Plasmid Transfer Research Articles

Staphylococcus equorum plasmid pKS1030-3 encodes auxiliary biofilm formation and trans-acting gene mobilization systems

The foodborne bacterium Staphylococcus equorum strain KS1030 harbours plasmid pSELNU1, which encodes a lincomycin resistance gene. pSELNU1 undergoes horizontal transfer between bacterial strains, thus spreading antibiotic resistance. However, the genes required for horizontal plasmid transfer are not encoded in pSELNU1. Interestingly, a relaxase gene, a type of gene related to horizontal plasmid transfer, is encoded in another plasmid of S. equorum KS1030, pKS1030-3. The complete genome of pKS1030-3 is 13,583 bp long and encodes genes for plasmid replication, biofilm formation (the ica operon), and horizontal gene transfer. The replication system of pKS1030-3 possesses the replication protein-encoding gene repB, a double-stranded origin of replication, and two single-stranded origins of replication. The ica operon, relaxase gene, and a mobilization protein-encoding gene were detected in pKS1030-3 strain-specifically. When expressed in S. aureus RN4220, the ica operon and relaxase operon of pKS1030-3 conferred biofilm formation ability and horizontal gene transfer ability, respectively. The results of our analyses show that the horizontal transfer of pSELNU1 of S. equorum strain KS1030 depends on the relaxase encoded by pKS1030-3, which is therefore trans-acting. Genes encoded in pKS1030-3 contribute to important strain-specific properties of S. equorum KS1030. These results could contribute to preventing the horizontal transfer of antibiotic resistance genes in food.

Construction and characterization of a novel miniaturized filamentous phagemid for targeted mammalian gene transfer

BackgroundAs simplistic proteinaceous carriers of genetic material, phages offer great potential as targeted vectors for mammalian transgene delivery. The filamentous phage M13 is a single-stranded DNA phage with attractive characteristics for gene delivery, including a theoretically unlimited DNA carrying capacity, amenability to tropism modification via phage display, and a well-characterized genome that is easy to genetically modify. The bacterial backbone in gene transfer plasmids consists of elements only necessary for amplification in prokaryotes, and, as such, are superfluous in the mammalian cell. These problematic elements include antibiotic resistance genes, which can disseminate antibiotic resistance, and CpG motifs, which are inflammatory in animals and can lead to transgene silencing.ResultsHere, we examined how M13-based phagemids could be improved for transgene delivery by removing the bacterial backbone. A transgene cassette was flanked by isolated initiation and termination elements from the phage origin of replication. Phage proteins provided in trans by a helper would replicate only the cassette, without any bacterial backbone. The rescue efficiency of “miniphagemids” from these split origins was equal to, if not greater than, isogenic “full phagemids” arising from intact origins. The type of cassette encoded by the miniphagemid as well as the choice of host strain constrained the efficiency of phagemid rescue.ConclusionsThe use of two separated domains of the f1 ori improves upon a single wildtype origin while still resulting in high titres of miniphagemid gene transfer vectors. Highly pure lysates of miniaturized phagemids could be rapidly obtained in a straightforward procedure without additional downstream processing.

Genomic epidemiology of mcr carrying multidrug-resistant ST34 Salmonella enterica serovar Typhimurium in a one health context: The evolution of a global menace

The rapid global dissemination of Salmonella enterica sequence type 34 (ST34) has sparked significant concern due to its resistance to critical antimicrobials and its ability to spread across various sectors. In order to investigate the evolution and transmission dynamics of this epidemic clonal lineage, as well as the horizontal transfer of mcr-carrying plasmids within the One Health framework, we conducted a comprehensive genomic epidemiological study. This study focused on the 11 mcr-carrying S. enterica isolates obtained from clinical settings in China, while also considering 2337 publicly available genomes of mcr-carrying S. enterica collected from 20 countries and diverse sources spanning over a 22-year period. Among the mcr-positive Salmonella isolates, ST34 was found to be the predominant lineage, comprising 30.12 % (704/2337) of the total collection. These isolates were identified as either serovar Typhimurium or its monophasic variant, which were obtained from both clinical and non-clinical sources. Phylogeographic analyses traced the global spread of the mcr-carrying ST34 lineage, which was divided into three distinct clusters, with 83.10 % of them carrying mcr-1 or/and mcr-9 genes. Notably, the mcr-1 positive ST34 isolates were primarily found in China (190/298, 63.76 %), with only four from the United States. Conversely, mcr-9 positive ST34 isolates were predominantly identified in the United States (261/293, 89.08 %), while none were observed in China. The mcr-1 positive ST34 isolates was predicted to have originated from clinical sources in United Kingdom, whereas mcr-9 positive ST34 isolates was likely derived from environmental sources in Germany. The most recent common ancestor for mcr-1 and mcr-9 carrying ST34 S. enterica was estimated to have emerged around 1983 and 1951. These findings provided thorough and intuitive insights into the intercontinental spread of mcr-carrying S. enterica ST34 lineage in a One Health context. Ongoing surveillance is crucial for effectively monitoring the worldwide dissemination of this multidrug-resistant high-risk clone.

