1. Enteric Gram negative aerobes rods
4.6 Enterobacteriaceae
Enteric Gram negative aerobes rods

2. Sub-grouping LACTOSE FERMENTING (PINK PIGMENT IN MAC AGAR)
NON-LACTOSE FERMENTING
(NO PINK PIGMENT IN MAC AGAR)
ESCHERICHIA
KLEBSIELLA
ENTEROBACTER
CITROBACTER
SALMONELLA
SHIGELLA
PROTEUS
MORGANELLA
PROVIDENCIA
SERRATIA

3. Morphology Belongs to Gamma Proteobacteria Gram negative Rods
1-4 X 0.6µm
Non-sporing
Have simple nutrition requirements
Facultative anaerobes

4. 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

5. 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.

6. Klebsiella
E.coli

7. 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

8. 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

9. E.coli
In nutrient agar
In MacConkey agar

10. Klebsiella sp
In macconkey agar
In blood agar

11. Salmonella sp
In ss agar
In xld agar

12. 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

13. 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:

14. 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.

15. Antigenic Structure of Enterobacteriaceae

16. Antigenic structure of Enterobacteriacea

17. Assignments
Prepare notes on each of genus under enterobacteriaceae group. (10 genus)
All the notes should have the following criteria:
morphology,b)culture, c)biochemical tests d) serology test
Submit on 3rd of September 2012

18. 4.7 BRUCELLA

19. Main species
Brucella melintensis
Brucella abortus
Brucella suis

20. Normal habitat Obligate intracellular pathogens of animals
B. melitensis mainly found in goat and sheep
B. abotus infects cattle
B. suis found in pigs and occasionally in goat
Other animal including horse, camel, eland and wild rodents

21. 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.

22. Microscopic observation
Non-motile
Gram negative
Coccobacili
Show bipolar staining
Rarely found in direct smear from uncultured specimen
On Gram stain they appear as dense clumps of Gram-negative coccobacilli and are exceedingly difficult to see.

23. 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

24. 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

25. 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.