4.6 Enterobacteriaceae

1. 4.6 Enterobacteriaceae Enteric Gram negative aerobes rods

2. Sub-grouping

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 ShigellaOn 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 Shigelladysenteriae and toxigenic strain of Escherichia coli (ETEC) • When lysed, enterobacteria will releaseendotoxin 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. melitensismainly found in goat and sheep • B. abotusinfects cattle • B. suisfound in pigs and occasionally in goat • Other animal including horse, camel, eland and wild rodents

21. Routes of infection • Mosquitoes helps in transfer Brucellafrom 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-negativecoccobacilli 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 Brucellaand negative for the Salmonella.