Overview of methodologies for the culturing, recovery and detection of Campylobacter.

Campylobacter species are responsible for human gastroenteritis with diverse clinical spectra, ranging from acute watery or bloody diarrhea to life-threatening autoimmune disorders. Given the importance of Campylobacter in causing human illness, this article has reviewed the transmission and attribution sources as well as methodologies for the detection and virulence characterization of campylobacteria. The recovery and detection of Campylobacter from clinical, food and environmental samples has been achieved by the combinatorial use of selective enrichment and culturing methods. Biochemical, immunological, and nucleic acid-based methodologies have enabled the detection and differentiation of closely related Campylobacter isolates in foodborne outbreak investigations and have assessed the diversity and phylogenetic relationships of these bacterial pathogens. Analyses of motility, adherence, and invasiveness in host cells have assessed the pathogenic potential of campylobacteria. Further examination of determinants conferring antimicrobial resistance in Campylobacter have supported the growing need to closely monitor antimicrobials use in clinical and agricultural sectors.

Genomic analysis of high copy-number sequences for the targeted detection of Listeria species using a flow-through surveillance system.

The bacterial foodborne pathogen Listeria monocytogenes has been implicated in fresh produce outbreaks with a significant economic impact. Given that L. monocytogenes is widespread in the environment, food production facilities constantly monitor for the presence of Listeria species. To develop a surveillance platform for food processing facilities, this study conducted a comparative genomic analysis for the identification of conserved high copy sequences in the ribosomal RNA of Listeria species. Simulated folding was performed to assess RNA accessibility in the identified genomic regions targeted for detection, and the developed singleplex assay accurately detected cell amounts lower than 5 cells, while no signals were detected for non-targeted bacteria. The singleplex assay was subsequently tested with a flow-through system, consisting of a DNA aptamer-capture step, followed by sample concentration and mechanical lysis for the detection of Listeria species. Validation experiments indicated the continuous flow-through system accurately detected Listeria species at low cell concentrations.

Bimodal Response to Shiga Toxin 2 Subtypes Results from Relatively Weak Binding to the Target Cell.

There are two major antigenic forms of Shiga toxin (Stx), Stx1 and Stx2, which bind the same receptor and act on the same target but nonetheless differ in potency. Stx1a is more toxic to cultured cells, but Stx2 subtypes are more potent in animal models. To understand this phenomenon in cultured cells, we used a system that combines flow cytometry with a fluorescent reporter to monitor the Stx-induced inhibition of protein synthesis in single cells. We observed that Vero cells intoxicated with Stx1a behave differently than those intoxicated with Stx2 subtypes: cells challenged with Stx1a exhibited a population-wide loss of protein synthesis, while cells exposed to Stx2a or Stx2c exhibited a dose-dependent bimodal response in which one subpopulation of cells was unaffected (i.e., no loss of protein synthesis). Cells challenged with a hybrid toxin containing the catalytic subunit of Stx1a and the cell-binding subunit of Stx2a also exhibited a bimodal response to intoxication, while cells challenged with a hybrid toxin containing the catalytic subunit of Stx2a and the cell-binding subunit of Stx1a exhibited a population-wide loss of protein synthesis. Other experiments further supported a primary role for the subtype of the B subunit in the outcome of host-Stx interactions. Our collective observations indicate that the bimodal response to Stx2 subtypes is due to relatively weak binding between Stx2 and the host cell that reduces the total functional pool of Stx2 in comparison to that of Stx1a. This explains, in part, the molecular basis for the differential cellular toxicity between Stx1a and Stx2 subtypes.

Sensitive Genotyping of Foodborne-Associated Human Noroviruses and Hepatitis A Virus Using an Array-Based Platform.

