1. UNIVERSITY OF COLORADO SCHOOL OF MEDICINE DEPARTMENT OF PATHOLOGY RESIDENCY TRAINING PROGRAM MICROBIOLOGY Case Studies: Beta Hemolytic Streptococci ANSWERS TO QUESTIONS

2. The colonies appearing on the surface of the sheep blood agar plate were recovered from each of a pair of blood culture bottles obtained from a 65 year-old man with low-grade fever, recent weight loss and mild hypo- chromic anemia. The gram stain features are illustrated in the right frame. Based on the colony morphology and the gram stain features, what presumptive identification can be made? What confirmatory tests should be performed? Gram-Positive Sepsis

3. AnswerS to Questions The colony morphology is non-specific. The colonies are relatively small, entire, slightly convex, and have a slight off- white yellow pigmentation. Hemolysis is absent. The gram stain, however, showing gram positive cocci in chains, indicates that the isolate is a streptococcus or possibly an enterococcus. The relatively long length of the chains rules against the latter. The lack of hemolysis makes presumptive identification more difficult. Several test options are open to establish an identification—PYR, esculin hydrolysis, growth in 6.5% NaCl, or set up of a commercial kit system. A first step might be to determine if the isolate carries one of the Lancefield group antigens.

4. Gram-Positive Sepsis A latex agglutination test was performed. This test employs polystyrene latex beads as the carriers for the group of specific antisera, which produces a flocculation of the latex particles if the unknown isolate possesses the complementary antigen. The latex panel illustrated here reveals a fine granulation in the Group D well (weak reaction). Latex Test What bacterial species should be considered? What confirmatory tests might be performed?

5. Answer to Questions The latex agglutination test indicates that this isolate is carrying the streptococcus Group D antigen. The two possibilities include a Group D streptococcus (Streptococcus bovis) or an Enterococcus species. To separate these two possibilities, tests to set up would include PYR, esculin hydrolysis, and growth in 6.5% NaCl.

6. Gram-Positive Sepsis The hydrolysis of PYR is helpful in separating Group D streptococci (negative) from the Enterococci (positive—see photograph). Group A streptococci are also PYR positive. The evolution of a red color indicates the presence of free beta-naphthalymide and a positive test. The reaction in this case was neg. In this case, the 6.5% NaCl (lack of growth in left tube) was negative; the bile esc- ulin reaction (black pigment in the right tube) was positive. WHAT IS THE MOST LIKELY GENUS ID?

7. Answer to Questions Following the legends for the images on this page, the PYR reaction was read as negative, no growth was observed in 6.5% NaCl and the black pigment in the bile/esculin tube indicates that esculin was hydrolyzed. These reactions are characteristic of a Group D streptococcus. Further testing is required to confirm this presumptive identification. The identification system used in your laboratory should be used to make the final definitive identification. In this case, an API Rapid Strep strip was used (next page).

8. The API Rapid Strep System is used in the identification of Streptococcus sp. This system consists of 20 cupules containing modified conventional and chromogenic substrates (see photograph). Each cupule is inoculated with a suspension of the unknown organism and incubated for 4 hours. The positive and negative reactions are converted into a biotype number using the octal coding system. The profile number is: 5240473 = Streptococcus bovis. What comment should be added to the report? Septicemia: API Rapid Strep Assay

9. Answer to Question The last step in the diagnosis and work-up of this case is to append the report of Streptococcus bovis with the comment: “Bacteremias with Streptococcus bovis are commonly associated with carcinoma of the colon. Follow-up diagnostic procedures are indicated.” The more advanced age of the patient makes this diagnosis a real possibility.

10. Abbreviated Identification of Streptococcus bovis Gram Positive Cocci in chains PYR Negative STREPTOCOCCUS BOVIS (GROUP D) Small, dry, gray-white, non or alpha hemolytic colonies on sheep blood agar Group D antigen in latex agglutination test Bile Esculin positive Set up commercial identification system (API 20 E Biocode # 5240473 keys out to): No Growth in 6.5% NaCl