Evaluation of the use of ampicillin‐ and streptomycin‐resistant Shiga toxin‐producing Escherichia coli to reduce the burden of background microbiota during food safety studies

2021 ◽  
Author(s):  
Kaylan Hayman ◽  
Ikechukwu Chukwuma Oguadinma ◽  
Abhinav Mishra ◽  
Govindaraj Dev Kumar
2020 ◽  
Author(s):  
Meghan Maguire ◽  
Julie A. Kase ◽  
Dwayne Roberson ◽  
Tim Muruvanda ◽  
Eric W. Brown ◽  
...  

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.


2013 ◽  
Vol 58 (14) ◽  
pp. 1625-1631 ◽  
Author(s):  
YuJun Cui ◽  
DongFang Li ◽  
RuiFu Yang

2013 ◽  
Vol 76 (9) ◽  
pp. 1513-1522 ◽  
Author(s):  
RONG WANG ◽  
NORASAK KALCHAYANAND ◽  
JOHN W. SCHMIDT ◽  
DAYNA M. HARHAY

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.


2019 ◽  
Vol 12 (10) ◽  
pp. 1584-1590
Author(s):  
Maria Kristiani Epi Goma ◽  
Alvita Indraswari ◽  
Aris Haryanto ◽  
Dyah Ayu Widiasih

Background and Aim: The feasibility assessment of food products on the market becomes one of the milestones of food safety. The quality of food safety of animal origin especially pork need to get attention and more real action from the parties related and concerned. Since pork is also a source of transmission for the contagion of foodborne disease so that the study of the existence of several agents in the pork and its products become the benchmark of safety level. This study aimed to isolate, identify, and detect the Shiga toxin 2a (stx2a) gene from Escherichia coli O157:H7 in pork, pig feces, and clean water in the Jagalan slaughterhouse. Materials and Methods: A total of 70 samples consisting of 32 pork samples, 32 pig fecal samples, and 6 clean water samples were used to isolate and identify E. coli O157:H7 and the stx2a gene. Isolation and identification of E. coli O157:H7 were performed using culture on eosin methylene blue agar and Sorbitol-MacConkey agar media and confirmed molecularly with polymerase chain reaction to amplify the target genes rfbE (317 bp) and fliC (381 bp). The isolates, which were identified as E. coli O157:H7, were investigated for the stx2a gene (553 bp). Results: The results of this study show that of the total collected samples, E. coli O157:H7 was 28.6% in Jagalan slaughterhouse and consisted of 25% of pork samples, 31.25% of pig fecal samples, and 33.3% of clean water samples. The isolates that were identified to be E. coli O157:H7 mostly contained the stx2a gene, which was equal to 75%, and consisted of seven isolates from pork samples, seven isolates from fecal samples, and one isolate from clean water samples. Conclusion: E. coli O157:H7 was found in 28.6% of pork, pig feces, and clean water in Jagalan slaughterhouse and 75% of identified E. coli O157:H7 contained the stx2a gene.


Author(s):  
Espen Rimstad ◽  
Leiv Sigve Håvarstein ◽  
Georg Kapperud ◽  
Jørgen Lassen ◽  
Bjørn-Tore Lunestad ◽  
...  

coli is part of the normal gastrointestinal microbial flora of humans and animals. E. coli bacteria causing enteric/diarrhoeal disease are categorized into different groups based on their virulence properties and pathogenic features in humans. Enterohaemorrhagic E. coli (EHEC) are E. coli strains that  cause bloody diarrhoea  and haemolytic uraemic  syndrome  (HUS)  in humans, and have a defined zoonotic association. The major virulence factor of EHEC (and the actual  cause  of HUS)  is  the  ability  to  produce Shiga  toxins  (Stx),  thus  the  name Shiga Toxin Producing E. coli (STEC).  With enteropathogenic Escherichia  coli  (EPEC),  the diarrhoea in these  patients  is  due  to  attaching  and  effacing  (A/E)  lesions  in  the  enteric epithelium.    This risk assessment  was  conducted  after  a  human  outbreak  of  STEC  O103  in  2006, associated with contaminated dry-fermented sausages.   The Norwegian Scientific Committee for Food Safety (Vitenskapskomitéen for mattrygghet), Panel on  Biological  Hazards,  was  asked  by  the  Norwegian  Food  Safety  Authority (Mattilsynet) for  a  risk  assessment  regarding  shiga  toxin-producing  E.  coli  (STEC)  in  the Norwegian meat chain, with emphasis on dry-cured sausages. In response, an ad hoc Working Group  of  experts was  appointed with  the mandate  to  draft  a  risk  assessment  regarding  this issue.   The current report approaches the task by following and analysing the entire process, from the origin of the meats at farm level, to the final production and storage of dry-cured sausages. An overall  aim of  the  report has been  to  identify  and describe potential  intervention options  in various parts of this chain.     The main conclusions from the risk assessment are as follows:   It is  not  possible  to  give  any  reliable  quantitative  estimates  of  the  current  risk associated with consumption of dry-cured sausages.    There are  no  clear  indications  of  any  general  change  in  the  epidemiology  of  STEC  infections in humans in Norway over the last decade.   There is no documentation that there has been any change in the occurrence of various STEC in the domestic animal reservoir during the last decade.   The combination of proper slaughter hygiene and use of  thermal decontamination of sheep,  cattle  and  pig  carcasses  represents  an  efficient  way  to  reduce  STEC contamination. This approach would not only cause a reduction in the contamination level of STEC, but also provide a general beneficial effect on the level of other enteric pathogens, such as Salmonella and Yersinia enterocolitica.   Proper use  of  starter  cultures  in  fermentation,  combined  with  higher  fermentation temperatures,  will  reduce  the  probability  of  growth  of  STEC  in  contaminated  drycured sausages.   A combination  of  higher  fermentation  temperatures,  a  lower  pH  during  the  process, and heat-treatment of  the  final product should effectively eliminate  the potential  risk for transmission of STEC infections from consumption of dry-cured sausages. A 5 log reduction is possible.   Technological options  are  available  to  reduce  significantly  the  transfer  of  potential pathogens through meats in general, and specifically through dry-cured sausages.    The most important data gap is the lack of information about the actual occurrence of STEC infections  in  humans  in Norway.  Improved  laboratory  diagnostic  procedures and  epidemiological  surveillance,  combined with  better  reporting  and  tracing  in  the health care system are necessary.    The implementation  of  properly  designed  base-line  studies  of  various  domestic animals,  to  provide  data  on  the  occurrence  of  various  serotypes  and  their  virulence factors  present  is  recommended.  Also,  this  would  provide  a  better  basis  for comparison with human isolates. 


