Dataset Information

Functional analysis of ycfR and ycfQ in Escherichia coli O157:H7 linked to outbreaks of illness associated with fresh produce.

ABSTRACT: Fresh produce has been associated with multiple outbreaks of illness caused by Escherichia coli O157:H7. The mechanism of E. coli O157:H7 survival through postharvest processing of fresh produce needs to be understood to help develop more effective interventions. In our recent transcriptomic study of strain Sakai, an isolate from the 1996 sprout outbreak in Japan, and strain TW14359, an isolate from the 2006 spinach outbreak in the United States, we showed that ycfR was the most significantly upregulated gene in response to chlorine-based oxidative stress. YcfR is known to be a multiple stress resistance protein and a biofilm regulator in E. coli K-12 strains; however, its role in the pathogenic E. coli O157:H7 has not been clearly defined. In this study, ycfR was replaced with a chloramphenicol resistance cassette oriented in two different directions to construct polar and nonpolar ycfR::cat mutants of Sakai and TW14359. Chlorine resistance and survival on spinach leaf surfaces were assessed in the wild-type strains and the ycfR mutants. Both polar and nonpolar ycfR mutants of Sakai showed significantly less chlorine resistance than their parent strain. In contrast, deletion of ycfR in TW14359 did not change chlorine resistance, indicating that ycfR in these two outbreak-related E. coli O157:H7 strains may function differently. In addition, after a 24-h incubation on spinach leaves in a sublethal concentration of chlorine, the Sakai nonpolar ycfR mutant exhibited lower survival compared to the wild type. The results suggest a role for ycfR in survival of Sakai during chlorine exposure. We also found that the upstream ycfQ, which is annotated as a DNA-binding regulator, acted as a repressor of ycfR. These findings suggest that gene regulation may be a mechanism by which E. coli O157:H7 strain Sakai could survive in the postharvest processing environment.

SUBMITTER: Deng K

PROVIDER: S-EPMC3131662 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Functional analysis of ycfR and ycfQ in Escherichia coli O157:H7 linked to outbreaks of illness associated with fresh produce.

Deng Kaiping K Wang Siyun S Rui Xiaoqian X Zhang Wei W Tortorello Mary Lou ML

Applied and environmental microbiology 20110415 12

Fresh produce has been associated with multiple outbreaks of illness caused by Escherichia coli O157:H7. The mechanism of E. coli O157:H7 survival through postharvest processing of fresh produce needs to be understood to help develop more effective interventions. In our recent transcriptomic study of strain Sakai, an isolate from the 1996 sprout outbreak in Japan, and strain TW14359, an isolate from the 2006 spinach outbreak in the United States, we showed that ycfR was the most significantly up ...[more]

PMID: 21498759

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Project description:Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and long-term evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies.

Project description:Fresh vegetables have become associated with outbreaks caused by Escherichia coli O157:H7 (EcO157). Between 1995-2006, 22 produce outbreaks were documented in the United States, with nearly half traced to lettuce or spinach grown in California. Outbreaks between 2002 and 2006 induced investigations of possible sources of pre-harvest contamination on implicated farms in the Salinas and San Juan valleys of California, and a survey of the Salinas watershed. EcO157 was isolated at least once from 15 of 22 different watershed sites over a 19 month period. The incidence of EcO157 increased significantly when heavy rain caused an increased flow rate in the rivers. Approximately 1000 EcO157 isolates obtained from cultures of>100 individual samples were typed using Multi-Locus Variable-number-tandem-repeat Analysis (MLVA) to assist in identifying potential fate and transport of EcO157 in this region. A subset of these environmental isolates were typed by Pulse Field Gel Electrophoresis (PFGE) in order to make comparisons with human clinical isolates associated with outbreak and sporadic illness. Recurrence of identical and closely related EcO157 strains from specific locations in the Salinas and San Juan valleys suggests that transport of the pathogen is usually restricted. In a preliminary study, EcO157 was detected in water at multiple locations in a low-flow creek only within 135 meters of a point source. However, possible transport up to 32 km was detected during periods of higher water flow associated with flooding. During the 2006 baby spinach outbreak investigation, transport was also detected where water was unlikely to be involved. These results indicate that contamination of the environment is a dynamic process involving multiple sources and methods of transport. Intensive studies of the sources, incidence, fate and transport of EcO157 near produce production are required to determine the mechanisms of pre-harvest contamination and potential risks for human illness.

Dataset Information

Functional analysis of ycfR and ycfQ in Escherichia coli O157:H7 linked to outbreaks of illness associated with fresh produce.

Publications

Functional analysis of ycfR and ycfQ in Escherichia coli O157:H7 linked to outbreaks of illness associated with fresh produce.

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