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Development of a robust method for isolation of shiga toxin-positive Escherichia coli (STEC) from fecal, plant, soil and water samples from a leafy greens production region in California.

Cooley MB, Jay-Russell M, Atwill ER, Carychao D, Nguyen K, Quiñones B, Patel R, Walker S, Swimley M, Pierre-Jerome E, Gordus AG, Mandrell RE - PLoS ONE (2013)

Bottom Line: Non-O157 STEC were at approximately 5-fold higher incidence compared to O157 STEC: cattle (37.9%), feral swine (21.4%), birds (2.4%), small mammals (3.5%), deer or elk (8.3%), water (14.0%), sediment (12.3%), produce (0.3%) and soil adjacent to produce (0.6%). stx1, stx2 and stx1/stx2 genes were detected in 63%, 74% and 35% of STEC isolates, respectively.The initial method was modified twice during the study revealing evidence of culture bias based on differences in virulence and O-antigen profiles.These results emphasize the importance of multiple approaches for isolation of non-O157 STEC, that livestock and wildlife are common sources of potentially virulent STEC, and evidence of STEC persistence and movement in a leafy greens production environment.

View Article: PubMed Central - PubMed

Affiliation: Produce Safety and Microbiology Research Unit, United States Department of Agriculture-Agricultural Research Service, Albany, California, United States of America. michael.cooley@ars.usda.gov

ABSTRACT
During a 2.5-year survey of 33 farms and ranches in a major leafy greens production region in California, 13,650 produce, soil, livestock, wildlife, and water samples were tested for Shiga toxin (stx)-producing Escherichia coli (STEC). Overall, 357 and 1,912 samples were positive for E. coli O157:H7 (2.6%) or non-O157 STEC (14.0%), respectively. Isolates differentiated by O-typing ELISA and multilocus variable number tandem repeat analysis (MLVA) resulted in 697 O157:H7 and 3,256 non-O157 STEC isolates saved for further analysis. Cattle (7.1%), feral swine (4.7%), sediment (4.4%), and water (3.3%) samples were positive for E. coli O157:H7; 7/32 birds, 2/145 coyotes, 3/88 samples from elk also were positive. Non-O157 STEC were at approximately 5-fold higher incidence compared to O157 STEC: cattle (37.9%), feral swine (21.4%), birds (2.4%), small mammals (3.5%), deer or elk (8.3%), water (14.0%), sediment (12.3%), produce (0.3%) and soil adjacent to produce (0.6%). stx1, stx2 and stx1/stx2 genes were detected in 63%, 74% and 35% of STEC isolates, respectively. Subtilase, intimin and hemolysin genes were present in 28%, 25% and 79% of non-O157 STEC, respectively; 23% were of the "Top 6″ O-types. The initial method was modified twice during the study revealing evidence of culture bias based on differences in virulence and O-antigen profiles. MLVA typing revealed a diverse collection of O157 and non-O157 STEC strains isolated from multiple locations and sources and O157 STEC strains matching outbreak strains. These results emphasize the importance of multiple approaches for isolation of non-O157 STEC, that livestock and wildlife are common sources of potentially virulent STEC, and evidence of STEC persistence and movement in a leafy greens production environment.

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Venn diagram of samples positive for non-O157 STEC by NT-RA, C-O157 and mSBA.The enrichment broths for 4,160 samples (sampling period Jan – Oct 2010) were processed by M3 corresponding to plating on C-O157 (PCR method; “C”), NT-RA agar (IMS method; “R”) and mSBA (IMS method; “B”). The figure shows the number of samples that were positive for non-O157 STEC on only 1 of the 3 media (R, C, B) and any combination of the 3 media (BC, RB, RC, RBC).
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pone-0065716-g005: Venn diagram of samples positive for non-O157 STEC by NT-RA, C-O157 and mSBA.The enrichment broths for 4,160 samples (sampling period Jan – Oct 2010) were processed by M3 corresponding to plating on C-O157 (PCR method; “C”), NT-RA agar (IMS method; “R”) and mSBA (IMS method; “B”). The figure shows the number of samples that were positive for non-O157 STEC on only 1 of the 3 media (R, C, B) and any combination of the 3 media (BC, RB, RC, RBC).

Mentions: Our method for isolating non-O157 STEC involved parallel procedures designated as “PCR method” (with C-O157) and “IMS method” (with both NT-RA and mSBA) (Figure 1). Although the three methods (M1, M2, M3) were not tested with the same samples obtained at the same time, samples processed by M3 provided comparison of the efficiency of isolation of non-O157 STEC from the same samples on the three media yielding STEC. The data summarized in Figure 5 illustrate that 377, 112 and 110 samples were positive only on NT-RA, C-O157 and mSBA, respectively. Only 56 samples were positive on all 3 media (Figure 5, “RBC”) and <90 of the 4160 samples tested on all 3 media were positive for STEC on any 2 media (Figure 5: “BC” = 81, “RB” = 67, “RC” = 77). The results indicating possible differences in the fitness of some strains on different media (“culture bias”).


