Escherichia coli Isolated from Bovine Sources Encoding the Fosfomycin Resistance Gene fosA7.5.

Dairy animals are reservoirs of antimicrobial-resistant Escherichia coli that are frequently resistant to tetracycline, aminoglycoside, ß-lactam, sulfonamide, and macrolide-lincosamide-streptogramin B antibiotics. However, resistance to other classes of antimicrobials is less frequently observed, and resistance to fosfomycin is rarely observed in E. coli. In this study, we describe the genomic characteristics of E. coli encoding fosA7.5 that have been recovered from bovine sources in the United States. Most isolates only encoded the fosA7.5 gene, whereas 37% encoded at least one other resistance gene, and 25% were genotypically multidrug-resistant. Most (112 isolates, 93%) belonged to phylogenetic group B1 and were assigned to 19 sequence types (STs), the most frequently identified being ST1727, ST2307, and ST3234. Results of this study indicate that fosA-encoding E. coli from bovine sources is very rare in the United States with isolates demonstrating a high level of similarity across a broad geographic region.

Microdiversity of Salmonella Kentucky During Long-Term Colonization of a Dairy Herd.

Salmonella enterica subsp. enterica serovar Kentucky was repeatedly isolated from a commercial dairy herd that was enrolled in a longitudinal study where feces of asymptomatic dairy cattle were sampled intensively over an 8-year period. The genomes of 5 Salmonella Kentucky isolates recovered from the farm 2 years before the onset of the long-term colonization event and 13 isolates collected during the period of endemicity were sequenced. A phylogenetic analysis inferred that the Salmonella Kentucky strains from the farm were distinct from poultry strains collected from the same region, and three subclades (K, A1, and A2) were identified among the farm isolates, each appearing at different times during the study. Based on the phylogenetic analysis, three separate lineages of highly similar Salmonella Kentucky were present in succession on the farm. Genomic heterogeneity between the clades helped identify regions, most notably transcriptional regulators, of the Salmonella Kentucky genome that may be involved in competition among highly similar strains. Notably, a region annotated as a hemolysin expression modulating protein (Hha) was identified in a putative plasmid region of strains that colonized a large portion of cows in the herd, suggesting that it may play a role in asymptomatic persistence within the bovine intestine. A cell culture assay of isolates from the three clades with bovine epithelial cells demonstrated a trend of decreased invasiveness of Salmonella Kentucky isolates over time, suggesting that clade-specific interactions with the animals on the farm may have played a role in the dynamics of strain succession. Results of this analysis further demonstrate an underappreciated level of genomic diversity within strains of the same Salmonella serovar, particularly those isolated during a long-term period of asymptomatic colonization within a single dairy herd.

Differences between the global transcriptomes of Salmonella enterica serovars Dublin and Cerro infecting bovine epithelial cells.

BACKGROUND: The impact of S. enterica colonization in cattle is highly variable and often serovar-dependent. The aim of this study was to compare the global transcriptomes of highly pathogenic bovine-adapted S. enterica serovar Dublin and the less pathogenic, bovine-adapted, serovar Cerro during interactions with bovine epithelial cells, to identify genes that impact serovar-related outcomes of S. enterica infections in dairy animals. RESULT: Bovine epithelial cells were infected with S. enterica strains from serovars Dublin and Cerro, and the bacterial RNA was extracted and sequenced. The total number of paired-end reads uniquely mapped to non-rRNA and non-tRNA genes in the reference genomes ranged between 12.1 M (Million) and 23.4 M (median: 15.7 M). In total, 360 differentially expressed genes (DEGs) were identified with at least two-fold differences in the transcript abundances between S. Dublin and S. Cerro (false discovery rate ≤ 5%). The highest number of DEGs (17.5%, 63 of 360 genes) between the two serovars were located on the genomic regions potentially associated with Salmonella Pathogenicity Islands (SPIs). DEGs potentially located in the SPI-regions that were upregulated (≥ 2-fold) in the S. Dublin compared with S. Cerro included: 37 SPI-1 genes encoding mostly Type 3 Secretion System (T3SS) apparatus and effectors; all of the six SPI-4 genes encoding type I secretion apparatus (siiABCDEF); T3SS effectors and chaperone (sopB, pipB, and sigE) located in SPI-5; type VI secretion system associated protein coding genes (sciJKNOR) located in SPI-6; and T3SS effector sopF in SPI-11. Additional major functional categories of DEGs included transcription regulators (n = 25), amino acid transport and metabolism (n = 20), carbohydrate transport and metabolism (n = 20), energy production and metabolism (n = 19), cell membrane biogenesis (n = 18), and coenzyme transport and metabolism (n = 15). DEGs were further mapped to the metabolic pathways listed in the KEGG database; most genes of the fatty acid ß-oxidation pathway were upregulated/uniquely present in the S. Dublin strains compared with the S. Cerro strains. CONCLUSIONS: This study identified S. enterica genes that may be responsible for symptomatic or asymptomatic infection and colonization of two bovine-adapted serovars in cattle.

