scholarly journals Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producing Escherichia coli in irrigation water

2020 ◽  
Author(s):  
Meghan Maguire ◽  
Julie A. Kase ◽  
Dwayne Roberson ◽  
Tim Muruvanda ◽  
Eric W. Brown ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
Shiga Toxin ◽  
Irrigation Water ◽  
Foodborne Illness ◽  
Nanopore Sequencing ◽  
E Coli ◽  
Antimicrobial Resistance Genes ◽  
Metagenomic Sample

ABSTRACTShiga toxin-producing Escherichia coli (STEC) contamination of agricultural water might be an important factor to recent foodborne illness and outbreaks involving leafy greens. Whole genome sequencing generation of closed bacterial genomes plays an important role in source tracking. We aimed to determine the limits of detection and classification of STECs by qPCR and nanopore sequencing using enriched irrigation water artificially contaminated with E. coli O157:H7 (EDL933). We determined the limit of STEC detection by qPCR to be 30 CFU/reaction, which is equivalent to 105 CFU/ml in the enrichment. By using Oxford Nanopore’s EPI2ME WIMP workflow and de novo assembly with Flye followed by taxon classification with a k-mer analysis software (Kraken), E. coli O157:H7 could be detected at 103 CFU/ml (68 reads) and a complete fragmented E. coli O157:H7 metagenome-assembled genome (MAG) was obtained at 105-108 CFU/ml. Using a custom script to extract the E. coli reads, a completely closed MAG was obtained at 107-108 CFU/ml and a complete, fragmented MAG was obtained at 105-106 CFU/ml. In silico virulence detection for E. coli MAGs for 105-108 CFU/ml showed that the virulotype was indistinguishable from the spiked E. coli O157:H7 strain. We further identified the bacterial species in the un-spiked enrichment, including antimicrobial resistance genes, which could have important implications to food safety. We propose this workflow could be used for detection and complete genomic characterization of STEC from a complex microbial sample and could be applied to determine the limit of detection and assembly of other foodborne bacterial pathogens.IMPORTANCEFoodborne illness caused by Shiga toxin-producing E. coli (STEC) ranges in severity from diarrhea to hemolytic uremic syndrome and produce-related incidence is increasing. The pervasive nature of E. coli requires not only detection, but also a complete genome to determine potential pathogenicity based on stx and eae genes, serotype, and other virulence factors. We have developed a pipeline to determine the limits of nanopore sequencing for STECs in a metagenomic sample. By utilizing the current qPCR in the FDA Bacteriological Analytical Manual (BAM) Chapter 4A, we can quantify the amount of STEC in the enrichment and then sequence and classify the STEC in less than half the time as current protocols that require a single isolate. These methods have wide implications for food safety, including decreased time to STEC identification during outbreaks, characterization of the microbial community, and the potential to use these methods to determine the limits for other foodborne pathogens.

scholarly journals Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producing Escherichia coli in irrigation water

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245172
Author(s):  
Meghan Maguire ◽  
Julie A. Kase ◽  
Dwayne Roberson ◽  
Tim Muruvanda ◽  
Eric W. Brown ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
Shiga Toxin ◽  
Irrigation Water ◽  
De Novo ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Source Tracking ◽  
Antimicrobial Resistance Genes ◽  
Custom Script

Shiga toxin-producing Escherichia coli (STEC) contamination of agricultural water might be an important factor to recent foodborne illness and outbreaks involving leafy greens. Closed bacterial genomes from whole genome sequencing play an important role in source tracking. We aimed to determine the limits of detection and classification of STECs by qPCR and nanopore sequencing using 24 hour enriched irrigation water artificially contaminated with E. coli O157:H7 (EDL933). We determined the limit of STEC detection by qPCR to be 30 CFU/reaction, which is equivalent to 105 CFU/ml in the enrichment. By using Oxford Nanopore’s EPI2ME WIMP workflow and de novo assembly with Flye followed by taxon classification with a k-mer analysis software (Kraken2), E. coli O157:H7 could be detected at 103 CFU/ml (68 reads) and a complete fragmented E. coli O157:H7 metagenome-assembled genome (MAG) was obtained at 105−108 CFU/ml. Using a custom script to extract the E. coli reads, a completely closed MAG was obtained at 107−108 CFU/ml and a complete, fragmented MAG was obtained at 105−106 CFU/ml. In silico virulence detection for E. coli MAGs for 105−108 CFU/ml showed that the virulotype was indistinguishable from the spiked E. coli O157:H7 strain. We further identified the bacterial species in the un-spiked enrichment, including antimicrobial resistance genes, which could have important implications to food safety. We propose this workflow provides proof of concept for faster detection and complete genomic characterization of STECs from a complex microbial sample compared to current reporting protocols and could be applied to determine the limit of detection and assembly of other foodborne bacterial pathogens.

