Multidrug resistance plasmids commonly reprogram the expression of metabolic genes in Escherichia coli.

Multidrug-resistant Escherichia coli is a leading cause of global mortality. Transfer of plasmids carrying genes encoding beta-lactamases, carbapenamases, and colistin resistance between lineages is driving the rising rates of hard-to-treat nosocomial and community infections. Multidrug resistance (MDR) plasmid acquisition commonly causes transcriptional disruption, and while a number of studies have shown strain-specific fitness and transcriptional effects of an MDR plasmid across diverse bacterial lineages, fewer studies have compared the impacts of different MDR plasmids in a common bacterial host. As such, our ability to predict which MDR plasmids are the most likely to be maintained and spread in bacterial populations is limited. Here, we introduced eight diverse MDR plasmids encoding resistances against a range of clinically important antibiotics into E. coli K-12 MG1655 and measured their fitness costs and transcriptional impacts. The scale of the transcriptional responses varied substantially between plasmids, ranging from >650 to <20 chromosomal genes being differentially expressed. However, the scale of regulatory disruption did not correlate significantly with the magnitude of the plasmid fitness cost, which also varied between plasmids. The identities of differentially expressed genes differed between transconjugants, although the expression of certain metabolic genes and functions were convergently affected by multiple plasmids, including the downregulation of genes involved in L-methionine transport and metabolism. Our data show the complexity of the interaction between host genetic background and plasmid genetic background in determining the impact of MDR plasmid acquisition on E. coli. IMPORTANCE: The increase in infections that are resistant to multiple classes of antibiotics, including those isolates that carry carbapenamases, beta-lactamases, and colistin resistance genes, is of global concern. Many of these resistances are spread by conjugative plasmids. Understanding more about how an isolate responds to an incoming plasmid that encodes antibiotic resistance will provide information that could be used to predict the emergence of MDR lineages. Here, the identification of metabolic networks as being particularly sensitive to incoming plasmids suggests the possible targets for reducing plasmid transfer.

Parallel loss of type VI secretion systems in two multi-drug-resistant Escherichia coli lineages.

The repeated emergence of multi-drug-resistant (MDR) Escherichia coli clones is a threat to public health globally. In recent work, drug-resistant E. coli were shown to be capable of displacing commensal E. coli in the human gut. Given the rapid colonization observed in travel studies, it is possible that the presence of a type VI secretion system (T6SS) may be responsible for the rapid competitive advantage of drug-resistant E. coli clones. We employed large-scale genomic approaches to investigate this hypothesis. First, we searched for T6SS genes across a curated dataset of over 20 000 genomes representing the full phylogenetic diversity of E. coli. This revealed large, non-phylogenetic variation in the presence of T6SS genes. No association was found between T6SS gene carriage and MDR lineages. However, multiple clades containing MDR clones have lost essential structural T6SS genes. We characterized the T6SS loci of ST410 and ST131 and identified specific recombination and insertion events responsible for the parallel loss of essential T6SS genes in two MDR clones.

The highly diverse plasmid population found in Escherichia coli colonizing travellers to Laos and its role in antimicrobial resistance gene carriage.

Increased colonization by antimicrobial-resistant organisms is closely associated with international travel. This study investigated the diversity of mobile genetic elements involved with antimicrobial resistance (AMR) gene carriage in extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli that colonized travellers to Laos. Long-read sequencing was used to reconstruct complete plasmid sequences from 48 isolates obtained from the daily stool samples of 23 travellers over a 3 week period. This method revealed a collection of 105 distinct plasmids, 38.1 % (n=40) of which carried AMR genes. The plasmids in this population were diverse, mostly unreported and included 38 replicon types, with F-type plasmids (n=23) the most prevalent amongst those carrying AMR genes. Fine-scale analysis of all plasmids identified numerous AMR gene contexts and emphasized the importance of IS elements, specifically members of the IS6/IS26 family, in the evolution of complex multidrug resistance regions. We found a concerning convergence of ESBL and colistin resistance determinants, with three plasmids from two different F-type lineages carrying bla CTX-M and mcr genes. The extensive diversity seen here highlights the worrying probability that stable new vehicles for AMR will evolve in E. coli populations that can disseminate internationally through travel networks.

The role of potentiating mutations in the evolution of pandemic Escherichia coli clones.