FI: The Fecobiome Initiative.

Animal husbandry has been key to the sustainability of human societies for millennia. Livestock animals, such as cattle, convert plants to protein biomass due to a compartmentalized gastrointestinal tract (GIT) and the complementary contributions of a diverse GIT microbiota, thereby providing humans with meat and dairy products. Research on cattle gut microbial symbionts has mainly focused on the rumen (which is the primary fermentation compartment) and there is a paucity of functional insight on the intestinal (distal end) microbiota, where most foodborne zoonotic bacteria reside. Here, we present the Fecobiome Initiative (or FI), an international effort that aims at facilitating collaboration on research projects related to the intestinal microbiota, disseminating research results, and increasing public availability of resources. By doing so, the FI can help mitigate foodborne and animal pathogens that threaten livestock and human health, reduce the emergence and spread of antimicrobial resistance in cattle and their proximate environment, and potentially improve the welfare and nutrition of animals. We invite all researchers interested in this type of research to join the FI through our website: www.fecobiome.com.

Genomic Analysis of Antibiotic-Resistant and -Susceptible Escherichia coli Isolated from Bovine Sources in Maputo, Mozambique.

This study reports a genomic analysis of Escherichia coli isolates recovered from 25 bovine fecal composite samples collected from four different production units in Maputo city and around Maputo Province, Mozambique. The genomes were analyzed to determine the presence of antibiotic resistance genes (ARGs), genetic relatedness, and virulence factors known to cause diseases in humans. Whole-genome sequencing was conducted on 28 isolates using an Illumina NextSeq 500 sequencing platform. The genomes were analyzed using BLASTN for the presence of resistance genes and virulence factors, as well as to determine their phylogenetic groups, sequence types (ST), and ST complexes (ST Cplxs). The majority of the isolates (85%) were identified as members of phylogenetic groups B1, with fewer isolates identified as members of group A, and a single isolate identified as group "E/Clade I." The ST analysis demonstrated a higher level of diversity than the phylogenetic group analysis. Sixteen different STs, five ST Cplxs, and seven singleton complexes were identified. A strain identified as a novel ST (ST9215) showed a high level of similarity with an isolate recovered from a wild animal in the Gambia. Seven different ARGs were identified, with tet(B) being the most frequently detected, followed by aph(3″)-Ib, aph(6)-Id, sul2, blaTEM-1B, and dfrA1. Three isolates encoded ß-lactam-conferring point mutations in the ampC promoter (-42C>T). In total, 51 different virulence factors were identified among the genomes. This study demonstrates that E. coli from bovine sources in Mozambique encoded multiple antibiotic resistance elements, plasmids, and virulence factors. To the best of our knowledge, this is the first genomic description of antibiotic-resistant E. coli isolated from bovine sources in Mozambique.

Differences in the Microbial Community and Resistome Structures of Feces from Preweaned Calves and Lactating Dairy Cows in Commercial Dairy Herds.

