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An epidemiological investigation on occurrence of enterohemorrhagic Escherichia coli in raw milk.

ABSTRACT: Aim:The aim of the present investigation was to study the epidemiology of enterohemorrhagic Escherichia coli (EHEC) in raw milk and molecular characterization of isolates using multiplex polymerase chain reaction (PCR). Materials and Methods:A total of 125 raw milk samples were subjected to isolation, identification, and confirmation of virulence-associated genes by multiplex PCR (mPCR). The samples were collected from a milk cooperative society of Thrissur district, Kerala. For further epidemiological investigation, samples such as dung (126), hair coat of cow (60), udder swab (60), udder wash (60), milking utensil wash (36), Milker's hand wash (36), water (36), soil (36), and feed (36) were collected from the households from which the raw milk tested positive for EHEC. Results:The occurrence of EHEC in individual raw milk samples was found to be 8.8%. The major source of contamination to raw milk was found to be dung (19.84%) followed by udder swab (16.67%), hair coat of cow (15%), Milker's hand and milking utensils and water (11.11% each), and udder wash and soil (8.33% each). For identification of virulence genes, all the isolates were subjected to mPCR, of 75 isolates 73.33% of isolates harbored stx 2 gene while 53.33, 36, and 36% of isolates were encoded by stx 1, eae A, and hly A genes, respectively. On epidemiological survey, the multiple risk factors accountable for occurrence of EHEC in raw milk were found to be the quality of water used, improper and inadequate udder preparation, unhygienic hands of Milker's, use of insufficiently cleaned milking utensils, and using common utensil for washings of udder and milking purposes. Conclusion:The result of the present study signifies that raw milk was contaminated with EHEC and possesses a high public health threat. As dairy cattle and its environment serve as a potential niche for EHEC, hygienic milking practices should be adopted to curb the occurrence of EHEC in raw milk.

SUBMITTER: Vanitha HD

PROVIDER: S-EPMC6141287 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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An epidemiological investigation on occurrence of enterohemorrhagic <i>Escherichia coli</i> in raw milk.

Vanitha H D HD Sethulekshmi C C Latha C C

Veterinary world 20180825 8

<h4>Aim</h4>The aim of the present investigation was to study the epidemiology of enterohemorrhagic <i>Escherichia coli</i> (EHEC) in raw milk and molecular characterization of isolates using multiplex polymerase chain reaction (PCR).<h4>Materials and methods</h4>A total of 125 raw milk samples were subjected to isolation, identification, and confirmation of virulence-associated genes by multiplex PCR (mPCR). The samples were collected from a milk cooperative society of Thrissur district, Kerala ...[more]

PMID: 30250379

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Project description:IntroductionBy adhering to host cells and colonizing tissues, bacterial pathogens can successfully establish infection. Adhesion is considered the first step of the infection process and bacterial adhesion to anti-adhesive compounds is now seen as a promising strategy to prevent infectious diseases. Among the natural sources of anti-adhesive molecules, the membrane of milk fat globules (MFGs) is of interest because of its compositional diversity of proteins and glycoconjugates. However, few studies have focused on the bacterial molecules involved in MFG- mediated inhibition of bacterial adhesion to enterocytes.MethodsWe used three pathogenic Shiga toxin-producing Escherichia coli (STEC) strains (O26:H11 str. 21765, O157:H7 str. EDL933, and O103:H3 str. PMK5) as models to evaluate whether STEC surface proteins are involved in the affinity of STEC for MFG membrane proteins (MFGMPs). The affinity of STEC for MFGMPs was assessed both indirectly by a natural raw milk creaming test and directly by an adhesion test. Mass spectrometry was used to identify enriched STEC proteins within the protein fraction of MFGMs. Bacterial mutants were constructed and their affinity to MFGs were measured to confirm the role of the identified proteins.ResultsWe found that free STEC surface proteins inhibit the concentration of the pathogen in the MFG-enriched cream in a strain-dependent manner. Moreover, the OmpA and FliC proteins were identified within the protein fraction of MFGMs. Our results suggest that FliC protein participates in STEC adhesion to MFGMPs but other STEC molecules may also participate.DiscussionFor the first time, this study highlighted, the involvement of STEC surface proteins in the affinity for MFGs. The mechanism of STEC-MFG association is still not fully understood but our results confirm the existence of receptor/ligand type interactions between the bacteria and MFGs. Further studies are needed to identify and specify the molecules involved in this interaction. These studies should consider the likely involvement of several factors, including adhesion molecules, and the diversity of each STEC strain.

