Differential Localization and Functional Specialization of parS Centromere-Like Sites in repABC Replicons of Alphaproteobacteria.

Partitioning systems ensure the stable inheritance of bacterial low-copy-number replicons, such as chromosomes, chromids, and megaplasmids. These loci consist of two genes encoding partition proteins A and B, and at least one parS centromere-like sequence. In chromids and megaplasmids, partitioning systems are often located in the vicinity of replication systems. An extreme example of this co-localization are alphaproteobacterial repABC replicons, where the partition (repAB) and replication (repC) genes form a single operon, with parS sequences usually positioned in close proximity to these genes. In this study, we characterized a more complex repABC system found in Paracoccus aminophilus (Rhodobacterales) megaplasmid pAMI4 (438 kb). Besides the repABC operon with a single parS site, this replicon has a 2-kb non-coding locus positioned 11.5 kb downstream of repC, which contains three additional parS repeats (3parS). We demonstrated that 3parS is bound by partition protein B in vitro and is essential for proper pAMI4 partitioning in vivo. In search of similar loci, we conducted a comparative analysis of parS distribution in other repABC replicons. This revealed different patterns of parS localization in Rhodobacterales and Rhizobiales. However, in both these taxonomic orders, parS sites are almost always located inside or close to the repABC operon. No other 3parS-like loci were found in the closest relatives of pAMI4. Another evolutionarily-independent example of such a locus was identified as a conserved feature in chromosome 2 of Allorhizobium vitis and related replicons. IMPORTANCE The repABC replication/partitioning loci are widespread in extrachromosomal replicons of Alphaproteobacteria. They are evolutionarily diverse, subject to multi-layer self-regulation, and are responsible for the maintenance of different types of replicons, such as plasmids (e.g., Agrobacterium pTi and pRi tumorigenic and rhizogenic plasmids), megaplasmids (e.g., Sinorhizobium pSymA and pSymB) and essential chromids (e.g., secondary chromosomes of Agrobacterium, Brucella and Rhodobacter). In this study, we functionally analyzed an atypical partition-related component of repABC systems, the 3parS locus, found in the P. aminophilus megaplasmid pAMI4. We also identified parS centromere-like site distribution patterns in different groups of repABC replicons and found other unrelated 3parS-like loci, which had been overlooked. Our findings raise questions concerning the biological reasons for differential parS distribution, which may reflect variations in repABC operon regulation as well as different replication and partition modes of replicons belonging to the repABC family.

Evolution of Acinetobacter baumannii plasmids carrying the oxa58 carbapenemase resistance gene via plasmid fusion, IS26-mediated events and dif module shuffling.

Acinetobacter baumannii RepAci1-RepAci10 plasmids pA388 from a global clone 1 (GC1) isolate from Greece, and pACICU1 and variant pACICU1b from an Italian GC2 isolate were found to share a common ancestor. The ancestor formed via recombination between pdif sites in the widely-distributed RepAci1 plasmid pA1-1 and in a RepAci10 plasmid carrying the oxa58 carbapenem-resistance gene in a dif module. Each plasmid includes copies of IS26 and multiple dif modules surrounded by 28 bp pdif sites resembling the chromosomal dif site, including one carrying the oxa58 gene. Plasmid sequences were compared to identify factors driving their evolution and divergence. IS26-mediated events, recombination between pdif sites and homologous recombination have all contributed. A translocatable unit that includes oxa58, generated by an IS26-mediated adjacent deletion, had been re-inserted by IS26 adjacent to an IS26 in pACICU1b to create the oxa58 gene duplication previously found in pACICU1. The oxa58 duplication has been lost from pACICU1b and the Tn6020 variant carrying the aphA1 (kanamycin, neomycin resistance) gene in pA388 has been lost from pACICU1/1b via recombination between directly-oriented IS26 copies. Two dif modules located between directly-oriented pdif sites in pA388 have been lost from pACICU1/1b and the product of this and other deletion events as well as inversion of dif modules located between inversely-oriented pdif sites were detected experimentally in pA388 DNA by PCR. Also, the new junctions were detected in a minority of reads in pA388 long-read sequence data. Inversion and deletion were only detected when the spacers in the pdif sites were identical and equivalent events involving mismatched spacers were not detected.

Primordial origin and diversification of plasmids in Lyme disease agent bacteria.

