Search Results (79)

The production of hydroponic fresh produce presents unique food safety and intervention challenges. A systematic approach was used to map and characterize the evidence on hydroponic food safety. Quantitative data describing the effectiveness of intervention studies were extracted, synthesized, and assessed for quality. A search of electronic databases yielded 131 relevant papers related to hydroponic food safety. Thirty-two studies focusing on food safety interventions reported 53 different interventions using chemical (n = 39), physical (n = 10), multiple-hurdle (n = 2), and biological (n = 2) approaches. Human pathogen indicators and surrogates were most often studied (n = 19), while pathogenic strains like Salmonella spp. (n = 9), Shiga toxin-producing Escherichia coli (STEC) (n = 5), Listeria monocytogenes (n = 2), and viruses (Hepatitis A virus (HAV), n = 1; norovirus (NoV), n = 1) were studied less frequently. Of fourteen articles (43.8%) investigating pre-harvest interventions, most (42.9%) did not specify the hydroponic system type. Gaps remain in the available evidence regarding the efficacy of interventions for controlling human pathogens in near-commercial hydroponic systems. The quality assessment revealed a significant lack of detailed reporting on methods and outcomes, making it difficult to translate the findings into practical recommendations for the industry; therefore, this review provides recommendations for the scientific community to improve future research design and reporting in this field. Full article

Early determination of the Shiga toxin type of Shiga toxin-producing Escherichia coli (STEC) is crucial for guiding STEC-infected patients for proper and timely treatment and patient care. Most diagnostic microbiology laboratories rely on PCR assays to detect the presence of stx1 and/or stx2 and enzymatic immunoassays (EIA) to detect the presence of the Shiga toxins 1 and/or 2 in STEC-positive stool samples. Occasionally, the stool samples test positive for STEC by PCR assays but test negative for the presence of Shiga toxins. Insufficient toxin production under laboratory conditions is the main culprit of this discordance. To test whether EIA-based STEC detection could be improved, various clinical STEC strains were treated with mitomycin C, which is a commonly used inducer of Shiga toxin production. A dose-dependent increase in Shiga toxin production, in response to mitomycin C doses of up to 500 ng/mL, was observed without any bactericidal effects. Depending on the serotype, 5–50 times more Shiga toxin 2 was produced than Shiga toxin 1. Shiga toxin production was not induced by the mitomycin C treatment in certain STEC serotypes carrying the toxin subtypes stx1a, stx2a, 2b, 2f, or 2h. This diversity in toxin production indicates that other factors may determine toxin expression in certain STEC strains, which warrant further exploration. Full article

Background/Objectives: Livestock species, particularly dairy animals, can serve as important reservoirs of E. coli, carrying antibiotic resistance and virulence genes under constant selective pressure and their spread in the environment. In this study, we performed the pathogenomic analysis of seven multidrug resistant (MDR) E. coli strains carrying efflux-associated and virulence genes from the dairy farm environment in Xinjiang Province, China. Methods: First, we processed the samples using standard microbiological techniques followed by species identification with MALDI-TOF MS. Then, we performed whole genome sequencing (WGS) on the Illumina NovaSeq PE150 platform and conducted pathogenomic analysis using multiple bioinformatics tools. Results: WGS analysis revealed that the E. coli strains harbored diverse antibiotic efflux-associated genes, including conferring resistance to fluoroquinolones, aminoglycosides, aminocoumarins, macrolides, peptides, phosphonic acid, nitroimidazole, tetracyclines, disinfectants/antiseptics, and multidrug resistance. The phylogenetic analysis classified seven E. coli strains into B1 (n = 4), C (n = 2), and F (n = 1) phylogroups. PathogenFinder predicted all E. coli strains as potential human pathogens belonging to distinct serotypes and carrying broad virulence genes (ranging from 12 to 27), including the Shiga toxin-producing gene (stx1, n = 1). However, we found that a few of the virulence genes were associated with prophages and genomic islands in the E. coli strains. Moreover, all E. coli strains carried a diverse bacterial secretion systems and biofilm-associated genes. Conclusions: The present study highlights the need for large-scale genomic surveillance of antibiotic-resistant bacteria in dairy farm environments to identify AMR reservoir spillover and pathogenic risks to humans and design targeted interventions to further stop their spread under a One Health framework. Full article

