A global medical crisis is unfolding as antibiotics lose effectiveness against a growing number of bacterial pathogens. Horizontal gene transfer (HGT) contributes significantly to the rapid spread of ...resistance, yet the transmission dynamics of genes that confer antibiotic resistance are poorly understood. Multiple mechanisms of HGT liberate genes from normal vertical inheritance. Conjugation by plasmids, transduction by bacteriophages, and natural transformation by extracellular DNA each allow genetic material to jump between strains and species. Thus, HGT adds an important dimension to infectious disease whereby an antibiotic resistance gene (ARG) can be the agent of an outbreak by transferring resistance to multiple unrelated pathogens. Here, we review the small number of cases where HGT has been detected in clinical environments. We discuss differences and synergies between the spread of plasmid-borne and chromosomal ARGs, with a special consideration of the difficulties of detecting transduction and transformation by routine genetic diagnostics. We highlight how 11 of the top 12 priority antibiotic-resistant pathogens are known or predicted to be naturally transformable, raising the possibility that this mechanism of HGT makes significant contributions to the spread of ARGs. HGT drives the evolution of untreatable “superbugs” by concentrating ARGs together in the same cell, thus HGT must be included in strategies to prevent the emergence of resistant organisms in hospitals and other clinical settings.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Pathogenicity Island 1 (SPI-1) encodes a type three secretion system (T3SS), effector proteins, and associated transcription factors that together enable invasion of epithelial cells in animal ...intestines. The horizontal acquisition of SPI-1 by the common ancestor of all
is considered a prime example of how gene islands potentiate the emergence of new pathogens with expanded niche ranges. However, the evolutionary history of SPI-1 has attracted little attention. Here, we apply phylogenetic comparisons across the family Enterobacteriaceae to examine the history of SPI-1, improving the resolution of its boundaries and unique architecture by identifying its composite gene modules. SPI-1 is located between the core genes
and
, a hotspot for the gain and loss of horizontally acquired genes. Despite the plasticity of this locus, SPI-1 demonstrates stable residency of many tens of millions of years in a host genome, unlike short-lived homologous T3SS and effector islands including
ETT2,
YSA,
PSI-2,
SSR2, and
CPI-1. SPI-1 employs a unique series of regulatory switches, starting with the dedicated transcription factors HilC and HilD, and flowing through the central SPI-1 regulator HilA. HilA is shared with other T3SS, but HilC and HilD may have their evolutionary origins in
. The
,
, and
gene promoters are the most AT-rich DNA in SPI-1, placing them under tight control by the transcriptional repressor H-NS. In all
lineages, these three promoters resist amelioration towards the genomic average, ensuring strong repression by H-NS. Hence, early development of a robust and well-integrated regulatory network may explain the evolutionary stability of SPI-1 compared to T3SS gene islands in other species.
Public health emergencies like SARS, MERS, and COVID-19 have prioritized surveillance of zoonotic coronaviruses, resulting in extensive genomic characterization of coronavirus diversity in bats. ...Sequencing viral genomes directly from animal specimens remains a laboratory challenge, however, and most bat coronaviruses have been characterized solely by PCR amplification of small regions from the best-conserved gene. This has resulted in limited phylogenetic resolution and left viral genetic factors relevant to threat assessment undescribed. In this study, we evaluated whether a technique called hybridization probe capture can achieve more extensive genome recovery from surveillance specimens. Using a custom panel of 20,000 probes, we captured and sequenced coronavirus genomic material in 21 swab specimens collected from bats in the Democratic Republic of the Congo. For 15 of these specimens, probe capture recovered more genome sequence than had been previously generated with standard amplicon sequencing protocols, providing a median 6.1-fold improvement (ranging up to 69.1-fold). Probe capture data also identified five novel
and
in these specimens, and their full genomes were recovered with additional deep sequencing. Based on these experiences, we discuss how probe capture could be effectively operationalized alongside other sequencing technologies for high-throughput, genomics-based discovery and surveillance of bat coronaviruses.