BACKGROUND: With approximately one-third of their genomes consisting of linear and circular plasmids, the Lyme disease agent cluster of species has the most complex genomes among known bacteria. We report here a comparative analysis of plasmids in eleven Borreliella (also known as Borrelia burgdorferi sensu lato) species. RESULTS: We sequenced the complete genomes of two B. afzelii, two B. garinii, and individual B. spielmanii, B. bissettiae, B. valaisiana and B. finlandensis isolates. These individual isolates carry between seven and sixteen plasmids, and together harbor 99 plasmids. We report here a comparative analysis of these plasmids, along with 70 additional Borreliella plasmids available in the public sequence databases. We identify only one new putative plasmid compatibility type (the 30th) among these 169 plasmid sequences, suggesting that all or nearly all such types have now been discovered. We find that the linear plasmids in the non-B. burgdorferi species have undergone the same kinds of apparently random, chaotic rearrangements mediated by non-homologous recombination that we previously discovered in B. burgdorferi. These rearrangements occurred independently in the different species lineages, and they, along with an expanded chromosomal phylogeny reported here, allow the identification of several whole plasmid transfer events among these species. Phylogenetic analyses of the plasmid partition genes show that a majority of the plasmid compatibility types arose early, most likely before separation of the Lyme agent Borreliella and relapsing fever Borrelia clades, and this, with occasional cross species plasmid transfers, has resulted in few if any species-specific or geographic region-specific Borreliella plasmid types. CONCLUSIONS: The primordial origin and persistent maintenance of the Borreliella plasmid types support their functional indispensability as well as evolutionary roles in facilitating genome diversity. The improved resolution of Borreliella plasmid phylogeny based on conserved partition-gene clusters will lead to better determination of gene orthology which is essential for prediction of biological function, and it will provide a basis for inferring detailed evolutionary mechanisms of Borreliella genomic variability including homologous gene and plasmid exchanges as well as non-homologous rearrangements.

Survival and Evolution of a Large Multidrug Resistance Plasmid in New Clinical Bacterial Hosts.

Large conjugative plasmids are important drivers of bacterial evolution and contribute significantly to the dissemination of antibiotic resistance. Although plasmid borne multidrug resistance is recognized as one of the main challenges in modern medicine, the adaptive forces shaping the evolution of these plasmids within pathogenic hosts are poorly understood. Here we study plasmid-host adaptations following transfer of a 73 kb conjugative multidrug resistance plasmid to naïve clinical isolates of Klebsiella pneumoniae and Escherichia coli. We use experimental evolution, mathematical modelling and population sequencing to show that the long-term persistence and molecular integrity of the plasmid is highly influenced by multiple factors within a 25 kb plasmid region constituting a host-dependent burden. In the E. coli hosts investigated here, improved plasmid stability readily evolves via IS26 mediated deletions of costly regions from the plasmid backbone, effectively expanding the host-range of the plasmid. Although these adaptations were also beneficial to plasmid persistence in a naïve K. pneumoniae host, they were never observed in this species, indicating that differential evolvability can limit opportunities of plasmid adaptation. While insertion sequences are well known to supply plasmids with adaptive traits, our findings suggest that they also play an important role in plasmid evolution by maintaining the plasticity necessary to alleviate plasmid-host constrains. Further, the observed evolutionary strategy consistently followed by all evolved E. coli lineages exposes a trade-off between horizontal and vertical transmission that may ultimately limit the dissemination potential of clinical multidrug resistance plasmids in these hosts.

Mutations in an antisense RNA, involved in the replication control of a repABC plasmid, that disrupt plasmid incompatibility and mediate plasmid speciation.

The maintenance of large plasmid in a wide variety of alpha-proteobacteria depends on the repABC replication/segregation unit. The intergenic repB-repC region of these plasmids encodes a countertranscribed RNA (ctRNA) that modulates the transcription/translation rate of RepC, the initiator protein. The ctRNA acts as a strong incompatibility factor when expressed in trans. We followed a site directed mutagenesis approach to map those sequences of the ctRNA that are required for plasmid incompatibility and for plasmid replication control. We found that the first three nucleotides of the 5'-end of the ctRNA are essential for interactions with its target RNA. We also found that stretches of 4-5 nucleotides of non-complementarity within the first 10 nucleotides of the left arm of the ctRNA and the target RNA are sufficient to avoid plasmid incompatibility. Additionally, miniplasmid derivatives expressing ctRNAs with mutations in the 5' end or small deletions in the ctRNA are capable of controlling their own replication and coexisting with the parental plasmid. We suggest that a mechanism that could have a crucial role in the speciation process of repABC plasmids is to accumulate enough changes in this small region of the ctRNA gene to disrupt heteroduplex formation between the target RNA of one plasmid and the ctRNA of the other. Plasmids carrying these changes will not have defects in their maintenance.

Emergence and plasmid cointegration-based evolution of NDM-1-producing ST107 Citrobacter freundii high-risk resistant clone in China.

