ENTEROBACTERIACEAE

Enterobacteriaceae comprises a variety of gram negative bacilli referred to as enteric

bacilli or simply enterics.
*Most of them are ubiquitous, commensal organisms that exists in large numbers in
the human and animal large bowel, but may also be found on the skin, in the
oropharynx, and in water.
*Most are opportunistic pathogens (E.coli, Citrobacter, Enterobacter, Serratia,
Providencia, Arizona, Klebsiella, Proteus, and Yersinia enterocolitica).
*Some of them are true pathogens: Salmonella, Shigella, Yersinia pestis and some
pathogenic strains of E.coli ( EAEC- enteroadherent E.coli, ETEC-enterotoxigenic
E.coli, EHEC enterohemorrhagic E.coli), they usually cause gastrointestinal
infection.
General properties
1.Gram negative rods, non-spore forming
2.Aerobs, facultative anaerobic, grow in simple media
3.Ferment glucose and produce acid
4.Have peritrichous flagella and are motile ( some are immobile-Shigella)
5.Posses a capsule (e.g.,Klebsiella)
Organism

Major reservoirs

E.coli

Human GI tract

Diarrhea, urinary tract inf. neonatal

Animal carriers

meningitis____________________

---//---

Pneumonia, septicemia, inf. in IC or

Klebsiella

Citrobacter,Enterobacter Environment

Major Clinical Diseases

hospitalized patients

Serratia__________________________________________________________________________________________
Proteus spp.

Environment

Urinary tract inf._______________

Yersinia enterocolitica animal and human carriers Enterocolitis____________

ETEC

Human carriers

Travelers diarrhea

EHEC

---//----

Diarrhea, hemolytic-uremic syndrome

EAEC

---//----

Diarrhea______________________

Shigella

---//---

Dysentery_____________________

Salmonella typhy____---//---

Typhoid fever__________________

Salmonella enteritidis_Cattle, poultry______Gastroenteritis__________________

Determinants of pathogenicity
a)major antigens
K antigen (K Ag) : associated with polysaccharide capsules
H antigen (H Ag): flagellar (important for the serotyping of Salmonella
O antigen (O Ag): somatic. Differences in the polysaccharide chain of the
lipopolysaccharide (LPS, endotoxin) determine the type of O antigen.
b)Virulence factors:
Endotoxin (LPS)
Exotoxins (enterotoxins produced by E.coli, the Shiga toxins and the Shiga-like
toxins)
adhesion colonization factors
capsules and other protective surface antigens
The differentiation between different genus and species of enterics can be made
according with their biochemical properties.
I.Energetic metabolism
1.Oxidative and fermentative degradation of glucose => acids are formed and the
pH indicator change the color of the media from blue to yellow
2.Usage of ammonia as a source of energy (nitrate reduction) => red color appears
around the place where the microorganism grow
3.Citocromoxidaze negative.
If 1 and 2 are (+) and 3 is (-) than the microorganism is an enteric.
II.Metabolism of ammonia compounds
4.If the enteric produces L-phenilalanin-dezaminaze(E) it will degrade phenilalanin
(PhenAla) with the formation of phenyl piruvic acid. At the addition of a reactive,
FeCl3, a green color reesults
PhenAla +(E) =>phenyl piruvic acid + FeCl3 => green
Reaction (+) for Proteus spp.
5.Urease release ammonia from urea: ammonia causes the pH to rise and changes the
indicator from yellow to red
Reaction (+) for Proteus , Klebsiella, etc.
6.Lysine decarboxylase transforms lysine into a basic primary amine, cadaverine.
This amine causes a pH rise and a change of the indicator.

7.Indole test: Metabolism of tryptophane results in the formation of indole. Kovacs

