Litter Commensal Bacteria Can Limit the Horizontal Gene Transfer of Antimicrobial Resistance to Salmonella in Chickens.

Fostering a "balanced" gut microbiome through the administration of beneficial microbes that can competitively exclude pathogens has gained a lot of attention and use in human and animal medicine. However, little is known about how microbes affect the horizontal gene transfer of antimicrobial resistance (AMR). To shed more light on this question, we challenged neonatal broiler chicks raised on reused broiler chicken litter-a complex environment made up of decomposing pine shavings, feces, uric acid, feathers, and feed-with Salmonella enterica serovar Heidelberg (S. Heidelberg), a model pathogen. Neonatal chicks challenged with S. Heidelberg and raised on reused litter were more resistant to S. Heidelberg cecal colonization than chicks grown on fresh litter. Furthermore, chicks grown on reused litter were at a lower risk of colonization with S. Heidelberg strains that encoded AMR on IncI1 plasmids. We used 16S rRNA gene sequencing and shotgun metagenomics to show that the major difference between chicks grown on fresh litter and those grown on reused litter was the microbiome harbored in the litter and ceca. The microbiome of reused litter samples was more uniform and enriched in functional pathways related to the biosynthesis of organic and antimicrobial molecules than that in fresh litter samples. We found that Escherichia coli was the main reservoir of plasmids encoding AMR and that the IncI1 plasmid was maintained at a significantly lower copy per cell in reused litter compared to fresh litter. These findings support the notion that commensal bacteria play an integral role in the horizontal transfer of plasmids encoding AMR to pathogens like Salmonella. IMPORTANCE Antimicrobial resistance spread is a worldwide health challenge, stemming in large part from the ability of microorganisms to share their genetic material through horizontal gene transfer. To address this issue, many countries and international organizations have adopted a One Health approach to curtail the proliferation of antimicrobial-resistant bacteria. This includes the removal and reduction of antibiotics used in food animal production and the development of alternatives to antibiotics. However, there is still a significant knowledge gap in our understanding of how resistance spreads in the absence of antibiotic selection and the role commensal bacteria play in reducing antibiotic resistance transfer. In this study, we show that commensal bacteria play a key role in reducing the horizontal gene transfer of antibiotic resistance to Salmonella, provide the identity of the bacterial species that potentially perform this function in broiler chickens, and also postulate the mechanism involved.

Sustained Transmission of Neisseria gonorrhoeae with High-Level Resistance to Azithromycin, in Indianapolis, Indiana, 2017-2018.

BACKGROUND: Since 2014, Neisseria gonorrhoeae azithromycin (AZM) susceptibility has declined in the United States, but high-level AZM resistance (HL-AZMR) has been infrequent and sporadic. We describe a cluster of 14 N. gonorrhoeae isolates with HL-AZMR identified in Indianapolis over 13 months. METHODS: N. gonorrhoeae culture specimens (genital and extragenital) were collected from attendees of the Bell Flower Clinic. Isolates underwent antimicrobial susceptibility testing (AST) using Etest. AZM minimum inhibitory concentrations ≥256 µg/mL were classified as HL-AZMR. Local disease intervention specialists interviewed patients whose isolates demonstrated HL-AZMR and conducted partner services. Relatedness of isolates was investigated by genomic analyses. RESULTS: During 2017-2018, AST was performed in 1016 N. gonorrhoeae isolates collected at the Bell Flower Clinic. Fourteen isolates (1.4%) from 12 men collected over 13 months demonstrated HL-AZMR; all were cephalosporin susceptible. Of the 12 men, 9 were white and reported male sex partners. Nine of the men were able to be retested; all were cured with 250-mg ceftriaxone plus 1-g AZM. Two men named each other as partners; no other partners in common were reported. Genomic analysis demonstrated close relatedness of the HL-AZMR isolates and a novel combination of a mosaic-mtrR promoter along with 23S ribosomal RNA mutations that appear to have emerged from circulating strains. CONCLUSIONS: The close genetic relatedness with limited epidemiologic linkages between patients highlights the challenges of gonorrhea partner investigations and suggests undetected local transmission. Local AST, rapid public health action, and epidemiologic investigations combined with genomic analysis provides a multipronged approach to understanding an outbreak of sexually transmitted disease.