Detection of Multiple-Antimicrobial Resistance and Characterization of the Implicated Genes in Escherichia coli Isolates from Foods of Animal Origin in Tunis

2009 ◽  
Vol 72 (5) ◽  
pp. 1082-1088 ◽  
Author(s):  
AHLEM JOUINI ◽  
KARIM BEN SLAMA ◽  
YOLANDA SÁENZ ◽  
NAOUEL KLIBI ◽  
DANIELA COSTA ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Antimicrobial Agents ◽  
Food Samples ◽  
Animal Origin ◽  
E Coli ◽  
Antimicrobial Resistance Genes ◽  
Class 1 ◽  
Agar Dilution

Phenotypic and genotypic characterization of antimicrobial resistance was conducted for 98 Escherichia coli isolates recovered from 40 food samples of animal origin (poultry, sheep, beef, fish, and others) obtained in supermarkets and local butcheries in Tunis during 2004 and 2005. Susceptibility to 15 antimicrobial agents was tested by disk diffusion and agar dilution methods, the mechanisms of resistance were evaluated using PCR and sequencing methods, and the clonal relationship among isolates was evaluated using pulsed-field gel electrophoresis. High resistance was detected to tetracycline, sulphonamides, nalidixic acid, ampicillin, streptomycin, and trimethoprim-sulfamethoxazole (29 to 43% of isolates), but all isolates were susceptible to cefotaxime, ceftazidime, cefoxitin, azthreonam, and amikacin. One-third of the isolates had multiresistant phenotypes (resistance to at least five different families of antimicrobial agents). Different variants of blaTEM, tet, sul, dfrA, aadA, and aac(3) genes were detected in most of the strains resistant to ampicillin, tetracycline, sulphonamide, trimethoprim, streptomycin, and gentamicin, respectively. The presence of class 1 and class 2 integrons was studied in 15 sulphonamide-resistant unrelated E. coli strains, and 14 of these strains harbored class 1 integrons with five different arrangements of gene cassettes, and a class 2 integron with the dfrA1 + sat + aadA1 arrangement was found in one strain. This study revealed the high diversity of antimicrobial resistance genes, some of them included in integrons, in E. coli isolates of food origin.

scholarly journals Prevalence and molecular characterization of Shiga toxin-producing escherichia coli isolates from human and sheep in Al-Madinah Al-Munawarah

Infectio ◽  
2017 ◽  
Vol 21 (2) ◽  
Author(s):  
Eman Fathi Sharafa ◽  
Iman I. Shabanaa
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
Shiga Toxin ◽  
Genetic Relatedness ◽  
Control Measures ◽  
Health Concern ◽  
Global Public Health ◽  
E Coli ◽  
Life Threatening

Shiga toxin-producing Escherichia coli (STEC) strains have emerged as important foodborne pathogens of global public health concern, causing life-threatening diseases. Sheep and their products have been documented as important reservoirs for STECs, especially E. coli O157. The aim of this study was to investigate STECs from diarrheal human and sheep in Al-Madinah Al-Munawarah, Saudi Arabia. Fecal samples were collected between June and August, 2015 from diarrheal humans (n = 134) and sheep (n = 87). Presumptive E. coli human-and sheep-isolated strains were identified for their serotypes, the associated virulence genes (Shiga toxin [stx1 , stx2 ], haemolysin [ehxA] and intimin [eae]) by polymerase chain reaction and their susceptibility to antibiotics. Pulsed-field gel electrophoresis (PFGE) was used to demonstrate the genetic relatedness between Serotype O157:H7 human- and sheep-isolated strains. Forty eight (48/221; 21.7%) STECs were recovered from both human and sheep, their serotypes were as follows: O157:H7, O26:H11, O157:HNM, O26:HNM, O128:H2, O48:HNM, O111:HNM and OUT:HUT. Various virulence profiles and multiple antibiotic resistance were observed among the isolates. Twenty eight O157:H7 serotypes (17 human isolates and 11 sheep isolates) were identified in 13 PFGE pulsotypes, where human and sheep isolates were highly related. PFGE banding profiles together with serotypes and genotypes afford proof that human and sheep can be colonized and infected with similar E. coli O157:H7 strains. Our findings highlight the importance of epidemiological and microbiological surveillance of STECs; as well as the development of control measures to decrease risks associated with zoonotic O157:H7.

