Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli. - Related Documents




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997501.0000Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli. Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the study of the mobilization of naturally occurring conjugative plasmids, using two naturally-occurring plasmids as an example. These protocols rely on the differential presence of selectable markers in donor, recipient, and conjugative plasmid. Specifically, the methods described include 1) the identification of natural conjugative plasmids, 2) the quantification of conjugation rates in solid culture, and 3) the diagnostic detection of the antibiotic resistance genes and plasmid replicon types in transconjugant recipients by polymerase chain reaction (PCR). The protocols described here have been developed in the context of studying the evolutionary ecology of horizontal gene transfer, to screen for the presence of conjugative plasmids carrying antibiotic-resistance genes in bacteria found in the environment. The efficient transfer of conjugative plasmids observed in these experiments in culture highlights the biological relevance of conjugation as a mechanism promoting horizontal gene transfer in general and the spread of antibiotic resistance in particular.202337036197
997410.9999Role of Plasmids in Co-Selection of Antimicrobial Resistances Among Escherichia coli Isolated from Pigs. Co-selection is thought to occur when resistance genes are located on the same mobile genetic element. However, this mechanism is currently poorly understood. In this study, complete circular plasmids from swine-derived Escherichia coli were sequenced with short and long reads to confirm that resistance genes involved in co-resistance were co-transferred by the same plasmid. Conjugative transfer tests were performed, and multiple resistance genes were transmitted. The genes possessed by the donor, transconjugant, and plasmid of the donor were highly similar. In addition, the sequences of the plasmid of the donor and the plasmid of the transconjugant were almost identical. Resistance genes associated with statistically significant combinations of antimicrobial use and resistance were co-transmitted by the same plasmid. These results suggest that resistance genes may be involved in co-selection by their transfer between bacteria on the same plasmid.202337540099
995220.9998Detection and Quantification of Conjugative Transfer of Mobile Genetic Elements Carrying Antibiotic Resistance Genes. Multidrug resistance, due to acquired antimicrobial resistance genes, is increasingly reported in the zoonotic pathogen Streptococcus suis. Most of these resistance genes are carried by chromosomal Mobile Genetic Elements (MGEs), in particular, Integrative and Conjugative Elements (ICEs) and Integrative and Mobilizable Elements (IMEs). ICEs and IMEs frequently form tandems or nested composite elements, which make their identification difficult. To evaluate their mobility, it is necessary to (i) select the suitable donor-recipient pairs for mating assays, (ii) do PCR excision tests to confirm that the genetic element is able to excise from the chromosome as a circular intermediate, and (iii) evaluate the transfer of the genetic element by conjugation by doing mating assays. In addition to a dissemination of resistance genes between S. suis strains, MGEs can lead to a spreading of resistance genes in the environment and toward pathogenic bacteria. This propagation had to be considered in a One Health perspective.202438884912
988330.9998Plasmids in Gram negatives: molecular typing of resistance plasmids. A plasmid is defined as a double stranded, circular DNA molecule capable of autonomous replication. By definition, plasmids do not carry genes essential for the growth of host cells under non-stressed conditions but they have systems which guarantee their autonomous replication also controlling the copy number and ensuring stable inheritance during cell division. Most of the plasmids confer positively selectable phenotypes by the presence of antimicrobial resistance genes. Plasmids evolve as an integral part of the bacterial genome, providing resistance genes that can be easily exchanged among bacteria of different origin and source by conjugation. A multidisciplinary approach is currently applied to study the acquisition and spread of antimicrobial resistance in clinically relevant bacterial pathogens and the established surveillance can be implemented by replicon typing of plasmids. Particular plasmid families are more frequently detected among Enterobacteriaceae and play a major role in the diffusion of specific resistance genes. For instance, IncFII, IncA/C, IncL/M, IncN and IncI1 plasmids carrying extended-spectrum beta-lactamase genes and acquired AmpC genes are currently considered to be "epidemic resistance plasmids", being worldwide detected in Enterobacteriaceae of different origin and sources. The recognition of successful plasmids is an essential first step to design intervention strategies preventing their spread.201121992746
