Application of MinION Long-Read Sequencer for Semi-targeted Detection of Foodborne Pathogens.

Foodborne pathogens remain a serious health issue in developed and developing countries. Safeness of food products has been assured for years with culture-based microbiological methods; however, these present several limitations such as turnaround time and extensive hands-on work, which have been typically address taking advantage of DNA-based methods such as real-time PCR (qPCR). These, and other similar techniques, are targeted assays, meaning that they are directed for the specific detection of one specific microbe. Even though reliable, this approach suffers from an important limitation that unless specific assays are design for every single pathogen potentially present, foods may be considered erroneously safe. To address this problem, next-generation sequencing (NGS) can be used as this is a nontargeted method; thus it has the capacity to detect every potential threat present. In this chapter, a protocol for the simultaneous detection and preliminary serotyping of Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Escherichia coli O157:H7 is described.

Identifying Putative Biomarkers of Foodborne Pathogens Using a Metabolomic Approach.

Metabolomics is the study of low molecular weight biochemical molecules (typically <1500 Da) in a defined biological organism or system. In case of food systems, the term "food metabolomics" is often used. Food metabolomics has been widely explored and applied in various fields including food analysis, food intake, food traceability, and food safety. Food safety applications focusing on the identification of pathogen-specific biomarkers have been promising. This chapter describes a nontargeted metabolite profiling workflow using gas chromatography coupled with mass spectrometry (GC-MS) for characterizing three globally important foodborne pathogens, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica, from selective enrichment liquid culture media. The workflow involves a detailed description of food spiking experiments followed by procedures for the extraction of polar metabolites from media, the analysis of the extracts using GC-MS, and finally chemometric data analysis using univariate and multivariate statistical tools to identify potential pathogen-specific biomarkers.

Sensitive Detection of Escherichia coli O157:H7 Using Allosteric Probe and Hairpin Switches-Based Isothermal Transcription Amplification.

Pathogens pose a serious threat to public and population health, leading to serious outbreak and spread of diseases irrespective of the region. The capability to directly, sensitively, and specifically detect viable pathogens in low numbers in food and clinical samples is very desirable but remains a challenge. In this work, we present a novel assay of a combination of an aptamer-based allosteric probe and hairpin switch-controlled T7 RNA polymerase-based isothermal transcription amplification, which enables rapid, ultrasensitive, label-free detection of direct pathogens. It can detect Escherichia coli as low as 73.2 CFU/mL. Moreover, with the usage of the proposed assay, sensitive quantification of E. coliO157:H7 in milk samples has been achieved, showing significant potential as a simple and sensitive tool to quantify pathogens in milk and other foods.

A meta-analysis of factors influencing the inactivation of Shiga toxin-producing Escherichia coli O157:H7 in leafy greens.

Recent advancements in modeling suggest that microbial inactivation in leafy greens follows a nonlinear pattern, rather than the simple first-order kinetics. In this study, we evaluated 17 inactivation models commonly used to describe microbial decline and established the conditions that govern microbial survival on leafy greens. Through a systematic review of 65 articles, we extracted 530 datasets to model the fate of Shiga toxin-producing Escherichia coli O157:H7 on leafy greens. Various factor analysis methods were employed to evaluate the impact of identified conditions on survival metrics. A two-parameter model (jm2) provided the best fit to most of both natural and antimicrobial-induced persistence datasets, whereas the one-parameter exponential model provided the best fit to less than 20% of the datasets. The jm2 model (adjusted R2 = .89) also outperformed the exponential model (adjusted R2 = .58) in fitting the pooled microbial survival data. In the context of survival metrics, the model averaging approach generated higher values than the exponential model for >4 log reduction times (LRTs), suggesting that the exponential model may be overpredicting inactivation at later time points. The random forest technique revealed that temperature and inoculum size were common factors determining inactivation in both natural and antimicrobial-induced die-offs.. The findings show the limitations of relying on the first-order survival metric of 1 LRT and considering nonlinear inactivation in produce safety decision-making.

A review of conditions influencing fate of Shiga toxin-producing Escherichia coli O157:H7 in leafy greens.

