Development of pVCR94?X from Vibrio cholerae, a prototype for studying multidrug resistant IncA/C conjugative plasmids.
ABSTRACT: Antibiotic resistance has grown steadily in Vibrio cholerae over the last few decades to become a major threat in countries affected by cholera. Multi-drug resistance (MDR) spreads among clinical and environmental V. cholerae strains by lateral gene transfer often mediated by integrative and conjugative elements (ICEs) of the SXT/R391 family. However, in a few reported but seemingly isolated cases, MDR in V. cholerae was shown to be associated with other self-transmissible genetic elements such as conjugative plasmids. IncA/C conjugative plasmids are often found associated with MDR in isolates of Enterobacteriaceae. To date, IncA/C plasmids have not been commonly found in V. cholerae or other species of Vibrio. Here we present a detailed analysis of pVCR94?X derived from pVCR94, a novel IncA/C conjugative plasmid identified in a V. cholerae clinical strain isolated during the 1994 Rwandan cholera outbreak. pVCR94 was found to confer resistance to sulfamethoxazole, trimethoprim, ampicillin, streptomycin, tetracycline, and chloramphenicol and to transfer at very high frequency. Sequence analysis revealed its mosaic nature as well as high similarity of the core genes responsible for transfer and maintenance with other IncA/C plasmids and ICEs of the SXT/R391 family. Although IncA/C plasmids are considered a major threat in antibiotics resistance, their basic biology has received little attention, mostly because of the difficulty to genetically manipulate these MDR conferring elements. Therefore, we developed a convenient derivative from pVCR94, pVCR94? X, a 120.5-kb conjugative plasmid which only codes for sulfamethoxazole resistance. Using pVCR94? X, we identified the origin of transfer (oriT) and discovered an essential gene for transfer, both located within the shared backbone, allowing for an annotation update of all IncA/C plasmids. pVCR94? X may be a useful model that will provide new insights on the basic biology of IncA/C conjugative plasmids.
Project description:Nowadays, healthcare systems are challenged by a major worldwide drug resistance crisis caused by the massive and rapid dissemination of antibiotic resistance genes and associated emergence of multidrug resistant pathogenic bacteria, in both clinical and environmental settings. Conjugation is the main driving force of gene transfer among microorganisms. This mechanism of horizontal gene transfer mediates the translocation of large DNA fragments between two bacterial cells in direct contact. Integrative and conjugative elements (ICEs) of the SXT/R391 family (SRIs) and IncA/C conjugative plasmids (ACPs) are responsible for the dissemination of a broad spectrum of antibiotic resistance genes among diverse species of Enterobacteriaceae and Vibrionaceae. The biology, diversity, prevalence and distribution of these two families of conjugative elements have been the subject of extensive studies for the past 15 years. Recently, the transcriptional regulators that govern their dissemination through the expression of ICE- or plasmid-encoded transfer genes have been described. Unrelated repressors control the activation of conjugation by preventing the expression of two related master activator complexes in both types of elements, i.e., SetCD in SXT/R391 ICEs and AcaCD in IncA/C plasmids. Finally, in addition to activating ICE- or plasmid-borne genes, these master activators have been shown to specifically activate phylogenetically unrelated mobilizable genomic islands (MGIs) that also disseminate antibiotic resistance genes and other adaptive traits among a plethora of pathogens such as Vibrio cholerae and Salmonella enterica.
