ABSTRACT: Shiga toxin-producing Escherichia coli (STEC) are a group of E. coli that cause food- and waterborne enteric illness. STEC are genotypically and phenotypically diverse as they do not have a single evolutionary origin; instead, multiple STEC lineages have appeared independently following horizontal gene transfer events. The diversity of STEC is also reflected in an apparent variability in the potential of individual STEC strains to cause severe illness (bloody diarrhea or hemolytic uremic syndrome) or be involved in large foodborne outbreaks. In the context of food safety decision-making, the variable characteristics of STEC strains and their potential health outcomes have resulted in an ongoing debate on how STEC should be categorized into risk groups. The aim of this review was to clarify that debate by examining how STEC characterization and risk categorization strategies have evolved over time in response to changes in analytical techniques, understanding of STEC epidemiology, and pathogenicity mechanisms. The origin of established perspectives and practices is explored, from the recognition of STEC O157:H7 as an emerging pathogen in the 1980s, through serotype-based identification and characterization of STEC, to contemporary approaches based on molecular markers for virulence. The evidence for the role of specific virulence factors in STEC pathogenicity is reviewed, and their utility in STEC risk categorization is assessed. Finally, a binary categorization scheme is proposed, based on evidence for the correlation of hemolytic uremic syndrome with the presence of Shiga toxin subtypes Stx2a, Stx2c, Stx2d, and colonization factors including the Locus of Enterocyte Effacement ( intimin/eae ) and Enteroaggregative attachment ( aggR/aaiC ).
ABSTRACT: Foodborne diseases represent a major global public health problem, causing significant morbidity and mortality, and substantial economic losses. Fruits and vegetables are considered among the main vehicles for transmission of infectious agents like bacteria, viruses, fungi, and parasites, including Cryptosporidium sp. This protozoan parasite comprises many species that infect a wide range of hosts, is environmentally and geographically ubiquitous, and has become one of the leading causes of diarrheal disease; in immunocompromised hosts, the infection can result in a chronic, life-threatening disease. Considering the importance of fruit and vegetable consumption in the daily diet and the need to ensure their safety, the aim of the study was to evaluate the presence of Cryptosporidium sp. in strawberries, lettuce, and cilantro marketed in the Metropolitan Area of Costa Rica and to assess the degree of fecal contamination in these foods. For this purpose, 96 samples were analyzed using parasitological and molecular methods that included a nested PCR to amplify the Cryptosporidium sp. gp60 gene. Moreover, the presence of total and thermotolerant coliforms and Escherichia coli was also determined by culture methods and counts, for further identification using the VITEK system. Direct examination of wet mount and Ziehl-Neelsen stains revealed five Cryptosporidium sp. positive samples by microscopy and 22 positive samples by PCR; the identified species was Cryptosporidium parvum . Cilantro samples presented the highest counts of total and thermotolerant coliforms, and E. coli was identified in14 samples among 82 samples subjected to bacteriological analyses. The detection of C. parvum in raw vegetables and fruits from different provinces in Costa Rica, particularly using molecular techniques, demonstrates the need to strengthen surveillance and control in the production and marketing of these foods, to improve risk assessment and its relevance to public health.
Conventional confirmation of Listeria monocytogenes from food samples involves enrichment, selective plating, and biochemical identification. Colony confirmation is a process that can take 3-5 days. We validated a multiplex PCR assay for the rapid identification of Listeria colonies at the genus level and the specific detection of L. monocytogenes directly from both selective and non-selective agars. The assay targets three markers: hlyA (152 bp, L. monocytogenes-specific), prs (370 bp, Listeria genus-specific), and 16S rRNA (107 bp, internal amplification control). Validation using 226 isolates (117 L. monocytogenes, 44 other Listeria spp., 65 non-Listeria) demonstrated 100% inclusivity and 100% exclusivity. Results were consistent across Oxford, Agar Listeria media, RAPID'L.mono, PALCAM, with isolate identifications confirmed by whole-genome sequencing. Although MALDI-TOF mass spectrometry also provides rapid identification, it requires specialized instrumentation and may not be available in all food microbiology laboratories. In contrast, this PCR assay reduces turnaround time to less than 4 hours and aligns with ISO 16140-6 and Health Canada guidelines for confirmatory testing. Additional evaluation of recently described Listeria species and haemolytic L. innocua strains, presented separately, identified atypical amplification profiles that highlight potential imitation in the broader applicability of the assay.
Recurring outbreaks of Shiga toxin-producing Escherichia coli (STEC) associated with fresh produce have increased attention to preharvest contamination pathways, including the potential movement of material from livestock operations through surface water. We developed an event-based hydrologic screening framework to characterize relative livestock-associated runoff exposure for produce farms in the Salinas Valley, California. The framework combines rainfall-derived runoff estimates with modeled surface-water pathways connecting upstream livestock sources to downstream farms. Forty-eight rainfall events during 2018-2021 were evaluated, with runoff from livestock source areas estimated using the Curve Number method, a standard approach for estimating runoff from rainfall and land-surface characteristics. Source-to-farm connections were identified using terrain-based drainage routing. Upstream pasture contributions were reduced with increasing flow-path distance to represent decreasing source influence over longer travel paths. The resulting runoff-exposure metric represents relative hydrologic influence from connected upstream livestock sources and does not represent pathogen presence, concentration, contamination probability, or food-safety risk. Hydrologic routing identified 1,818 pasture-farm connections involving 277 downstream farms and 45 animal feeding operation (AFO)-farm connections involving 45 farms. Most farms (86.3%) had no modeled upstream pasture contribution under the baseline attenuation assumptions, and examples of farms intersecting pasture polygons but lacking routed downstream connections demonstrated that geographic proximity alone did not determine runoff-exposure. Farm-level rankings were broadly stable across alternative assumptions about how rapidly upstream influence decreased with flow-path distance, although the identity of the highest-ranked farms was more sensitive to this assumption. The framework provides a transparent approach for identifying hydrologically connected farms and upstream source areas that may warrant targeted environmental investigation, monitoring, or mitigation following substantial rainfall events.
ABSTRACT: Salmonella is a major foodborne pathogen, and chicken meat is an important source of infection. This study investigated Salmonella prevalence, serovar distribution, antimicrobial resistance, and genomic characteristics in 150 retail chicken meat samples collected in Japan in 2023. The predominant serovar was S. Schwarzengrund (42.7%), followed by S. Infantis (7.3%), which has long been the predominant serovar in Japan. The globally expanding S. Infantis sequence type (ST) 32 harboring pESI-like plasmids has declined in Japan. In contrast, S. Schwarzengrund ST241 was isolated more frequently in winter (66%) than in summer (31%, P < 0.001) and more often from chicken meat produced in eastern Japan (47.1%) than in western Japan (29.4%, P = 0.042). Antimicrobial resistance was most common to tetracycline (50.6%) and streptomycin (45.7%), whereas kanamycin resistance (33.3%) had declined compared with 2015 to 2019, consistent with reduced use of this antimicrobial in broiler production. Moreover, the proportion of S. Schwarzengrund isolates lacking antimicrobial resistance genes (ARGs) increased markedly from summer (19.4%) to winter (45.5%). cgMLST analysis revealed that antimicrobial-susceptible S. Schwarzengrund isolates formed the largest clade and, notably, retained pESI-like plasmids despite apparent loss of ARG-containing regions from these plasmids. These findings demonstrate ongoing genomic shifts in Salmonella populations in Japan and underscore the need for continuous genomic surveillance to mitigate their public health impact.