413340.9998Importance of integrons in the diffusion of resistance. Horizontal transfer of resistance genes is a successful mechanism for the transmission and dissemination of multiple drug resistance among bacterial pathogens. The impact of horizontally transmitted genetic determinants in the evolution of resistance is particularly evident when resistance genes are physically associated in clusters and transferred en bloc to the recipient cell. Recent advances in the molecular characterisation of antibiotic resistance mechanisms have highlighted the existence of genetic structures. called integrons, involved in the acquisition of resistance genes. These DNA elements have frequently been reported in multi-drug resistant strains isolated from animals and humans, and are located either on the bacterial chromosome or on broad-host-range plasmids. The role of integrons in the development of multiple resistance relies on their unique capacity to cluster and express drug resistance genes. Moreover, the spread of resistance genes among different replicons and their exchange between plasmid and bacterial chromosome are facilitated by the integration of integrons into transposable elements. The association of a highly efficient gene capture and expression system, together with the capacity for vertical and horizontal transmission of resistance genes represents a powerful weapon used by bacteria to combat the assault of antibiotics.200111432416
465850.9998Class 1 integrons potentially predating the association with tn402-like transposition genes are present in a sediment microbial community. Integrons are genetic elements that contribute to lateral gene transfer in bacteria as a consequence of possessing a site-specific recombination system. This system facilitates the spread of genes when they are part of mobile cassettes. Most integrons are contained within chromosomes and are confined to specific bacterial lineages. However, this is not the case for class 1 integrons, which were the first to be identified and are one of the single biggest contributors to multidrug-resistant nosocomial infections, carrying resistance to many antibiotics in diverse pathogens on a global scale. The rapid spread of class 1 integrons in the last 60 years is partly a result of their association with a specific suite of transposition functions, which has facilitated their recruitment by plasmids and other transposons. The widespread use of antibiotics has acted as a positive selection pressure for bacteria, especially pathogens, which harbor class 1 integrons and their associated antibiotic resistance genes. Here, we have isolated bacteria from soil and sediment in the absence of antibiotic selection. Class 1 integrons were recovered from four different bacterial species not known to be human pathogens or commensals. All four integrons lacked the transposition genes previously considered to be a characteristic of this class. At least two of these integrons were located on a chromosome, and none of them possessed antibiotic resistance genes. We conclude that novel class 1 integrons are present in a sediment environment in various bacteria of the beta-proteobacterial class. These data suggest that the dispersal of this class may have begun before the "antibiotic era."200616885440
447260.9998Conjugative plasmids in bacteria of the 'pre-antibiotic' era. Antibiotic resistance is common in bacteria that cause disease in man and animals and is usually determined by plasmids. The prevalence of such plasmids, and the range of drugs to which they confer resistance, have increased greatly in the past 25 yr. It has become clear from work in many laboratories that plasmids have acquired resistance genes, of ultimately unknown origin, as insertions into their circular DNA. The intensive use of antibiotics since their introduction in the 1940s can explain the spread of plasmids that have acquired such genes but little is known of the incidence of plasmids in pathogenic bacteria before the widespread use of antibiotics in medicine. E.D.G. Murray collected strains of Enterobacteriaceae from 1917 to 1954; we now report that 24% of these encode information for the transfer of DNA from one bacterium to another. From at least 19% of the strains, conjugative plasmids carrying no antibiotic resistance were transferred to Escherichia coli K-12.19836835408