Project description:Integrative and Conjugative Elements (ICEs) of the SXT/R391 family disseminate multidrug resistance among pathogenic Gammaproteobacteria such as Vibrio cholerae. SXT/R391 ICEs are mobile genetic elements that reside in the chromosome of their host and eventually self-transfer to other bacteria by conjugation. Conjugative transfer of SXT/R391 ICEs involves a transient extrachromosomal circular plasmid-like form that is thought to be the substrate for single-stranded DNA translocation to the recipient cell through the mating pore. This plasmid-like form is thought to be non-replicative and is consequently expected to be highly unstable. We report here that the ICE R391 of Providencia rettgeri is impervious to loss upon cell division. We have investigated the genetic determinants contributing to R391 stability. First, we found that a hipAB-like toxin/antitoxin system improves R391 stability as its deletion resulted in a tenfold increase of R391 loss. Because hipAB is not a conserved feature of SXT/R391 ICEs, we sought for alternative and conserved stabilization mechanisms. We found that conjugation itself does not stabilize R391 as deletion of traG, which abolishes conjugative transfer, did not influence the frequency of loss. However, deletion of either the relaxase-encoding gene traI or the origin of transfer (oriT) led to a dramatic increase of R391 loss correlated with a copy number decrease of its plasmid-like form. This observation suggests that replication initiated at oriT by TraI is essential not only for conjugative transfer but also for stabilization of SXT/R391 ICEs. Finally, we uncovered srpMRC, a conserved locus coding for two proteins distantly related to the type II (actin-type ATPase) parMRC partitioning system of plasmid R1. R391 and plasmid stabilization assays demonstrate that srpMRC is active and contributes to reducing R391 loss. While partitioning systems usually stabilizes low-copy plasmids, srpMRC is the first to be reported that stabilizes a family of ICEs.
Project description:Integrative and conjugative elements (ICEs) of the SXT/R391 family have been recognized as key drivers of antibiotic resistance dissemination in the seventh-pandemic lineage of Vibrio cholerae. SXT/R391 ICEs propagate by conjugation and integrate site-specifically into the chromosome of a wide range of environmental and clinical Gammaproteobacteria. SXT/R391 ICEs bear setC and setD, two conserved genes coding for a transcriptional activator complex that is essential for activation of conjugative transfer. We used chromatin immunoprecipitation coupled with exonuclease digestion (ChIP-exo) and RNA sequencing (RNA-seq) to characterize the SetCD regulon of three representative members of the SXT/R391 family. We also identified the DNA sequences bound by SetCD in MGIVflInd1, a mobilizable genomic island phylogenetically unrelated to SXT/R391 ICEs that hijacks the conjugative machinery of these ICEs to drive its own transfer. SetCD was found to bind a 19-bp sequence that is consistently located near the promoter -35 element of SetCD-activated genes, a position typical of class II transcriptional activators. Furthermore, we refined our understanding of the regulation of excision from and integration into the chromosome for SXT/R391 ICEs and demonstrated that de novo expression of SetCD is crucial to allow integration of the incoming ICE DNA into a naive host following conjugative transfer.
Project description:Integrative and conjugative elements (ICEs) of the SXT/R391 family are key drivers of the spread of antibiotic resistance in <i>Vibrio cholerae</i>, the infectious agent of cholera, and other pathogenic bacteria. The SXT/R391 family of ICEs was defined based on the conservation of a core set of 52 genes and site-specific integration into the 5' end of the chromosomal gene <i>prfC</i> Hence, the integrase gene <i>int</i> has been intensively used as a marker to detect SXT/R391 ICEs in clinical isolates. ICEs sharing most core genes but differing by their integration site and integrase gene have been recently reported and excluded from the SXT/R391 family. Here we explored the prevalence and diversity of atypical ICEs in GenBank databases and their relationship with typical SXT/R391 ICEs. We found atypical ICEs in <i>V. cholerae</i> isolates that predate the emergence and expansion of typical SXT/R391 ICEs in the mid-1980s in seventh-pandemic toxigenic <i>V. cholerae</i> strains O1 and O139. Our analyses revealed that while atypical ICEs are not associated with antibiotic resistance genes, they often carry cation efflux pumps, suggesting heavy metal resistance. Atypical ICEs constitute a polyphyletic group likely because of occasional recombination events with typical ICEs. Furthermore, we show that the alternative integration and excision genes of atypical ICEs remain under the control of SetCD, the main activator of the conjugative functions of SXT/R391 ICEs. Together, these observations indicate that substitution of the integration/excision module and change of specificity of integration do not preclude atypical ICEs from inclusion into the SXT/R391 family.<b>IMPORTANCE</b><i>Vibrio cholerae</i> is the causative agent of cholera, an acute intestinal infection that remains to this day a world public health threat. Integrative and conjugative elements (ICEs) of the SXT/R391 family have played a major role in spreading antimicrobial resistance in seventh-pandemic <i>V. cholerae</i> but also in several species of <i>Enterobacteriaceae</i> Most epidemiological surveys use the integrase gene as a marker to screen for SXT/R391 ICEs in clinical or environmental strains. With the recent reports of closely related elements that carry an alternative integrase gene, it became urgent to investigate whether ICEs that have been left out of the family are a liability for the accuracy of such screenings. In this study, based on comparative genomics, we broaden the SXT/R391 family of ICEs to include atypical ICEs that are often associated with heavy metal resistance.