If lettuce is contaminated in the field, Shiga toxin-producing E. coli (STEC) O157:H7 can survive through the distribution chain. Prolonged cold storage during transportation may impact pathogen physiology, affecting subsequent stress survival and virulence. Greenhouse-grown Romaine lettuce, inoculated with three STEC O157:H7 strains, was harvested after 24 h and stored at 2 °C for 5 d following 4 h at harvest temperature (9 °C or 17 °C). Culturable, persister, and viable but non-culturable (VBNC) cells were quantified. Virulence was evaluated using Galleria mellonella and acid tolerance at pH 2.5 and tolerance to 20–25 ppm free chlorine were quantified. Colder harvest temperature (9 °C) before cold storage led to greater transformation of STEC O157:H7 into dormant states and decreased virulence in most cases. Increasing length of cold storage led to decreased virulence and acid tolerance of STEC O157:H7 on lettuce, while having no significant effect on chlorine tolerance. These findings highlight that entry of STEC O157:H7 into dormant states during harvest and transportation at cold temperatures leads to decreased stress tolerance and virulence with increasing cold storage. Changes in STEC O157:H7 physiology on lettuce during cold storage can be integrated into risk assessment tools for producers, which can assist in identifying practices that minimize risk of STEC O157:H7 from consumption of lettuce. Full article

Background/Objectives: Enterobacteriaceae, including pathogenic Shigella (S.) flexneri and Escherichia (E.) coli, cause severe gastrointestinal infections through toxins like Shiga and Shiga-like toxins. Antibiotic use is often discouraged due to its potential to increase toxin effects or contribute to the development of resistance. The host protease furin is capable of activating several viral glycoproteins and bacterial toxins, thus enhancing pathogen infectivity. Methods: To assess the therapeutic potential of furin inhibitors, cultured epithelial cell models (IPEC-J2 and MDCK) were used. The effects of MI-1851 and MI-2415 were evaluated after short-term (2 h) and long-term (6 h) exposure to S. flexneri, enterohemorrhagic E. coli (EHEC), and enteropathogenic E. coli (EPEC). Cytotoxicity was determined using the CCK-8 assay, and the inflammatory response was assessed by measuring interleukin (IL)-6 and IL-8 levels. Additionally, extracellular hydrogen peroxide production was monitored in IPEC-J2 cells to evaluate the potential alterations in redox status. Results: Infections with EHEC, EPEC, and S. flexneri significantly reduced the viability of epithelial cells after 6 h of incubation. Furin inhibitors MI-1851 and MI-2415 decreased cytotoxicity and compensated for IL-6 and IL-8 overproduction in cells during infection with EHEC and S. flexneri, but not in cells exposed to EPEC. In addition, they alleviated oxidative stress, particularly during S. flexneri addition. Conclusions: The development of new antimicrobial drugs that act via alternative mechanisms and effectively manage life-threatening enterobacterial infections is of key importance. Targeting furin with inhibitors MI-1851 and MI-2415, thus blocking toxin activation, could prevent the development of antimicrobial resistance, reduce the need for antibiotics and enhance overall treatment outcomes. Full article