Escaping the fate of Sisyphus: assessing resistome hybridization baits for antimicrobial resistance gene capture.

Finding, characterizing and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19 933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement.

Genomic Characterization of Neisseria gonorrhoeae Strains from 2016 U.S. Sentinel Surveillance Displaying Reduced Susceptibility to Azithromycin.

In 2016, the proportion of Neisseria gonorrhoeae isolates with reduced susceptibility to azithromycin rose to 3.6%. A phylogenetic analysis of 334 N. gonorrhoeae isolates collected in 2016 revealed a single, geographically diverse lineage of isolates with MICs of 2 to 16 µg/ml that carried a mosaic-like mtr locus, whereas the majority of isolates with MICs of ≥16 µg/ml appeared sporadically and carried 23S rRNA mutations. Continued molecular surveillance of N. gonorrhoeae isolates will identify new resistance mechanisms.

Microbiota of Four Tissue Types in American Alligators (Alligator mississippiensis) Following Extended Dietary Selenomethionine Exposure.

Selenium represents an essential trace nutrient that is necessary for biological functions. Deficiencies can induce disease, but excess can induce toxicity. Selenium deficiency is a major concern in underdeveloped countries, while also posing as a toxic pollutant in waterways surrounding landfills, agricultural areas, and fossil fuel production sites. We examined the microbiome of selenomethionine (SeMet) fed American alligators (Alligator mississippiensis) at the beginning and end of a 7-week exposure experiment. Alligators were randomly divided into three groups: control and 1000 or 2000 ppm SeMet. DNA from before exposure (oral and cloaca swabs) and post-exposure (oral, cloaca, small & large intestines) sampling were extracted and amplified for bacterial 16 s rRNA. While treatment did not seem to have much effect, we observed a predominance of Fusobacteriaceae and Porpyromonodaceae across all tissue types. Cetobacterium and Clostridium are the most abundant genera as potential indicators of the aquatic and carrion feeding lifestyle of alligators.

Evidence of Recent Genomic Evolution in Gonococcal Strains With Decreased Susceptibility to Cephalosporins or Azithromycin in the United States, 2014-2016.

BACKGROUND: Given the lack of new antimicrobials or a vaccine, understanding the evolutionary dynamics of Neisseria gonorrhoeae is a significant public and global health priority. We investigated the emergence and spread of gonococcal strains with decreased susceptibility to cephalosporins and azithromycin using detailed genomic analyses of gonococcal isolates collected in the United States, 2014-2016. METHODS: We sequenced genomes of 649 isolates collected through the Gonococcal Isolate Surveillance Project. We examined the genetic relatedness of isolates and assessed associations between clades and various genotypic and phenotypic combinations. RESULTS: We identified a large and clonal lineage of strains (MLST ST9363) associated with elevated azithromycin minimum inhibitory concentration (AZIem), characterized by a mosaic mtr locus (C substitution in the mtrR promoter, mosaic mtrR and mtrD). Mutations in 23S rRNA were sporadically distributed among AZIem strains. Another clonal group (MLST ST1901) possessed 7 unique PBP2 patterns, and it shared common mutations in other genes associated with cephalosporin resistance. CONCLUSIONS: Whole-genome sequencing methods can enhance monitoring of antimicrobial resistant gonococcal strains by identifying gonococcal populations containing mutations of concern. These methods could inform the development of point-of-care diagnostic tests designed to determine the specific antibiotic susceptibility profile of a gonococcal infection in a patient.

Horizontal Gene Transfer and Acquired Antibiotic Resistance in Salmonella enterica Serovar Heidelberg following In Vitro Incubation in Broiler Ceca.