Carbapenem-resistant Citrobacter freundii (CRCF) poses an enormous challenge in the health care setting. However, the epidemiology and plasmid dynamic evolution of this species have not been well studied, especially for the novel high-risk resistant clones in the intensive care units (ICUs). Here, we characterised the cointegration-based plasmid dynamic evolution of the emerging ST107 CRCF clone in China. Twenty CRCF strains were identified, including ST22 (30%), ST107 (25%), ST396 (10%) and ST116 (10%). Interestingly, the tigecycline (TGC) resistance gene cluster tmexCD2-toprJ2 and blaNDM-1 and blaKPC-2 were simultaneously found in one ST107 strain. Epidemiological analysis showed that ST107 clone contained human- and environment-derived strains from five countries. Notably, 93.75% (15/16) of the isolates harboured blaNDM-1 or blaKPC-2. Plasmid fusion among various ST107 strains of two patients occurred in the same ICU, mediated by Tn5403 and IS26-based insertion and deletion events. pCF1807-2 carried blaNDM-1 while pCF1807-3 carried both tmexCD2-toprJ2 and blaKPC-2 in the CF1807 strain. Importantly, the cointegrate plasmid pCF1807-2 exhibited higher transfer efficiency and could remain stable after serial passage. Notably, no fitness cost was observed for the host. In conclusion, ST107 CRCF is a high-risk resistant clone due to its ability to integrate resistant plasmids. Our findings elucidated the potential threat and global transmission of the ST107 lineage, and reasonable monitoring should be performed to prevent its further spread in hospitals.

Genome sequencing unveils blaKPC-2-harboring plasmids as drivers of enhanced resistance and virulence in nosocomial Klebsiella pneumoniae.

The threat posed by Klebsiella pneumoniae in healthcare settings has worsened due to the evolutionary advantages conferred by blaKPC-2-harboring plasmids (pKPC-2). However, the specific evolutionary pathway of nosocomial K. pneumoniae carrying pKPC-2 and its transmission between patients and healthcare environments are not yet well understood. Between 1 August and 31 December 2019, 237 ST11 KPC-2-producing-carbapenem-resistant K. pneumoniae (CRKP) (KPC-2-CRKP) were collected from patient or ward environments in an intensive care unit and subjected to Illumina sequencing, of which 32 strains were additionally selected for Nanopore sequencing to obtain complete plasmid sequences. Bioinformatics analysis, conjugation experiments, antimicrobial susceptibility tests, and virulence assays were performed to identify the evolutionary characteristics of pKPC-2. The pKPC-2 plasmids were divided into three subgroups with distinct evolutionary events, including Tn3-mediated plasmid homologous recombination, IS26-mediated horizontal gene transfer, and dynamic duplications of antibiotic resistance genes (ARGs). Surprisingly, the incidence rates of multicopy blaKPC-2, blaSHV-12, and blaCTX-M-65 were quite high (ranging from 27.43% to 67.01%), and strains negative for extended-spectrum ß-lactamase tended to develop multicopy blaKPC-2. Notably, the presence of multicopy blaSHV-12 reduced sensitivity to ceftazidime/avibactam (CZA), and the relative expression level of blaSHV-12 in the CZA-resistant group was 6.12 times higher than that in the sensitive group. Furthermore, a novel hybrid pKPC-2 was identified, presenting enhanced virulence levels and decreased susceptibility to CZA. This study emphasizes the notable prevalence of multicopy ARGs and provides a comprehensive insight into the intricate and diverse evolutionary pathways of resistant plasmids that disseminate among patients and healthcare environments.IMPORTANCEThis study is based on a CRKP screening program between patients and ward environments in an intensive care unit, describing the pKPC-2 (blaKPC-2-harboring plasmids) population structure and evolutionary characteristics in clinical settings. Long-read sequencing was performed in genetically closely related strains, enabling the high-resolution analysis of evolution pathway between or within pKPC-2 subgroups. We revealed the extremely high rates of multicopy antibiotic resistance genes (ARGs) in clinical settings and its effect on resistance profile toward novel ß-lactam/ß-lactamase inhibitor combinations, which belongs to the last line treatment choices toward CRKP infection. A novel hybrid pKPC-2 carrying CRKP with enhanced resistance and virulence level was captured during its clonal spread between patients and ward environment. These evidences highlight the threat of pKPC-2 to CRKP treatment and control. Thus, surveillance and timely disinfection in clinical settings should be practiced to prevent transmission of CRKP carrying threatful pKPC-2. And rational use of antibiotics should be called for to prevent inducing of pKPC-2 evolution, especially the multicopy ARGs.