Preweaned dairy calves and lactating dairy cows are known reservoirs of antibiotic-resistant bacteria. To further understand the differences in the resistomes and microbial communities between the two, we sequenced the metagenomes of fecal composite samples from preweaned dairy calves and lactating dairy cows on 17 commercial dairy farms (n = 34 samples). Results indicated significant differences in the structures of the microbial communities (analysis of similarities [ANOSIM] R = 0.81, p = 0.001) and resistomes (ANOSIM R = 0.93 to 0.96, p = 0.001) between the two age groups. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were the predominant members of the communities, but when the groups were compared, Bacteroidetes and Verrumicrobia were significantly more abundant in calf fecal composite samples, whereas Firmicutes, Spirochaetes, Deinococcus-Thermus, Lentisphaerae, Planctomycetes, Chlorofexi, and Saccharibacteria-(TM7) were more abundant in lactating cow samples. Diverse suites of antibiotic resistance genes (ARGs) were identified in all samples, with the most frequently detected being assigned to tetracycline and aminoglycoside resistance. When the two groups were compared, ARGs were significantly more abundant in composite fecal samples from calves than those from lactating cows (calf median ARG abundance = 1.8 × 100 ARG/16S ribosomal RNA [rRNA], cow median ARG abundance = 1.7 × 10-1 ARG/16S rRNA) and at the antibiotic resistance class level, the relative abundance of tetracycline, trimethoprim, aminoglycoside, macrolide-lincosamide-streptogramin B, ß-lactam, and phenicol resistance genes was significantly higher in calf samples than in cow samples. Results of this study indicate that composite feces from preweaned calves harbor different bacterial communities and resistomes than composite feces from lactating cows, with a greater abundance of resistance genes detected in preweaned calf feces.

Interaction of Salmonella enterica with Bovine Epithelial Cells Demonstrates Serovar-Specific Association and Invasion Patterns.

Dairy cows are known reservoirs of Salmonella enterica and human salmonellosis has been attributed to the consumption of contaminated dairy and beef products as well as poultry meat and eggs. Although many S. enterica serovars are known to colonize the gastrointestinal tract of cattle, the interactions between dairy commensal (or persistent) and transient Salmonella serovars with bovine epithelial cells are not well understood. Association-invasion assays were used to characterize the interactions of 26 S. enterica strains from bovine origins, comprising serovars Anatum, Cerro, Dublin, Give, Kentucky, Mbandaka, Meleagridis, Montevideo, Muenster, Newport, Oranienburg, Senftenberg, and Typhimurium, with cultured bovine epithelial cells. There were significant differences in the association with and invasion of bovine epithelial cells within and across Salmonella serovars (Tukey's Honestly Significant Difference test, p < 0.05). Salmonella enterica serovar Dublin strains were the most invasive, whereas Kentucky, Mbandaka, Cerro, and Give strains were the least invasive (p < 0.05). Significant differences in motility on semisolid medium were also observed between strains from different serovars. Findings from this study demonstrate an underappreciated level of phenotypic diversity among Salmonella strains within and across serovars and serve as a baseline for future studies that may identify the molecular mechanisms of asymptomatic Salmonella carriage and bovine salmonellosis.

Antimicrobial Resistance in Fecal Escherichia coli and Salmonella enterica from Dairy Calves: A Systematic Review.