The Mechanism of ITGB3 and PPIF Plasmid Construction on the Offspring of Rats with Intrauterine Adhesion and Bioinformatics Analysis

This study aimed to develop an animal model of pathologic regeneration of intrauterine adhesions (IUA) and assess the impact of ITGB3 and PPIF plasmid construction on the offspring of rats with IUA. Thirty female SD rats were divided into an experimental group and a control group. In the experimental group, the rats’ left and right endometrium underwent mild and severe mechanical damage using a self-made curette. The control group underwent a sham operation without endometrial injury. At various time points after surgery, uteruses were collected for analysis. Immunohistochemical staining was performed to evaluate changes in major histocompatibility complex II molecule (MHC II) and 5-bromodeoxyuridine (BrdU) in endometrial cells. The study observed the inflammatory and hyperplastic changes in endometrial tissue and the repair process in rats with different endometrial gland injuries. RNA interference (RNAi) sequences targeting the rat PPIF gene were designed and cloned into a lentivirus vector transfer plasmid, ITGB3. The number of endometrial glands decreased with increased mechanical injury. The PPIF short hairpin RNA (shRNA) fragment was successfully cloned into the lentiviral vector. After mild mechanical injury, the endometrial tissue regenerated to a basic repair level, while severe mechanical injury led to incomplete repair and tissue fibrosis, resulting in IUA.

Antibiotic-degrading resistance changes bacterial community structure via species-specific responses

Some bacterial resistance mechanisms degrade antibiotics, potentially protecting neighbouring susceptible cells from antibiotic exposure. We do not yet understand how such effects influence bacterial communities of more than two species, which are typical in nature. Here, we used experimental multispecies communities to test the effects of clinically important pOXA-48-plasmid-encoded resistance on community-level responses to antibiotics. We found that resistance in one community member reduced antibiotic inhibition of other species, but some benefitted more than others. Further experiments with supernatants and pure-culture growth assays showed the susceptible species profiting most from detoxification were those that grew best at degraded antibiotic concentrations (greater than zero, but lower than the starting concentration). This pattern was also observed on agar surfaces, and the same species also showed relatively high survival compared to most other species during the initial high-antibiotic phase. By contrast, we found no evidence of a role for higher-order interactions or horizontal plasmid transfer in community-level responses to detoxification in our experimental communities. Our findings suggest carriage of an antibiotic-degrading resistance mechanism by one species can drastically alter community-level responses to antibiotics, and the identities of the species that profit most from antibiotic detoxification are predicted by their intrinsic ability to survive and grow at changing antibiotic concentrations.

Extensive Diversity in Escherichia coli Group 3 Capsules Is Driven by Recombination and Plasmid Transfer from Multiple Species.