Insights into the Evolution of IncR Plasmids Found in the Southern European Clone of the Monophasic Variant of Salmonella enterica Serovar Typhimurium.

Salmonella enterica subspecies enterica serovar 4,[5],12:i:- is a monophasic variant of S. Typhimurium which has emerged as a world-wide distributed pathogen in the last decades. Several clones have been identified within this variant, the European clone, the Spanish clone, the Southern European clone and the U.S./American clone. The present study focused on isolates of the Southern European clone that were obtained from clinical samples at Spanish hospitals. The selected isolates were multidrug resistant, with most resistance genes residing on IncR plasmids that also carried virulence genes. These plasmids had a mosaic structure, comprising a highly reduced IncR backbone, which has acquired a large amount of exogenous DNA mostly derived from pSLT and IncI1-I(alfa) plasmids. Although composed of approximately the same elements, the investigated plasmids displayed a high diversity, consistent with active evolution driven by a wealth of mobile genetic elements. They comprise multiple intact or truncated insertion sequences, transposons, pseudo-compound transposons and integrons. Particularly relevant was the role of IS26 (with six to nine copies per plasmid) in generating insertions, deletions and inversions, with many of the rearrangements uncovered by tracking the patterns of eight bp target site duplications. Most of the resistance genes detected in the analyzed isolates have been previously associated with the Southern European clone. However, erm(B), lnu(G) and blaTEM-1B are novel, with the last two carried by a second resistance plasmid found in one of the IncR-positive isolates. Thus, evolution of resistance in the Southern European clone is not only mediated by diversification of the IncR plasmids, but also through acquisition of additional plasmids. All isolates investigated in the present study have the large deletion affecting the fljBA region previously found to justify the monophasic phenotype in the Southern European and U.S./American clones. An SNP-based phylogenetic analysis revealed the close relationship amongst our isolates, and support that those sharing the large fljBA deletion could be more heterogeneous than previously anticipated.

Evolution of IncHI1 plasmids: two distinct lineages.

The IncHI1 plasmid pSRC27-H from Salmonella enterica serovar Typhimurium carries a region containing several genes that confer resistance to different antibiotics, and this resistance region is in the same position as related resistance regions in a group of sequenced IncHI1 plasmids from various sources that includes pHCM1. Four further additional segments are found in pHCM1 relative to another IncHI1 plasmid, R27. Using PCR or DNA sequencing to detect the presence or absence of each of these additional segments in the same position in the IncHI1 backbone, plasmid pSRC27-H was found to include them. However, in one case the additional segment was smaller in pSRC27-H, lacking a transposon carrying a second resistance region in pHCM1. The sequences of IncHI1 plasmids, pO111_1 and pMAK1, were also examined and found to share the same or closely related additional segments. The structure of the additional material in pHCM1, pO111_1 and pMAK1 was examined, and potential novel transposons were identified. These additional segments define an IncHI1 lineage (pHCM1, pO111_1, pMAK1, pSRC27-H) which we designated type 2 to distinguish it from type 1 (R27, pAKU_1, pP-stx-12). A segment from the Escherichia coli genome and an adjacent copy of IS1 in pHCM1 was defined by comparison to pO111_1 and pMAK1, which lack it. pSRC27-H also lacks it. This structure is present in the same position in R27 and type 1 plasmids, but in the opposite orientation, and appears to have been incorporated via IS1-mediated transposition. The PCRs developed provide a simple means of distinguishing type 1 and type 2 IncHI1 plasmids based on the presence or absence of variable regions.

Dynamic evolution and transmission of a blaNDM-1-bearing fusion plasmid in a clinical Escherichia coli.

Plasmids are the main driving forces for the rapid dissemination of blaNDM-1. In recent years, blaNDM-1-carrying fusion plasmids have been frequently reported. However, the evolutionary patterns of blaNDM-1-carrying fusion plasmids remain largely unknown. Herein, we reported a blaNDM-1-bearing fusion plasmid pZX35-269k possessing IncFII and IncA/C2 replicons from clinical ST349 E. coli 13ZX35. The backbone of pZX35-269k was structurally unstable, which was manifested in different types of structural dissociation during conjugation and passage, thereby forming various daughter plasmids. Moreover, the same events were observed in the clinical setting as well. We found that pZX35-269k exhibited highly identical to two plasmids (pZX30-70k and pZX30-192k) in 13ZX30, both of which were isolated from the same hospital. Sequence analysis highlighted that two plasmids in 13ZX30 evolved from pZX35-269k through homologous recombination of a 4856-bp fragment. Collectively, this study confirmed the transmission and structural evolution of a blaNDM-1-bearing fusion plasmid in both laboratory and clinical settings, and provided clear evidence of plasmid spread and evolution in clinical settings. Such versatile plasmids may represent a potential risk for the public health.