reagent (reagent indole, which is yellow) forms a colored complex (pink to red) with
indole. (+) for E.coli
III. The fermentative type
8.Metil red reaction shows the mixed acid fermentation of glucose => red color
Reaction (+) for E.coli, Enterobacter, etc.
9.Voges-Proskauer reaction shows the acetoinic fermentation if a reagent (-naftol)
is added a red ring is formed at the interface of the two liquids (the media with
bacterial grow and reagent).
Reaction (+) for Klebsiella
10.Utilization of carbohydrate (Glu, Man, and Lac) results in acid formation and a
consequent pH decrease. The indicator changes to yellow.
IV.Minimal nutritional requirements
11.Growing on citrate media: citrate is the sole carbon source for some species.
Citrate utilization results in a pH rise and a change of the indicator from green to blue.
Reaction (+) for Klebsiella, Salmonella, etc.
Reaction (-) for E.coli and Shigella. These two may be differentiated by the
development on media with sodium acetate: Shigella (-) and E.coli (+)
For classification into tribes and genus, reactions 7, 8, 9, 11 (I.M.V.I.C) and H2S
production are considered.
12. Hydrogen sulfide is produced from thisulfate, reacts with iron salts and produces
a black precipitate.
Reaction (+) for Salmonella and Proteus spp.
ESCHERICHIA
E.coli is the most common cause of urinary tract infection, "traveler's diarrhea",
neonatal meningitis and sepsis with gram-negative.
General properties
-the most abundant facultative anaerobe in the colon and feces
-ferments lactose (property that distinguishes from the 2 major intestinal pathogens,
Shigella and Salmonella)
Determinants of pathogenicity

E.coli has several well known components that contribute to its ability to cause
disease: pili, capsule, endotoxin and 2 exotoxins (enterotoxins).
Clinical disease
1.Community -acquired urinary tract infections
-occurs primarily in women
2.Nosocomial (hospital-acquired) urinary tract infections
-equally frequent in men and women
-associated with the use of urinary catheters
Both infections may be limited to the bladder (designated as cystitis) or extended to
kidneys (designated as pyelonephritis).
Cystitis has as main symptoms pain (dysuria) and frequency of urination.
Pyelonephritis is characterized by fever, chills and flank pain.
3.Neonatal meningitis. Contamination took place during birth, due to the presence of
E.coli in the vagina of 25% of pregnant women.
4.Diarrhea caused by enterotoxigenic (ETEC) E.coli is usually watery, non-bloody,
self0limited, and of short duration (1-3 days). It is associated with travel.
5.Infection with enteropathogenic E.coli (EPEC) results in a dysenterialike syndrome,
with bloody diarrhea, abdominal cramping, and fever similar to that caused by
Shigella.
LABORATORY DIAGNOSTIC IN INFECTIONS WITH OPPORTUNISTIC
ENTERICS
It is only a direct diagnosis.
1.Sample collection: feces, urine, blood, CSF, pus, sputum
2.Microscopic examination : gram negative bacilli
-capsulated (in CSF, sputum, otic secretion) => may be Klebsiella
-gram negative rods in urine => urinary infection
-no relevance in feces
3.Inoculation on media for enterics
-differential media like MacConkey agar, which contains lactose and a pH indicator. If
lactose is fermented (Lac+), acid will be generated and the colonies will turn pink
-Drigalski (violet media and if the enteric is a Lac(+) one the media turn to yellow),
-Istrate-Meitert (green media and if the enteric is a Lac (+) turn to yellow ),
-blood-agar
4.Identification based on:

-morphological features: G(-) rods = red; if capsulated may be Klebsiella spp.; if

polymorphs may be Proteus spp.; if cocobacillar may be Yersinia spp.
-cultural features:
-Klebsiella colonies are mucous
-Proteus colonies look like cat eyes on Istrate media (green with a black
middle); they grow in concentric waves on Drigalski media ( invasion); they climb on
oblique agar, even in the condensed water.
-biochemical analysis and serotyping. Final identification of the species requires
biochemical analysis (glucose fermentation, nitrates reduction, production of indole
and H2S).
Some major clues are:
-Indole, metil red, H2S (-) and Voges-Proskauer, citrate (+) for Klebsiella
-Proteus spp. Are the only that produce phenylalanin-dezaminaze, so it is Phen

(+)

-generally E.coli has to be differentiated by Shigella;

-Salmonella species have to be differentiated by Citrobacter and Arizona.
Biochemical characteristics of E.coli
-produces indole from tryptophan (indole +)
-it decarboxylates lysine (+)
-it utilizes acetate as its only source of carbon (acetat +)
-it is motile (differentiation of Shigella)
Specific serotypes are indicated by the initial designation of the major antigen, and
then (for E.coli) by a number. Ex. O111:K55:H3
Treatment
Antimycrobial sensitivity test has to be performed because resistance is widespread
between this family of bacteria by means of conjugation.