The discovery of antibiotics brought with it many advances in the health and well-being of humans and animals; however, in recent years development of antimicrobial resistance (AMR) has increasingly become a concern. Much of the antibiotic use on dairy farms is for disease management in mature cattle, and AMR in fecal organisms is relatively rare in this group. However, young dairy calves often carry high levels of AMR in their fecal Escherichia coli and Salmonella enterica, which could provide a potential reservoir of AMR genes on dairy farms. To develop practical and effective antibiotic stewardship policies for dairy calf rearing, it is vital to have a solid understanding of the current state of knowledge regarding AMR in these animals. A systematic review process was used to summarize the current scientific literature regarding AMR in fecal S. enterica and E. coli and associations between management practices and AMR prevalence in dairy calves in the United States and Canada. Seven online databases were searched for literature published from 1997 to 2018. Multiple studies indicated an association between preweaned calves and increased risk of fecal shedding of resistant bacteria, compared to other animal groups on dairy farms. There also was evidence, although less consistent, of an impact of antibiotic treatment, antibiotic-containing milk replacer feeding, and feeding nonsalable or waste milk (WM) on the presence of AMR bacteria. Overall, the research summarized in this systematic review highlights the need for continued research on the impact of management practices, including antibiotic use, WM feeding, and disease prevention practices in reducing AMR in E. coli and S. enterica in dairy calves. In addition, few data were available on physiological and microbiological factors that may contribute to the high relative populations of resistant bacteria in young calves, suggesting another valuable area of future research.

Diversity of Extended-Spectrum Cephalosporin-Resistant Escherichia coli in Feces from Calves and Cows on Pennsylvania Dairy Farms.

The global incidence of human infections associated with extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli is increasing. Dairy animals are reservoirs of ESBL-producing E. coli, especially, third-generation cephalosporin (3GC)-resistant strains. To further understand the diversity of 3GC-resistant E. coli across animals of different age groups (e.g., pre- and postweaned calves, lactating cows, and dry cows) and farms, we used pulsed-field gel electrophoresis (PFGE) to characterize 70 fecal isolates from 14 dairy farms located in nine Pennsylvania counties. Results of this analysis indicated that 3GC-resistant E. coli were highly diverse and grouped into 27 PFGE clades (80% similarity cutoff) and 24 unique antimicrobial resistance patterns were observed among the isolates. For eight farms, clonal E. coli with the same resistance patterns were isolated from two or more age groups, indicating that strains were carried in both the calves and adult cows within the same herd. However, there were also several isolates with the same resistance pattern that were distributed to different clades, including isolates from different animal age groups on the same farm, suggesting different strains of E. coli within a farm harbored the same resistance-conferring elements. Results of this analysis indicated that 3GC-resistant E. coli were highly diverse, associated with multidrug resistance, and circulated through different (noncommingled) animal groups on individual farms.

Antimicrobial Resistance Among Escherichia coli Isolated from Veal Calf Operations in Pennsylvania.

Antimicrobial resistance (AR) is a pressing public health concern, and agricultural operations such as dairy and beef cattle production have been implicated as potential sources of resistant bacteria or genetic elements. This study aimed to determine the prevalence of antimicrobial-resistant Escherichia coli from calf pens in 6 auction houses (56 manure composite samples) and 12 veal calf operations (240 fecal samples in 2 visits: after the calves arrived at the farm and shortly before the animals were sent to slaughter) in the Commonwealth of Pennsylvania. A total of 1567 generic E. coli were isolated and screened for resistance phenotypes. Resistant E. coli were isolated from all auction houses and farms sampled. Based on nonparametric Kruskal-Wallis tests, incremental prevalence of E. coli resistant to ampicillin, azithromycin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, streptomycin, sulfisoxazole, trimethoprim-sulfamethoxazole, and tetracycline in the samples from auction houses and the first and second farm visits was observed (χ2 6.98-15.91, p < 0.05). Multidrug-resistant E. coli (resistant to more than three antimicrobial classes) were identified in 76.8%, 90.8%, and 100% of samples collected from the auction houses, first farm visits, and second farm visits, respectively. The presence of blaCTX-M-E. coli in 11 of the 12 farms presents the possibility of veal production environments being a reservoir for resistant genetic materials that may pose a risk to human health if they are transferred to human pathogens. Additional research on the impact of various management strategies in veal calf rearing is needed for a complete scenario of AR in these production environments.

Age-Associated Distribution of Antimicrobial-Resistant Salmonella enterica and Escherichia coli Isolated from Dairy Herds in Pennsylvania, 2013-2015.