Human noroviruses (NoV) are the leading cause of human gastroenteritis in populations of all ages and are linked to most of the foodborne outbreaks worldwide. Hepatitis A virus (HAV) is another important foodborne enteric virus and is considered the most common agent causing acute liver disease worldwide. In the present study, a focused, low-density DNA microarray was developed and validated for the simultaneous identification of foodborne-associated genotypes of NoV and HAV. By employing a novel algorithm, capture probes were designed to target variable genomic regions commonly used for typing these foodborne viruses. Validation results showed that probe signals, specific for the tested NoV or HAV genotypes, were on average 200-times or 38-times higher than those detected for non-targeted genotypes, respectively. To improve the analytical sensitivity of this method, a 12-mer oligonucleotide spacer sequence was added to the capture probes and resulted in a detection threshold of less than 10 cRNA transcripts. These findings have indicated that this array-based typing sensor has the accuracy and sensitivity for identifying NoV and HAV genotypic profiles predominantly linked to food poisoning. The implementation of this typing sensor would thus provide highly relevant and valuable information for use in surveillance and outbreak attribution.

An Environmental Shiga Toxin-Producing Escherichia coli O145 Clonal Population Exhibits High-Level Phenotypic Variation That Includes Virulence Traits.

Shiga toxin-producing Escherichia coli (STEC) serotype O145 is one of the major non-O157 serotypes associated with severe human disease. Here we examined the genetic diversity, population structure, virulence potential, and antimicrobial resistance profiles of environmental O145 strains recovered from a major produce production region in California. Multilocus sequence typing analyses revealed that sequence type 78 (ST-78), a common ST in clinical strains, was the predominant genotype among the environmental strains. Similarly, all California environmental strains belonged to H28, a common H serotype in clinical strains. Although most environmental strains carried an intact fliC gene, only one strain retained swimming motility. Diverse stx subtypes were identified, including stx1a, stx2a, stx2c, and stx2e. Although no correlation was detected between the stx genotype and Stx1 production, high Stx2 production was detected mainly in strains carrying stx2a only and was correlated positively with the cytotoxicity of Shiga toxin. All environmental strains were capable of producing enterohemolysin, whereas only 10 strains were positive for anaerobic hemolytic activity. Multidrug resistance appeared to be common, as nearly half of the tested O145 strains displayed resistance to at least two different classes of antibiotics. The core virulence determinants of enterohemorrhagic E. coli were conserved in the environmental STEC O145 strains; however, there was large variation in the expression of virulence traits among the strains that were highly related genotypically, implying a trend of clonal divergence. Several cattle isolates exhibited key virulence traits comparable to those of the STEC O145 outbreak strains, emphasizing the emergence of hypervirulent strains in agricultural environments.

Serum Shiga toxin 2 values in patients during acute phase of diarrhoea-associated haemolytic uraemic syndrome.

AIM: Shiga toxins are delivered via systemic circulation and are considered to be the cause of diarrhoea-associated haemolytic uraemic syndrome (HUS), as they injure endothelial cells, particularly in the glomeruli. This study measured Shiga toxin 2 (Stx2) in the serum of children affected in by HUS due to Stx2 producing Escherichia coli. METHODS: The concentration of free Stx2 was measured in the serum of 16 children, collected immediately after admission to the clinic in the acute phase of HUS, using a sandwich enzyme-linked immunosorbent assay. The family members of two children were also investigated, with the relative toxicity of Stx2 assessed by a Vero cell-based fluorescent assay. RESULTS: Stx2 was found in the serum of eight of the 16 children who were investigated. It was also detected in four of the six family members not showing symptomatic HUS, with an extremely high level in two. CONCLUSION: An absent or rather low concentration of Stx2 was found in the serum of children admitted to the clinic with diarrhoea-associated HUS. The high concentration of Stx2 in family members without HUS, but mostly with watery diarrhoea and raised functional activity, was in line with the concept of early injury by Stx2.

Top-down proteomic identification of Shiga toxin 2 subtypes from Shiga toxin-producing Escherichia coli by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry.

