ENTEROBACTERIACEAE-

III
SALMONELLA
1
Narayan Parajuli
 ENTEROBACTERIACEAE: COLIFORMS

• Group of gram negative, non sporing

rods occurring in the intestinal tract of
human and animals ,on the plants and
soils ,and leading a Saprophytic,
commensal or pathogenic existence
• Theobald Smith (1890)
• Coliforms capable of fermenting
lactose
• Normal resident gut flora

Narayan Parajuli MS (CMID)

 ENTEROBACTERIACEAE DEFINED

1. Small gram-negative rods (2-5 by 0.5 microns) except

Arsenophonous (7-10µm in length) with parallel sides

and rounded ends

2. Non spore forming

3. Capsulated or non capsulated

4. Non-acid fast

5. All ferment glucose

6. All reduce nitrates to nitrites except Saccahrobacter

fermentatus, some strains of Yersinia & Erwinia

Narayan Parajuli MS (CMID) 3
7. None produce cytochrome oxidase. except Plesiomonas, some

strains of Serratia fecaria

8. All except Klebsiella and Shigella and Yersinia, others are motile:

with rare exception Tatumella (single polar or lateral or subpolar)

or Non-motile

9. Facultative anaerobic bacilli

10. Grow well on peptone, meat extract, MacConkey agar except

Calymmatobacterium,

11. Most grow well at 22-35ºC; optimal growth, biochemical capacity

at 25-28ºC, Yersinia, Hafnia, Xenorhabdus, Photorhabdus, many
Erwinia

12. Some grow on D-glucose as the sole carbon source; some require
4
vitamins and amino acids
Narayan Parajuli MS (CMID)
13.Non – halophilic
14.Most are catalase positive except Shigella dysenteriae 1 and
Xenorhabdus.
15.G + C of the DNA:49-59 mol%, but in Proteus and
Providencia 37-42mol% Yersinia 46-47 mol%, (63.5 for
Saccharobacter fermentatus)
16. Some strains opportunistic pathogens: Escherichia coli ,
Klebsiella pneumoniae , Enterobacter aerogenes , Serratia
marcescens, Proteus spp., Providencia spp., Citrobacter
spp.
17. Some strains true pathogens : Salmonella spp. , Shigella
spp., Yersinia spp. and Some E. coli strains
Narayan Parajuli MS (CMID) 5
 GENERA
 Many genera: Citrobacter, Edwardsiella, Enterobacter,
Escherichia, Hafnia, Klebsiella, Kluyvera, Morganella, Proteus,
Providencia, Salmonalla, Serratia, Shigella, and Yersinia

 In addition, the more recently proposed genera:

Cedecea,Ewingella,Leclercia,Leminorella,Moellerella,Pantoea,Rah
nella,
Tatumella,Yokenella,Alterococcus,Buchnera,Calymmatobacterium
,Erwinia,Plesiomonas,Saccharobacter,Sodalis,Trabulsiella,,Xenorh
abdus

6
Narayan Parajuli MS (CMID)
 Classification of Enterobacteriaceae
• Complex and controversial : more than 38 genera and
120 species
• About 96% of the medically important isolates belong to
14 genera and constitute 38 species
• Oldest method classify these bacteria into 3 groups based
on their action on lactose:-

I. Lactose fermenters Escherichia ,Klebsiella

II. Late lactose fermenters Shigella sonnei

III. Nonlactose fermenters Salmonella ,Shigella

Narayan Parajuli MS (CMID) 7

• Most Enterobacteria fall into one of 3 divisions, whether or not
they ferment lactose

a. MR+,VP-,Citrate - Includes the faecal coliform

group and dysentery
bacteria

b. MR+,VP-,Citrate + Contains intermediate

coliform bacteria and the
Salmonellae

c. MR-,VP+,Citrate + Includes all the

aerogenes like organisms

• Morganella,Proteus and Providencia

Narayan were generally believed
Parajuli MS (CMID) 8 to
 Modern
Classification
Edward & Ewing (1986)
• Major genera within a family sharing similar
biochemical properties of similar diagnostic
importance
• Not a recognized taxonomic rank
• Convenient for learning, clinicians
TribeI Escherichieae Escherichia, Shigella
Tribe II Ewardsielleae Edwardsiella
Tribe III Salmonelleae Salmonella
Tribe IV Citrobacterieae Citrobacter
Tribe V Klebsielleae Klebsiella,
Enterobacter,