SHIGELLA
General characteristics of Shigella species (spp.):
-nonmotile
-do not ferment lactose readily on MacConkey agar
-do not produce gas from fermentable carbohydrates

-do not produce H2S from thiosulfate

Classification is made according to differences in somatic (O) antigens:
a. Group A (S.dysenteriae)
b. Group B (S.flexneri)
c. Group C (S.boydii)
d. Group D (S.sonnei)
According with the others antigens, these groups are classified as follows:
S.dysenteriae is split into 10 antigenic types
1 is Shiga, 2 is Schmitzii, and from 3 to 10 are the Large-Sachs types.
S.flexneri is split into 6 antigenic types, some subtypes and 2 variants (x and y).
S.boydii is split into 15 antigenic types.
S.sonnei has only 1 antigenic type but two variants (S and R).
Although all species share the properties of being invasive and causing mucosal cell
death, the production of Shiga toxin is limited to S.dysenteriae type 1, which is only
released when bacteria die, causing the hemolytic-uremic syndrome by inducing
vascular damage.
Pathogenesis
Shigellae, which cause disease almost exclusively in the gastrointestinal tract, produce
bloody diarrhea 9dysentery) by invading the mucosa of the distal ileum and colon.
Local inflammation and ulceration occurs, but the organism rarely penetrate the wall
or enter the bloodstream, unlike salmonellae. Although some strains produce
enterotoxin, the critical factor in pathogenesis is the invasion.
Epidemiology:
Shigella is a true human pathogen, for which there is no animal reservoir. It is
transmitted by the fecal-oral route by direct contact (including sexual contact) or
through contaminated food and water.
Clinical disease
Shigella is the agent of bacterial dysentery (shigellosis).
The main symptoms of dysentery are:
-severe abdominal cramps
-frequent, painful passage of low volume stools containing blood and mucus
Laboratory diagnosis
To confirm the diagnosis, Shigella must be isolated from an adequate sample.
1.Sample collection: feces (obtained spontaneous or by Nelaton sonda) or rectal swab

-the samples have to be isolated on media in the first 2 hours after collection, if not
they have to be placed on a transport media (Cary-Blair)
2.Microscopic examination is only orientative: immobile, G(-) rods and maybe
hematia or leukocytes may be seen in a Gram smear
3.Inoculation on media for enterics (e.g. MacConkey, Drigalski, Leifsen, ShigellaSalmonella)
4.Identification based on:
-morphological properties: gram negative, non-motile rods,
-cultural characteristics: round, smooth (S), lactose (-) colonies
-biochemical characteristics provide the correct identification:
-

do not produce gas by fermentation,

do not ferment lactose,

do not have phenildesaminaze,

do not produce H2S,

do not grow on citrate or sodium acetate,

Voges Proskauer reaction is negative (-)

but indole and metil red are positive (+).

-antigenic characteristics allow the classification ( e.g. S.disenteriae, S.flexneri and so

on) and it is performed as an agglutination reaction on the slide and then, for further
classification in test tubes.
-pathogenic properties: Sereny test can confirm the invasiveness of isolated Shigella,
however, this test is not routinely used. A drop of invasive isolate deposited in the
cornea of a guinea pig or rabbit will produce severe keratoconjunctivitis within few
days, followed by ulceration and then cure within 2-3 weeks
5.Treatment: according with sensitivity.
Drugs of choice are Ampicillin and Amoxicillin, but if the antibiotic susceptibility of
the infecting strain is unknown, trimetoprim-sulfamethoxazole may be preferred,
because resistance to this drug is less common.
Prophilaxis
Prevention is based on proper personal and colectivity hygiene for the interruption of
fecal-oral transmission by proper sewage disposal, chlorination of water and hand
washing.
SALMONELLA
General properties:

-do not ferment lactose on MacConkey agar (Lac (-)