A study was conducted to validate the plain bagel baking process as an effective kill-step in controlling Shiga toxin-producing Escherichia coli (STEC) in the event of pre-baking contamination originating from flour. Unbleached bread flour was inoculated with five strains of STEC and dried back to its original water activity levels. The inoculated flour was used to prepare the bagel dough, proofed, boiled for 2 min, and baked at 232.2 °C (450 °F) for 14 min mimicking the commercial manufacturing process. Additionally, water activity (a_w) and pH in plain bagels during baking, and thermal inactivation kinetics (D- and z-values) of STEC in plain bagel dough were studied. The results clearly demonstrated that baking plain bagels at 232.2 °C (450 °F) for 14 min will result in at least a >5 log reduction in the STEC population, thus providing an effective kill-step assuring the safety of the finished food products. The pH of plain bagels increased significantly from pre-proofed plain bagel dough to seven min into the baking process, reaching a final value of 5.83. The water activity of the crust and crumb portions of plain bagels was significantly different during the baking process. The D-values of STEC in plain bagels at, 56, 59, and 62 °C were 26.3 ± 1.55, 9.0 ± 0.27, and 2.50 ± 0.15 min with a z-value of 5.8 ± 0.16 °C. Full article

Background: Escherichia coli is one of the most studied bacteria worldwide due to its genetic plasticity. Recently, in addition to characterizing its pathogenic potential, research has focused on understanding its resistance profile to inhibitory agents, whether these be antibiotics or sanitizers. Objectives: The present study aimed to investigate six of the main serogroups of foodborne infection (O26, O45, O103, O111, O121, and O157) and to understand the dynamics of heterogeneity in resistance to sanitizers derived from quaternary ammonium compounds (QACs) and peracetic acid (PAA) using whole-genome sequencing (WGS). Methods: Twenty-four E. coli strains with varied resistance profiles to QACs and PAA were analyzed by WGS using NovaSeq6000 (150 bp Paired End reads). Bioinformatic analyses included genome assembly (Shovill), annotation via Prokka, antimicrobial resistance gene identification using Abricate, and core-genome analysis using Roary. A multifactorial multiple correspondence analysis (MCA) was conducted to explore gene–sanitizer relationships. In addition, a large-scale analysis utilizing the NCBI Pathogen Detection database involved a 2 × 2 chi-square test to examine associations between the presence of qac and stx genes. Results: The isolates exhibited varying antimicrobial resistance profiles, with O45 and O157 being the most resistant serogroups. In addition, the qac gene was identified in only one strain (S22), while four other strains carried the stx gene. Through multifactorial multiple correspondence analysis, the results obtained indicated that strains harboring genes encoding Shiga toxin (stx) presented profiles that were more likely to be sensitive to QACs. To further confirm these results, we analyzed 393,216 E. coli genomes from the NCBI Pathogen Detection database. Our results revealed a significant association (p < 0.001) between the presence of qac genes and the absence of stx1, stx2, or both toxin genes. Conclusion: Our findings highlight the complexity of bacterial resistance mechanisms and suggest that non-pathogenic strains may exhibit greater tolerance to QAC sanitizer than those carrying pathogenicity genes, particularly Shiga toxin genes. Full article