The chicken gastrointestinal tract harbors microorganisms that play a role in the health and disease status of the host. The cecum is the part of the gut that carries the highest microbial densities, has the longest residence time of digesta, and is a vital site for urea recycling and water regulation. Therefore, the cecum provides a rich environment for bacteria to horizontally transfer genes between one another via mobile genetic elements such as plasmids and bacteriophages. In this study, we used broiler chicken cecum as a model to investigate antibiotic resistance genes that can be transferred in vitro from cecal flora to Salmonella enterica serovar Heidelberg. We used whole-genome sequencing and resistome enrichment to decipher the interactions between S Heidelberg, the gut microbiome, and acquired antibiotic resistance. After 48 h of incubation of ceca under microaerophilic conditions, we recovered one S Heidelberg isolate with an acquired IncK2 plasmid (88 kb) carrying an extended-spectrum-ß-lactamase gene (blaCMY-2). In vitro, this plasmid was transferable between Escherichia coli and S Heidelberg strains but transfer was unsuccessful between S Heidelberg strains. An in-depth genetic characterization of transferred plasmids suggests that they share significant homology with P1-like phages. This study contributes to our understanding of horizontal gene transfer between an important foodborne pathogen and the chicken gut microbiome.IMPORTANCES. Heidelberg is a clinically important serovar, linked to foodborne illness and among the top 5 serovars isolated from poultry in the United States and Canada. Acquisition of new genetic material from the microbial flora in the gastrointestinal tract of food animals, including broilers, may contribute to increased fitness of pathogens like S. Heidelberg and may increase their level of antibiotic tolerance. Therefore, it is critical to gain a better understanding of the interactions that occur between important pathogens and the commensals present in the animal gut and other agroecosystems. In this report, we show that the native flora in broiler ceca were capable of transferring mobile genetic elements carrying the AmpC ß-lactamase (blaCMY-2) gene to an important foodborne pathogen, S Heidelberg. The potential role for bacteriophage transduction is also discussed.

Promoting Value and Reducing Costs in Medicaid Transformation.

The success of Medicaid transformation in North Carolina depends on participating health plans' ability to bring about better value to deliver on the Triple Aim of health care. Blue Cross and Blue Shield of North Carolina, working in collaboration with Amerigroup Partnership Plan, LLC, is making value-based care a cornerstone of its approach to serving the state's Medicaid population.

Prodrugs of imidazotriazine and pyrrolotriazine C-nucleosides can increase anti-HCV activity and enhance nucleotide triphosphate concentrations in vitro.

A number of prodrugs of HCV-active purine nucleoside analogues 2'-C-methyl 4-aza-9-deaza adenosine 1, 2'-C-methyl 4-aza-7,9-dideaza adenosine 2, 2'-C-methyl 4-aza-9-deaza guanosine 3 and 2'-C-methyl 4-aza-7,9-dideaza guanosine 4 were prepared and evaluated to improve potency, selectivity and liver targeting. Phosphoramidate guanosine prodrugs (3a-3k and 4a, b) showed insufficient cell activity for further profiling. Striking enhancement in replicon activity relative to the parent was observed for phosphoramidate imidazo[2,1-f][1,2,4]triazine-4-amine adenosine prodrugs (1a-1p), but this was accompanied by an increase in cytotoxicity. Improved or similar potency without a concomitant increase in toxicity relative to the parent was demonstrated for phosphoramidate pyrrolo[2,1-f][1,2,4]triazine-4-amine adenosine prodrugs (2a-2k). Carbamate, ester and mixed prodrugs of 2 showed mixed results. Selected prodrugs of 2 were analysed for activation to the triphosphate, with most demonstrating much better activation in hepatocytes over replicon cells. The best activation was observed for a mixed phosphoramidate-3'ester (11) followed by a simple 3'-ester (10).

