Biology subjects

Roberts, A. P.

Publications and source records attributed to Roberts, A. P..

29 records · Page 2Linked to original sources

bioRxiv2020

Integron Gene Cassettes Harboring Novel Variants of D-Alanine-D-Alanine Ligase Confer High-level Resistance to D-Cycloserine

During a PCR-based screen of antibiotic resistance genes (ARGs) associated with integrons in saliva-derived metagenomic DNA of healthy human volunteers, two novel variants of genes encoding a D-alanine-D-alanine ligase (ddl6 and ddl7) located within gene cassettes in the first position of a reverse integron were identified. Treponema denticola was identified as the likely host of the ddl cassettes. Both ddl6 and ddl7 conferred high level resistance to D-cycloserine when expressed in Escherichia coli with ddl7 conferring four-fold higher resistance to D-cycloserine compared to ddl6. A SNP was found to be responsible for this difference in resistance phenotype between both ddl variants. Molecular dynamics simulations were used to explain the mechanism of this phenotypic change at the atomic scale. A hypothesis for the evolutionary selection of ddl containing integron gene cassettes is proposed, based on molecular docking of plant metabolites within the ATP and D-cycloserine binding pockets of Ddl. Significance StatementThe drivers of antimicrobial resistance are varied and numerous. One such hypothesis is that compounds not normally considered to be antibiotics may be able to select for genes that can provide resistance to conventional antibiotics. Here we provide evidence that dietary flavonoids are likely to provide the selective pressure for two novel variants of the ddl gene, encoding D-alanine-D-alanine ligases, to be maintained as the first gene cassette of a reverse integron detectable in the human oral cavities from both the UK and Bangladesh. We show that Ddl is functional, able to confer resistance to D-cycloserine and that the dietary flavonoids quercetin and apigenin are likely able to compete with both ATP and D-cycloserine within their Ddl binding sites.

microbiology↗

bioRxiv2020

A novel hemA mutation is responsible for small colony variant phenotype in Escherichia coli.

We identified a small colony variant (SCV) of a clinical isolate of Escherichia coli from Malawi following sequential in vitro selection in sub-inhibitory concentrations of amoxicillin-clavulanic acid and gentamicin. The SCV was auxotrophic for hemin and had impaired biofilm formation compared to the ancestral isolates. A single novel nucleotide polymorphism (SNP) in hemA, which encodes a glutamyl-tRNA reductase responsible for the initial step of porphyrin biosynthesis leading to the production of haem, was responsible for the SCV phenotype. We showed this phenotype was stable over multiple generations and the SNP in hemA resulted in a significant fitness cost to the isolate which persisted even in the presence of hemin. As hemA is not found in mammalian cells, and disruption of the gene results in impaired biofilm formation and a significant fitness cost, it represents a potential target for novel drug development specifically for the treatment of catheter-associated urinary tract infections caused by biofilm-producing E. coli.

microbiology↗

bioRxiv2020

Expanding magnetic organelle biogenesis in the domain Bacteria

The discovery of membrane-enclosed, metabolically functional organelles in Bacteria and Archaea has transformed our understanding of the subcellular complexity of prokaryotic cells. However, whether prokaryotic organelles emerged early or late in evolutionary history remains unclear and limits understanding of the nature and cellular complexity of early life. Biomineralization of magnetic nanoparticles within magnetosomes by magnetotactic bacteria (MTB) is a fascinating example of prokaryotic organelles. Here, we reconstruct 168 metagenome-assembled MTB genomes from various aquatic environments and waterlogged soils. These genomes represent nearly a 3-fold increase over the number currently available, and more than double the known MTB species. Phylogenomic analysis reveals that these newly described genomes belong to 13 Bacterial phyla, six of which were previously not known to include MTB. These findings indicate a much wider taxonomic distribution of magnetosome organelle biogenesis across the domain Bacteria than previously thought. Comparative genome analysis reveals a vast diversity of magnetosome gene clusters involved in magnetosomal biogenesis in terms of gene content and synteny residing in distinct taxonomic lineages. These gene clusters therefore represent a promising, diverse genetic resource for biosynthesizing novel magnetic nanoparticles. Finally, our phylogenetic analyses of the core magnetosome proteins in this largest available and taxonomically diverse dataset support an unexpectedly early evolutionary origin of magnetosome biomineralization, likely ancestral to the origin of the domain Bacteria. These findings emphasize the potential biological significance of prokaryotic organelles on the early Earth and have important implications for our understanding of the evolutionary history of cellular complexity.