Genomics, Transcriptomics, and Metabolomics Reveal That Minimal Modifications in the Host Are Crucial for the Compensatory Evolution of ColE1-Like Plasmids.

Plasmid-mediated antimicrobial resistance is one of the major threats to public health worldwide. The mechanisms involved in the plasmid/host coadaptation are still poorly characterized, and their understanding is crucial to comprehend the genesis and evolution of multidrug-resistant bacteria. With this purpose, we designed an experimental evolution using Haemophilus influenzae RdKW20 as the model strain carrying the ColE1-like plasmid pB1000. Five H. influenzae populations adapted previously to the culture conditions were transformed with pB1000 and subsequently evolved to compensate for the plasmid-associated fitness cost. Afterward, we performed an integrative multiomic analysis combining genomics, transcriptomics, and metabolomics to explore the molecular mechanisms involved in the compensatory evolution of the plasmid. Our results demonstrate that minimal modifications in the host are responsible for plasmid adaptation. Among all of them, the most enriched process was amino acid metabolism, especially those pathways related to serine, tryptophan, and arginine, eventually related to the genesis and resolution of plasmid dimers. Additional rearrangements occurred during the plasmid adaptation, such as an overexpression of the ribonucleotide reductases and metabolic modifications within specific membrane phospholipids. All these findings demonstrate that the plasmid compensation occurs through the combination of diverse host-mediated mechanisms, of which some are beyond genomic and transcriptomic modifications. IMPORTANCE The ability of bacteria to horizontally transfer genetic material has turned antimicrobial resistance into one of the major sanitary crises of the 21st century. Plasmid conjugation is considered the main mechanism responsible for the mobilization of resistance genes, and its understanding is crucial to tackle this crisis. It is generally accepted that the acquisition and maintenance of mobile genetic elements entail a fitness cost to its host, which is susceptible to be alleviated through a coadaptation process or compensatory evolution. Notwithstanding, despite recent major efforts, the underlying mechanisms involved in this adaptation remain poorly characterized. Analyzing the plasmid/host coadaptation from a multiomic perspective sheds light on the physiological processes involved in the compensation, providing a new understanding on the genesis and evolution of plasmid-mediated antimicrobial-resistant bacteria.

Characterization of a Conjugative Multidrug Resistance IncP-2 Megaplasmid, pPAG5, from a Clinical Pseudomonas aeruginosa Isolate.

The spread of resistance genes via horizontal plasmid transfer plays a significant role in the formation of multidrug-resistant (MDR) Pseudomonas aeruginosa strains. Here, we identified a megaplasmid (ca. 513 kb), designated pPAG5, which was recovered from a clinical multidrug-resistant P. aeruginosa PAG5 strain. The pPAG5 plasmid belonged to the IncP-2 incompatibility group. Two large multidrug resistance regions (MDR-1 and MDR-2) and two heavy metal resistance operons (merEDACPTR and terZABCDE) were identified in the pPAG5 plasmid. Genetic analysis demonstrated that the formation of MDR regions was mediated by several homologous recombination events. Further conjugation assays identified that pPAG5 could be transferred to P. aeruginosa but not Escherichia coli. Antimicrobial susceptibility testing on transconjugants demonstrated that pPAG5 was capable of transferring resistance genes to transconjugants and producing a multidrug-resistant phenotype. Comparative analysis revealed that pPAG5 and related plasmids shared an overall similar backbone, including genes essential for replication (repA), partition (par), and conjugal transfer (tra). Further phylogenetic analysis showed that pPAG5 was closely related to plasmids pOZ176 and pJB37, both of which are members of the IncP-2-type plasmid group. IMPORTANCE The emergence and spread of plasmid-associated multidrug resistance in bacterial pathogens is a key global threat to public health. It is important to understand the mechanisms of the formation and evolution of these plasmids in patients, hospitals, and the environment. In this study, we detailed the genetic characteristics of a multidrug resistance IncP-2 megaplasmid, pPAG5, and investigated the formation of its MDR regions and evolution. To the best of our knowledge, plasmid pPAG5 is the largest multidrug resistance plasmid ever sequenced in the Pseudomonas genus. Our results may provide further insight into the formation of multidrug resistance plasmids in bacteria and the molecular evolution of plasmids.

Plasmid Viability Depends on the Ecological Setting of Hosts within a Multiplasmid Community.

