Enterobacteriaceae

Discovery of causal agentsa history

1850s correlations between enteric
disease and contaminated water (Snow
and Budd)
1880s discovery of microbial disease
agents (Koch)
1880s use of Bacillus coli as indicator
for fecal contamination (Escherich)

assessment methodologiesa background

1904 assays for E. coli using glucose
broths (Eijkman)
1920s multiple tube fermentation with
lactose broths (Leiter)
1951 membrane filtration developed
(Goetz & Tsuneishi)
1988 defined substrates developed
(Edberg et al.)

Enterobacteriaceae
The largest, most heterogeneous
collection of medically important gramnegative rods
>40 genera and 150 species
Fewer than 20 species are responsible for
more than 95% of the infections
Ubiquitous organisms, found worldwide in
soil, water, and vegetation

 part of the normal intestinal flora

30% to 35% of all septicemias, more than
70% of urinary tract infections (UTIs), and
many intestinal infections
Salmonella typhi, Shigella species,
Yersinia pestis
Escherichia coli, Klebsiella pneumoniae,
Proteus mirabilis

 become pathogenic when they acquire

virulence factor
can originate from an animal
or from a human carrier
or through the endogenous spread of
organisms

Enterobacteriaceae
Opportunistic pathogens

Meningitis
Pneumonia

Escherichia coli
Klebsiella pneumoniae

Sepsis

Enterobacter aerogenes
Serratia marcescens

Diarrhea

Proteus spp.
Providencia spp.
Citrobacter spp.
Obligate pathogens

Salmonella spp.
Shigella spp.
Yersinia spp.
Some E. coli strains

UTI

Dr Praveg Gupta MD

Enterobacteriaceae
moderately sized (0.3-1.0 1.0-6.0 m)
gram-negative rods
either nonmotile or motile with peritrichous
flagella
do not form spores
facultative anaerobes
have simple nutritional requirements

Gram stain of Salmonella typhi from a positive blood culture.

Note the intense staining at the ends of the bacteria.
This "bipolar staining" is characteristic of the Enterobacteriaceae

Enterobacteriaceae

Resistance to bile salts

Some have capsules

Common Medically Important Enterobacteriaceae

Citrobacter freundii, Citrobacter koseri
Enterobacter aerogenes, Enterobacter cloacae
Escherichia coli
Klebsiella pneumoniae, Klebsiella oxytoca
Morganella morganii
Proteus mirabilis, Proteus vulgaris
Salmonella enterica
Serratia marcescens
Shigella sonnei, Shigella flexneri
Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis

 LPS: Consists of three components:

the outermost somatic O polysaccharide
a core polysaccharide common to all
Enterobacteriaceae (enterobacterial common
antigen)
lipid A

Lipopolysaccharide (LPS)
is also called endotoxin.
LPS is composed of lipid A,
core polysaccharide, and Ospecific polysaccharide.
Lipid A anchors LPS in the lipid
bilayer of outer membrane. It
causes symptoms associated
with endotoxin.
O-specific polysaccharide can
be used to identify certain
species and strains.
Back

 serologic
classification
O polysaccharides
capsular K antigens
(type-specific
polysaccharides)
the flagellar H proteins

Antigenic structure of Enterobacteriaceae

Enterobacteriaceae
Common Virulence Factors Associated with
Enterobacteriaceae
Endotoxin
Capsule
Antigenic phase variation
Type III secretion systems
Sequestration of growth factors
Resistance to serum killing
Antimicrobial resistance

ENTEROBACTERIACEAE
A large Family of aerobic bacterial flora of intestine of
humans and other animals.
Its members are nonsporting, non acid-fast, gram
negative bacilli.
Capsule
Motility
General features aerobic and facultatively anaerobic,
grow readily on ordinary media, ferment glucose, reduce
nitrates to nitrites and form catalase but not oxidase.
.

16

Catalase, glucose, nitrate +ve; oxidase -ve

17

 Wide biochemical and antigenic heterogeneity.

Genetic mechanisms like conjugation and transduction in
these bacteria contribute to their infinite variety.
Various classifications of Enterobacteriaceae have been
put forward.
Two important classifications are 1. based on taxonomy
and 2. based on lactose fermentation.

18

Lactose fermenter v/s non fermenter

19

TAXONOMICAL CLASSIFICATION
ENTEROBACTERIACEAE
Tribe I: Escherichia

Tribe III: Proteae

Genus

Genus

Escherichia
Edwardsville
Citrobacter
Salmonella
Shigella

Proteus
Morganella
Providencia

Tribe IV: Erwinieae

Genus

Tribe II: Klebsiella

Erwinia

Genus

Klebsiella
Enterobacter
Hafnia
Serratia

20

 Genus Escherichia named after Theodor Escherich

who was the first to describe the colon bacillus under the
name Bacterium coli commune (1885).

Species:
E.coli,
E. fergusonii,
E. hermanii,
E. vulneris,
E. blattae etc.