scholarly journals Characterization of commensal Escherichia coli isolates from slaughtered sheep in Mexico

2021 ◽  
Vol 15 (11) ◽  
pp. 1755-1760
Author(s):  
Jorge Acosta-Dibarrat ◽  
Edgar Enriquez-Gómez ◽  
Martín Talavera-Rojas ◽  
Edgardo Soriano-Vargas ◽  
Armando Navarro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Multidrug Resistant ◽  
Phylogenetic Group ◽  
Phylogenetic Groups ◽  
E Coli ◽  
New Information ◽  
Antimicrobial Resistance Genes ◽  
Uremic Syndrome

Introduction: Commensal Escherichia coli is defined as bacteria without known virulence factors that could be playing a specific role in some diseases; however, they could be responsible to disseminate antimicrobial resistance genes to other microorganisms. This study aimed to characterize the commensal E. coli isolates obtained from slaughtered sheep in the central region of Mexico. Methodology: Isolates were classified as commensal E. coli when distinctive genes related to diarrheagenic pathotypes (stx1, stx2, eae, bfp, LT, stp, ipaH, and aggR) were discarded by PCR. Identification of serotype, phylogenetic group, and antimicrobial resistance was also performed. Results: A total of 41 isolates were characterized. The phylogenetic groups found were B1 in 37 isolates (90.2%), A in 2 (4.8%), and 1 isolate (2.4%) for C and D groups. Serotypes associated with diarrhea in humans (O104:H2 and O154:NM) and hemolytic uremic syndrome (O8:NM) were detected. Thirty-three isolates (80%) were resistant to ceftazidime, 23 (56%), to tetracycline 8 (19.5%) to ampicillin, and 1 to amikacin. Six isolates (14.6%) were multidrug-resistant. Conclusions: This study provides new information about commensal E. coli in slaughtered sheep, high percentages of resistance to antibiotics, and different profiles of antimicrobial resistance were found, their dissemination constitute a risk factor towards the consuming population.

scholarly journals Longitudinal Assessment of the Dynamics of Escherichia coli, Total Coliforms, Enterococcus spp., and Aeromonas spp. in Alternative Irrigation Water Sources: a CONSERVE Study

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Sultana Solaiman ◽  
Sarah M. Allard ◽  
Mary Theresa Callahan ◽  
Chengsheng Jiang ◽  
Eric Handy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
Irrigation Water ◽  
Reclaimed Water ◽  
The United States ◽  
Water Sources ◽  
Agricultural Water ◽  
Content Type ◽  
E Coli ◽  
Alternative Irrigation