Antimicrobial resistance has become a major global public health concern, and agricultural operations are often implicated as a source of resistant bacteria. This study characterized the prevalence of antimicrobial-resistant Salmonella enterica and Escherichia coli from a total of 443 manure composite samples from preweaned calves, postweaned calves, dry cows, and lactating cows from 80 dairy operations in Pennsylvania. A total of 1095 S. enterica and 2370 E. coli isolates were screened and tested for resistance to 14 antimicrobials on the National Antimicrobial Resistance Monitoring System Gram-negative (NARMS GN) panel. Salmonellae were isolated from 67% of dairy operations, and 99% of the isolates were pan-susceptible. Salmonella were isolated more frequently from lactating and dry cow samples than from pre- and postweaned calf samples. Overall, the most prevalent serotypes were Cerro, Montevideo, Kentucky, and Newport. E. coli were isolated from all the manure composite samples, and isolates were commonly resistant to tetracyclines, sulfonamides, and aminoglycosides. Resistance was detected more frequently in the E. coli isolates from pre- and postweaned calf samples than in isolates from dry and lactating cow samples (p < 0.05). Multidrug-resistant E. coli (i.e., resistant to >3 antimicrobial classes) were isolated from 66 farms (83%) with significantly greater prevalence in preweaned calves (p < 0.05) than in the older age groups. The blaCTX-M and blaCMY genes were detected in the cephalosporin-resistant E. coli from 4% and 35% of the farms, respectively. These findings indicate that dairy animals, especially the calf population, serve as significant reservoirs for antimicrobial-resistant bacteria. Additional research on the colonization and persistence of resistant E. coli in calves is warranted to identify potential avenues for mitigation.

Deep-sea hydrothermal vent bacteria related to human pathogenic Vibrio species.

Vibrio species are both ubiquitous and abundant in marine coastal waters, estuaries, ocean sediment, and aquaculture settings worldwide. We report here the isolation, characterization, and genome sequence of a novel Vibrio species, Vibrio antiquarius, isolated from a mesophilic bacterial community associated with hydrothermal vents located along the East Pacific Rise, near the southwest coast of Mexico. Genomic and phenotypic analysis revealed V. antiquarius is closely related to pathogenic Vibrio species, namely Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio vulnificus, but sufficiently divergent to warrant a separate species status. The V. antiquarius genome encodes genes and operons with ecological functions relevant to the environment conditions of the deep sea and also harbors factors known to be involved in human disease caused by freshwater, coastal, and brackish water vibrios. The presence of virulence factors in this deep-sea Vibrio species suggests a far more fundamental role of these factors for their bacterial host. Comparative genomics revealed a variety of genomic events that may have provided an important driving force in V. antiquarius evolution, facilitating response to environmental conditions of the deep sea.

Prevalence of Salmonella enterica, Listeria monocytogenes, and pathogenic Escherichia coli in bulk tank milk and milk filters from US dairy operations in the National Animal Health Monitoring System Dairy 2014 study.

The dairy farm environment is a well-documented reservoir for zoonotic pathogens such as Salmonella enterica, Shiga-toxigenic Escherichia coli, and Listeria monocytogenes, and humans may be exposed to these pathogens via consumption of unpasteurized milk and dairy products. As part of the National Animal Health Monitoring System Dairy 2014 study, bulk tank milk (BTM, n = 234) and milk filters (n = 254) were collected from a total of 234 dairy operations in 17 major dairy states and analyzed for the presence of these pathogens. The invA gene was detected in samples from 18.5% of operations and Salmonella enterica was isolated from 18.0% of operations. Salmonella Dublin was detected in 0.7% of operations. Sixteen Salmonella serotypes were isolated, and the most common serotypes were Cerro, Montevideo, and Newport. Representative Salmonella isolates (n = 137) were tested against a panel of 14 antimicrobials. Most (85%) were pansusceptible; the remaining were resistant to 1 to 9 antimicrobials, and within the resistant strains the most common profile was resistance to ampicillin/clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline. Listeria spp. were isolated from 19.9% of operations, and L. monocytogenes was isolated from 3.0% of operations. Serogroups 1/2a and 1/2b were the most common, followed by 4b and 4a. One or more E. coli virulence genes were detected in the BTM from 30.5% of operations and in the filters from 75.3% of operations. A combination of stx2, eaeA, and γ-tir genes was detected in the BTM from 0.5% of operations and in the filters from 6.6% of operations. The results of this study indicate an appreciable prevalence of bacterial pathogens in BTM and filters, including serovars known to infect humans.