21

ESCHERICHIA COLI

22

Escherichia coli

five species
sepsis, UTIs, meningitis, gastroenteritis
Gram-negative, facultative anaerobic rods
Fermenter; oxidase negative
Outer membrane makes the organisms
susceptible to drying
Lipopolysaccharide consists of outer somatic O
polysaccharide, core polysaccharide (common
antigen), and lipid A (endotoxin)

Escherichia coli
the most common gram-negative rods
isolated from patients with sepsis
responsible for causing more than 80% of
all community-acquired UTIs
gastroenteritis in developing countries
Most infections are endogenous

Escherichia coli

Incidence of Enterobacteriaceae associated with bacteremia

 Diseases
Bacteremia (most commonly isolated gram-negative rod)
Urinary tract infection (most common cause of bacterial
UTIs); limited to bladder (cystitis) or can spread to kidneys
(pyelonephritis) or prostate (prostatitis)
At least five different pathogenic groups cause
gastroenteritis (EPEC, ETEC, EHEC, EIEC, EAEC); most
cause diseases in developing countries, although EHEC is
an important cause of hemorrhagic colitis (HC) and
hemolytic uremic syndrome (HUS)
Neonatal meningitis (usually with strains carrying the K1
capsular antigen)
Intraabdominal infections (associated with intestinal
perforation)
Most infections are endogenous

Escherichia coli
Neonatal Meningitis: E. coli and group B
streptococci
Gastroenteritis

enteropathogenic (EPEC)
enterotoxigenic (ETEC)
enterohemorrhagic (EHEC)
enteroinvasive (EIEC)
enteroaggregative (EAEC) E. coli

Pathogenic E. coli

Escherichia coli-UTI
Colon
contaminate urethra
ascend into bladder and may migrate to
kidney or prostate
UTIs: adhesins (primarily P pili) and
hemolysin HlyA

Escherichia coli-Neonatal
Meningitis
E.coli and group B streptococci major
CNS pathogens
K1 capsular antigen

Escherichia coli-Septicemia
May be originated from UT
Mortality is high

MORPHOLOGY
Gram negative bacilli
1-3 x 0.4-0.7 m
Single, pairs
Motile by peritrichate flagella
Found in some capsules, fimbriae, immobility
Non spore forming
.

32

SHOWING GRAM NEGATIVE BACILLI

AND PERITRICHOUS FLAGELLA

33

CULTURE CHARACTERISTICS
Aerobe and facultative anaerobe
10-40C (37C)
S = smooth forms seen in fresh isolates, easily
emulsifiable in saline.
R = rough forms seen in older cultures, with irregular dull
surface, often autoagglutinable in saline.
S-R variation occurs as a result of repeated subcultures
and is associated with the loss of surface antigens and
usually of virulence.
.

34

 Many pathogenic isolates have polysaccharide capsules.

Some strains may occur in the mucoid form.
Nutrient agar colonies are large, thick, greyish white,
moist, smooth, opaque or partially translucent discs.
Blood agar - Many strains esp. pathogenic ones are
hemolytic on blood agar.
MacConkey medium - colonies are bright pink due to
lactose fermentation.
Broth general turbidity, heavy deposit.
.

35

ECOLI ON NUTRIENT AGAR

36

ECOLI ON BLOOD AGAR

37

ECOLI IN BROTH

38

E.COLI ON MACCONKEY AGAR

39

BIOCHEMICAL REACTIONS
Sugar fermentation glucose, lactose, manitol, maltose
and many other sugars fermented with acid and gas
production.
Sucrose generally not fermented.
IMViC ++- Gelatin -, H2S -, urease -.
.

40

BIOCHEMICAL REACTIONS OF E.coli

41

ANTIGENIC STRUCTURE
O = somatic antigen
K = capsular antigen
H = flagellar antigen
So far, >170 types of O, 100 types of H and 75 types of K
have been identified.
Antigenic pattern of an organism based on these antigens
is written as eg. O111:K58:H2, O54:K27:H41 etc.
K antigen is the acidic polysaccharide antigen located in
the envelope or microcapsule (K for kapsel, german for
capsule).
It encloses the O antigen and renders the strain
inagglutinable by the O antiserum.
It may also contribute to virulence by inhibiting
phagocytosis.
.

42

VIRULENCE FACTORS

Surface antigens: O and K

O antigen somatic lipopolysaccharide surface O
antigen has endotoxic activity, protects the bacteria from
phagocytosis and bactericidal effects of complement.
K antigen affords protection against phagocytosis and
antibacterial factors in normal serum.

43

 Fimbriae plasmid coded, found in small numbers and

mediate mannose resistant hem agglutinins, act as
virulence factors.
Examples:
CFA = colonization factor antigens in enterotoxigenic
E.coli causing human diarrhea.
P fimbriae which bind to uroepithelial cells and P blood
group substance on human erythrocytes, have a role in
urinary tract infection.
.