Global Emergence and Dissemination of Neisseria gonorrhoeae ST-9363 Isolates with Reduced Susceptibility to Azithromycin.

Neisseria gonorrhoeae multilocus sequence type (ST) 9363 core-genogroup isolates have been associated with reduced azithromycin susceptibility (AZMrs) and show evidence of clonal expansion in the United States. Here, we analyze a global collection of ST-9363 core-genogroup genomes to shed light on the emergence and dissemination of this strain. The global population structure of ST-9363 core-genogroup falls into three lineages: Basal, European, and North American; with 32 clades within all lineages. Although, ST-9363 core-genogroup is inferred to have originated from Asia in the mid-19th century; we estimate the three modern lineages emerged from Europe in the late 1970s to early 1980s. The European lineage appears to have emerged and expanded from around 1986 to 1998, spreading into North America and Oceania in the mid-2000s with multiple introductions, along with multiple secondary reintroductions into Europe. Our results suggest two separate acquisition events of mosaic mtrR and mtrR promoter alleles: first during 2009-2011 and again during the 2012-2013 time, facilitating the clonal expansion of this core-genogroup with AZMrs in the United States. By tracking phylodynamic evolutionary trajectories of clades that share distinct demography as well as population-based genomic statistics, we demonstrate how recombination and selective pressures in the mtrCDE efflux operon granted a fitness advantage to establish ST-9363 as a successful gonococcal lineage in the United States and elsewhere. Although it is difficult to pinpoint the exact timing and emergence of this young core-genogroup, it remains critically important to continue monitoring it, as it could acquire additional resistance markers.

Phylogenomic Comparison of Neisseria gonorrhoeae Causing Disseminated Gonococcal Infections and Uncomplicated Gonorrhea in Georgia, United States.

Disseminated gonococcal infection (DGI) is a rare complication caused by the systemic dissemination of Neisseria gonorrhoeae to normally sterile anatomical sites. Little is known about the genetic diversity of DGI gonococcal strains and how they relate to other gonococcal strains causing uncomplicated mucosal infections. We used whole genome sequencing to characterize DGI isolates (n = 30) collected from a surveillance system in Georgia, United States, during 2017-2020 to understand phylogenetic clustering among DGI as well as uncomplicated uro- and extragenital gonococcal infection (UGI) isolates (n = 110) collected in Fulton County, Georgia, during 2017-2019. We also investigated the presence or absence of genetic markers related to antimicrobial resistance (AMR) as well as surveyed the genomes for putative virulence genetic factors associated with normal human-serum (NHS) resistance that might facilitate DGI. We found that DGI strains demonstrated significant genetic variability similar to the population structure of isolates causing UGI, with sporadic incidences of geographically clustered DGI strains. DGI isolates contained various AMR markers and genetic mechanisms associated with NHS resistance. DGI isolates had a higher frequency of the porB1A allele compared with UGI (67% vs 9%, P < .0001); however, no single NHS resistance marker was found in all DGI isolates. Continued DGI surveillance with genome-based characterization of DGI isolates is necessary to better understand specific factors that promote systemic dissemination.

Phylogenomic analysis reveals persistence of gonococcal strains with reduced-susceptibility to extended-spectrum cephalosporins and mosaic penA-34.

The recent emergence of strains of Neisseria gonorrhoeae associated with treatment failures to ceftriaxone, the foundation of current treatment options, has raised concerns over a future of untreatable gonorrhea. Current global data on gonococcal strains suggest that several lineages, predominately characterized by mosaic penA alleles, are associated with elevated minimum inhibitory concentrations (MICs) to extended spectrum cephalosporins (ESCs). Here we report on whole genome sequences of 813 N. gonorrhoeae isolates collected through the Gonococcal Isolate Surveillance Project in the United States. Phylogenomic analysis revealed that one persisting lineage (Clade A, multi-locus sequence type [MLST] ST1901) with mosaic penA-34 alleles, contained the majority of isolates with elevated MICs to ESCs. We provide evidence that an ancestor to the globally circulating MLST ST1901 clones potentially emerged around the early to mid-20th century (1944, credibility intervals [CI]: 1935-1953), predating the introduction of cephalosporins, but coinciding with the use of penicillin. Such results indicate that drugs with novel mechanisms of action are needed as these strains continue to persist and disseminate globally.

