Santos García

Salmonella outbreaks are often linked to contaminated cantaloupes, and bacterial multiple antibiotic resistance is frequently observed. Research is needed to understand the growth and survival of Salmonella on cantaloupe surfaces, and the mechanisms of antibiotic resistance in that environment. This study investigated the growth, survival, and capacity for horizontal gene transfer of antibiotic resistance genes in Salmonella on the surface of cantaloupe. Bovine-derived Salmonella isolates were inoculated onto the surface of cantaloupe and incubated at 35°C under 65–75% humidity for 5 days. Each isolate was co-inoculated with donor E. coli CSH56 harboring helper plasmid RP4-8 and mobilizable plasmid pSC27, which carries kanamycin, neomycin, and ampicillin resistance genes. One hundred microliters of the Salmonella suspension (1 × 10 ⁸ CFU/mL) and 100 µL of the E. coli suspension (1 × 10 ⁷ CFU/mL) was applied to each cantaloupe (1:10 ratio). Salmonella survival and growth were monitored on Hektoen agar, transconjugants were enumerated on Hektoen agar with kanamycin, and donor E. coli were quantified on MacConkey agar. All Salmonella isolates survived and exhibited growth within the 5-day period, with increases ranging from 0.94 log ₁₀ CFU/mL to 3.43 log ₁₀ CFU/mL. Notably, all Salmonella isolates possess the capability to accept and integrate sequences derived from the pSC27 plasmid from E. coli into their chromosome, both in vitro and on cantaloupe. After conjugation, the count of Salmonella mutants carrying the plasmid was 2.79 log ₁₀ CFU/mL in vitro . The survival, growth, and horizontal gene transfer of Salmonella on the surface of cantaloupe pose significant risks to public health. IMPORTANCE: Salmonella , a leading cause of foodborne diseases, poses a global health threat, with multidrug resistance emerging as a severe problem. Cantaloupes have been implicated in salmonellosis outbreaks in multiple countries over the last three decades. We hypothesize that acquisition of antibiotic resistance genes from other bacteria may occur on the surface of the fruit. This study established that Salmonella can survive, proliferate, and acquire antibiotic resistance genes on cantaloupe surfaces. This observation underscores the potential risks on the growth and transfer of genes between pathogens such as Salmonella and E. coli in foods. This impact goes beyond disease production and contributes to the rapid escalation of antimicrobial resistance and transmission of virulence genes in foods and the environment, which is a significant threat to public health.

The consumption of fresh produce (fruits and vegetables) provides several health benefits, ranging from promoting overall well-being to preventing severe illnesses, such as cancer, diabetes, cardio...

Subinhibitory concentrations of various antibiotics can exacerbate microbial virulence. Acinetobacter baumannii is often resistant to oxacillin; however, the effect of low oxacillin concentrations on oxacillinase-producing bacteria remains unclear. Herein, oxacillinase producer A. baumannii ATCC-strains 2093 (motile) and 19606 (non-motile) were pre-exposed to sub-bactericidal concentrations (subMBC) of oxacillin (0.25, 0.5, and 1.0 mg/mL) and incubated at 37 °C. Growth kinetics, twitching and swarming-like motility, biofilm formation, exopolysaccharide production, transformation capability, and gene expression were determined. All oxacillin subMBC conditions exerted bacteriostatic effects in both strains. Pre-exposing the motile strain with 0.25 and 0.5 mg/mL of oxacillin for 1 h increased the twitching motility (4.2 ± 0.3 cm; control = 3.5 cm), whereas pre-exposure for 2 h it increased swarming-like motility (2.95 ± 0.15 cm; control = 2.7 cm). This strain also increased the biofilm production by effect of all 6 h-oxacillin subMBC treated bacteria (≤ 1.42 Biofilm Formation Index (BFI); control = 0.6 BFI), whereas the strain 19606 reduced biofilm up to 1 BFI (control = 2 BFI). The antibiotic also reduced the exopolysaccharide production in almost all treated cells from both strains. The transformation efficiency (TE) of strain 19606 increased X̄ = 19 ± 11% more than the control by effect of all DNA and oxacillin conditions studied; however, the TE for strain 2093 was lower than the control. The expression of genes for resistance (bla-OXA), twitching (pilA/tonB) and swarming-like motilities (dat/ddc), and biofilm production (csuE) were altered by the oxacillin subMBC. Overall, exposure of oxacillinase-producing A. baumannii to oxacillin subMBC increases several virulence factors, representing a potential public health risk.

