Gram-Negative Rods
General Classification
Based on source or site of infection
1. Enteric tract
2. Respiratory tract
3. Animal sources
Source or site of infection
Enteric tract
Primarily within:
Shigella, vibrio, Campylobacter
Both within and outside:
Escherichia, Salmonella
Enteric but outside only:
Klebsiella, Enterobacter, Serratia, Proteus, Providencia,
Morganella, Pseudomonas, Bacteroides
Source or site of infection (continued)
Respiratory tract:
Haemophilus, Legionella, Bordetella
Animal sources:
Brucella, Francisella, Pasteurella, Yersinia
Classification
Based on morphology, biochemical traits and
genetic (phylogenetic) relationship
Cocobacilli
Vibrionaceae
Pseudomonaseae
Enterobacteriaceae
Enterobacteriaceae
A heterogenous family
Mostly found in colon of human and other animals
Different pathogenetic mechanisms
Facultative anaerobic
Glucose fermentation
None have cytochrome oxidase
Reduce nitrates to nitrites
Most important genuses
in Enterobacteriaceae family
Escherichia
Shigella
Salmonella
Klebsiella
Enterobacter
Serratia
Proteus
Yersinia
In contrast to
Enterobacteriaceae
Pseudomonaceae
Gram negative rods:
Non-fermenting (Strict aerobic)
Not reduce nitrate
Oxidase-positive
General structure of cells in
Enterobacteriaceae
All have Endotoxin
Some have Exotoxins, mostly called enterotoxins
Three surface antigens:
O antigen: outer polysaccharide portion of the lipopolysaccharide (repeating
3-4 oligosaccharide sugars 15-20 times). A basis for the serologic typing
(about 2000 types of Salmonella and 150 types of E. coli).
H antigen: on the flagellar protein (in E.coli and Salmonella and not in
Klebsiella and shigella). Unusual H antigens in Salmonella called phase
1 and phase 2. The organism can reversibly change in antigenicity to
evade the immune response.
K polysaccharide antigen: In encapsulated organisms such as Klebsiella.
Identified by quellung (capsular swelling) reaction in the presence of
specific antisera used for epidemiologic purposes. In S. typhi, it is called
Vi (or virulence) antigen.
Quellung (capsular swelling) reaction
Coliforms
That part of this family which are normal
inhabitants of the colon:
E. coli
Enterobacter
Klebsiella
Citrobacter
So, E.coli is the indicator for
fecal contamination of water supply:
Coliforms
Lactose fermentation, Acid and gas production, growth
at 44.5 C and typical colony on EMB.
4 colony count per dL in drinking water is indicative of
unacceptable fecal contamination.
Antibiotic therapy
Must be individually tailored to the antibiotic
sensitivity test (Antibiogram).
Penicillin and cephalosporin families.
Aminoglycosides (Gentamicin, amikacin, kanamycin,
streptomycin …), Chloramphenicol, tetracyclines,
quinolones and sulfonamides.
Laboratory diagnosis
Culture for isolation
Suspected specimens are inoculated onto 2 media: 1.
Blood Agar 2. A selective differential medium
(MacConkey’s agar or Eosin-methylene blue, EMB
agar. The differential ability is based on lactose
fermentation as the most important criterion in
identification of these organism. Non lactose
fermenters form colorless colonies. Selective effect
is exerted by bile salts or bacteriostatic dyes.
Laboratory diagnosis (continued)
Culture for identification
Screening biochemical tests for a final definitive
identification:
Array of 20 or more biochemical tests to identify the
species.
Serology
Usually in Salmonella, Shigella and E.coli the final
detection is by serotyping using agglutination Ag+Ab
test.
Triple Sugar Iron Agar
Almost enough to identify the genus
Indicator : Phenol red
Components:
 Iron or Ferrous sulfate
FeSO4 (ferrous sulfate) + Solfate reductase  SH2  Sodium
tiosufate
Ferrous sulphide (FeS)
Black FeS indicates the production of SH2
 3 sugars: glucose (0.1%), lactose (1%), and sucrose (1%)
 Pepton and yeast extract
Different observations for TSI
Reactions
Slant
Button
Ga
s
H2 Representative genera
S
Acid
Acid
+
-
Escherichia, Enterobacter, Klebsiella
Alkaline
Acid
-
-
Shigella, Serratia
Alkalin
Acid
+
+
Salmonella, Proteus
Alkalin
Alkali
n
-
-
Pseudomonas
Kligler Iron Agar can be used as alternative with two
sugers: glucose & lactose
Urea Agar
Indicator: Phenol red
Component : Urea
If the bacterium Produces
urease:
Urea (NH2)2CO is hydrolyzed to
NH3 and CO2
light orange
changes to reddish purple (in
Proteus and K pneumoniae
Amonium citrate (Simmons Citrate)
Indicator: Bromothymol blue
If the bacterium can utilizes
ammonium dihydrogen phosphate
(a salt of ammonium) and
sodium citrate as sole source of
nitrogen and carbon, the indicator
turns to blue at alkalin pH due to
releasing ammonia.
