Diversity Of Plasmids Research Articles

Bacterial Community Composition and Presence of Plasmids in the Endosphere- and Rhizosphere-Associated Microbiota of Sea Fig (Carpobrotus aequilaterus)

The plant microbiome is one of the most important environments for ecological interactions between bacteria that impact the plant and the ecosystem. However, studies on the diversity of mobile genetic elements (such as plasmids) associated with the plant microbiome are very scarce. Here, we determined the bacterial community composition and the occurrence of plasmids in the microbiota associated with sea fig, Carpobrotus aequilaterus (N.E. Br.), a succulent species widely used as an ornamental plant in Chile. The abundance and composition of the endophytic and rhizospheric bacterial communities were determined by quantitative PCR (qPCR) and DNA metabarcoding analysis. Plasmid diversity in the plant microbiome was determined by plasmid DNA extraction and screened by endpoint PCR of backbone genes for four different incompatibility groups (Inc). The results showed about 106 copies of the 16S rRNA gene in the endosphere and rhizosphere, showing significant differences according to the diversity index. Proteobacteria (Pseudomonadota; 43.4%), Actinobacteria (Actinomycetota; 25.7%), and Bacteroidetes (Bacteroidota; 17.4%) were the most dominant taxa in both plant compartments, and chemoheterotrophy (30%) was the main predicted function assigned to the microbiota. Plasmid diversity analysis showed the presence of transferable plasmids in the endosphere and rhizosphere of C. aequilaterus, particularly among environmental plasmids belonging to the IncP and IncN incompatibility groups.

Plants acquired mitochondrial linear plasmids horizontally from fungi likely during the conquest of land

Mitochondrial linear plasmids have been sporadically reported in fungi and plants. Yet, much remains obscure about the diversity, distribution, and evolution of mitochondrial linear plasmids. Here, through phylogenomic analyses across 7,163 cellular organisms (including 991 plants), we find that mitochondrial linear plasmids are widely present in land plants and fungi. Phylogenetic analyses indicate that plants are likely to have acquired mitochondrial linear plasmids horizontally from fungi before or during the conquest of terrestrial environments by plants. Gene content analyses show that mitochondrial linear plasmids harbor a highly dynamic and promiscuous repertoire of genes. Our study refines the understanding of the origin and evolution of mitochondrial linear plasmids.

FRI410 ESR1 Variants Reduce Estrogen Receptor Alpha Expression In Women With Unexplained Infertility

Abstract Disclosure: R.A. Roman: None. H. Liu: None. L.P. Chorich: None. Y. Li: None. J.E. Hall: None. M.P. Diamond: None. K.S. Korach: None. L.C. Layman: None. Objective: Estrogen receptor alpha (ERα), encoded by ESR1, is vital to human reproduction. DNA sequencing previously identified four ESR1 missense variants of uncertain significance in 197 cisgender women with well-characterized unexplained infertility, including a p.Thr313Met variant with impaired estrogen signaling in vitro. The p.His6Tyr, p.Ser118Pro, and p.Arg269Cys ESR1 variants did not affect estrogen signaling. We hypothesize that milder ESR1 variants could decrease ERα expression in women with unexplained infertility. Methods: Clinically identified ESR1 variant plasmids were constructed using site-directed mutagenesis and confirmed by Sanger sequencing. HepG2 cells were transiently transfected with either empty vector, wild type (WT) ESR1, p.His6Tyr, p.Ser118Pro, p.Arg269Cys, p.Thr313Met, or p.Gln375His (ESR1 missense variant control) estrogen receptor variant expression plasmids using Lipofectamine 3000 transfection reagent. Cell lysis was isolated from HepG2 cells 24 hours after transfection using RIPA lysis buffer. ERα expression was assessed by western blot fluorescence imaging and normalized to β-actin on LI-COR Odyssey DLx. Quantitative analysis was performed using Empiria Studio Software v.2. Results: Western blot analysis showed that the ESR1 p.His6Tyr variant had 87% decreased and the p.Ser118Pro variant had 75.6% decreased expression compared to WT ERα in vitro. The p.Arg269Cys, p.Thr313Met, and p.Gln375His variants had comparable expression to WT. Conclusion: Although the p.His6Tyr and p.Ser118Pro variant receptors have conserved estrogen signaling, our preliminary data show that they decrease ERα expression in vitro. This could be an underlying pathophysiologic mechanism affecting some women with unexplained infertility. Additional in vitro and in vivo analyses can further characterize their pathogenicity. Presentation: Friday, June 16, 2023