We have analyzed 26 Shiga toxin-producing Escherichia coli (STEC) strains for Shiga toxin 2 (Stx2) production using matrix-assisted laser desorption ionization (MALDI)-tandem time of flight (TOF-TOF) tandem mass spectrometry (MS/MS) and top-down proteomic analysis. STEC strains were induced to overexpress Stx2 by overnight culturing on solid agar supplemented with either ciprofloxacin or mitomycin C. Harvested cells were lysed by bead beating, and unfractionated bacterial cell lysates were ionized by MALDI. The A2 fragment of the A subunit and the mature B subunit of Stx2 were analyzed by MS/MS. Sequence-specific fragment ions were used to identify amino acid subtypes of Stx2 using top-down proteomic analysis using software developed in-house at the U.S. Department of Agriculture (USDA). Stx2 subtypes (a, c, d, f, and g) were identified on the basis of the mass of the A2 fragment and the B subunit as well as from their sequence-specific fragment ions by MS/MS (postsource decay). Top-down proteomic identification was in agreement with DNA sequencing of the full Stx2 operon (stx2) for all strains. Top-down results were also compared to a bioassay using a Vero-d2EGFP cell line. Our results suggest that top-down proteomic identification is a rapid, highly specific technique for distinguishing Stx2 subtypes.

Antimicrobial Activity of Green Synthesized Silver and Copper Oxide Nanoparticles against the Foodborne Pathogen Campylobacter jejuni.

Campylobacter jejuni is a major cause of global foodborne illnesses. To develop alternative antimicrobial strategies against C. jejuni, this study designed and optimized the green synthesis of metallic nanoparticles (NPs) with intracellular components of the medicinal fungus Ganoderma sessile to provide the needed reducing and stabilizing agents. NPs were characterized by transmission electron microscopy and dynamic light scattering, and the quasi-spherical NPs had sizes of 2.9 ± 0.9 nm for the copper oxide NPs and 14.7 ± 0.6 nm for the silver NPs. Surface charge assessment revealed zeta potentials of -21.0 ± 6.5 mV and -24.4 ± 7.9 mV for the copper oxide and silver NPs, respectively. The growth inhibition of C. jejuni by the NPs occurred through attachment to the outer cell membrane and subsequent intracellular internalization and resulted in minimum inhibitory concentrations of the silver NPs at 6 µg/mL and copper oxide NPs at 10 µg/mL. On the other hand, a differential ROS production caused by silver and copper NPs was observed. In summary, this research presents the first demonstration of using green synthesis with the medicinal fungus G. sessile to produce metallic NPs that effectively inhibit C. jejuni growth, providing a sustainable and effective approach to the traditional use of antimicrobials.

Development and Implementation of an eHealth Oncohematonootric Program: Descriptive, Observational, Prospective Cohort Pilot Study.