Plasmids are extrachromosomal genetic elements, some of which disperse horizontally between different strains and species of bacteria. They are a major factor in the dissemination of virulence factors and antibiotic resistance. Understanding the ecology of plasmids has a notable anthropocentric value, and therefore, the interactions between bacterial hosts and individual plasmids have been studied in detail. However, bacterial systems often carry multiple genetically distinct plasmids, but dynamics within these multiplasmid communities have remained unstudied. Here, we set to investigate the survival of 11 mobilizable or conjugative plasmids under five different conditions where the hosts had a differing ecological status in comparison to other bacteria in the system. The key incentive was to determine whether plasmid dynamics are reproducible and whether there are tradeoffs in plasmid fitness that stem from the ecological situation of their initial hosts. Growth rates and maximum population densities increased in all communities and treatments over the 42-day evolution experiment, although plasmid contents at the end varied notably. Large multiresistance-conferring plasmids were unfit when the community also contained smaller plasmids with fewer resistance genes. This suggests that restraining the use of a few antibiotics can make bacterial communities sensitive to others. In general, the presence or absence of antibiotic selection and plasmid-free hosts (of various fitnesses) has a notable influence on which plasmids survive. These tradeoffs in different settings can help explain, for example, why some resistance plasmids have an advantage during a rapid proliferation of antibiotic-sensitive pathogens whereas others dominate in alternative situations. IMPORTANCE Conjugative and mobilizable plasmids are ubiquitous in bacterial systems. Several different plasmids can compete within a single bacterial community. We here show that the ecological setting of the host bacteria has a notable effect on the survival of individual plasmids. Selection for opportunistic genes such as antibiotic resistance genes and the presence of plasmid-free hosts can determine which plasmids survive in the system. Host bacteria appear to adapt specifically to a situation where there are multiple plasmids present instead of alleviating the plasmid-associated fitness costs of individual plasmids. Plasmids providing antibiotic resistance survived under all conditions even if there was a constant migration of higher-fitness plasmid-free hosts and no selection via antibiotics. This study is one of the first to observe the behavior of multiple genetically different plasmids as a part of a single system.

Emerging Opportunity and Destiny of mcr-1- and tet(X4)-Coharboring Plasmids in Escherichia coli.

The emergence of the plasmid-mediated colistin resistance gene mcr-1 and the plasmid-mediated tigecycline resistance gene tet(X4) represents a significant threat to public health. Although mcr-1 and tet(X4) have been reported to coexist in the same isolate, there are no reports on the emergence of plasmids coharboring mcr-1 and tet(X4). In this study, we aimed to investigate the opportunities for the emergence of mcr-1- and tet(X4)-coharboring plasmids and their destiny in Escherichia coli. Two plasmids carrying both mcr-1 and tet(X4) were constructed through conjugation assays and confirmed by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Nanopore long-read sequencing. Seven evolved plasmids carrying mcr-1 and tet(X4) from one of the two plasmids were acquired after continuous evolutionary processes. The fitness effects of mcr-1- and tet(X4)-coharboring plasmids were studied by stability experiments, competition experiments, and growth curve measurements. A plasmid carrying mcr-1 and tet(X4) and conferring no fitness cost to its host strain E. coli C600 emerged after evolution during serial passages of bacteria. We proved that it can be anticipated that mcr-1 and tet(X4) could appear in a single plasmid, and the possibility of occurrence in field strains should be monitored constantly. The originally formed cointegrate plasmids coharboring mcr-1 and tet(X4) could evolve into a plasmid with lower fitness costs. This will undoubtedly accelerate the transmission of mcr-1 and tet(X4) globally. The findings highlighted the great possibility of novel hybrid plasmids positive for mcr-1 and tet(X4), and the risk is worthy of increasing attention and public concern globally. IMPORTANCE Tigecycline and colistin are used as last-resort therapies to treat infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, the emergence of the plasmid-mediated tigecycline resistance gene tet(X4) and the plasmid-mediated colistin resistance gene mcr-1 represents a significant threat to human health. A plasmid coharboring mcr-1 and tet(X4) has not emerged so far, but the potential risk should not be ignored. Plasmids coharboring such vital resistance genes will greatly accelerate the progression of pan-drug resistance among pathogens globally. Therefore, evaluation of the emerging opportunity for the mcr-1- and tet(X4)-coharboring plasmids and their destiny in E. coli is of great significance. We provide important insight into the contributions of intI1, IS26, a truncated ISCR2 (ΔISCR2), and IS4321R during the generation of cointegrate plasmids carrying mcr-1 and tet(X4) and highlight the importance of antimicrobials in the evolution and diversity of mcr-1- and tet(X4)-coharboring plasmids. We show that monitoring of the occurrence of mcr-1-carrying MDR plasmids and tet(X4)-bearing MDR plasmids in the same strain should be strengthened to avoid the formation of mcr-1- and tet(X4)-coharboring plasmids.

