At least 14 separate outbreaks of food poisoning attributed to either Salmonella enterica or Esch... more At least 14 separate outbreaks of food poisoning attributed to either Salmonella enterica or Escherichia coli O157:H7 have been traced to sprouts in the past decade. Seeds contaminated with human pathogens caused most of these outbreaks, thus many sprout growers are now treating alfalfa seeds with the sanitizing agent, calcium hypochlorite (Ca[OCl]2), prior to sprouting. The efficacy of alfalfa seed sanitation varies between seed lots and between seeds within each lot. Alfalfa seeds from different seed lots were sorted by type in an effort to determine if certain seed types carry more aerobic bacteria than other seed types. Seeds with a wrinkled type, characteristic of lygus bug damage, had significantly higher levels of culturable aerobic bacteria and were more difficult to sanitize than smooth, healthy seeds. After sanitation, wrinkled alfalfa seeds that had been inoculated with S. enterica ser. Newport carried significantly higher levels of Salmonella Newport than smooth seeds. If S. enterica is present on wrinkled seeds in naturally contaminated seed lots, it may be difficult to chemically sanitize the seed lot. Removal of the wrinkled alfalfa seeds from the seed lots, perhaps by adapting color sorting equipment similar to that used to sort rice grains and other seeds, should reduce the level of aerobic bacteria in seed lots and may result in lower levels of human pathogens on contaminated alfalfa seeds.
F+ RNA coliphages (FRNA) are used to source-track fecal contamination and as surrogates for enter... more F+ RNA coliphages (FRNA) are used to source-track fecal contamination and as surrogates for enteric pathogen persistence in the environment. However, the environmental persistence of FRNA is not clearly understood and necessitates the evaluation of the survival of prototype and environmental isolates of FRNA representing all four genogroups in surface waters from the central coast of California. Water temperature played a significant role in persistence-all prototype and environmental strains survived significantly longer at 10 °C compared to 25 °C. Similarly, the availability of host bacterium was found to be critical in FRNA survival. In the absence of E. coli F(amp), all prototypes of FRNA disappeared rapidly with a D-value (days for one log reduction) of <1.2 d from water samples incubated at 25 °C; the longest surviving prototype was SP. However, in the presence of the host, the order of persistence at 25 °C was QB>MS2>SP>GA and at 10 °C it was QB = MS2>GA>SP. Significant differences in survival were observed between prototypes and environmental isolates of FRNA. While most environmental isolates disappeared rapidly at 25 °C and in the absence of the host, members of genogroups GIII and GI persisted longer with the host compared to members of GII and GIV. Consequentially, FRNA based source tracking methods can be used to detect phages from recent fecal contamination along with those that persist longer in the environment as a result of cooler temperatures and increased host presence.
To provide data for traditional trace-back studies from fork to farm, it is necessary to determin... more To provide data for traditional trace-back studies from fork to farm, it is necessary to determine the environmental sources for Shiga-toxigenic Escherichia coli. We developed SYBR green based reverse-transcriptase PCR methods to determine the prevalence of F+ RNA coliphages (FRNA) as indicators of fecal contamination. Male-specific coliphages, determined using a single-agar overlay method, were prevalent in all surface waters sampled for 8 months. F+ DNA coliphages (FDNA) were predominant compared to FRNA in water samples from majority of sampling locations. Most (90%) of the FRNA were sourced to humans and originated from human-impacted sites. Members of genogroup III represented 77% of FRNA originated from human sources. Furthermore, 93% of FRNA sourced to animals were also detected in water samples from human-impacted sites. Eighty percent of all FRNA were isolated during the winter months indicating seasonality in prevalence. In contrast, FDNA were more prevalent during summer months. E. coli O157:H7 and Shiga-toxigenic E. coli were detected in water samples from locations predominantly influenced by agriculture. Owing to their scarcity, their numbers could not be correlated with the prevalence of FRNA or FDNA in water samples. Both coliform bacteria and generic E. coli from agricultural or human-impacted sites were similar in numbers and thus could not be used to determine the sources of fecal contamination. Data on the prevalence of male-specific coliphages may be invaluable for predicting the sources of fecal contamination and aid in developing methods to prevent enteric pathogen contamination from likely sources during produce production.