The accuracy of predictive microbial models used in quantitative microbial risk assessment (QMRA) relies on the relevancy of conditions influencing growth or inactivation. The continued use of log-linear models in studies remains widespread, despite evidence that they fail to accurately account for biphasic kinetics or include parameters to account for the effect of environmental conditions within the model equation. Although many experimental studies detail conditions of interest, studies that do not do so lead to uncertainty in QMRA modeling because the applicability of the predictive microbial models to the conditions in the risk scenarios is questionable or must be extrapolated. The current study systematically reviewed 65 articles that provided quantitative data and documented the conditions influencing the inactivation or growth of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in leafy greens. The conditions were identified and categorized as environmental, biological, chemical, and/or processing. Our study found that temperature (n = 37 studies) and sanitizing and washing procedures (n = 12 studies) were the most studied conditions in the farm-to-table continuum of leafy greens. In addition, relative humidity was also established to affect growth and inactivation in more than one stage in the continuum. This study proposes the evaluation of the interactive effects of multiple conditions in processing and storage stages from controlled experiments as they relate to the fate of STEC O157:H7 in leafy greens for future quantitative analysis.

An outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 associated with contaminated lettuce and the cascading risks from climate change, the United Kingdom, August to September 2022.

Shiga-toxin producing Escherichia coli (STEC) O157 is a food-borne pathogen which causes gastrointestinal illness in humans. Ruminants are considered the main reservoir of infection, and STEC exceedance has been associated with heavy rainfall. In September 2022, a large outbreak of STEC O157:H7 was identified in the United Kingdom (UK). A national-level investigation was undertaken to identify the source of the outbreak and inform risk mitigation strategies. Whole genome sequencing (WGS) was used to identify outbreak cases. Overall, 259 cases with illness onset dates between 5 August and 12 October 2022, were confirmed across the UK. Epidemiological investigations supported a UK grown, nationally distributed, short shelf-life food item as the source of the outbreak. Analytical epidemiology and food chain analysis suggested lettuce as the likely vehicle of infection. Food supply chain tracing identified Grower X as the likely implicated producer. Independent of the food chain investigations, a novel geospatial analysis triangulating meteorological, flood risk, animal density and land use data was developed, also identifying Grower X as the likely source. Novel geospatial analysis and One Health approaches are potential tools for upstream data analysis to predict and prevent contamination events before they occur and to support evidence generation in outbreak investigations.

Variability in the acid adaptation of ten different O157:H7 and non-O157 Escherichia coli strains in orange juice and the impact on UV radiation resistance.

This study aimed to assess the impact of adaptation of ten strains of O157:H7 and non-O157 Escherichia coli to low pH (acid shock or slow acidification) and the effects of this exposure or not on the resistance of E. coli strains to UV radiation in orange juice (pH 3.5). The acid-shocked cells were obtained through culture in tryptic soy broth (TSB) with a final pH of 4.8, which was adjusted by hydrochloric, lactic, or citric acid and subsequently inoculated in orange juice at 4 °C for 30 days. No significant differences (p > 0.05) in survival in orange juice were observed between the serotypes O157:H7 and non-O157:H7 for acid-shocked experiments. After slow acidification, where the cells were cultured in TSB supplemented with glucose 1% (TSB + G), a significant increase (p < 0.05) in survival was observed for all strains evaluated. The D-values (radiation dose (J/cm2) necessary to decrease the microbial population by 90%) were determined as the inverse of the slopes of the regressions (k) obtained by plotting log (N/N0). The results show that among the strains tested, E. coli O157:H7 (303/00) and O26:H11 were the most resistant and sensitive strains, respectively. According to our results, the method of acid adaptation contributes to increasing the UV resistance for most of the strains tested.

Establishment of LAMP-CRISPR/Cas12a for rapid detection of Escherichia coli O157:H7 and one-pot detection.

