treatment of salmonella enteritis in neonates is important
In enterocolitis, clinical symptoms and excretion of the salmonellae may be prolonged by
antimicrobial therapy
In severe diarrhea, replacement of fluids and electrolytes is essential
Antimicrobial therapy of invasive salmonella infections - ampicillin, trimethoprimsulfamethoxazole, or a third-generation cephalosporin
In most carriers, the organisms persist in the gallbladder (particularly if gallstones are present)
and in the biliary tract; in most cases cholecystectomy must be combined with drug treatment
Salmonella
EPIDEMIOLOGY
The feces of persons who have unsuspected subclinical disease or are carriers are a more
important source of contamination than frank clinical cases that are promptly isolated
Many animals, including cattle, rodents, and fowl, are naturally infected with a variety of
salmonellae and have the bacteria in their tissues (meat), excreta, or eggs
The problem probably is aggravated by the widespread use of animal feeds containing
antimicrobial drugs that favor the proliferation of drug-resistant salmonellae and their potential
transmission to humans
Carriers:
After manifest or subclinical infection, some individuals continue to harbor salmonellae in their tissues for
variable lengths of time (convalescent carriers or healthy permanent carriers)
Three percent of survivors of typhoid become permanent carriers, harboring the organisms in the
gallbladder, biliary tract, or, rarely, the intestine or urinary tract
Sources of Infection:
Contaminated food and drink
o

 Water, Milk and Other Dairy Products (Ice Cream, Cheese, Custard)
Shellfish, Dried or Frozen Eggs
Meats and Meat Products
"Recreational" Drugs (Marijuana and other drugs)
Animal Dyes
Household Pets
PREVENTION AND CONTROL
Sanitary measures must be taken to prevent contamination of food and water by rodents or other
animals that excrete salmonellae
Infected poultry, meats, and eggs must be thoroughly cooked
Carriers must not be allowed to work as food handlers and should observe strict hygienic
precautions.
Two injections of acetone-killed bacterial suspensions of Salmonella Typhi, followed by a booster
injection some months later, give partial resistance to small infectious inocula of typhoid bacilli but
not to large ones
Oral administration of a live avirulent mutant strain of Salmonella Typhi has given significant
protection in areas of high endemicity
DIAGNOSTIC LABORATORY TESTS
Among the host factors that contribute to resistance to salmonella infection are:
gastric acidity
normal intestinal microbial flora
local intestinal immunity
MacConkey Agar
Isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli
Selective (Gram negative bacteria)
Differential (Lactose fermenters and non-lactose fermenters)
Principle
Inhibitors of Gram Positive bacteria
Bile Salts, Crystal violet, Neutral Red
Lactose - only carbohydrate source
Neutral red
Indicator - Brown at pH 6.8-8.0
Pink-red at pH <6.8
Rapid Lactose Fermenters
(Pink)
Enterobacter spp.
Escherichia spp.
Klebsiella spp.

Slow Lactose Fermenters (Pink

48h)
Serratia spp.
Citrobacter spp.

Non- Lactose Fermenters

(Colorless)
Shigella spp.
Salmonella spp.
Proteus spp.

Triple Sugar Iron Agar (TSI)

Composition:
Triple Sugar: Lactose, Sucrose and Glucose (10:10:1 conc.)
Iron: Ferrous sulfate (Indicator of H2S formation)
Phenol red: Indicator of acidification (pH)
yellow - acidic condition
red - alkaline condition
Tissue extracts (Peptone - source of nitrogen)
when peptone is utilized under aerobic condition,ammonia is produced)
Procedure
With a sterilized straight inoculation needle touch the top of a well-isolated colony
Inoculate TSI Agar by first stabbing through the center of the medium to the bottom of the tube
and then streaking the surface of the agar slant.
Leave the cap on loosely and incubate the tube at 35 o in ambient air for 18 to 24 hours.
Interpretation
If lactose (or sucrose) is fermented large amount of acid produced red (A) both in butt and
in the slant
Some organisms generate gases, which produces bubbles/cracks on the medium.
If lactose is not fermented but the small amount of glucose is butt will be yellow (K); on the
slant, acid will be oxidized to carbon dioxide and water by the organism & by oxidative
decarboxylation of proteins the slant will be red (alkaline or neutral pH)
If neither lactose/sucrose nor glucose is fermented both the butt and the slant will be red

The slant can become a deeper red-purple (more alkaline) as a result of production of ammonia
from the oxidative deamination of amino acids (peptone)
if H2S is produced, the black color of ferrous sulfide is seen

Eosin Methylene Blue Agar (EMB)

Isolation and differentiation of lactose-fermenting and non-lactose fermenting enteric bacilli
For Escherichia coli identification
Principle

Inhibitors of Gram Positive bacteria (Eosin , Methylene blue)

Lactose - only carbohydrate source
Lactose Fermenters
(Purple)
Escherichia coli
(Green metallic sheen)
Klebsiella spp.
Serratia marcescens
Enterobacter aerogenes

Non-Lactose Fermenters
(Colorless)
Proteus spp.
Pseudomonas aeruginosa

Expected Results
Alkaline slant/no change in butt (K/NC)
Red/Red = glucose, lactose and sucrose nonfermenter
Alkaline slant/Alkaline butt (K/K)
Red/Red = glucose, lactose and sucrose nonfermenter
Alkaline slant/acidic butt (K/A)
Red/Yellow = glucose fermentation only, gas (+ or -), H2S (+ or -)
Acidic slant/acidic butt (A/A)
Yellow/Yellow = glucose, lactose and/or sucrose fermenter, gas (+ or -), H2S (+ or -).
Salmonella-Shigella Agar
Selective for Salmonella spp.
Principle
Inhibitors of Gram Positive bacteria - Bile Salts
Inhibitors of other Gram Negative bacteria - Brilliant green,Bile Salts
Lactose - only carbohydrate source
Sodium thiosulfate - Sulfur source (H2S production)
Ferric sulfide - black color (colonies with black centers)

** H2S + Ferric Ammonium citrate = Ferric sulfide

Organisms which ferment lactose produce acid which,
in the presence of the neutral red indicator, results in the formation of red colonies
Lactose-nonfermenters form colorless colonies (intestinal
pathogens, including Salmonella and Shigella)