BACKGROUND: In oncohematology, both the development of the disease and the side effects of antineoplastic treatment often take a toll on patients' physical and nutritional well-being. In this era of digital transformation, we launched a pioneering project for oncohematologic patients to promote adherence to a healthy lifestyle and improve their physical and nutritional well-being. We aim to achieve this goal by involving doctors and nutritionists through the Nootric app. OBJECTIVE: This study aims to assess the impact of the use of eHealth tools to facilitate nutrition and well-being in oncohematologic patients. We also aim to determine the usefulness of physical-nutritional management in improving tolerance to chemotherapy treatments within routine clinical practice. METHODS: We designed a descriptive, observational, longitudinal, prospective cohort pilot study that included a total of 22 patients from March to May 2022 in the Vinalopó University Hospital. The inclusion criteria were adults over 18 years of age diagnosed with oncohematological pathology in active chemotherapy treatment. An action plan was created to generate alerts between the doctor and the nutritionist. In the beginning, the patients were trained to use the app and received education highlighting the importance of nutrition and physical exercise. Sociodemographic, clinical-biological-analytical (eg, malnutrition index), health care impact, usability, and patient adherence data were collected. Tolerance to chemotherapy treatment and its health care impact were evaluated. RESULTS: We included 22 patients, 11 (50%) female and 11 (50%) male, ranging between 42 and 84 years of age. Among them, 13 (59%) were adherents to the program. The most frequent diseases were lymphoproliferative syndromes (13/22, 59%) and multiple myeloma (4/22, 18%). Moreover, 15 (68%) out of 22 patients received immunochemotherapy, while 7 (32%) out of 22 patients received biological treatment. No worsening of clinical-biological parameters was observed. Excluding dropouts and abandonments (n=9/22, 41%), the adherence rate was 81%, established by calculating the arithmetic mean of the adherence rates of 13 patients. No admission was observed due to gastrointestinal toxicity or discontinuation of treatment related to alterations in physical and nutritional well-being. In addition, only 5.5% of unscheduled consultations were increased due to incidents in well-being, mostly telematic (n=6/103 consultation are unscheduled). Additionally, 92% of patients reported an improvement in their nutritional habits (n=12/13), and up to 45% required adjustment of medical supportive treatment (n=5/11). There were no cases of grade 3 or greater gastrointestinal toxicity. All of this reflects improved tolerance to treatments. Patients reported a satisfaction score of 4.3 out of 5, while professionals rated their satisfaction at 4.8 out of 5. CONCLUSIONS: We demonstrated the usefulness of integrating new technologies through a multidisciplinary approach. The Nootric app facilitated collaboration among the medical team, nutritionists, and patients. It enabled us to detect health issues related to physical-nutritional well-being, anticipate major complications, and mitigate potentially avoidable risks. Consequently, there was a decrease in unscheduled visits and admissions related to this condition.

Pathogenicity assessment of Shiga toxin-producing Escherichia coli strains isolated from wild birds in a major agricultural region in California.

Shiga toxin-producing Escherichia coli (STEC) consists of diverse strains differing in genetic make-up and virulence potential. To better understand the pathogenicity potential of STEC carried by the wildlife, three STEC and one E. coli strains isolated from wild birds near a major agricultural region in California were selected for comparative pathogenomic analyses. Three American crow (Corvus brachyrhynchos) strains, RM9088, RM9513, and RM10410, belonging to phylogroup A with serotypes O109:H48, O9:H30, and O113:H4, respectively, and a red-winged blackbird (Agelaius phoeniceus) strain RM14516 in phylogroup D with serotype O17:H18, were examined. Shiga toxin genes were identified in RM9088 (stx1a), RM10410 (stx1a + stx2d), and RM14516 (stx2a). Unlike STEC O157:H7 strain EDL933, none of the avian STEC strains harbored the pathogenicity islands OI-122, OI-57, and the locus of enterocyte effacement, therefore the type III secretion system biogenesis genes and related effector genes were absent in the three avian STEC genomes. Interestingly, all avian STEC strains exhibited greater (RM9088 and RM14516) or comparable (RM10410) cytotoxicity levels compared with EDL933. Comparative pathogenomic analyses revealed that RM9088 harbored numerous genes encoding toxins, toxins delivery systems, and adherence factors, including heat-labile enterotoxin, serine protease autotransporter toxin Pic, type VI secretion systems, protein adhesin Paa, fimbrial adhesin K88, and colonization factor antigen I. RM9088 also harbored a 36-Kb high pathogenicity island, which is related to iron acquisition and pathogenicity in Yersinia spp. Strain RM14516 carried an acid fitness island like the one in EDL933, containing a nine gene cluster involved in iron acquisition. Genes encoding extracellular serine protease EspP, subtilase cytotoxin, F1C fimbriae, and inverse autotransporter adhesin IatC were only detected in RM14516, and genes encoding serine protease autotransporter EspI and P fimbriae were only identified in RM10410. Although all curli genes were present in avian STEC strains, production of curli fimbriae was only detected for RM9088 and RM14516. Consistently, strong, moderate, and little biofilms were observed for RM9088, RM14516, and RM10410, respectively. Our study revealed novel combinations of virulence factors in two avian strains, which exhibited high level of cytotoxicity and strong biofilm formation. Comparative pathogenomics is powerful in assessing pathogenicity and health risk of STEC strains.