Horizontal gene transfer by plasmids can confer metabolic capabilities that expand a host cell's niche. Yet, it is less understood whether the coalescence of specialized catabolic functions, ...antibiotic resistances and metal resistances on plasmids provides synergistic benefits. In this study, we report whole-genome assembly and phenotypic analysis of five
strains isolated in the 1980s from milk powder in Munich, Germany. All strains exhibited the unusual phenotype of lactose-fermentation and encoded either of two variants of the
operon. Surprisingly, all strains encoded the mobilized colistin resistance gene 9 (
), long before the first report of this gene in the literature. In two cases, the
gene and the
locus were linked within a large gene island that formed an IncHI2A-type plasmid in one strain but was chromosomally integrated in the other strain. In two other strains, the
gene was found on a large IncHI1B/IncP-type plasmid, whereas the
locus was encoded on a separate chromosomally integrated plasmidic island. The
sequences were identical and genomic contexts could not explain the wide range of colistin resistances exhibited by the
strains. Nucleotide variants did explain phenotypic differences in motility and exopolysaccharide production. The observed linkage of
to lactose metabolism, an array of heavy-metal detoxification systems, and other antibiotic resistance genes may reflect a coalescence of specialized phenotypes that improve the spread of colistin resistance in dairy facilities, much earlier than previously suspected.
Antimicrobial resistance is a disseminated global health challenge because many of the genes that cause resistance can transfer horizontally between bacteria. Despite the central role of ...extrachromosomal DNA elements called plasmids in driving the spread of resistance, the detection and surveillance of plasmids remains a significant barrier in molecular epidemiology. We assessed two DNA sequencing platforms alone and in combination for laboratory diagnostics in Botswana by annotating antibiotic resistance genes and plasmids in extensively drug resistant bacteria from diarrhea in Botswana. Long-read Nanopore DNA sequencing and high accuracy basecalling effectively estimated the architecture and gene content of three plasmids in Escherichia coli HUM3355 and two plasmids in Klebsiella pneumoniae HUM7199. Polishing the assemblies with Illumina reads increased base calling precision with small improvements to gene prediction. All five plasmids encoded one or more antibiotic resistance genes, usually within gene islands containing multiple antibiotic and metal resistance genes, and four plasmids encoded genes associated with conjugative transfer. Two plasmids were almost identical to antibiotic resistance plasmids sequenced in Europe and North America from human infection and a pig farm. These One Health connections demonstrate how low-, middle-, and high-income countries collectively benefit from increased whole genome sequencing capacity for surveillance and tracking of infectious diseases and antibiotic resistance genes that can transfer between animal hosts and move across continents.
The best whole genome assemblies are currently built from a combination of highly accurate short-read sequencing data and long-read sequencing data that can bridge repetitive and problematic regions. ...Oxford Nanopore Technologies (ONT) produce long-read sequencing platforms and they are continually improving their technology to obtain higher quality read data that is approaching the quality obtained from short-read platforms such as Illumina. As these innovations continue, we evaluated how much ONT read coverage produced by the Rapid Barcoding Kit v14 (SQK-RBK114) is necessary to generate high-quality hybrid and long-read-only genome assemblies for a panel of carbapenemase-producing
