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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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Flow chart for STEC isolation (O157 and non-O157) and examples of typical colony morphologies.The final isolation protocol (M3) incorporates the M1 and M2 methods and starts with enrichment in TSB and plating anti-O157 magnetic beads on three different media (“IMS”; media A, B, C) and direct plating of stx-positive enrichment broths on C-O157 (“PCR”; medium D). O157 suspect colonies appear as pale and steel blue colonies on SMAC and NT-RA, respectively. Suspect STEC colonies from any media are subcultured on LB and confirmed as either O157 or non-O157 STEC by PCR. Anti-O157 magnetic beads bind other bacteria present in enrichment broths of environmental samples, but, fortuitously, also many non-O157 STEC. Typical non-O157 STEC colonies are shown from enrichments growing on C-O157 (Indicator Media, panel D, blue colonies), NT-RA agar (panel B, pink colonies). Non-O157 STEC colonies expressing beta-galactosidase and hemolysin are indicated by blue colonies with a clearing zone of hemolysis on mSBA (panel C). The parts of the final method for isolating O157 and non-O157 STEC are shown by an orange box (O157), blue box (M1), green box (M2) and red box (M3).
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pone-0065716-g001: Flow chart for STEC isolation (O157 and non-O157) and examples of typical colony morphologies.The final isolation protocol (M3) incorporates the M1 and M2 methods and starts with enrichment in TSB and plating anti-O157 magnetic beads on three different media (“IMS”; media A, B, C) and direct plating of stx-positive enrichment broths on C-O157 (“PCR”; medium D). O157 suspect colonies appear as pale and steel blue colonies on SMAC and NT-RA, respectively. Suspect STEC colonies from any media are subcultured on LB and confirmed as either O157 or non-O157 STEC by PCR. Anti-O157 magnetic beads bind other bacteria present in enrichment broths of environmental samples, but, fortuitously, also many non-O157 STEC. Typical non-O157 STEC colonies are shown from enrichments growing on C-O157 (Indicator Media, panel D, blue colonies), NT-RA agar (panel B, pink colonies). Non-O157 STEC colonies expressing beta-galactosidase and hemolysin are indicated by blue colonies with a clearing zone of hemolysis on mSBA (panel C). The parts of the final method for isolating O157 and non-O157 STEC are shown by an orange box (O157), blue box (M1), green box (M2) and red box (M3).

Mentions: All samples in this study were processed for generic E. coli and for E. coli O157 by the same methods (described below) throughout the 2.5 year survey. However, to increase the robustness of our method for isolating non-O157 STEC, we modified our initial method twice during the study. This resulted in three sets of samples, collected over three sequential periods, cultured separately by three different methods (Figure 1). Thus, our initial method for isolating non-O157 STEC (designated M1,4/28/08 to 10/15/08) involved PCR of the TSB enrichment broth to detect stx genes, plating samples on Chromagar O157 that reached a threshold Ct value, and isolating suspect STEC colonies. The second isolation method (M2, 7/19/08 to 1/13/10) continued theM1method and added the step of selecting suspect STEC colonies from the same Rainbow Agar plate where suspect O157 STEC were isolated. Finally, the third isolation method (M3, 1/8/10 to 10/26/10) continued the M2 method and added the step of selecting colonies from a modified sheep’s blood agar medium (mSBA).


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)

Flow chart for STEC isolation (O157 and non-O157) and examples of typical colony morphologies.The final isolation protocol (M3) incorporates the M1 and M2 methods and starts with enrichment in TSB and plating anti-O157 magnetic beads on three different media (“IMS”; media A, B, C) and direct plating of stx-positive enrichment broths on C-O157 (“PCR”; medium D). O157 suspect colonies appear as pale and steel blue colonies on SMAC and NT-RA, respectively. Suspect STEC colonies from any media are subcultured on LB and confirmed as either O157 or non-O157 STEC by PCR. Anti-O157 magnetic beads bind other bacteria present in enrichment broths of environmental samples, but, fortuitously, also many non-O157 STEC. Typical non-O157 STEC colonies are shown from enrichments growing on C-O157 (Indicator Media, panel D, blue colonies), NT-RA agar (panel B, pink colonies). Non-O157 STEC colonies expressing beta-galactosidase and hemolysin are indicated by blue colonies with a clearing zone of hemolysis on mSBA (panel C). The parts of the final method for isolating O157 and non-O157 STEC are shown by an orange box (O157), blue box (M1), green box (M2) and red box (M3).
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Related In: Results  -  Collection

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pone-0065716-g001: Flow chart for STEC isolation (O157 and non-O157) and examples of typical colony morphologies.The final isolation protocol (M3) incorporates the M1 and M2 methods and starts with enrichment in TSB and plating anti-O157 magnetic beads on three different media (“IMS”; media A, B, C) and direct plating of stx-positive enrichment broths on C-O157 (“PCR”; medium D). O157 suspect colonies appear as pale and steel blue colonies on SMAC and NT-RA, respectively. Suspect STEC colonies from any media are subcultured on LB and confirmed as either O157 or non-O157 STEC by PCR. Anti-O157 magnetic beads bind other bacteria present in enrichment broths of environmental samples, but, fortuitously, also many non-O157 STEC. Typical non-O157 STEC colonies are shown from enrichments growing on C-O157 (Indicator Media, panel D, blue colonies), NT-RA agar (panel B, pink colonies). Non-O157 STEC colonies expressing beta-galactosidase and hemolysin are indicated by blue colonies with a clearing zone of hemolysis on mSBA (panel C). The parts of the final method for isolating O157 and non-O157 STEC are shown by an orange box (O157), blue box (M1), green box (M2) and red box (M3).
Mentions: All samples in this study were processed for generic E. coli and for E. coli O157 by the same methods (described below) throughout the 2.5 year survey. However, to increase the robustness of our method for isolating non-O157 STEC, we modified our initial method twice during the study. This resulted in three sets of samples, collected over three sequential periods, cultured separately by three different methods (Figure 1). Thus, our initial method for isolating non-O157 STEC (designated M1,4/28/08 to 10/15/08) involved PCR of the TSB enrichment broth to detect stx genes, plating samples on Chromagar O157 that reached a threshold Ct value, and isolating suspect STEC colonies. The second isolation method (M2, 7/19/08 to 1/13/10) continued theM1method and added the step of selecting suspect STEC colonies from the same Rainbow Agar plate where suspect O157 STEC were isolated. Finally, the third isolation method (M3, 1/8/10 to 10/26/10) continued the M2 method and added the step of selecting colonies from a modified sheep’s blood agar medium (mSBA).

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