Motility
SIM medium: SH2, Indole, Motility
Proteus: Swarms
Differentiation between Enterobacter cloacae (motile)
from Klebsiella pneumoniae (Non motile)
Indole (in SIM)
Tryptophan + Tryptophanase  Deamination 
Intermediate products: Indole + ….
Detection: 5 drops Kovac’s reagent (contains paradimethyl-amino-benzaldehyde = PDAB) is added 
Benzyl pyrol  Red ring
MR-VP
1. Methyl Red
MR-VP medium
Components: Glucose, Phosphate
After 48 hours  Methyl-red (1 droplet to 1 ml of
medium) :
I. If pH < 4.4  Mixed acidic fermentation (Red
color).
II. If pH ≥ 5  Butylen glycol fermentation (Yellow
color).
MR-VP
2. Voges Proskauer
Butylen glycol fermentation
MR-VP medium: After 24-48 hours  Alcoholic alpha
naftool (15 droplets to 1 ml medium) + KOH 40%
(10 droplets) (may come along cratin)  15-30 min:
I. Red (If Acetoin “Acetyl methyl carbynol” exists.)
II. No color change (If no Acetoin exist)
API 20E system
A plastic strips consist of 20 small wells containing
dehydrated media components
1. The bacterium is suspended in sterile saline and
added to each well.
2. The strip is incubated for 16-24 hours.
3. The colour reactions are noted as either positive or
negative.
4. The test results can be entered into a computer
programme to identify the bacterium.
API 20E system
Escherichia coli
Diseases
1. Diarrhea or dysentery
2. UTI
3. Sepsis (The most common cause among negative
rods)
4. Neonatal meningititis (One of the 2 important
agents. The other is the group B streptococci due to
colonization of vagina by these organisms in about
25% of pregnant women ).
5. Nosocomial infection
UTI
Sepsis
Neonatal
meningititis
Virolence factors:
Pili
Capsule
Endotoxin
Two exotoxins (enterotoxins).
Pathogenesis
E. coli attaches to the surface of jejunum and ileum
by Pili  Bacteria synthesize enterotoxins
(exotoxins determined by plasmids)  Diarrhea
The toxins are strikingly cell-specific. Cells of colon
are lack of receptors for the toxins.
E. coli pili (fimbriae)
mannose
galactose
glycolipids
glycoproteins
43
Most important sub species of
pathogenic E. coli
Enteropathogenic E. coli (EPEC)
Enterotoxigenic E. coli (ETEC)
Enteroinvasive E. coli (EIEC)
Enterohemorrhagic E. coli (EHEC)
Enteropathogenic E. coli (EPEC)
destruction of surface microvilli
Gut lumen
fever
diarrhea
vomiting
nausea
non-bloody stools
Diarrhea is self-limited and short duration (1-3 days)
45
Enterotoxigenic E. coli
Travellers diarrhea
Diarrhea like cholera but milder
Diarrhea is self-limited and short duration (1-3
days)
46
Enterotoxigenic E. coli (ETEC)
Heat labile toxin (LT)
like choleragen
Activation of Adenylate cyclase
Cyclic AMP concentration
Secretion water/ions (potassium and chloride)
Heat stable toxin (ST)
Activation of Guanylate cyclase
Cyclic GMP concentration
Uptake water/ions (Sodium and Chloride)
47
Enterohemorrhagic E. coli (EHEC)
Produce verotoxin which works like Shiga toxin
Hemorrhagic
bloody, copious diarrhea
few leukocytes
Hemolytic-uremic syndrome
thrombocytopenia (low platelets)
hemolytic anemia
kidney failure
Enterohemorrhagic E. coli
Usually O157:H7
Flagella
Transmission electron
micrograph
49
Enteroinvasive E. coli (EIEC)
Very similar to shigella species
(in biochemical and morphological traits)
Invades to epithelial mucosal cells
Cause enteric inflammation
Non lactose fermentative
Non motile
E. coli Transmission
By:
Meat products or sewage-contaminated vegetables
51
UTI
The most common agent for UTI and nosocomial UTI.
