Search Results (28)

Zoonotic pathogens could persist in their environment and be introduced into the food-chain. With careful screening and selection, lactic acid bacteria (LAB) could be used as direct-fed microbials (DFMs) to control these pathogens in food animals. Previously isolated LAB (n = 250) were evaluated for inhibition against Shiga-toxigenic Escherichia coli (STEC) and Salmonella enterica, using agar spot test. Tests revealed that LAB were more effective against Salmonella than STEC, with 67% showing excellent (>15 mm) inhibition. LAB (n = 65) exhibiting significant pathogen inhibition (zones > 10 mm) were tested for acid (pH: 2, 4, 5, 7) and bile (0, 0.1, 0.3, 0.5%) tolerance, and biofilm-forming capabilities. About half of the tested LAB exhibited excellent to very good tolerance. All LAB formed biofilms, with 33% forming strong biofilms. LAB (n = 59) were also examined for susceptibility to commonly used antibiotics due to their intrinsic or acquired antibiotic resistance (AR), transferrable to pathogens. Only S. thermophilus S-2 showed susceptibility to all the antibiotics. The majority were susceptible to erythromycin (88%), followed by ampicillin (85%), clindamycin (64%), tetracycline (58%), vancomycin (44%), streptomycin (15%), and gentamicin (9%). Overall, LAB showed strong inhibition against pathogens, along with survival capabilities for environmental stress conditions, and could be considered for potential DFM applications. Full article

The concept of using ‘acid-adapted’ challenge cultures in the microbial validation of food processes that incorporate an acidic treatment is that they would be more resistant to acid and require a robust process to obtain targeted log reductions. The recent confirmation that acid-adapted Salmonella challenge cultures for droëwors and biltong processes are more sensitive to those processes than non-adapted cultures changes that preference for the use of non-adapted cultures for validation studies with these specific processes. However, it is difficult to achieve > 5-log reductions with non-adapted cultures, one of two USDA-FSIS parameters available for validation of processes that are not aligned with traditional process conditions for dried beef products in the USA (i.e., beef jerky). A natural multipurpose (flavor, antimicrobial) commercial product, described as a refined liquid smoke flavorant, provided >7-log reductions with droëwors when challenged with non-adapted cultures of Salmonella (5-serovar mixture), Shiga toxigenic Escherichia coli (STEC, four-strain mixture), and Listeria monocytogenes (four-strain mixture) as well as a >7-log reduction with biltong processing (vs Salmonella). Comparisons between standard droëwors and biltong processes (all <5-log reductions) using non-adapted challenge cultures vs. the same formulation plus 0.75% pyrolyzed liquid smoke extracts (Flavoset) showed greater and significant (p < 0.05) reductions in duplicate trials with triplicate samples at each sampling point in each trial (total n = 6) when analyzed by repeated measures analysis of variance (RM-ANOVA). Although sold as a flavorant, this study examines the antimicrobial properties of Flavoset 5400L to improve the safety of droëwors and biltong by achieving a >5-log reduction with non-adapted pathogenic challenge cultures. Validation processes for droëwors and biltong established with these parameters should result in greater safety of marinaded, non-thermally processed meats from traditional foodborne pathogens commonly associated with meats or meat processing environments. Full article

Background: Diarrheagenic Escherichia coli (DEC) remains relevant to public health and agri-food chains. The context in Brazil, as a major food producer and exporter, reinforces the need for genomic surveillance. Objective: We aimed to characterize Brazilian diffusely adhering (DAEC), enteropathogenic (EPEC), and Shiga toxin-producing E. coli (STEC) sequences in silico across O-serogroups, in addition to sequence-type (ST), virulence, resistome, and phylogenomic relationships. Methodology: We retrieved 973 genomes assigned to Brazil from NCBI Pathogen Detection Database and performed virtual-PCR screening for key DEC-genes. We then typed O-serogroups (ABRicate/EcOH), Multi-Locus Sequencing Type (MLST), virulome (Ecoli_VF), resistome (ResFinder), and characterized stx genes. Results: DEC represented 18.7% of genomes, driven primarily by EPEC. In EPEC, the eae β-1 subtype was most common; we detected, for the first time in Brazilian sequences, ξ-eae subtype and ST583/ST301. Seventy-eight percent of DAEC isolates were multidrug-resistant (MDR), and two ST were newly reported in the country (ST2141/ST500). In STEC, O157 formed a largely susceptible clade with uniform eae γ-1, whereas 57% of non-O157 were MDR. New STs (ST32/ST1804) were observed, and three genomes were closely related to international isolates. Conclusions: Despite the low DEC representation in the dataset, new STs and eae subtypes were detected in Brazil. Also, MDR in DAEC and non-O157 STEC reinforces the need for antimicrobial-resistance genomic surveillance. Full article

