Clinical presentation, antimicrobial resistance, and treatment outcomes of Aeromonas human infections: A 14-year retrospective study and comparative genomics of two isolates from fatal cases.

BACKGROUND: Aeromonas virulence may not be entirely dependent on the host immune status. Pathophysiologic determinants of disease progression and severity remain unclear. METHODS: One hundred five patients with Aeromonas infections and 112 isolates were identified, their clinical presentations and outcomes analyzed, and their antimicrobial resistance (AMR) patterns assessed. Two isolates (A and B) from fatal cases of Aeromonas dhakensis bacteremia were characterized using whole genome sequence analysis. Virulence factor- and AMR-encoding genes from these isolates were compared with a well-characterized diarrheal isolate A. dhakensis SSU, and environmental isolate A. hydrophila ATCC_7966T. RESULTS: Skin and soft tissue infections, traumatic wound infections, sepsis, burns, and intraabdominal infections were common. Diabetes, malignancy, and cirrhosis were frequent comorbidities. Male sex, age ≥ 65 years, hospitalization, burns, and intensive care were associated with complicated disease. High rates of AMR to carbapenems and piperacillin-tazobactam were found. Treatment failure was observed in 25.7% of cases. Septic shock and hospital-acquired infections were predictors of treatment failure. All four isolates harbored assorted broad-spectrum AMR genes including blaOXA, ampC, cphA, and efflux pumps. Only clinical isolates possessed both polar and lateral flagellar genes, genes for various surface adhesion proteins, type 3- and -6 secretion systems and their effectors, and toxin genes, including exotoxin A. Both isolates A and B were resistant to colistin and harbored the mobile colistin resistance-3 (mcr-3) gene. CONCLUSIONS: Empirical therapy tailored to local Aeromonas antibiograms may facilitate more favorable outcomes, while advanced diagnostic methods may aid in identifying correct Aeromonas spp. of significant clinical importance.

Microbiome signatures of progression toward celiac disease onset in at-risk children in a longitudinal prospective cohort study.

Other than exposure to gluten and genetic compatibility, the gut microbiome has been suggested to be involved in celiac disease (CD) pathogenesis by mediating interactions between gluten/environmental factors and the host immune system. However, to establish disease progression markers, it is essential to assess alterations in the gut microbiota before disease onset. Here, a prospective metagenomic analysis of the gut microbiota of infants at risk of CD was done to track shifts in the microbiota before CD development. We performed cross-sectional and longitudinal analyses of gut microbiota, functional pathways, and metabolites, starting from 18 mo before CD onset, in 10 infants who developed CD and 10 matched nonaffected infants. Cross-sectional analysis at CD onset identified altered abundance of six microbial strains and several metabolites between cases and controls but no change in microbial species or pathway abundance. Conversely, results of longitudinal analysis revealed several microbial species/strains/pathways/metabolites occurring in increased abundance and detected before CD onset. These had previously been linked to autoimmune and inflammatory conditions (e.g., Dialister invisus, Parabacteroides sp., Lachnospiraceae, tryptophan metabolism, and metabolites serine and threonine). Others occurred in decreased abundance before CD onset and are known to have anti-inflammatory effects (e.g., Streptococcus thermophilus, Faecalibacterium prausnitzii, and Clostridium clostridioforme). Additionally, we uncovered previously unreported microbes/pathways/metabolites (e.g., Porphyromonas sp., high mannose-type N-glycan biosynthesis, and serine) that point to CD-specific biomarkers. Our study establishes a road map for prospective longitudinal study designs to better understand the role of gut microbiota in disease pathogenesis and therapeutic targets to reestablish tolerance and/or prevent autoimmunity.

Environmental Factors Influencing Occurrence of Vibrio parahaemolyticus and Vibrio vulnificus.