Comparative Genomic Analyses of Virulence and Antimicrobial Resistance in Citrobacter werkmanii, an Emerging Opportunistic Pathogen.

Citrobacter werkmanii is an emerging and opportunistic human pathogen found in developing countries and is a causative agent of wound, urinary tract, and blood infections. The present study conducted comparative genomic analyses of a C. werkmanii strain collection from diverse geographical locations and sources to identify the relevant virulence and antimicrobial resistance genes. Pangenome analyses divided the examined C. werkmanii strains into five distinct clades; the subsequent classification identified genes with functional roles in carbohydrate and general metabolism for the core genome and genes with a role in secretion, adherence, and the mobilome for the shell and cloud genomes. A maximum-likelihood phylogenetic tree with a heatmap, showing the virulence and antimicrobial genes' presence or absence, demonstrated the presence of genes with functional roles in secretion systems, adherence, enterobactin, and siderophore among the strains belonging to the different clades. C. werkmanii strains in clade V, predominantly from clinical sources, harbored genes implicated in type II and type Vb secretion systems as well as multidrug resistance to aminoglycoside, beta-lactamase, fluoroquinolone, phenicol, trimethoprim, macrolides, sulfonamide, and tetracycline. In summary, these comparative genomic analyses have demonstrated highly pathogenic and multidrug-resistant genetic profiles in C. werkmanii strains, indicating a virulence potential for this commensal and opportunistic human pathogen.

Genomic and Phenotypic Characterization of Shiga Toxin-Producing Escherichia albertii Strains Isolated from Wild Birds in a Major Agricultural Region in California.

Escherichia albertii is an emerging foodborne pathogen. To better understand the pathogenesis and health risk of this pathogen, comparative genomics and phenotypic characterization were applied to assess the pathogenicity potential of E. albertii strains isolated from wild birds in a major agricultural region in California. Shiga toxin genes stx2f were present in all avian strains. Pangenome analyses of 20 complete genomes revealed a total of 11,249 genes, of which nearly 80% were accessory genes. Both core gene-based phylogenetic and accessory gene-based relatedness analyses consistently grouped the three stx2f-positive clinical strains with the five avian strains carrying ST7971. Among the three Stx2f-converting prophage integration sites identified, ssrA was the most common one. Besides the locus of enterocyte effacement and type three secretion system, the high pathogenicity island, OI-122, and type six secretion systems were identified. Substantial strain variation in virulence gene repertoire, Shiga toxin production, and cytotoxicity were revealed. Six avian strains exhibited significantly higher cytotoxicity than that of stx2f-positive E. coli, and three of them exhibited a comparable level of cytotoxicity with that of enterohemorrhagic E. coli outbreak strains, suggesting that wild birds could serve as a reservoir of E. albertii strains with great potential to cause severe diseases in humans.

Implementing Telemedicine in Clinical Practice in the First Digital Hematology Unit: Feasibility Study.

BACKGROUND: Currently, there are no telemedicine models that fully integrate all areas of hematology into daily practice. OBJECTIVE: The objectives of this feasibility study were to assess the practicality of implementing telemedicine into our clinical practice in the first Digital Hematology Unit and propose an innovative integrative design for clinical practice. METHODS: We designed the Digital Hematology Unit, which is a specific physical space dedicated to carrying out telemedicine and monitoring patients in a holistic way. Also, a satisfaction questionnaire was performed and health care indicators were measured. RESULTS: In 2021, there were 1331 first visits and 7534 follow-up visits. Of the first visits, 12.2% (n=163) were face-to-face and 87.8% (n=1168) were telematic. For follow-up visits, 29.9% (n=2251) were face-to-face and 70.1% (n=5283) were telematic. The health care management indicators showed that we had a waiting time of less than 4 days and took less than 4 hours to answer interconsultations among specialists. Moreover, patients reported a high level of satisfaction with the services provided. CONCLUSIONS: Our Digital Hematology Unit, as a case of success, serves as an example of how innovative digital solutions can contribute to the quality of care and excellence in health care achieved through a digital transformation process led by hematologists.

