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BACKGROUND: Pseudomonas aeruginosa is an opportunistic bacterium that causes serious hospital-acquired infections. To assess the risk of clinically isolated P. aeruginosa to human health, we analyzed the resistance and virulence mechanisms of a collection of clinical isolates. METHODS: This was a retrospective study in which P. aeruginosa isolates collected from January 1, 2018 to August 31, 2019 were analyzed using phenotypic and whole-genome sequencing (WGS) methods. The analysis included 48 clinical samples. Median patient age was 54.0 (29.5) years, and 58.3% of patients were women. Data from the microbiology laboratory database were reviewed to identify P. aeruginosa isolates. All unique isolates available for further testing were included, and related clinical data were collected. Infections were defined as hospital acquired if the index culture was obtained at least 48 h after hospitalization. RESULTS: High-risk P. aeruginosa clones, including sequence types (STs) ST235 and ST111, were identified, in addition to 12 new STs. The isolates showed varying degrees of biofilm formation ability when evaluated at room temperature, along with reduced metabolic activity, as measured by metabolic staining, suggesting their ability to evade antimicrobial therapy. Most isolates (77.1%) were multidrug resistant (MDR), with the highest resistance and susceptibility rates to beta-lactams and colistimethate sodium, respectively. CONCLUSIONS: The MDR phenotypes of the examined isolates can be explained by the high prevalence of efflux-mediated resistance- and hydrolytic enzyme-encoding genes. These isolates had high cytotoxic potential, as indicated by the detection of toxin production-related genes.

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This study describes the hybrid genome assemblies of four Shiga toxin-producing Escherichia coli strains isolated from the recto-anal junction of slaughter-age Irish sheep. In silico serotyping and genome analysis determined that each of the strains harbored a Shiga-toxin subtype, a complete locus of enterocyte effacement, and a rare O-island 122.

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The diagnostic protocol currently used globally to identify Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is RT-qPCR. The spread of these infections and the epidemiological imperative to describe variation across the virus genome have highlighted the importance of sequencing. SARS-CoV-2 rapid antigen diagnostic tests (RADTs) are designed to detect viral nucleocapsid protein with positive results suggestive of the presence of replicating virus and potential infectivity. In this study, we developed a protocol for recovering SARS-CoV-2 RNA from "spent" RADT devices of sufficient quality that can be used directly for whole virus genome sequencing. The experimental protocol included the spiking of RADTs at different concentrations with viable SARS-CoV-2 variant Alpha (lineage B.1.1.7), lysis for direct use or storage. The lysed suspensions were used for RNA extraction and RT-qPCR. In parallel, we also tested the stability of the viral RNA in the RADTs and the RNA extracted from the RADTs was used as a template for tiling-PCR and whole virus genome sequencing. RNA recovered from RADTs spiked with SARS-CoV-2 was detected through RT-qPCR with Ct values suitable for sequencing and the recovery from RADTs was confirmed after 7 days of storage at both 4 and 20°C. The genomic sequences obtained at each time-point aligned to the strain used for the spiking, demonstrating that sufficient SARS-CoV-2 viral genome can be readily recovered from positive-RADT devices in which the virus has been safely inactivated and genomically conserved. This protocol was applied to obtain whole virus genome sequence from RADTs ran in the field where the omicron variant was detected. The study demonstrated that viral particles of SARS-CoV-2 suitable for whole virus genome sequencing can be recovered from positive spent RADTs, extending their diagnostic utility, as a risk management tool and for epidemiology studies. In large deployment of the RADTs, positive devices could be safely stored and used as a template for sequencing allowing the rapid identification of circulating variants and to trace the source and spread of outbreaks within communities and guaranteeing public health.