Improved Microbial Community Characterization of 16S rRNA via Metagenome Hybridization Capture Enrichment.

Environmental microbial diversity is often investigated from a molecular perspective using 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics. While amplicon methods are fast, low-cost, and have curated reference databases, they can suffer from amplification bias and are limited in genomic scope. In contrast, shotgun metagenomic methods sample more genomic regions with fewer sequence acquisition biases, but are much more expensive (even with moderate sequencing depth) and computationally challenging. Here, we develop a set of 16S rRNA sequence capture baits that offer a potential middle ground with the advantages from both approaches for investigating microbial communities. These baits cover the diversity of all 16S rRNA sequences available in the Greengenes (v. 13.5) database, with no sequence having <78% sequence identity to at least one bait for all segments of 16S. The use of our baits provide comparable results to 16S amplicon libraries and shotgun metagenomic libraries when assigning taxonomic units from 16S sequences within the metagenomic reads. We demonstrate that 16S rRNA capture baits can be used on a range of microbial samples (i.e., mock communities and rodent fecal samples) to increase the proportion of 16S rRNA sequences (average > 400-fold) and decrease analysis time to obtain consistent community assessments. Furthermore, our study reveals that bioinformatic methods used to analyze sequencing data may have a greater influence on estimates of community composition than library preparation method used, likely due in part to the extent and curation of the reference databases considered. Thus, enriching existing aliquots of shotgun metagenomic libraries and obtaining modest numbers of reads from them offers an efficient orthogonal method for assessment of bacterial community composition.

Unveiling the Gut Microbiota and Resistome of Wild Cotton Mice, Peromyscus gossypinus, from Heavy Metal- and Radionuclide-Contaminated Sites in the Southeastern United States.

The prevalence of antibiotic resistance genes (ARGs) can be driven by direct selection from antibiotic use and indirect selection from substances such as heavy metals (HMs). While significant progress has been made to characterize the influence of HMs on the enrichment and dissemination of ARGs in the environment, there is still much we do not know. To fill this knowledge gap, we present a comprehensive analysis of gut bacteria associated with wild cotton mice (Peromyscus gossypinus) trapped from several areas affected by legacies of HM and radionuclide contamination. We explore how these contaminants affect gut microbial community (GMC) composition and diversity and the enrichment of antibiotic, biocide, and metal resistance genes. Although we were able to identify that a myriad of co-occurring antimicrobial and HM resistance genes appear in mice from all areas, including those without a history of contamination, the proportions of co-occurring ARGs and metal resistance genes (MRGs) are higher in sites with radionuclide contamination. These results support those from several previous studies and enhance our understanding of the coselection process, while providing new insights into the ubiquity of antimicrobial resistance in the resistome of wild animals. IMPORTANCE Antimicrobial resistance is a serious global public health concern because of its prevalence and ubiquitous distribution. The rapid dissemination of antibiotic resistance genes is thought to be the result of the massive overuse of antibiotics in agriculture and therapeutics. However, previous studies have demonstrated that the spread of antibiotic resistance genes can also be influenced by heavy metal contamination. This coselection phenomenon, whereby different resistance determinants are genetically linked on the same genetic element (coresistance) or a single genetic element provides resistance to multiple antimicrobial agents (cross-resistance), has profound clinical and environmental implications. In contrast to antibiotics, heavy metals can persist in the environment as a selection pressure for long periods of time. Thus, it is important to understand how antibiotic resistance genes are distributed in the environment and to what extent heavy metal contaminants may be driving their selection, which we have done in one environmental setting.