The impact of Oedema Disease produced by Shiga toxigenic Escherichia coli (STEC) in swine is increasing in some production countries due to increasing limitations on treatment with antimicrobials and zinc oxide, either because of the increased prevalence of multi-resistant strains or because of legal restrictions. The main pathological effect of Shiga toxin 2e is represented by damage to the endothelial cells of the blood vessel walls, leading to liquid extravasation and oedema formation in multiple tissues. These oedemas are generally easily identifiable in acute clinical cases. However, disease caused by Shiga toxin can occur without any externally visible oedema in the pigs, as observed in the subclinical presentation of Oedema Disease. It also causes productive losses, so it is important to identify and/or diagnose cases to set up control measures in order to optimize production and health. This article includes a comprehensive review of lesions and signs caused by Shiga toxin toxicosis in pigs, as well as other insights about the aetiology and epidemiology of STEC in pigs, and the effect of Shiga toxin recombinant toxoid vaccines in reducing these clinical and subclinical signs under field conditions. Full article

Background/Objectives: Increasing levels of antimicrobial resistance (AMR) have recently been observed at the human–domestic animal–wildlife interface. Wild birds have been identified as carriers of antimicrobial-resistant bacteria and serve as excellent biomarkers for epidemiological studies. This study assessed the current AMR presence in Eastern Spain’s commensal Escherichia coli isolated from free-ranging Bonelli’s eagles (Aquila fasciata). Methods: Nestlings and their nests were intensively sampled between 2022 and 2024 to determine their AMR profile and characterize E. coli. AMR testing was conducted using the broth microdilution method, following the European Committee on Antimicrobial Susceptibility Testing guidelines. Additionally, the presence of eaeA (intimin gene) and stx-1 and stx-2 (shiga toxins) was analyzed by real-time PCR to classify E. coli strains into enteropathogenic (EPEC) and Shiga-toxigenic (STEC) pathotypes. Results: Of all E. coli isolates, 41.7% were resistant to at least one antimicrobial, and 30% were multidrug-resistant. Only two strains were classified as EPEC and none as STEC. The highest resistance rates were observed for amoxicillin and tetracycline (19.6% each). Alarmingly, resistance to colistin and meropenem, last-resort antibiotics in human medicine, was also detected. Conclusions: Although the mechanisms of resistance acquisition remain unclear, transmission is likely to occur through the food chain, with synanthropic prey acting as intermediary vectors. These results highlight the role of Bonelli’s eagles as essential sentinels of environmental AMR dissemination, even in remote ecosystems. Strengthening One Health-based surveillance is necessary to address AMR’s ecological and public health risks in wildlife. Full article