Antibiotics Interfere with the Evolution of Plasmid Stability.

Extra-chromosomal genetic elements are important drivers of bacterial evolution, and their evolutionary success depends on positive selection for the genes they encode. Examples are plasmids encoding antibiotic resistance genes that are maintained in the presence of antibiotics (e.g., [1-3]). Plasmid maintenance is considered a metabolic burden to the host [4]; hence, when the cost of plasmid carriage outweighs its benefit, plasmid-free segregants are expected to outcompete plasmid-carrying cells, eventually leading to plasmid loss [5-7]. Thus, in the absence of positive selection, plasmid survival hinges upon stable persistence in the population. The ubiquity of plasmids in nature suggests that plasmids having a negligible effect on host fitness may evolve stable inheritance and thus gain a long-term persistence in the population, also in the absence of positive selection [8]. Nonetheless, the transition of plasmids into stably inherited genetic elements remains understudied. Here, we show that positive selection for a plasmid-encoded gene interferes with the evolution of plasmid stability. Evolving plasmids under different selection regimes in Escherichia coli, we find that antibiotics led to plasmid amplification, resulting in plasmid instability. Thus, under positive selection, suboptimal solutions for plasmid stability were maintained in the population hindering long-term plasmid persistence. Indeed, a survey of Escherichia plasmids confirms that antibiotic resistance genes are rarely found on small plasmids. Our results show that a plasmid-mediated advantage for the host may manifest in reduced plasmid evolutionary success. Considering plasmids as autonomously evolving entities holds promise for understanding the factors that govern their evolution.

Tracking Recombination Events That Occur in Conjugative Virulence Plasmid p15WZ-82_Vir during the Transmission Process.

We recently reported the recovery of a conjugative virulence plasmid, p15WZ-82_Vir, from a clinical Klebsiella variicola strain. In this study, we found that several new plasmid types were generated due to genetic rearrangement. Partial integration of plasmid p15WZ-82_Vir with existing plasmids such as resistance plasmids by different homologous recombination events was observable in three recipient strains. Such recombination events enable the formation of various types of mosaic plasmids simultaneously carrying virulence-encoding and antibiotic resistance-encoding genes as well as genes involved in plasmid conjugation, which promote transmission of various virulence-encoding and resistance-encoding elements among pathogens. Our data also suggest that these conjugative events may play an integral role in the development of novel mosaic plasmids, which is vital for plasmid evolution.IMPORTANCE Although they are often nonconjugative, large virulence plasmids are increasingly detected in clinical K. pneumoniae and contribute to the hypervirulence phenotype of this organism. In this study, we demonstrated that the virulence-encoding region that originated from virulence plasmid pLVPK actively interacted with different types of plasmids via homologous recombination to generate new conjugative plasmids. This report provides insights into the evolution of self-transmissible plasmids carrying genetic elements encoding both hypervirulent and multidrug-resistant phenotypes, which facilitate the rapid development of clinical K. pneumoniae strains that are hypervirulent and multidrug resistant.

Co-occurrence of a novel VIM-1 and FosA3-encoding multidrug-resistant plasmid and a KPC-2-encoding pKP048-like plasmid in a clinical isolate of Klebsiella pneumoniae sequence type 11.

The spread of carbapenem-resistant Enterobacteriaceae (CRE) worldwide remains a major threat to public health. Notably, carbapenemase-encoding genes are usually located in plasmids harboring other resistance determinants, and isolates having multiple plasmids are often highly resistant to carbapenems. In this study, we characterized the genetic context of coproduction of KPC-2, VIM-1, and FosA3 via two plasmids in the multidrug-resistant Klebsiella pneumoniae sequence type 11 (ST11) isolate JS187, recovered during an outbreak of KPC-2-producing K. pneumoniae in a Chinese teaching hospital in 2008. Plasmid p187-1, coharboring blaVIM-1 and fosA3, consisted of a pKOX-R1-like backbone and two multidrug resistant (MDR) regions separated by pKHS1-like backbone sequences involving plasmid replication and stability. The MDR region 1 was a chimera composed of the blaVIM-1-bearing In916-like integron and Tn1721-like transposon, and was disrupted by sequential insertion of an IS26-based transposition unit carrying blaCTX-M-3 and a ΔTn3-like transposon bearing blaTEM-1. MDR region 2 was an IS26-array structure with fosA3 and blaSHV-12. Plasmid p187-2 harboring blaKPC-2 was closely related to pKP048. blaKPC-2 in p187-2 was carried by a Tn1721 variant, which differed from the prototype Tn1721-blaKPC-2 transposon observed in pKP048 by disruption of an IS26 at Tn3 and deletion of a 31-kb MDR fragment. Co-existence of the novel VIM-1- and FosA3-encoding MDR plasmid p187-1 and the KPC-2-encoding pKP048-like plasmid p187-2 made K. pneumoniae JS187 highly resistant to carbapenems. Moreover, p187-1 still carried genes conferring resistance to cephalosporins, fosfomycin, chloramphenicol, and quaternary ammonium, posing substantial challenges for the treatment of Enterobacteriaceae infections. Thus, monitoring the prevalence and evolution of these plasmids and/or strains is critical.