Horizontal Gene Transfer Is the Main Driver of Antimicrobial Resistance in Broiler Chicks Infected with Salmonella enterica Serovar Heidelberg.

The overuse and misuse of antibiotics in clinical settings and in food production have been linked to the increased prevalence and spread of antimicrobial resistance (AR). Consequently, public health and consumer concerns have resulted in a remarkable reduction in antibiotics used for food animal production. However, there are no data on the effectiveness of antibiotic removal in reducing AR shared through horizontal gene transfer (HGT). In this study, we used neonatal broiler chicks and Salmonella enterica serovar Heidelberg, a model food pathogen, to test if chicks raised antibiotic free harbor transferable AR. We challenged chicks with an antibiotic-susceptible S. Heidelberg strain using various routes of inoculation and determined if S. Heidelberg isolates recovered carried plasmids conferring AR. We used antimicrobial susceptibility testing and whole-genome sequencing (WGS) to show that chicks grown without antibiotics harbored an antimicrobial resistant S. Heidelberg population at 14 days after challenge and chicks challenged orally acquired AR at a higher rate than chicks inoculated via the cloaca. Using 16S rRNA gene sequencing, we found that S. Heidelberg infection perturbed the microbiota of broiler chicks, and we used metagenomics and WGS to confirm that a commensal Escherichia coli population was the main reservoir of an IncI1 plasmid acquired by S. Heidelberg. The carriage of this IncI1 plasmid posed no fitness cost to S. Heidelberg but increased its fitness when exposed to acidic pH in vitro. These results suggest that HGT of plasmids carrying AR shaped the evolution of S. Heidelberg and that antibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella enterica. IMPORTANCE The reported increase in antibiotic-resistant bacteria in humans has resulted in a major shift away from antibiotic use in food animal production. This shift has been driven by the assumption that removing antibiotics will select for antibiotic susceptible bacterial taxa, which in turn will allow the currently available antibiotic arsenal to be more effective. This change in practice has highlighted new questions that need to be answered to assess the effectiveness of antibiotic removal in reducing the spread of antibiotic resistance bacteria. This research demonstrates that antibiotic-susceptible Salmonella enterica serovar Heidelberg strains can acquire multidrug resistance from commensal bacteria present in the gut of neonatal broiler chicks, even in the absence of antibiotic selection. We demonstrate that exposure to acidic pH drove the horizontal transfer of antimicrobial resistance plasmids and suggest that simply removing antibiotics from food animal production might not be sufficient to limit the spread of antimicrobial resistance.

Azithromycin susceptibility of Neisseria gonorrhoeae in the USA in 2017: a genomic analysis of surveillance data.

BACKGROUND: The number of cases of gonorrhoea in the USA and worldwide caused by Neisseria gonorrhoeae is increasing (555 608 reported US cases in 2017, and 87 million cases worldwide in 2016). Many countries report declining in vitro susceptibility of azithromycin, which is a concern because azithromycin and ceftriaxone are the recommended dual treatment in many countries. We aimed to identify strain types associated with decreased susceptibility to azithromycin. METHODS: We did a genomic analysis of N gonorrhoeae isolates obtained by the US Gonococcal Isolate Surveillance Project. Isolates were whole-genome sequenced based on decreased susceptibility to azithromycin (minimal inhibitory concentration [MIC] ≥2 µg/mL, using agar dilution antibiotic susceptibility testing) and geographical representation. Bioinformatic analyses established genomic diversity, strain population dynamics, and antimicrobial resistance profiles. FINDINGS: 410 isolates were sorted into more than 20 unique phylogenetic clades. One predominant persistent clade (consisting of 97 isolates) included the most isolates with azithromycin MICs of 2 µg/mL or higher (61 of 97 [63%] vs 59 of 311 [19%]; p<0·0001) and carried a mosaic mtr (multiple transferable resistance) locus (68 of 97 [70%] vs two of 313 [1%]; p<0·0001). Of the remaining 313 isolates, 57 (18%) had decreased susceptibility to azithromycin (MIC ≥4 µg/mL), which was attributed to 23S rRNA variants (56 of 57 [98%]) and formed phylogenetically diverse clades, showing various levels of clonal expansion. INTERPRETATION: Reduced azithromycin susceptibility was associated with expanding and persistent clades harbouring two well described resistance mechanisms, mosaic mtr locus and 23S rRNA variants. Understanding the role of recombination, particularly within the mtr locus, on the fitness and expansion of strains with decreased susceptibility has important implications for the public health response to minimise gonorrhoea transmission. FUNDING: US Centers for Disease Control and Prevention (CDC), CDC Combating Antibiotic Resistant Bacteria initiative, Oak Ridge Institute for Science Education, US Department of Energy/CDC/Emory University, National Institutes of Health, and Biomedical Laboratory Research and Development Service of the US Department of Veterans Affairs.