The Escherichia coli species exhibits a vast array of variable lifestyles, including environmental, commensal, and pathogenic organisms. Many of these E. coli contribute significantly to the global threat of antimicrobial resistance (AMR). Multidrug-resistant (MDR) clones of E. coli have arisen multiple times over varying timescales. The repeated emergence of successful pandemic clones, including the notorious ST131 lineage, highlights a desperate need to further study the evolutionary processes underlying their emergence and success. Here, we review the evolutionary emergence of E. coli ST131 pandemic clones and draw parallels between their evolutionary trajectories and those of other lineages. From colonization and expansion to the acquisition of multidrug resistance plasmids, potentiating mutations are present at each stage, leading to a proposed sequence of events that may result in the formation of an antimicrobial-resistant pandemic clone.

Dynamics of intestinal multidrug-resistant bacteria colonisation contracted by visitors to a high-endemic setting: a prospective, daily, real-time sampling study.

BACKGROUND: Antimicrobial resistance is highly prevalent in low-income and middle-income countries. International travel contributes substantially to the global spread of intestinal multidrug-resistant Gram-negative bacteria. Hundreds of millions of annual visitors to low-income and middle-income countries are all exposed to intestinal multidrug-resistant Gram-negative bacteria resulting in 30-70% of them being colonised at their return. The colonisation process in high-exposure environments is poorly documented because data have only been derived from before travel and after travel sampling. We characterised colonisation dynamics by exploring daily stool samples while visiting a low-income and middle-income countries. METHODS: In this prospective, daily, real-time sampling study 20 European visitors to Laos volunteered to provide daily stool samples and completed daily questionnaires for 22 days. Samples were initially assessed at Mahosot Hospital, Vientiane, Laos, for acquisition of extended-spectrum ß-lactamase-producing (ESBL) Gram-negative bacteria followed by whole-genome sequencing of isolates at MicrobesNG, University of Birmingham, Birmingham, UK. The primary outcome of the study was to obtain data on the dynamics of intestinal multidrug-resistant bacteria acquisition. FINDINGS: Between Sept 18 and Sept 20, 2015, 23 volunteers were recruited, of whom 20 (87%) European volunteers were included in the final study population. Although colonisation rates were 70% at the end of the study, daily sampling revealed that all participants had acquired ESBL-producing Gram-negative bacteria at some point during the study period; the colonisation status varied day by day. Whole-genome sequencing analysis ascribed the transient pattern of colonisation to sequential acquisition of new strains, resulting in a loss of detectable colonisation by the initial multidrug-resistant Gram-negative strains. 19 (95%) participants acquired two to seven strains. Of the 83 unique strains identified (53 Escherichia coli, 10 Klebsiella spp, and 20 other ESBL-producing Gram-negative bacteria), some were shared by as many as four (20%) participants. INTERPRETATION: To our knowledge, this is the first study to characterise in real-time the dynamics of acquiring multidrug-resistant Gram-negative bacterial colonisation during travel. Our data show multiple transient colonisation events indicative of constant microbial competition and suggest that travellers are exposed to a greater burden of multidrug-resistant bacteria than previously thought. The data emphasise the need for preventing travellers' diarrhoea and limiting antibiotic use, addressing the two major factors predisposing colonisation. FUNDING: The Finnish Governmental Subsidy for Health Science Research, The Scandinavian Society for Antimicrobial Chemotherapy, the Sigrid Jusélius Foundation, Biotechnology and Biological Sciences Research Council; Wellcome Trust, Medical Research Council; The Royal Society; Joint Programming Initiative on Antimicrobial Resistance, and European Research Council.

Transcriptomic and Functional Screens Reveal MicroRNAs That Modulate Prostate Cancer Metastasis.

Identifying new mechanisms that underlie the complex process of metastasis is vital to combat this fatal step in prostate cancer (PCa) progression. Small non-coding RNAs are emerging as important regulators of tumor cell biology. Here we take an integrative approach to elucidate the contribution of microRNAs to metastatic progression, combining transcriptomic analysis with functional screens for migration and morphology. We developed high-content microscopy, high-throughput functional screens for migration and morphology in PCa cells using a microRNA library. RNA-Seq analysis of paired epithelial and mesenchymal PCa cells identified differential expression of 200 microRNAs. Data integration identified two microRNAs that inhibited migration, induced an epithelial-like morphology and were increased in epithelial PCa cells. An overrepresentation of the AAGUGC seed sequence was detected in all three datasets. Analysis of published datasets of patients with PCa identified microRNAs of clinical relevance. The integration of high-throughput functional and expression analyses identifies microRNAs with clinical significance that modulate metastatic behavior in PCa.