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen and known to reside naturally in cattle. The application of untreated biological soil amendments of animal origin on fresh produce fields results in unique food safety challenges. It is critical to identify farm manure management practices to mitigate pre-harvest pathogen contamination. The objective of this study was to quantify the prevalence and level of STEC in cattle manure in the Mid-Atlantic region of the United States. A total of 161 bovine manure samples were collected from 13 cattle farms between 2016 and 2018. The samples were enriched with non-selective and selective media and quantified following a Most-Probable Number (MPN) assay. Among the recovered STEC isolates, PCR was performed to determine the presence of stx, eae, and rfbE. Clermont PCR was performed to identify phylogenetic groups of isolates. Of the 13 farms, 11 had STEC populations between <1.0 and >5.6 log MPN/g. Farm, humidity, and sampling year significantly (p < 0.05) influenced STEC populations in bovine manure. Of the 108 isolates, 50% were stx+ and 14% eae+. Phylogenetic group analysis revealed that 46% of the isolates belonged to group A, 19% to B1, 7% to B2, and 28% to D. Group D had the highest prevalence of stx+ and eae+ and group B1 had the lowest prevalence. Results suggest STEC geographical distribution in the Mid-Atlantic region is farm-specific, and climatic conditions can be critical for its survival and dissemination. Full article

E. coli is considered one of the most important zoonotic pathogens worldwide. Highly virulent and antimicrobial-resistant strains of E. coli have been reported in recent years, making it essential to understand their ecological origins. In this study, we analyzed the characteristics of E. coli strains present in the natural population of American bison (Bison bison) in Mexico. We sampled 123 individuals and determined the presence of E. coli using standard bacteriological methods. The isolated strains were characterized using molecular techniques based on PCR. To evaluate the diversity of E. coli strains in this population, we analyzed 108 suggestive colonies from each fecal sample. From a total of 13,284 suggestive colonies, we isolated 33 E. coli strains that contained at least one virulence gene. The virotypes of these strains were highly varied, including strains with atypical patterns or combinations compared to classical pathotypes, such as the presence of escV, eae, bfpB, and ial genes in E. coli strain LMA-26-6-6, or stx2, eae, and ial genes in E. coli strain LMA-16-1-32. Genotype analysis of these strains revealed a previously undescribed phylogenetic group. Serotyping of all strains showed that serogroups O26 and O22 were the most abundant. Interestingly, strains belonging to these groups exhibited different patterns of virulence genes. Finally, the isolated E. coli strains demonstrated broad resistance to antimicrobials, including various beta-lactam antibiotics. Full article

Shiga toxin-producing Escherichia coli are zoonotic pathogens that cause food-borne human disease. Among these, the O157:H7 serotype has evolved from an enteropathogenic O55:H7 ancestor through the displacement of the somatic gene cluster and recurrent toxigenic conversion by Shiga toxin-converting bacteriophages. However, atypical strains that lack the Shiga toxin, the characteristic virulence hallmark, are circulating in this lineage. For this study, we analyzed the pathogenome and virulence inventories of the stx+ strain, TT12A, isolated from a patient with hemorrhagic colitis, and its respective co-isolated stx− strain, TT12B. Sequencing the genomes to closure proved critical to the cataloguing of subtle strain differentiating sequence and structural polymorphisms at a high-level of phylogenetic accuracy and resolution. Phylogenomic profiling revealed SNP and MLST profiles similar to the near clonal outbreak isolates. Their prophage inventories, however, were notably different. The attenuated atypical non-shigatoxigenic status of TT12B is explained by the absence of both the ΦStx_1a- and ΦStx_2a-prophages carried by TT12A, and we also recorded further alterations in the non-Stx prophage complement. Phenotypic characterization indicated that culture growth was directly impacted by the strains’ distinct lytic phage complement. Altogether, our phylogenomic and phenotypic analyses show that these intimately related isogenic strains are on divergent Stx(+/stx−) evolutionary paths. Full article

Shiga-toxin-producing Escherichia coli (STEC) is typically detected on food products mainly due to cross-contamination with faecal matter. The serotype O157:H7 has been of major public health concern due to the severity of illness caused, prevalence, and management. In the food chain, the main methods of controlling contamination by foodborne pathogens often involve the application of antimicrobial agents, which are now becoming less efficient. There is a growing need for the development of new approaches to combat these pathogens, especially those that harbour antimicrobial resistant and virulent determinants. Strategies to also limit their presence on food contact surfaces and food matrices are needed to prevent their transmission. Recent studies have revealed that bacteriophages are useful non-antibiotic options for biocontrol of E. coli O157:H7 in both animals and humans. Phage biocontrol can significantly reduce E. coli O157:H7, thereby improving food safety. However, before being certified as potential biocontrol agents, the safety of the phage candidates must be resolved to satisfy regulatory standards, particularly regarding phage resistance, antigenic properties, and toxigenic properties. In this review, we provide a general description of the main virulence elements of E. coli O157:H7 and present detailed reports that support the proposals that phages infecting E. coli O157:H7 are potential biocontrol agents. This paper also outlines the mechanism of E. coli O157:H7 resistance to phages and the safety concerns associated with the use of phages as a biocontrol. Full article

