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ABSTRACT: Background: The rapid and global spread of Escherichia coli carrying mcr-type genes at the human-animal-environmental interface has become a serious global public health problem. Objective: To perform a genomic investigation of a colistin-resistant E. coli strain (14005RM) causing urinary tract infection, using a hybrid de novo assembly of Illumina/Nanopore sequence data, presenting phylogenomic insights into the relationship with mcr-1-positive strains circulating at the human-animal-environmental interface, in Brazil. Methods: Genomic DNA was sequenced using both the Illumina NexSeq and Nanopore MinION platforms. De novo hybrid assembly was performed by Unicycler. Genomic data were assessed by in silico prediction and bioinformatic tools. Results: The genome assembly size was 5 333 039 bp. The mcr-1.5-positive E. coli strain 14005RM belongs to the sequence type ST354 and presented a broad resistome (antibiotics, heavy metals, disinfectants, and glyphosate) and virulome. The mcr-1.5 gene was carried by an IncI2 plasmid (p14005RM, sizing 65,458 kb). Full genome SNP-based phylogenetic analysis reveals that mcr-1.5-producing E. coli strain 14005RM is highly related (> 98% identity) to colistin-resistant mcr-1.1-positive ST354 lineages associated with urinary tract infections in Brazil since 2015. Conclusion: Mobile colistin resistance within the Brazilian One Health microbiosphere is mediated by mcr gene variants propagated by IncX4, IncHI2, and IncI2 plasmids, circulating among global clones of E. coli.

The emergence of Klebsiella (K.) pneumoniae as a leading cause of nosocomial infections in Southeast Asia is of concern. Vietnam has an outstanding position with regard to antimicrobialresistant (AMR) pathogens and is a hotspot for carbapenem-resistant K. pneumoniae. In the current study, 19 clinical K. pneumoniae strains isolated from patients in Vietnam were tested for antibiotic susceptibility, and their genome sequences were analyzed to investigate potential resistance profiles, genotypes, AMR determinants, and virulence-associated genes. More than half of the isolates were multidrug-resistant (MDR), displaying resistance to ciprofloxacin, levofloxacin, chloramphenicol, piperacillin, cefotaxime, ceftazidime, and cefepime. Carbapenem- resistance was detected in 47% (n=9) of isolates. Five isolates were assigned to sequence type (ST) 23 by multi-locus sequence typing (MLST). These ST23 strains exhibited determinants characteristic of hypervirulent strains and were sensitive to all antibiotics tested except for one strain, which was resistant to chloramphenicol and trimethoprim/sulfamethoxazole. All isolates had AMR determinants that conferred resistance to aminoglycosides and ß-lactams. Single nucleotide variants of oqxA and oqxB conferring resistance to phenicol/quinolone, ompK37 and ompA conferring resistance to beta-lactams, and lptD conferring resistance to rifamycin, were found in all isolates. Additionally, 95% of the isolates (n=18) carried fosA genes; however, only two were resistant to fosfomycin. Five isolates carried genes conferring resistance to colistin; only one carried mcr-1.1 and showed resistance to colistin. One hundred thirty virulence-associated genes were identified. The current study demonstrated an inverse relationship between the number of detected virulence determinants and antibiotic resistance. Several well-known resistance genes have been identified but did not mediate resistance, which could be due to insufficient levels or lack of expression. A comprehensive study of the genotypic findings of AMR determinants and virulence with phenotypic data is required. The presence of hypervirulent K. pneumoniae carrying specific virulomes is a growing problem, and monitoring of virulence characteristics of K. pneumoniae is important. [ABSTRACT FROM AUTHOR]