Project description:Integrating and conjugative elements (ICEs) are one of the three principal types of self-transmissible mobile genetic elements in bacteria. ICEs, like plasmids, transfer via conjugation; but unlike plasmids and similar to many phages, these elements integrate into and replicate along with the host chromosome. Members of the SXT/R391 family of ICEs have been isolated from several species of gram-negative bacteria, including Vibrio cholerae, the cause of cholera, where they have been important vectors for disseminating genes conferring resistance to antibiotics. Here we developed a plasmid-based system to capture and isolate SXT/R391 ICEs for sequencing. Comparative analyses of the genomes of 13 SXT/R391 ICEs derived from diverse hosts and locations revealed that they contain 52 perfectly syntenic and nearly identical core genes that serve as a scaffold capable of mobilizing an array of variable DNA. Furthermore, selection pressure to maintain ICE mobility appears to have restricted insertions of variable DNA into intergenic sites that do not interrupt core functions. The variable genes confer diverse element-specific phenotypes, such as resistance to antibiotics. Functional analysis of a set of deletion mutants revealed that less than half of the conserved core genes are required for ICE mobility; the functions of most of the dispensable core genes are unknown. Several lines of evidence suggest that there has been extensive recombination between SXT/R391 ICEs, resulting in re-assortment of their respective variable gene content. Furthermore, our analyses suggest that there may be a network of phylogenetic relationships among sequences found in all types of mobile genetic elements.
Project description:Integrative and conjugative elements (ICEs) of the SXT/R391 family are the main contributors to acquired multidrug resistance in the seventh pandemic lineage of Vibrio cholerae, the etiological agent of the diarrheal disease cholera. Conjugative transfer of SXT/R391 ICEs is triggered by antibiotics and agents promoting DNA damage through RecA-dependent autoproteolysis of SetR, an ICE-encoded ? CI-like repressor. Here, we describe the role of CroS, a distant ? Cro homolog, as a key component contributing to the regulation of expression of the activator SetCD that orchestrates the expression of the conjugative transfer genes. We show that deletion of croS abolishes the SOS response-dependent induction of SXT despite the presence of a functional setR gene. Using quantitative reverse transcription-PCR and lacZ reporter assays, we also show that CroS represses setR and setCD expression by binding to operator sites shared with SetR. Furthermore, we provide evidence of an additional operator site bound by SetR and CroS. Finally, we show that SetCD expression generates a positive feedback loop due to SXT excision and replication in a fraction of the cell population. Together, these results refine our understanding of the genetic regulation governing the propagation of major vectors of multidrug resistance.Healthcare systems worldwide are challenged by an alarming drug resistance crisis caused by the massive and rapid propagation of antibiotic resistance genes and the associated emergence of multidrug-resistant pathogenic bacteria. SXT/R391 ICEs contribute to this phenomenon not only in clinical and environmental vibrios but also in several members of the family Enterobacteriaceae. We have identified and characterized here the regulator CroS as a key factor in the stimulation of conjugative transfer of these ICEs in response to DNA-damaging agents. We have also untangled conflicting evidence regarding autoactivation of transfer by the master activator of SXT/R391 ICEs, SetCD. Discovery of CroS provides a clearer and more complete understanding of the regulatory network that governs the dissemination of SXT/R391 ICEs in bacterial populations.