Hafnia, Pantoea,
Serratia
Tribe VI Proteeae
Narayan Parajuli MS (CMID) Proteus, Morganella,
9
SALMONELLA
 Introduction
• Salmonellae are ubiquitous human and animal pathogens
• Salmonella infections in humans typically produce one of three
clinical syndromes, such as gastroenteritis, enteric fever, or
focal disease
• Gram-negative, flagellated, and facultative anaerobic bacilli
characterized by the presence of O, H, and Vi antigens
• Based on DNA homology and host range, the genus Salmonella
is classified into two species: Salmonella enterica and
Salmonella bongori
• Most of the salmonellae that are pathogenic to human beings
belong to S. enterica subsp. enterica
• According to the presence
Narayan of particular
Parajuli MS (CMID) somatic O, flagellar
11 H,
 DEFINITION
1. Gram negative rod-shaped bacteria

2. Usually motile by peritrichous flagella.

3. Aerobes and facultative anaerobes.

4. Non sporing and non capsulated.

5. Ferment glucose with the formation of acid or of

acid and gas.

6. Most produce H2S.

7. Catalase positive .

8. Oxidase negative.
12
Narayan Parajuli MS (CMID)
9. KCN negative

10. Citrate positive

11. Indole negative

12. Urea not hydrolyzed

13. Non lactose fermenter

14. MR positive

15. VP negative

13
Narayan Parajuli MS (CMID)
 HISTORY
 The genus Salmonella was named after
Daniel Elmer Salmon, an American veterinary pathologist
 While Theobald Smith was the actual discoverer of the

type bacterium (Salmonella enterica var. choleraesuis) in

1885, the organism was named after him by Smith
 Smith and Salmon had been searching for the cause of

common hog cholera and proposed this organism as the

causal agent
 The genus Salmonella was finally formally adopted in

1900 by J. Lignières for the many species of Salmonella

14
Narayan Parajuli MS (CMID)
 TAXONOMY

Domain: Bacteria

Phylum: Proteobacteria

Class: γ-Proteobacteria

Order: Enterobacteriales

Family: Enterobacteriaceae
Genus: Salmonella (Lignieres 1900)

Species: bongori, enterica

Narayan Parajuli MS (CMID) 15
 Morphology
 Gram-negative, nonsporing, non capsulated bacillus
measuring 2-4 µm x 0.6 µm
 Most serotypes are motile by peritrichous flagella
except S. gallinarum and S. pullorum
 Fimbriae are present in most strains

16
Narayan Parajuli MS (CMID)
 CULTURAL CHARACTERS
• Aerobes and facultative anaerobes

• Grow on ordinarily culture media

• Optimum temperature of 37ºC in a pH of 6–8 on a variety of media

A. Liquid Media: most strains give abundant growth with uniform turbidity

• A thin surface Pellicle on prolonged incubation

B. Nonselective solid media:

• On nutrient and blood agar, colonies are moderately large(2-3 mm),grey

white, moist, circular disc with a smooth convex surface and entire edge.

• On Mac Conkey agar ,colonies are pale due to non lactose fermentation.

• Salmonella is resistant to low concentration of bile (0.05% sodium

deoxycholate).

• Killed at 60 º c for 15 min or 55º c for 60 min.