CRISPR-Cas systems and diversity of targeting phages in Lactobacillus johnsonii strains; insights from genome mining approach.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes make up bacteria's adaptive immune system. These genes protect bacteria from being eaten by bacteriophages. In this study, CRISPR-Cas systems were characterized using a genomic approach. For this purpose, genome sequences of Lactobacillus johnsonii strains were retrieved, and the diversity, occurrence, and evolution of the CRISPR-Cas systems were analyzed. Then, homology analyses of spacer sequences in identified CRISPR arrays were performed to analyze and characterize the diversity of target phages and plasmids. Finally, the evolutionary paths of spaceromes in each subtype of CRISPR arrays were performed using acquisition and deletion events surveyed under the selective pressure of foreign plasmids and phages. Results showed that 138 strains contain valid CRISPR-Cas structures (CRISPR loci together with the Cas genes) in their genomes, which accounted for about 17% of the L. johnsonii studied strains belonging to subtypes II-A, I-E, and I-C. Moreover, results indicated that some specific groups of plasmids were targeted with specific CRISPR array systems. Homology analysis of spacer sequences with phage genomes also revealed that spacers of strains that harbored only CRISPR-Cas subtype-II targeted a greater diversity of foreign phages. In conclusion, the current study indicates that there is great diversity between the CRISPR-Cas systems identified in L. johnsonii strains. Such diverse CRISPR-Cas systems indicate that these systems are naturally active and important in terms of adaptive immunity and evolutionary relationships.

Emergence of a clinical isolate of E. coil ST297 co-carrying blaNDM-13 and mcr-1.1 in China

BackgroundThe coexistence of carbapenem resistance genes and mcr-1.1 in Enterobacterales has been an urgent and persistent threat to global public health. In this study, we isolated a clinical NB4833, an Escherichia coli isolate that co-carries mcr-1.1 and blaNDM-13. ObjectivesThis study aimed to isolate a clinical NB4833, an Escherichia coli isolate that co-carries mcr-1.1 and blaNDM-13 and investigated the phenotypic and genotypic characteristics of plasmids harbored by E. coli isolate NB4833. MethodsAntimicrobial susceptibility testing and conjugation assay were performed on E. coli isolate NB4833. Stability of the plasmid and growth rate determination were used to characterize the plasmids harboring mcr-1.1 and blaNDM-13. In addition, the genetic characteristics of the plasmids were analyzed based on whole-genome sequencing of the strain and comparative genetic analysis with related plasmids. ResultsWhole-genome sequencing showed that the isolate carried multiple resistance genes and possessed phenotypes indicative of all antibiotic resistance except tigecycline. And the mcr-1.1- and blaNDM-13-harbouring plasmids showed relatively high similarity to the related plasmids. The pNB4833-NDM-13 plasmid was capable of trans conjugation with an efficiency of 1.04 × 10−2 in a filter mating experiment and the transconjugant J53/ pNB4833-NDM-13 was able to be stably inherited after 10 days of passage. ConclusionsTo our knowledge, this is the first report of the coexistence of the IncI2 plasmid carrying mcr-1.1 and a blaNDM-13-carrying integrated IncFIB/IncFII plasmid in an ST297 clinical E. coli isolate. In addition, we investigated a novel plasmid carrying blaNDM-13. Our study expands the diversity of plasmids carrying blaNDM-13, which exhibits epidemic importance in bacterial resistance. Therefore, there are important measures that should be taken to prevent the spread of these plasmids.