Bacterial capsules provide protection against environmental challenges and host immunity. Historically, Escherichia coli K serotyping scheme, which relies on the hypervariable capsules, has identified around 80 K forms that fall into four distinct groups. Based on recent work by us and others, we predicted that E. coli capsular diversity is grossly underestimated. We exploited group 3 capsule gene clusters, the best genetically defined capsule group in E. coli, to analyze publicly available E. coli sequences for overlooked capsular diversity within the species. We report the discovery of seven novel group 3 clusters that fall into two distinct subgroups (3A and 3B). The majority of the 3B capsule clusters were found on plasmids, contrary to the defining feature of group 3 capsule genes localizing at the serA locus on the E. coli chromosome. Other new group 3 capsule clusters were derived from ancestral sequences through recombination events between shared genes found within the serotype variable central region 2. Intriguingly, flanking regions 1 and 3, known to be conserved areas among capsule clusters, showed considerable intra-subgroup variation in clusters from the 3B subgroup, containing genes of shared ancestry with other Enterobacteriaceae species. Variation of group 3 kps clusters within dominant E. coli lineages, including multidrug-resistant pathogenic lineages, further supports that E. coli capsules are undergoing rigorous change. Given the pivotal role of capsular polysaccharides in phage predation, our findings raise attention to the need of monitoring kps evolutionary dynamics in pathogenic E. coli in supporting phage therapy. IMPORTANCE Capsular polysaccharides protect pathogenic bacteria against environmental challenges, host immunity, and phage predations. The historical Escherichia coli K typing scheme, which relies on the hypervariable capsular polysaccharide, has identified around 80 different K forms that fall into four distinct groups. Taking advantage of the supposedly compact and genetically well-defined group 3 gene clusters, we analyzed published E. coli sequences to identify seven new gene clusters and revealed an unexpected capsular diversity. Genetic analysis revealed that group 3 gene clusters shared closely related serotype-specific region 2 and were diversified through recombination events and plasmid transfer between multiple Enterobacteriaceae species. Overall, capsular polysaccharides in E. coli are undergoing rigorous change. Given the pivotal role capsules play in phage interactions, this work highlighted the need to monitor the evolutionary dynamics of capsules in pathogenic E. coli for effective phage therapy.

Genomic Evidence of mcr-1.26 IncX4 Plasmid Transmission between Poultry and Humans.

Colistin is still commonly used and misused in animal husbandry driving the evolution and dissemination of transmissible plasmid-mediated colistin resistance (mcr). mcr-1.26 is a rare variant and, so far, has only been detected in Escherichia coli obtained from a hospitalized patient in Germany in 2018. Recently, it was also notified in fecal samples from a pigeon in Lebanon. We report on the presence of 16 colistin-resistant, mcr-1.26-carrying extended-spectrum beta-lactamase (ESBL)-producing and commensal E. coli isolated from poultry samples in Germany, of which retail meat was the most common source. Short- and long-read genome sequencing and bioinformatic analyses revealed the location of mcr-1.26 exclusively on IncX4 plasmids. mcr-1.26 was identified on two different IncX4 plasmid types of 33 and 38 kb and was associated with an IS6-like element. Based on the genetic diversity of E. coli isolates, transmission of the mcr-1.26 resistance determinant is mediated by horizontal transfer of IncX4 plasmids, as confirmed by conjugation experiments. Notably, the 33-kb plasmid is highly similar to the plasmid reported for the human sample. Furthermore, we identified the acquisition of an additional beta-lactam resistance linked to a Tn2 transposon on the mcr-1.26 IncX4 plasmids of three isolates, indicating progressive plasmid evolution. Overall, all described mcr-1.26-carrying plasmids contain a highly conserved core genome necessary for colistin resistance development, transmission, replication, and maintenance. Variations in the plasmid sequences are mainly caused by the acquisition of insertion sequences and alteration in intergenic sequences or genes of unknown function. IMPORTANCE Evolutionary events causing the emergence of new resistances/variants are usually rare and challenging to predict. Conversely, common transmission events of widespread resistance determinants are quantifiable and predictable. One such example is the transmissible plasmid-mediated colistin resistance. The main determinant, mcr-1, has been notified in 2016 but has successfully established itself in multiple plasmid backbones in diverse bacterial species across all One Health sectors. So far, 34 variants of mcr-1 are described, of which some can be used for epidemiological tracing-back analysis to identify the origin and transmission dynamics of these genes. Here, we report the presence of the rare mcr-1.26 gene in E. coli isolated from poultry since 2014. Based on the temporal occurrence and high similarity of the plasmids between poultry and human isolates, our study provides first indications for poultry husbandry as the primary source of mcr-1.26 and its transmission between different niches.