microbiology↗

bioRxiv2020

Within-patient evolution to piperacillin/tazobactam resistance in a clinical isolate of Escherichia coli due to IS26-mediated amplification of blaTEM-1B

A novel phenotype of Escherichia coli and Klebsiella pneumoniae resistant to piperacillin/tazobactam (TZP), but susceptible to carbapenems and 3rd generation cephalosporins has recently emerged. The resistance mechanism of this phenotype has been identified as hyperproduction of the {beta}-lactamase blaTEM, however the mechanism of hyperproduction in isolates lacking promoter region mutations is not well understood. We sought to understand this mechanism by focussing on a pair of isolates obtained from an individual patient across two infection episodes and displaying within-patient evolution to TZP resistance. Following confirmation that the two isolates were clonal, we found that the TZP-resistant isolate hyperproduced a {beta}-lactamase but lacked mutations within {beta}-lactamase promoter regions. Hybrid assembly of long and short sequencing reads of the two isolates revealed both harboured a novel IS26-flanked composite transposon containing several antibiotic resistance genes, including blaTEM-1B, which was designated Tn6762. These resistance genes are also found to be present on a translocatable unit which had excised from Tn6762 in the TZP-resistant isolate. By replicating the evolutionary event leading to TZP resistance we were able to observe excision of the translocatable unit from Tn6762 following exposure to TZP and capture the TU in a plasmid containing a copy of IS26. Subsequent amplification of the TU, and by extension blaTEM-1B, leads to {beta}-lactamase hyperproduction and TZP resistance. Despite a significant increase in gene copy number (P value = <0.0001), we found that the TZP-resistant isolate was as fit as the susceptible ancestor. This mechanism of gene amplification, and the subsequent hyperproduction, of blaTEM-1B is an important consideration when using genomic data to predict resistance/susceptibility to TZP.

microbiology↗

bioRxiv2019

Integrative and conjugative elements, the neglected drivers of carbapenemase-encoding genes

Carbapenemases inactivate most {beta}-lactam antibiotics, including carbapenems and have been frequently reported among Enterobacteriaceae, Acinetobacter spp. and Pseudomonas spp. Traditionally, the horizontal gene transfer of carbapenemase encoding genes (CEGs) has been linked to plasmids. However, given that integrative and conjugative elements (ICEs) are possibly the most abundant conjugative elements among prokaryotes, we conducted an in-silico analysis to ascertain the likely role of ICEs in the spread of CEGs among all bacterial genomes (n=182,663). We detected 17,520 CEGs, of which 66 were located within putative ICEs among several bacterial species (including clinically-relevant bacteria as Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli). Most CEGs detected within ICEs belong to the IMP, NDM and SPM metallo-beta-lactamase families, and the serine beta-lactamase KPC and GES families. Different mechanisms were likely responsible for acquisition of these genes. The majority of CEG-bearing ICEs belong to the MPFG, MPFT and MPFF classes and often encode resistance to other antibiotics (e.g., aminoglycosides and fluoroquinolones). This study provides a snapshot of the different CEGs associated with ICEs among available bacterial genomes and sheds light on the underappreciated contribution of ICEs to the spread of carbapenem resistance globally. Author NotesAll supporting data has been provided within the article or through supplementary data files. Supplementary material is available with the online version of this article. Impact StatementCarbapenems are commonly used to treat severe infections in humans. Resistance is often mediated by carbapenemases. These enzymes degrade carbapenems and are frequently present in plasmids. Here, we demonstrate that common carbapenemase-encoding genes (CEGs) found in clinical isolates (e.g. blaKPC, blaGES, blaIMP, blaNDM, blaVIM) can also be located within integrative and conjugative elements (ICEs). CEG-bearing ICEs belong to three mating-pair formation families. These mobile elements may be particularly important in bacteria where plasmids do not seem to play a significant role in the spread of antibiotic resistance genes, as Pseudomonas spp. This study considerably expands the knowledge of the repertoire of CEGs-bearing ICEs among clinically-relevant bacterial pathogens, such as Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli. Data SummaryAll the bacterial genomes scanned in this study have been deposited previously in the National Center for Biotechnology Information genome database and are listed on the supplementary tables. The extracted 66 ICEs in fasta format and the outputs for the profile HMMs scanned on the 386 putative MGEs identified in this study are deposited on figshare at https://figshare.com/projects/_Comprehensive_genome_data_analysis_establishes_a_triple_whammy_of_carbapenemases_ICEs_and_multiple_clinically-relevant_bacteria/78369.