ABSTRACT As climate change continues to stress freshwater resources, we have a pressing need to identify alternative (nontraditional) sources of microbially safe water for irrigation of fresh produce. This study is part of the center CONSERVE, which aims to facilitate the adoption of adequate agricultural water sources. A 26-month longitudinal study was conducted at 11 sites to assess the prevalence of bacteria indicating water quality, fecal contamination, and crop contamination risk (Escherichia coli, total coliforms [TC], Enterococcus, and Aeromonas). Sites included nontidal freshwater rivers/creeks (NF), a tidal brackish river (TB), irrigation ponds (PW), and reclaimed water sites (RW). Water samples were filtered for bacterial quantification. E. coli, TC, enterococci (∼86%, 98%, and 90% positive, respectively; n = 333), and Aeromonas (∼98% positive; n = 133) were widespread in water samples tested. Highest E. coli counts were in rivers, TC counts in TB, and enterococci in rivers and ponds (P < 0.001 in all cases) compared to other water types. Aeromonas counts were consistent across sites. Seasonal dynamics were detected in NF and PW samples only. E. coli counts were higher in the vegetable crop-growing (May-October) than nongrowing (November-April) season in all water types (P < 0.05). Only one RW and both PW sites met the U.S. Food Safety Modernization Act water standards. However, implementation of recommended mitigation measures of allowing time for microbial die-off between irrigation and harvest would bring all other sites into compliance within 2 days. This study provides comprehensive microbial data on alternative irrigation water and serves as an important resource for food safety planning and policy setting. IMPORTANCE Increasing demands for fresh fruit and vegetables, a variable climate affecting agricultural water availability, and microbial food safety goals are pressing the need to identify new, safe, alternative sources of irrigation water. Our study generated microbial data collected over a 2-year period from potential sources of irrigation (rivers, ponds, and reclaimed water sites). Pond water was found to comply with Food Safety Modernization Act (FSMA) microbial standards for irrigation of fruit and vegetables. Bacterial counts in reclaimed water, a resource that is not universally allowed on fresh produce in the United States, generally met microbial standards or needed minimal mitigation. We detected the most seasonality and the highest microbial loads in river water, which emerged as the water type that would require the most mitigation to be compliant with established FSMA standards. This data set represents one of the most comprehensive, longitudinal analyses of alternative irrigation water sources in the United States.

Prevalence and Characterization of Escherichia coli O157:H7 on Pork Carcasses and in Swine Colon Contents from Provincially Licensed Abattoirs in Alberta, Canada

2020 ◽  
Vol 83 (11) ◽  
pp. 1909-1917
Author(s):  
SAIDA ESSENDOUBI ◽  
XIANQIN YANG ◽  
ROBIN KING ◽  
JULIA KEENLISIDE ◽  
JAVIER BAHAMON ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Gel Electrophoresis ◽  
Mean Value ◽  
Pulsed Field Gel Electrophoresis ◽  
Escherichia Coli O157 ◽  
Pulsed Field ◽  
E Coli ◽  
Swine Farms

ABSTRACT The objective of this study was to determine the prevalence of Shiga toxin–producing Escherichia coli (STEC) O157:H7 in colon contents and on carcasses from pigs slaughtered at provincially licensed abattoirs (PLAs) in Alberta, Canada. In 2017, carcass sponge samples and colon content samples were collected from 504 healthy market hogs at 39 PLAs and analyzed for E. coli O157:H7. Carcass samples were also analyzed for E. coli and aerobic colony count (ACC). Nine (1.8%) of 504 carcass samples were confirmed positive for E. coli O157:H7. Seven (1.4%) of 504 colon content samples were confirmed positive for E. coli O157:H7. These positives were found in 5 (12.8%) of 39 PLAs from hogs originating from eight farms. The E. coli O157:H7 isolates recovered from the positive samples (n = 1 isolate per sample) were clonal, as determined by pulsed-field gel electrophoresis. Six E. coli O157:H7 isolates obtained over 8 months from one PLA that only processed hogs and sourced hogs from one farm had indistinguishable pulsed-field gel electrophoresis patterns. All 16 E. coli O157:H7 isolates harbored eae and ehxA and were of stx2a subtype, suggesting that swine can carry E. coli O157:H7 of importance to human health. All carcass sponge swabs (100%) were positive for ACC. E. coli was present in 72% of carcass swabs. Carcasses from PLAs slaughtering both beef and hogs had a numerically higher ACC mean value but not statistically different compared with the carcasses from PLAs slaughtering only swine (2,799 and 610 CFU/cm2, respectively). E. coli showed a similar trend with a mean value of 0.88 CFU/cm2 in PLAs slaughtering both species and 0.26 CFU/cm2 in PLAs slaughtering only swine (P ≤ 0.05). This study provides evidence that healthy market hogs from different producers and farms in Alberta can carry E. coli O157:H7, and some strains of the organism may be able to establish persistence on some swine farms. HIGHLIGHTS

scholarly journals Mixed Biofilm Formation by Shiga Toxin–Producing Escherichia coli and Salmonella enterica Serovar Typhimurium Enhanced Bacterial Resistance to Sanitization due to Extracellular Polymeric Substances†