Incidence of vibriosis is rising globally, with evidence that changing climatic conditions are influencing environmental factors that enhance growth of pathogenic Vibrio spp. in aquatic ecosystems. To determine the impact of environmental factors on occurrence of pathogenic Vibrio spp., samples were collected in the Chesapeake Bay, Maryland, during 2009 to 2012 and 2019 to 2022. Genetic markers for Vibrio vulnificus (vvhA) and Vibrio parahaemolyticus (tlh, tdh, and trh) were enumerated by direct plating and DNA colony hybridization. Results confirmed seasonality and environmental parameters as predictors. Water temperature showed a linear correlation with vvhA and tlh, and two critical thresholds were observed, an initial increase in detectable numbers (>15°C) and a second increase when maximum counts were recorded (>25°C). Temperature and pathogenic V. parahaemolyticus (tdh and trh) were not strongly correlated; however, the evidence showed that these organisms persist in oyster and sediment at colder temperatures. Salinity (10 to 15 ppt), total chlorophyll a (5 to 25 µg/L), dissolved oxygen (5 to 10 mg/L), and pH (8) were associated with increased abundance of vvhA and tlh. Importantly, a long-term increase in Vibrio spp. numbers was observed in water samples between the two collection periods, specifically at Tangier Sound (lower bay), with the evidence suggesting an extended seasonality for these bacteria in the area. Notably, tlh showed a mean positive increase that was ca. 3-fold overall, with the most significant increase observed during the fall. In conclusion, vibriosis continues to be a risk in the Chesapeake Bay region. A predictive intelligence system to assist decision makers, with respect to climate and human health, is warranted. IMPORTANCE The genus Vibrio includes pathogenic species that are naturally occurring in marine and estuarine environments globally. Routine monitoring for Vibrio species and environmental parameters influencing their incidence is critical to provide a warning system for the public when the risk of infection is high. In this study, occurrence of Vibrio parahaemolyticus and Vibrio vulnificus, both potential human pathogens, in Chesapeake Bay water, oysters, and sediment samples collected over a 13-year period was analyzed. The results provide a confirmation of environmental predictors for these bacteria, notably temperature, salinity, and total chlorophyll a, and their seasonality of occurrence. New findings refine environmental parameter thresholds of culturable Vibrio species and document a long-term increase in Vibrio populations in the Chesapeake Bay. This study provides a valuable foundation for development of predicative risk intelligence models for Vibrio incidence during climate change.

Genomic and evolutionary study from SARS-CoV-2 virus isolates from Bangladesh during the early stage of pandemic strongly correlate with European origin and not with China.

The goal of this study was to identify the genomic variants and determine molecular epidemiology of SARS-CoV-2 virus during the early pandemic stage in Bangladesh. Viral RNA was extracted, converted to cDNA, and amplified using Ion AmpliSeq™ SARS-CoV-2 Research Panel. 413 unique mutants from 151 viral isolates were identified. 80% of cases belongs to 8 mutants: 241C toT, 1163A toT, 3037C toT, 14408C toT, 23403A toG, 28881G toA, 28,882 G toA, and 28883G toC. Observed dominance of GR clade variants that have strong presence in Europe, suggesting European channel a possible entry route. Among 37 genomic mutants significantly associated with clinical symptoms, 3916CtoT (associated with sore-throat), 14408C to T (associated with cough-protection), 28881G to A, 28882G to A, and 28883G to C (associated with chest pain) were notable. These findings may inform future research platforms for disease management and epidemiological study.

T6SS and ExoA of flesh-eating Aeromonas hydrophila in peritonitis and necrotizing fasciitis during mono- and polymicrobial infections.