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Shiga toxin-producing Escherichia coli (STEC) organisms are a diverse group of pathogenic bacteria capable of causing serious human illness, and serogroups O157 and O26 are frequently implicated in human disease. Ruminant hosts are the primary STEC reservoir, and small ruminants are important contributors to STEC transmission. This study investigated the prevalence, serotypes, and shedding dynamics of STEC, including the supershedding of serogroups O157 and O26, in Irish sheep. Recto-anal mucosal swab samples (n = 840) were collected over 24 months from two ovine slaughtering facilities. Samples were plated on selective agars and were quantitatively and qualitatively assessed via real-time PCR (RT-PCR) for Shiga toxin prevalence and serogroup. A subset of STEC isolates (n = 199) were selected for whole-genome sequencing and analyzed in silico. In total, 704/840 (83.8%) swab samples were Shiga toxin positive following RT-PCR screening, and 363/704 (51.6%) animals were subsequently culture positive for STEC. Five animals were shedding STEC O157, and three of these were identified as supershedders. No STEC O26 was isolated. Post hoc statistical analysis showed that younger animals are more likely to harbor STEC and that STEC carriage is most prevalent during the summer months. Following sequencing, 178/199 genomes were confirmed as STEC. Thirty-five different serotypes were identified, 15 of which were not yet reported for sheep. Serotype O91:H14 was the most frequently reported. Eight Shiga toxin gene variants were reported, two stx1 and six stx2, and three novel Shiga-toxin subunit combinations were observed. Variant stx1c was the most prevalent, while many strains also harbored stx2b. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) bacteria are foodborne, zoonotic pathogens of significant public health concern. All STEC organisms harbor stx, a critical virulence determinant, but it is not expressed in most serotypes. Sheep shed the pathogen via fecal excretion and are increasingly recognized as important contributors to the dissemination of STEC. In this study, we have found that there is high prevalence of STEC circulating within sheep and that prevalence is related to animal age and seasonality. Further, sheep harbor a variety of non-O157 STEC, whose prevalence and contribution to human disease have been underinvestigated for many years. A variety of Stx variants were also observed, some of which are of high clinical importance.

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Shiga-toxin producing Escherichia coli (STEC) are zoonotic foodborne pathogens that are capable of causing serious human illness. Ovine ruminants are recognized as an important source of STEC and a notable contributor to contamination within the food industry. This review examined the prevalence of STEC in the ovine food production chain from farm-to-fork, reporting carriage in sheep herds, during abattoir processing, and in raw and ready-to-eat meats and meat products. Factors affecting the prevalence of STEC, including seasonality and animal age, were also examined. A relative prevalence can be obtained by calculating the mean prevalence observed over multiple surveys, weighted by sample number. A relative mean prevalence was obtained for STEC O157 and all STEC serogroups at multiple points along the ovine production chain by using suitable published surveys. A relative mean prevalence (and range) for STEC O157 was calculated: for feces 4.4% (0.2-28.1%), fleece 7.6% (0.8-12.8%), carcass 2.1% (0.2-9.8%), and raw ovine meat 1.9% (0.2-6.3%). For all STEC independent of serotype, a relative mean prevalence was calculated: for feces 33.3% (0.9-90.0%), carcass 58.7% (2.0-81.6%), and raw ovine meat 15.4% (2.7-35.5%). The prevalence of STEC in ovine fleece was reported in only one earlier survey, which recorded a prevalence of 86.2%. Animal age was reported to affect shedding in many surveys, with younger animals typically reported as having a higher prevalence of the pathogen. The prevalence of STEC decreases significantly along the ovine production chain after the application of postharvest interventions. Ovine products pose a small risk of potential STEC contamination to the food supply chain.

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Molecular methods may reveal the presence of pathogens in samples through the detection of specific target gene(s) associated with microorganisms, but often, the subsequent cultural isolation of the pathogen is not possible. This discrepancy may be related to low concentration of the cells, presence of dead cells, competitive microflora, injured cells and cells in a viable but non-culturable state, free DNA and the presence of free bacteriophages which can carry the target gene causing the PCR-positive/culture-negative results. Shiga-toxigenic Escherichia coli (STEC) was used as a model for studying this phenomenon, based on the phage-encoded cytotoxins genes (Stx family) as the detection target in samples through real-time qPCR. Stx phages can be integrated in the STEC chromosome or can be isolated as free particles in the environment. In this study, a combination of PCR with culturing was used for investigating the presence of the stx1 and stx2 genes in 155 ovine recto-anal junction swab samples (method (a)-PCR). Samples which were PCR-positive and culture-negative were subjected to additional analyses including detection of dead STEC cells (method (b)-PCR-PMA dye assay), presence of Stx phages (method (c)-plaque assays) and inducible integrated phages (method (d)-phage induction). Method (a) showed that even though 121 samples gave a PCR-positive result (78%), only 68 samples yielded a culturable isolate (43.9%). Among the 53 (34.2%) PCR-positive/culture-negative samples, 21 (39.6%) samples were shown to have STEC dead cells only, eight (15.1%) had a combination of dead cells and inducible stx phage, while two samples (3.8%) had a combination of dead cells, inducible phage and free stx phage, and a further two samples had Stx1 free phages only (3.8%). It was thus possible to reduce the samples with no explanation to 20 (37.7% of 53 samples), representing a further step towards an improved understanding of the STEC PCR-positive/culture-negative phenomenon.

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A quantitative PCR method is described for the detection and quantification of E. coli O157 and O26 in sheep recto-anal junction swabs. The method incorporated a short enrichment step (5 h) and the use of a developed standard calibration curve relating the real time PCR cycle threshold (Ct) values to the initial concentration of pathogen in the sheep sample.

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