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"Thomas, Jesse"
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Co‐occurrence of antibiotic, biocide, and heavy metal resistance genes in bacteria from metal and radionuclide contaminated soils at the Savannah River Site
Summary 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. We present a comprehensive analysis of the soil microbiome in several areas affected by legacies of heavy‐metal and radionuclide contamination. We explore how these contaminants affect bacterial community composition and diversity, and the enrichment of antibiotic, biocide, and metal‐resistance genes. We find that these contaminants can not only have a profound impact on soil microbiome composition, but it can also affect how it functions.
Journal Article
Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix)
Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I , we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5′ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html . A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy .
Journal Article
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.
Journal Article
Carbapenem-Resistant, Virulence Plasmid–Harboring Klebsiella pneumoniae, United States
Carbapenem-resistant and virulence plasmid-harboring Klebsiella pneumoniae (pVir-CRKP) has emerged and spread globally, yet clinical investigations from the United States remain limited. We conducted a genomic analysis of 884 unique carbapenem-resistant K. pneumoniae isolates from a multicenter US cohort and identified 6 pVir-CRKP isolates, including 2 sequence type (ST) 23, 2 ST893, and 2 ST11 isolates. Patients infected with pVir-CRKP experienced high Pitt bacteremia scores and a 33% 30-day mortality rate. The pVir-CRKP isolates exhibited significant sequence variation in virulence genes and plasmids, along with differences in mucoviscosity, capsule production, survival in normal human serum, resistance to killing by human polymorphonuclear neutrophils, and in vivo pathogenicity. Phylogenetic analyses showed that most pVir-CRKP isolates were genetically similar to strains reported from other global regions. The emergence of pVir-CRKP with higher virulence potential and carbapenem resistance in the United States than the predominant carbapenem-resistant K. pneumoniae clone underscores the need for active global surveillance.
Journal Article
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.
Resistance of Neisseria gonorrhoeae to extended spectrum cephalosporins is an increasing concern. Here, the authors conduct whole genome sequencing of isolates from the United States and find that most resistant isolates were associated with a persistent circulating lineage.
Journal Article
Evidence of Recent Genomic Evolution in Gonococcal Strains With Decreased Susceptibility to Cephalosporins or Azithromycin in the United States, 2014–2016
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.
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.
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.
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.
Journal Article
Molecular Surveillance for Lymphoproliferative Disease Virus in Wild Turkeys (Meleagris gallopavo) from the Eastern United States
Lymphoproliferative disease virus (LPDV) is a poorly understood, oncogenic avian retrovirus of domestic turkeys that has historically been restricted to Europe and Israel. However, a recent study reported LPDV in multiple wild turkey diagnostic cases from throughout the eastern United States of America (USA). To better understand the distribution of LPDV in the eastern USA, we surveyed 1,164 reportedly asymptomatic hunter-harvested wild turkeys from 17 states for the presence of LPDV proviral DNA by PCR. In total, 564/1,164 (47%) turkeys were positive for LPDV. Wild turkeys from each state had a relatively high prevalence of LPDV, although statewide prevalence varied from 26 to 83%. Phylogenetic analysis revealed two major clades of LPDV in the USA, although one was at a low frequency suggesting restricted transmission, as well as significant clustering by state of isolation. To determine the best tissue to target for diagnostic purposes, liver, spleen, and bone marrow were tested from a subset of 15 hunter-harvested wild turkeys and 20 wild turkey diagnostic cases. Overall, bone marrow provided the highest level of detection for both hunter-harvested turkeys and diagnostic cases. The sensitivity of LPDV detection between tissues was not significantly different for diagnostic cases, but was for hunter-harvested birds. These results indicate that LPDV infection is common and widespread in wild turkey populations throughout the eastern USA, even without overt signs of disease.
Journal Article
Evaluation of a New Carbon Dioxide System for Autonomous Surface Vehicles
Current carbon measurement strategies leave spatiotemporal gaps that hinder the scientific understanding of the oceanic carbon biogeochemical cycle. Data products and models are subject to bias because they rely on data that inadequately capture mesoscale spatiotemporal (kilometers and days to weeks) changes. High-resolution measurement strategies need to be implemented to adequately evaluate the global ocean carbon cycle. To augment the spatial and temporal coverage of ocean–atmosphere carbon measurements, an Autonomous Surface Vehicle CO 2 (ASVCO 2 ) system was developed. From 2011 to 2018, ASVCO 2 systems were deployed on seven Wave Glider and Saildrone missions along the U.S. Pacific and Australia’s Tasmanian coastlines and in the tropical Pacific Ocean to evaluate the viability of the sensors and their applicability to carbon cycle research. Here we illustrate that the ASVCO 2 systems are capable of long-term oceanic deployment and robust collection of air and seawater p CO 2 within ±2 μ atm based on comparisons with established shipboard underway systems, with previously described Moored Autonomous p CO 2 (MAPCO 2 ) systems, and with companion ASVCO 2 systems deployed side by side.
Journal Article