Whole strains vs MGEs in short and longterm transmission of ESBL genes between healthcare and community settings in Uganda

Multidrug-resistant ESBL-producing Escherichia coli are a leading cause of infections in hospital and community settings. Based on samples from two hospitals in Uganda and households of inpatients we tested the hypothesis that ESBL E. coli and/or their resistance determinants could spread within the healthcare and community settings through discharged patients that were still colonized. We used bacterial culture, susceptibility testing whole genome sequencing and detailed bioinformatics analysis to test the above hypothesis. Genome analysis revealed presence of predominantly blaCTX-M-15 and blaOXA-1 genes with a total resistome with genes belonging to 14 different classes of antimicrobials. Short-term cases of strain sharing were reported within each setting and strains from the two settings were found to cluster together based on their overall resistome. Long-term horizontal transfer of ESBL genes by various IncF and IncY types of plasmids shared between healthcare and community settings was demonstrated. Based on hybrid assembly, plasmid reconstruction and phylogenetic analyses, our study suggests that while the dissemination of AMR between healthcare and community settings in the short-term is possible at whole strain level, the long-term transmission between healthcare and communities is sustained by the transfer of plasmids circulating across niches and disseminating related resistomes.

Differences in carbon metabolic capacity fuel co-existence and plasmid transfer between Salmonella strains in the mouse gut

Antibiotic resistance plasmids can be disseminated between different Enterobacteriaceae in the gut. Here, we investigate how closely related Enterobacteriaceae populations with similar nutrient needs can co-bloom in the same gut and thereby facilitate plasmid transfer. Using different strains of Salmonella Typhimurium (S.Tm SL1344 and ATCC14028) and mouse models of Salmonellosis, we show that the bloom of one strain (i.e., recipient) from very low numbers in a gut pre-occupied by the other strain (i.e., donor) depends on strain-specific utilization of a distinct carbon source, galactitol or arabinose. Galactitol-dependent growth of the recipient S.Tm strain promotes plasmid transfer between non-isogenic strains and between E.coli and S.Tm. In mice stably colonized by a defined microbiota (OligoMM12), galactitol supplementation similarly facilitates co-existence of two S.Tm strains and promotes plasmid transfer. Our work reveals a metabolic strategy used by Enterobacteriaceae to expand in a pre-occupied gut and provides promising therapeutic targets for resistance plasmids spread.

Prevalence and genetic diversity of multidrug-resistant Salmonella Typhimurium monophasic variant in a swine farm from China.

Salmonella 4,[5],12:i:-, a monophasic variant of S. Typhimurium, has become a global serovar causing animal and human infections since its first emergence in the late 1980's. Several previous studies showed the increasing prevalence of S. 4,[5],12:i:- in China, most of which were from swine with multidrug resistance (MDR) profiles. However, the molecular characteristic and evolution of S. 4,[5],12:i:- in the same swine farm are still unknown. In this study, a total of 54 S. enterica strains were isolated from different fattening pigs aged 1, 3, and 6 months, most of which belonged to S. 4,[5],12:i:-. Whole-genome sequencing revealed that all 45 S. 4,[5],12:i:- strains belonged to ST34 and were further divided into two different ribosomal STs and nine different core-genome STs. Phylogenetic analysis of 286 S. 4,[5],12:i:- strains in China, including 241 from the EnteroBase Salmonella database, revealed the genetic diversity of S. 4,[5],12:i:- and indicated that S. 4,[5],12:i:- in this swine farm might have multiple origins. Three different IncHI2 plasmids carrying various resistance genes were characterized by nanopore sequencing and could be conjugated to Escherichia coli. The colistin resistance gene mcr-1 and ESBLs gene blaCTX - M-14 were co-located on the chromosome of one strain. The dynamic changes in antimicrobial resistance regions and transferability of IncHI2 plasmids, as well as the chromosomal location of resistance genes, facilitated the diversity of the antimicrobial resistance characteristics in S. 4,[5],12:i:-. Since the swine farm is regarded as the important reservoir of MDR S. 4,[5],12:i:-, the prevalence and evolution of S. 4,[5],12:i:- from swine farms to pig products and humans should be continually monitored.

Timing of antibiotic administration determines the spread of plasmid-encoded antibiotic resistance during microbial range expansion

Plasmids are the main vector by which antibiotic resistance is transferred between bacterial cells within surface-associated communities. In this study, we ask whether there is an optimal time to administer antibiotics to minimize plasmid spread in new bacterial genotypes during community expansion across surfaces. We address this question using consortia of Pseudomonas stutzeri strains, where one is an antibiotic resistance-encoding plasmid donor and the other a potential recipient. We allowed the strains to co-expand across a surface and administered antibiotics at different times. We find that plasmid transfer and transconjugant proliferation have unimodal relationships with the timing of antibiotic administration, where they reach maxima at intermediate times. These unimodal relationships result from the interplay between the probabilities of plasmid transfer and loss. Our study provides mechanistic insights into the transfer and proliferation of antibiotic resistance-encoding plasmids within microbial communities and identifies the timing of antibiotic administration as an important determinant.

