Next-generation sequencing of a multi-drug resistance plasmid captured from stream sediment

Plasmids in agriculturally-impacted bodies of water may play a significant role in the dissemination of antibiotic resistance. Previously, Erika Gehr, as part of her M.S. thesis work in our laboratory, captured environmental plasmids without cultivation of host bacteria from stream sediment into Escherichia coli. Individual plasmids were capable of conferring resistance to a surprising array of antibiotics including aminoglycosides and extended-spectrum β-lactams. In this study, we developed a method to sequence multi-drug resistance plasmids using both Oxford Nanopore MinION and Ion Torrent Personal Genome Machine sequencers. Plasmid pEG1-1 was sequenced on both platforms and a hybrid assembly utilizing data from both sequencing platforms generated a single 73,320 bp contig that was annotated using automated and manual techniques. Analysis of the genome revealed pEG1-1 to be an IncP-1β plasmid with two mobile genetic elements – a a tn21-related transposon and an in104 complex integron – both of which carry multiple antibiotic resistance genes. These findings suggest that plasmids in stream sediment are prone to the incorporation of mobile genetic elements that introduce a broad range of antibiotic resistance genes into their genome. This could cause serious risk to human health since IncP-1β plasmids are capable of transferring into nearly all Gram-negative bacteria, including fecal pathogens that get introduced to stream sediment

Development of an amplicon-based sequencing approach in response to the global emergence of mpox

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.This publication was made possible by CTSA Grant Number UL1 TR001863 from the National Center for Advancing Translational Science (NCATS), a component of the National Institutes of Health (NIH) awarded to CBFV. INSA was partially funded by the HERA project (Grant/ 2021/PHF/23776) supported by the European Commission through the European Centre for Disease Control (to VB).info:eu-repo/semantics/publishedVersio

Assessment of mold contamination in hurricane-damaged homes in Houston, Texas after sanitization by volunteers

The purpose of this pilot study was to evaluate the effectiveness of mold sanitation in homes that suffered hurricane-related water damage. After a home is flooded, sanitation of the structure for mold is necessary before the interior of the home can be rebuilt. In this study, homes (n = 6) in Houston, Texas that had been flooded by Hurricane Harvey were sanitized by volunteers. At either 6, 8, 15, 25, 34, or 56 days after the sanitation was completed, a Button™ sampler was used to collect a 48-hour air sample, so that the mold cells in the air could be quantified. Each air sample was then analyzed by quantitative PCR (qPCR) assays for the 36 molds in the Environmental Relative Moldiness Index (ERMI) panel of indicator molds. Quantifying the 36-ERMI molds in air samples results in “ERMI-like” values. The ERMI-like values in the sanitized homes were inversely correlated (Pearson p - value 0.04) with the log of the number of days after the sanitation was completed, an indication that it takes time after sanitation for the mold levels to stabilize. This pilot study demonstrated that the ERMI-like metric was useful in assessing post-sanitation mold levels in previously flooded homes.&nbsp

Nanopore + Ion Torrent sequencing, assembly, and annotation of culture-free streambed plasmids reveals hitchhiking genes for resistance to multiple human clinical antibiotics

<p>Poster presented at the 2015 ASM NGS meeting.</p

MinION Sequencing of a captured Antibiotic Resistance Plasmid

<p>MinION and Ion Torrent sequencing of a captured environmental antibiotic resistance plasmid. </p

Nanopore Sequencing of Transmissible Tetracyline Plasmids Captured without Cultivation from Stream Sediment Reveals Linked Genes Encoding Resistance to Multiple Human Clinical Antibiotics

<p><strong>Background</strong>. Transmissible plasmids in environmental ecosystems may facilitate the exchange of antibiotic resistance genes between introduced fecal and native bacterial populations. Bacteria in aquatic and soil habitats may thus act as incubators and sites for recombination for genes subsequently transferred to human pathogens.</p> <p><strong>Methods</strong>. Transmissible plasmids were captured “exogenously” from uncultivated stream bacteria by conjugation with E. coli. After electroporation, plasmids were tested for decreased antibiotic susceptibility using a modified Stokes disk diffusion method. In addition to tetracycline, antibiotics tested were chosen based upon their use in human clinical settings. Two multiresistance plasmids were sequenced using an Oxford Nanopore MinION DNA sequencer, assembled using the PBcR pipeline of the Celera assembler, and annotated using Prokka. Both plasmids were also sequenced on an Ion Torrent Personal Genome Machine.</p> <p><strong>Results</strong>. Twenty-three of 30 plasmids conferred decreased susceptibility to multiple antibiotics in addition to tetracycline. The most common phenotypic resistance profiles were tetR/kanamycinR/ticarcillinR/piperacillinR, and tetR/kanR/ticR/pipR/cefipimeR. One plasmid conferred decreased susceptibility to seven of 12 tested antibiotics (tet, kan, tic, tzp, pip, fep, and tobramycin). Nanopore sequencing and assembly of this plasmid resulted in two ca. 15-kb contigs. Contigs were screened for resistance genes against the ResFinder database. The following genes were identified with >93% identify: sul1 (2X) – suggesting the presence of one or more Class 1 integrons – tetC, tetG, aadA9, aadA2, sfloR, aph(3')-Ic, strB, and blaCARB-2. A second plasmid exhibiting decreased susceptibility to tet, kan, tic, pip, and ciprofloxacin was also sequenced.</p> <p><strong>Conclusions</strong>. The presence of genes encoding resistance to multiple human clinical antibiotics on transmissible plasmids selected using tetracycline suggests that there may be a significant reservoir of such genes in stream sediments, that they can be selected by tetracycline use, and that they may be capable of transmission to pathogenic Enterobacteriaceae.</p