Project description:Enteric pathogens with low infectious doses rely on the ability to orchestrate the expression of virulence and metabolism-associated genes in response to environmental cues for successful infection. Accordingly, the human pathogen enterohemorrhagic Escherichia coli (EHEC) employs a complex multifaceted regulatory network to link the expression of type III secretion system (T3SS) components to nutrient availability. While phosphorylation of histidine and aspartate residues on two-component system response regulators is recognized as an integral part of bacterial signaling, the involvement of phosphotyrosine-mediated control is minimally explored in Gram-negative pathogens. Our recent phosphotyrosine profiling study of E. coli identified 342 phosphorylated proteins, indicating that phosphotyrosine modifications in bacteria are more prevalent than previously anticipated. The present study demonstrates that tyrosine phosphorylation of a metabolite-responsive LacI/GalR family regulator, Cra, negatively affects T3SS expression under glycolytic conditions that are typical for the colonic lumen environment where production of the T3SS is unnecessary. Our data suggest that Cra phosphorylation affects T3SS expression by modulating the expression of ler, which encodes the major activator of EHEC virulence gene expression. Phosphorylation of the Cra Y47 residue diminishes DNA binding to fine-tune the expression of virulence-associated genes, including those of the locus of enterocyte effacement pathogenicity island that encode the T3SS, and thereby negatively affects the formation of attaching and effacing lesions. Our data indicate that tyrosine phosphorylation provides an additional mechanism to control the DNA binding of Cra and other LacI/GalR family regulators, including LacI and PurR. This study describes an initial effort to unravel the role of global phosphotyrosine signaling in the control of EHEC virulence potential.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) causes outbreaks of hemorrhagic colitis and the potentially fatal hemolytic-uremic syndrome. Successful host colonization by EHEC relies on the ability to coordinate the expression of virulence factors in response to environmental cues. A complex network that integrates environmental signals at multiple regulatory levels tightly controls virulence gene expression. We demonstrate that EHEC utilizes a previously uncharacterized phosphotyrosine signaling pathway through Cra to fine-tune the expression of virulence-associated genes to effectively control T3SS production. This study demonstrates that tyrosine phosphorylation negatively affects the DNA-binding capacity of Cra, which affects the expression of genes related to virulence and metabolism. We demonstrate for the first time that phosphotyrosine-mediated control affects global transcription in EHEC. Our data provide insight into a hitherto unexplored regulatory level of the global network controlling EHEC virulence gene expression.

Project description:BackgroundEnterohemorrhagic Escherichia coli (EHEC) are zoonotic agents associated with outbreaks worldwide. Growth of EHEC strains in ground beef could be inhibited by background microbiota that is present initially at levels greater than that of the pathogen E. coli. However, how the microbiota outcompetes the pathogenic bacteria is unknown. Our objective was to identify metabolic pathways of EHEC that were altered by natural microbiota in order to improve our understanding of the mechanisms controlling the growth and survival of EHECs in ground beef.ResultsBased on 16S metagenomics analysis, we identified the microbial community structure in our beef samples which was an essential preliminary for subtractively analyzing the gene expression of the EHEC strains. Then, we applied strand-specific RNA-seq to investigate the effects of this microbiota on the global gene expression of EHEC O2621765 and O157EDL933 strains by comparison with their behavior in beef meat without microbiota. In strain O2621765, the expression of genes connected with nitrate metabolism and nitrite detoxification, DNA repair, iron and nickel acquisition and carbohydrate metabolism, and numerous genes involved in amino acid metabolism were down-regulated. Further, the observed repression of ftsL and murF, involved respectively in building the cytokinetic ring apparatus and in synthesizing the cytoplasmic precursor of cell wall peptidoglycan, might help to explain the microbiota's inhibitory effect on EHECs. For strain O157EDL933, the induced expression of the genes implicated in detoxification and the general stress response and the repressed expression of the peR gene, a gene negatively associated with the virulence phenotype, might be linked to the survival and virulence of O157:H7 in ground beef with microbiota.ConclusionIn the present study, we show how RNA-Seq coupled with a 16S metagenomics analysis can be used to identify the effects of a complex microbial community on relevant functions of an individual microbe within it. These findings add to our understanding of the behavior of EHECs in ground beef. By measuring transcriptional responses of EHEC, we could identify putative targets which may be useful to develop new strategies to limit their shedding in ground meat thus reducing the risk of human illnesses.

Dataset Information

An epidemiological investigation on occurrence of enterohemorrhagic Escherichia coli in raw milk.

Publications

An epidemiological investigation on occurrence of enterohemorrhagic <i>Escherichia coli</i> in raw milk.

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