Evolutionary Relatedness and Classification of Tumor-Inducing and Opine-Catabolic Plasmids in Three Rhizobium rhizogenes Strains Isolated from the Same Crown Gall Tumor.

Plasmids play a crucial role in the ecology of agrobacteria. In this study, we sequenced tumor-inducing (Ti) and opine-catabolic (OC) plasmids in three Rhizobium rhizogenes (Agrobacterium biovar 2) strains isolated from the same crown gall tumor on "Colt" cherry rootstock and conducted comparative genomic analyses. Tumorigenic strains C5.7 and C6.5 carry nopaline-type Ti plasmids pTiC5.7/pTiC6.5, whereas the nonpathogenic strain Colt5.8 carries the nopaline-type OC plasmid pOC-Colt5.8. Overall, comparative genomic analysis indicated that pTiC5.7/pTiC6.5 and related Ti plasmids described before (pTiC58 and pTi-SAKURA) originate from a common ancestor, although they have diverged during evolution. On the other hand, plasmid pOC-Colt5.8 was most closely related to the well-known OC plasmid pAtK84b; however, analysis suggested that they had different evolutionary histories and seem to share a more distant common ancestor. Although the reconstruction of the evolutionary history of Ti and OC plasmids is still speculative, we hypothesized that nopaline-type Ti plasmid might originate from the nopaline-type OC plasmid. Our results suggested that OC plasmids are widespread and closely associated with crown gall tumors. Finally, we proposed a thorough scheme for classification of Ti and OC plasmids that is based on separate comparative analysis of each functional element of the plasmid studied.

The toxic guardians - multiple toxin-antitoxin systems provide stability, avoid deletions and maintain virulence genes of Pseudomonas syringae virulence plasmids.

BACKGROUND: Pseudomonas syringae is a γ-proteobacterium causing economically relevant diseases in practically all cultivated plants. Most isolates of this pathogen contain native plasmids collectively carrying many pathogenicity and virulence genes. However, P. syringae is generally an opportunistic pathogen primarily inhabiting environmental reservoirs, which could exert a low selective pressure for virulence plasmids. Additionally, these plasmids usually contain a large proportion of repeated sequences, which could compromise plasmid integrity. Therefore, the identification of plasmid stability determinants and mechanisms to preserve virulence genes is essential to understand the evolution of this pathogen and its adaptability to agroecosystems. RESULTS: The three virulence plasmids of P. syringae pv. savastanoi NCPPB 3335 contain from one to seven functional stability determinants, including three highly active toxin-antitoxin systems (TA) in both pPsv48A and pPsv48C. The TA systems reduced loss frequency of pPsv48A by two orders of magnitude, whereas one of the two replicons of pPsv48C likely confers stable inheritance by itself. Notably, inactivation of the TA systems from pPsv48C exposed the plasmid to high-frequency deletions promoted by mobile genetic elements. Thus, recombination between two copies of MITEPsy2 caused the deletion of an 8.3 kb fragment, with a frequency of 3.8 ± 0.3 × 10- 3. Likewise, one-ended transposition of IS801 generated plasmids containing deletions of variable size, with a frequency of 5.5 ± 2.1 × 10- 4, of which 80% had lost virulence gene idi. These deletion derivatives were stably maintained in the population by replication mediated by repJ, which is adjacent to IS801. IS801 also promoted deletions in plasmid pPsv48A, either by recombination or one-ended transposition. In all cases, functional TA systems contributed significantly to reduce the occurrence of plasmid deletions in vivo. CONCLUSIONS: Virulence plasmids from P. syringae harbour a diverse array of stability determinants with a variable contribution to plasmid persistence. Importantly, we showed that multiple plasmid-borne TA systems have a prominent role in preserving plasmid integrity and ensuring the maintenance of virulence genes in free-living conditions. This strategy is likely widespread amongst native plasmids of P. syringae and other bacteria.