An earlier report described a human case of necrotizing fasciitis (NF) caused by mixed infection with 4 Aeromonas hydrophila strains (NF1-NF4). While the NF2, NF3, and NF4 strains were clonal and possessed exotoxin A (ExoA), the NF1 strain was determined to be phylogenetically distinct, harboring a unique type 6 secretion system (T6SS) effector (TseC). During NF1 and NF2 mixed infection, only NF1 disseminated, while NF2 was rapidly killed by a contact-dependent mechanism and macrophage phagocytosis, as was demonstrated by using in vitro models. To confirm these findings, we developed 2 NF1 mutants (NF1ΔtseC and NF1ΔvasK); vasK encodes an essential T6SS structural component. NF1 VasK and TseC were proven to be involved in contact-dependent killing of NF2 in vitro, as well as in its elimination at the intramuscular injection site in vivo during mixed infection, with overall reduced mouse mortality. ExoA was shown to have an important role in NF by both NF1-exoA (with cis exoA) and NF2 during monomicrobial infection. However, the contribution of ExoA was more important for NF2 than NF1 in the murine peritonitis model. The NF2∆exoA mutant did not significantly alter animal mortality or NF1 dissemination during mixed infection in the NF model, suggesting that the ExoA activity was significant at the injection site. Immunization of mice to ExoA protected animals from NF2 monomicrobial challenge, but not from polymicrobial infection because of NF2 clearance. This study clarified the roles of T6SS and ExoA in pathogenesis caused by A. hydrophila NF strains in both mouse peritonitis and NF models in monomicrobial and polymicrobial infections.

Evaluation of a Combined Multilocus Sequence Typing and Whole-Genome Sequencing Two-Step Algorithm for Routine Typing of Clostridioides difficile.

Multilocus sequence typing (MLST) is a low-resolution but rapid genotyping method for Clostridioides difficile Whole-genome sequencing (WGS) has emerged as the new gold standard for C. difficile typing, but cost and lack of standardization still limit broad utilization. In this study, we evaluated the potential to combine the portability of MLST with the increased resolution of WGS for a cost-saving approach to routine C. difficile typing. C. difficile strains from two New York City hospitals (hospital A and hospital B) were selected. WGS single-nucleotide polymorphism (wgSNP) was performed using established methods. Sequence types (ST) were determined using PubMLST, while wgSNP analysis was performed using the Bionumerics software. An additional analysis of a subset of data (hospital A) was made comparing the Bionumerics software to the CosmosID pipeline. Cost and turnaround time to results were compared for the algorithmic approach of MLST followed by wgSNP versus direct wgSNP. Among the 202 C. difficile isolates typed, 91% (n = 185/203) clustered within the representative ST, showing a high agreement between MLST and wgSNP. While clustering was similar between the Bionumerics and CosmosID pipelines, large differences in the overall number of SNPs were noted. A two-step algorithm for routine typing results in significantly lower cost than routine use of WGS. Our results suggest that using MLST as a first step in routine typing of C. difficile followed by WGS for MLST concordant strains is a less technically demanding, cost-saving approach for performing C. difficile typing than WGS alone without loss of discriminatory power.

Nanopore-based metagenomics analysis reveals prevalence of mobile antibiotic and heavy metal resistome in wastewater.

In-depth studies of the microbiome and mobile resistome profile of different environments is central to understanding the role of the environment in antimicrobial resistance (AMR), which is one of the urgent threats to global public health. In this study, we demonstrated the use of a rapid (and easily portable) sequencing approach coupled with user-friendly bioinformatics tools, the MinION (Oxford Nanopore Technologies), on the evaluation of the microbial as well as mobile metal and antibiotic resistome profile of semi-rural wastewater. A total of 20 unique phyla, 43 classes, 227 genera, and 469 species were identified in samples collected from the Amherst Wastewater Treatment Plant, both from primary and secondary treated wastewater. Alpha diversity indices indicated that primary samples were significantly richer and more microbially diverse than secondary samples. A total of 1041 ARGs, 68 MRGs, and 17 MGEs were detected in this study. There were more classes of AMR genes in primary than secondary wastewater, but in both cases multidrug, beta-lactam and peptide AMR predominated. Of note, OXA ß-lactamases, some of which are also carbapenemases, were enriched in secondary samples. Metal resistance genes against arsenic, copper, zinc and molybdenum were the dominant MRGs in the majority of the samples. A larger proportion of resistome genes were located in chromosome-derived sequences except for mobilome genes, which were predominantly located in plasmid-derived sequences. Genetic elements related to transposase were the most common MGEs in all samples. Mobile or MGE/plasmid-associated resistome genes that confer resistance to last resort antimicrobials such as carbapenems and colistin were detected in most samples. Worryingly, several of these potentially transferable genes were found to be carried by clinically-relevant hosts including pathogenic bacterial species in the orders Aeromonadales, Clostridiales, Enterobacterales and Pseudomonadales. This study demonstrated that the MinION can be used as a metagenomics approach to evaluate the microbiome, resistome, and mobilome profile of primary and secondary wastewater.