Genome diversity of Borrelia garinii in marine transmission cycles does not match host associations but reflects the strains evolutionary history

Borrelia burgdorferi sensu lato is a species complex of spirochetal bacteria that occupy different ecological niches which is reflected in their reservoir host- and vector-associations. Borrelia genomes possess numerous linear and circular plasmids. Proteins encoded by plasmid genes play a major role in host- and vector-interaction and are important for Borrelia niche adaptation. However, the plasmid composition and therewith the gene repertoire may vary even in strains of a single species.Borrelia garinii, one of the six human pathogenic species, is common in Europe (vector Ixodes ricinus), Asia (vector Ixodes persulcatus) and in marine birds (vector Ixodes uriae). For the latter, only a single culture isolate (Far04) and its genome were previously available. The genome was rather small containing only one circular and six linear plasmids with a notable absence of cp32 plasmids. To further investigate B. garinii from marine transmission cycles and to explore i) whether the small number of plasmids found in isolate Far04 is a common feature in B. garinii from marine birds and presents an adaptation to this particular niche and ii) whether there may be a correlation between genome type and host species, we initiated in vitro cultures from live I. uriae collected in 2017 and 2018 from marine avian hosts and their nests. Hosts included common guillemots, Atlantic Puffin, razorbill, and kittiwake.We obtained 17 novel isolates of which 10 were sequenced using Illumina technology, one also with Pacific Bioscience technology. The 10 genomes segregated into five different genome types defined by plasmid types (based on PFam32 loci). We show that the genomes of seabird associated B. garinii contain fewer plasmids (6–9) than B. garinii from terrestrial avian species (generally ≥10), potentially suggesting niche adaptation. However, genome type did not match an association with the diverse avian seabird hosts investigated but matched the clonal complex they originated from, perhaps reflecting the isolates evolutionary history.Questions that should be addressed in future studies are (i) how is plasmid diversity related to host- and/or vector adaptation; (ii) do the different seabird species differ in reservoir host competence, and (iii) can the genome types found in seabirds use terrestrial birds as reservoir hosts.

Characterisation and comparative genomics of three new Varanus-associated Borrelia spp. from Indonesia and Australia

BackgroundBorrelia are important disease-causing tick- and louse-borne spirochaetes than can infect a wide variety of vertebrates, including humans and reptiles. Reptile-associated (REP) Borrelia, once considered a peculiarity, are now recognised as a distinct and important evolutionary lineage, and are increasingly being discovered worldwide in association with novel hosts. Numerous novel Borrelia spp. associated with monitor lizards (Varanus spp.) have been recently identified throughout the Indo-Pacific region; however, there is a lack of genomic data on these Borrelia.MethodsWe used metagenomic techniques to sequence almost complete genomes of novel Borrelia spp. from Varanus varius and Varanus giganteus from Australia, and used long- and short-read technologies to sequence the complete genomes of two strains of a novel Borrelia sp. previously isolated from ticks infesting Varanus salvator from Indonesia. We investigated intra- and interspecies genomic diversity, including plasmid diversity and relatedness, among Varanus-associated Borrelia and other available REP Borrelia and, based on 712 whole genome orthologues, produced the most complete phylogenetic analysis, to the best of our knowledge, of REP Borrelia to date.ResultsThe genomic architecture of Varanus-associated Borrelia spp. is similar to that of Borrelia spp. that cause relapsing fever (RF), and includes a highly conserved megaplasmid and numerous smaller linear and circular plasmids that lack structural consistency between species. Analysis of PF32 and PF57/62 plasmid partitioning genes indicated that REP Borrelia plasmids fall into at least six distinct plasmid families, some of which are related to previously defined Borrelia plasmid families, whereas the others appear to be unique. REP Borrelia contain immunogenic variable major proteins that are homologous to those found in Borrelia spp. that cause RF, although they are limited in copy number and variability and have low sequence identities to RF variable major proteins. Phylogenetic analyses based on single marker genes and 712 single copy orthologs also definitively demonstrated the monophyly of REP Borrelia as a unique lineage.ConclusionsIn this work we present four new genomes from three novel Borrelia, and thus double the number of REP Borrelia genomes publicly available. The genomic characterisation of these Borrelia clearly demonstrates their distinctiveness as species, and we propose the names Borrelia salvatorii, ‘Candidatus Borrelia undatumii’, and ‘Candidatus Borrelia rubricentralis’ for them.Graphical

Conjugative Plasmid pPPUT-Tik1-1 from a Permafrost Pseudomonas putida Strain and Its Present-Day Counterparts Inhabiting Environments and Clinics.

