Survival of Escherichia coli O157:H7 in water of animal-drinking troughs in dairy farm.

ROSANA POLIFRONI; GUILLERMO H. ARROYO; ANALÍA I. ETCHEVERRÍA; NORA L. PADOLA; ALBERTO E. PARMA

Centro Cultural Borges - C.A.B.A

Congreso; VTEC2009 -7th. International Symposium on Shiga Toxin (verocytotoxin)- Producing Escherichia coli Infections – Buenos Aires; 2009

Asociación Argentina de Microbiología

This study was carried out with the aim to determine how long Escherichia coli O157:H7 can survive in water from animal-drinking troughs of dairy farm. This ability was observed by many authors. E. coli could survive over more than 200 days in water from river and lake at temperature lower than 4° C, particularly in countries with cold weather. Recent microbiological studies demonstrated that E. coli O157:H7 and other bacteria may become viable but nonculturable (VBNC) when exposed to sublethal stress, such as changes in temperature, nutrients availability, pH or exposure to chlorine. Under simple starvation conditions, E. coli O157:H7 can also enter into a VBNC state. In this state, the bacteria can not be growth in regular enrichment media used in the microbiology laboratory and could become culturable again in the presence of sodium pyruvate. We inoculated steril water from animal-drinking troughs with 103 CFU/ml of a strain of E. coli O157:H7 isolated from a feedlot cattle resistant to nalidixic acid (characteristic virulence factors of this strain: ehlyA, eae, vt2). The bacteria’s presence was periodically controlled by the colony quantifycation in SMAC plates with nalidixic acid added at a concentration of 50 ìg/ml (SMAC-Nal), until the 117th day when the UFC/ml in water was lower than 1. At day 0, 50 and 56, we took sample to compare the colony counts (CC) with the optic density at 600 nm (OD600). The result at day 0 was 104 CFU/ml by CC and 103 by OD600. At day 50 and 56, the water showed 104 UFC/ml by CC and 107 by OD600 and 103 CFU/ml by CC and 106 by OD600 , respectively. One sample was taken on day 92 , the CC in SMAC-Nal. was 10 CFU/ml. We compared the colony count in LB agar containing 0.1% of sodium pyruvate (LB+ P) and SMAC, without antibiotic to reduce the stress condition, and observing if the cells could be in VBNC state. The result was an enhanced count in the medium containing pyruvate (>500 CFU/ml in LB+P vs. 150 CFU/ml in SMAC ). We confirm positive result to E. coli O157:H7 in LB+P by using agglutination test with latex particles (OXOID) in 5 random selected colonies, multiplex PCR (ehlyA, eae, vt2) and a new culture in LB+P+Nal from the sample took on 92th day. This last colony count was 110 CFU/ml and positive for OXOID test. These results suggest that it was possible to recover cells of E. coli O157:H7 that could have been in VBNC state due the starvation. The presence of VBNC E. coli cells is a concern regarding in water from animal-drinking troughs in the dairy farm and a potential health human risk. Keys words: Survival, O157:H7, dairy, PCR.