Cross-talk among flesh-eating Aeromonas hydrophila strains in mixed infection leading to necrotizing fasciitis.

Necrotizing fasciitis (NF) caused by flesh-eating bacteria is associated with high case fatality. In an earlier study, we reported infection of an immunocompetent individual with multiple strains of Aeromonas hydrophila (NF1-NF4), the latter three constituted a clonal group whereas NF1 was phylogenetically distinct. To understand the complex interactions of these strains in NF pathophysiology, a mouse model was used, whereby either single or mixed A. hydrophila strains were injected intramuscularly. NF2, which harbors exotoxin A (exoA) gene, was highly virulent when injected alone, but its virulence was attenuated in the presence of NF1 (exoA-minus). NF1 alone, although not lethal to animals, became highly virulent when combined with NF2, its virulence augmented by cis-exoA expression when injected alone in mice. Based on metagenomics and microbiological analyses, it was found that, in mixed infection, NF1 selectively disseminated to mouse peripheral organs, whereas the other strains (NF2, NF3, and NF4) were confined to the injection site and eventually cleared. In vitro studies showed NF2 to be more effectively phagocytized and killed by macrophages than NF1. NF1 inhibited growth of NF2 on solid media, but ExoA of NF2 augmented virulence of NF1 and the presence of NF1 facilitated clearance of NF2 from animals either by enhanced priming of host immune system or direct killing via a contact-dependent mechanism.

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.

Microbial diversity changes with rhizosphere and hydrocarbons in contrasting soils.

In the ecotoxicological assessment of petroleum hydrocarbon-contaminated soil, microbial community profile is important aspect due to their involvement in soil functions. However, soil physicochemical properties and the inhabiting plants could dictate the microbial composition. A question remains unanswered is, how an integrated approach may be utilized to account for various contrasting soil properties, plant types (reference vs. native) and the nature of the hydrocarbon contamination. In this study, we utilized bacterial DNA profiling techniques to investigate the relationship between soil properties, contaminant and plant species. Results identified that Proteobacteria and Actinobacteria were the most abundant bacteria of the 45 phyla identified in the hydrocarbon-contaminated soil. The bulk and rhizosphere microbiome showed that the contaminated soil originally had quite distinct bacterial communities compared to the artificially contaminated soil (mine soil = 95 genera vs. other soils = 2-29 genera). In these cases, not significantly but the native plant slightly increased bacterial diversity and relative abundance in the same soils. Also, within each site, the bacterial community was significantly altered with the hydrocarbon concentration. In this instance, the influence of the contaminant was strong and also with the soil pH and organic matter. These results would significantly contribute to the novel insights on the molecular technique-based hydrocarbon toxicity assessment and the development of the further integrative approach with other microbial community and their metabolic profile in the contaminated sites.

Distinct consequences of amoxicillin and ertapenem exposure in the porcine gut microbiome.