A novel group of conjugative plasmids of Pseudomonas is characterized. The prototype plasmid pPPUT-Tik1-1 (153,663 bp), isolated from a permafrost strain of P. putida Tik1, carries a defective mercury transposon, Tn501, and a streptomycin resistance transposon, Tn5393. Ten plasmids and 34 contigs with backbone regions closely related to pPPUT-Tik1-1 have been found in GenBank. Two of these plasmids from clinical strains of P. putida and P. fulva are almost identical to the ancient plasmid. A characteristic feature of this group of plasmids is the presence of two genes encoding the initiators of replication (repA1 and repA2). None of these genes have high similarity with plasmid replication genes belonging to known incompatibility groups. It has been demonstrated that while pPPUT-Tik1-1-like plasmids have homologous backbone regions, they significantly differ by the molecular structure and the predicted functions of their accessory regions. Some of the pPPUT-Tik1-1-related plasmids carry determinants of antibiotic resistance and/or heavy metal salts. Some plasmids are characterized by the ability to degrade xenobiotics. Plasmids related to pPPUT-Tik1-1 are characterized by a narrow host range and are found in various species of the Pseudomonas genus. Interestingly, we also found shorter plasmid variants containing the same replication module, but lacking conjugation genes and containing other structural changes that strongly distinguish them from plasmids related to pPPUT-Tik1-1, indicating that the structure of the replication module cannot be used as the sole criterion for classifying plasmids. Overall, the results suggest that the plasmids of the novel group can be spread using conjugation in environmental and clinical strains of Pseudomonas and may play diverse adaptive functions due to the presence of various accessory regions.

Detection of resistance and virulence plasmids in Campylobacter coli and Campylobacter jejuni isolated from North Carolina food animal production, 2018–2019

Campylobacter remains the leading cause of bacterial foodborne illness in the U.S. and worldwide. Campylobacter plasmids may play a significant role in antimicrobial resistance (AMR) and virulence factor distribution, and potentially drive rapid adaptation. C. coli (n = 345) and C. jejuni (n = 199) isolates collected from live cattle, swine, turkey, and chickens, poultry carcasses at production, and retail meat in N.C. were analyzed to determine plasmid prevalence, extrachromosomal virulence and AMR genes, and the phylogeny of assembled plasmids. Putative plasmids ranging from <2 kb to 237kb were identified with virulence factors present in 66.1% (228/345) C. coli and 88.4% (176/199) C. jejuni plasmids (promoting adherence, invasion, exotoxin production, immune modulation, chemotaxis, mobility, and the type IV secretion system). AMR genes were identified in 21.2% (73/345) C. coli and 28.1% C. jejuni plasmids (conferring resistance to tetracyclines, aminoglycosides, beta-lactams, nucleosides, and lincosamides). Megaplasmids (>100 kb) were present in 25.7% (140/544) of the isolates and carried genes previously recognized to be involved with interspecies recombination. Our study highlights the extensive distribution and diversity of Campylobacter plasmids in food animal production and their role in the dissemination of biomedically important genes. Characterizing Campylobacter plasmids within the food animal production niche is important to understanding the epidemiology of potential emerging strains.

There are three major Neisseria gonorrhoeae β-lactamase plasmid variants which are associated with specific lineages and carry distinct TEM alleles.

Neisseria gonorrhoeae is a significant threat to global health with an estimated incidence of over 80 million cases each year and high levels of antimicrobial resistance. The gonococcal β-lactamase plasmid, pbla, carries the TEM β-lactamase, which requires only one or two amino acid changes to become an extended-spectrum β-lactamase (ESBL); this would render last resort treatments for gonorrhoea ineffective. Although pbla is not mobile, it can be transferred by the conjugative plasmid, pConj, found in N. gonorrhoeae. Seven variants of pbla have been described previously, but little is known about their frequency or distribution in the gonococcal population. We characterised sequences of pbla variants and devised a typing scheme, Ng_pblaST that allows their identification from whole genome short-read sequences. We implemented Ng_pblaST to assess the distribution of pbla variants in 15 532 gonococcal isolates. This demonstrated that only three pbla variants commonly circulate in gonococci, which together account for >99 % of sequences. The pbla variants carry different TEM alleles and are prevalent in distinct gonococcal lineages. Analysis of 2758 pbla-containing isolates revealed the co-occurrence of pbla with certain pConj types, indicating co-operativity between pbla and pConj variants in the spread of plasmid-mediated AMR in N. gonorrhoeae. Understanding the variation and distribution of pbla is essential for monitoring and predicting the spread of plasmid-mediated β-lactam resistance in N. gonorrhoeae.

