Dataset Information

Genetic determinants of heat resistance in Escherichia coli.

ABSTRACT: Escherichia coli AW1.7 is a heat resistant food isolate and the occurrence of pathogenic strains with comparable heat resistance may pose a risk to food safety. To identify the genetic determinants of heat resistance, 29 strains of E. coli that differed in their of heat resistance were analyzed by comparative genomics. Strains were classified as highly heat resistant strains, exhibiting a D60-value of more than 6 min; moderately heat resistant strains, exhibiting a D60-value of more than 1 min; or as heat sensitive. A ~14 kb genomic island containing 16 predicted open reading frames encoding putative heat shock proteins and proteases was identified only in highly heat resistant strains. The genomic island was termed the locus of heat resistance (LHR). This putative operon is flanked by mobile elements and possesses >99% sequence identity to genomic islands contributing to heat resistance in Cronobacter sakazakii and Klebsiella pneumoniae. An additional 41 LHR sequences with >87% sequence identity were identified in 11 different species of ?- and ?-proteobacteria. Cloning of the full length LHR conferred high heat resistance to the heat sensitive E. coli AW1.7?pHR1 and DH5?. The presence of the LHR correlates perfectly to heat resistance in several species of Enterobacteriaceae and occurs at a frequency of 2% of all E. coli genomes, including pathogenic strains. This study suggests the LHR has been laterally exchanged among the ?- and ?-proteobacteria and is a reliable indicator of high heat resistance in E. coli.

SUBMITTER: Mercer RG

PROVIDER: S-EPMC4563881 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Genetic determinants of heat resistance in Escherichia coli.

Mercer Ryan G RG Zheng Jinshui J Garcia-Hernandez Rigoberto R Ruan Lifang L Gänzle Michael G MG McMullen Lynn M LM

Frontiers in microbiology 20150909

Escherichia coli AW1.7 is a heat resistant food isolate and the occurrence of pathogenic strains with comparable heat resistance may pose a risk to food safety. To identify the genetic determinants of heat resistance, 29 strains of E. coli that differed in their of heat resistance were analyzed by comparative genomics. Strains were classified as highly heat resistant strains, exhibiting a D60-value of more than 6 min; moderately heat resistant strains, exhibiting a D60-value of more than 1 min; ...[more]

PMID: 26441869

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Project description:Microbial resistance to processing treatments poses a food safety concern, as treatment tolerant pathogens can emerge. Occasional foodborne outbreaks caused by pathogenic Escherichia coli have led to human and economic losses. Therefore, this study screened for the extreme heat resistance (XHR) phenotype as well as one known genetic marker, the locus of heat resistance (LHR), in 4,123 E. coli isolates from diverse meat animals at different processing stages. The prevalences of XHR and LHR among the meat-borne E. coli were found to be 10.3% and 11.4%, respectively, with 19% agreement between the two. Finished meat products showed the highest LHR prevalence (24.3%) compared to other processing stages (0 to 0.6%). None of the LHR+ E. coli in this study would be considered pathogens based on screening for virulence genes. Four high-quality genomes were generated by whole-genome sequencing of representative LHR+ isolates. Nine horizontally acquired LHRs were identified and characterized, four plasmid-borne and five chromosomal. Nine newly identified LHRs belong to ClpK1 LHR or ClpK2 LHR variants sharing 61 to 68% nucleotide sequence identity, while one LHR appears to be a hybrid. Our observations suggest positive correlation between the number of LHR regions present in isolates and the extent of heat resistance. The isolate exhibiting the highest degree of heat resistance possessed four LHRs belonging to three different variant groups. Maintenance of as many as four LHRs in a single genome emphasizes the benefits of the LHR in bacterial physiology and stress response.IMPORTANCE Currently, a "multiple-hurdle" approach based on a combination of different antimicrobial interventions, including heat, is being utilized during meat processing to control the burden of spoilage and pathogenic bacteria. Our recent study (M. Guragain, G. E. Smith, D. A. King, and J. M. Bosilevac, J Food Prot 83:1438-1443, 2020, https://doi.org/10.4315/JFP-20-103) suggests that U.S. beef cattle harbor Escherichia coli that possess the locus of heat resistance (LHR). LHR seemingly contributes to the global stress tolerance in bacteria and hence poses a food safety concern. Therefore, it is important to understand the distribution of the LHRs among meat-borne bacteria identified at different stages of different meat processing systems. Complete genome sequencing and comparative analysis of selected heat-resistant bacteria provide a clearer understanding of stress and heat resistance mechanisms. Further, sequencing data may offer a platform to gain further insights into the genetic background that provides optimal bacterial tolerance against heat and other processing treatments.

