1. UNIVERSITY OF COLORADO SCHOOL OF MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM MICROBIOLOGY V: Enterobacteriaceae: Non-Lactose Fermenters Case Study 5a: Acute Diarrheal Syndrome Adapted from the CACMLE Teleconference first presented by: Paul C. Schreckenberger, Ph. D. Director of Clinical Microbiology: Loyola University, Chicago August 26, 1992

2. Acute Diarrheal Syndrome Illustrated in this split-frame photograph is the blood agar and MacConkey agar growth obtained from a patient who presented with acute diarrhea. He had no travel outside the Chicago area and had no contact with cold-blooded animals or exotic pets. The colonies on blood agar (left) are flat, gray, and non- hemolytic. What does the appearance of the colonies on MacConkey agar indicate? What additional media reactions would be helpful in making a presumptive identification? (Next Page)

3. Acute Diarrheal Syndrome The lack of yellow pigment production by the colonies growing on MacConkey agar indicates that the isolate is a non-lactose fermenter. Although no longer in common laboratory practice, it is still highly recommended that any such non-lactose fermenter be transferred to Kligler’s iron agar or to triple sugar iron agar at the time a test kit or automated instrument is inoculated. These iron agar media provide a quality control check for the biocode read-out. Describe the reaction on the KIA tube shown in the photograph to the right. What does the pink-red slant indicate? What does the diffuse black deep indicate? What genera of the Family Enterobacteriaceae must be considered? What biochemical characteristics are helpful in making this differentiation? ANSWERS

4. Acute Diarrheal Syndrome The urease activity of an unknown isolate can be easily determined by inoculating a Christensen’s urea agar slant. Proteus sp. will turn the media pink-red within a few hours. Lysine decarboxylase activity can be most easily determined by using Xylose Lysine Deocycholate agar (XLD), a photograph of which is shown to the right. The surface had been inoculated with the isolate 24 hours earlier. The black centers of the colonies indicates H 2 S production. The light pink halo around each of the colonies indicates a drop in pH and the decarboxylation of lysine, indicating a Salmonella species.

5. DIAGNOSIS: Acute Diarrheal Syndrome The API 20E strip produced the biocode number 7504552k, which keyed out as “Salmonella subgroup 3, excellent identification”. The GNI card produced a biocode number of 3030721766, indicated “Salmonella arizonae” With 99% confidence

6. Abbreviated Identification of Salmonella arizonae Non-pigmented colonies on MacConkey agar XLD and HE agars--H 2 S-producing colonies Light pink-red halo around colonies on XLD agar Dry, gray-white colonies on sheep blood agar. Alk/Acid reaction on KIA or TSI with H 2 S in deep Salmonella subgroup 3 (serogroup arizonae) ONPG + Suspect Salmonella species Biocode confirmation of Salmonella sp. Malonate +Dulcitol -

7. Added Pearl As an added bonus, presented in the photograph is a KIA slant indicating the reaction of another H 2 S-producing bacterial species. The narrow rim of H 2 S at the junction of the slant and the deep, as seen in the left hand tube, is characteristic of the weak Salmonella typhosa, allowing a presumptive differentiation from other H 2 S-producing strains where the entire deep is pigmented. Notice that the deep is yellow in color, indicating glucose fermentation. Even when the deep is diffusely black from H 2 S production, the pH can be read as acid (glucose fermentation) because H 2 S does not react in at an alkaline pH.

8. “THE RULE OF STOOL” For the optimum recovery of bacterial pathogens from stool specimens, a non-inhibitory medium such as sheep blood agar, and a differential, moderately selective medium such as Hektoen or XLD are recommended. Although the high concentration of bile salts in Salmonella/Shigella (SS) medium is satisfactory for the recovery of Salmonella species, its routine use is discouraged because of its inhibitory effect on certain Shigella species. In addition, a Campylobacter selective media (CMS agar or CAMPY BAP) must be included in certain circumstances. Selective enrichment broth has been used for the recovery of Salmonella or Shigella species from heavily contaminated specimens, such as feces or sewage. Selenite broth is more inhibitory to the growth of E. coli and other coliforms than is Gram- negative (GN) broth. The latter is less inhibitory to the growth of certain fastidious strains of Shigella sp. Enrichment of fecal specimens in GN broth for 7 – 6 hours and then subculture to HE or XLD agar is the optimal technique for the recovery of Shigella species in suspected cases of bacillary dysentery.

9. Salmonella arizonae: RECAPITULATION Salmonella arizonae, formerly Arizona hinshwaii, is a gram-negative bacillus in the family Enterobacteriaceae and resembles the genus Salmonella antigenically, clinically, and epidemiologically. It was first recovered in 1939 from diseased reptiles in Arizona and was initially called “Dar-es-Salaam type variety from Arizona”. It was distinguished from Salmonella as Arizona hinshawaii, but as of July 1, 1983, was reclassified by the Centers for Disease Control as a serotype within the genus Salmonella. Although most Salmonella species serotypes cannot be distinguished by biochemical reactions, Salmonella serotype arizonae can be easily differentiated on the basis of having positive malonate and negative dulcitol reactions. Some strains ferment lactose; all are ONPG positive. Thus, with these unique biochemical reactions, a correct serotype designation can easily be made using most commercially available identification systems.

10. ANSWERS The pink-red slant indicates that lactose has not been fermented (thus KIA provides a ready means to separate lactose and non-lactose fermenting species of the Enterobacteriaceae). The diffuse black pigmentation of the deep indicates the production of H 2 S, which reacts with the ferric ions in the medium to produce ferrous sulfide. Of the non-lactose fermenting Enterobacteriaceae, only Salmonella species and Proteus species produce H 2 S. Two key biochemical reactions are commonly used to separate these two genera (remember, however, that Proteus species swarm on the surface of blood agar): Urease activity (Proteus positive) Lysine decarboxylase activity (Salmonella positive) BACK