381570.9998Development of a high-throughput platform to measure plasmid transfer frequency. Antibiotic resistance represents one of the greatest threats to global health. The spread of antibiotic resistance genes among bacteria occurs mostly through horizontal gene transfer via conjugation mediated by plasmids. This process implies a direct contact between a donor and a recipient bacterium which acquires the antibiotic resistance genes encoded by the plasmid and, concomitantly, the capacity to transfer the acquired plasmid to a new recipient. Classical assays for the measurement of plasmid transfer frequency (i.e., conjugation frequency) are often characterized by a high variability and, hence, they require many biological and technical replicates to reduce such variability and the accompanying uncertainty. In addition, classical conjugation assays are commonly tedious and time-consuming because they typically involve counting colonies on a large number of plates for the quantification of donors, recipients, and transconjugants (i.e., the bacteria that have received the genetic material by conjugation). Due to the magnitude of the antibiotic resistance problem, it is critical to develop reliable and rapid methods for the quantification of plasmid transfer frequency that allow the simultaneous analysis of many samples. Here, we present the development of a high-throughput, reliable, quick, easy, and cost-effective method to simultaneously accomplish and measure multiple conjugation events in 96-well plates, in which the quantification of donors, recipients, and transconjugants is estimated from the time required to reach a specific threshold value (OD(600) value) in the bacterial growth curves. Our method successfully discriminates different plasmid transfer frequencies, yielding results that are equivalent to those obtained by a classical conjugation assay.202337886666
382180.9998Persistence of transferable extended-spectrum-β-lactamase resistance in the absence of antibiotic pressure. The treatment of infections caused by antibiotic-resistant bacteria is one of the great challenges faced by clinicians in the 21st century. Antibiotic resistance genes are often transferred between bacteria by mobile genetic vectors called plasmids. It is commonly believed that removal of antibiotic pressure will reduce the numbers of antibiotic-resistant bacteria due to the perception that carriage of resistance imposes a fitness cost on the bacterium. This study investigated the ability of the plasmid pCT, a globally distributed plasmid that carries an extended-spectrum-β-lactamase (ESBL) resistance gene (bla(CTX-M-14)), to persist and disseminate in the absence of antibiotic pressure. We investigated key attributes in plasmid success, including conjugation frequencies, bacterial-host growth rates, ability to cause infection, and impact on the fitness of host strains. We also determined the contribution of the bla(CTX-M-14) gene itself to the biology of the plasmid and host bacterium. Carriage of pCT was found to impose no detectable fitness cost on various bacterial hosts. An absence of antibiotic pressure and inactivation of the antibiotic resistance gene also had no effect on plasmid persistence, conjugation frequency, or bacterial-host biology. In conclusion, plasmids such as pCT have evolved to impose little impact on host strains. Therefore, the persistence of antibiotic resistance genes and their vectors is to be expected in the absence of antibiotic selective pressure regardless of antibiotic stewardship. Other means to reduce plasmid stability are needed to prevent the persistence of these vectors and the antibiotic resistance genes they carry.201222710119
988190.9998Plasmids and the spread of resistance. Plasmids represent one of the most difficult challenge for counteracting the dissemination of antimicrobial resistance. They contribute to the spread of relevant resistance determinants, promoting horizontal gene transfer among unrelated bacteria. Undistinguishable plasmids were identified in unrelated bacterial strains isolated at huge geographically distant area, with no apparent epidemiological links. These plasmids belong to families that are largely prevalent in naturally occurring bacteria, usually carry multiple physically linked genetic determinants, conferring resistance to different classes of antibiotics simultaneously. Plasmids also harbour virulence factors and addiction systems, promoting their stability and maintenance in the bacterial host, in different environmental conditions. The characteristics of the most successful plasmids that were at the origin of the spread of carbapenemase, expanded-spectrum β-lactamase, and plasmid-mediated quinolone resistance genes are discussed in this review.201323499304
4660100.9998Recovery of new integron classes from environmental DNA. Integrons are genetic elements known for their role in the acquisition and expression of genes conferring antibiotic resistance. Such acquisition is mediated by an integron-encoded integrase, which captures genes that are part of gene cassettes. To test whether integrons occur in environments with no known history of antibiotic exposure, PCR primers were designed to conserved regions of the integrase gene and the gene cassette recombination site. Amplicons generated from four environmental DNA samples contained features typical of the integrons found in antibiotic-resistant and pathogenic bacteria. The sequence diversity of the integrase genes in these clones was sufficient to classify them within three new classes of integron. Since they are derived from environments not associated with antibiotic use, integrons appear to be more prevalent in bacteria than previously observed.200111166996
