Comparison of two methods for detection and isolation of Shiga toxin-producing Escherichia coli from feces in commercial beef cattle processing plants.

LEOTTA G.A., DEL CASTILLO L., GALLI L., PALLADINO M., CARBONARI C., SOSA-ESTANI S., MASANA M., RIVAS, M.

Buenos Aires

Simposio; 7th International Symposium on Shiga Toxin (Verocytotoxin) - Producing Escherichia coli infections.; 2009

Shiga toxin-producing Escherichia coli (STEC) strains have caused major outbreaks and sporadic cases of human illnesses, including mild diarrhea, bloody diarrhea, hemorrhagic colitis, and the life-threatening hemolytic uremic syndrome. These illnesses have been traced to both O157 and non-O157 STEC. In a large number of STEC-associated outbreaks, infections were attributed to consumption of ground beef or other beef products contaminated with microorganisms harbored in cattle feces. The aim of this work was to compare two methods for the detection and isolation of STEC from feces of beef cattle at the start of the operation of commercial processing plants of Argentina. Between November 2006 and March 2007, 251 fecal samples were collected from the descending colon near the rectum in nine abattoirs. Two experimental approaches were evaluated for their ability to detect stx genes by PCR in order to isolate STEC in bovine fecal samples. Method 1: 10 g of the feces were incubated in 90 ml of GN Hajna at 37ºC for 5 h; after primary enrichment 10 ml were added to 90 ml of MacConkey broth and incubated at 37ºC for 18 h. Enriched samples were then screened for stx genes by MK-PCR. Each positive sample was plated on MacConkey agar and EMB-Levine agar and incubated at 37ºC for 18 h. Confluent growth zone and colony pools were screened for stx1, stx2 and rfbO157 genes by multiplex PCR. Method 2: a fecal sample swab was directly plated onto MacConkey agar and incubated at 37°C for 18 h. Confluent growth zone and colony pools were screened for stx1, stx2 and rfbO157 genes by multiplex PCR. Of 251 samples, 110 (43.8%) were positive by PCR: 19 (17.2%) were positive using method 1, 56 (51%) using method 2, and 35 (31.8%) were positive by both methods. However, STEC strains were recovered in 69 (27.5%) samples: 10 (14.5%) by method 1, 43 (62.3%) by method 2, and 16 (23.2%) samples using both methods. The comparison of the two methods was based on the relation of method 2 with method 1, with method 1 as the reference method. The concordance of both methods was 73.71%            (CI 95%, 68.06-79.35), sensitivity was 66.67% (CI 95%, 52.29-81.04), and specificity was 75.37% (CI 95%, 69.20-81.54). Both methods can be used to detect and isolate STEC in bovine fecal samples. However, method 2 was able to detect and isolate a higher number of positive samples. Our study confirms and emphasizes that PCR is an effective tool for screening of STEC in bovine feces, but the recovery of STEC strains depends on the coupled isolation methodology used. These methods could be used as appropriate alternatives for detection and confirmation of STEC strains from bovine fecal samples.