At least 14 separate outbreaks of food poisoning attributed to either Salmonella enterica or Esch... more At least 14 separate outbreaks of food poisoning attributed to either Salmonella enterica or Escherichia coli O157:H7 have been traced to sprouts in the past decade. Seeds contaminated with human pathogens caused most of these outbreaks, thus many sprout growers are now treating alfalfa seeds with the sanitizing agent, calcium hypochlorite (Ca[OCl]2), prior to sprouting. The efficacy of alfalfa seed sanitation varies between seed lots and between seeds within each lot. Alfalfa seeds from different seed lots were sorted by type in an effort to determine if certain seed types carry more aerobic bacteria than other seed types. Seeds with a wrinkled type, characteristic of lygus bug damage, had significantly higher levels of culturable aerobic bacteria and were more difficult to sanitize than smooth, healthy seeds. After sanitation, wrinkled alfalfa seeds that had been inoculated with S. enterica ser. Newport carried significantly higher levels of Salmonella Newport than smooth seeds. If S. enterica is present on wrinkled seeds in naturally contaminated seed lots, it may be difficult to chemically sanitize the seed lot. Removal of the wrinkled alfalfa seeds from the seed lots, perhaps by adapting color sorting equipment similar to that used to sort rice grains and other seeds, should reduce the level of aerobic bacteria in seed lots and may result in lower levels of human pathogens on contaminated alfalfa seeds.
F+ RNA coliphages (FRNA) are used to source-track fecal contamination and as surrogates for enter... more F+ RNA coliphages (FRNA) are used to source-track fecal contamination and as surrogates for enteric pathogen persistence in the environment. However, the environmental persistence of FRNA is not clearly understood and necessitates the evaluation of the survival of prototype and environmental isolates of FRNA representing all four genogroups in surface waters from the central coast of California. Water temperature played a significant role in persistence-all prototype and environmental strains survived significantly longer at 10 °C compared to 25 °C. Similarly, the availability of host bacterium was found to be critical in FRNA survival. In the absence of E. coli F(amp), all prototypes of FRNA disappeared rapidly with a D-value (days for one log reduction) of <1.2 d from water samples incubated at 25 °C; the longest surviving prototype was SP. However, in the presence of the host, the order of persistence at 25 °C was QB>MS2>SP>GA and at 10 °C it was QB = MS2>GA>SP. Significant differences in survival were observed between prototypes and environmental isolates of FRNA. While most environmental isolates disappeared rapidly at 25 °C and in the absence of the host, members of genogroups GIII and GI persisted longer with the host compared to members of GII and GIV. Consequentially, FRNA based source tracking methods can be used to detect phages from recent fecal contamination along with those that persist longer in the environment as a result of cooler temperatures and increased host presence.
To provide data for traditional trace-back studies from fork to farm, it is necessary to determin... more To provide data for traditional trace-back studies from fork to farm, it is necessary to determine the environmental sources for Shiga-toxigenic Escherichia coli. We developed SYBR green based reverse-transcriptase PCR methods to determine the prevalence of F+ RNA coliphages (FRNA) as indicators of fecal contamination. Male-specific coliphages, determined using a single-agar overlay method, were prevalent in all surface waters sampled for 8 months. F+ DNA coliphages (FDNA) were predominant compared to FRNA in water samples from majority of sampling locations. Most (90%) of the FRNA were sourced to humans and originated from human-impacted sites. Members of genogroup III represented 77% of FRNA originated from human sources. Furthermore, 93% of FRNA sourced to animals were also detected in water samples from human-impacted sites. Eighty percent of all FRNA were isolated during the winter months indicating seasonality in prevalence. In contrast, FDNA were more prevalent during summer months. E. coli O157:H7 and Shiga-toxigenic E. coli were detected in water samples from locations predominantly influenced by agriculture. Owing to their scarcity, their numbers could not be correlated with the prevalence of FRNA or FDNA in water samples. Both coliform bacteria and generic E. coli from agricultural or human-impacted sites were similar in numbers and thus could not be used to determine the sources of fecal contamination. Data on the prevalence of male-specific coliphages may be invaluable for predicting the sources of fecal contamination and aid in developing methods to prevent enteric pathogen contamination from likely sources during produce production.
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Papers by Chester Sarreal