• Resistant to certain dyes brilliant green(0.004G/L) and higher concentration of

deoxycholate (0.25%). 17
Narayan Parajuli MS (CMID)
C. Selective solid media:
Wilson and Blair’s bismuth sulfite agar- medium of choice
• Salmonellae produce jet black colonies surrounded by a
metallic sheen due to production of hydrogen sulphide
• S. Paratyphi A and other species, which do not produce H2S,
form green colonies
• XLD (xylose, lysine deoxycholate agar)
• Salmonella spp. produce pink colonies with black centers as
a result of H2S production
• H2S-negative Salmonella serotypes produce transparent
colonies without black centers
D. Enrichment Media- Selenite F and Tetrathionate broth
18
Narayan Parajuli MS (CMID)
 BIOCHEMICALS
• Salmonellae ferment glucose, Mannitol, and maltose, forming
acid and gas
• Do not ferment lactose, sucrose, or salicin and do not produce
indole
• Most salmonellae except S. Paratyphi A, S. Choleraesuis, and
some other species produce H2S
• Do not hydrolyze urea and MR positive and VP negative
• S. Typhi and S. Paratyphi, however, do not grow in Simmon’s
citrate media as they need tryptophan as the growth factor
• Salmonellae decarboxylate lysine, ornithine, and arginine, S.
Typhi do not decarboxylate ornithine andS. Paratyphi A does
not decarboxylate lysine 19
Narayan Parajuli MS (CMID)
 Cell Wall Components and Antigenic Structure
 Lipopolysaccharide (LPS)

• Cell wall contains a complex lipopolysaccharide (LPS) structure which is

liberated during lysis of the cell
• LPS moiety functions as an endotoxin and is an important component of
the virulence of the bacteria

• Repeating sugar units in the outer O polysaccharide

• chain are responsible for O-antigen specificity and virulence

• Endotoxin component cause fever, activate the serum complement

kinin and clotting systems, and depresses myocardiac functions
• Antibodies produced against R core (common enterobacterial
antigen) are protective against infection caused by wide variety of
GN bacteria
20
Narayan Parajuli MS (CMID)
 ANTIGENS

• Salmonella possess three major antigens:

• 1. H or flagellar antigen
• 2. O or somatic antigen
• 3. Surface antigens (Vi antigen, M and N antigen,
and
• F antigens)

21
Narayan Parajuli MS (CMID)
 H or flagellar antigen -‘Hauch’
• Present on the flagella, and is heat and alcohol labile

• Destroyed by boiling or by treatment with alcohols and

acid, but they are preserved in 0.2–0.4% formaldehyde
• H antigens of Salmonella are genus specific and are not
shared by other enterobacteria
• H antigen is strongly immunogenic and is associated with
the formation of antibodies following infection or
immunization
• H antigen may occur in either or both the forms called
phase I and phase II
• When mixed with antisera, H suspension agglutinate
22
rapidly, producing large, loose,
Narayan fluffy
Parajuli MS (CMID) clumps
 O or somatic antigen -‘Ohne ’
• Occur on the surface of the outer membranes and are
determined by specific sugar sequences on the cell surface
• Heat stable, resistant to boiling up to 2 hours and 30
minutes
• It is also alcohol stable, resistant to treatment with 96%
ethanol at 37ºC for 4 hours and is also resistant to 0.2%
formaldehyde
• Can be extracted from cell wall by treatment with
trichloroacetic acid
• Antigen is less immunogenic than H antigen
• O antigen is not a single factor but a mosaic of two or more
antigenic factors 23
Narayan Parajuli MS (CMID)
 Surface antigens
 Vi antigen

• Surface antigen overlying the “O” antigen

• Felix and Pitt, who first described this antigen, believed that it
was related to virulence and gave it the name ‘Vi antigen
• Antigen is present only in few serotypes, the most important
being S. Typhi: Paratyphi C, Dublin, and Citrobacter freundii
• Inagglutinable by their specific O antiserum but agglutinable
by Vi antisera
• Heat labile and is destroyed by boiling within 1 hour and by
treatment with phenol hydrochloric acid and 0.5 sodium
hydroxide,
• Antigen is lost on serial subculture 24
Narayan Parajuli MS (CMID)
 M and N antigens

• Present on the surface of bacteria and are polysaccharide

in nature
• Prevent agglutination by O antiserum
• Boiling for two and half hours destroys these antigens
• Responsible for mucoid nature of Salmonella colonies
 F antigen

• Present on the fimbriae

• Occur in two phases: one with F antigen and the other
without F antigen
25
Narayan Parajuli MS (CMID)
 Antigenic variations