Assessing the Incidence of Plasmid-borne Resistance to Clinically-Significant Antibiotics in Stream Sediments

<p>Plasmids in agriculturally-impacted bodies of water may play a significant role in the dissemination of antibiotic resistance (AR). High bacterial loads in stream sediment and selective pressures introduced by agricultural practices may facilitate the exchange and recombination of genetic material, creating reservoirs of AR genes that can potentially be accessed by fecal and other animal and human pathogens. </p> <p>  Transmissible plasmids were captured “exogenously” from stream sediment samples by conjugating sediment cells with a rifampicin-resistant strain of Escherichia coli. Transconjugants were tested for decreased antibiotic susceptibility using a modified Stokes disk diffusion method.  Twenty-three of thirty captured plasmids conferred decreased susceptibility to multiple antibiotics in addition to tetracycline. </p> <p>  One plasmid, pEG1-1, conferred resistance to tetracycline, tobramycin, kanamycin, ticarcilin, piperacillin, piperacillin-tazobactam, and cefepime. A method to sequence multi-drug resistance plasmids using both Oxford Nanopore MinION and Ion Torrent Personal Genome Machine sequencers was developed to sequence plasmid pEG1-1. A hybrid assembly generated a single 73,320 bp contig. Analysis of the genome revealed pEG1-1 to be an IncP-1β plasmid with two mobile genetic elements – a tn21-related transposon and an in104 complex integron – both of which carry multiple antibiotic resistance genes. </p> <p>  These findings suggest that plasmids in stream sediment are prone to the incorporation of mobile genetic elements that introduce a broad range of antibiotic resistance genes into their genome. This could cause serious risk to human health since IncP-1β plasmids are capable of transferring into nearly all Gram-negative bacteria, including fecal pathogens that get introduced to stream sediment.</p

GAMBIT (Genomic Approximation Method for Bacterial Identification and Tracking): A methodology to rapidly leverage whole genome sequencing of bacterial isolates for clinical identification.

Whole genome sequencing (WGS) of clinical bacterial isolates has the potential to transform the fields of diagnostics and public health. To realize this potential, bioinformatic software that reports identification results needs to be developed that meets the quality standards of a diagnostic test. We developed GAMBIT (Genomic Approximation Method for Bacterial Identification and Tracking) using k-mer based strategies for identification of bacteria based on WGS reads. GAMBIT incorporates this algorithm with a highly curated searchable database of 48,224 genomes. Herein, we describe validation of the scoring methodology, parameter robustness, establishment of confidence thresholds and the curation of the reference database. We assessed GAMBIT by way of validation studies when it was deployed as a laboratory-developed test in two public health laboratories. This method greatly reduces or eliminates false identifications which are often detrimental in a clinical setting

Effect of Manure Application on Abundance of Antibiotic Resistance Genes and Their Attenuation Rates in Soil: Field-Scale Mass Balance Approach

The development of models for understanding antibiotic resistance gene (ARG) persistence and transport is a critical next step toward informing mitigation strategies to prevent the spread of antibiotic resistance in the environment. A field study was performed that used a mass balance approach to gain insight into the transport and dissipation of ARGs following land application of manure. Soil from a small drainage plot including a manure application site, an unmanured control site, and an adjacent stream and buffer zone were sampled for ARGs and metals before and after application of dairy manure slurry and a dry stack mixture of equine, bovine, and ovine manure. Results of mass balance suggest growth of bacterial hosts containing ARGs and/or horizontal gene transfer immediately following slurry application with respect to <i>ermF</i>, <i>sul1</i>, and <i>sul2</i> and following a lag (13 days) for dry-stack-amended soils. Generally no effects on <i>tet</i>(G), <i>tet</i>(O), or <i>tet</i>(W) soil concentrations were observed despite the presence of these genes in applied manure. Dissipation rates were fastest for <i>ermF</i> in slurry-treated soils (logarithmic decay coefficient of −3.5) and for <i>sul1</i> and <i>sul2</i> in dry-stack-amended soils (logarithmic decay coefficients of −0.54 and −0.48, respectively), and evidence for surface and subsurface transport was not observed. Results provide a mass balance approach for tracking ARG fate and insights to inform modeling and limiting the transport of manure-borne ARGs to neighboring surface water