(Cystitis, pyelonephritis): fever, chills, flank pain
Occurs primarily in women
Systemic infection
Capsule and endotoxin play a prominent role
Capsular polysaccharide interferes with phagocytosis
(Serotype having K1 causes neonatal meningitis).
LPS during sepsis causes fever, hypotension and
disseminated intravascular coagulation.
Treatment
Antibiogram for most infections
A combination of ampicillin and gentamicin in
neonatal meningitis
Rehydration for diarrhea
Prevention
No passive or active immunization
Prompt withdraw of catheters and intravenous
lines
Caution regarding uncooked food and unpurified
water while traveling.
Klebsiella
Klebsiella ozaenae
- Ozena (the atrophy of nose with bad smelling)
K. rhinoscleromatis
- Rhinoscleroma (A granulomatosis in nose and
pharynx)
Klebsiella penomoniea
- 5% in upper respiratory and GI systems
- Nosocomial infection
Klebsiella oxytoca
- Nosocomial infection
Lab detection
Large mocoidal colonies.
Lac pos.
Not motile
Proteus
P. mirabilis
UTI (Alkalic environment
in urinary tracts)
due to urease  kidney stone
P. vulgaris
Nosocomial infection
Shigella
Species:
S. dysenteriae
S. sonnei
S. flexeneri
S. bouedi
There are more than 40 shigella serotypes.
Important properties
- Non lactose fermenting
- Distiguishable from Salmonella by: no gas, no H2S,
nonmotile.
- Having an enterotoxin called Shiga toxin
Shigellosis
Only a human disease
Transmitted from person to person by asymptomatic
carriers (oral-fecal transmission)
4 F’s – fingers, flies, food, feces
Food-born outbreaks outnumber water-born
outbreaks by 2 to 1.
In mental hospitals and day-care nurseries
Children <7 accounts for half of shigella positive stool
culture
Shigella disentery type 1 (Shiga bacillus):
- Labile Exotoxin
(effective both on intestine and CNS works like
verotoxin in E.coli)
Pathogenesis
Exclusively in gastrointestinal tract
Bloody diarrhea (dysentery): Invading the mucosa
of the distal ileum and colon.
Local inflammation accompanied by ulceration
occurs
The organisms rarely penetrate the wall or enter
the bloodstream unlike salmonellae.
Invasion to epithelial cells (M cells)  spreading to
next cells  microabcess formation in the wall of
colon and distal ileum  necrosis of mocusal layer
 ulcer  bleeding  psudomemberane.
Clinical findings
Incubation period: 1-2 days
Symptoms: Fever, abdominal cramps, followed by
diarrhea (watery at first but later contains blood and
mucus).
Mild or severe disease depending:
 - The species of Shigella
 - The age of the patient
Most invasive species
S. dysenteriae causes the most severe disease
S. sonnei causes mild disease but more frequent
Shiga toxin neurotoxic effects
Shiga toxin CNS abnormalities can include
lethargy
disorientation
paralysis
coma
Neurotoxicity occurs most often in children and the
elderly and is often fatal.
Clinical findings
Resolves in 2-3 days but antibiotic can shorten the
course.
Serum antibodies appear after recovery but are not
protective.
Most patients spread bacteria only short time after
recovery but few people stay chronic carriers.
Most shigellosis cases are in children less than 10
years.
Treatment
In 50% cases self recovery is in 2-5 days.
The main treatment: Fluid and electrolyte
replacement.
No antibiotic in mild cases
Antibiogram test: Trimethoprim - sulfamethoxazole
or Ampiciln.
Prevention
Interruption of fecal-oral transmission by proper
sewage disposal, chlorination of water and personal
hygiene.
No vaccine
Antibiotic prophylactic is not recommended.
Lab diagnosis
Samples:
feces, rectal swabs
Culture and biochemical tests: Non-lactose
fermenter
Serology tests:
Slide agglutination to detect its species
Lab diagnosis
Invasive enteric infections:
Shigella, Salmonella or Campylobacter
Toxin-producing organism:
V. cholerae, E. coli, Clostridium perfringens or
certain viruses or Entamoeba histolytica
Methylene blue stain of a fecal sample to determine
whether PMNs are present.
Salmonella
Important properties
Not lactose - fermentive
Produce H2S, Gas, motile
Salmonella nomenclature
Is complicated.
Currently there are two recognized species:
S. enterica and S. bongori
S. enterica has 5 main subspecies.
S. enterica enterica is relevant with human infections
(specific or zoonotic).