Prevalence, antimicrobial resistance, and molecular characterization of Campylobacter spp. in bulk tank milk and milk filters from US dairies.

Campylobacter spp. are frequently isolated from dairy cows as commensal organisms. Sporadic Campylobacter infections in humans in the United States are generally attributed to poultry, but outbreaks are also commonly associated with dairy products, particularly unpasteurized or raw milk. Bulk tank milk samples and milk filters from US dairy operations were collected during the National Animal Health Monitoring System Dairy 2014 study and analyzed using real-time PCR and traditional culture techniques for the presence of thermophilic Campylobacter species. The weighted prevalence of operations from which we detected Campylobacter spp. in either bulk tank milk or milk filters was 24.9%. We detected Campylobacter spp. in a higher percentage of operations with 100-499 cows (42.8%) and 500 or more cows (47.5%) than in operations with 30-99 cows (6.5%). Campylobacter spp. were also more frequently detected in operations in the west than the east (45.9 and 22.6%, respectively). We isolated Campylobacter spp. from approximately half of PCR-positive samples, representing 12.5% (weighted prevalence) of operations. The majority (91.8%) of isolates were C. jejuni, but C. lari and C. coli were also isolated. We detected resistance to tetracycline in 68.4% of C. jejuni isolates, and resistance to ciprofloxacin and nalidixic acid in 13.2% of C. jejuni isolates. Based on pulsed-field gel electrophoresis, we found that dairy-associated C. jejuni were genotypically diverse, although clonal strains were isolated from different geographic regions. These results suggest that bulk tank milk can be contaminated with pathogenic Campylobacter spp., and that the consumption of unpasteurized or raw milk presents a potential human health risk.

Genomic diversity of 2010 Haitian cholera outbreak strains.

The millions of deaths from cholera during the past 200 y, coupled with the morbidity and mortality of cholera in Haiti since October 2010, are grim reminders that Vibrio cholerae, the etiologic agent of cholera, remains a scourge. We report the isolation of both V. cholerae O1 and non-O1/O139 early in the Haiti cholera epidemic from samples collected from victims in 18 towns across eight Arrondissements of Haiti. The results showed two distinct populations of V. cholerae coexisted in Haiti early in the epidemic. As non-O1/O139 V. cholerae was the sole pathogen isolated from 21% of the clinical specimens, its role in this epidemic, either alone or in concert with V. cholerae O1, cannot be dismissed. A genomic approach was used to examine similarities and differences among the Haitian V. cholerae O1 and V. cholerae non-O1/O139 strains. A total of 47 V. cholerae O1 and 29 V. cholerae non-O1/O139 isolates from patients and the environment were sequenced. Comparative genome analyses of the 76 genomes and eight reference strains of V. cholerae isolated in concurrent epidemics outside Haiti and 27 V. cholerae genomes available in the public database demonstrated substantial diversity of V. cholerae and ongoing flux within its genome.

Diversity of Listeria monocytogenes within a U.S. dairy herd, 2004-2010.