Coexistence of blaIMP-4, blaNDM-1 and blaOXA-1 in blaKPC-2-producing Citrobacter freundii of clinical origin in China.

To explore the genetic characteristics of the IMP-4, NDM-1, OXA-1, and KPC-2 co-producing multidrug-resistant (MDR) clinical isolate, Citrobacter freundii wang9. MALDI-TOF MS was used for species identification. PCR and Sanger sequencing analysis were used to identify resistance genes. In addition to agar dilution, broth microdilution was used for antimicrobial susceptibility testing (AST). We performed whole genome sequencing (WGS) of the strains and analyzed the resulting data for drug resistance genes and plasmids. Phylogenetic trees were constructed with maximum likelihood, plotted using MAGA X, and decorated by iTOL. Citrobacter freundii carrying blaKPC-2, blaIMP-4, blaOXA-1, and blaNDM-1 are resistant to most antibiotics, intermediate to tigecycline, and only sensitive to polymyxin B, amikacin, and fosfomycin. The blaIMP-4 coexists with the blaNDM-1 and the blaOXA-1 on a novel transferable plasmid variant pwang9-1, located on the integron In1337, transposon TnAS3, and integron In2054, respectively. The gene cassette sequence of integron In1337 is IntI1-blaIMP-4-qacG2-aacA4'-catB3Δ, while the gene cassette sequence of In2054 is IntI1-aacA4cr-blaOXA-1-catB3-arr3-qacEΔ1-sul1. The blaNDM-1 is located on the transposon TnAS3, and its sequence is IS91-sul-ISAba14-aph (3')-VI-IS30-blaNDM-1-ble-trpF-dsbD-IS91. The blaKPC-2 is located on the transposon Tn2 of plasmid pwang9-1, and its sequence is klcA-korC-ISkpn6-blaKPC-2-ISkpn27-tnpR-tnpA. Phylogenetic analysis showed that most of the 34°C. freundii isolates from China were divided into three clusters. Among them, wang1 and wang9 belong to the same cluster as two strains of C. freundii from environmental samples from Zhejiang. We found C. freundii carrying blaIMP-4, blaNDM-1, blaOXA-1, and blaKPC-2 for the first time, and conducted in-depth research on its drug resistance mechanism, molecular transfer mechanism and epidemiology. In particular, we found that blaIMP-4, blaOXA-1, and blaNDM-1 coexisted on a new transferable hybrid plasmid that carried many drug resistance genes and insertion sequences. The plasmid may capture more resistance genes, raising our concern about the emergence of new resistance strains.

An overview of plasmid transfer in the plant microbiome

Plant microbiomes are pivotal for healthy plant physiological development. Microbes live in complex co-association with plant hosts, and interactions within these microbial communities vary with plant genotype, plant compartment, phenological stage, and soil properties, among others. Plant microbiomes also harbor a substantial and diverse pool of mobile genes encoded on plasmids. Several plasmid functions attributed to plant-associated bacteria are relatively poorly understood. Additionally, the role of plasmids in disseminating genetic traits within plant compartments is not well known. Here, we present the current knowledge on the occurrence, diversity, function, and transfer of plasmids in plant microbiomes, emphasizing the factors that could modulate gene transfer in-planta. We also describe the role of the plant microbiome as a plasmid reservoir and the dissemination of its genetic material. We include a brief discussion on the current methodological limitations in studying plasmid transfer within plant microbiomes. This information could be useful to elucidate the dynamics of the bacterial gene pools, the adaptations different organisms have made, and variations in bacterial populations that might have never been described before, particularly in complex microbial communities associated with plants in natural and anthropogenic impacted environments.

Conditions Necessary for the Transfer of Antimicrobial Resistance in Poultry Litter.