Naming the salmonella
S. enterica enterica:
1. S. typhi
2. S. paratyphi (A, B, C…)
3. S. typhimurium
4. S. choleraesuis
5. S. enteritidis (1500 serotypes)
Diseases
Enterocolitis (S. typhimurium & S. enteritidis)
Enteric fever (typhoid fever) (S. typhi and S.
paratyphi)
Septicemia with metastatic abscesses (S.
choleraesuis)
Diseases
Enterocolitis (S. typhimurium & S. enteritidis)
An invasion of epithelial and subepithelial tissue of
small and large intestines.
Penetration both through and between the mucosal
cells: Inflammation and diarrhea.
PMN response limits the infection to the gut and the
adjacent mesenteric lymph nodes.
The dose of Salmonella required: at least 105 - 108
while for Shigella: 103 organisms.
Clinical findings of Enterocolitis
Incubation period: 6-48 hours
Symptoms:
Nausea, Vomiting, Abdominal pain and diarrhea with or
without blood
The disease is self-limited. Treatment only in very
young and very old.
S. Typhimurium & S. enteritidis : the most common
cause of enterocolitis.
S. typhimurium colonies
Typhoid or Enteric fever
Typhoid or Enteric fever caused by S. typhi and S.
paratyphi (A, B and C).
 The illness is slow, with fever and constipation
rather than vomiting and diarrhea.
 After the first week, bacteremia becomes sustained.
High fever, tender abdomen, and enlarged spleen
occur.
Typhoid (Enteric fevers)
Infection begins in small intestine but few
gastrointestinal symptoms occur.
The organisms multiply in the mononuclear
phagocytes of peyer’s patches, then spread to the
phagocytes of the liver, gallbladder and spleen
leading to bacteremia and then fever.
Typhoid (continued)
Rose spot (rose-coloured papules) on the abdomen
are associated with typhoid fever but occur only
rarely.
The disease begins to resolve by the third week but
intestinal hemorrage or perforation can occur.
3% of typhoid fever patients become chronic
carriers. The carrier rate is higher among women.
Septicemia
S. choleraesuis: most often cause septicemia.
Symptoms:
Fever
Little or no enterocolitis
Focal symptoms: bone, lung, or meninges.
Septicemia (S. choleraesuis)
Accounts for only about 5-10% of Salmonella
infections and occurs:
More common in patients with chronic disease or
children with enterocolitis.
It leads to seeding of many organs commonly:
osteomyelitis, pneumonia, and meningitis
Typhoid vaccine
Two types:
Live vaccine
Subunit vaccine (a vi capsular polysaccharide
vaccine)
Transmission of salmonella
Ingestion of food and water contaminated by human
and animal wastes.
S. typhi, transmitted only by humans, but other
species have a significant animal reservoir.
Human sources:
 1. Temporarily excrete the organism during or
shortly after enterocolitis
 2. Chronic carriers
Transmission
The most frequent animal source is poultry and
eggs, but meat products that are inadequately
cooked have been implicated as well.
Dogs and other pets including turtles are additional
sources.
Lab. diagnosis
Enterocolitis: isolated from stool
Enteric fever: blood culture during first 2 weeks of
illness.
Septicemia: Blood culture
Lab. diagnosis
MacConkey, EMB, Hekton Enteric agar, XLD, Endo
agar
Lactose -, other biochemical tests
Gas and H2S (S. type: no gas)
Serological tests by their O, H and Vi antigens
Serological detection for Ab if culture is negative:
Vidal test
Treatment
Enterocolitis:
Self-limited.
Fluid and electrolyte replacement.
Antimicrobial agents are indicated only for
neonates or persons with chronic disease who are at
risk of septicemia and disseminated abscesses.
Treatment
Enteric fever and Septicemia:
Ampicillin or chloramphenicol
Ampicillin: in patients who are chronic carriers of
S. typhi.
Cholecystectomy may be necessary to abolish the
chronic carrier state.
Focal absesses should be drained.
Prevention
Public health and personal hygiene measures.
Proper sewage treatment
A chlorinated water supply
Cultures of stool samples from food handlers
Two vaccine confer protection against S. typhi but no
common
Proper cooking of poultry and meat
Pasteurization of milk
Genus: Yersinia
Species: Yersinia pestis
and Yersinia enterocolitica
Small gram negative bacillus, bipolar staining
(like a safety pin)
Capsule in freshly isolated organism but lost
with passage
Plague or black death
None motile
Disease
Virulence factors
Capsule antigen (F-1) which protects against
phagocytosis.