Viruses are ubiquitous microbiome components, shaping ecosystems via strain-specific predation, horizontal gene transfer and redistribution of nutrients through host lysis. Viral impacts are important in groundwater ecosystems, where microbes drive many nutrient fluxes and metabolic processes; however, little is known about the diversity of viruses in these environments. We analyzed four groundwater plasmidomes (the entire plasmid content of an environment) and identified 200 viral sequences, which clustered into 41 genus-level viral clusters (approximately equivalent to viral genera) including 9 known and 32 putative new genera. We used publicly available bacterial whole-genome sequences (WGS) and WGS from 261 bacterial isolates from this groundwater environment to identify potential viral hosts. We linked 76 of the 200 viral sequences to a range of bacterial phyla, the majority associated with Proteobacteria, followed by Firmicutes, Bacteroidetes, and Actinobacteria. The publicly available WGS enabled mapping bacterial hosts to several viral sequences. The WGS of groundwater isolates increased the depth of host prediction by allowing host identification at the strain level. The latter included 4 viruses that were almost entirely (>99% query coverage, >99% identity) identified as integrated in the genomes of Pseudomonas, Acidovorax, and Castellaniella strains, resulting in high-confidence host assignments. Lastly, 21 of these viruses carried putative auxiliary metabolite genes for metal and antibiotic resistance, which might drive their infection cycles and/or provide selective advantage to infected hosts. Exploring the groundwater virome provides a necessary foundation for integration of viruses into ecosystem models where they are key players in microbial adaption to environmental stress. IMPORTANCE To our knowledge, this is the first study to identify the bacteriophage distribution in a groundwater ecosystem shedding light on their prevalence and distribution across metal-contaminated and background sites. Our study is uniquely based on selective sequencing of solely the extrachromosomal elements of a microbiome followed by analysis for viral signatures, thus establishing a more focused approach for phage identifications. Using this method, we detected several novel phage genera along with those previously established. Our approach of using the whole-genome sequences of hundreds of bacterial isolates from the same site enabled us to make host assignments with high confidence, several at strain levels. Certain phage genes suggest that they provide an environment-specific selective advantage to their bacterial hosts. Our study lays the foundation for future research on directed phage isolations using specific bacterial host strains to further characterize groundwater phages, their life cycles, and their effects on groundwater microbiome and biogeochemistry.

ABSTRACT Plasmids contribute to microbial diversity and adaptation, providing microorganisms with the ability to thrive in a wide range of conditions in extreme environments. However, while the number of marine microbiome studies is constantly increasing, very little is known about marine plasmids, and they are very poorly represented in public databases. To extend the repertoire of environmental marine plasmids, we established a pipeline for the de novo assembly of plasmids in the marine environment by analyzing available microbiome metagenomic sequencing data. By applying the pipeline to data from the Red Sea, we identified 362 plasmid candidates. We showed that the distribution of plasmids corresponds to environmental conditions, particularly, depth, temperature, and physical location. At least 7 of the 362 candidates are most probably real plasmids, based on a functional analysis of their open reading frames (ORFs). Only one of the seven has been described previously. Three plasmids were identified in other public marine metagenomic data from different locations all over the world; these plasmids contained different cassettes of functional genes at each location. Analysis of antibiotic and metal resistance genes revealed that the same positions that were enriched with genes encoding resistance to antibiotics were also enriched with resistance to metals, suggesting that plasmids contribute site-dependent phenotypic modules to their ecological niches. Finally, half of the ORFs (50.8%) could not be assigned to a function, emphasizing the untapped potential of the unique marine plasmids to provide proteins with multiple novel functions. IMPORTANCE Marine plasmids are understudied and hence underrepresented in databases. Plasmid functional annotation and characterization is complicated but, if successful, may provide a pool of novel genes and unknown functions. Newly discovered plasmids and their functional repertoire are potentially valuable tools for predicting the dissemination of antimicrobial resistance, providing vectors for molecular cloning and an understanding of plasmid-bacterial interactions in various environments.