Escherichia coli serotype O55:H7 diversity supports parallel acquisition of bacteriophage at Shiga toxin phage insertion sites during evolution of the O157:H7 lineage.

Enteropathogenic Escherichia coli (EPEC) continues to be a leading cause of mortality and morbidity in children around the world. Two EPEC genomes have been fully sequenced: those of EPEC O127:H6 strain E2348/69 (United Kingdom, 1969) and EPEC O55:H7 strain CB9615 (Germany, 2003). The O55:H7 serotype is a recent precursor to the virulent enterohemorrhagic E. coli O157:H7. To explore the diversity of O55:H7 and better understand the clonal evolution of O157:H7, we fully sequenced EPEC O55:H7 strain RM12579 (California, 1974), which was collected 1 year before the first U.S. isolate of O157:H7 was identified in California. Phage-related sequences accounted for nearly all differences between the two O55:H7 strains. Additionally, O55:H7 and O157:H7 strains were tested for the presence and insertion sites of Shiga toxin gene (stx)-containing bacteriophages. Analysis of non-phage-associated genes supported core elements of previous O157:H7 stepwise evolutionary models, whereas phage composition and insertion analyses suggested a key refinement. Specifically, the placement and presence of lambda-like bacteriophages (including those containing stx) should not be considered stable evolutionary markers or be required in placing O55:H7 and O157:H7 strains within the stepwise evolutionary models. Additionally, we suggest that a 10.9-kb region (block 172) previously believed unique to O55:H7 strains can be used to identify early O157:H7 strains. Finally, we defined two subsets of O55:H7 strains that share an as-yet-unobserved or extinct common ancestor with O157:H7 strains. Exploration of O55:H7 diversity improved our understanding of the evolution of E. coli O157:H7 and suggested a key revision to accommodate existing and future configurations of stx-containing bacteriophages into current models.

Comparative genomic and phenotypic analyses of the virulence potential in Shiga toxin-producing Escherichia coli O121:H7 and O121:H10.

Shiga toxin-producing Escherichia coli (STEC) O121 is among the top six non-O157 serogroups that are most frequently associated with severe disease in humans. While O121:H19 is predominant, other O121 serotypes have been frequently isolated from environmental samples, but their virulence repertoire is poorly characterized. Here, we sequenced the complete genomes of two animal isolates belonging to O121:H7 and O121:H10 and performed comparative genomic analysis with O121:H19 to assess their virulence potential. Both O121:H7 and O121:H10 strains carry a genome comparable in size with the O121:H19 genomes and belong to phylogroup B1. However, both strains appear to have evolved from a different lineage than the O121:H19 strains according to the core genes-based phylogeny and Multi Locus Sequence Typing. A systematic search of over 300 E. coli virulence genes listed in the Virulence Factor DataBase revealed a total of 73 and 71 in O121:H7 and O121:H10 strains, respectively, in comparison with an average of 135 in the O121:H19 strains. This variation in the virulence genes repertoire was mainly attributed to the reduction in the number of genes related to the Type III Secretion System in the O121:H7 and O121:H10 strains. Compared to the O121:H19 strains, the O121:H7 strain carries more adherence and toxin genes while the O121:H10 strain carries more genes related to the Type VI Secretion System. Although both O121:H7 and O121:H10 strains carry the large virulence plasmid pEHEC, they do not harbor all pEHEC virulence genes in O121:H19. Furthermore, unlike the O121:H19 strains, neither the O121:H7 nor O121:H10 strain carried the Locus of Enterocyte Effacement, OI-122, nor the tellurite resistance island. Although an incomplete Locus of Adhesion and Autoaggregation (LAA) was identified in the O121:H7 and O121:H10 strains, a limited number of virulence genes were present. Consistently, both O121:H7 and O121:H10 strains displayed significant reduced cytotoxicity than either the O157:H7 strain EDL933 or the O121:H19 strain RM8352. In fact, the O121:H7 strain RM8082 appeared to cause minimal cytotoxicity to Vero cells. Our study demonstrated distinct evolutionary lineages among the strains of serotypes O121:H19, O121:H10, and O121:H7 and suggested reduced virulence potentials in STEC strains of O121:H10 and O121:H7.