44

 TOXINS:
E.coli produce two kinds of exotoxins haemolysins and
enterotoxins.

Three distinct types of E.coli enterotoxins have been

identified
LT = heat labile toxin
ST = heat stable toxin
VT = serotoxin (also known as SLT = shiga like toxin).

45

LT = HEAT LABILE TOXIN

Resembles cholera toxin in its structure, antigenic
properties and mode of action.
It is a complex of polypeptide subunits.
Each unit of toxin has 1 subunit A and 5 subunits B.
LT is a powerful antigen and can be detected by
serological and biological tests.

46

LT

1 SUBUNIT A

bind to GM1 ganglioside

Receptors on intestinal
epithelial cells

5 SUBUNITS B

activation of A subunit into A1 and A2

A1 activates adenyl cyclase in the enterocyte to form

cAMP, leading to increased outflow of water and electrolytes
into the gut lumen, with consequent. diarrhoea.

47

ST = HEAT STABLE TOXIN

LMW polypeptide, poorly antigenic.
Two types known STA/ST1 and STB/ST2.
ST genes are carried on plasmids which may also carry
other genes, such as for LT and drug resistance.
STA

STB

STA acts by activation of cGMP in

the intestine.

M/A not known.

Infant mouse test STA acts very

rapidly and induces fluid
accumulation in the intestines of
infant mice within four hours of
intragastric administration.

STB causes fluid accumulation in

young piglets but not in infant mice.

Methanol soluble

Not methanol soluble.

.

48

VT = VEROTOXIN = VEROCYTOTOXIN

Named so because it was first detected by its cytotoxic

effect on Vero cells, a cell line derived from African
green monkey kidney cells.
It is also known as SLT = shiga like toxin because it is
similar to the shigella dysenteriae type 1 toxin in its
physical, antigenic and biological properties.
Has A and B subunits.
VT genes appear to be phage encoded.
VT1, VT2 identified.
.

49

CLINICAL INFECTIONS

URINARY TRACT INFECTIONS

DIARRHEA
PYOGENIC INFECTIONS
SEPTICAEMIA

50

URINARY TRACT INFECTIONS

Community acquired: E.coli and other
coliforms account for the large majority of
naturally acquired UTIs.
Hospital acquired: Those acquired in the
hospital, following instrumentation, are more
often caused by other bacteria such as
pseudomonas and proteus.
The E.coli serotypes commonly responsible for
UTI are those normally found in the feces, O

groups 1,2,4,6,7,etc.
.

51

Ascending urinary tract infection

 Asymptomatic bacteriuria:
Observed in some pregnant women, it means urinary
infection without any symptoms.
If it progresses, it may lead to symptomatic infection later
in pregnancy, pyelonephritis and hypertension in the
pregnant woman, as well as to prematurity and perinatal
death of the fetus.
Urinary tract infection can be:
Ascending = via urethra
Descending = haematogenous route
.

53

KASS CONCEPT OF SIGNIFICANT BACTERIURIA

Normal urine is sterile, but during voiding may get
contaminated by genital commensals.
Hence presence of bacteria in microscopy and culture of a
urine sample need not necessarily mean UTI by that
organism.
To differentiate between actual pathogen and contaminant,
Kass etc. devised the following formula.
Colony count in urine:
<10000/ml = contaminant
10000 to 100000/ml = indecisive, repeat test.
>100000/ml = significant bacteriuria.
Exceptions: in patients on antibacterial or diuretic drugs
and with some bacteria like Staph. aureus, even low
counts may be significant. .

54

URINE SAMPLE COLLECTION AND TRANSPORT

Inform the patient about the procedure.
Proper cleaning of area.
Clean voided midstream urine sample is collected in a
sterile leak-proof container.
Sample container is labeled, request form filled and send
along with the sample to the laboratory within an hour or
two.
If delay of more than 1-2 hours is there, the sample
should be refrigerated.
Boric acid can be used as a preservative to prevent the
change in count of bacteria in the sample.
.

55

56

57

QUANTITATIVE AND SEMIQUANTITATIVE

METHODS
Quantitative culture:
Serial ten fold dilutions of urine are tested by the pour
plate or surface culture methods. Complicated method.
Semiquantitative culture:
Fixed volume of urine placed on a noninhibitory medium
like blood agar with a standard loop for quantitative
measurement.
Another loopful of urine placed on a indicator medium like
MacConkey agar for presumptive diagnosis of causative
organism.
Culture is followed by biochemical tests etc for
identification of the pathogen.
.

58

DIARRHEA CAUSING E.coli

EPEC = ENTEROPATHOGENIC E.coli
ETEC = ENTEROTOXIGENIC E.coli
EIEC = ENTEROINVASIVE E.coli
EHEC = ENTEROHAEMORRHAGIC E.coli
EAEC = ENTEROAGGREGATIVE E.coli

59

EPEC = ENTEROPATHOGENIC E.COLI

Associated with diarrhea in infants and children.
Institutional outbreaks, sporadic diarrhea.
Do not produce enterotoxins.
Not invasive.
Attach to mucosa of upper small intestine, cause
disruption of brush border microvilli.
Enter adherent E.coli is another name given to them
because they can adhere to cells.
.