Escherichia coli O157:H7 is a pathogenic serotype of Escherichia coli. Consumption of food contaminated with E. coli O157:H7 could cause a range of diseases. Therefore, it is of great importance to establish rapid and accurate detection methods for E. coli O157:H7 in food. In this study, based on LAMP and combined with the CRISPR/cas12a system, a sensitive and specific rapid detection method for E. coli O157:H7 was established, and One-Pot detection method was also constructed. The sensitivity of this method could stably reach 9.2 × 10° CFU/mL in pure culture, and the whole reaction can be completed within 1 h. In milk, E. coli O157:H7 with an initial contamination of 7.4 × 10° CFU/mL only needed to be cultured for 3 h to be detected. The test results can be judged by the fluorescence curve or by visual observation under a UV lamp, eliminating instrument limitations and One-Pot detection can effectively prevent the problem of false positives. In a word, the LAMP-CRISPR/cas12a system is a highly sensitive and convenient method for detecting E. coli O157:H7.

Ultrasensitive Analysis of Escherichia coli O157:H7 Based on Immunomagnetic Separation and Labeled Surface-Enhanced Raman Scattering with Minimized False Positive Identifications.

It is a big challenge to monitor pathogens in food with high selectivity. In this study, we reported an ultrasensitive method for Escherichia coli O157:H7 detection based on immunomagnetic separation and labeled surface-enhanced Raman scattering (SERS). The bacterium was identified by heterogeneous recognition elements, monoclonal antibody (mAb), and aptamer. E. coli O157:H7 was separated and enriched by magnetic nanoparticles modified by mAb, and then a plasmonic nanostructure functionalized by aptamers with embedded Raman tags and interior gaps was utilized for further discrimination and detection. The selectivity was enhanced by two binding sites. The higher Raman enhancement was obtained by strong local electromagnetic field oscillation in the gap and the firm embedment of 4-mercaptopyridine (4-Mpy). Optimum experiments created that SERS signals of 4-Mpy at 1010 cm-1 had a good linearity with E. coli O157:H7 at a large range of 10 to 107 CFU/mL with a limit of detection of 2 CFU/mL. This method has great potential for on-site food pathogenic bacterial detection.

Phenotypic and genomic comparison of three human outbreak and one cattle-associated Shiga toxin-producing Escherichia coli O157:H7.

Escherichia coli O157:H7-adulterated food products are associated with disease outbreaks in humans. Although cattle feces are a source for E. coli O157:H7 contamination, it is unclear if human-associated outbreak isolates differentially colonize and shed in the feces of cattle from that of non-outbreak isolates. It is also unclear if phenotypes, such as biofilm formation, cell attachment, or toxin production, differentiate environmental E. coli O157:H7 isolates from those associated with human illness. The objective of this study was to compare the genotypes and phenotypes of a diverse set of E. coli O157:H7 isolates, with the intent of identifying differences that could inform cattle colonization and fecal shedding, along with virulence potential in humans. Isolates differed in attachment phenotypes on human Caco-2 cells and bovine-derived recto-anal junction squamous epithelial cells, with curli having a strong impact on attachment to the human-derived cell line. The prototypical E. coli O157 isolate EDL933 had the greatest expression of the adhesin gene iha, yet it had decreased expression of the virulence genes stx2, eae, and ehxA compared the lineage I/II isolates RM6067W and/or FRIK1989. Strong or weak biofilm production was not associated with significant differences in cattle colonization or shedding, suggesting biofilms may not play a major role in cattle colonization. No significant differences in cattle colonization and fecal shedding were detected, despite genomic and in vitro phenotypic differences. The outbreak isolate associated with the greatest incidence of hemolytic uremic syndrome, RM6067W, induced the greatest Vero cell cytotoxicity and had the greatest stx2 gene expression. IMPORTANCE: Foodborne illness has major impacts on global health and imposes financial hardships on food industries. Escherichia coli serotype O157:H7 is associated with foodborne illness. Cattle feces are a source of E. coli O157:H7, and routine surveillance has led to an abundance of E. coli O157:H7 genomic data. The relationship between E. coli O157:H7 genome and phenotype is not clearly discerned for cattle colonization/shedding and improved understanding could lead to additional strategies to limit E. coli O157:H7 in the food chain. The goal of the research was to evaluate genomic and phenotypic attributes of E. coli O157:H7 associated with cattle colonization and shedding, environmental persistence, and human illness. Our results indicate variations in biofilm formation and in vitro cellular adherence was not associated with differences in cattle colonization or shedding. Overall, processes involved in cattle colonization and various phenotypes in relation to genotype are complex and remain not well understood.