Undergo phenotypic and genotypic variation

1. H-O variation

• Loss of flagella, when grown on agar containing

phenol(1;800), flagella are inhibited

• Phenotypic and temporary change, reappear when

the strain is subcultured on media without phenol

• Loss flagella by mutation

• A Stable non motile mutant of S. typhi is the 901-O

strain- use to prepare O antigen

• Not totally lost the flagella , only
diminution the number of flagella

• Craige’s tube method is done to obtain the

motile cells rich in H antigen from small
numbered motile cells

• Consists of a short, narrow tube opened at

both ends, is embedded in to soft
agar(0.2%).

• Inoculate

• U tube can be used

 Phase Variation

• H antigens of salmonellae occur in one of two phases due to

one or other alternative sets of antigens( defined by 2 separate

sets of genes in the bacterial genome)

• Phase 1 antigens: Either specific for a species or shared by a

few species only- called specific phase. Designated by a, b, c, d

etc, and after z, as z1, z2 etc

• Phase 2 antigens: Widely shared – called nonspecific or group

phase. Fewer than phase 1 antigens and termed as 1, 2, 3, 4, 5

etc.

• Monophasic: S. typhi
 V-W variation
• V- form: O antigen of Fresh isolate of S. typhi is completely
masked by Vi antigen. Such only agglutinate with Vi antiserum
not with O antiserum- called V form
• W form: But after a number of subculture, Vi is completely
lost, which can agglutinate the O antiserum , not with Vi
antiserum- this is called W form
• VW form: Intermediate stage during the loss of the Vi antigen,
when the bacilli can agglutinate both O and Vi antiserum
• Other Vi containing bacilli such as S. paratyphi C and S. dublin
seldom have the O antigen completely masked by the Vi
antigen
 S-R variation
 The smooth to rough variation

- Change in the colony morphology(large,

rough and irregular)
- Loss of the O antigen
- Loss of virulence
 Causes: Mutation ( Due to loss of glucose moiety)

 Maintenance of R variant: Serial sub cultivation in

lab
 Prevention S-R : Maintaining culture on Dorset egg’s
media in cold or ideally by lyophilization
 PATHOGENESIS
• Salmonellosis- Any of several bacterial infections
caused by species of Salmonella, ranging from mild to
serious infections
• S. Typhi is an invasive bacterium

• Invades the intestinal mucosa or multiplies for several

days within the mononuclear phagocytic cells in the
liver, spleen, lymph nodes, and Peyer patches of the
ileum before invasion
• Bacteria subsequently enter the blood stream and
cause the disease manifestations
31
Narayan Parajuli MS (CMID)
 Virulence factors

• Endotoxin – may play a role in intracellular survival

• Capsule (for S. typhi and some strains of S. paratyphi)
• Adhesions – both fimbrial and non-fimbrial
• Type III secretion systems and effector molecules – 2 different
systems may be found:
– One type is involved in promoting entry into intestinal epithelial
cells
– Other type is involved in the ability of Salmonella to survive
inside macrophages
• Outer membrane proteins - involved in the ability of Salmonella
to survive inside macrophages
• Flagella – help bacteria to move through intestinal mucous
• Enterotoxin - may be involved in gastroenteritis
VIRULENCE FACTORS

33
Narayan Parajuli MS (CMID)
 ENTERIC FEVER- Pathobiology
• Severity of disease dependent on the virulence factors
of the infecting strain as well as on the human host
• Infective dose: Infection is acquired by ingestion of
food or water contaminated with salmonellae The
infective dose (ID50) in human volunteer experiments
has been found to be about 10 ˄3–10 ˄ 6 bacilli
• Although the infectious dose varies among strains, a
large inoculum is necessary to overcome stomach
acidity and to compete with normal intestinal flora
• Large inocula are also associated with higher rates of
illness and shorter incubation
Narayan Parajuli MSperiods
(CMID) 34
 Contd….
• On reaching the intestine, the salmonellae attach themselves