microbiology↗

bioRxiv2018

Promoter activity of ORF-less gene cassettes isolated from the oral metagenome

Integrons are genetic elements consisting of a functional platform for recombination and expression of gene cassettes (GCs). GCs usually carry promoter-less open reading frames (ORFs), encoding proteins with various functions including antibiotic resistance. The transcription of GCs relies mainly on a cassette promoter (PC), located upstream of an array of GCs. Some integron GCs, called ORF-less GCs, contain no identifiable ORF with a small number shown to be involved in antisense mRNA mediated gene regulation.\n\nIn this study, promoter sequences were identified, using in silico analysis, within GCs PCR amplified from the oral metagenome. The promoter activity of ORF-less GCs was verified by cloning them upstream of a gusA reporter, proving they can function as a promoter, presumably allowing bacteria to adapt to multiple stresses within the complex physico-chemical environment of the human oral cavity. A bi-directional promoter detection system was also developed allowing direct identification of clones with promoter-containing GCs on agar plates. Novel promoter-containing GCs were identified from the human oral metagenomic DNA using this construct, called pBiDiPD.\n\nThis is the first demonstration and detection of promoter activity of ORF-less GCs and the development of an agar plate-based detection system will enable similar studies in other environments.

microbiology↗

bioRxiv2018

Carbapenemases on the move: it’s good to be on ICE

The evolution and spread of antibiotic resistance is often mediated by mobile geneticelements. Integrative and conjugative elements (ICEs) are the most abundant conjugativeelements among prokaryotes. However, the contribution of ICEs to horizontal gene transferof antibiotic resistance has been largely unexplored. Here we report that ICEs belonging tomating-pair formation (MPF) classes G and T are highly prevalent among the opportunisticpathogen Pseudomonas aeruginosa, contributing to the spread of carbapenemase-encodinggenes (CEGs). Most CEGs of the MPFG class were encoded within class I integrons, which co-harbour genes conferring resistance to other antibiotics. The majority of the integrons werelocated within Tn3-like and composite transposons. A conserved attachment site could bepredicted for the MPFGclass ICEs. MPFTclass ICEs carried the CEGs within compositetransposons which were not associated with integrons. The data presented here provides aglobal snapshot of the different CEG-harbouring ICEs and sheds light on the underappreciatedcontribution of these elements for the evolution and dissemination of antibiotic resistanceon P. aeruginosa.

microbiology↗

bioRxiv2018

Evolutionary trajectories to amoxicillin-clavulanic acid resistance in Escherichia coli are affected by growth media.

In the face of an accelerating global antimicrobial resistance crisis, the determination of bacterial fitness following acquisition of resistance is an expanding area of research, and increased understanding of this process will be crucial to translate in vitro fitness data to successful therapies. Given that crucial clinical treatment situations are guided by in vitro diagnostic testing in an artificial environment far removed from human physiological niches, we used Escherichia coli and amoxicillin-clavulanic acid (AMC) resistance as a model to understand how such environments could affect the emergence of resistance, associated fitness costs and the predictive value of this data when strains were grown in the more physiologically relevant environments of urine and urothelial organoids. Resistant E. coli isolates were selected for following 24-hour exposure to sub-inhibitory concentrations of AMC in either M9, ISO or LB broth, followed by growth on LB agar containing AMC. No resistant colonies emerged following growth in M9, whereas resistant isolates were detected from cultures grown in ISO and LB broth. We observed both within and between media-type variability in the levels of resistance and fitness of the resistant mutants grown in LB. MICs and fitness of these resistant strains in different media (M9, ISO, LB, human urine and urothelial organoids) showed considerable variation. Media can therefore have a direct effect on the isolation of mutants that confer resistance to AMC and these mutants can exhibit unpredictable MIC and fitness profiles under different growth conditions. This study highlights the risks in relying on a single culture protocol to predict the behaviour and treatment response of bacteria in vivo and highlights the importance of developing comprehensive experimental designs to ensure effective translation of diagnostic procedures to successful clinical outcomes.

microbiology↗

bioRxiv2018

Conserved collateral susceptibility networks in diverse clinical strains of Escherichia coli.