Naturally occurring plasmids constitute a major category of mobile genetic elements responsible for harboring and transferring genes important in survival and fitness. A targeted evaluation of plasmidomes can reveal unique adaptations required by microbial communities. We developed a model system to optimize plasmid DNA isolation procedures targeted to groundwater samples which are typically characterized by low cell density (and likely variations in the plasmid size and copy numbers). The optimized method resulted in successful identification of several hundred circular plasmids, including some large plasmids (11 plasmids more than 50 kb in size, with the largest being 1.7 Mb in size). Several interesting observations were made from the analysis of plasmid DNA isolated in this study. The plasmid pool (plasmidome) was more conserved than the corresponding microbiome distribution (16S rRNA based). The circular plasmids were diverse as represented by the presence of seven plasmid incompatibility groups. The genes carried on these groundwater plasmids were highly enriched in metal resistance. Results from this study confirmed that traits such as metal, antibiotic, and phage resistance along with toxin-antitoxin systems are encoded on abundant circular plasmids, all of which could confer novel and advantageous traits to their hosts. This study confirms the ecological role of the plasmidome in maintaining the latent capacity of a microbiome, enabling rapid adaptation to environmental stresses.IMPORTANCE Plasmidomes have been typically studied in environments abundant in bacteria, and this is the first study to explore plasmids from an environment characterized by low cell density. We specifically target groundwater, a significant source of water for human/agriculture use. We used samples from a well-studied site and identified hundreds of circular plasmids, including one of the largest sizes reported in plasmidome studies. The striking similarity of the plasmid-borne ORFs in terms of taxonomical and functional classifications across several samples suggests a conserved plasmid pool, in contrast to the observed variability in the 16S rRNA-based microbiome distribution. Additionally, the stress response to environmental factors has stronger conservation via plasmid-borne genes as marked by abundance of metal resistance genes. Last, identification of novel and diverse plasmids enriches the existing plasmid database(s) and serves as a paradigm to increase the repertoire of biological parts that are available for modifying novel environmental strains.

Abstract Background Our interconnected world and the ability of bacteria to quickly swap antibiotic resistance genes (ARGs) make it particularly important to establish the epidemiological links of multidrug resistance (MDR) transfer between wastewater treatment plant (WWTP)- and human/animal-associated bacteria, under the One Health framework. However, evidence of ARGs exchange and potential factors that contribute to this transfer remain limited. Results Here, by combining culture-based population genomics and genetic comparisons with publicly available datasets, we reconstructed the complete genomes of 82 multidrug-resistant isolates from WWTPs and found that most WWTP-associated isolates were genetically distinct from their closest human/animal-associated relatives currently available in the public database. Even in the minority of lineages that were closely related, WWTP-associated isolates were characterized by quite different plasmid compositions. We identified a high diversity of circular plasmids (264 in total, of which 141 were potentially novel), which served as the main source of resistance, and showed potential horizontal transfer of ARG-bearing plasmids between WWTP- and humans/animal-associated bacteria. Notably, the potentially transferred ARGs and virulence factors (VFs) with different genetic backgrounds were closely associated with flanking insertion sequences (ISs), suggesting the importance of synergy between plasmids and ISs in mediating a multilayered hierarchical transfer of MDR and potentiating the emergence of MDR-hypervirulent clones. Conclusion Our findings advance the current efforts to establish potential epidemiological links of MDR transmission between WWTP- and human/animal-associated bacteria. Plasmids play an important role in mediating the transfer of ARGs and the IS-associated ARGs that are carried by conjugative plasmids should be prioritized to tackle the spread of resistance. Video Abstract