9886110.9998Development of an antimicrobial resistance plasmid transfer gene database for enteric bacteria. Introduction: Type IV secretion systems (T4SSs) are integral parts of the conjugation process in enteric bacteria. These secretion systems are encoded within the transfer (tra) regions of plasmids, including those that harbor antimicrobial resistance (AMR) genes. The conjugal transfer of resistance plasmids can lead to the dissemination of AMR among bacterial populations. Methods: To facilitate the analyses of the conjugation-associated genes, transfer related genes associated with key groups of AMR plasmids were identified, extracted from GenBank and used to generate a plasmid transfer gene dataset that is part of the Virulence and Plasmid Transfer Factor Database at FDA, serving as the foundation for computational tools for the comparison of the conjugal transfer genes. To assess the genetic feature of the transfer gene database, genes/proteins of the same name (e.g., traI/TraI) or predicted function (VirD4 ATPase homologs) were compared across the different plasmid types to assess sequence diversity. Two analyses tools, the Plasmid Transfer Factor Profile Assessment and Plasmid Transfer Factor Comparison tools, were developed to evaluate the transfer genes located on plasmids and to facilitate the comparison of plasmids from multiple sequence files. To assess the database and associated tools, plasmid, and whole genome sequencing (WGS) data were extracted from GenBank and previous WGS experiments in our lab and assessed using the analysis tools. Results: Overall, the plasmid transfer database and associated tools proved to be very useful for evaluating the different plasmid types, their association with T4SSs, and increased our understanding how conjugative plasmids contribute to the dissemination of AMR genes.202338033626
9889120.9998Evolution and dissemination of L and M plasmid lineages carrying antibiotic resistance genes in diverse Gram-negative bacteria. Conjugative, broad host-range plasmids of the L/M complex have been associated with antibiotic resistance since the 1970s. They are found in Gram-negative bacterial genera that cause human infections and persist in hospital environments. It is crucial that these plasmids are typed accurately so that their clinical and global dissemination can be traced in epidemiological studies. The L/M complex has previously been divided into L, M1 and M2 subtypes. However, those types do not encompass all diversity seen in the group. Here, we have examined 148 complete L/M plasmid sequences in order to understand the diversity of the complex and trace the evolution of distinct lineages. The backbone sequence of each plasmid was determined by removing translocatable genetic elements and reversing their effects in silico. The sequence identities of replication regions and complete backbones were then considered for typing. This supported the distinction of L and M plasmids and revealed that there are five L and eight M types, where each type is comprised of further sub-lineages that are distinguished by variation in their backbone and translocatable element content. Regions containing antibiotic resistance genes in L and M sub-lineages have often formed by initial rare insertion events, followed by insertion of other translocatable elements within the inceptive element. As such, islands evolve in situ to contain genes conferring resistance to multiple antibiotics. In some cases, different plasmid sub-lineages have acquired the same or related resistance genes independently. This highlights the importance of these plasmids in acting as vehicles for the dissemination of emerging resistance genes. Materials are provided here for typing plasmids of the L/M complex from complete sequences or draft genomes. This should enable rapid identification of novel types and facilitate tracking the evolution of existing lineages.202132781088
4464130.9998Class 1 integrons, gene cassettes, mobility, and epidemiology. Integrons are genetic elements that, although unable to move themselves, contain gene cassettes that can be mobilized to other integrons or to secondary sites in the bacterial genome. The majority of approximately 60 known gene cassettes encode resistance to antibiotics. Recently, a number of gene cassettes encoding extended-spectrum beta-lactamases or carbapenemases have been described. Up to at least five cassettes may be present in an integron, which leads to multiresistance. Frequently, more than one integron is observed within the same bacterial cell. Integrons are widespread in their species distribution. Although integrons are normally reported from Enterobacteriaceae and other gram-negative bacteria, an integron has been described in Corynebacterium glutamicum, a gram-positive species. The gene cassette in this integron showed even higher expression when compared to the expression in Escherichia coli. Integrons have been reported from all continents and are found frequently. The widespread occurrence of integrons is thought to be due to their association with transposon plasmids, conjugative plasmids, or both. Integrons form an important source for the spread of antibiotic resistance, at least in gram-negative bacteria but also potentially in gram-positive bacteria. The aim of this review is to describe the versatility of integrons, especially their mobility and their ability to collect resistance genes.199910614949