2004 ◽  
Vol 132 (2) ◽  
pp. 297-302 ◽  
Author(s):  
D. R. SMITH ◽  
J. T. GRAY ◽  
R. A. MOXLEY ◽  
S. M. YOUNTS-DAHL ◽  
M. P. BLACKFORD ◽  
...  

Although cattle are reservoirs, no validated method exists to monitor Shiga toxin-producing Escherichia coli O157 (STEC O157) on farms. In 29 Midwestern United States feedlot pens we compared culturing faeces from the individual cattle to: (1) culturing rope devices that cattle rub or chew; and (2) culturing a composite of faecal pats. Eighty-six per cent (68–96%) of pens were classified correctly using rope devices to detect pens with at least 16% of the cattle shedding STEC O157 [sensitivity=82% (57–96%); specificity=92% (62–100%)]. Ninety per cent of pens (73–98%) were classified correctly using composite faeces to detect pens with at least 37% of the cattle shedding STEC O157 [sensitivity=86% (42–100%); specificity=91% (71–99%)]. Ranking pens into three risk levels based on parallel interpretation of the pen-test results correlated (Spearman's r=0·76, P<0·0001) with the pen's prevalence. This strategy could identify pens of cattle posing a higher risk to food safety.


2015 ◽  
Vol 81 (23) ◽  
pp. 8183-8191 ◽  
Author(s):  
Susan R. Leonard ◽  
Mark K. Mammel ◽  
David W. Lacher ◽  
Christopher A. Elkins

ABSTRACTCulture-independent diagnostics reduce the reliance on traditional (and slower) culture-based methodologies. Here we capitalize on advances in next-generation sequencing (NGS) to apply this approach to food pathogen detection utilizing NGS as an analytical tool. In this study, spiking spinach with Shiga toxin-producingEscherichia coli(STEC) following an established FDA culture-based protocol was used in conjunction with shotgun metagenomic sequencing to determine the limits of detection, sensitivity, and specificity levels and to obtain information on the microbiology of the protocol. We show that an expected level of contamination (∼10 CFU/100 g) could be adequately detected (including key virulence determinants and strain-level specificity) within 8 h of enrichment at a sequencing depth of 10,000,000 reads. We also rationalize the relative benefit of static versus shaking culture conditions and the addition of selected antimicrobial agents, thereby validating the long-standing culture-based parameters behind such protocols. Moreover, the shotgun metagenomic approach was informative regarding the dynamics of microbial communities during the enrichment process, including initial surveys of the microbial loads associated with bagged spinach; the microbes found included key genera such asPseudomonas,Pantoea, andExiguobacterium. Collectively, our metagenomic study highlights and considers various parameters required for transitioning to such sequencing-based diagnostics for food safety and the potential to develop better enrichment processes in a high-throughput manner not previously possible. Future studies will investigate new species-specific DNA signature target regimens, rational design of medium components in concert with judicious use of additives, such as antibiotics, and alterations in the sample processing protocol to enhance detection.


2012 ◽  
Vol 78 (17) ◽  
pp. 6341-6344 ◽  
Author(s):  
Rong Wang ◽  
Norasak Kalchayanand ◽  
James L. Bono ◽  
John W. Schmidt ◽  
Joseph M. Bosilevac

ABSTRACTEscherichia coliO26:H11 strains were able to outgrow O157:H7 companion strains in planktonic and biofilm phases and also to effectively compete with precolonized O157:H7 cells to establish themselves in mixed biofilms.E. coliO157:H7 strains were unable to displace preformed O26:H11 biofilms. Therefore,E. coliO26:H11 remains a potential risk in food safety.


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