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Non-typhoidal Salmonella enterica is a leading cause of gastrointestinal illnesses in the United States. Among the 2,600 different S . enterica serovars, Infantis has been significantly linked to human illnesses and is frequently recovered from broilers and chicken parts in the U.S. A key virulence determinant in serovar Infantis is the presence of the megaplasmid pESI, conferring multidrug resistance. To further characterize the virulence potential of this serovar, the present study identified the types of plasmids harbored by Infantis strains, recovered from surface waters adjacent to leafy greens farms in California. Sequencing analysis showed that each of the examined 12 Infantis strains had a large plasmid ranging in size from 78 kb to 125 kb. In addition, a second 4-kb plasmid was detected in two strains. Plasmid nucleotide queries did not identify the emerging megaplasmid pESI in the examined Infantis strains; however, the detected plasmids each had similarity to a plasmid sequence already cataloged in the nucleotide databases. Subsequent comparative analyses, based on gene presence or absence, divided the detected plasmids into five distinct clusters, and the phylogram revealed these Infantis plasmids were clustered based either on the plasmid conjugation system, IncI and IncF, or on the presence of plasmid phage genes. Assignment of the putative genes to functional categories revealed that the large plasmids contained genes implicated in cell cycle control and division, replication and recombination and defense mechanisms. Further analysis of the mobilome, including prophages and transposons, demonstrated the presence of genes implicated in the release of the bactericidal peptide microcin in the IncF plasmids and identified a Tn10 transposon conferring tetracycline resistance in one of the IncI1 plasmids. These findings indicated that the plasmids in the environmental S . enterica serovar Infantis strains from surface waters harbored a wide variety of genes associated with adaptation, survivability and antimicrobial resistance.

The rapid spreading of polymyxin E (colistin) resistance among bacterial strains through the horizontally transmissible mcr-1 and mcr-2 plasmids has become a serious concern. The emergence of these genes in Shiga toxin-producing Escherichia coli (STEC), a group of human pathogenic bacteria was even more worrisome, urging us to investigate the prevalence of mcr genes among STEC isolates. A total of 1000 STEC isolates, recovered from livestock, wildlife, produce and other environmental sources in a major production region for leafy vegetables in California during 2006-2014, were screened by PCR for the presence of plasmid-borne mcr-1 and mcr-2. All isolates tested yielded negative results, indicating if any, the occurrence rate of mcr-1/mcr-2 among STEC was very low in this agricultural region. This study provides valuable information such as sample size needed and methodologies for future surveillance programs of antimicrobial resistance.

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.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic enteric pathogens linked to human gastroenteritis worldwide. To aid the development of pathogen control efforts, the present study characterized the genotypic diversity and pathogenic potential of STEC recovered from sources near agricultural fields in Northwest Mexico. Samples were collected from irrigation river water and domestic animal feces in farms proximal to agricultural fields and were subjected to enrichment followed by immunomagnetic separation and plating on selective media for the recovery of the STEC isolates. Comparative genomic analyses indicated that the recovered STEC with the clinically relevant serotypes O157:H7, O8:H19, and O113:H21 had virulence genes repertoires associated with host cell adherence, iron uptake and effector protein secretion. Subsequent phenotypic characterization revealed multidrug resistance against aminoglycoside, carbapenem, cephalosporin, fluoroquinolone, penicillin, phenicol, and tetracycline, highlighting the need for improved surveillance on the use of antimicrobials. The present study indicated for the first time that river water in the agricultural Culiacan Valley in Mexico is a relevant key route of transmission for STEC O157 and non-O157 with a virulence potential. In addition, feces from domestic farm animals near surface waterways can act as potential point sources of contamination and transport of diverse STEC with clinically relevant genotypes.