4906140.9998Factors that affect transfer of the IncI1 β-lactam resistance plasmid pESBL-283 between E. coli strains. The spread of antibiotic resistant bacteria worldwide presents a major health threat to human health care that results in therapy failure and increasing costs. The transfer of resistance conferring plasmids by conjugation is a major route by which resistance genes disseminate at the intra- and interspecies level. High similarities between resistance genes identified in foodborne and hospital-acquired pathogens suggest transmission of resistance conferring and transferrable mobile elements through the food chain, either as part of intact strains, or through transfer of plasmids from foodborne to human strains. To study the factors that affect the rate of plasmid transfer, the transmission of an extended-spectrum β-lactamase (ESBL) plasmid from a foodborne Escherichia coli strain to the β-lactam sensitive E. coli MG1655 strain was documented as a function of simulated environmental factors. The foodborne E. coli isolate used as donor carried a CTX-M-1 harboring IncI1 plasmid that confers resistance to β-lactam antibiotics. Cell density, energy availability and growth rate were identified as factors that affect plasmid transfer efficiency. Transfer rates were highest in the absence of the antibiotic, with almost every acceptor cell picking up the plasmid. Raising the antibiotic concentrations above the minimum inhibitory concentration (MIC) resulted in reduced transfer rates, but also selected for the plasmid carrying donor and recombinant strains. Based on the mutational pattern of transconjugant cells, a common mechanism is proposed which compensates for fitness costs due to plasmid carriage by reducing other cell functions. Reducing potential fitness costs due to maintenance and expression of the plasmid could contribute to persistence of resistance genes in the environment even without antibiotic pressure. Taken together, the results identify factors that drive the spread and persistence of resistance conferring plasmids in natural isolates and shows how these can contribute to transmission of resistance genes through the food chain.201525830294
9912150.9998Comprehensive Genomic Investigation of Coevolution of mcr genes in Escherichia coli Strains via Nanopore Sequencing. Horizontal gene transfer facilitates the spread of antibiotic resistance genes, which constitutes a global challenge. However, the evolutionary trajectory of the mobile colistin resistome in bacteria is largely unknown. To investigate the coevolution and fitness cost of the colistin resistance genes in wild strains, different assays to uncover the genomic dynamics of mcr-1 and mcr-3 in bacterial populations are utilized. Escherichia coli strains harboring both mcr-1 and mcr-3.1/3.5 are isolated and mcr genes are associated with diverse mobile elements. Under exposure to colistin, the mcr-1-bearing resistome is stably inherited during bacterial replication, but mcr-3 is prone to be eliminated in populations of certain strains. In the absence of colistin, the persistence rates of the mcr-1 and mcr-3-bearing subclones varies depending on the genomic background. The decay of the mcr-bearing bacterial populations can be mediated by the elimination of mcr-containing segments, large genomic deletions, and plasmid loss. Mobile elements, including plasmids and transposons, are double-edged swords in the evolution of the resistome. The findings support the idea that antibiotic overuse accounts for global spread of multidrug-resistant (MDR) bacteria. Therefore, stringent regulation of antibiotic prescription for humans and animals should be performed systematically to alleviate the threat of MDR bacteria.202133728052
3910160.9998Characterization and Abundance of Plasmid-Dependent Alphatectivirus Bacteriophages. Antimicrobial resistance (AMR) is a major public health threat, exacerbated by the ability of bacteria to rapidly disseminate antimicrobial resistance genes (ARG). Since conjugative plasmids of the incompatibility group P (IncP) are ubiquitous mobile genetic elements that often carry ARG and are broad-host-range, they are important targets to prevent the dissemination of AMR. Plasmid-dependent phages infect plasmid-carrying bacteria by recognizing components of the conjugative secretion system as receptors. We sought to isolate plasmid-dependent phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncP plasmid pKJK5. Irrespective of the site, we only obtained bacteriophages belonging to the genus Alphatectivirus. Eleven isolates were sequenced, their genomes analyzed, and their host range established using S. enterica, Escherichia coli, and Pseudomonas putida carrying diverse conjugative plasmids. We confirmed that Alphatectivirus are abundant in domestic and hospital wastewater using culture-dependent and culture-independent approaches. However, these results are not consistent with their low or undetectable occurrence in metagenomes. Therefore, overall, our results emphasize the importance of performing phage isolation to uncover diversity, especially considering the potential of plasmid-dependent phages to reduce the spread of ARG carried by conjugative plasmids, and to help combat the AMR crisis.202438935220
