Standard Operating Procedure Subject Culture: Routine Stool Index Number Lab-3105 Section Laboratory Subsection Microbiology Category Departmental Contact Sarah Stoner Last Revised 3/18/2019 References Required document for Laboratory Accreditation by the College of American Pathologists. Applicable To Employees of the Gundersen Health Systems laboratories. Detail This document establishes guidelines for isolation and identification of stool pathogens. PRINCIPLE: Gastroenteritis can be caused by bacteria, parasites or viruses. Physician input can help the lab determine which tests are appropriated for detecting the etiological agent. When only a routine fecal culture is requested, however, the lab should search for the most common or more easily detected bacterial agents of diarrhea. Our laboratory looks for Salmonella, Shigella, E.coli O157:H7 and Campylobacter. CLINICAL SIGNIFICANCE: Salmonella, Shigella, Campylobacter and E.coli O157:H7 are the most prevalent stool pathogens isolated by our laboratory. They are implicated as agents of foodborne illness and isolates need to be reported to the state for epidemiological purposes. Salmonella outbreaks have been associated with raw eggs, reptiles, and poor hand washing. E.coli O157:H7 outbreaks have been traced to hamburger, raw milk, sausage, roast beef, un-chlorinated municipal water, apple cider, raw vegetables, salads, and mayonnaise. E.coli O157:H7 spreads easily from person to person because the infectious dose is low: outbreaks associated with person to person spread have occurred in schools, long term care institutions, families, and day-care facilities. Campylobacter has been associated with poultry products and contaminated water. SPECIMEN: Refer to Lab-1215 Microbiology Specimen Collection and Transport. Fresh stool received within 1-2 hours of passage. 1. Stool in Cary-Blair transport media (store at 4-6°C). 2. Routine bacterial fecal cultures should not be performed on patients hospitalized for > 3 days. The exception to this is a hospital outbreak. REAGENTS/MATERIALS: 1. BBL Mac Conkey II with sorbitol (commercial) SMAC for E.coli O157:H7. 2. Hektoen Agar Plate (commercial). Page 1 of 5 Standard Operating Procedure 3. Mac Conkey Agar Plate (commercial). 4. Campylobacter Agar Plate (commercial). 5. TSI slant 6. KIA slant 7. LIA slant 8. Urea slant 9. Blood Agar Plate (BAP) 10. VITEK2 GNI, AST-GN66 (Salmonella and Shigella only), GPI and AST-GP67 cards 11. 0.9% sterile saline 12. Salmonella serotyping antisera 13. Shigella serotyping antisera 14. RIM Test Kit EQUIPMENT/INSTRUMENTATION: N/A QUALITY CONTROL: 1. Refer to Lab-3257 User Quality Assurance Procedure for Culture Media. 2. The stock isolate of Campylobacter jejuni is subbed daily to BAP, for each jar in use. This serves as a growth indicator to ensure that the correct atmospheric conditions were used during incubation. Implementation Inoculate the following media: SMAC, Hektoen, Mac Conkey and Campylobacter agar. If blood or mucus is present in the stool, use this portion to inoculate the plates. If a gram stain is requested make a smear also. 1. Streak plates for isolation. 2. Incubation: a. SMAC, HEK and MAC agars should be incubated in the non-CO2 incubator at 35 to 37°C. Hold plates for 48 hours before discarding. b. Media for isolation of Campylobacter spp. should be incubated in a microaerobic atmosphere (5% oxygen) at 42°C for 72 hours before discarding. PROCEDURAL NOTES: Routine stool cultures do not include screening for Yersinia. If Yersinia is incidentally recovered on routine cultures, work up, and report to physician. (Physicians should order a culture for Yersinia if suspected.) CALCULATIONS: N/A INTERPRETATION OF GROWTH ON CULTURE MEDIA FOR STOOL SAMPLES: Medium Pathogen Colony Morphology Identification Procedure Mac Conkey Colorless or transparent TULN (see chart) and BAP SMAC Colorless or transparent TULN plus sorbitol, dulcitol and BAP (refer to identification scheme for E.coli O157:H7) Page 2 of 5 Standard Operating Procedure Hektoen Blue or blue green with or without black centers TULN (see chart) and BAP Campy plate Grey to pinkish, flat to mucoid to convex to Gram stain, Wet Prep and spreading all over plate Oxidase (see Table 1) ENTERIC SCREENING: 1. Suspicious colonies should be first spotted to MALDI-TOF for rapid identification. Colonies identified as E. coli should be further tested by biochemical methods. 