Listeria monocytogenes, the causative agent of listeriosis, is frequently isolated from the environment. Dairy cows and dairy farm environments are reservoirs of this pathogen, where fecal shedding contributes to its environmental dispersal and contamination of milk, dairy products, and meat. The molecular diversity of 40 L. monocytogenes isolates representing 3 serogroups (1/2a, 1/2b, and 4b) collected between 2004 and 2010 from the feces of dairy cattle on a single dairy farm was assessed using a multivirulence locus sequence typing (MVLST) assay. The dairy farm L. monocytogenes MVLST patterns were compared to those from 138 strains isolated globally from clinical cases, foods, and the environment. Results of the study demonstrated that several distantly related L. monocytogenes strains persisted among members of the herd over the course of the study while other strains were transient. Furthermore, some strains isolated during this study appear to be distantly related to previously isolated L. monocytogenes while others are closely related to Epidemic Clones associated with human illness. This work demonstrates that dairy cows can be reservoirs of a diverse population of potentially human pathogenic L. monocytogenes that represents a risk to consumers of milk, dairy products, and meat.

Vibrio metoecus sp. nov., a close relative of Vibrio cholerae isolated from coastal brackish ponds and clinical specimens.

A Gram-staining-negative, curved-rod-shaped bacterium with close resemblance to Vibrio cholerae, the aetiological agent of cholera, was isolated over the course of several years from coastal brackish water (17 strains) and from clinical cases (two strains) in the United States. 16S rRNA gene identity with V. cholerae exceeded 98 % yet an average nucleotide identity based on genome data of around 86 % and multi locus sequence analysis of six housekeeping genes (mdh, adk, gyrB, recA, pgi and rpoB) clearly delineated these isolates as a distinct genotypic cluster within the V. cholerae-V. mimicus clade. Most standard identification techniques do not differentiate this cluster of isolates from V. cholerae. Only amplification of the ompW gene using V. cholerae-specific primers and a negative Voges-Proskauer test showed a difference between the two clusters. Additionally, all isolated strains differed phenotypically from V. cholerae in their ability to utilize N-acetyl-d-galactosamine and d-glucuronic acid as sole carbon sources. Furthermore, they were generally unable to infect the slime mould Dictyostelium discoideum, a widespread ability in V. cholerae. Based on these clear phenotypic differences that are not necessarily apparent in standard tests as well as average nucleotide identity and phylogeny of protein-coding genes, we propose the existence of a novel species, Vibrio metoecus sp. nov. with the type strain OP3H(T) ( = LMG 27764(T) = CIP 110643(T)). Due to its close resemblance to V. cholerae and the increasing number of strains isolated over the past several years, we suggest that V. metoecus sp. nov. is a relatively common species of the genus Vibrio, isolates of which have been identified as atypical isolates of V. cholerae in the past. Its isolation from clinical samples also indicates that strains of this species, like V. cholerae, are opportunistic pathogens.

Virulome analysis of Escherichia coli ST117 from bovine sources identifies similarities and differences with strains isolated from other food animals.

Escherichia coli ST117 is a pandemic extraintestinal pathogenic E. coli (ExPEC) causing significant morbidity globally. Poultry are a known reservoir of this pathogen, but the characteristics of ST117 strains from other animal sources have not been adequately investigated. Here we characterize the genomes of 36 ST117 strains recovered primarily from preweaned dairy calves, but also from older postweaned calves and lactating cows, in the context of other bovine-associated strains and strains from poultry, swine, and humans. Results of this study demonstrate that bovine-associated ST117 genomes encode virulence factors (VFs) known to be involved in extraintestinal infections, but also occasionally encode the Shiga toxin, a virulence factor (VF) involved in severe gastrointestinal infections and more frequently identified in E. coli from ruminants than other animals. Bovine-associated ST117 genomes were also more likely to encode afa-VIII (adhesins), pap (P-fimbriae), cdt (cytolethal distending toxin), and stx (Shiga toxins) than were poultry and swine-associated genomes. All of the ST117 genomes were grouped into seven virulence clusters, with bovine-associated genomes grouping into Clusters 1, 2, 4, 5, but not 3, 6, or 7. Major differences in the presence of virulence factors between clusters were observed as well. Antimicrobial resistance genes were detected in 112 of 122 (91%) bovine-associated genomes, with 103 of these being multidrug-resistant (MDR). Inclusion of genomes that differed from ST117 by one multi-locus sequence type (MLST) allele identified 31 STs, four of these among the bovine-associated genomes. These non-ST117 genomes clustered with the ST117 genomes suggesting that they may cause similar disease as ST117. Results of this study identify cattle as a reservoir of ST117 strains, some of which are highly similar to those isolated from other food animals and some of which have unique bovine-specific characteristics.

