1. UNIVERSITY OF COLORADO SCHOOL OF MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM MICROBIOLOGY V: Enterobacteriaceae: Non-Lactose Fermenters Case Study 5b: “Thigh Abscess with Septicemia” Abstracted from a CACMLE Teleconference with Frank Kocka, Ph. D. Cook County Hospital, Chicago, Illinois

2. Thigh Abscess with Septicemia The colonies illustrated in this photograph were recovered after 24 hours incubation at 35 o C from a thigh abscess of an infant on day 120 after premature delivery, complicating a femoral artery line. Prior medical history revealed previous episodes of E. coli and Staphylococcus aureus septicemia related to indwelling lines. The colonies illustrated here are large, off-white, entire, and dome- shaped. Hemolysis is absent. A musty odor of unpeeled potatoes was detected.

3. Thigh Abscess with Septicemia Photograph of the appearance of the colonies of this isolate growing on the surface of MacConkey agar after 24 hours incubation at 35 o C. The colonies are large, entire, smooth, and dome-shaped. Note the distinct light rose-pink pigmentation; however, note in particular that the pigment is confined to the colonies and does not leach out into the surrounding agar. What might this colony pigmentation signify? What Genus within the Family Enterobacteriaceae is most likely? What biochemical characteristics help in making the identification? ANSWERS

4. Thigh Abscess with Septicemia The reactions observed on a Microtiter Neg ID Panel Type 2 plate inoculated with a suspension of this isolate produced a biocode number of 77604356, coding to “Enterobacter aerogenes”. The positive DNAse and negative ornithine reactions, however, are inconsistent with this species. Using an API 20- E strip, the biocode of 5247753 coded out to an identification of: Serratia odorifera This case illustrates the need to recognize characteristics that may indicate when the identification provided by a given system cannot be correct. In such cases, additional tests or an alternate system may be required.

5. Abbreviated Identification of Serratia odorifera Pink colonies on MacConkey agar—no leaching into agar — weak or late lactose fermenter DNAse test Positive Lysine Positive Smooth, gray-white, entire, convex colonies on sheep blood agar. Odor of unpeeled potatoes Citrate + Serratia odorifera, Biogroup 2 (Confirm with biotype profile) Urea Negative Ornithine Negative

6. Serratia odorifera: RECAPITULATION An initial clue to the presumptive identification of Serratia odorifera is the detection of the musty, unpeeled potato odor emanating from mature colonies. The observation of a rose-pink pigment confined to colonies growing on MacConkey agar may also be an initial clue. A positive DNA’se reaction points to Serratia species. Serratia odorifera is an uncommon cause of opportunistic human infections. Serratia marcescens is the Serratia species most commonly recovered from clinical specimens. However, the lack of the characteristic odor and the negative ornithine reaction rules against this identification. Demonstrating the inability to produce acid from sucrose would also be in favor of Serratia odorifera. In this case, the source of the infection was most likely nosocomial, with extension into the site of the catheter wound from the adjacent skin where the organism undoubtedly resided as a commensal.

7. ANSWERS TO QUESTIONS The pigment confined to the colonies, without leaching into the adjacent agar, indicates that indiginous pigment has been produced and lactose has not been fermented. Among the Enterobacteriaceae, the genus Serratia, particularly, S. marcescens is singled out. Serratia species are unique among the Enterobacteriaceae in producing three hydrolytic enzymes: lipase, gelatinase, and DNAse. Illustrated in this photograph is the surface of DNA agar on which are growing the colonies of this isolate after 24 hours incubation at 35 o C. Note the distinct sunset-red pigmentation of the agar adjacent to the growth of the colonies along the streak line. BACK This pigment change indicates hydrolysis of the DNA under the action of DNAse, with clearing and a pH shift in the toluidine O blue indicator in the medium. This positive reaction points to an identification of Serratia species.