bacterial isolates. We found that 30× long-read coverage is sufficient if Illumina data are available, and that more (at least 100× long-read coverage is recommended for long-read-only assemblies. Illumina polishing is still improving single nucleotide variants (SNVs) and INDELs in long-read-only assemblies. We also examined if antimicrobial resistance genes could be accurately identified in long-read-only data, and found that Flye assemblies regardless of ONT coverage detected >96% of resistance genes at 100% identity and length. Overall, the Rapid Barcoding Kit v14 and long-read-only assemblies can be an optimal sequencing strategy (i.e., plasmid characterization and AMR detection) but finer-scale analyses (i.e., SNV) still benefit from short-read data.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Carbapenems are considered last-resort antibiotics for the treatment of infections caused by multidrug-resistant Enterobacterales, but carbapenem resistance due to acquisition of carbapenemase genes ...is a growing threat that has been reported worldwide. Klebsiella pneumoniae carbapenemase (blaKPC) is the most common type of carbapenemase in Canada and elsewhere; it can hydrolyze penicillins, cephalosporins, aztreonam, and carbapenems and is frequently found on mobile plasmids in the Tn4401 transposon. This means that alongside clonal expansion, blaKPC can disseminate through plasmid- and transposon-mediated horizontal gene transfer. We applied whole genome sequencing to characterize the molecular epidemiology of 829 blaKPC carbapenemase-producing isolates collected by the Canadian Nosocomial Infection Surveillance Program from 2010 to 2021. Using a combination of short-read and long-read sequencing, we obtained 202 complete and circular blaKPC-encoding plasmids. Using MOB-suite, 10 major plasmid clusters were identified from this data set which represented 87% (175/202) of the Canadian blaKPC-encoding plasmids. We further estimated the genomic location of incomplete blaKPC-encoding contigs and predicted a plasmid cluster for 95% (603/635) of these. We identified different patterns of carbapenemase mobilization across Canada related to different plasmid clusters, including clonal transmission of IncF-type plasmids (108/829, 13%) in K. pneumoniae clonal complex 258 and novel repE(pEh60-7) plasmids (44/829, 5%) in Enterobacter hormaechei ST316, and horizontal transmission of IncL/M (142/829, 17%) and IncN-type plasmids (149/829, 18%) across multiple genera. Our findings highlight the diversity of blaKPC genomic loci and indicate that multiple, distinct plasmid clusters have contributed to blaKPC spread and persistence in Canada.
Carbapenems are last-resort antibiotics for treatment of infections caused by multidrug-resistant Enterobacterales , but carbapenem resistance is a rising global threat due to the acquisition of ...carbapenemase genes. Oxacillinase-48 ( bla OXA-48 )-type carbapenemases are increasing in abundance in Canada and elsewhere; these genes are frequently found on mobile genetic elements and are associated with specific transposons. This means that alongside clonal dissemination, bla OXA-48-type genes can spread through plasmid-mediated horizontal gene transfer. We applied whole genome sequencing to characterize 249 bla OXA-48-type -producing Enterobacterales isolates collected by the Canadian Nosocomial Infection Surveillance Program from 2010 to 2021. Using a combination of short- and long-read sequencing, we obtained 70 complete and circular bla OXA-48-type -encoding plasmids. Using MOB-suite, four major plasmids clustered were identified, and we further estimated a plasmid cluster for 91.9 % (147/160) of incomplete bla OXA-48-type -encoding contigs. We identified different patterns of carbapenemase mobilization across Canada, including horizontal transmission of bla OXA-181 /IncX3 plasmids (75/249, 30.1 %) and bla OXA-48 /IncL/M plasmids (47/249, 18.9 %), and both horizontal transmission and clonal transmission of bla OXA-232 for Klebsiella pneumoniae ST231 on ColE2-type/ColKP3 plasmids (25/249, 10.0 %). Our findings highlight the diversity of OXA-48-type plasmids and indicate that multiple plasmid clusters and clonal transmission have contributed to bla OXA-48-type spread and persistence in Canada.
Carbapenems are considered last-resort antibiotics for the treatment of infections caused by multidrug-resistant
, but carbapenem resistance due to acquisition of carbapenemase genes is a growing ...threat that has been reported worldwide.