Inactivation of Escherichia coli, Salmonella enterica, and Listeria monocytogenes using the Contamination Sanitization Inspection and Disinfection (CSI-D) device.

The Contamination Sanitization Inspection and Disinfection (CSI-D) device is a handheld fluorescence-based imaging system designed to disinfect food contact surfaces using ultraviolet-C (UVC) illumination. This study aimed to determine the optimal CSI-D parameters (i.e., UVC exposure time and intensity) for the inactivation of the following foodborne bacteria plated on non-selective media: generic Escherichia coli (indicator organism) and the pathogens enterohemorrhagic E. coli, enterotoxigenic E. coli, Salmonella enterica, and Listeria monocytogenes. Each bacterial strain was spread-plated on non-selective agar and exposed to high-intensity (10 mW/cm2) or low-intensity (5 mW/cm2) UVC for 1-5 s. Control plates were not exposed to UVC. The plates were incubated overnight at 37 °C and then enumerated. Three trials for each bacterial strain were conducted. Statistical analysis was carried out to determine if there were significant differences in bacterial growth between UVC intensities and exposure times. Overall, exposure to low or high intensity for 3-5 s resulted in consistent inhibition of bacterial growth, with reductions of 99.9-100 % for E. coli, 96.8-100 % for S. enterica, and 99.2-100 % for L. monocytogenes. The 1 s exposure time showed inconsistent results, with a 66.0-100 % reduction in growth depending on the intensity and bacterial strain. When the results for all strains within each species were combined, the 3-5 s exposure times showed significantly greater (p < 0.05) growth inhibition than the 1 s exposure time. However, there were no significant differences (p > 0.05) in growth inhibition between the high and low UVC intensities. The results of this study show that, in pure culture conditions, exposure to UVC with the CSI-D device for ≥3 s is required to achieve consistent reduction of E. coli, S. enterica, and L. monocytogenes.

Comparative genomics of clinical and environmental Vibrio mimicus.

Whether Vibrio mimicus is a variant of Vibrio cholerae or a separate species has been the subject of taxonomic controversy. A genomic analysis was undertaken to resolve the issue. The genomes of V. mimicus MB451, a clinical isolate, and VM223, an environmental isolate, comprise ca. 4,347,971 and 4,313,453 bp and encode 3,802 and 3,290 ORFs, respectively. As in other vibrios, chromosome I (C-I) predominantly contains genes necessary for growth and viability, whereas chromosome II (C-II) bears genes for adaptation to environmental change. C-I harbors many virulence genes, including some not previously reported in V. mimicus, such as mannose-sensitive hemagglutinin (MSHA), and enterotoxigenic hemolysin (HlyA); C-II encodes a variant of Vibrio pathogenicity island 2 (VPI-2), and Vibrio seventh pandemic island II (VSP-II) cluster of genes. Extensive genomic rearrangement in C-II indicates it is a hot spot for evolution and genesis of speciation for the genus Vibrio. The number of virulence regions discovered in this study (VSP-II, MSHA, HlyA, type IV pilin, PilE, and integron integrase, IntI4) with no notable difference in potential virulence genes between clinical and environmental strains suggests these genes also may play a role in the environment and that pathogenic strains may arise in the environment. Significant genome synteny with prototypic pre-seventh pandemic strains of V. cholerae was observed, and the results of phylogenetic analysis support the hypothesis that, in the course of evolution, V. mimicus and V. cholerae diverged from a common ancestor with a prototypic sixth pandemic genomic backbone.