Project description:Some chlorine-resistant Escherichia coli isolates harbor the locus of heat resistance (LHR), a genomic island conferring heat resistance. In this study, the protective effect of the LHR for cells challenged by chlorine and oxidative stress was quantified. Cloning of the LHR protected against NaClO (32 mM; 5 min), H2O2 (120 mM; 5 min), and peroxyacetic acid (105 mg/liter; 5 min) but not against 5.8 mM KIO4, 10 mM acrolein, or 75 mg/liter allyl isothiocyanate. The lethality of oxidizing treatments for LHR-negative strains of E. coli was about 2 log10 CFU/ml higher than that for LHR-positive strains of E. coli The oxidation of cytoplasmic proteins and membrane lipids was quantified with the fusion probe roGFP2-Orp1 and the fluorescent probe BODIPY581/591, respectively. The fragment of the LHR coding for heat shock proteins protected cytoplasmic proteins but not membrane lipids against oxidation. The middle fragment of the LHR protected against the oxidation of membrane lipids but not of cytoplasmic proteins. The addition of H2O2, NaClO, and peroxyacetic acid also induced green fluorescent protein (GFP) expression in the oxidation-sensitive reporter strain E. coli O104:H4 Δstx2::gfp::amp Cloning of pLHR reduced phage induction in E. coli O104:H4 Δstx2::gfp::amp after treatment with oxidizing chemicals. Screening of 160 strains of Shiga toxin-producing E. coli (STEC) revealed that none of them harbors the LHR, additionally suggesting that the LHR and Stx prophages are mutually exclusive. Taking our findings together, the contribution of the LHR to resistance to chlorine and oxidative stress is based on the protection of multiple cellular targets by different proteins encoded by the genetic island.IMPORTANCE Chlorine treatments are used in water and wastewater sanitation; the resistance of Escherichia coli to chlorine is thus of concern to public health. We show that a genetic island termed the locus of heat resistance (LHR) protects E. coli not only against heat but also against chlorine and other oxidizing chemicals, adding to our knowledge of the tools used by E. coli to resist stress. Specific detection of the oxidation of different cellular targets in combination with the cloning of fragments of the LHR provided insight into mechanisms of protection and demonstrated that different fragments of the LHR protect different cellular targets. In E. coli, the presence of the LHR virtually always excluded other virulence factors. It is tempting to speculate that the LHR is maintained by strains of E. coli with an environmental lifestyle but is excluded by pathogenic strains that adapted to interact with vertebrate hosts.

Project description:The locus of heat resistance (LHR) can confer heat resistance to Escherichia coli to various extents. This study investigated the phylogenetic relationships and the genomic and phenotypic characteristics of E. coli with or without LHR recovered from beef by direct plating or from enrichment broth at 42°C. LHR-positive E. coli isolates (n = 24) were subjected to whole-genome sequencing by short and long reads. LHR-negative isolates (n = 18) from equivalent sources as LHR-positive isolates were short-read sequenced. All isolates were assessed for decimal reduction time at 60°C (D60°C) and susceptibility to the sanitizers E-SAN and Perox-E. Selected isolates were evaluated for growth at 42°C. The LHR-positive and -negative isolates were well separated on the core genome tree, with 22/24 positive isolates clustering into three clades. Isolates within clade 1 and 2, despite their different D60°C values, were clonal, as determined by subtyping (multilocus sequence typing [MLST], core genome MLST, and serotyping). Isolates within each clade are of one serotype. The LHR-negative isolates were genetically diverse. The LHR-positive isolates had a larger (P < 0.001) median genome size by 0.3 Mbp (5.0 versus 4.7 Mbp) and overrepresentation of genes related to plasmid maintenance, stress response, and cryptic prophages but underrepresentation of genes involved in epithelial attachment and virulence. All LHR-positive isolates harbored a chromosomal copy of LHR, and all clade 2 isolates had an additional partial copy of LHR on conjugative plasmids. The growth rates at 42°C were 0.71 ± 0.02 and 0.65 ± 0.02 log(OD) h-1 for LHR-positive and -negative isolates, respectively. No meaningful difference in sanitizer susceptibility was noted between LHR-positive and -negative isolates. IMPORTANCE Resistant bacteria are serious food safety and public health concerns. Heat resistance conferred by the LHR varies largely among different strains of E. coli. The findings in this study show that genomic background and composition of LHR, in addition to the presence of LHR, play an important role in the degree of heat resistance in E. coli and that strains with certain genetic backgrounds are more likely to acquire and maintain the LHR. Also, caution should be exercised when recovering E. coli at elevated temperatures, as the presence of LHR may confer growth advantages to some strains. Interestingly, the LHR-harboring strains seem to have evolved further from their primary animal host to adapt to their secondary habitat, as reflected by fewer genes involved in virulence and epithelial attachment. The phylogenetic relationships among the isolates point toward multiple mechanisms for acquisition of LHR by E. coli, likely prior to its being deposited on meat.

Dataset Information

Genetic determinants of heat resistance in Escherichia coli.

Publications

Genetic determinants of heat resistance in Escherichia coli.

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