Project description:UNLABELLED:SXT-R391 Integrative conjugative elements (ICEs) are self-transmissible mobile genetic elements able to confer multidrug resistance and other adaptive features to bacterial hosts, including Vibrio cholerae, the causative agent of cholera. ICEs are arranged in a mosaic genetic structure composed of a conserved backbone interspersed with variable DNA clusters located in conserved hot spots. In this study, we investigated ICE acquisition and subsequent microevolution in pandemic V. cholerae. Ninety-six ICEs were retrieved from publicly available sequence databases from V. cholerae clinical strains and were compared to a set of reference ICEs. Comparative genomics highlighted the existence of five main ICE groups with a distinct genetic makeup, exemplified by ICEVchInd5, ICEVchMoz10, SXT, ICEVchInd6, and ICEVchBan11. ICEVchInd5 (the most frequent element, represented by 70 of 96 elements analyzed) displayed no sequence rearrangements and was characterized by 46 single nucleotide polymorphisms (SNPs). SNP analysis revealed that recent inter-ICE homologous recombination between ICEVchInd5 and other ICEs circulating in gammaproteobacteria generated ICEVchMoz10, ICEVchInd6, and ICEVchBan11. Bayesian phylogenetic analyses indicated that ICEVchInd5 and SXT were independently acquired by the current pandemic V. cholerae O1 and O139 lineages, respectively, within a period of only a few years. IMPORTANCE:SXT-R391 ICEs have been recognized as key vectors of antibiotic resistance in the seventh-pandemic lineage of V. cholerae, which remains a major cause of mortality and morbidity on a global scale. ICEs were acquired only recently in this clade and are acknowledged to be major contributors to horizontal gene transfer and the acquisition of new traits in bacterial species. We have reconstructed the temporal dynamics of SXT-R391 ICE acquisition and spread and have identified subsequent recombination events generating significant diversity in ICEs currently circulating among V. cholerae clinical strains. Our results showed that acquisition of SXT-R391 ICEs provided the V. cholerae seventh-pandemic lineage not only with a multidrug resistance phenotype but also with a powerful molecular tool for rapidly accessing the pan-genome of a large number of gammaproteobacteria.
Project description:The self-transferring integrative and conjugative elements (ICEs) are large genomic segments carrying several bacterial adaptive functions including antimicrobial resistance (AMR). SXT/R391 family is one of the ICEs extensively studied in cholera-causing pathogen Vibrio cholerae. The genetic characteristics of ICE-SXT/R391 in V. cholerae are dynamic and region-specific. These ICEs in V. cholerae are strongly correlated with resistance to several antibiotics such as tetracycline, streptomycin and trimethoprim-sulfamethoxazole. We screened V. cholerae O1 strains isolated from cholera patients in Kolkata, India from 2008 to 2015 for antibiotic susceptibility and the presence of ICEs, and subsequently sequenced their conserved genes. Resistance to tetracycline, streptomycin and trimethoprim-sulfamethoxazole was detected in strains isolated during 2008-2010 and 2014-2015. The genes encoding resistance to tetracycline (tetA), trimethoprim-sulfamethoxazole (dfrA1 and sul2), streptomycin (strAB), and chloramphenicol (floR) were detected in the ICEs of these strains. There was a decrease in overall drug resistance in V. cholerae associated with the ICEs in 2011. DNA sequence analysis also showed that AMR in these strains was conferred mainly by two types of ICEs, i.e., ICETET (comprising tetA, strAB, sul2, and dfrA1) and ICEGEN (floR, strAB, sul2, and dfrA1). Based on the genetic structure, Kolkata strains of V. cholerae O1 had distinct genetic traits different from the ICEs reported in other cholera endemic regions. Transfer of AMR was confirmed by conjugation with sodium azide resistant Escherichia coli J53. In addition to the acquired resistance to streptomycin and trimethoprim-sulfamethoxazole, the conjugally transferred (CT) E. coli J53 with ICE showed higher resistance to chloramphenicol and tetracycline than the donor V. cholerae. Pulsed-field gel electrophoresis (PFGE) based clonal analysis revealed that the V. cholerae strains could be grouped based on their ICEs and AMR patterns. Our findings demonstrate the epidemiological importance of ICEs and their role in the emergence of multidrug resistance (MDR) in El Tor vibrios.