2. E.coli identified by MALDI-TOF should be set up to the following tubes and plate. a. TSI: stab the butt and streak the slant b. KIA: stab the butt and streak the slant c. LIA: stab the butt x 2 and streak the slant d. Urea: streak the slant e. BAP (to be used for indole, oxidase, PYRr, serotyping and gni/gns if necessary) If the SMAC plate has a large number of clear colonies of the same morphologic colony type, you can do a RIM test from the SMAC plate and do not do a TULN. If positive sub to a BAP to get a pure isolate for the MALDI-TOF or Vitek ID card. Refer to Lab-3106 RIM Test for E.coli O157:H7 3. Incubate tubes overnight at 35-37°C (non-CO2 incubator) 4. Determine if the organism is a potential pathogen by comparing reactions and doing quick tests. Follow the flow chart provided. 5. Suggestive screening results should have complete biochemical identification. a. Vitek 2 gni b. Serology – Refer to Lab-3326 Serotyping Salmonella; Lab-3328 Serotyping Shigella; Lab-3106 RIM Test for E.coli O157 6. Do sensitivities on Salmonella and Shigella species. 7. Confirmed pathogens should be called to the RN or doctor. 8. Streak a nutrient slant x 2. Send all stool pathogens to the applicable state lab for food borne illness surveillance. (See binder for list of desired surveillance isolates) CAMPYLOBACTER SPECIES 1. Examine plates at 24, 48 and 72 hours for characteristic colonies, which can range from flat, spreading colonies that cover the entire surface of the plate to very small, convex, translucent colonies. Colony color can range from gray to yellowish or pinkish. 2. Presumptive identification a. Gram stain shows small, slightly convex, gram negative rods that are “s” or seagull shape. b. Wet prep shows darting or tumbling motility. c. Oxidase positive. d. Catalase positive. 3. Definitive identification: spot to MALDI-TOF E.COLI O157:H7 1. If the TULN is interpreted as E.coli, proceed with RIM test. If the SMAC plate has a large number of clear colonies of the same morphologic colony type, you can do a RIM test from the SMAC Page 3 of 5 Standard Operating Procedure plate and do not do a TULN. If positive, sub to a BAP to a pure isolate for the MALDI-TOF or Vitek ID card. Refer to Lab-3106 RIM Test for E.coli O157. 2. Isolate must be confirmed to be an E.coli before sending out as E.coli O157. Perform Vitek ID card to confirm. DIFFERENTIAL CHART: TULN Reading TSI, LIA, Urea, KIA (On suspected Salmonella and Shigella – non-lactose fermenting colonies) Non- TSI H2S LIA H2S UREA KIA Pathogens Citrobacter A / A + or K / A + - or A / A K / A trace - trace K / A Klebsiella K / A - K / A - Trace A / A Enterobacter Or or Or Or Serratia A / A K / K + K / A Proteus K / A + / - K / A - / + + K / A R / A Providencia K / A - R / A - - K / A E.coli K / A - K / K - - A / A A / A K / A K / A Pathogens TSI H2S LIA H2S Urea KIA other than E.coli O157 and Camylobacter Salmonella or K / A Trace or K / K + - K / A Edwardsiella + Arizona sp. A / A + K / K + - K / A or A / A Possible K / A - K / A - - K / A Shigella Yersinia A / A - K / A - - or K with acid enterocolitica Trace (yellow Or orange Salmonella A / A - K / A - - color) paratyphi K / A K / A Refer to Lab-3105.1 Stool Culture Work Aide for additional guidance from the Clinical Microbiology Procedure Handbook. Copy is in the drawer by the R3 desk. REPORTING: 1. A preliminary culture report will be sent out at 24 hours. Additional preliminary reports should be sent out on anything significant after 24’. 2. A final culture report will be sent out when all identifying tests are completed. A final report negative report will be sent out as: No Salmonella, Shigella, E.coli O157:H7 or Campylobacter isolated. Notify microbiology at ext. 53138 if other pathogens are suspected. Page 4 of 5 Standard Operating Procedure 3. The following are considered pathogen in a stool culture: a. Campylobacter b. Salmonella c. Shigella d. Yersinia enterocolitica (Dr. must specifically request) e. E.coli O157:H7 f. Vibrio (Dr. must specifically request) LIMITATIONS: N/A REVIEW AND CHANGES: This document and all attached forms should be reviewed optimally on an annual basis, with 2 years as the maximum review date. Review will be done by the Technical Leader, Medical Director or designated person. Changes require retyping the document or form and review by the Medical Director. REFERENCES: Pillai, DR (ed). 2016. Fecal Culture for Aerobic Pathogens of Gastroenteritis: Aerobic Bacteriology. Section 3.8.1.1-3.8.1.20. In Leber, A. L.(ed). Clinical Microbiology Procedures Handbook, 4th ed, vol 1. ASM Press, Washington, DC. 2016. Page 5 of 5 .