by fimbriae or pili to cells lining the ileal mucosa

• Bacteria selectively attach to specialized epithelial cells (M

cells) of the Peyer patches

• Salmonella TTSS mediate the initial invasion of S. Typhi into

the intestinal mucosa

• SPI-1 introduces Salmonella secreted invasion proteins (Sips

or Ssps) into the M cells resulting in membrane ruffling

• Ruffled membranes surround and swallow salmonellae,

leading to intracellular replication in the phagosome with

subsequent death of Narayan

hostParajuli
cells and
MS (CMID)
spread to adjacent
35
• An ATR gene also protects Salmonella spp. from stomach acids

and the acidic pH of the phagosome

• Catalase and superoxide dismutase are other factors that protect

the bacteria from intracellular killing

• Bacteria are then transported within phagosomes to the lamina

propria, where they are released

• In the lamina propria, typhoidal strains of salmonellae induce

production of macrophages, while nontyphoidal strains induce

production of neutrophils (nontyphoidal strains)

• Organisms travel from the submucosa to the mesenteric lymph

nodes, multiply, and then enter the blood stream via the thoracic

duct (transient primary Narayan

bacteremia) to spread to other tissues
Parajuli MS (CMID) 36
 37
Narayan Parajuli MS (CMID)
• During this bacteremic phase, the bacteria may invade liver, spleen,
bone marrow, gallbladder, and Peyer patches in the terminal ileum
• Gallbladder is infected via the liver, and the resultant cholecystitis
usually is subclinical and the infected bile renders stool cultures
positive
• Invasion of Peyer patches occurs during either the primary intestinal
infection or secondary bacteremia, and further spread of bacteria
occurs through infected bile
• Peyer patches become hyperplastic with infiltration of chronically
inflamed cells, which may lead to necrosis of the superficial layer
and ulcer formation, with potential hemorrhage from blood vessel
erosion or peritonitis from transmural perforation
• Pyrogens and mediators produced at the sites of inflammation have
been suggested as factors responsible for the prolonged fever 38
Narayan Parajuli MS (CMID)
 Enteric fever
• An acute illness manifested by fever, headache, and abdominal symptoms

• Incubation period is usually from 7 to 14 days, but may range from 3 to 56

days
• Onset of the disease is usually gradual, with headache, malaise, anorexia, a
coated tongue, and abdominal discomfort with either constipation or
diarrhea
• A step-ladder pyrexia with relative bradycardia and toxemia is the typical
feature
• Intestinal perforation, severe hemorrhage, and circulatory collapse are most
important complications
• Toxic encephalopathy, cerebral thrombosis, hepatitis, pancreatitis, arthritis,
and myocarditis are other complications
• Convalescence is slow- Relapse is common after initial recovery in 10–20%
of patients treated with antibiotics 39
Narayan Parajuli MS (CMID)
 NON- TYPHOIDAL SALMONELLA
• General Incubation: 6 hrs-10 days; Duration: 2-7 days
• Infective Dose = usually millions to billions of cells
• Transmission occurs via contaminated food and water
• Reservoir:

a) multiple animal reservoirs

b) mainly from poultry and eggs (80% cases from eggs)
c) fresh produce and exotic pets are also a source of
contamination (> 90% of reptile stool contain salmonella
bacterium); small turtles ban
• General Symptoms: diarrhea with fever, abdominal cramps,
nausea and sometimes vomiting
• Caused by S. typhimurium and S. enteritidis
 NONTYPHOIDAL SALMONELLA: GASTROENTERITIS

• Incubation: 8-48 hrs ; Duration: 3-7 days for diarrhea &

72 hrs. for fever

• Inoculum: large

• Limited to GI tract

• Symptoms include: diarrhea, nausea, abdominal

cramps and fevers of 100.5-102.2ºF. Also accompanied
by loose, bloody stool; Pseudoappendicitis (rare)