Phylogenetic group and virulence profile classification in Escherichia coli from distinct isolation sources in Mexico.

Escherichia coli is a leading cause of human enteric diseases worldwide. The rapid and accurate causal agent identification to a particular source represents a crucial step in the establishment of safety and health measures in the affected human populations and would thus provide insights into the relationship of traits that may contribute for pathogen persistence in a particular reservoir. The objective of the present study was to characterize over two hundred E. coli strains from different isolation sources in Mexico by conducting a correspondence analysis to explore associations with the detected phylogenetic groups. The results indicated that E. coli strains, recovered from distinct sources in Mexico, were classified into phylogroups B1 (35.8%), A (27.8%), and D (12.3%) and were clustered to particular clades according to the predicted phylogroups. The results from correspondence analysis showed that E. coli populations from distinct sources in Mexico, belonging to different phylogroups, were not dispersed randomly and were associated with a particular isolation source. Phylogroup A was strongly associated with human sources, and the phylogroup B1 showed a significant relationship with food sources. Additionally, phylogroup D was also related to human sources. Phylogroup B2 was associated with herbivorous and omnivorous mammals. Moreover, common virulence genes in the examined E. coli strains, assigned to all phylogroups, were identified as essential markers for survival and invasion in the host. Although virulence profiles varied among the detected phylogroups, E. coli strains belonging to phylogroup D, associated with humans, were found to contain the largest virulence gene repertoire conferring for persistence and survival in the host. In summary, these findings provide fundamental information for a better characterization of pathogenic E. coli, recovered from distinct isolation sources in Mexico and would assist in the development of better tools for identifying potential transmission routes of contamination.

Prevalence and Genomic Diversity of Salmonella enterica Recovered from River Water in a Major Agricultural Region in Northwestern Mexico.

Salmonella enterica is a leading cause of human gastrointestinal disease worldwide. Given that Salmonella is persistent in aquatic environments, this study examined the prevalence, levels and genotypic diversity of Salmonella isolates recovered from major rivers in an important agricultural region in northwestern Mexico. During a 13-month period, a total of 143 river water samples were collected and subjected to size-exclusion ultrafiltration, followed by enrichment, and selective media for Salmonella isolation and quantitation. The recovered Salmonella isolates were examined by next-generation sequencing for genome characterization. Salmonella prevalence in river water was lower in the winter months (0.65 MPN/100 mL) and significantly higher in the summer months (13.98 MPN/100 mL), and a Poisson regression model indicated a negative effect of pH and salinity and a positive effect of river water temperature (p = 0.00) on Salmonella levels. Molecular subtyping revealed Oranienburg, Anatum and Saintpaul were the most predominant Salmonella serovars. Single nucleotide polymorphism (SNP)-based phylogeny revealed that the detected 27 distinct serovars from river water clustered in two major clades. Multiple nonsynonymous SNPs were detected in stiA, sivH, and ratA, genes required for Salmonella fitness and survival, and these findings identified relevant markers to potentially develop improved methods for characterizing this pathogen.

Sensitive detection of Shiga Toxin 2 and some of its variants in environmental samples by a novel immuno-PCR assay.