The gut microbiome influences many, if not all, aspects of human health. Antibiotics, while lifesaving, have the unintended consequence of killing commensal microbiota inhabiting the gastrointestinal (GI) tract, which can lead to overgrowth of opportunistic pathogens such as Clostridium difficile and emergence of antibiotic-resistant organisms. Here, porcine models were developed to evaluate changes to the gut microbiome caused by two distinct types of beta-lactam antibiotics delivered via common administration routes, oral amoxicillin and intravenous ertapenem. Amoxicillin is one of the most often used broad-spectrum antibiotics, frequently prescribed to young children. Ertapenem, a carbapenem considered a last resort antibiotic, is used sparingly in humans and prohibited for use in animals. Cohorts of normal pigs (n = 5) were treated with amoxicillin (20 mg/kg, PO, BID) or ertapenem (30 mg/kg, IV, SID) for seven days. Microbiomes were evaluated using whole genome shotgun metagenomics analyses of fecal DNA collected prior to, during, and after antibiotic treatment. Each antibiotic resulted in significant and distinct changes in the microbiome, causing elimination of key commensal bacterial species and overgrowth of other, potentially pathogenic taxa. In addition, amoxicillin promoted propagation of a broad range of antibiotic resistance genes, many encoding efflux pump components and beta-lactamases, while ertapenem triggered emergence of genes encoding vancomycin resistance, and beta-lactamases, including the carbapenemase, IMP-27. Notably, microbiota alterations and antibiotic resistance gene propagation displayed unique patterns following exposure to amoxicillin or ertapenem. These data underscore the importance of understanding consequences of individual antibiotic use to predict and potentially mitigate adverse outcomes. The porcine models developed here can facilitate evaluation of therapeutic interventions to prevent antibiotic-mediated microbiome disruption.

Comparative genomic analysis and characterization of incompatibility group FIB plasmid encoded virulence factors of Salmonella enterica isolated from food sources.

BACKGROUND: The degree to which the chromosomal mediated iron acquisition system contributes to virulence of many bacterial pathogens is well defined. However, the functional roles of plasmid encoded iron acquisition systems, specifically Sit and aerobactin, have yet to be determined for Salmonella spp. In a recent study, Salmonella enterica strains isolated from different food sources were sequenced on the Illumina MiSeq platform and found to harbor the incompatibility group (Inc) FIB plasmid. In this study, we examined sequence diversity and the contribution of factors encoded on the IncFIB plasmid to the virulence of S. enterica. RESULTS: Whole genome sequences of seven S. enterica isolates were compared to genomes of serovars of S. enterica isolated from food, animal, and human sources. SeqSero analysis predicted that six strains were serovar Typhimurium and one was Heidelberg. Among the S. Typhimurium strains, single nucleotide polymorphism (SNP)-based phylogenetic analyses revealed that five of the isolates clustered as a single monophyletic S. Typhimurium subclade, while one of the other strains branched with S. Typhimurium from a bovine source. DNA sequence based phylogenetic diversity analyses showed that the IncFIB plasmid-encoded Sit and aerobactin iron acquisition systems are conserved among bacterial species including S. enterica. The IncFIB plasmid was transferred to an IncFIB plasmid deficient strain of S. enterica by conjugation. The transconjugant SE819::IncFIB persisted in human intestinal epithelial (Caco-2) cells at a higher rate than the recipient SE819. Genes of the Sit and aerobactin operons in the IncFIB plasmid were differentially expressed in iron-rich and iron-depleted growth media. CONCLUSIONS: Minimal sequence diversity was detected in the Sit and aerobactin operons in the IncFIB plasmids present among different bacterial species, including foodborne Salmonella strains. IncFIB plasmid encoded factors play a role during infection under low-iron conditions in host cells.

Occurrence in Mexico, 1998-2008, of Vibrio cholerae CTX+ El Tor carrying an additional truncated CTX prophage.