Animal manures contain a large and diverse reservoir of antimicrobial resistance (AMR) genes that could potentially spillover into the general population through transfer of AMR to antibiotic-susceptible pathogens. The ability of poultry litter microbiota to transmit AMR was examined in this study. Abundance of phenotypic AMR was assessed for litter microbiota to the antibiotics: ampicillin (Ap; 25 μg/mL), chloramphenicol (Cm; 25 μg/mL), streptomycin (Sm; 100 μg/mL), and tetracycline (Tc; 25 μg/mL). qPCR was used to estimate gene load of streptomycin-resistance and sulfonamide-resistance genes aadA1 and sul1, respectively, in the poultry litter community. AMR gene load was determined relative to total bacterial abundance using 16S rRNA qPCR. Poultry litter contained 108 CFU/g, with Gram-negative enterics representing a minor population (<104 CFU/g). There was high abundance of resistance to Sm (106 to 107 CFU/g) and Tc (106 to 107 CFU/g) and a sizeable antimicrobial-resistance gene load in regards to gene copies per bacterial genome (aadA1: 0.0001-0.0060 and sul1: 0.0355-0.2455). While plasmid transfer was observed from Escherichia coli R100, as an F-plasmid donor control, to the Salmonella recipient in vitro, no AMR Salmonella were detected in a poultry litter microcosm with the inclusion of E. coli R100. Confirmatory experiments showed that isolated poultry litter bacteria were not interfering with plasmid transfer in filter matings. As no R100 transfer was observed at 25 °C, conjugative plasmid pRSA was chosen for its high plasmid transfer frequency (10-4 to 10-5) at 25 °C. While E. coli strain background influenced the persistence of pRSA in poultry litter, no plasmid transfer to Salmonella was ever observed. Although poultry litter microbiota contains a significant AMR gene load, potential to transmit resistance is low under conditions commonly used to assess plasmid conjugation.

Genome-based characterization of conjugative IncHI1B plasmid carrying carbapenemase genes blaVIM-1, blaIMP-23, and truncated blaOXA-256 in Klebsiella pneumoniae NTU107224

The wide dissemination of plasmids carrying antibiotic resistance determinants among bacteria is a severe threat to global public health. Here, we characterized an extensively drug-resistant (XDR) Klebsiella pneumoniae NTU107224 by whole genome sequencing (WGS) in combination with phenotypic tests. Broth dilution method was used to determine the minimal inhibitory concentrations (MICs) of NTU107224 to 24 antibiotics. The whole genome sequence of NTU107224 was determined by Nanopore/Illumina hybrid genome sequencing. Conjugation assay was performed to determine the transferability of plasmids in NTU107224 to recipient K. pneumoniae 1706. Larvae infection model was used to determine the effect(s) of conjugative plasmid pNTU107224-1 on bacterial virulence. Among the 24 antibiotics tested, XDR K. pneumoniae NTU107224 had low MICs only for amikacin (≤1μg/mL), polymyxin B (0.25μg/mL), colistin (0.25μg/mL), eravacycline (0.25μg/mL), cefepime/zidebactam (1μg/mL), omadacycline (4μg/mL), and tigecycline (0.5μg/mL). Whole genome sequencing showed that the closed NTU107224 genome comprises a 5,076,795-bp chromosome, a 301,404-bp plasmid named pNTU107224-1, and a 78,479-bp plasmid named pNTU107224-2. IncHI1B plasmid pNTU107224-1 contained three class 1 integrons accumulated various antimicrobial resistance genes (including carbapenemase genes blaVIM-1, blaIMP-23, and truncated blaOXA-256) and the blast results suggested the dissemination of IncHI1B plasmids in China. By day 7 after infection, larvae infected with K. pneumoniae 1706 and transconjugant had 70% and 15% survival rates, respectively. We found that the conjugative plasmid pNTU107224-1 is closely related to IncHI1B plasmids disseminated in China and contributes to the virulence and antibiotic resistance of pathogens.

Restriction-modification systems have shaped the evolution and distribution of plasmids across bacteria.

Many novel traits such as antibiotic resistance are spread by plasmids between species. Yet plasmids have different host ranges. Restriction-modification systems (R-M systems) are by far the most abundant bacterial defense system and therefore represent one of the key barriers to plasmid spread. However, their effect on plasmid evolution and host range has been neglected. Here we analyse the avoidance of targets of the most abundant R-M systems (Type II) for complete genomes and plasmids across bacterial diversity. For the most common target length (6 bp) we show that target avoidance is strongly correlated with the taxonomic distribution of R-M systems and is greater in plasmid genes than core genes. We find stronger avoidance of R-M targets in plasmids which are smaller and have a broader host range. Our results suggest two different evolutionary strategies for plasmids: small plasmids primarily adapt to R-M systems by tuning their sequence composition, and large plasmids primarily adapt through the carriage of additional genes protecting from restriction. Our work provides systematic evidence that R-M systems are important barriers to plasmid transfer and have left their mark on plasmids over long evolutionary time.

Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool.

Antimicrobial resistance (AMR) genes are widely disseminated on plasmids. Therefore, interventions aimed at blocking plasmid uptake and transfer may curb the spread of AMR. Previous studies have used CRISPR-Cas-based technology to remove plasmids encoding AMR genes from target bacteria, using either phage- or plasmid-based delivery vehicles that typically have narrow host ranges. To make this technology feasible for removal of AMR plasmids from multiple members of complex microbial communities, an efficient, broad host-range delivery vehicle is needed. We engineered the broad host-range IncP1-plasmid pKJK5 to encode cas9 programmed to target an AMR gene. We demonstrate that the resulting plasmid pKJK5::csg has the ability to block the uptake of AMR plasmids and to remove resident plasmids from Escherichia coli. Furthermore, due to its broad host range, pKJK5::csg successfully blocked AMR plasmid uptake in a range of environmental, pig- and human-associated coliform isolates, as well as in isolates of two species of Pseudomonas. This study firmly establishes pKJK5::csg as a promising broad host-range CRISPR-Cas9 delivery tool for AMR plasmid removal, which has the potential to be applied in complex microbial communities to remove AMR genes from a broad range of bacterial species.

Isolation of Hv-CRKP with co-production of three carbapenemases (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and a virulence plasmid: a study from a Chinese tertiary hospital.

The worldwide dissemination of K. pneumoniae isolates is a significant public health concern, as these organisms possess a unique capacity to acquire genetic elements encoding both resistance and hypervirulence. This study aims to investigate the epidemiological, resistance, and virulence characteristics of K. pneumoniae isolates that carry both virulence plasmids and blaOXA-48-like genes in a tertiary hospital in China. A total of 217 clinical isolates of carbapenem-resistant K. pneumoniae (CRKP) were collected between April 2020 and March 2022. The antimicrobial susceptibility test was conducted to evaluate the drug resistance profile. All isolates were screened for the presence of genes encoding carbapenemases (blaKPC, blaNDM, blaIMP, blaVIM, and blaOXA-48-like), ESBLs genes (blaCTX-M, blaSHV, blaTEM), and virulence plasmid pLVPK-borne genes (rmpA, rmpA2, iucA, iroB, and peg344) using polymerase chain reaction (PCR) amplification. Clonal lineages were assigned using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The plasmid incompatibility groups were identified using PCR-based replicon typing (PBRT). The transferability of carbapenemase-encoding plasmids and pLVPK-like virulence plasmids was assessed via conjugation. The plasmid location of rmpA2 was determined using S1-Pulsed Field Gel Electrophoresis (S1-PFGE) and southern blotting hybridization. The virulence potential of the isolates was assessed using the string test, capsular serotyping, serum killing assay and a Galleria mellonella larval infection model. Of the 217 CRKP clinical isolates collected, 23% were identified as carrying blaOXA-48-like genes. All blaOXA-48-like isolates exhibited resistance to commonly used clinical antimicrobial agents, except for ceftazidime/avibactam, colistin, tigecycline, trimethoprim-sulfamethOXAzole, polymyxin B, and nitrofurantoin. The main common OXA-48-like carbapenemase enzymes were found to be blaOXA-181 and blaOXA-232. MLST and PFGE fingerprinting analysis revealed clonal transmission and plasmid transmission. OXA-48-like producing CRKP isolates mainly clustered in K64 ST11 and K47 ST15. Results of the string Test, serum killing assay (in vitro) and Galleria mellonella infection model (in vivo) indicated hypervirulence. PBRT showed that the blaOXA-181 and blaOXA-232 producing hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP) were mainly carried on ColE-type, IncF, and IncX3. Eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1). Moreover, Southern blotting hybridization revealed that all eight isolates had a pLVPK-like virulent plasmid (138.9-216.9 kb) with an uneven number and size of plasmid. In our investigation, we have observed the emergence of hv-CRKP carrying blaOXA-48-like genes, which identified two genetic relationships: clonal transmission and plasmid transmission. PBRT analysis showed that these genes were mainly carried on ColE-type, IncF, and IncX3 plasmids. These isolates have been shown to be hypervirulent in vitro and in vivo. Additionally, eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and carrying a pLVPK-like virulent plasmid. Hence, our findings highlight the need for further investigation and active surveillance of hypervirulent OXA-48-like producing Hv-CRKP isolates to control their transmission.