Fast and sensitive detection of viable Escherichia coli O157:H7 using a microwell-confined and propidium monoazide-assisted digital CRISPR microfluidic platform.

Escherichia coli O157:H7 is a major foodborne pathogen that poses a significant threat to food safety and human health. Rapid and sensitive detection of viable Escherichia coli O157:H7 can effectively prevent food poisoning. Here, we developed a microwell-confined and propidium monoazide-assisted digital CRISPR microfluidic platform for rapid and sensitive detection of viable Escherichia coli O157:H7 in food samples. The reaction time is significantly reduced by minimizing the microwell volume, yielding qualitative results in 5 min and absolute quantitative results in 15 min. With the assistance of propidium monoazide, this platform can eliminate the interference from 99% of dead Escherichia coli O157:H7. The direct lysis method obviates the need for a complex nucleic acid extraction process, offering a limit of detection of 3.6 × 101 CFU mL-1 within 30 min. Our results demonstrated that the platform provides a powerful tool for rapid detection of Escherichia coli O157:H7 and provides reliable guidance for food safety testing.

Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability.

Escherichia coli O157:H7 (E. coli O157) is known for causing severe foodborne illnesses such as hemorrhagic colitis and hemolytic uremic syndrome. Although E. coli O157 is typically regarded as an extracellular pathogen and a weak biofilm producer, some E. coli O157 strains, including a clinical strain ATCC 43895, exhibit a notable ability to invade bovine crypt cells and other epithelial cells, as well as to form robust biofilm. This invasive strain persists in the bovine host significantly longer than non-invasive strains. Various surface-associated factors, including lipopolysaccharides (LPS), flagella, and other adhesins, likely contribute to this enhanced invasiveness and biofilm formation. In this study, we constructed a series of LPS-core deletion mutations (waaI, waaG, waaF, and waaC) in E. coli O157 ATCC 43895, resulting in stepwise truncations of the LPS. This approach enabled us to investigate the effects on the biosynthesis of key surface factors, such as flagella and curli, and the ability of this invasive strain to invade host cells. We confirmed the LPS structure and found that all LPS-core mutants failed to form biofilms, highlighting the crucial role of core oligosaccharides in biofilm formation. Additionally, the LPS inner-core mutants ΔwaaF and ΔwaaC lost the ability to produce flagella and curli. Furthermore, these inner-core mutants exhibited a dramatic reduction in adherence to and invasion of epithelial cells (MAC-T), showing an approximately 100-fold decrease in cell invasion compared with the outer-core mutants (waaI and waaG) and the wild type. These findings underscore the critical role of LPS-core truncation in impairing flagella and curli biosynthesis, thereby reducing the invasion capability of E. coli O157 ATCC 43895.

Regulation of gene transcription in Escherichia coli O157:H7 in response to a natural derivate peptide of esculentin-1a used in combination with essential oils from plants of the Cymbopogon genus.

INTRODUCTION: We analyzed the expression of several genes implicated in the pathogenicity of Escherichia coli O157:H7, treating bacteria with Esc(1-21), a derivative of peptide esculentin-1 in combination with three essential oils obtained from plants from the Cympopogon genus. METHODS: We used the checkerboard assay to determine the antimicrobial activity of the combinations. We analyzed the expression of some genes implicated in the pathogenicity and quorum sensing system of E. coli O157:H7 by real-time RT-PCR technique. RESULTS: Treatment of the bacteria with the peptide combined with oils had an efficacious antimicrobial activity. The analysis of gene expression showed that all used combinations regulate positively the espAD and ler genes, located in the pathogenicity island, named the locus of enterocyte effacement. None of the combinations affects the quorum sensing genes: lsrABCFKR and qseBC. CONCLUSIONS: This study demonstrates that the use of essential oil/peptide combinations can be effective in fighting microbial infections.

Phage-induced "one-to-many" FRET sensor for highly sensitive detection of Escherichia coli O157:H7.