• Stool culture will remain positive for 4-5 weeks

• < 1% will become carriers

 Nontyphoidal Salmonella:
Bacteremia and Endovascular Infections

• 5% develop septicemia; 5-10% of septicemia patients

develop localized infections
• Endocarditis: Salmonella often infect vascular sites;
preexisting heart valve disease risk factor
• Arteritis: Elderly patients with a history of back/chest +
prolonged fever or abdominal pain proceeding
gastroenteritis are particularly at risk.
• Both are rare, but can cause complications that may lead
to death
• Serotype S. choleraesius causes septicemia:- prolonged
state of fever, chills, anorexia, and anemia
 Epidemiology of Enteric fever

• Problem of poor nations

• Due to marked geographical differences

distribution of paratyphoid bacilli, the control
has not been successful
• Source of infections: Mainly carrier

• Typhoid Mary” Mallon was the first famous

carrier of typhoid fever in the U.S.
 Epidemiology …
• Mary Mallon, who was hired as a

cook at several private homes in the

new York area in the early 1900’s.

• Mary Mallon caused several typhoid

outbreaks, moving from household to

household, always disappearing

before an epidemic could be traced

back to the particular household

Mary was working in. All together,

she had worked for 7 families, with

22 cases of typhoid and one death.

 Nomenclature
• Several modifications over the years
• Inclusion in the genus is based on common
biochemical properties
1) on antigenic characterization based on the
Kauffman and White scheme
2) Bacteriophage typing
3) Biotyping
4) Molecular Methods

45
Narayan Parajuli MS (CMID)
 Kauffmann and White Scheme
• Based on structural formulae of the O and H antigens of the
strains which are identified by agglutination with specific antisera
 Somatic O antigen, the phase- 1 H antigen and phase- 2 H
antigen in this order
 Three parts are separated by colons and components of each
part by commas
 The somatic O antigen are given arabic numerals
 The phase -1 H antigens are designated a – z, a series that is
complete except for j. After z are designated as z with subcripts,
eg. z1 to z83
 The phase-2 H antigens are designated by arabic numerals(1-12)
but phases 2 may also contain antigenic components of the e
Narayan Parajuli MS (CMID) 46
series or one of the z series
 Antigenic notation…..
 __ = Underlined O factors are determined by phage conversion
 [ ] = O (not underlined) or H factor that may be present or absent
without relation to phage conversion
 {} = O-factors indicated in curly brackets are exclusive. In a given
serovar, factors in curly brackets cannot coexist with other factors
in curly brackets. Some factors may be phage-determined
(underlined). In group O:3,10, factors O:15 or O:15,34, when
present, replace O:10. To indicate this fact, the following symbols
are used,O :3,{10},{15},{15, 34}
 ( ) = O or H factor weakly agglutinable H factor k, in S. enterica
subsp. arizonae, is weakly agglutinable by the standard k serum
(prepared against S. enterica subsp.enterica), but is normally
agglutinable by polyvalent k serum
KAUFFMANN AND WHITE SCHEME

