Enterobacteriaceae & Brucella

4.6 Enterobacteriaceae

Enteric Gram negative aerobes rods

Sub-grouping

LACTOSE FERMENTING(PINK PIGMENT IN MAC AGAR)

NON-LACTOSE FERMENTING(NO PINK PIGMENT IN MAC AGAR)

1. ESCHERICHIA2. KLEBSIELLA3. ENTEROBACTER4. CITROBACTER

1. SALMONELLA2. SHIGELLA3. PROTEUS4. MORGANELLA5. PROVIDENCIA6. SERRATIA

Morphology

Belongs to Gamma Proteobacteria Gram negativeRods1-4 X 0.6µmNon-sporing Have simple nutrition requirementsFacultative anaerobes

Important bacterial group, they are called enteric bacteria as it reflects the fact that they inhabit the intestinal tracts of humans and other animals.

Differentiation is based on biochemical reactions and differences in antigenic structure

Motility

Mostly surrounded by flagella (peritrichous) and are motile

Non-motile enterobacteriaceae are Shigella and Klebsiella

In Escherichia and Morganella, most of the strain are motile, but some are non-motile

Special case like Yersinia, contain species that are motile at 25oC but non-motile at 35 – 37oC.

Klebsiella E.coli

Culture

Most will grow in wide temperature range in ordinary culture media including NA and BA and selective media.

The selective media is incorporated with dyes and bile salts that inhibit G+ organisms and may suppress the growth of nonpathogenic species of Enterobacteriaceae

Eg: Selective media is required to recover Salmonella and Shigella On BA, enterobacteria produce large, shiny, grey colonies that may be hemolytic. Most grow well on a variety of lab media including a lot of selective and differential media originally developed for the selective isolation of enteric pathogens

Enterobacteriaceae

Many are differential on the basis of whether or not the organisms ferment lactose and/or produce H2S. Species that produce hydrogen sulphide often show a green colour around the subsurface colonies (Klebsiella) and capsulated strains (Escherichia) produce large mucoid colonies.

Catalase reaction vary among Enterobactericeae

E.coli

In nutrient agar In MacConkey agar

Klebsiella sp

In macconkey agar In blood agar

Salmonella sp

In ss agar In xld agar

Toxin production

Exotoxin (enterotoxin) is produced by Shigella dysenteriae and toxigenic strain of Escherichia coli (ETEC)

When lysed, enterobacteria will release endotoxin from their cell wall

*The feature above is applied to all Gram negative rods

Enterobacteriaceae

On CBA they all produce similar colonies that are relatively large and dull gray. They may or may not be hemolytic.

The three most useful media for screening stool cultures for potential pathogens are TSI, Lysine iron agar (LIA), and urea or phenylalanine agar.

The antigenic structure is used to differentiate organisms within a genus or species.

Three major classes of antigens are found:

Enterobacteriaceae

Somatic O antigens – these are the heat stable polysaccharide part of the LPS.

Variation from smooth to rough colonial forms is accompanied by progressive loss of smooth O Antigen.

Flagellar H antigens – are heat labile Envelope or capsule K antigens – overlay the surface O

antigen and may block agglutination by O specific antisera.

Boiling for 15 minutes will destroy the K antigen and unmask O antigens.

The K antigen is called the Vi (virulence) antigen in Salmonella typhi.

Antigenic Structure of Enterobacteriaceae

Antigenic structure of Enterobacteriacea

Assignments

Prepare notes on each of genus under enterobacteriaceae group. (10 genus)

All the notes should have the following criteria:

a) morphology,b)culture, c)biochemical tests d) serology test

Submit on 3rd of September 2012

4.7 BRUCELLA

Main species

Brucella melintensisBrucella abortusBrucella suis

Normal habitat

Obligate intracellular pathogens of animalsB. melitensis mainly found in goat and

sheepB. abotus infects cattleB. suis found in pigs and occasionally in

goatOther animal including horse, camel, eland

and wild rodents

Routes of infection

Mosquitoes helps in transfer Brucella from animal to human

Also by ingesting unpastuerized milk, meat or milk products, enter damaged skin or eyes, inhaled in airborne particles or aerosols and close contact with secretions.

Microscopic observation

Non-motileGram negativeCoccobaciliShow bipolar stainingRarely found in direct smear from

uncultured specimenOn Gram stain they appear as dense

clumps of Gram-negative coccobacilli and are exceedingly difficult to see.

Culture characteristics

Mostly cultured from blood of high fever patient(Brucellosis) Isolation is extremely rare in chronic brucellosis In all blood culture, they need carbon dioxide Blood culture should be kept in 4 – 6 weeks before result as no

organisms isolated To reduce the risk of contamination, use the diphasic medium

such as Castaneda or tryptic soy broth or agar Brucellae are aerobic with enriched of carbon dioxide

Biochemical tests Serology tests

Urease and hydrogen sulphide production

All brucella strains are catalase positive

Possess two antigens called A and M

Famous test serum: Rapid slide

agglutination test Tube agglutination

titration test

Serology test

It is crucial to be able to differentiate Brucella from Salmonella which could also be isolated from blood cultures and are Gram-negative. Testing for urease would successfully accomplish the task; as it is positive for the Brucella and negative for the Salmonella.