Beth Schweitzer MT(ASCP), SM Nebraska Public Health Laboratory Enteric Pathogens Beth Schweitzer MT(ASCP), SM Nebraska Public Health Laboratory

Pathogens Routinely Tested Salmonella spp. Shigella spp. Campylobacter spp. Aeromonas Plesiomonas Vibro This is a list of pathogens that will be detected by a routine Stool Culture. A Stool Culture must be held for 48 hours before it can be determined negative. However, a positive may take slightly longer to fully identify the organism involved.

Pathogens Tested for upon request Yersinia entercolitica E. coli 0157:H7 Vibrio Must be specifically requested This is a list of organisms that must be specifically requested in order for them to be identified. There are special medias required for their identification. A physician must order a test for these organisms.

Testing for Salmonella/Shigella Look for appearance of colonies on specialized media

Normal Enteric Flora Stool is not a sterile body site, as we all know. The lab uses special medias that let us see what sugars the bacteria can use as a food source, as a way to help identify them. These are 2 pictures of normal enteric flora. See the pink colonies and the yellow colonies.

Salmonella/ Shigella This is a picture of an isolate of Salmonella or Shigella. Notice the colonies are clear, as compared to the pink colonies on the previous slide.

E. coli/ Shigella This is a picture of E coli on the top and Shigella on the bottom. Notice the yellow colonies as compared to the dark green colonies on the bottom.

Salmonella This is a picture of an isolate of Salmonella. Salmonella is able to perform a biochemical reaction on this media that causes the colonies to become black. Notice the colonies with the black centers.

Campylobacter testing Look for colonies on media after incubation at specific temperatures and atmospheric conditions Campylobacter needs special atmospheric conditions that help inhibit the other normal enteric flora. It is also able to grow at warmer temperatures than the normal enteric flora. Because of the special atmospheric needs, this is why we need to get the stool specimen as soon as possible or it may die. There are special enteric preservatives you can put the stool into if there is going to be a delay in transport.

Campylobacter screening This is baggie we fill with gas so the Campylobacter can grow.

Campylobacter spp. This is an electron micrograph picture of Campylobacter.

Gram- Negative cell structure Once we have a organism identified we now have to use structures on the cell surface to serogroup and serotype the organism.

There are proteins sticking off the surface of the cell wall, they are known as the O antigens

Salmonella Serogrouping Based on Cell Wall Antigens (O) Somatic Each antigen identified by a number designation Multiple antigens can be present on each cell’s surface Serogroup is determined by pattern of O antigens present 95% of isolates belong to serogroups A-E (O2-O10) Remaining 5% O antigens O11-O67 Commercial kits available Latex agglutination These O antigens are what determine the serogroup.

Next we look at the proteins that are on the flagellum.

Salmonella serotyping Based on Flagellar antigens (H) Each antigen identified by a letter or number designation Multiple antigens can be present on each isolate Most isolates can posses 2 sets (phase1/phase 2) Serotype is determined by O antigens present and phase 1/phase 2 H antigens Over 2400 different serotypes Over 1400 common to humans No commercial kit available

Serotyping End up with an antigenic formula O antigen: Phase 1 H antigens: Phase 2 H antigens Most common formula’s have a name Salmonella serotype Typhimurium (4,5:i:2) Serogroup B Salmonella serotype Enteritidis (9:gm:-) Serogroup D

E. coli O157:H7 First look for E. coli in stool Sorbitol negative Test organism for presence of O and H antigens Commercial kit available In order to find E coli in stool we use special media to help us tell the difference between the normal healthy E coli, and the E coli O157:H7. E coli O157:H7 cannot use a sugar called sorbitol as food, where normal E coli can. Once we find a sorbitol negative E coli, we then test for the specific O and H antigens.

This is a basic antigen/ antibody reaction, and is the basis for our latex agglutination tests. As the antibodies bind up the antigens they begin to clump up. If the antibodies are bound to latex beads we then can visualize the clumping.

Circle 2 contains a positive latex agglutination test you can see the beads are all clumped together as compared to the homogenous texture of circle 1.

Further testing All E coli O157:H7 isolates have Pulse field gel electrophoresis (PFGE) performed on them Any other organisms can have PFGE performed, at the request of the state Once we have identified an E coli O157:H7 Pulse Field Gel Electrophoresis is performed. The state of Nebraska has decided that PFGE is to be performed on all E coli O157:H7 that are found in the state. We can perform PFGE on any organism, but it must be requested by the state.

Any questions?? Contact information bschweitzer@unmc.edu 559-6098