Background Antimicrobial resistance in Shiga toxin-producing Escherichia coli (STEC) O157 and non-O157 is a matter of increasing concern. The aim of the present study was to investigate the antimicrobial resistance profiles of STEC O157 and non-O157 recovered from feces of domestic farm animals in the agricultural Culiacan Valley in Northwestern Mexico. Findings All of the examined STEC strains showed susceptibility to five antimicrobials, ceftazidime, ceftriaxone, ciprofloxacin, nalidixic acid, and trimethoprim-sulfamethoxazole. However, resistance to the four antimicrobials, ampicillin, cephalothin, chloramphenicol, and kanamycin was commonly observed. Interestingly, non-susceptibility to cephalothin was predominant among the examined STEC strains, corresponding to 85 % (22/26) of the O157:H7 from cattle, sheep and chicken and 73 % (24/33) of the non-O157 strains from cattle and sheep. Statistical analyses revealed that resistance to ampicillin was significantly correlated to 38 % (10/26) of STEC O157:H7 strains from multiple animal sources. Another significant correlation was found between serotype, source, and antimicrobial resistance; all of the O20:H4 strains, recovered from sheep, were highly resistant to tetracycline. Multidrug resistance profiles were identified in 42 % (22/53) of the non-susceptible STEC strains with clinically-relevant serotypes O8:H9, O75:H8, O146:H21, and O157:H7. Conclusions STEC O157 and non-O157 strains, recovered from domestic farm animals in the Culiacan Valley, exhibited resistance to classes of antimicrobials commonly used in Mexico, such as aminoglycosides, tetracyclines, cephalosporins and penicillin but were susceptible to fluoroquinolones, quinolones, and sulfonamides. These findings provide fundamental information that would aid in the surveillance of antimicrobial resistance in an important agricultural region in Northwestern Mexico.

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.

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.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic enteric pathogen that causes human gastrointestinal illnesses. The present study characterized the virulence profiles of O157 and non-O157 STEC strains, recovered from domestic animals in small rural farms within the agricultural Culiacan Valley in Mexico. Virulence genes coding for adhesins, cytotoxins, proteases, subtypes of Shiga toxin (Stx), and other effectors were identified in the STEC strains by PCR. The genotyping analysis revealed the presence of the effectors nleA, nleB, nleE, and nleH1-2, espK, and espN in the O157:H7 and O111:H8 STEC strains. Furthermore, the genes encoding the autoagglutinating adhesin (Saa) and subtilase (SubA) were exclusively identified in the O8:H19 eae-negative strains. The adhesin (iha) and the silent hemolysin (sheA) genes were detected in 79% of the O157 and non-O157 strains. To examine the relative toxicities of the STEC strains, a fluorescent Vero cell line, Vero-d2EGFPs, was employed to measure the inhibition of protein synthesis by Stx. Analysis of culture supernatants from serotype O8:H19 strains with the stx gene profile stx 1a, stx 2a, and stx 2c and serotypes O75:H8 and O146:H8 strains with the stx gene profile stx 1a, stx 1c, and stx 2b, resulted in a significant reduction in the Vero-d2EGFP fluorescent signal. These observations suggest that these non-O157 strains may have an enhanced ability to inhibit protein synthesis in Vero cells. Interestingly, analysis of the stx 2c-positive O157:H7 strains resulted in a high fluorescent signal, indicating a reduced toxicity in the Vero-d2EGFP cells. These findings indicate that the O157 and non-O157 STEC strains, recovered in the Culiacan Valley, display distinct virulence profiles and relative toxicities in mammalian cells and have provided information for evaluating risks associated with zoonotic STEC in this agricultural region in Mexico.

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.

Aflatoxins are toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus and cause toxin contamination in food chain worldwide. Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu. Koji mold species are generally perceived of as being nontoxigenic and are generally recognized as safe (GRAS). Fungal isolates were collected from a California orchard and a few were initially identified to be A. sojae using β-tubulin gene sequences blasted against NCBI data base. These new isolates all produced aflatoxins B1, B2, G1, and G2 and were named as Pistachio Winter Experiment (PWE) strains. Thus, it is very important to further characterize these strains for food safety purposes. The full length of aflR gene of these new isolates was sequenced. Comparison of aflR DNA sequences of PWE, A. parasiticus and A. sojae, showed that the aflatoxigenic PWE strains had the six base insertion (CTCATG) similar to domesticated A. sojae, but a pre-termination codon TGA at nucleotide positions 1153–1155 was absent due to a nucleotide codon change from T to C. Colony morphology and scanning microscopic imaging of spore surfaces showed similarity of PWE strains to both A. parasiticus and A. sojae. Concordance analysis of multi locus DNA sequences indicated that PWE strains were closely linked between A. parasiticus and A. sojae. The finding documented the first report that such unique strains have been found in North America and in the world.