48
Narayan Parajuli MS (CMID)
 LABORATORY DIAGNOSIS

• Laboratory diagnosis of enteric fever is based on the

following methods:
• 1. Isolation of Salmonella spp. by culture,

• 2. Serodiagnosis by demonstration Salmonella

antibodies and antigens
• 3. Molecular diagnosis by DNA probes and PCR

Narayan Parajuli MS (CMID) 49

 CLINICAL SPECIMENS

• Common specimens- Blood, blood clot, bone marrow,

and stool

• Isolation of typhoidal bacilli for culture

• Others - cerebrospinal fluid, peritoneal fluid,

mesenteric lymph nodes, resected intestine, pharynx,

tonsils, abscess, bone, and urine

Narayan Parajuli MS (CMID) 50

 CULTURE /ISOLATION OF BACILLI……

1. Ist weeks: Blood culture, Bone marrow culture

2. Second weeks: Serology; Widal test
3. Third weeks; Stool culture
4. Fourth weeks: Urine culture
 Culture medias: XLD Agar, DCA, SS Agar, Rambach’s
agar, McConkey agar
 Enrichment media: Tetrathionate broth, Selenite F
broth
 Blood Culture
• Very useful procedure for diagnosis of enteric fever
• Positive in 90% of cases in the first week of fever, 75%
of cases in the second week, 60% in the third week, and
25% thereafter
• Approximately 5–10 mL of blood inoculated into a
culture bottle containing 50–100 mL of 0.5% bile broth
• 10-fold dilution of blood to neutralize the bactericidal
action of many substances that are present in the blood
• Addition of liquid (sodium polyanethol sulfonate)
• Blood culture bottle is incubated at 37ºC for up to 7
days
• After incubation overnight at 37ºC, the bile broth is
subcultured on Mac Conkey agar
• Castaneda's biphasic method - better method of
culture to reduce the risk of contamination during
repeated subcultures Narayan Parajuli MS (CMID) 52
 CLOT CULTURE
• More sensitive method - certain inhibitory substances that are found
in the serum are absent in the clot proper

• Serum - can be used for demonstration of Salmonella antigens or

antibodies

• 5 mL of blood is collected and allowed to clot; serum is pipetted off

and used for serological tests

• Clot is broken up with a sterile glass rod and added to a bottle of bile
broth containing streptokinase (100 units/mL)

• Bile broth is incubated and subcultured on media in the same way as

described for blood culture

• Bone marrow culture is a most sensitive- positive in most cases

even when blood cultures are negative and positive even in case of
Narayan Parajuli MS (CMID) 53
antibiotic use
 FECES CULTURE
• Useful because salmonellae are excreted in feces throughout the
disease and even during convalescence with varying frequency

• Collected in a sterile container and sent immediately to the laboratory

• Fecal samples are inoculated directly on MacConkey, DCA, and Wilson–

Blair media

• For enrichment, one tube each of selenite and tetrathionate broth is

inoculated and incubated at for 12–18 hours before subculture onto
selective media

• Salmonellae may be isolated from several specimens by culture, but

they are not usually employed

• Urine, bile, rose spots, pus from suppurative lesions, CSF, and sputum-
other specimens
Narayan Parajuli MS (CMID) 54
 IDENTIFICATION

Narayan Parajuli MS (CMID) 55

 Slide agglutination test

• Performed with a loopful of growth from nutrient agar

plate or slope emulsified in two drops of saline on a clean
slide
• If S. Typhi is suspected, that is, when no gas is formed
from glucose, a loopful of typhoid O antiserum (factor
9/group D)
• Development of immediate agglutination suggests that
Salmonella strain tested belongs to Salmonella group D
• Identity of S. Typhi is established by agglutination with
the flagellar antiserum (anti-d
Narayan Parajuli MS serum)
(CMID) 56
 SERODIAGNOSIS
• Based on detection of specific Salmonella antibodies in
the serum, or antigen in the serum and also in urine by
various serological tests
A. Demonstration of serum antigens
• Circulating antigen present in the serum and urine,
but absent in serum of a cured case
• Demonstration of serum antigen always indicates an
active or recent typhoid fever
• Counter-current Immunoelectrophoresis, co-
agglutination test, and ELISA are frequently employed
for detection of circulating antigen in the serum
Narayan Parajuli MS (CMID) 57
 DEMONSTRATION OF SERUM ANTIBODIES
Widal test
• Traditional serologic test - measures agglutinating antibodies

against flagellar (H) and somatic (O) antigens in the patient’s sera

• Two tubes- Dreyer’s narrow agglutination tube with a conical

bottom is used for H agglutination, and Felix’s short round

bottomed tube for O agglutination

• O antigen is prepared by culture of S. Typhi on phenol agar (1:800)

and harvesting the growth in a small volume of saline

• Chloroform is used as a preservative

• H antigens are prepared by adding 0.1% formalin to a 24-hour

broth culture (Hagna broth) or saline suspension of an agar culture

58
Narayan Parajuli MS (CMID)
Narayan Parajuli MS (CMID) 59
• Test is performed by taking equal volumes (0.4 mL) of serial dilutions

of the serum (from 1/20 to 1/640) and H antigen of S. Typhi (TH), S.