The seventh cholera pandemic caused by Vibrio cholerae O1 El Tor (ET) has been superseded in Asia and Africa by altered ET possessing the cholera toxin (CTX) gene of classical (CL) biotype. The CL biotype of V. cholerae was isolated, along with prototypic and altered ET, during the 1991 cholera epidemic in Mexico and subsequently remained endemic until 1997. Microbiological, molecular, and phylogenetic analyses of clinical and environmental V. cholerae isolated in Mexico between 1998 and 2008 revealed important genetic events favoring predominance of ET over CL and altered ET. V. cholerae altered ET was predominant after 1991 but not after 2000. V. cholerae strains isolated between 2001 and 2003 and a majority isolated in 2004 lacked CTX prophage (Φ) genes encoding CTX subunits A and B and repeat sequence transcriptional regulators of ET and CL biotypes: i.e., CTXΦ(-). Most CTXΦ(-) V. cholerae isolated in Mexico between 2001 and 2003 also lacked toxin coregulated pili tcpA whereas some carried either tcpA(ET) or a variant tcpA with noticeable sequence dissimilarity from tcpA(CL). The tcpA variants were not detected in 2005 after CTXΦ(+) ET became dominant. All clinical and environmental V. cholerae O1 strains isolated during 2005-2008 in Mexico were CTXΦ(+) ET, carrying an additional truncated CTXΦ instead of RS1 satellite phage. Despite V. cholerae CTXΦ(-) ET exhibiting heterogeneity in pulsed-field gel electrophoresis patterns, CTXΦ(+) ET isolated during 2004-2008 displayed homogeneity and clonal relationship with V. cholerae ET N16961 and V. cholerae ET isolated in Peru.

Nontoxigenic Vibrio cholerae non-O1/O139 isolate from a case of human gastroenteritis in the U.S. Gulf Coast.

An occurrence of Vibrio cholerae non-O1/O139 gastroenteritis in the U.S. Gulf Coast is reported here. Genomic analysis revealed that the isolate lacked known virulence factors associated with the clinical outcome of a V. cholerae infection but did contain putative genomic islands and other accessory virulence factors. Many of these factors are widespread among environmental strains of V. cholerae, suggesting that there might be additional virulence factors in non-O1/O139 V. cholerae yet to be determined. Phylogenetic analysis revealed that the isolate belonged to a phyletic lineage of environmental V. cholerae isolates associated with sporadic cases of gastroenteritis in the Western Hemisphere, suggesting a need to monitor non-O1/O139 V. cholerae in the interest of public health.

Non-O1/non-O139 Vibrio cholerae carrying multiple virulence factors and V. cholerae O1 in the Chesapeake Bay, Maryland.

Non-O1/non-O139 Vibrio cholerae inhabits estuarine and coastal waters globally, but its clinical significance has not been sufficiently investigated, despite the fact that it has been associated with septicemia and gastroenteritis. The emergence of virulent non-O1/non-O139 V. cholerae is consistent with the recognition of new pathogenic variants worldwide. Oyster, sediment, and water samples were collected during a vibrio surveillance program carried out from 2009 to 2012 in the Chesapeake Bay, Maryland. V. cholerae O1 was detected by a direct fluorescent-antibody (DFA) assay but was not successfully cultured, whereas 395 isolates of non-O1/non-O139 V. cholerae were confirmed by multiplex PCR and serology. Only a few of the non-O1/non-O139 V. cholerae isolates were resistant to ampicillin and/or penicillin. Most of the isolates were sensitive to all antibiotics tested, and 77 to 90% carried the El Tor variant hemolysin gene hlyAET, the actin cross-linking repeats in toxin gene rtxA, the hemagglutinin protease gene hap, and the type 6 secretion system. About 19 to 21% of the isolates carried the neuraminidase-encoding gene nanH and/or the heat-stable toxin (NAG-ST), and only 5% contained a type 3 secretion system. None of the non-O1/non-O139 V. cholerae isolates contained Vibrio pathogenicity island-associated genes. However, ctxA, ace, or zot was present in nine isolates. Fifty-five different genotypes showed up to 12 virulence factors, independent of the source of isolation, and represent the first report of both antibiotic susceptibility and virulence associated with non-O1/non-O139 V. cholerae from the Chesapeake Bay. Since these results confirm the presence of potentially pathogenic non-O1/non-O139 V. cholerae, monitoring for total V. cholerae, regardless of serotype, should be done within the context of public health.

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.

GENOTYPE AND DRUG RESISTANCE OF CLINICAL AND ENVIRONMENTAL VIBRIO CHOLERAE NON-O1/NON-O139 IN NORTHEASTERN THAILAND.