Co-occurrence of antibiotic, biocide, and heavy metal resistance genes in bacteria from metal and radionuclide contaminated soils at the Savannah River Site.

Contaminants such as heavy metals may contribute to the dissemination of antimicrobial resistance (AMR) by enriching resistance gene determinants via co-selection mechanisms. In the present study, a survey was performed on soils collected from four areas at the Savannah River Site (SRS), South Carolina, USA, with varying contaminant profiles: relatively pristine (Upper Three Runs), heavy metals (Ash Basins), radionuclides (Pond B) and heavy metal and radionuclides (Tim's Branch). Using 16S rRNA gene amplicon sequencing, we explored the structure and diversity of soil bacterial communities. Sites with legacies of metal and/or radionuclide contamination displayed significantly lower bacterial diversity compared to the reference site. Metagenomic analysis indicated that multidrug and vancomycin antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) including those associated with copper, arsenic, iron, nickel and zinc were prominent in all soils including the reference site. However, significant differences were found in the relative abundance and diversity of certain ARGs and MRGs in soils with metal/radionuclide contaminated soils compared to the reference site. Co-occurrence patterns revealed significant ARG/MRG subtypes in predominant soil taxa including Acidobacteriaceae, Bradyrhizobium, Mycobacterium, Streptomyces, Verrumicrobium, Actinomadura and Solirubacterales. Overall, the study emphasizes the potential risk of human activities on the dissemination of AMR in the environment.

Regional biogeography of microbiota composition in the Chagas disease vector Rhodnius pallescens.

BACKGROUND: Triatomine bugs are vectors of the protozoan parasite Trypanosoma cruzi, which causes Chagas disease. Rhodnius pallescens is a major vector of Chagas disease in Panama. Understanding the microbial ecology of disease vectors is important in the development of vector management strategies that target vector survival and fitness. In this study we examined the whole-body microbial composition of R. pallescens from three locations in Panama. METHODS: We collected 89 R. pallescens specimens using Noireau traps in Attalea butyracea palms. We then extracted total DNA from whole-bodies of specimens and amplified bacterial microbiota using 16S rRNA metabarcoding PCR. The 16S libraries were sequenced on an Illumina MiSeq and analyzed using QIIME2 software. RESULTS: We found Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes to be the most abundant bacterial phyla across all samples. Geographical location showed the largest difference in microbial composition with northern Veraguas Province having the most diversity and Panama Oeste Province localities being most similar to each other. Wolbachia was detected in high abundance (48-72%) at Panama Oeste area localities with a complete absence of detection in Veraguas Province. No significant differences in microbial composition were detected between triatomine age class, primary blood meal source, or T. cruzi infection status. CONCLUSIONS: We found biogeographical regions differ in microbial composition among R. pallescens populations in Panama. While overall the microbiota has bacterial taxa consistent with previous studies in triatomine microbial ecology, locality differences are an important observation for future studies. Geographical heterogeneity in microbiomes of vectors is an important consideration for future developments that leverage microbiomes for disease control.