Project description:Integrating conjugative elements (ICEs) are mobile genetic elements that can transfer from the chromosome of a host to the chromosome of a new host through the process of excision, conjugation, and integration. Although SXT/R391-related ICEs, originally demonstrated in Vibrio cholerae O139 isolates, have become prevalent among V. cholerae isolates in Asia, the prevalence of the ICEs among gram-negative bacteria other than Vibrio spp. remains unknown. In addition, SXT/R391-related ICEs carrying genes conferring resistance to extended-spectrum cephalosporins have never been described. Here we carried out a genetic analysis of a cefoxitin-resistant Proteus mirabilis clinical isolate, TUM4660, which revealed the presence of a novel SXT/R391-related ICE, ICEPmiJpn1. ICEPmiJpn1 had a core genetic structure showing high similarity to that of R391 and carried xis and int genes completely identical to those of R391, while an IS10-mediated composite transposon carrying bla(CMY-2) was integrated into the ICE. A nucleotide sequence identical to the 3' part of ISEcp1 was located upstream of the bla(CMY-2) gene, and other genes observed around bla(CMY-2) in earlier studies were also present. Furthermore, the nucleotide sequences of hot spot 2 and hot spot 4 in ICEPmiJpn1 showed high similarity to that of hot spot 2 in SXT(MO10) and with a part of the nucleotide sequence found in P. mirabilis ATCC 29906, respectively. ICEPmiJpn1 was successfully transferred to Escherichia coli, Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Citrobacter koseri in conjugation experiments. These observations suggest that ICEs may contribute to the dissemination of antimicrobial resistance genes among clinically relevant Enterobacteriaceae, which warrants careful observation of the prevalence of ICEs, including SXT/R391-related ICEs.
Project description:Integrating conjugative elements (ICEs) are a class of bacterial mobile genetic elements that disseminate via conjugation and then integrate into the host cell genome. The SXT/R391 family of ICEs consists of more than 30 different elements that all share the same integration site in the host chromosome but often encode distinct properties. These elements contribute to the spread of antibiotic resistance genes in several gram-negative bacteria including Vibrio cholerae, the agent of cholera. Here, using comparative analyses of the genomes of several SXT/R391 ICEs, we found evidence that the genomes of these elements have been shaped by inter-ICE recombination. We developed a high throughput semi-quantitative method to explore the genetic determinants involved in hybrid ICE formation. Recombinant ICE formation proved to be relatively frequent, and to depend on host (recA) and ICE (s065 and s066) loci, which can independently and potentially cooperatively mediate hybrid ICE formation. s065 and s066, which are found in all SXT/R391 ICEs, are orthologues of the bacteriophage lambda Red recombination genes bet and exo, and the s065/s066 recombination system is the first Red-like recombination pathway to be described in a conjugative element. Neither ICE excision nor conjugative transfer proved to be essential for generation of hybrid ICEs. Instead conjugation facilitates the segregation of hybrids and could provide a means to select for functional recombinant ICEs containing novel combinations of genes conferring resistance to antibiotics. Thus, ICEs promote their own diversity and can yield novel mobile elements capable of disseminating new combinations of antibiotic resistance genes.