As a foodborne pathogen capable of causing severe illnesses, early detection of Escherichia coli O157:H7 (E. coli O157:H7) is crucial for ensuring food safety. While Förster resonance energy transfer (FRET) is an efficient and precise detection technique, there remains a need for amplification strategies to detect low concentrations of E. coli O157:H7. In this study, we presented a phage (M13)-induced "one to many" FRET platform for sensitively detecting E. coli O157:H7. The aptamers, which specifically recognize E. coli O157:H7 were attached to magnetic beads as capture probes for separating E. coli O157:H7 from food samples. The peptide O157S, which specifically targets E. coli O157:H7, and streptavidin binding peptide (SBP), which binds to streptavidin (SA), were displayed on the P3 and P8 proteins of M13, respectively, to construct the O157S-M13K07-SBP phage as a detection probe for signal output. Due to the precise distance (≈3.2 nm) between two neighboring N-terminus of P8 protein, the SA-labeled FRET donor and acceptor can be fixed at the Förster distance on the surface of O157S-M13K07-SBP via the binding of SA and SBP, inducing FRET. Moreover, the P8 protein, with ≈2700 copies, enabled multiple FRET (≈605) occurrences, amplifying FRET in each E. coli O157:H7 recognition event. The O157S-M13K07-SBP-based FRET sensor can detect E. coli O157:H7 at concentration as low as 6 CFU/mL and demonstrates excellent performance in terms of selectivity, detection time (≈3 h), accuracy, precision, practical application, and storage stability. In summary, we have developed a powerful tool for detecting various targets in food safety, environmental monitoring, and medical diagnosis.

Impact of Operational Parameters on Pathogen Lethality in Dry and Semi-dry Uncooked Fermented Sausages.

The safety of uncooked fermented, dried sausages relies upon controlled fermentation and drying that inactivates pathogenic bacteria. Current guidelines for the production of fermented sausages by the United States Department of Agriculture (USDA) Food Safety Inspection Services (FSIS) and related research highlight specific safety parameters. The confidence that processing steps, which do not include cooking, inherently mitigate microbial risks, is challenged by the resilience of pathogens in the dry and acidic environments of these food products. The aim of this work was to examine the length of drying required to achieve a target pathogen reduction across a range of sausage diameters. This study investigated the relationship between product diameter and time required to achieve target reductions of Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes, as well as the attainment of specific water activity (aw). The research utilized salami and summer sausage with diameters of 18 mm, 30 mm, 60 mm, 90 mm, and 110 mm. Sausage batter was inoculated with 5 strains each of E. coli O157:H7, L. monocytogenes, and S. enterica. Inoculated sausages were processed with fermentation and drying protocols for each sausage type. Smaller diameter sausages reached both the desired pathogen reduction and target aw of 0.85 sooner than larger ones. However, the time to achieve the target aw did not align with the time to achieve the pathogen reduction targets, suggesting that aw alone is not a reliable indicator of safety. Another finding was larger sausages achieved the target pathogen reduction without reaching the target aw, suggesting complex relationship between aw, diameter, and pathogen inactivation. These data support the need for food safety guidelines that consider drying duration, aw, and pathogen behavior for varying sausage diameters. This research contributes to developing more precise safety protocols for producing dry and semi-dry fermented sausages.

Successful application of eculizumab in typical haemolytic uraemic syndrome.

A woman in her 40s with no medical history presented on hospital day #0 with 3 days of epigastric pain, nausea, vomiting and bloody diarrhoea. Initial blood work demonstrated acute kidney injury with metabolic acidosis with an elevated anion gap, thrombocytopenia, an elevated lactate dehydrogenase, and an undetectable haptoglobin. She was quickly diagnosed with haemolytic uraemic syndrome from Shiga toxin-producing O157:H7 Escherichia coli Her microangiopathic haemolytic anaemia and renal failure progressively worsened and only improved after the initiation of eculizumab, a monoclonal antibody directed against complement component C5. We report a case of Shiga toxin-producing E. coli-haemolytic uraemia syndrome with a complement-mediated component.

