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.

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.

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.

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.

Draft Genome Sequences of Shiga Toxin-Producing Escherichia coli O157:H7 Strains Recovered from a Major Production Region for Leafy Greens in California.

Shiga toxin-producing Escherichia coli O157:H7 is a foodborne pathogen and is responsible for outbreaks of human gastroenteritis. This report documents the draft genome sequences of nine O157:H7 cattle strains, which were identified to be PCR positive for a Shiga toxin gene but displayed different levels of functional toxin activity.

Using Nanospray Liquid Chromatography and Mass Spectrometry to Quantitate Shiga Toxin Production in Environmental Escherichia coli Recovered from a Major Produce Production Region in California.

A set of 45 environmental strains of Shiga toxin producing Escherichia coli (STEC) from three California counties were analyzed for Shiga toxin production by nanospray liquid chromatography-mass spectrometry and Vero cell bioassay. The STEC in this set comprised six serotypes ((O113:H21, O121:H19, O157:H7, O6:H34, O177:H25, and O185:H7) each containing either the stx2a or stx2c operon. Six of the seven O113:H21 were found to contain two distinct stx2a operons. Eight strains of O157:H7 possessed a stx2c operon whose A subunit gene was interrupted by an insertion sequence (IS1203v). Shiga toxin production was induced by nutrient depletion and quantitated by mass spectrometry. The 37 strains produced Shiga toxins in a near 50-fold range (1.4-49 ng/mL). The IS-interrupted strains expressed low but measurable amounts of the B subunits (0.5-1.9 ng/mL). Another strain possessed an identical stx operon without an IS interruption and produced intact Stx2c (5.7 ng/mL).

Clinically-relevant Shiga toxin 2 subtypes from environmental Shiga toxin-producing Escherichia coli identified by top-down/middle-down proteomics and DNA sequencing.

Thirty-five environmental isolates of Shiga toxin-producing Escherichia coli (STEC) were analyzed by MALDI-TOF-TOF mass spectrometry, top-down/middle-down proteomics and DNA sequencing. Clinically-relevant Shiga toxin 2 (Stx2) produced by these STEC strains were subtyped based on MS and MS/MS (tandem mass spectrometry) of the intact B-subunit (top-down) and A2 fragment (middle-down) of the A-subunit using antibiotic-induced protein expression. Antibiotic induction of Stx2 was found to be strain dependent. By proteomic analysis, seventeen strains were identified as Stx2a, six strains as Stx2c, four strains as either Stx2a or 2c and eight strains as either Stx2a, 2c or 2d. DNA sequencing indicated only stx 2a and stx 2c genes as being present in these strains. Weak induction of Stx2 for certain strains made it difficult to distinguish between clinical subtypes by proteomic analysis. Very weak toxin induction in eight strains was consistent with a ∼1300 bp transposon insertion in the stx 2c A-subunit gene identified by DNA sequencing. DNA sequencing also revealed the presence of two bacteriophage (BP) in three strains with a stx 2a gene in each BP genome. Middle-down proteomic analysis of the A2 fragment confirmed expression of two stx 2a genes present in one of these strains based on a slight difference in the amino acid sequence (D ↔ E substitution) in the two A2 fragments.

First report of an atypical new Aspergillus parasiticus isolates with nucleotide insertion in aflR gene resembling to A. sojae.

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 ß-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.

Isolation, genotyping and antimicrobial resistance of Shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen linked to outbreaks of human gastroenteritis with diverse clinical spectra. In this review, we have examined the currently methodologies and molecular characterization techniques for assessing the phenotypic, genotypic and functional characteristics of STEC O157 and non-O157. In particular, traditional culture and isolation methods, including selective enrichment and differential plating, have enabled the effective recovery of STEC. Following recovery, immunological serotyping of somatic surface antigens (O-antigens) and flagellum (H-antigens) are employed for the classification of the STEC isolates. Molecular genotyping methods, including multiple-locus variable-number tandem repeat analysis, arrays, and whole genome sequencing, can discriminate the isolate virulence profile beyond the serotype level. Virulence profiling is focused on the identification of chromosomal and plasmid genes coding for adhesins, cytotoxins, effectors, and hemolysins to better assess the pathogenic potential of the recovered STEC isolates. Important animal reservoirs are cattle and other small domestic ruminants. STEC can also be recovered from other carriers, such as mammals, birds, fish, amphibians, shellfish and insects. Finally, antimicrobial resistance in STEC is a matter of growing concern, supporting the need to monitor the use of these agents by private, public and agricultural sectors. Certain antimicrobials can induce Shiga toxin production and thus promote the onset of severe disease symptoms in humans. Together, this information will provide a better understanding of risks associated with STEC and will aid in the development of efficient and targeted intervention strategies.

Draft Genome Sequences of Escherichia coli O113:H21 Strains Recovered from a Major Produce Production Region in California.

Shiga toxin-producing Escherichia coli is a foodborne and waterborne pathogen and is responsible for outbreaks of human gastroenteritis. This report documents the draft genome sequences of seven O113:H21 strains recovered from livestock, wildlife, and soil samples recovered from a major agricultural region for leafy greens in California, USA.

Screening for the presence of mcr-1/mcr-2 genes in Shiga toxin-producing Escherichia coli recovered from a major produce-production region in California.

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.

Concurrent Detection of Human Norovirus and Bacterial Pathogens in Water Samples from an Agricultural Region in Central California Coast.

Bacterial pathogens and human norovirus (HuNoV) are major cause for acute gastroenteritis caused by contaminated food and water. Public waterways can become contaminated from a variety of sources and flood after heavy rain events, leading to pathogen contamination of produce fields. We initiated a survey of several public watersheds in a major leafy green produce production region of the Central California Coast to determine the prevalence of HuNoV as well as bacterial pathogens. Moore swabs were used to collect environmental samples bi-monthly at over 30 sampling sites in the region. High prevalence of HuNoV and bacterial pathogens were detected in environmental water samples in the region. The overall detection rates of HuNoV, O157 Shiga toxin-producing Escherichia coli (STEC), non-O157 STEC, Salmonella, and Listeria were 25.58, 7.91, 9.42, 59.65, and 44.30%, respectively. The detection rates of Salmonella and L. monocytogenes were significantly higher in the spring. Fall and spring had elevated detection rates of O157 STEC. The overall detection rates of non-O157 STEC in the fall were lower than the other seasons but not significant. The overall detection rates of HuNoV were highest in fall, followed by spring and winter, with summer being lowest and significantly lower than other seasons. This study presented the first study of evaluating the correlation between the detection rate of HuNoV and the detection rates of four bacterial pathogens from environmental water. Overall, there was no significant difference in HuNoV detection rates between samples testing positive or negative for the four bacterial pathogens tested. Pathogens in animal-impacted and human-impacted areas were investigated. There were significant higher detection rates in animal-impacted areas than that of human-impacted areas for bacterial pathogens. However, there was no difference in HuNoV detection rates between these two areas. The overall detection levels of generic E. coli and detection rate of HuNoV showed no correlation.

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.

Antimicrobial resistance profiles of Shiga toxin-producing Escherichia coli O157 and Non-O157 recovered from domestic farm animals in rural communities in Northwestern Mexico.

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.

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.

Virulence profiling of Shiga toxin-producing Escherichia coli recovered from domestic farm animals in Northwestern Mexico.

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.