Citrobacter freundii resistant to novel β-lactamase inhibitor combinations and cefiderocol, co-producing class A, B and D carbapenemases encoded by transferable plasmids.

To characterize a carbapenem-resistant Citrobacter freundii (Cf-Emp) co-producing class A, B and D carbapenemases, resistant to novel β-lactamase inhibitor combinations (BLICs) and cefiderocol. Carbapenemase production was tested by an immunochromatography assay. Antibiotic susceptibility testing (AST) was performed by broth microdilution. WGS was performed using short- and long-read sequencing. Transfer of carbapenemase-encoding plasmids was assessed by conjugation experiments. Cf-Emp was isolated on selective medium for carbapenem-resistant Enterobacterales from the surveillance rectal swab taken at hospital admission from a patient of Moroccan origin. Cf-Emp produced three different carbapenemases, including KPC-2, OXA-181 and VIM-1, and was resistant to all β-lactams including carbapenems, novel BLICs (ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam) and cefiderocol. MIC of aztreonam/avibactam was 0.25 mg/L. The strain belonged to ST22, one of the C. freundii lineages of global diffusion, known to be associated with carbapenemase production. Each carbapenemase gene was located aboard a different plasmid (named pCf-KPC, pCf-OXA and pCf-VIM, respectively), which also carried other clinically relevant resistance genes, such as armA (pCf-KPC), blaSHV-12 (pCf-VIM) and qnrS1 (pCf-OXA). Transferability to Escherichia coli J53 by conjugation was observed for all plasmids. The finding of enterobacterial strains carrying multiple carbapenemase genes on transferable plasmids is alarming, because similar strains could provide an important reservoir for disseminating these clinically relevant resistance determinants.

Characterization of blaKPC-2 and blaNDM-1 Plasmids of a K. pneumoniae ST11 Outbreak Clone.

The most common resistance mechanism to carbapenems is the production of carbapenemases. In 2021, the Pan American Health Organization warned of the emergence and increase in new carbapenemase combinations in Enterobacterales in Latin America. In this study, we characterized four Klebsiella pneumoniae isolates harboring blaKPC and blaNDM from an outbreak during the COVID-19 pandemic in a Brazilian hospital. We assessed their plasmids' transference ability, fitness effects, and relative copy number in different hosts. The K. pneumoniae BHKPC93 and BHKPC104 strains were selected for whole genome sequencing (WGS) based on their pulsed-field gel electrophoresis profile. The WGS revealed that both isolates belong to ST11, and 20 resistance genes were identified in each isolate, including blaKPC-2 and blaNDM-1. The blaKPC gene was present on a ~56 Kbp IncN plasmid and the blaNDM-1 gene on a ~102 Kbp IncC plasmid, along with five other resistance genes. Although the blaNDM plasmid contained genes for conjugational transfer, only the blaKPC plasmid conjugated to E. coli J53, without apparent fitness effects. The minimum inhibitory concentrations (MICs) of meropenem/imipenem against BHKPC93 and BHKPC104 were 128/64 and 256/128 mg/L, respectively. Although the meropenem and imipenem MICs against E. coli J53 transconjugants carrying the blaKPC gene were 2 mg/L, this was a substantial increment in the MIC relative to the original J53 strain. The blaKPC plasmid copy number was higher in K. pneumoniae BHKPC93 and BHKPC104 than in E. coli and higher than that of the blaNDM plasmids. In conclusion, two ST11 K. pneumoniae isolates that were part of a hospital outbreak co-harbored blaKPC-2 and blaNDM-1. The blaKPC-harboring IncN plasmid has been circulating in this hospital since at least 2015, and its high copy number might have contributed to the conjugative transfer of this particular plasmid to an E. coli host. The observation that the blaKPC-containing plasmid had a lower copy number in this E. coli strain may explain why this plasmid did not confer phenotypic resistance against meropenem and imipenem.