Canada is a leading producer of wild lowbush blueberries, most of which are mechanically harvested, washed, individually quick frozen (IQF), and bulk packaged. Still, some berries are harvested by more gentle methods and sold as fresh-packed products. These berries do not undergo a wash step, nor are subjected to antimicrobial treatments. The purpose of this study was to conduct a microbiological survey of berries harvested in the province of Nova Scotia to assess their potential for harborage of bacterial foodborne pathogens. A combination of standardized plate count methods and 3M-Petrifilm protocols were used to enumerate total aerobic mesophilic bacteria (APC), yeasts and molds (YMC), coliforms, and generic E. coli, the latter being an indicator of fecal contamination. Overall, APC and YMC levels were 1.2 and 0.5 log greater, respectively, for berries collected early in the harvest season versus those acquired late season and varied significantly (p < 0.05) between farm (location) and harvest practices used. Berries harvested by our team using sanitized hand rakes (SH) had consistently lower APC and YMC levels than those harvested by farm crews. Yet, when gentle harvesting (GH) methods (hand-raking, walk-behind or modified mechanical harvesters) were employed on farms, lower numbers were generally observed compared to berries harvested by traditional tractor-mounted mechanized harvesters (MH). The presence of coliforms (and their levels) was also impacted by the harvest method, with detection rates of ~29%, 73%, and 92% in SH, GH, and MH samples, respectively. Mean counts were < 2.5 log₁₀ CFU/g for both SH and GH berries, but significantly higher (p < 0.05) on MH berries (3.6 log₁₀ CFU/g). Although ~56% of all berry samples collected (n = 350) contained coliforms, only 12 were positive for E. coli, 9 of which were MH samples. Only the latter had numbers > 2 log₁₀ CFU/g, but none tested positive for Shiga toxin-producing serotype O157 (STEC O157) or Salmonella spp. when using internationally recognized selective enrichment and plating methods. ATP luminescence was used to assess the general hygiene of processing lines, whereby “hot spots” for microbial activity were identified, even after cleaning., Standard selective enrichment and plating methods were used for the detection of Listeria monocytogenes on 61 swab samples taken from berry totes or conveyor belts at different times during processing; 4 swabs tested positive for L. monocytogenes. However, the pathogen could not be detected by direct plating on selective agar without prior enrichment; this indicated its numbers were low. The results from this work demonstrated that alternative gentle harvest methods can reduce microbial numbers on wild blueberries. Although the frequency of fecal contamination in berry samples appeared to be low and targeted human pathogens were not detected; this represents a single study conducted over one harvest season. Therefore, it would be prudent for processors to seek effective antimicrobial technologies prior to packaging, while consumers should use caution and thoroughly wash produce before consumption. Where sporadic detection of L. monocytogenes was observed on environmental samples from the processing line, processors must ensure that effective sanitation programs are implemented to avoid potential food safety risks. Full article

Background/Objectives: Hemolytic uremic syndrome (HUS) is a known cause of acute kidney injury in children, but there are few recent reports on its epidemiology and outcome. We aimed to investigate trends in the incidence and the long-term outcomes of both Shiga toxin-producing Escherichia coli -HUS (STEC-HUS) and atypical HUS (aHUS) in Poland over the last 12 years (2012—2023), based on the Polish Pediatric HUS and Pediatric Renal Replacement Therapy (RRT) Registries. Methods: A total of 436 patients (301 with STEC-HUS and 135 with aHUS) were included. Results: The incidence of STEC-HUS increased during the observation period, with a mean of 3.9 cases per million age-related population (marp). The incidence of aHUS was relatively constant with a mean of 1.8/marp. The majority of patients fully recovered, although kidney sequelae were observed at 5-year follow-ups in 31% of children with STEC-HUS, 57% of aHUS subjects in the pre-eculizumab era, and 37% of aHUS subjects who had received eculizumab. The overall mortality rate was 2% for STEC-HUS and 3.7% for aHUS, with no deaths reported in children on eculizumab and mortality mainly attributed to neurological damage. A decreasing incidence of chronic kidney disease stage 5 (CKD5) due to HUS was observed. Conclusions: Despite an unchanging incidence of aHUS and an increasing incidence of STEC-HUS, the kidney outcomes of both diseases have improved significantly over the last 12 years. Mortality from HUS has dropped due to improved symptomatic treatment and the introduction of anti-C5 therapy. The development of CKD5 in childhood as a consequence of HUS has become exceptional. Full article

Loop-mediated isothermal amplification (LAMP) is a cost-effective, rapid, and highly specific method of replicating nucleic acids. Adding multiple targets into a single LAMP assay to create a multiplex format is highly desirable for clinical applications but has been challenging due to a need to develop specific detection techniques and strict primer design criteria. This study describes the evaluation of a rapid triplex LAMP assay, MAST ISOPLEX^® VTEC, for the simultaneous detection of Shiga toxin/verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) genes in verotoxigenic Escherichia coli (E. coli) (VTEC) isolates with inhibition control (IC) synthetic DNA using a single fluorophore–oligonucleotide probe, MAST ISOPLEX^® Probes, integrated into the primer set of each target. MAST ISOPLEX^® Probes used in the MAST ISOPLEX^® VTEC kit produce fluorescent signals as they integrate with reaction products specific to each target, allowing tracking of multiple amplifications in real time using a real-time analyzer. Initial validation on DNA extracts from fecal cultures and synthetic DNA sequences (gBlocks) showed that the MAST ISOPLEX^® VTEC kit provides a method for sensitive simultaneous triplex detection in a single assay with a limit of detection (LOD) of less than 100 target copies/assay and 96% and 100% sensitivity and specificity, respectively. Full article

