ISOLATION AND

IDENTIFICATION OF
ENTERIC PATHOGENS

PRESENTED BY
AMRUTHA S
MSC 1ST YEAR
 ENTERIC BACTERIA

Members of Enterobacteriaceae

Non-sporing, non-acid fast, Gram-negative

Capsulated/non-capsulated

Motile by peritrichate flagella, or non-motile

Aerobic/facultative anaerobe, grow readily in ordinary media

Ferment glucose, produce acid & gas (or acid only)

Reduce nitrates to nitrites, form catalase but not oxidase

Vary in biochemical & antigenic properties

 CLASSIFICATION

Oldest method of Three widely used

classification: systems for the
• Lactose classification of
fermenters(Escherichia and Enterobacteriaceae Further classified into
Klebsiella) Majority of commensal
are: types-biotypes,
• Non-lactose intestinal bacilli are
fermenters(Salmonella, • Bergey's classification serotypes,
lactose fermenters,
Shigella and Proteus) • Kauffmann and White's bacteriophage types
called coliform bacilli classification applied to
Salmonella
and colicin types.
• Edwards-Ewing classification
 ESCHERICHIA COLI
• Gram negative, straight rod
• Arranged singly or in pairs
• Motile by peritrichate flagella
• Capsules & fimbriae found in virulent strains
• Aerobic/facultatively anaerobic
 Selective media (DCA Broth – uniform
Ordinary media – MacConkey medium
Blood agar – or SS agar) – turbidity & heavy
large, greyish white, – bright pink
hemolytic(associated isolation of deposit, disperse
moist, smooth colonies(Lactose
with infection) salmonellae & completely on
colonies fermentation)
shigellae shaking
 ISOLATION & IDENTIFICATION
1. Isolates grown at 37°C, 150rpm for 10hours
• Two types of growth media:
i. Luria-Bertani (LB) broth
ii. Tryptone Bile X-glucuronide (TBX) agar
medium
2. Standard microbiological methods – nutrient
broth, MacConkey Agar, Gram staining,
biochemical tests & EMB agar LB Broth TBX Agar
3. PCR after pre-enrichment
4. Electrophoresed in a 1.5% agarose gel for
one hour at 120V
5. Data analysed using chi-square method
 SHIGELLAE
• Short, Gram negative rods
• Non-motile, non-sporing & non-capsulated
• Fimbriae may be present
• Causative agent of bacillary dysentery
• Aerobes & facultative anaerobes
• Growth temp – 10-40°C (optimum temp –
37°C)
• pH – 7.4
• Small, circular, convex, smooth and
translucent colonies
 MacConkey agar -
colourless due to the Deoxycholate citrate On DCA agar Growth is inhibited
absence of lactose agar (DCA) and xylose on Wilson and Blair
fermentation lysine deoxycholate
Grow on ordinary bismuth sulphite
(XLD) - shigella do not
media but less readily • Except S.sonnei which medium
have a black centre
than other ferments lactose late and
forms pale pink colonies but Salmonella which
enterobacteria
appears red with a
black centre (selective
media)
 ISOLATION & IDENTIFICATION
• Specimen – feces • Serotyping – using commercial antisera to
• Suitable medium – Sachs’ buffered glycerol LPS O-antigen
saline or Gram negative broth(pH 7.0-7.4) Laborious, time-consuming & impractical
Enrichment media – selenite F broth or Salmonella Shigella
broth
• MALDI-TOF MS – rapid, cost-effective,
high throughput & reliable microbial
• Microscopy – plenty of pus cells
identification
• Culture in MacConkey & DCA or XLD agar,
Hektoen enteric agar and SS agar • PCR – target plasmid virulence gene such
as virA, ial, she & tuf.
• Commercial biochemical identification:
• pH based reactions Primer gene targets – ipaH
• Utilization of carbon source
• Enzyme-based reactions
• Visual detection of bacterial growth
• Detection of volatile or non-volatile fatty acids
 SALMONELLAE
• Gram-negative rods
• Aerobic and facultatively anaerobic
• Motile with peritrichate flagella, except S.Gallinarum
and S.Pullorum
• Do not form capsules or spores but may possess
fimbriae
• The most important member of the genus is Salmonella
Typhi, the causative agent of typhoid fever
• Salmonellae currently comprise above 2000 serotypes
or species, all of them potentially pathogenic
• Salmonellae may be divided into two groups:
Typhoidal & non-Typhoidal.
Grows readily on Wilson and Blair
simple media over a bismuth sulphite
pH range of 6-8 and Deoxycholate citrate medium - jet black
temperature 15-41 °C media and XLD (xylose colonies with a Selenite F and
(optimum 37°C) MacConkey agar - lysine deoxycholate) - metallic sheen are tetrathionate broth -
grow as non-lactose colonies show black formed due to the
• Large(2-3 mm in diameter), commonly employed
circular, low convex and fermenting colonies production of H2S
heads due to H2S as enrichment media
smooth colonies
• More translucent than production • S.Paratyphi A and other
coliform colonies species that do not form H,S
produce green colonies
 ISOLATION & IDENTIFICATION
• Enrichment & plating technique with subsequent • Individual colonies streaked onto conventional
estimation using MPN tables(traditional method) growth media & confirmation using biochemical
tests, serological identification or confirmation using
• Peptone & lactose broth found to be most effective nucleic acid methods
for resuscitation
• Slide agglutination test – Widal test
• Rappaport-Vassiliadis(RV) broth – Selection based
on resistance to malachite green, MgCl2 and ability • Pulse field gel electrophoresis for characterisation
to grow at pH 5 • Other immunological assays – ELISA, latex
• NR10 broth(modified RV broth) – isolation of agglutination & immunochromatography
Salmonella from marine water • Direct detection by fluorogenic & chromogenic
• Selective or differential medium – growth media
• Hektoen enteric agar • Rapid methods – molecular based assays(PCR),
• Brilliant green bile agar mass spectrometry & spectroscopy based detection,
• Xylose-lysine-deoxycholate agar optical phenotyping & electrochemical biosensors
 MEMBRANE FILTRATION
TECHNIQUE
• For detection of fecal coliforms in water sample
• Membrane incubated on m-FC agar at 45°C for
24hours
• Appearance- fecal coliforms: blue colonies
non-fecal coliforms: grey-cream colonies
• Viewed & counted under microscope
• Enriched lactose medium – 1% rosalic acid – inhibit
non coliforms with elevated incubation temperature
• To recover injured bacteria –
1. Membrane incubated on non selective media at 35°C
2. Rosalic acid removed from m-FC medium
3. Incubated on m-FC medium
4. Confirmed as coliforms
 CONCLUSION
Enteric infections caused by Salmonella, only half of individuals develop clinical signs of illness

Mode of transmission : fecal-oral route

Infect gastrointestinal tract

Stable in water & food materials, can grow outside host under favourable conditions

Isolation & identification includes-

• Sample collection
• Selective isolation
• Cultural characterization
• Biochemical & molecular testing
• Confirmation

Essential for diagnosing infections, ensuring food safety & monitoring environmental health

Advancement in molecular biology enhanced accuracy & efficiency of techniques

THANK YOU