There is urgent need to develop novel treatment strategies to reduce antimicrobial resistance. Collateral sensitivity (CS), where resistance to one antimicrobial increases susceptibility to other drugs, is a uniquely promising strategy that enables selection against resistance during treatment. However, using CS-informed therapy depends on conserved CS networks across genetically diverse bacterial strains. We examined CS conservation in 10 clinical strains of E. coli resistant to four clinically relevant antibiotics. Collateral susceptibilities of these 40 resistant mutants were then determined against a panel of 16 antibiotics. Multivariate statistical analyses demonstrate that resistance mechanisms, in particular efflux-related mutations, as well as relative fitness were principal contributors to collateral changes. Moreover, collateral responses shifted the mutant selection window suggesting that CS-informed therapies could affect evolutionary trajectories of antimicrobial resistance. Our data allow optimism for CS-informed therapy and further suggest that early detection of resistance mechanisms is important to accurately predict collateral antimicrobial responses.

microbiology↗

bioRxiv2017

A helicase-containing module defines a family of pCD630-like plasmids in Clostridium difficile

Clostridium difficile is a Gram-positive and sporulating enteropathogen that is a major cause of healthcare-associated infections. Even though a large number of genomes of this species have been sequenced, only a few plasmids have been described in the literature. Here, we use a combination of in silico analyses and laboratory experiments to show that plasmids are common in C. difficile. We focus on a group of plasmids that share similarity with the plasmid pCD630, from the reference strain 630. The family of pCD630-like plasmids is defined by the presence of a conserved putative helicase that is likely part of the plasmid replicon. This replicon is compatible with at least some other C. difficile replicons, as strains can carry pCD630-like plasmids in addition to other plasmids. We find two distinct sub-groups of pCD630-like plasmids that differ in size and accessory modules. This study is the first to describe a family of plasmids in C. difficile.

microbiology↗

bioRxiv2017

The human oral microbiome is shaped by shared environment rather than genetics: evidence from a large family of closely-related individuals

The human microbiome is affected by multiple factors, including the environment and host genetics. In this study, we analyzed the oral microbiome of an extended family of Ashkenazi Jewish individuals living in several cities and investigated associations with both shared household and host genetic similarities. We found that environmental effects dominated over genetic ones. While there was weak evidence of geographic structuring at the level of cities, we observed a large and significant effect of shared household on microbiome composition, supporting the role of immediate shared environment in dictating the presence or absence of taxa. This effect was also seen when including adults who had grown up in the same household but moved out prior to the time of sampling, suggesting that the establishment of the oral microbiome earlier in life may affect its long-term composition. We found weak associations between host genetic relatedness and microbiome dissimilarity when using family pedigrees as proxies for genetic similarity. However this association disappeared when using more accurate measures of kinship based on genome-wide genetic markers, indicating that environment rather than host genetics is the dominant factor affecting the composition of the oral microbiome in closely-related individuals. Our results support the concept that there is a consistent core microbiome conserved across global scales, but that small-scale effects due to shared living environment significantly affect microbial community composition.\n\nIMPORTANCEPrevious research shows that relatives have a more similar oral microbiome composition than non-relatives, but it remains difficult to distinguish the effects of relatedness and shared household environment. Furthermore, pedigree measures may not accurately measure host genetic similarity. In this study, we include genetic relatedness based on genome-wide SNPs (rather than pedigree measures) and shared environment in the same analysis. We quantify the relative importance of these factors by studying the oral microbiome in members of a large extended Ashkenazi Jewish family who share a similar diet and lifestyle despite living in different locations. We find that host genetics plays no significant role and that the dominant factor is shared environment at the household level. We also find that this effect appears to persist in individuals who have moved out of the parental household, suggesting that the oral microbiome established earlier in life persists long-term.

microbiology↗