carbapenemase (
) is the most common type of carbapenemase in Canada and elsewhere; it can hydrolyze penicillins, cephalosporins, aztreonam, and carbapenems and is frequently found on mobile plasmids in the Tn
transposon. This means that alongside clonal expansion,
can disseminate through plasmid- and transposon-mediated horizontal gene transfer. We applied whole genome sequencing to characterize the molecular epidemiology of 829
carbapenemase-producing isolates collected by the Canadian Nosocomial Infection Surveillance Program from 2010 to 2021. Using a combination of short-read and long-read sequencing, we obtained 202 complete and circular
-encoding plasmids. Using MOB-suite, 10 major plasmid clusters were identified from this data set which represented 87% (175/202) of the Canadian
-encoding plasmids. We further estimated the genomic location of incomplete
-encoding contigs and predicted a plasmid cluster for 95% (603/635) of these. We identified different patterns of carbapenemase mobilization across Canada related to different plasmid clusters, including clonal transmission of IncF-type plasmids (108/829, 13%) in
clonal complex 258 and novel repE(pEh60-7) plasmids (44/829, 5%) in
ST316, and horizontal transmission of IncL/M (142/829, 17%) and IncN-type plasmids (149/829, 18%) across multiple genera. Our findings highlight the diversity of
genomic loci and indicate that multiple, distinct plasmid clusters have contributed to
spread and persistence in Canada.
Bacteria have dynamic genomes that allow them to adapt and survive almost anywhere on Earth. This genetic flexibility is facilitated by mobile DNA elements, which can transfer within or between ...genomes independently of cell lineages and drive bacterial evolution. Mobile genetic elements influence many aspects of bacterial life by encoding and transferring antimicrobial resistance genes, pathogenicity factors, and toxin-antitoxin modules. Plasmids, integrons, and genomic islands are several types of mobile DNA elements that can carry beneficial genes which help host cells adapt to new environments or provide new ecological functions. However, much is still unknown about how mobile genetic elements persist in the environment, how they mediate cell survival, and how they impact interactions in bacterial communities. In this thesis, genomic and experimental techniques were used to investigate the ecology and evolution of mobile genetic elements at three different scales: genes, species, and ecosystems.At the gene level, I performed a literature review to determine how the main processes of horizontal gene transfer (conjugation, transduction, and natural transformation) impact antimicrobial resistance gene transmission in clinical environments. Conjugation of antimicrobial resistance gene-encoding plasmids does occur within clinics and patients, but there is less evidence for transduction and transformation. This may be due to low transfer rates or difficulty detecting the transfer of core genes with key mutations that provide resistance. In another project, I used comparative genomics to assess the evolutionary history of the Salmonella pathogenicity island SPI-1, which encodes a type three secretion system for invasion of mammalian intestinal cells and is remarkably conserved across the Salmonella genus. I defined the multiple genomic islands that comprise the mosaic structure of SPI-1, with some islands having arrived in the Salmonella clade earlier than others. Related pathogenicity islands possess homologs to SPI1 transcriptional regulator hilA, but are missing homologs to hilD and hilC; the high nucleotide identity between hilD and hilCsuggests they may be paralogs.To assess how mobile genetic elements impact communities at the species level, I designed a synthetic multi-species community wherein only Escherichia coli can access a carbon source and Salmonella enterica must rely on cross-feeding to survive while simultaneously killing the E. coliwith a plasmid-encoded colicin toxin. Despite relying on cross-feeding to survive, S. enterica consistently emerged as the dominant community member. Experimental results and mathematical modeling confirmed that the colicin liberates nutrients through cell lysis and benefits the colicin-producing population, which had not been previously described for this type of toxin.At the ecosystem level, I attempted to isolate all of the plasmids (“plasmidome”) from surface water of agricultural ponds (dugouts) to determine how the gene content changes over time using long-read DNA sequencing. However, ongoing technical difficulties caused by environmental contaminants resulted in limited DNA sequencing data and interference in enzymatic reactions during library preparation. Nevertheless, additional insights into these ecosystems were possible through 16S rRNA bacterial community profiling and qPCR of select antimicrobial resistance genes and mobile elements. Antimicrobial resistance genes are detectable and persist over time in dugouts, and regression modeling demonstrated that their abundance was explained by the abundance of the mobile integron integrase intI1 and cattle presence.
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