Shiga toxin-producing Escherichia coli (STEC) in the environment has been reported frequently. However, robust detection of STEC in environmental samples remains difficult because the numbers of bacteria in samples are often below the detection threshold of the method. We developed a novel and sensitive immuno-PCR (IPCR) assay for the detection of Shiga toxin 2 (Stx2) and Stx2 variants. The assay involves immunocapture of Stx2 at the B subunit and real-time PCR amplification of a DNA marker linked to a detection antibody recognizing the Stx2 A subunit. The qualitative detection limit of the assay is 0.1 pg/ml in phosphate-buffered saline (PBS), with a quantification range of 10 to 100,000 pg/ml. The IPCR method was 10,000-fold more sensitive than an analogue conventional enzyme-linked immunosorbent assay (ELISA) in PBS. Although the sensitivity of the IPCR for detection of Stx2 was affected by environmental sample matrices of feces, feral swine colons, soil, and water from watersheds, application of the IPCR assay to 23 enriched cultures of fecal, feral swine colon, soil, and watershed samples collected from the environment revealed that the IPCR detected Stx2 in all 15 samples that were shown to be STEC positive by real-time PCR and culture methods, demonstrating a 100% sensitivity and specificity. The modification of the sandwich IPCR we have described in this study will be a sensitive and specific screening method for evaluating the occurrence of STEC in the environment.

Identification of Escherichia coli O157 by using a novel colorimetric detection method with DNA microarrays.

Shiga toxin-producing Escherichia coli O157 is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype E. coli O157 strains, the present study evaluated the use of ampliPHOX, a novel colorimetric detection method based on photopolymerization, for pathogen identification with DNA microarrays. A low-density DNA oligonucleotide microarray was designed to target stx1 and stx2 genes encoding Shiga toxin production, the eae gene coding for adherence membrane protein, and the per gene encoding the O157-antigen perosamine synthetase. Results from the validation experiments demonstrated that the use of ampliPHOX allowed the accurate genotyping of the tested E. coli strains, and positive hybridization signals were observed for only probes targeting virulence genes present in the reference strains. Quantification showed that the average signal-to-noise ratio values ranged from 47.73 ± 7.12 to 76.71 ± 8.33, whereas average signal-to-noise ratio values below 2.5 were determined for probes where no polymer was formed due to lack of specific hybridization. Sensitivity tests demonstrated that the sensitivity threshold for E. coli O157 detection was 100-1000 CFU/mL. Thus, the use of DNA microarrays in combination with photopolymerization allowed the rapid and accurate genotyping of E. coli O157 strains.

Comparison of genotypes of Salmonella enterica serovar Enteritidis phage type 30 and 9c strains isolated during three outbreaks associated with raw almonds.

In 2000 to 2001, 2003 to 2004, and 2005 to 2006, three outbreaks of Salmonella enterica serovar Enteritidis were linked with the consumption of raw almonds. The S. Enteritidis strains from these outbreaks had rare phage types (PT), PT30 and PT9c. Clinical and environmental S. Enteritidis strains were subjected to pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem repeat analysis (MLVA), and DNA microarray-based comparative genomic indexing (CGI) to evaluate their genetic relatedness. All three methods differentiated these S. Enteritidis strains in a manner that correlated with PT. The CGI analysis confirmed that the majority of the differences between the S. Enteritidis PT9c and PT30 strains corresponded to bacteriophage-related genes present in the sequenced genomes of S. Enteritidis PT4 and S. enterica serovar Typhimurium LT2. However, PFGE, MLVA, and CGI failed to discriminate between S. Enteritidis PT30 strains related to outbreaks from unrelated clinical strains or between strains separated by up to 5 years. However, metabolic fingerprinting demonstrated that S. Enteritidis PT4, PT8, PT13a, and clinical PT30 strains metabolized L-aspartic acid, L-glutamic acid, L-proline, L-alanine, and D-alanine amino acids more efficiently than S. Enteritidis PT30 strains isolated from orchards. These data indicate that S. Enteritidis PT9c and 30 strains are highly related genetically and that PT30 orchard strains differ from clinical PT30 strains metabolically, possibly due to fitness adaptations.