60

EPEC brush border disruption

61

ETEC = ENTEROTOXIGENIC E.coli

Endemic in developing countries in tropics, all age groups.
Mild watery diarrhea to fatal disease indistinguishable from
cholera.
Persons from developed countries visiting endemic areas
often suffer from ETEC diarrhea a condition known as
TRAVELERS DIARRHEA.
Adhere to intestinal epithelium via fimbrial or colonization
factor antigens (CFA I,II,III,IV,etc.).
Produce LT or ST or both.
Diagnosis done by demonstration of the toxin.
.

62

TRAVELERS DIARRHOEA

63

ETEC TRAVELERS DIARRHOEA

64

EIEC = ENTEROINVASIVE E.coli

Resemble Shigella
Many are nonmotile, do not ferment lactose or
ferment it late with only acid production, and do not
form lysine decarboxylase.
Many of these show O antigen cross reactivity with
Shigella.
Earlier names given Shigella alkalescens, Shigella
dispar and were grouped under Alkalescens-Dispar
group.
Named EIEC because they have the capacity to invade
interstitial epithelial cells in vivo and penetrate HeLa cells
in tissue culture.
.
65

 Clinically EIEC infection resembles shigellosis, ranging

from mild diarrhea to frank dysentery.
Sereny test:
Instillation of a suspension of freshly isolated EIEC or
Shigella into the eyes of guinea pigs leads to
mucopurulent conjunctivitis and severe keratitis.
Mice can also be used.
Cell Penetration in HeLa or HEP-2 cells.
Plasmid detection:
VMA ELISA: The plasmid codes for outer membrane
antigens called the virulence marker antigens (VMA) which
can be detected by the ELISA (VMA ELISA) test.
.

66

EHEC = ENTEROHAEMORRHAGIC E.coli

Produce VT
Mild diarrhea to fatal hemorrhagic colitis and
hemorrhagic uremic syndrome (HUS) particularly in
young children and elderly.
Primary target of VT = vascular endothelial cells.
O157:H7, O26:H1 etc
The disease may occur sporadically or as outbreaks
of food poisoning.
Changing lifestyle and eating habits.
Salad vegetables such as radish and alfalfa sprouts, in
which bacteria were found beneath the skin and in the
deeper tissues.
Diagnosis: demonstration of VT.
.

67

EHEC ATTACKS VASCULAR ENDOTHELIAL

CELLS, ALSO PRODUCES VT

68

EAEC = ENTEROAGGREGATIVE E.coli

Appear aggregated in a stacked brick formation on
Hep-2 cells or glass.
They have been associated with persistent diarrhea,
especially in developing countries.
They form a LMW heat stable enterotoxin called EAST1
(enter aggregative heat stable enterotoxin-1).

70

EAEC FORM STACKED BRICK LIKE

FORMATION

71

 PYOGENIC INFECTIONS:
E.coli form the most common cause of intra-abdominal
infections, such as peritonitis and abscess resulting from
spillage of bowel contents.
Pyogenic infections in the perianal area.
Neonatal meningitis
SEPTICAEMIA:
Blood stream invasion by E.coli may lead to fatal
conditions like septic shock and systemic
inflammatory response syndrome (SIDS).

HeLa = Human carcinoma of cervix cell line (named after a lady

named Hela)
HEP-2 = Human epithelioma of larynx cell line
Vero = vervet monkey kidney cell line
.

72

E. coli and other opportunistic

Enterobacteriaceae
Treatment
Diarrhea patients usually need only symptomatic relief.
Antibiotic treatment may prolong the fecal carriage or
increase the risk of secondary complications.
Treatment of bacteremia and septic shock: prompt
antibiotic treatment, restoration of fluid and electrolyte
balance, and treatment of disseminated IV coagulation.
Variation in drug susceptibility is great in opportunistic
infections, and antibiotic sensitivity tests are essential.

E. coli and other opportunistic

Enterobacteriaceae
Prevention and control
Enterobacteriaceae are a major part of normal flora and
a common contaminant of the environment. Food safety
plays an important role in prevention of GI infections.
In hospitals, opportunistic Enterobacteriaceae are
commonly transmitted by personnel, instruments, or
parenteral medications. Their control depends on hand
washing, rigorous asepsis, sterilization of equipment,
disinfection, restraint in IV therapy, and strict precautions
in keeping the urinary tract sterile.