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.

Genetic Polymorphisms in SLCO2B1 and ABCC1 Conjointly Modulate Atorvastatin Intracellular Accumulation in HEK293 Recombinant Cell Lines.

Although atorvastatin (ATV) is well-tolerated, patients may report muscle complaints. These are difficult to predict owing to high interindividual variability. Such side effects are linked to intramuscular accumulation of ATV. This study aimed to investigate the relative role of transporters expressed in muscle tissue in promoting or limiting drug access to cells. The impact of common single nucleotide polymorphisms (SNPs) in SLCO2B1 coding for OATP2B1 and ABCC1 coding for MRP1 on ATV transport was also evaluated. HEK293 cells were stably transfected with plasmids containing cDNA encoding wild-type or variant SLCO2B1 and/or ABCC1 to generate single and double stable transfectant HEK293 recombinant models overexpressing variant or wild-type OATP2B1 (influx) and/or MRP1 (efflux) proteins. Variant plasmids were generated by site-directed mutagenesis. Expression analyses were performed to validate recombinant models. Accumulation and efflux experiments were performed at different concentrations. ATV was quantified by LC-MS/MS, and kinetic parameters were compared between single and double HEK transfectants expressing wild-type and variant proteins. The results confirm the involvement of OATP2B1 and MRP1 in ATV cellular transport because it was demonstrated that intracellular accumulation of ATV was boosted by OATP2B1 overexpression, whereas ATV accumulation was decreased by MRP1 overexpression. In double transfectants, it was observed that increased ATV intracellular accumulation driven by OATP2B1 influx was partially counteracted by MRP1 efflux. The c.935G > A SNP in SLCO2B1 was associated with decreased ATV OATP2B1-mediated influx, whereas the c.2012G > T SNP in ABCC1 seemed to increase MRP1 efflux activity against ATV. Intracellular ATV accumulation is regulated by OATP2B1 and MRP1 transporters, whose functionality is modulated by natural genetic variants. This is significant because it may play a role in ATV muscle side-effect susceptibility.

Phylogenomics of Globally Spread Clonal Groups 14 and 15 of Klebsiella pneumoniae.

Klebsiella pneumoniae sequence type 14 (ST14) and ST15 caused outbreaks of CTX-M-15 and/or carbapenemase producers worldwide, but their phylogeny and global dynamics remain unclear. We clarified the evolution of K. pneumoniae clonal group 14 (CG14) and CG15 by analyzing the capsular locus (KL), resistome, virulome, and plasmidome of public genomes (n = 481) and de novo sequences (n = 9) representing main sublineages circulating in Portugal. CG14 and CG15 evolved independently within 6 main subclades defined according to the KL and the accessory genome. The CG14 (n = 65) clade was structured in two large monophyletic subclades, CG14-I (KL2, 86%) and CG14-II (KL16, 14%), whose emergences were dated to 1932 and 1911, respectively. Genes encoding extended-spectrum β-lactamase (ESBL), AmpC, and/or carbapenemases were mostly observed in CG14-I (71% versus 22%). CG15 clade (n = 170) was segregated into subclades CG15-IA (KL19/KL106, 9%), CG15-IB (variable KL types, 6%), CG15-IIA (KL24, 43%) and CG15-IIB (KL112, 37%). Most CG15 genomes carried specific GyrA and ParC mutations and emerged from a common ancestor in 1989. CTX-M-15 was especially prevalent in CG15 (68% CG15 versus 38% CG14) and in CG15-IIB (92%). Plasmidome analysis revealed 27 predominant plasmid groups (PG), including particularly pervasive and recombinant F-type (n = 10), Col (n = 10), and new plasmid types. While blaCTX-M-15 was acquired multiple times by a high diversity of F-type mosaic plasmids, other antibiotic resistance genes (ARGs) were dispersed by IncL (blaOXA-48) or IncC (blaCMY/TEM-24) plasmids. We first demonstrate an independent evolutionary trajectory for CG15 and CG14 and how the acquisition of specific KL, quinolone-resistance determining region (QRDR) mutations (CG15), and ARGs in highly recombinant plasmids could have shaped the expansion and diversification of particular subclades (CG14-I and CG15-IIA/IIB). IMPORTANCE Klebsiella pneumoniae represents a major threat in the burden of antibiotic resistance (ABR). Available studies to explain the origin, the diversity, and the evolution of certain ABR K. pneumoniae populations have mainly been focused on a few clonal groups (CGs) using phylogenetic analysis of the core genome, the accessory genome being overlooked. Here, we provide unique insights into the phylogenetic evolution of CG14 and CG15, two poorly characterized CGs which have contributed to the global dissemination of genes responsible for resistance to first-line antibiotics such as β-lactams. Our results point out an independent evolution of these two CGs and highlight the existence of different subclades structured by the capsular type and the accessory genome. Moreover, the contribution of a turbulent flux of plasmids (especially multireplicon F type and Col) and adaptive traits (antibiotic resistance and metal tolerance genes) to the pangenome reflect the exposure and adaptation of K. pneumoniae under different selective pressures.