2013 ◽  
Vol 76 (9) ◽  
pp. 1513-1522 ◽  
Author(s):  
RONG WANG ◽  
NORASAK KALCHAYANAND ◽  
JOHN W. SCHMIDT ◽  
DAYNA M. HARHAY
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
Foodborne Pathogens ◽  
Shiga Toxin ◽  
Biofilm Formation ◽  
Extracellular Polymeric Substances ◽  
Bacterial Resistance ◽  
Salmonella Enterica Serovar Typhimurium ◽  
E Coli ◽  
Serovar Typhimurium

Shiga toxin–producing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium are important foodborne pathogens capable of forming single-species biofilms or coexisting in multispecies biofilm communities. Bacterial biofilm cells are usually more resistant to sanitization than their planktonic counterparts, so these foodborne pathogens in biofilms pose a serious food safety concern. We investigated how the coexistence of E. coli O157:H7 and Salmonella Typhimurium strains would affect bacterial planktonic growth competition and mixed biofilm composition. Furthermore, we also investigated how mixed biofilm formation would affect bacterial resistance to common sanitizers. Salmonella Typhimurium strains were able to outcompete E. coli strains in the planktonic growth phase; however, mixed biofilm development was highly dependent upon companion strain properties in terms of the expression of bacterial extracellular polymeric substances (EPS), including curli fimbriae and exopolysaccharide cellulose. The EPS-producing strains with higher biofilm-forming abilities were able to establish themselves in mixed biofilms more efficiently. In comparison to single-strain biofilms, Salmonella or E. coli strains with negative EPS expression obtained significantly enhanced resistance to sanitization by forming mixed biofilms with an EPS-producing companion strain of the other species. These observations indicate that the bacterial EPS components not only enhance the sanitizer resistance of the EPS-producing strains but also render protections to their companion strains, regardless of species, in mixed biofilms. Our study highlights the potential risk of cross-contamination by multispecies biofilms in food safety and the need for increased attention to proper sanitization practices in food processing facilities.

scholarly journals Microbial Safety and Sanitary Quality of Strawberry Primary Production in Belgium: Risk Factors for Salmonella and Shiga Toxin-Producing Escherichia coli Contamination

2015 ◽  
Vol 81 (7) ◽  
pp. 2562-2570 ◽  
Author(s):  
Stefanie Delbeke ◽  
Siele Ceuppens ◽  
Claudia Titze Hessel ◽  
Irene Castro ◽  
Liesbeth Jacxsens ◽  
...  
Keyword(s):  
Risk Factors ◽  
Escherichia Coli ◽  
Shiga Toxin ◽  
Irrigation Water ◽  
Multivariable Logistic Regression Analysis ◽  
Content Type ◽  
E Coli ◽  
Production And Consumption

ABSTRACTStrawberries are an important fruit in Belgium in both production and consumption, but little information is available about the presence ofSalmonellaand Shiga toxin-producingEscherichia coli(STEC) in these berries, the risk factors in agricultural production, and possible specific mitigation options. In 2012, a survey was undertaken of three soil and three soilless cultivation systems in Belgium. NoSalmonellaspp. were isolated. No STEC was detected in the strawberry samples (0 of 72), but STEC was detected by PCR in 11 of 78 irrigation water and 2 of 24 substrate samples. Culture isolates were obtained for 2 of 11 PCR-positive irrigation water samples and 2 of 2 substrate samples. Multivariable logistic regression analysis revealed elevated genericE. colinumbers (the odds ratio [OR] for a 1 log increase being 4.6) as the most important risk factor for STEC, together with the berry-picking season (elevated risk in summer). The presence of genericE. coliin the irrigation water (≥1 CFU per 100 ml) was mainly influenced by the type of irrigation water (collected rainfall water stored in ponds was more often contaminated than groundwater pumped from boreholes [OR = 5.8]) and the lack of prior treatment (untreated water versus water subjected to sand filtration prior to use [OR = 19.2]). The follow-up study in 2013 at one of the producer locations indicated cattle to be the most likely source of STEC contamination of the irrigation water.

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