Pathogens Outside The

Enteric Tract

Klebsiella-Enterobacter-Serratia
Group

Usually opportunistic pathogens

Cause nosocomial infections
K. Pneumonia is a primary pathogen
Causes pneumonia and UTI
L.F on EMB
Hospital-acquired strains are multi-drug
resistance

Pathogenesis of
Klebsiella pneumoniae
Associated pneumonia
Frequently associated with
Necrotic destruction of alveolar spaces
Production of blood-tinged sputum

Can also cause wound, soft tissue, and

urinary tract infections

Proteus-Providencia-Morganella
Group

Phenylalanine deaminase & urease +ve

Usually highly motile swarming
Weil-Felix reaction
N.L.F on EMB,
Some Proteus spp. H2S +ve
DoC for P. mirabilis is penicillin where for
other spp is cefotaxime

KLEBSIELLA

79

INTRODUCTION
GENUS KLEBSIELLA:

K.pneumoniae
K.ozaenae
K.rhinoscleromatis
K.oxytoca etc.

80

GENERAL FEATURES
Non-motile
Capsulated
Grow on OM forming large dome shaped mucoid
colonies.
Short plump straight rods.
Capsular halo seen prominently in gram stain.
Commensals, saprophytes.
.

81

Klebsiella
K. pneumoniae and Klebsiella oxytoca
Klebsiella rhinoscleromatis (causes a
granulomatous disease of the nose)
Klebsiella ozaenae (causes chronic
atrophic rhinitis)
K. granulomatis is the etiologic agent of
granuloma inguinale

Klebsiella
K. pneumoniae and K. oxytoca are the most commonly isolated.
Can cause community-acquired primary lobar pneumonia
(frequently involves necrotic destruction of alveolar space), and
infections of wound, soft tissue, and urinary tract.
Risk factors for pneumonia: alcoholism; compromised pulmonary
function.
*In Taiwan: liver abscess is commonly seen in infection of
diabetes patients by K. pneumoniae.
K. granulomatis may cuase granuloma inguinale, a sexually
transmitted disease, in some countries.

Klebsiaella granulomatis

LARGE DOME SHAPED MUCOID

COLONIES AND CAPSULAR HALO

85

KLEBSIELLA PNEUMONIAE
(FRIEDLANDERS BACILLUS, BACILLUS MUCOSUS
CAPSULATUS)
Sugar fermentation acid + gas
IMViC --++
Urease +
Second most populous member of aerobic bacterial flora
of the intestine.
Important cause of nosocomial infections.
Pneumonia, UTI, pyogenic infections, septicemia, and
rarely diarrhea.
.

86

KLEBSIELLA PNEUMONIA
Serious disease with high case fatality.
Middle age or older persons.
Alcoholism, chronic bronchopulmonary disease, diabetes.
Massive mucoid inflammatory exudate of lobar or lobular
distribution, involving one or more lobes of the lung.
Necrosis and abscess formation.
Serotypes 1, 2, 3.
.

KLEBSIELLA - MICROSCOPY

Klebsiella on Nutrient agar and Blood agar

89

VIRULENCE FACTORS

CAPSULE: Mucoid capsule is ant phagocytic and acts

as a major virulence factor.

PLASMID EXCHANGE: Klebsiella participates in

exchange of plasmids with other Enterobacteriaceae.
The exchange of plasmid is presumed to be the basis
for two constant characteristics of Klebsiella species.
a. Antibiotic resistance
Many strains are highly resistant to most antibiotics.
b. Toxins
Some Klebsiella strains carry plasmids that code for
toxins similar to heat labile and heat stable exotoxins of
E. coli.
.

90

CLINICAL SYNDROME
1. PNEUMONIA

K. pneumoniae is found in 10% normal individuals as

normal flora of respiratory tract.

Pneumonia in diabetics, alcoholics and

immunocompromised patients.

Lung abscess

Lobar pneumonia necrotic destruction of the alveolar

spaces, cavity formation and production of thick blood
tinged viscus sputum. Prognosis is grave with 50%
mortality.
.
91

2.

URINARY TRACT INFECTIONS

3.

SEPTICAEMIA

4.

WOUND INFECTION

5.

MENINGITIS

6.

EPIDEMIC DIARRHOEA in newborns.

92

ENTEROBACTER, CITROBACTER,
MORGANELLA, SERRATIA
Primary infections are rare
Citrobacter koseri has a predilection for
causing meningitis and brain abscesses in
neonates
Resistance is a particularly serious
problem with Enterobacter species

SERRATIA

Serratia species
Seven species, but S. marcescens is the only
one clinically important
Frequently found in nosocomial infections of
urinary or respiratory tracts
Implicated in bacteremic outbreaks in
nurseries, cardiac surgery, and burn units
Fairly resistant to antibiotics

Serratia species (contd)

Major characteristics
Ferments lactose slowly
Produce characteristic pink pigment, especially
when cultures are left at room temperature
S. marscens on
nutrient agar