Extensive genome analysis identifies novel plasmid families in Clostridium perfringens.

Globally, the anaerobic bacterium Clostridium perfringens causes severe disease in a wide array of hosts; however, C. perfringens strains are also carried asymptomatically. Accessory genes are responsible for much of the observed phenotypic variation and virulence within this species, with toxins frequently encoded on conjugative plasmids and many isolates carrying up to 10 plasmids. Despite this unusual biology, current genomic analyses have largely excluded isolates from healthy hosts or environmental sources. Accessory genomes, including plasmids, also have often been excluded from broader scale phylogenetic investigations. Here we interrogate a comprehensive collection of 464 C. perfringens genomes and identify the first putative non-conjugative enterotoxin (CPE)-encoding plasmids and a putative novel conjugative locus (Bcp) with sequence similarity to a locus reported from Clostridium botulinum. We sequenced and archived 102 new C. perfringens genomes, including those from rarely sequenced toxinotype B, C, D and E isolates. Long-read sequencing of 11 C. perfringens strains representing all toxinotypes (A-G) identified 55 plasmids from nine distinct plasmid groups. Interrogation of the 464 genomes in this collection identified 1045 plasmid-like contigs from the nine plasmid families, with a wide distribution across the C. perfringens isolates. Plasmids and plasmid diversity play an essential role in C. perfringens pathogenicity and broader biology. We have expanded the C. perfringens genome collection to include temporal, spatial and phenotypically diverse isolates including those carried asymptomatically in the gastrointestinal microbiome. This analysis has resulted in the identification of novel C. perfringens plasmids whilst providing a comprehensive understanding of species diversity.

Insertion sequences mediate clinical ST34 monophasic Salmonella enterica serovar Typhimurium plasmid polymorphism

Hybrid plasmids can combine the genetic elements of multiple plasmids, with the potential to carry a variety of antibiotic resistance genes and virulence genes, causing a great public health concern. Hybrid plasmids formed by fusion events may further exacerbate the spread of antibiotic resistance genes as well as plasmid evolution. Salmonella enterica serovar 4,[5],12:i:- is a monophasic variant of S. Typhimurium, which is one of the major causes of foodborne disease outbreaks worldwide. To assess the risk of transmission due to plasmid structure changes, we investigated the structural diversity of plasmids in two S. 4,[5],12:i:- isolates. Nanopore long-read sequencing was performed for plasmid comparison between original plasmids (donor isolates) and reorganized plasmids. We found that the IncHI2-IncHI2A multidrug resistance (MDR) plasmids in S. 4,[5],12:i:- possessed high plasticity, and could undergo recombination with other plasmids to form fusion plasmids of different sizes. Plasmid structural polymorphisms were mainly mediated by insertion sequences such as IS26 and ISPa40, and led to the rearrangement of the plasmid internal structures. To the best of our knowledge, this is the first report of the fusion of the IncHI2-IncHI2A and IncB/O/K/Z plasmids in S. 4,[5],12:i:- mediated by IS26. In addition, we also found that the mcr-1 gene was able to generate duplication during conjugation. Polymorphic changes in MDR plasmids during conjugation may further reduce the choice of clinical therapeutic agents. Therefore, continuous monitoring regarding plasmid polymorphic changes during transmission in both in vitro and in vivo is urgently needed to decipher the MDR plasmid evolution.