To better understand the molecular genetics of the Shiga toxin type 2 subunit A gene (stx2A gene), we collected many subtypes of stx2A genes and performed detailed molecular evolutionary analyses of the gene. To achieve the aim of the study, we used several bioinformatics technologies, including time-scaled phylogenetic analyses, phylogenetic distance analyses, phylodynamics analyses, selective pressure analyses, and conformational epitope analyses. A time-scaled phylogeny showed that the common ancestor of the stx2A gene dated back to around 18,600 years ago. After that, the gene diverged into two major lineages (Lineage 1 and 2). Lineage 1 comprised the stx2a–2d subtypes, while Lineage 2 comprised the stx2e, 2g, 2h, and 2o subtypes. The evolutionary rates of the genes were relatively fast. Phylogenetic distances showed that the Lineage 2 strains had a wider genetic divergence than Lineage 1. Phylodynamics also indicated that the population size of the stx2A gene increased after the 1930s and spread globally. Moreover, negative selection sites were identified in the Stx2A proteins, and these sites were diffusely distributed throughout the protein. Two negative selection sites were located adjacent to an active site of the common Stx2A protein. Many conformational epitopes were also estimated in these proteins, while no conformational epitope was found adjacent to the active site. The results suggest that the stx2A gene has uniquely evolved and diverged over an extremely long time, resulting in many subtypes. The dominance of the strains belonging to Lineage 1 suggests that differences in virulence may be involved in the prosperity of the offspring. Furthermore, some subtypes of Stx2A proteins may be able to induce effective neutralizing antibodies against the proteins in humans. Full article

The current study outlines the advancement of an innovative technique for the simultaneous detection of E. coli O157:H7 and its Shiga-like toxins in food samples by utilizing a photonic label-free biosensor coupled with a microfluidic system. This detection method relies on ring resonator transduction that is functionalized with specific bioreceptors against O157:H7 on silicon nitride surfaces capable of binding specifically to the antigen bacterium and its verotoxins. This experiment included the characterization of selected monoclonal and polyclonal antibodies employed as detection probes through ELISA immunoassays exposed to target bacterial antigens. A thorough validation of photonic immunosensor detection was conducted on inoculated minced beef samples using reference standards for E. coli O157:H7 and its verotoxins (VTx1 and VTx2) and compared to gold-standard quantification. The lowest limit-of-detection values of 10 CFU/mL and 1 ppm were achieved for the detection of bacteria and its verotoxins. In this study, the lowest limit of quantification (LoQ) achieved for bacterial quantification was 100 CFU/mL, and, for verotoxins, it was 2 ppm. This study confirmed the effectiveness of a new quality control and food hygiene method, demonstrating the rapid and sensitive detection of E. coli O157:H7 and its verotoxins. This innovative approach has the potential to be applied in food production environments. Full article