A total of 124 V cholerae non-O1/non-O139 isolates were collected in Khon Kaen, Thailand from diarrheal patients, asymptomatic carriers and environmental water. The presence of virulence-associated and regulatory genes including ctxA, tcpA, zot, ace, ompU, stn, hlyA and toxR) were examined using multiplex PCR. The genomic diversity of the various V. cholerae isolates were differentiated using the random amplified polymorphic DNA (RAPD) method. Antimicrobial susceptibility was tested using disk diffusion. All of V. cholerae non-O/non-O139 isolates carried hlyA and toxR and none carried ctxA and tcpA. The zot, ace and both genes together were found in 1.6%, 4.7% and 4.7% of 64 clinical V. cholerae non-O1 isolates, respectively, while the environmental ones did not. The stn gene was found in 3.1% (2/64) of the clinical and 3.3% (2/60) of the environmental isolates. The RAPD patterns were differentiated into 45 types (A to 2S). RAPD type A (32.3%) was the most frequently found in both clinical and environmental V cholerae non-O1 strains (34.4% and 30.0%, respectively); indicating that there was a clonal relationship between some clinical and environmental isolates whereas almost all of the environmental isolates belonged to different clones. All strains were sensitive to ciprofloxacin and norfloxacin. The environmental isolates (30%) were more resistant than the clinical ones (21.9%). Resistance to sulfamethoxazole/trimethoprim and tetracycline among the clinical isolates occurred in 9.4% (6/64) in 2007, during which period the prevalence of V cholerae O1 increased. We conclude that V. cholerae non-O1/non-O139 from the aquatic environment are potentially pathogenic and this same aquatic environment may be a source of antimicrobial resistance in V. cholerae.

Dissemination of antibiotic resistance genes through soil-plant-earthworm continuum in the food production environment.

Treated municipal wastewater (TMW) can provide a reliable source of irrigation water for crops, which is especially important in arid areas where water resources are limited or prone to drought. Nonetheless, TMW may contain residual antibiotics, potentially exposing the crops to these substances. The goal of this study was to investigate the dissemination of antibiotics resistance genes (ARGs) in the soil-plant-earthworm continuum after irrigation of spinach and radish plants with TMW containing trimethoprim, sulfamethoxazole, and sulfapyridine in a greenhouse experiment, followed by feeding of earthworms with harvested plant materials. Our results showed that antibiotic resistance genes (ARGs) were enriched in the soil-plant-earthworm microbiomes irrigated with TMW and TMW spiked with higher concentrations of antibiotics. The number of ARGs and antibiotic-resistant bacteria (ARB) enrichment varied with plant type, with spinach harboring a significantly higher amount of ARGs and ARB compared to radish. Our data showed that bulk and rhizosphere soils of spinach and radish plants irrigated with MilliQ water, TMW, TMW10, or TMW100 had significant differences in bacterial community (p < 0.001), ARG (p < 0.001), and virulence factor gene (VFG) (p < 0.001) diversities. The abundance of ARGs significantly decreased from bulk soil to rhizosphere to phyllosphere and endosphere. Using metagenome assembled genomes (MAGs), we recovered many bacterial MAGs and a near complete genome (>90 %) of bacterial MAG of genus Leclercia adecarboxylata B from the fecal microbiome of earthworm that was fed harvested radish tubers and spinach leaves grown on TMW10 irrigated waters, and this bacterium has been shown to be an emerging pathogen causing infection in immunocompromised patients that may lead to health complications and death. Therefore, crops irrigated with TMW containing residual antibiotics and ARGs may lead to increased incidences of enrichment of ARB in the soil-plant-earthworm continuum.

Distribution of virulence genes in clinical and environmental Vibrio cholerae strains in Bangladesh.

Vibrio cholerae, an environmental organism, is a facultative human pathogen. Here, we report the virulence profiles, comprising 18 genetic markers, of 102 clinical and 692 environmental V. cholerae strains isolated in Bangladesh between March 2004 and January 2006, showing the variability of virulence determinants within the context of public health.

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.