Insight into the Mechanisms of Carbapenem Resistance in Klebsiella pneumoniae: A Study on IS26 Integrons, Beta-Lactamases, Porin Modifications, and Plasmidome Analysis.

The emergence of carbapenem-resistant Klebsiella pneumoniae poses a significant threat to public health. In this study, we aimed to investigate the distribution and genetic diversity of plasmids carrying beta-lactamase resistance determinants in a collection of carbapenem-resistant K. pneumoniae blood isolates. Blood isolates of carbapenem-resistant K. pneumoniae bacteremia were collected and identified. Whole-genome sequencing, assembly and analysis were performed for the prediction of antimicrobial resistance determinants. Plasmidome analysis was also performed. Our plasmidome analysis revealed two major plasmid groups, IncFII/IncR and IncC, as key players in the dissemination of carbapenem resistance among carbapenem-resistant K. pneumoniae. Notably, plasmids within the same group exhibited conservation of encapsulated genes, suggesting that these plasmid groups may serve as conservative carriers of carbapenem-resistant determinants. Additionally, we investigated the evolution and expansion of IS26 integrons in carbapenem-resistant K. pneumoniae isolates using long-read sequencing. Our findings revealed the evolution and expansion of IS26 structure, which may have contributed to the development of carbapenem resistance in these strains. Our findings indicate that IncC group plasmids are associated with the endemic occurrence of carbapenem-resistant K. pneumoniae, highlighting the need for targeted interventions to control its spread. Although our study focuses on the endemic presence of carbapenem-resistant K. pneumoniae, it is important to note that carbapenem-resistant K. pneumoniae is indeed a global problem, with cases reported in multiple regions worldwide. Further research is necessary to better understand the factors driving the worldwide dissemination of carbapenem-resistant K. pneumoniae and to develop effective strategies for its prevention and control.

Plasmids of the incompatibility group FIBK occur in Klebsiella variicola from diverse ecological niches.

Plasmids play a fundamental role in the evolution of bacteria by allowing them to adapt to different environments and acquire, through horizontal transfer, genes that confer resistance to different classes of antibiotics. Using the available in vitro and in silico plasmid typing systems, we analyzed a set of isolates and public genomes of K. variicola to study its plasmid diversity. The resistome, the plasmid multilocus sequence typing (pMLST), and molecular epidemiology using the MLST system were also studied.A high frequency of IncF plasmids from human isolates but lower frequency from plant isolates were found in our strain collection.Insilico detection revealed 297 incompatibility (Inc) groups, but the IncFIBK(216/297) predominated in plasmids from human and environmentalsamples, followed by IncFIIK(89/297) and IncFIA/FIA(HI1) (75/297). These Inc groups were associated with clinically important ESBL (CTX-M-15), carbapenemases (KPC-2 and NDM-1), and colistin-resistant genes which were associated withmajor sequence types (ST): ST60, ST20, andST10. Insilico MOB typing showed 76% (311/404) of the genomes contained one or more of the six relaxase families with MOBFbeing most abundant. We identified untypeable plasmids carrying blaKPC-2, blaIMP-1, and blaSHV-187 but for which a relaxase was found; this may suggest that novel plasmid structures could be emerging in this bacterial species. The plasmid content in K. variicola has limited diversity, predominantly composed of IncFIBK plasmids dispersed in different STs. Plasmid detection using the replicon and MOB typing scheme provide a broader context of the plasmids inK. variicola. This study showed that whole-sequence-basedtyping provides current insights of the prevalence of plasmid types and their association with antimicrobial resistant genes inK. variicolaobtained from humans and environmental niches.

Diversity of Bacterial Clones and Plasmids of NDM-1 Producing Escherichia coli Clinical Isolates in Central Greece.