Serratia
Members of the Serratia genus were once
known as harmless organisms that produced a
characteristic red pigment.
Today, Serratia marcescens is considered a
harmful human pathogen which has been known
to cause urinary tract infections, wound
infections, pneumonia and diarrhea.
Serratia bacteria also have many antibiotic
resistance properties which may become
important if the incidence of Serratia infections
dramatically increases.
Serratia can be distinguished from other genera
belonging to Enterobacteriaceae by its

SPECIES:
1. S. MARCESCENS
2. S. RUBIDDEA
3. S. LIQUEFACIENS

Biochemical Characteristics:
Non lactose fermenter
V-P: positive
Lysine: positive
TSI A/A: (NO gas)
Motile

Citrate: positive.
ODC: positive
Indole: Negative
DNase: positive

DIAGNOSIS
Specimen: Sputum, urine, and blood.
Culture: A red pigment is produced by colonies of Serratia
on routine laboratory media which is more clear when the
organism is grown at 22 C in the dark ( Prodiginines dye) .
= Some strains are hemolytic.

THE PICTURE WAS TAKEN BY AN ELECTRON

MICROSCOPE OF TYPICAL SERRATIA CELLS.

Klebsiella, Enterobacter, Serratia &

Hafnia sp.
Usually found in intestinal tract
Wide variety of infections, primarily pneumonia, wound,
and UTI
General characteristics:

Some species are non-motile

Simmons citrate positive
H2S negative
Phenylalanine deaminase negative
Some weakly urease positive
MR negative; VP positive

Enterobacter species
Comprised of 12 species; E. cloacae and E.
aerogenes are most common
Isolated from wounds, urine, blood and CSF
Major characteristics
Colonies resemble Klebsiella
Motile
MR negative; VP positive

Enterobacter species

(contd)

Hafnia species
Hafnia alvei is only species
Has been isolated from many anatomical sites
in humans and the environment
Occasionally isolated from stools
Delayed citrate reaction is major characteristic

Pathogens Primarily
within The Enteric Tract

Shigella
S. flexneri, S. boydii, S. sonnei,
S. dysenteriae
bacillary dysentery
shigellosis
bloody feces
intestinal pain
pus

Shigellosis
within 2-3 days
epithelial cell damage

Shigella sonnei

Shiga toxin

enterotoxic
cytotoxic
inhibits protein synthesis
lysing 28S rRNA

Shigellosis
man only "reservoir"
mostly young children
fecal to oral contact
children to adults
transmitted by adult food handlers
unwashed hands

Structure

Gram-negative,
nonmotile,
facultatively anaerobic,
non-spore-forming rods
failure to ferment lactose or decarboxylate
lysine
closely related with Escherichia coli

Shigella

Classification

four serogroups with multiple serotypes

A (S dysenteriae, 12 serotypes);
B (S flexneri, 6 serotypes);
C (S boydii, 18 serotypes);
D (S sonnei, 1 serotype).

Shigella

bacillary dysentery--shigellosis

Shigella

Virulence
1. invasin
encoded by large extra-chromosomal
elements
(plasmids)--Invasion
Plasmid
Antigens (Ipa) B and C.
induces the endocytic uptake of shigellae by
M cells, epithelial cells, and macrophages.
deform the plasma membrane of contiguous
cells.
IcsB plasmid-encoded protein lyses the
plasma membranes, resulting in intercellular
bacterial spread.

Shigella

Histopathology of acute colitis following peroral infectionwith shigellae

Shigella

Virulence
2. Endotoxin
cause fever, shock, bloody, mucoid stools,
and abdominal pain (cramps and
tenesmus) .

Shigella

Virulence
3.exotoxin- Shiga toxin(vero toxin)
chromosomally-encoded
neurotoxic, enterotoxic and cytotoxic
The toxin inhibits protein synthesis (acting
on the 70S ribosome and lysing 28S
rRNA). Its enterotoxicity can make the
disease clinically appear as a diarrhea.

Shigella

Diagnosis -- Sampling
Positive cultures are most often obtained
from blood-tinged plugs of mucus in
freshly passed stool specimens obtained
during the acute phase of disease
Rectal swabs may also be used if the
specimen is deposited in a buffered
glycerol saline holding solution

Shigella

Diagnosis -- Isolation
primary
differential/sel
ective media:
SalmonellaShigella (SS)
Agar(contain
bile salts& pH
indicators)
MacConkey,
Hektoen
Enteric Agar,

Shigella

Diagnosis -- Isolation
colorless, non-lactose-fermenting
colonies
Secondly tubed slants of Kligler's Iron
Agar or Triple Sugar Iron Agar.
Shigella species produce an alkaline
slant and an acid butt with no bubbles
of gas in the agar.