Standard food detection methods do not distinguish between infectious and non-infectious human norovirus leading to uncertainty in the management of a norovirus positive food sample. These methods also require expensive RT-qPCR-based equipment and reagents. In contrast, CRISPR-based, compared to RT-qPCR-based, detection methods are generally less expensive and yield similar sensitivity and specificity. Our goal was to detect norovirus with an intact capsid, a proxy for infectivity, through a CRISPR–Cas13a-based detection method together with an RNase-capsid integrity assay. We termed this assay: Foodborne RNA-virus Enzymatic Sensing for High-throughput on fresh produce (CRISPR FRESH) reflecting its potential to detect infectious or potentially infectious virus particles. Our CRISPR FRESH method detected murine norovirus (MNV-1), with an intact capsid, at a limit of detection of 2.59 log10 gc/25 g (5 gc/rx). This method did not cross-react with other targets (synthetic DNA targets for hepatitis A virus; human norovirus GI, GII; rotavirus). Compared with RT-qPCR, CRISPR FRESH showed an increased sensitivity when detecting low copy numbers of RNase-pre-treated MNV-1 in lettuce and blueberries samples. Viral detection with the RT-qPCR assay is quantifiable while the CRISPR assay is present/absent. This report describes a CRISPR-based detection of potentially infectious viruses in food samples.

Current standard food detection methods do not distinguish between infectious and non-infectious norovirus leading to uncertainty in the interpretation and management of a norovirus positive food sample. These methods also require expensive RT-qPCR based equipment. In contrast, CRISPR-based, compared to RT-qPCR based, detection methods yield similar sensitivity and specificity and are generally less expensive. The aim of this study was to detect norovirus with an intact capsid, a proxy for infectivity, through a CRISPR-Cas13a based detection method in conjunction with acapsid integrity assay. Our CRISPR method detected murine norovirus (MNV-1), with an intact capsid, at a limit of detection of 2.59 log10 gc/ 25 g (5 gc/ rx). This method did not cross-react with other targets (synthetic hepatitis A virus; human norovirus GI, GII; rotavirus). Compared with RT-qPCR, this CRISPR based method showed an increased sensitivity when detecting low copy numbers of RNase-pretreated MNV-1 in lettuce and blueberries samples. This is the first report describing a CRISPR-based detection of potentially infectious viruses in food samples.

Norovirus is the primary cause of acute gastroenteritis outbreaks, considerably impacting children under 5 years, followed by older adults and immunocompromised individuals. As an RNA virus, norovirus exhibits high genetic variability, driven by recombination events at the ORF1–ORF2 junction. This study reports the first detection of the rare norovirus GI.5 [P4] variant in Northeastern Mexico, identified in a single positive isolate (MTY0115; GenBank: PQ369661) from a sample group of 386 individuals, with a prevalence of 0.25%. Notably, norovirus GII was not detected. Phylogenetic analysis of the partial RdRp/VP1 region revealed clustering with global GI.5 [P4] sequences, revealing evolutionary relationships with isolates from Asia, Europe, and America. A recombination event was identified at position 5307 (breakpoint based on reference sequences of GI.5 [P5] and GI.4 [P4]) within ORF1, with genetic inheritance from a GI.5 [P5] isolate from Moscow, Russia, and a GI.4 [P4] isolate from France. Typing classification through sequencing of overlapping ORF1 and ORF2 regions is valuable for understanding genomic variations and their epidemiological impact on at-risk and non-risk populations.

The presence of antibiotic-resistant bacteria in the agro-environment is a growing international concern. In Mexico, few studies thus far have assessed the quality of water irrigation through the isolation of bacteria pathogens and the identification of antibiotic-resistant genes. Irrigation water samples obtained from rivers, wells, and ponds in different regions of Mexico were analyzed for the presence and antibiotic resistance of pathotypes of Escherichia coli, Salmonella spp., and Enterococcus spp. using selective agars and polymerase chain reaction. Two Enterococcus faecalis, one typical enteropathogenic (EPEC), and one atypical EPEC pathotypes were isolated from river water samples; two atypical EPEC and one enterotoxigenic pathotypes were isolated from pond water samples. There were no targeted pathogens detected in well water samples. All isolates were resistant to at least three antibiotics, with vancomycin being the most common; however, all isolates were sensitive to trimethoprim and ciprofloxacin. The presence of pathotypes of EPEC and ETEC and E. faecalis in water samples used for crop irrigation are a potential risk to public health because of their pathogenicity and antibiotic resistance.