The objective of the present study was to genetically characterize ten NDM-1 producing Escherichia coli isolates, recovered from patients in a hospital in Central Greece during the period 2017 to 2021.The isolates were studied by whole genome sequencing to obtain multi-locus sequencing typing (MLST), identification of blaNDM1-environment, resistome and plasmid content. MLST analysis showed the presence of eight sequence types: ST46* (two isolates), ST46, ST744, ST998, ST410, ST224, ST4380, ST683 and ST12 (one isolate each). Apart of the presence of blaNDM-1, the isolates carried a combination of various to β-lactams encoding resistance genes: blaTEM-1B, blaCTX-15, blaOXA-1, blaVIM-1, blaSHV-5, blaOXA-16, blaOXA-10 and blaVEB-1. Additionally, plurality of resistance genes to aminoglycosides, macrolides, rifamycin, phenicols, sulfonamides and tetracycline was detected. The presence of multiple replicons was observed, with predominance of IncFII and IncFIB. Analysis of blaNDM-1 genetic environment of the isolates showed that seven had 100% identity with the pS-3002cz plasmid (Accession Number KJ 958927), two with the pB-3002cz plasmid (Accession Number KJ958926) and one with the pEc19397-131 plasmid (Accession Number MG878866). Τhis latter plasmid was derived by the fusion of two, previously identified, plasmids, pAMPD2 and pLK75 (Accession Numbers CP078058 and KJ440076, respectively). The diversity of clones and plasmids of NDM-1 producing E. coli isolated from patients in Greece indicates a continuous horizontal gene transfer.

Whole-Genome and Plasmid Comparative Analysis of Campylobacter jejuni from Human Patients in Toyama, Japan, from 2015 to 2019.

Campylobacter jejuni is a major causative agent of food poisoning, and increasing antimicrobial resistance is a concern. This study investigated 116 clinical isolates of C. jejuni from Toyama, Japan, which were isolated from 2015 to 2019. Antimicrobial susceptibility testing and whole-genome sequencing were used for phenotypic and genotypic characterization to compare antimicrobial resistance (AMR) profiles and phylogenic linkage. The multilocus sequence typing approach identified 37 sequence types (STs) and 15 clonal complexes (CCs), including 7 novel STs, and the high frequency CCs were CC21 (27.7%), CC48 (10.9%), and CC354 (9.9%). The AMR profiles and related resistant factors were as follows: fluoroquinolones (51.7%), mutation in quinolone resistance-determining region (QRDRs) (GyrA T86I); tetracyclines (27.6%), acquisition of tet(O); ampicillin (7.8%), harboring blaOXA184 or a promoter mutation in blaOXA193; aminoglycosides (1.7%), acquisition of ant(6)-Ia and aph(3')-III; chloramphenicol (0.9%), acquisition of cat. The acquired resistance genes tet(O), ant(6)-Ia, aph(3')-III, and cat were located on pTet family plasmids. Furthermore, three pTet family plasmids formed larger plasmids that incorporated additional genes such as the type IV secretion system. Sequence type 4526 (ST4526; 10.9%), which is reported only in Japan, was the most predominant, suggesting continued prevalence. This study reveals the sequences of the pTet family plasmids harbored by C. jejuni in Japan, which had been unclear, and the acquisition of the insertion sequences in a part of the pTet family plasmids. Because pTet family plasmids can be horizontally transmitted and are a major factor in acquired resistance in Campylobacter, the risk of spreading pTet that has acquired further resistance should be considered. IMPORTANCE Campylobacter jejuni is among the major causes of enteritis and diarrhea in humans in many countries. Drug-resistant Campylobacter is increasing in both developing and developed countries, and in particular, fluoroquinolone-resistant Campylobacter was one of the species included on the priority list of antibiotic-resistant bacteria. Campylobacter drug resistance surveillance is important and has been conducted worldwide. In this study, we performed whole-genome analysis of Campylobacter jejuni isolated from diarrhea patients at a hospital in Toyama, Japan. This revealed the continued prevalence of Campylobacter jejuni ST4526, which has been reported to be prevalent in Japan, and the acquisition of resistance and virulence factors in the pTet family plasmids. The diversity of pTet family plasmids, the major resistance transmission factor, is expected to potentially increase the risk of Campylobacter. The usefulness of whole-genome sequencing in Campylobacter surveillance was also demonstrated.