Shigella

Diagnosis -- Identification
slide agglutination tests with antisera for
serogroup and serotype confirm the
identification
polymerase chain reaction (PCR).
Enzyme-linked immunosorbent assay
(ELISA)

Shigella

Epidemiology
occurring by fecal-oral contact
it can be transmitted by infected adult food
handlers who contaminate food.
Man is the only "reservoir"

Shigella

Treatment and Control

Managing of dehydration is of primary
concern.
Patients with severe dysentery are usually
treated with antibiotics (e.g. ampicillin).
Patients respond to antibiotic therapy and
disease duration is diminished

Treating shigellosis
manage dehydration
patients respond to antibiotics
disease duration diminished

Salmonella
Flagella

Salmonella

Structure & Antigenic Types

Gram-negative, flagellated, facultatively
anaerobic bacilli
three major antigens:
H or flagellar antigen;
O or somatic antigen;
Vi antigen (only a few serovars)

Salmonella

Virulence Factors
(1) the ability to invade cells
---- invasin: Vi (capsular) antigen
(2) a complete lipopolysaccharide coat
---- LPS (endotoxin)
(3) the ability to replicate intracellularly,
(4) possibly the elaboration of toxin(s)

Salmonella

Salmonella

Invasion of intestinal mucosa by Salmonella.

Salmonella -- ClinicalManifestations
1) Gastroenteritis--food poisoning

Symptoms usually begin 6 to 48 hours after

ingestion of contaminated food or water
the cardinal manifestation is diarrhea.
nausea, vomiting, abdominal cramps,
myalgia, headache, fever (38oC to 39oC)
and chills are common
The duration of fever and diarrhea is usually
2 to 7 days

Salmonella -- ClinicalManifestations

2) Septicemia
an intermediate stage of infection
no intestinal symptoms and the bacteria
cannot be isolated from fecal
specimens.
it may remain localized in the intestine
or disseminate to the bloodstream

Salmonella -- ClinicalManifestations

3) Enteric fevers ---- typhoid

severe systemic form, may be fatal
The best studied enteric fever is typhoid
fever, the form mainly caused by S. typhi
may be preceded by gastroenteritis
an incubation period of 10 to 14 days
symptoms of enteric fevers are nonspecific:
fever, anorexia, headache, myalgias, and
constipation

Salmonella -- ClinicalManifestations

3) Enteric fevers ---- typhoid

primary bacteraemic phase:
(7- 10 days of the incubation period )

invade the epithelium

spread to mesenteric lymph nodes &
throughout the body
be taken up by the reticuloendothelial cells
infect the liver, spleen, gallbladder, bones,
meninges
invade bloodstream via thoracic duct

Salmonella -- ClinicalManifestations

3) Enteric fevers ---- typhoid

second and heavier bacteraemic phase
(2-3 weeks)
pass into the blood( the onset of the fever and
other signs of clinical illness)
From the gallbladder a further invasion of the
intestine results.
Peyers patches & other gut lymphoid tissues
become involved in an inflammatory reaction
infiltration with mononuclear cells
followed by necrosis, sloughing and the
formation of characteristic typhoid ulcers

Salmonella -- ClinicalManifestations

3) Enteric fevers ---- typhoid

Onset: 2weeks/insidious/vagueearlysymptoms
vague
Progression :
thetemperatureshowsastepladderriseoverthe1st
weekoftheillness,remainshighfor7-10daysandthen
fallsbylysisduringthe3rdor4thweek.
physicalsignsincludearelativebradycardiaforthe
heightofthefever,hepatomegaly,splenomegalyand
oftenarashofrosespots.

Relapse: shorterandofmilder.
Complications:
severeintestinalhaemorrhageandintestinalperforation

Salmonella -- ClinicalManifestations

4) The prolonged carrier state

continue to excrete the salmonellae for
a year or more
The bacilli are most commonly present
in the gallbladder

Salmonella -- laboratory diagnosis

Isolation & identification 1

Blood culture
the definitive diagnosis of enteric fever
most commonly found during the first 7-10
days and during relapses
Stool and urine culture
In typhoid fever, stool cultures are usually
positive form the 2nd week and urine cultures
from the 3rd week of the infection.

Salmonella-laboratory diagnosis

Identification & identification

Specimens should
be plated on
several
nonselective and
selective agar(EMB)
media.
Laboratory
identification of the
genus Salmonella is
done by
biochemical tests.

Salmonella-laboratory diagnosis

Identification
Biochemicalreactionsofsuspicious
coloniesarethendeterminedontriple
sugarironagarandlysine-ironagar.
serologictypeisconfirmedbyserologic
testing.
Itcanbeconfirmedbyantigenicanalysis
ofOandHantigensusingpolyvalentand
specificantisera.

Salmonella-laboratory diagnosis

Widal test
a tube test for determining the quantity
of agglutinating antibodies, or
agglutinins, in the serum of a patient
with typhoid fever

Salmonella-laboratory diagnosis-widal test

Theprocedureinvolves Thereciprocalofthe
addingasuspensionof
highestdilutionat
deadtyphoidbacterial
whichagglutinationis
cellstoaseriesoftubes
seendesignatedasthe
containingthepatients
antibodytiterof
serum,whichhasbeen
patientsserum.
dilutedouttovarious
Naturally,thehigher
concentrations.
Afterthetubeshave
thetiter,thegreateris
beenincubatedfor30
theantibodyresponse
minutesat37,they
oftheindividualtothe
arecentrifugedand
disease
examinedtonotethe
amountofagglutination
thathasoccurred.