Endotoxin
Exotoxins (block beta adrenergic receptors)
Coagulase
Fibrinolysin
Pesticin I (a bacteriocin)
V antigen protein
W antigen protein
Pathogenesis
Urban cycle
Transmission of the bacteria among urban rats
with the rat flea as vector to human.
This cycle predominates during times of poor
sanitation, eg. Wartime, when rats proliferate and
come in contact with the fleas in the sylvatic cycle.
Sylvatic cycle
Humans are accidental hosts and cases of
plague occur as a result of being bitten by a flea
that is a part of the sylvatic cycle.
Event within the flea
The flea ingests the bacteria while taking a blood meal
from a bacteremic rodent.  The blood clots in the flea’s
stomach owing to the action of the enzyme coagulase
made by the bacteria  The bacteria are trapped in the
fibrin and proliferate to large numbers.  The mass of
organisms and fibrin block the proventriculus of the
flea’s intestinal tract. During its next blood meal the flea
regurgitates the organisms into the next animal 
Because the proventriculus is blocked, the flea gets no
nutrition.  becomes hungrier  loses its natural
host selectivity for rodents  more readily bites a
human  The bacteria inoculated by bite spread to the
regional lymph nodes
 become swollen and tender called buboes and this
plague is called bubonic plague. The organisms can
reach high concentrations in the blood and disseminate
to form abscesses in many organs including lungs. 
The endotoxin-related symptoms, including
disseminated intravascular coagulation and cutaneous
hemorrhages, probably were the genesis of the term
‘black death’.
Respiratory droplet transmission
Respiratory droplet transmission of the organism
from patients with pneumonic plague can occur.
Clinical findings
Bubonic plague, is the most frequent form, begins
with pain and swelling of the lymph nodes
draining the site of the flea bite and systemic
symptoms such as high fever, myalgia, and
prostration.
The buboes are an early characteristic finding.
Septic shock and pneumonia are the main lifethreatening subsequent events.
Epidemiology
Endemic in the wild rodents of Europe and Asia for
thousands of years.
99% of cases of plague occur in Southeast Asia.
Enzootic (sylvatic) cycle consists of transmission
among wild rodent by fleas.
Rodents are relatively resistant to disease.
Humans are accidental hosts and cases of plague
occur as a result of being bitten by a flea that is a part
of the sylvatic cycle.
Pneumonic plague can arise either from inhalation
of an aerosol or from septic emboli that reach the
lung.
Untreated bubonic plague is fatal in approximately
half of the cases.
Untreated pneumonic plague is invariably fatal.
Lab. diagnosis
The best procedure: Smear and culture of blood or
pus from the bubo.
Giemsa’s or Wayson’s stain reveals the typical
safety-pin appearance of the organism better than
does Gram’s stain.
Fluorescent-antibody staining can be used to
identify the organism in tissues.
A rise in antibody titer to the capsule antigen can be
useful.
Treatment
The treatment of choice is a combination of
streptomycin and tetracycline.
Due to the rapid progression of the disease,
treatment should not wait for the results of the
bacteriologic culture.
Incision and drainage of the buboes are not usually
necessary.
Prevention
Prevention
Controlling the spread of rats in urban areas.
Preventing rats from entering the country by ship
or airplane.
Avoiding flea bites
Avoiding contact with dead wild rodents.
A patient with plague must be placed in strict
isolation (quarantine) for 72 hours after antibiotic
therapy is started.
Only close contacts need to receive prophylactic
tetracycline.
There is no vaccine for citizens normally. But a
killed organism vaccine protecting bubonic but
not pneumonic plague was used by USA forces
during Vietnam wars.
Yersinia enterocolitica
Motile: flagella are present at 22 c.
No capsule
Primarily a zoonotic disease (cattle, deer, pigs, and
birds)
Yersiniosis : Incubation 1-2 days.
Acute Y. enterocolitica infections produce severe
diarrhea in humans, along with Peyer's patch
necrosis, chronic lymphadenopathy, and hepatic or
splenic abscesses. Fever and right-sided abdominal
pain.
Lab diagnosis
Culture: Cold enrichment
Culture on blood
Sample: Feces
Y. pseudotuberculosis
Motile: flagella are present at 22 c., No capsule.
Culture on blood.
Primarily a zoonotic disease (cattle, deer, pigs, and
birds).
In animals, can cause tuberculosis-like symptoms,
including localized tissue necrosis and granulomas in
the spleen, liver, and lymph node.
In humans, symptoms of Pseudotuberculosis
(Yersinia) are similar to those of infection with
Yersinia enterocolitica (fever and right-sided
abdominal pain), except that the diarrhea is often
absent.