(1) Background: Formation of biofilms on food-contact surfaces by Shiga-toxigenic Escherichia coli (STEC) can pose a significant challenge to the food industry, making conventional control methods insufficient. Targeted use of bacteriophages to disrupt these biofilms could reduce this problem. Previously isolated and characterized bacteriophages (n = 52) were evaluated against STEC biofilms in vitro and on food-contact surfaces. (2) Methods: Phage treatments (9 logs PFU/mL) in phosphate-buffered saline were used individually or as cocktails. Biofilms of STEC (O157, O26, O45, O103, O111, O121, and O145) were formed in 96-well micro-titer plates (7 logs CFU/mL; 24 h) or on stainless steel (SS) and high-density polyethylene (HDPE) coupons (9 logs CFU/cm²; 7 h), followed by phage treatment. Biofilm disruption was measured in vitro at 0, 3, and 6 h as a change in optical density (A₅₉₅). Coupons were treated with STEC serotype-specific phage-cocktails or a 21-phage cocktail (3 phages/serotype) for 0, 3, 6, and 16 h, and surviving STEC populations were enumerated. (3) Results: Of the 52 phages, 77% showed STEC biofilm disruption in vitro. Serotype-specific phage treatments reduced pathogen population within the biofilms by 1.9–4.1 and 2.3–5.6 logs CFU/cm², while the 21-phage cocktail reduced it by 4.0 and 4.8 logs CFU/cm² on SS and HDPE, respectively. (4) Conclusions: Bacteriophages can be used to reduce STEC and their biofilms. Full article

The most significant serotype of Shiga-toxigenic Escherichia coli that causes foodborne illnesses is Escherichia coli O157:H7. Elimination of E. coli O157:H7 during food processing and storage is a possible solution. Bacteriophages have a significant impact on bacterial populations in nature due to their ability to lyse their bacterial host. In the current study, a virulent bacteriophage, Ec_MI-02, was isolated from the feces of a wild pigeon in the United Arab Emirates (UAE) for potential future use as a bio-preservative or in phage therapy. Using a spot test and an efficiency of plating analysis, Ec_MI-02 was found to infect in addition to the propagation host, E. coli O157:H7 NCTC 12900, five different serotypes of E. coli O157:H7 (three clinical samples from infected patients, one from contaminated green salad, and one from contaminated ground beef). Based on morphology and genome analysis, Ec_MI-02 belongs to the genus Tequatrovirus under the order Caudovirales. The adsorption rate constant (K) of Ec_MI-02 was found to be 1.55 × 10⁻⁸ mL/min. The latent period was 50 min with a burst size of almost 10 plaque forming units (pfu)/host cell in the one-step growth curve when the phage Ec_MI-02 was cultivated using the propagation host E. coli O157:H7 NCTC 12900. Ec_MI-02 was found to be stable at a wide range of pH, temperature, and commonly used laboratory disinfectants. Its genome is 165,454 bp long with a GC content of 35.5% and encodes 266 protein coding genes. Ec_MI-02 has genes encoding for rI, rII, and rIII lysis inhibition proteins, which supports the observation of delayed lysis in the one-step growth curve. The current study provides additional evidence that wild birds could also be a good natural reservoir for bacteriophages that do not carry antibiotic resistance genes and could be good candidates for phage therapy. In addition, studying the genetic makeup of bacteriophages that infect human pathogens is crucial for ensuring their safe usage in the food industry. Full article