Shiga toxin-producing Escherichia coli (STEC) infection can cause a broad spectrum of symptoms spanning from asymptomatic shedding to mild and bloody diarrhea (BD) and even life-threatening hemolytic-uremic syndrome (HUS). As a member of the serine protease autotransporters of Enterobacteriaceae (SPATE) family, EspP has the ability to degrade human coagulation factor V, leading to mucosal bleeding, and also plays a role in bacteria adhesion to the surface of host cells. Here, we investigated the prevalence and genetic diversity of espP among clinical STEC isolates from patients with mild diarrhea, BD, and HUS, as well as from asymptomatic individuals, and assessed the presence of espP and its subtypes in correlation to disease severity. We found that 130 out of 239 (54.4%) clinical STEC strains were espP positive, and the presence of espP was significantly associated with BD, HUS, and O157:H7 serotype. Eighteen unique espP genotypes (GTs) were identified and categorized into four espP subtypes, i.e., espPα (119, 91.5%), espPγ (5, 3.8%), espPδ (4, 3.1%), and espPε (2, 1.5%). espPα was widely distributed, especially in strains from patients with BD and HUS, and correlated with serotype O157:H7. Serogroup O26, O145, O121, and O103 strains carried espPα only. Ten GTs were identified in espPα, and espPα/GT2 was significantly associated with severe disease, i.e., BD and HUS. Additionally, espP was strongly linked to the presence of eae gene, and the coexistence of espPα and stx2/stx2a + stx2c was closely related to HUS status. To sum up, our data demonstrated a high prevalence and genetic diversity of the espP gene in clinical STEC strains in Sweden and revealed an association between the presence of espP, espP subtypes, and disease severity. espP, particularly the espPα subtype, was prone to be present in more virulent STEC strains, e.g., “top-six” serotypes strains. Full article

Shiga-toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). The current Food Safety Inspection Service (FSIS) testing methods for STEC use the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) protocol, which includes enrichment, cell plating, and genomic sequencing and takes time to complete, thus delaying diagnosis and treatment. We wanted to develop a rapid, sensitive, and potentially portable assay that can identify STEC by detecting Shiga toxin (Stx) using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) B-cell based biosensor technology. Five potential biosensor cell lines were evaluated for their ability to detect Stx2. The results using the best biosensor cell line (T5) indicated that this biosensor was stable after reconstitution with assay buffer covered in foil at 4 °C for up to 10 days with an estimated limit of detection (LOD) of ≈0.1–0.2 ng/mL for days up to day 5 and ≈0.4 ng/mL on day 10. The assay detected a broad range of Stx2 subtypes, including Stx2a, Stx2b, Stx2c, Stx2d, and Stx2g but did not cross-react with closely related Stx1, abrin, or ricin. Additionally, this assay was able to detect Stx2 in culture supernatants of STEC grown in media with mitomycin C at 8 and 24 h post-inoculation. These results indicate that the STEC CANARY biosensor developed in this study is sensitive, reproducible, specific, rapid (≈3 min), and may be applicable for surveillance of the environment and food to protect public health. Full article

Foodborne pathogens are a significant cause of illness, and infection with Shiga toxin-producing Escherichia coli (STEC) may lead to life-threatening complications. The current methods to identify STEC in meat involve culture-based, molecular, and proteomic assays and take at least four days to complete. This time could be reduced by using long-read whole-genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate the use of long-read sequencing to detect STEC in ground beef. The objectives of the project included establishing optimal sequencing parameters, determining the limit of detection of all STEC virulence genes of interest in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on the Oxford Nanopore Technologies’ MinION sequencer. Optimal sequencing output was obtained using the default parameters in MinKNOW, except for setting the minimum read length to 1 kb. All genes of interest (eae, stx1, stx2, fliC, wzx, wzy, and rrsC) were detected in DNA extracted from STEC pure cultures within 1 h of sequencing, and 30× coverage was obtained within 2 h. All virulence genes were confidently detected in STEC DNA quantities as low as 12.5 ng. In STEC-inoculated ground beef, software-controlled selective sequencing improved virulence gene detection; however, several virulence genes were not detected due to high bovine DNA concentrations in the samples. The growth enrichment of inoculated meat samples in mTSB resulted in a 100-fold increase in virulence gene detection as compared to the unenriched samples. The results of this project suggest that further development of long-read sequencing protocols may result in a faster, less labor-intensive method to detect STEC in ground beef. Full article