Salmonella-laboratory diagnosis-widal test

Generally, in typhoid cases, it is
valuable that the titre of specific
O antibodies is 1:80 or the titre of
specific H antibodies is 1:160.
In paratyphoid cases, if the titre of
specific H antibodies is 1:80, the
result is positive.

Salmonella-laboratory diagnosis-widal test

Interpretation

Thelevelofentericantibodiesinthehealthy
population must be known and may be
variable.
Previous inoculation with TAB(typhoid,
paratyphoid A, paratyphoid B)vaccine can
giverelativelyhightitresofspecificantibodies,
ascanpreviousinfection,althoughonlytheH
antibodiestendtopersistatdetectablelevels.
Cross-reacting antibodies from previous
exposure to other salmonellae sometimes
confusethepicture.

Salmonella-laboratory diagnosis-widal test

Interpretation
As with other serological tests for acute
infections, the usefulness of the Widal test is
greatest when a four-fold or greater rise in
antibody levels is detected on testing a second
specimenofthepatientsserumsomedaysafter
specimen
the firstby this time the diagnosis has usually
been made by isolation of the offending
organismfrombloodculture.
ThedetectionofpersistingViantibodyin
significanttitremaybeoflimitedvalueinthe
searchforlong-termcarriersafteranoutbreak
ofinfection.

Salmonella

Control and Treatment

Vaccinesareavailablefortyphoidfeverand
arepartiallyeffective.
Typhoidfeverandentericfeversshouldbe
treatedwithantibiotics.

Salmonella
2000 antigenic "types
genetically single species
S. enterica
disease category
S. enteritidis
many serotypes

S. typhi

Salmonellosis
S. enteritidis
the common salmonella infection
poultry, eggs
no human reservoir
Gastroenteritis

nausea
vomiting
non-bloody stool
self-limiting (2 - 5 days)

Salmonella
Salmonella enterica and Salmonella
bongori
S. enterica is subdivided into six
subspecies, S. enterica subsp. enterica
the two species have been subdivided
into more than 2500 unique serotypes
S. enterica subspecies enterica serotype
Typhimurium or S.Typhimurium

Salmonellosis

uncomplicated cases (the vast majority)

antibiotic therapy not useful

Typhoid

enteric fever
severest salmonella disease
Salmonella typhi
rare in US
epidemics
third world
Europe
* historical

Salmonella typhi
human reservoir
carrier state common

contaminated food
water supply
poor sanitary conditions

Typhoid

septicemia
- occurs 10-14 days
lasts 7 days

gall bladder
shedding, weeks
acute phase, gastroenteritis

gastrointenteritis

S. typhi
Vi (capsular) antigen
protective

Typhoid -Therapy
Antibiotics
essential
Vaccines
ineffective

Proteus sp.

Dr Praveg Gupta MD

155

156

Pathogenesis of
Proteus mirabilis

Urinary tract infections

Produce large amounts of urease
Urea into carbon dioxide and ammonia
Changes renal pH

Facilitates formation of stones

Also toxic for uroepithelium

Presence of pili may decrease virulence

Better phagocytosis

166

170

171

172

173

174

Proteus strains are used in the Weil-Felix test for

Rickettsial diseases:
1. Proteus OX K
2. Proteus OX 19
3. Proteus OX 2
Weil and Felix isolated strains of Proteus vulgaris from
patients with typhus fever and found that the sera of
the patients agglutinated particular Proteus strains
designated OX19 and OX2.
The Proteus organism is not the cause of Typhus fever.
The cross agglutination seems to be caused by the
presence of an alkalistable polysaccharide which is also
present in R. prowazeki. The agglutination of these
particular strains by sera of patients with Rickettsial
infection is known as the Weil-Felix reaction .

177

Dr Praveg Gupta MD

178

Morganella &Providencia
Both are closely related to the Proteus group because
both elaborate urease and produce indole.
They however, don`t produce hydrogen sulfide.

Morganella morganii
Morganella morganii is the only important species of this
.genus
It can cause urinary tract and wound infections, as well
. as diarrhea
Chloramphenicol is a good choice for treating Morganella
.infections
+Ornithine
TSIA: A/A
: + + - -IMVIC reaction
179
motile

Providencia alcalifaciens
Although rare, Providencia species have been

associated with nosocomial (hospital acquired)

urinary tract infections. One species, P.
alcalifaciens, has been associated with some cases
of diarrhea in children. Since infection is so rare,
other genera of Enterobacteriaceae should be
considered before Providencia as being causative.
Hydrogen sulfide
Lysine
Lactose
TSIA: K/A
IMVIC reaction: + + - +
motile
Dr Praveg Gupta MD

180