-produce gas when fermenting glucose
-produce H2S from thiosulfate
Classification:
S.typi, S.paratyphi A and B, S.enteritidis (subclassified into hundreds of serotypes)
There are a lot of Salmonella that parasitate animals.
Diseases
Different species and serotypes of Salmonella cause different diseases.
a)Major salmonelosis: typhoid and paratyphoid fever, affects humans exclusively,
is acquired via fecal-oral transmission, contaminated water (following an earthquake
of flood) or food processed by asymptotic chronic carriers.
-first week: lethargy, malaise, fever, aches and pains, constipation
-second week: high fever, tender abdomen, rose-colored spots on the skin, diarrhea,
hepatosplenomegaly
b) Minor salmonelosis: are increased in number nowadays and they manifest as food
poisoning in which animal species of Salmonella are involved:
-S.typhimurium, S.anatum, S.enteritidis
-after ingestion of poultry meat, milk, eggs
Symptoms of minor salmonelosis have to be differentiated by the stafilococcic food
poisoning.
-longer incubation (24-48 hours)
-fever
-diarrhea
-self limiting after 2-5 days
Laboratory diagnosis in typhoid and paratyphoid fever
A. Direct diagnosis
1.Sample collection: blood and urine in the first week; feces beginning with the
second week; bile and feces after purgation in carriers.
In food poisoning: food, vomiting, feces, blood
2.Microscopic examination is irrelevant
3.Inoculation on media
* Blood (5-10ml) is isolated in 20 ml of sterile ox bile and is kept 7 days at incubation
(37C). Each day inoculations are made on solid media for enterics and if Lac (-)
colonies developed they are isolated in tubes with gelose.

* Feces are mixed with saline, a first isolation on solid media is performed. Then a
quantity is placed in enrichment media (sodium acid selenite) for 12 to16 hours and a
second isolation on solid media is performed.
* Bile is placed on media as it was collected and a second isolation is made after
incubation in enrichment media
* Urine (5-10ml) is first centrifuged and the sediment is inseminated on solid media,
or incubated in enrichment media and inseminated thereafter to obtain a pure culture
4.Idedntification is based on:
-morphologic properties: gram negative, mobile rods,
-cultural properties: the same as other enterics
-biochemical characteristics:
-

production of H2S (Black = +),

gas formation on TSI (triple sugar iron) agar

grows on citrate (+),

acid mix fermentation of glucose => methil red (+),

form lactose negative colonies (colorless) on MacConkey's or Drigaslky

agar (-),

Indole (-),

Do not have phenilalanindesaminase =>Phen-Ala(-)

Observation: S.typhi is the major exception; it does not form gas and produces only a
small amount of H2S.
-antigenic characteristics is based on Kauffman White antigenic scheme which is
applied in public health laboratories for epidemiologic purposes. Salmonela
serotyping is made according with :
-O Ag => 65 different factors (A, B,29,..65); more than one may be present
on a strain;
-H Ag => phase 1 (a, b, cz1, z2..z15)
and phase 2 (1, 2, 3..)
(these two phase are disjunctive on the same cell ( e.g. if one is present the other is
missing), but they may be present both in the culture.
-K (Vi) Ag is the virulence antigen ( give inaglutinability to the strain)
The serologic identification is performed by agglutination reaction on the slide with
progressively specific serum.
B. Indirect diagnosis
Serologic diagnosis is made by Widal reaction.

-2-3 blood test are collected at one week period and antibodies against S.typhi,
S.paratyphi A and B, separately for O and H antigens, are identified.
Technique: 6 rows with 6 test tubes
-each row has a progressively serum dilution and a specific Ag: TH, AH, BH,
(antigen H agains S.typhi, S.paratyphi A and B) and TO, AO, BO (antigen O against
S.typhi, S.paratyphi A and B).
-the agglutination titer is the greater dilution at which agglutination can be seen.
Observations: The titer is greater in week 3 to 4. Healthy people may have a titer as
great as 1/160, vaccinated people may have (in the first 3 months) a titer as big as
1/1000, against H Ag; if the person was treated with Chloramfenicol antibodies
against O Ag failed to form.
Treatment:
Treatment is made according to antibiotic sensitivity test. Otherwise Chloramfenicol
is the drug of choice. Ampicilline and trimethoprim-sulfametoxazole are alternatives
with less severe side effects.
Prophylaxis
Prevention is based on public health and personal hygiene measures: proper sewage
treatment, chlorinated water supply, periodic cultures of stool samples from food
handlers to detect carriers, hand washing prior to food handling, pasteurization of
milk, and proper cooking of poultry and meat.
Two vaccines are available but their protection is limited (50-80%) against S.typhi.
One is administered intramuscularly (acetone-killer microorganisms) and one is taken
orally (live, attenuated microorganisms).