Paratyphi A (AH), S. Paratyphi B (BH) and O antigens of S. Typhi (TO)

and mixing in Dreyer’s tubes and Felix’s tubes, respectively

• Tubes are then incubated in a water bath at 37ºC overnight; some

recommend incubation at 37ºC for 4 hours, followed by overnight

incubation at 4ºC

• Control tubes containing the antigen and normal saline are used to

check for autoagglutination

• H agglutination is characterized by the formation of loose, cotton
woolly clumps, while O agglutination by a disc-like pattern at the
bottom of the tube
• Titre: highest dilution of the serum showing agglutination with H or O
60
antigens suggests the antibody titer of the patient’s serum
 INTERPRETATION
• Antibodies against H and O antigens usually appear by 7th–10th day
of the illness and increase steadily till the third or the fourth week
• Demonstration of a fourfold or more rise in titer of antibodies

• A titer of 1/100 or more for O antibodies and 1/200 or more for H

antibodies is usually suggestive of enteric fever- necessary to obtain
basal antibody titer levels in “normal sera” in different areas
• Serum from an individual vaccinated with TAB vaccine may show
high titers of antibodies to S. Typhi and S. Paratyphi A and B
• Individuals who had suffered from enteric infections in past or who
have been immunized may develop an anamnestic response during
an unrelated fever, such as malaria, influenza, etc.
• Patients treated early with antibiotics, such as chloramphenicol,
may show a poor antibody response 61
Narayan Parajuli MS (CMID)
 Other serological tests

• Indirect hemagglutination

• Countercurrent immunoelectrophoresis

• Indirect fluorescent Vi antibody

• Indirect enzyme-linked immunosorbent assay (ELISA) for

Immunoglobulin M (IgM) and IgG

Narayan Parajuli MS (CMID) 62

 Molecular Diagnosis

• DNA probes and polymerase chain reaction

• (PCR) are being increasingly evaluated for the
diagnosis of typhoid fever
• DNA probes, although not commercially available,
have been developed for identifying S. Typhi from
bacterial culture isolates and directly from blood
• PCR for diagnosis is stillin experimental stage

Narayan Parajuli MS (CMID) 63

 Other laboratory tests

• Leucopenia with relative lymphocytosis

• Diazo test in urine positive: Generally between 5 th
and 14 th day of fever and remains positive until
fever subsides
 DIAGNOSIS OF CARRIER

• Detection is necessary for epidemiological and

public health purposes
• Repeated stool culture due to intermittent
shedding of bacilli
• Bile and duodenal drainage culture

• Repeated urine culture

• Antibody to Vi antigen in serum

 TREATMENT

• Chloramphenicol is the choice of drug for enteric fever

• Amoxycillin , ampicillin and cotrimoxazole are also effective

• Resistance to Chloramphenicol and other antibiotic have been

reported. So, Third generation cephalosporins or quinolones is

the current treatment

• IV or IM ceftriaxone (1-2g) is also prescribed; usually 10-14 days

(5-7 days for uncomplicated cases)

• Multi Drug Resistant (MDR) strains of S. typhi: quinolones are

the only effective oral treatment

• Nalidixic acid resistant S. typhi (NARST) must be tested for

sensitivity to determine course of treatment

 Prevention
• - Generally treated with antibiotics

- Vaccinations

- Education to general public

- Identification of all carriers and sources of contamination of

water supplies

- Avoid risky foods & drinks

- COOK and CLEAN food thoroughly, avoid raw vegetables

and fruits.

- WASH YOUR HANDS WITH SOAP AND WATER!!!

 SALMONELLA VACCINES
• Inactivated whole-cell vaccine: 2 doses/ 4wks. Apart;

single booster dose recommended every 3 years. Eg.

TAB( Trivalent)

• Ty21a- a live, attenuated S. typhi vaccine ( Oral

vaccine): avirulent mutant strain of S. typhi (TY21a)

lacking the enzyme UPD- galactose-4- empimerase has

been used. Administered orally (4 doses). Efficacy: 7 year:

Available in capsule form (Typhoral) contains 109 viable

mutant bacilli

• Vi polysaccharide vaccine: from purified Vi polysaccharide

from S. typhi. Administered subcutaneously or

ANY QUERIES?
Please…………..

Narayan Parajuli MS (CMID) 69