9973170.9998Spread and Persistence of Virulence and Antibiotic Resistance Genes: A Ride on the F Plasmid Conjugation Module. The F plasmid or F-factor is a large, 100-kbp, circular conjugative plasmid of Escherichia coli and was originally described as a vector for horizontal gene transfer and gene recombination in the late 1940s. Since then, F and related F-like plasmids have served as role models for bacterial conjugation. At present, more than 200 different F-like plasmids with highly related DNA transfer genes, including those for the assembly of a type IV secretion apparatus, are completely sequenced. They belong to the phylogenetically related MOB(F12)A group. F-like plasmids are present in enterobacterial hosts isolated from clinical as well as environmental samples all over the world. As conjugative plasmids, F-like plasmids carry genetic modules enabling plasmid replication, stable maintenance, and DNA transfer. In this plasmid backbone of approximately 60 kbp, the DNA transfer genes occupy the largest and mostly conserved part. Subgroups of MOB(F12)A plasmids can be defined based on the similarity of TraJ, a protein required for DNA transfer gene expression. In addition, F-like plasmids harbor accessory cargo genes, frequently embedded within transposons and/or integrons, which harness their host bacteria with antibiotic resistance and virulence genes, causing increasingly severe problems for the treatment of infectious diseases. Here, I focus on key genetic elements and their encoded proteins present on the F-factor and other typical F-like plasmids belonging to the MOB(F12)A group of conjugative plasmids.201830022749
9882180.9998Integrons in Enterobacteriaceae: diversity, distribution and epidemiology. Integrons are versatile gene acquisition systems that allow efficient capturing of exogenous genes and ensure their expression. Various classes of integrons possessing a wide variety of gene cassettes are ubiquitously distributed in enteric bacteria worldwide. The epidemiology of integrons associated multidrug resistance in Enterobacteriaceae is rapidly evolving. In the past two decades, the incidence of integrons in enteric bacteria has increased drastically with evolution of multiple gene cassettes, novel gene arrangements and complex chromosomal integrons such as Salmonella genomic islands. This review focuses on the distribution, versatility, spread and global trends of integrons among important members of the Enterobacteriaceae, including Escherichia coli, Klebsiella, Shigella and Salmonella, which are known to cause infections globally. Such a comprehensive understanding of integron-associated antibiotic resistance, their role in the spread of such resistance traits and their clinical relevance especially with regard to each genus individually is paramount to contain the global spread of antibiotic resistance.201829038087
4162190.9998Gene sharing among plasmids and chromosomes reveals barriers for antibiotic resistance gene transfer. The emergence of antibiotic resistant bacteria is a major threat to modern medicine. Rapid adaptation to antibiotics is often mediated by the acquisition of plasmids carrying antibiotic resistance (ABR) genes. Nonetheless, the determinants of plasmid-mediated ABR gene transfer remain debated. Here, we show that the propensity of ABR gene transfer via plasmids is higher for accessory chromosomal ABR genes in comparison with core chromosomal ABR genes, regardless of the resistance mechanism. Analysing the pattern of ABR gene occurrence in the genomes of 2635 Enterobacteriaceae isolates, we find that 33% of the 416 ABR genes are shared between chromosomes and plasmids. Phylogenetic reconstruction of ABR genes occurring on both plasmids and chromosomes supports their evolution by lateral gene transfer. Furthermore, accessory ABR genes (encoded in less than 10% of the chromosomes) occur more abundantly in plasmids in comparison with core ABR genes (encoded in greater than or equal to 90% of the chromosomes). The pattern of ABR gene occurrence in plasmids and chromosomes is similar to that in the total Escherichia genome. Our results thus indicate that the previously recognized barriers for gene acquisition by lateral gene transfer apply also to ABR genes. We propose that the functional complexity of the underlying ABR mechanism is an important determinant of ABR gene transferability. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.202234839702