Effect of Rheum ribes L. pulp enriched with eugenol or thymol on survival of foodborne pathogens and quality parameters of chicken breast fillets.

The aim of this study was to characterize the pulp of Rheum ribes L. and to determine the effect of the pulp enriched with eugenol (1 %) or thymol (1 %) on the microbiological and physico-chemical quality of chicken breast fillets. Chicken breast fillets, inoculated with Listeria monocytogenes, Salmonella enterica subsp. enterica serovar Typhimurium, and Escherichia coli O157:H7 (~6.0 log10), were marinated for 24 h in a mixture prepared from a combination of Rheum ribes L. pulp with eugenol or thymol. The quality parameters were analyzed for 15 days at +4 °C. The Rheum ribes L. pulp was found to have high antioxidant activity, high total phenolic content and contained 22 different phenolic substances, among which rutin ranked first. The pulp contained high levels of p-xylene and o-xylene as volatile substances and citric acid as an organic acid. The combination of Pulp + Eugenol + Thymol (PET) reduced the number of pathogens in chicken breast fillets by 2.03 to 3.50 log10 on day 0 and by 2.25 to 4.21 log10 on day 15, compared to the control group (P < 0.05). The marinating treatment significantly lowered the pH values of fillet samples on the first day of the study, compared to the control group (P < 0.05). During storage, TVB-N levels showed slower increase in the treatment groups compared to the control group (P < 0.05). In addition, the marinating process led to significant changes in physicochemical parameters such as water holding capacity, color, texture, cooking loss, and drip loss compared to the control group (P < 0.05). In conclusion, the results of this study showed that the pulp of Rheum ribes L., which has a high antioxidant capacity and contains various bioactive compounds. Furthermore, S. Typhimurium, E. coli O157:H7 and L. monocytogenes were inhibited considerably by marinating Rheum ribes L. pulp with a combination of eugenol and thymol.

Extended-Spectrum ß-Lactamase and carbapenemase-producing Escherichia coli O157:H7 among diarrheic patients in Shashemene, Ethiopia.

BACKGROUND: The worldwide increase in multidrug resistance is a major threat to public health. One particular concern is the presence of Escherichia coli strains that carry Extended-Spectrum ß-Lactamase (ESBL) and Carbapenemase enzymes, which can make multiple antibiotics ineffective. This complicates treatment strategies and raises the risk of illness and death. The aim of this study was to isolate E. coli O157:H7, assess its susceptibility against antimicrobial agents, and determine the presence of ESBL and Carbapenemase production in stool samples collected from diarrheic patients in Shashemene, west Arsi, Ethiopia from July to November 2022. METHODS: The samples were cultured McConkey Agar and E. coli were isolated and identified by standard biochemical tests using API 20E. E. coli O157:H7 was further identified using sorbitol McConkey Agar and antisera for O157 antigen test. The antimicrobial susceptibility test was performed using the Kirby-Bauer disc diffusion method using different antibiotics. Each identified isolate was screened and tested for phenotypical ESBL and Carbapenemase production using combined disc method and modified carbapenem inactivation method, respectively. Bivariant and multivariant analyses were employed using a logistic regression model for further analysis and were interpreted based on the odds ratio and level of statistical significance at a p-value <0.05 with 95% confidence interval. RESULTS: E. coli O157:H7 strain was found from 9% (38/423) study participants. The majority of the participants [61.9% (262/423)] were males; and 19.1% (81/ 423) of the participants were under five children. Living in urban areas, having domestic animals, and ≥5 family size in the household were identified as statistically significant factors associated with E. coli O157:H7. Twenty-seven (71.1%) and 12 (31.6%) of the 38 E. coli O157:H7 isolates were phenotypically confirmed to be ESBL and carbapenemase producers, respectively. All isolates were resistant against Ampicillin, but sensitive to ciprofloxacin. High resistance to Ampicillin and Amoxicillin/Clavulanic acid was observed among the ESBL and carbapenemase producing isolates also. The extent of detection of multidrug resistant E. coli O157:H7 isolates against three or more classes of antimicrobial agents tested was alarmingly very high (84%). CONCLUSION: The E. coli O157:H7 isolates in this study showed a significant resistance to certain antimicrobials that were tested. The level of ESBL and Carbapenemase production among these isolates was found to be quite high. We observed a high resistance to Ampicillin and Amoxicillin/Clavulanic acid among the ESBL and carbapenemase producing isolates. Ciprofloxacin was found to be the most effective drug against both the ESBL producers and nonproducers.

