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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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Virulence genes and O-antigen genes in a subset of STEC strains.A subset of strains isolated by the final prototype method (M3; all media) was analyzed by PCR as described previously (Quinones et al, 2012, Frontiers). This provided an opportunity to compare the types of strains isolated from samples exposed to all three media used for isolation of non-O157 STEC.
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pone-0065716-g006: Virulence genes and O-antigen genes in a subset of STEC strains.A subset of strains isolated by the final prototype method (M3; all media) was analyzed by PCR as described previously (Quinones et al, 2012, Frontiers). This provided an opportunity to compare the types of strains isolated from samples exposed to all three media used for isolation of non-O157 STEC.

Mentions: To determine the virulence profile of strains isolated from different media (Figure 5), O-types and virulence genes of STEC strains from each medium were assessed by PCR (Figure 6). O-types O26, O103, O111, O145 and O157 were isolated more frequently from IMS on NT-RA compared to the PCR method (no IMS, C-O157); conversely, O45, O91, O113 and O128 O-types were isolated more frequently from C-O157 and mSBA (P<0.001). Non-O157 STEC strains untypable by our typing assays (ELISA and PCR) were isolated at a similar proportion by the three methods. However, the most significant differences in strains from NT-RA, mSBA and C-O157 media were virulence gene incidence (Figure 6). Strains of the stx subtypes were detected at different proportions from RA, mSBA and C-O157 media (Figure 6). stx1c-, stx2b- and stx2g-positive strains were isolated only from C-O157, and subA-positive strains predominantly from mSBA. Strains positive for eae (“adhesion”), ent, espK, espN, katP, nleA, nleB, nleE, or nleH1–2, were isolated predominantly, or only, from NT-RA. The higher proportion of hlyA-positive strains from mSBA was consistent with blood hemolysis as a selection criterion. These results indicate a significant bias in the virulence profile of strains isolated from NT-RA, mSBA and C-O157 (Figures 5 and 6).


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)

Virulence genes and O-antigen genes in a subset of STEC strains.A subset of strains isolated by the final prototype method (M3; all media) was analyzed by PCR as described previously (Quinones et al, 2012, Frontiers). This provided an opportunity to compare the types of strains isolated from samples exposed to all three media used for isolation of non-O157 STEC.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065716-g006: Virulence genes and O-antigen genes in a subset of STEC strains.A subset of strains isolated by the final prototype method (M3; all media) was analyzed by PCR as described previously (Quinones et al, 2012, Frontiers). This provided an opportunity to compare the types of strains isolated from samples exposed to all three media used for isolation of non-O157 STEC.
Mentions: To determine the virulence profile of strains isolated from different media (Figure 5), O-types and virulence genes of STEC strains from each medium were assessed by PCR (Figure 6). O-types O26, O103, O111, O145 and O157 were isolated more frequently from IMS on NT-RA compared to the PCR method (no IMS, C-O157); conversely, O45, O91, O113 and O128 O-types were isolated more frequently from C-O157 and mSBA (P<0.001). Non-O157 STEC strains untypable by our typing assays (ELISA and PCR) were isolated at a similar proportion by the three methods. However, the most significant differences in strains from NT-RA, mSBA and C-O157 media were virulence gene incidence (Figure 6). Strains of the stx subtypes were detected at different proportions from RA, mSBA and C-O157 media (Figure 6). stx1c-, stx2b- and stx2g-positive strains were isolated only from C-O157, and subA-positive strains predominantly from mSBA. Strains positive for eae (“adhesion”), ent, espK, espN, katP, nleA, nleB, nleE, or nleH1–2, were isolated predominantly, or only, from NT-RA. The higher proportion of hlyA-positive strains from mSBA was consistent with blood hemolysis as a selection criterion. These results indicate a significant bias in the virulence profile of strains isolated from NT-RA, mSBA and C-O157 (Figures 5 and 6).

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