Development of a robust method for isolation of shiga toxin-positive Escherichia coli (STEC) from fecal, plant, soil and water samples from a leafy greens production region in California.

Cooley MB, Jay-Russell M, Atwill ER, Carychao D, Nguyen K, Quiñones B, Patel R, Walker S, Swimley M, Pierre-Jerome E, Gordus AG, Mandrell RE - PLoS ONE (2013)

Venn diagram of samples positive for non-O157 STEC by NT-RA, C-O157 and mSBA.The enrichment broths for 4,160 samples (sampling period Jan – Oct 2010) were processed by M3 corresponding to plating on C-O157 (PCR method; “C”), NT-RA agar (IMS method; “R”) and mSBA (IMS method; “B”). The figure shows the number of samples that were positive for non-O157 STEC on only 1 of the 3 media (R, C, B) and any combination of the 3 media (BC, RB, RC, RBC).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675059&req=5

pone-0065716-g005: Venn diagram of samples positive for non-O157 STEC by NT-RA, C-O157 and mSBA.The enrichment broths for 4,160 samples (sampling period Jan – Oct 2010) were processed by M3 corresponding to plating on C-O157 (PCR method; “C”), NT-RA agar (IMS method; “R”) and mSBA (IMS method; “B”). The figure shows the number of samples that were positive for non-O157 STEC on only 1 of the 3 media (R, C, B) and any combination of the 3 media (BC, RB, RC, RBC).
Mentions: Our method for isolating non-O157 STEC involved parallel procedures designated as “PCR method” (with C-O157) and “IMS method” (with both NT-RA and mSBA) (Figure 1). Although the three methods (M1, M2, M3) were not tested with the same samples obtained at the same time, samples processed by M3 provided comparison of the efficiency of isolation of non-O157 STEC from the same samples on the three media yielding STEC. The data summarized in Figure 5 illustrate that 377, 112 and 110 samples were positive only on NT-RA, C-O157 and mSBA, respectively. Only 56 samples were positive on all 3 media (Figure 5, “RBC”) and <90 of the 4160 samples tested on all 3 media were positive for STEC on any 2 media (Figure 5: “BC” = 81, “RB” = 67, “RC” = 77). The results indicating possible differences in the fitness of some strains on different media (“culture bias”).

Bottom Line: Non-O157 STEC were at approximately 5-fold higher incidence compared to O157 STEC: cattle (37.9%), feral swine (21.4%), birds (2.4%), small mammals (3.5%), deer or elk (8.3%), water (14.0%), sediment (12.3%), produce (0.3%) and soil adjacent to produce (0.6%). stx1, stx2 and stx1/stx2 genes were detected in 63%, 74% and 35% of STEC isolates, respectively.The initial method was modified twice during the study revealing evidence of culture bias based on differences in virulence and O-antigen profiles.These results emphasize the importance of multiple approaches for isolation of non-O157 STEC, that livestock and wildlife are common sources of potentially virulent STEC, and evidence of STEC persistence and movement in a leafy greens production environment.

View Article: PubMed Central - PubMed

Affiliation: Produce Safety and Microbiology Research Unit, United States Department of Agriculture-Agricultural Research Service, Albany, California, United States of America. michael.cooley@ars.usda.gov

ABSTRACT
During a 2.5-year survey of 33 farms and ranches in a major leafy greens production region in California, 13,650 produce, soil, livestock, wildlife, and water samples were tested for Shiga toxin (stx)-producing Escherichia coli (STEC). Overall, 357 and 1,912 samples were positive for E. coli O157:H7 (2.6%) or non-O157 STEC (14.0%), respectively. Isolates differentiated by O-typing ELISA and multilocus variable number tandem repeat analysis (MLVA) resulted in 697 O157:H7 and 3,256 non-O157 STEC isolates saved for further analysis. Cattle (7.1%), feral swine (4.7%), sediment (4.4%), and water (3.3%) samples were positive for E. coli O157:H7; 7/32 birds, 2/145 coyotes, 3/88 samples from elk also were positive. Non-O157 STEC were at approximately 5-fold higher incidence compared to O157 STEC: cattle (37.9%), feral swine (21.4%), birds (2.4%), small mammals (3.5%), deer or elk (8.3%), water (14.0%), sediment (12.3%), produce (0.3%) and soil adjacent to produce (0.6%). stx1, stx2 and stx1/stx2 genes were detected in 63%, 74% and 35% of STEC isolates, respectively. Subtilase, intimin and hemolysin genes were present in 28%, 25% and 79% of non-O157 STEC, respectively; 23% were of the "Top 6″ O-types. The initial method was modified twice during the study revealing evidence of culture bias based on differences in virulence and O-antigen profiles. MLVA typing revealed a diverse collection of O157 and non-O157 STEC strains isolated from multiple locations and sources and O157 STEC strains matching outbreak strains. These results emphasize the importance of multiple approaches for isolation of non-O157 STEC, that livestock and wildlife are common sources of potentially virulent STEC, and evidence of STEC persistence and movement in a leafy greens production environment.

Show MeSH
Related in: MedlinePlus