OBJECTIVES Describe the general characteristics of the Enterobacteriaceae family. Discuss the principle, purpose, and interpretation of the following tests: Citra te DN a se L ysine iron a ga r Methyl R ed MIO N itra te ON PG Oxida se Phenyl a nil i n e dea mina se TSI Urea se Voges - Pra sk a uer Distinguish between lactose fermenting and non -lactose fermenting Enterobacteriaceae on the MacConkey, Eosin Methylene Blue, Hektoen, and XLD agars. List the non-motile Enterobacteriaceae. L i s t t h e E n t e r o b a c t e r i a c e a e t h a t p r o d u c e H 2S . Differentiate the genera of Enterobacteriaceae based on biochemical tests. Explain the common modes of transmission and clinical significance of Salmonella, Shigella and E. coli 0157:H7. Differentiate the following species: E.coli and E. coli 0157:H7 Proteus vulgaris and Proteus mirabilis Klebsiella pneumoniae and Klebsiella oxytoca. Discuss the clinical significance of the Enterobacteriaceae in this unit.
LACTOSE FERMENTATION TESTS Lactose non-fermenters Cannot break down lactose Lactose fermenters Have enzymes that allow for transport of lactose into the cell for utilization Slow lactose fermenters Lack beta-galactosidase permease But have beta-galactosidase
TRIPLE SUGAR IRON AGAR (TSI) - 1 Media composition Sugars (Glucose, Lactose, Sucrose) Peptone - nutrient and energy pH indicator H 2 S indicator
TSI - 2 How to read the tube? FIRST - Slant reaction SECOND – Butt reaction THIRD - H 2 S production “/” separates the reactions Circling the butt reaction denotes gas production Rules for test interpretation Glucose is always the preferred sugar for fermenters Glucose fermentation results in acid production Peptone utilization results in alkaline by-products
ONPG Lactose fermenter have the β- galactosidase enzyme, which hydrolyzes ONPG into galactose and orthonitrophenol = Color change from colorless to yellow Non-lactose fermenters - do not have the beta-galactosidase enzyme to hydrolyze the ONPG = No color change
MOTILITY Motility is defined as any growth away from the stab 1 = Non-motile 2 = Motile 3 = Motile
PHENYLALANINE DEAMINASE (PDA) Organisms that produce the enzyme deaminase are able to removes the amine group from the amino acid phenylalanine and releases the amine group as free ammonia. As a result of this reaction, phenylpyruvic acid is also produced. Negative After incubation, 10% ferric chloride is added to the media. Positive If phenylpyruvic acid was produced, it will react with the ferric chloride and turn dark green. If the medium remains a straw color, the organism is negative for phenylalanine deaminase production.
CLINICAL SIGNIFICANCE Shegellosis or bacterial dysentery Transmitted by ingesting contaminated food or water Fever Abdominal cramping and pain Scant stools with blood, mucous and pus Few organisms are capable of causing disease