Understanding Shiga toxin subtypes in E. coli from reservoir hosts may give insight into their significance as human pathogens. The data also serve as an epidemiological tool for source tracking. We characterized Shiga toxin subtypes in 491 goat E. coli isolates (STEC) from the mid-Atlantic US region (stx1 = 278, stx2 = 213, and stx1/stx2 = 95). Their serogroups, phylogroups, M13RAPD genotypes, eae (intimin), and hly (hemolysin) genes were also evaluated. STEC-positive for stx1 harbored Stx1c (79%), stx1a (21%), and stx a/c (4%). Those positive for Stx2 harbored stx2a (55%) and Stx2b (32%), while stx2a/stx2d and stx2a/stx2b were each 2%. Among the 343 STEC that were serogrouped, 46% (n = 158) belonged to O8, 20% (n = 67) to 076, 12% (n = 42) to O91, 5% (n = 17) to O5, and 5% (n = 18) to O26. Less than 5% belonged to O78, O87, O146, and O103. The hly and eae genes were detected in 48% and 14% of STEC, respectively. Most belonged to phylogroup B1 (73%), followed by D (10%), E (8%), A (4%), B2 (4%), and F (1%). M13RAPD genotyping revealed clonality of 091, O5, O87, O103, and O78 but higher diversity in the O8, O76, and O26 serogroups. These results indicate goat STEC belonged to important non-O157 STEC serogroups, were genomically diverse, and harbored Shiga toxin subtypes associated with severe human disease. Full article

The objectives of the current study were first to determine the prevalence of non O₁₅₇:H₇E. coli, especially Shiga toxin-producing Escherichia coli (STEC) in retailed milk and dairy products in Egypt. Second, the antimicrobial resistance profiling and virulence genes of the isolated E. coli strains were screened. Third, evaluation of the inhibitory effects of D-tryptophan against E. coli O₂₆:H₁₁ was further performed. The results revealed that 20% (30/150) of the samples were STEC positive, with 64 isolates harboring some virulent genes, such as Stx1, Stx2, eaeA, and hylA. Serological identification revealed four different pathotypes belonging to EPEC, ETEC, EHEC, and EIEC. Antimicrobial susceptibility testing revealed that 100%, 98.44%, 92.19%, 71.87%, 65.63% and 64.06% of the isolates had a resistance against tetracycline, oxacillin, erythromycin, nalidixic acid, sulphamethoxazol, and ampicillin, respectively. D-tryptophan addition (40 mM) to E. coli O₂₆:H₁₁-inoculated soft cheese and ice cream revealed a significant reduction (p < 0.05) in bacterial growth, especially when accompanied with other food stressors. D-Tryptophan is considered as an effective food preservative and as a promising alternative candidate in the dairy industry. Full article

Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC₅₀) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes. Full article

The biofilms of the wooden tables used for the acidification of the curd were investigated for PDO Provola dei Nebrodi cheese, a traditional stretched cheese made in eastern Sicily (southern Italy) from raw cows’ milk. To this purpose the wooden tables of four dairy facilities were analysed for their microbiota by scanning electron microscopy (SEM) analysis and a combined culture-independent and -dependent microbiological approach. SEM inspection showed an almost continuous biofilm formation. MiSeq Illumina analysis identified 8 phyla, 16 classes, 25 orders, 47 families and 50 genera. Corynebacterium, Bifidobacterium and lactic acid bacteria (LAB) were detected in all samples. In particular, the LAB genera detected on all wooden tables were Lactobacillus, Streptococcus and Lactococcus. LAB dominated the surfaces of all wooden tables with levels higher than 7.0 Log CFU/cm². In particular, the LAB found at the highest levels were mesophilic cocci. Coagulase positive staphylococci, Salmonella spp., Listeria monocytogenes and Shiga-toxigenic Escherichia coli were never detected. Twenty-seven dominating LAB strains were identified within the genera Enterococcus, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus. This work showed that the wooden table used during the production of PDO Provola dei Nebrodi cheese is a safe system and a microbiologically active tool. Full article