Efficacy of a lytic bacteriophage vB_EcoM_SQ17 against Enterohemorrhagic Escherichia coli O157:H7 and Enterotoxigenic E. coli biofilms on cucumber.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) and Enterotoxigenic E. coli (ETEC) have been found to readily develop biofilms on cucumber (Cucumis sativus L.), presenting a significant risk to the safety of ready-to-eat vegetables. This study aimed to assess the effectiveness of the lytic bacteriophage vB_EcoM_SQ17 (SQ17) against EHEC O157:H7 and ETEC biofilms on cucumber. Here, we evaluated the efficacy of phage SQ17 on the formation and reduction of biofilms formed by EHEC O157:H7 and ETEC strains on various surfaces, including polystyrene, poly-d-lysine precoated films, and fresh-cut cucumber, at different temperatures. Phage SQ17 significantly inhibited ETEC biofilm formation, reducing the number of adhered cells by 0.15 log CFU/mL at 37 °C. Treatment with phage SQ17 also significantly decreased the number of adhered cells in established biofilms via SEM observation. Moreover, phage SQ17 effectively reduced the biomass of EHEC O157:H7 and ETEC biofilms by over 54.8 % at 37 °C after 24 h of incubation. Following phage treatment, the viability of adhered EHEC O157:H7 cells decreased by 1.37 log CFU/piece and 0.46 log CFU/piece in biofilms on cucumber at 4 °C and 25 °C, respectively. Similarly, the viability of ETEC cells decreased by 1.07 log CFU/piece and 0.61 log CFU/piece in biofilms on cucumber at 4 °C and 25 °C, respectively. These findings suggest that phage SQ17 shows promise as a potential strategy for eradicating pathogenic E. coli biofilms on cucumber.

The Use of Antimicrobial Washes to Inactivate Shiga Toxin-Producing Escherichia coli from In-Shell Pecans and Wash Water Contaminated by Different Inoculation Routes.

In-shell pecans are typically harvested after falling from trees to the ground, presenting a potential route of contamination of foodborne pathogens from soil contact. In-shell pecans are often subjected to various processing or washing steps prior to being shelled. This study determined Shiga toxin-producing Escherichia coli (STEC) reductions after treatment with antimicrobial washes on direct and soil-inoculated in-shell pecans and evaluated the cross-contamination potential of the spent pecan washes after treatment. Pecans were directly and soil-inoculated with an STEC cocktail (O157:H7, O157:NM, O121, O26). Direct inoculation was achieved by spraying the STEC cocktail on the pecans. For soil-inoculation pecans, autoclaved soil was sprayed with the STEC cocktail, homogenized for 2 min, and used to coat in-shell pecans. Inoculated pecans were washed in treatments of 2% lactic acid (LA), 1,000 ppm free chlorine (sodium hypochlorite; NaClO), hot water (HW; 85 ± 2 °C), or ambient water (C [control]; 18 ± 2 °C) for 2, 5, and 10 min and diluted to enumerate STEC populations. After treatments, 100 mL of the spent wash was vacuum filtered through a 0.45-µm membrane and plated on selective agar. HW significantly reduced STEC populations from pecans with and without soil regardless of treatment time (p < 0.05), NaClO reduced STEC populations more than the ambient control wash on directly inoculated pecans, but there were no significant differences between STEC reductions from ambient water (C), LA, and NaClO treatments on soil-inoculated pecans (p > 0.05). Larger STEC populations were enumerated from ambient water wash compared to the antimicrobial washes (p < 0.05). The HW, LA, and NaClO treatments were effective at maintaining the quality of the wash water, with STEC levels being generally at or below the detection limit (<1 CFU/100 mL), while HW was the most effective at reducing STEC from in-shell pecans with and without a soil coating (>5-log CFU/mL reductions).