To characterize environmental antimicrobial resistance (AMR) in urban areas, extended-spectrum β-lactamase- (ESBL)/carbapenemase-producing bacteria (EPB/CPB, respectively) from urban wastewater treatment plant effluents in Tokyo were isolated on CHROMagar ESBL plate. Complete genome sequence analysis, including plasmids, indicated that 126 CTX-M-positive isolates (31%) were identified among the 404 obtained isolates. The CTX-M-9 group was predominant (n = 65, 52%), followed by the CTX-M-1 group (n = 44, 35%). Comparative genome analysis revealed that CTX-M-27-positive E. coli O16:H5-ST131-fimH41 exhibited a stable genome structure and clonal-global dissemination. Plasmidome network analysis revealed that 304 complete plasmid sequences among 85 isolates were grouped into 14 incompatibility (Inc) network communities (Co1 to Co14). Co10 consisted of primarily IncFIA/IncFIB plasmids harboring blaCTX-M in E. coli, whereas Co12 consisted primarily of IncFIA(HI1)/Inc FIB(K) plasmids harboring blaCTX-M, blaKPC, and blaGES in Klebsiella spp. Co11 was markedly located around Co10 and Co12. Co11 exhibited blaCTX-M, blaKPC, and blaNDM, and was mainly detected in E. coli and Klebsiella spp. from human and animal sources, suggesting a mutual role of Co11 in horizontal gene transfer between E. coli and Klebsiella spp. This comprehensive resistome analysis uncovers the mode of relational transfer among bacterial species, highlighting the potential source of AMR burden on public health in urban communities. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Enterobacter cloacae complex is a significant global healthcare threat, particularly carbapenemase-producing Enterobacter hormaechei (CPEH). From January 2017 to January 2021, twenty-two CPEH isolates from a regional teaching hospital in central Taiwan were identified with the carriage of carbapenemase genes bla KPC-2 , bla IMP-8 , and predominantly bla OXA-48 . Over 80% of these CPEH strains clustered into the high-risk ST78 lineage, carrying a bla OXA-48 IncL plasmid (pOXA48-CREH), nearly identical to the endemic plasmid pOXA48-KP in ST11 Klebsiella pneumoniae . This OXA-48-producing ST78 lineage disseminated clonally from 2018 to 2021 and transferred pOXA48-CREH to ST66 and ST90 E. hormaechei . An IMP-8-producing ST78 strain harbouring a bla IMP-8 -carrying pIncHI2 plasmid appeared in 2018, and by late 2020, a KPC-2-producing ST78 strain was identified after acquiring a novel bla KPC-2 -carrying IncFII plasmid. These findings suggest that the high-risk ST78 lineage of E . hormaechei has emerged as the primary driver behind the transmission of CPEH . ST78 has not only acquired various carbapenemase-gene-carrying plasmids but has also facilitated the transfer of pOXA48-CREH to other lineages. Continuous genomic surveillance and targeted interventions are urgently needed to control the spread of emerging CPEH clones in hospital settings.

Although previous studies using phenotypic or metagenomic approaches have revealed the patterns of antibiotic resistance genes (ARGs) in hospital effluents in local regions, limited information is available regarding the antibiotic resistome and plasmidome in human pathogenic bacteria in hospital effluents of megacity in China. To address this knowledge gap, we analyzed effluent samples from 166 hospitals across 13 geographical districts in Shanghai, China, using both cultivation-based approaches and metagenomics. A total of 357 strains were isolated from these samples, with the predominant species being Escherichia coli (n = 61), Aeromonas hydrophila (n = 57), Klebsiella pneumoniae (n = 48), and Aeromonas caviae (n = 42). Those identified indicator bacteria were classified into biosafety level 1 (BSL-1, 60 %) and BSL-2 (40 %). We identified 1237 ARG subtypes across 22 types, predominantly including beta-lactam, tetracycline, multidrug, polymyxin, and aminoglycoside resistance genes, using culture-enriched phenotypic metagenomics. Mobile genetic elements such as plasmids, transposons (tnpA), integrons (intI1), and insertion sequences (IS91) were abundant. We recovered 135 plasmids classified into mobilizable (n = 94) and non-mobilizable (n = 41) types. Additionally, 80 metagenome-assembled genomes (MAGs) were reconstructed from the hospital effluents for the assessment of ARG transmission risks, including genes for last-line antibiotics such as bla NDM , bla KPC , bla imiH , and mcr. This study is the first to comprehensively characterize and assess the risk of antimicrobial resistance levels and plasmidome in the hospital effluents of China's megacity, providing city-wide surveillance data and evidence to inform public health interventions., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Swine manure treatment plants are important reservoirs of plasmid-harboring antibiotic resistance genes (ARGs) and physicochemical contaminants, but the changes in the abundances of plasmids and ARGs, and their interactions with the physicochemical properties of manure, are still unclear. Thus, in the present study, plasmidome and metagenome analyses were conducted for samples collected at different stages in the swine manure treatment process. The results indicated that anaerobic digestion and aerobic digestion were the most efficient stages for reducing the abundances of ARGs in swine manure. However, the plasmids associated with ARGs were not effectively removed in these stages. Through the whole treatment process, the IncL/M, IncQ1, IncHI2A, IncA/C, and IncN plasmid groups had strong correlations (r > 0.8, P < 0.01) with most ARG types, thereby indicating that these plasmids play important roles in the persistence of ARGs in this environment. Furthermore, the pH, total nitrogen, total phosphorus, and four heavy metals (Cu, Zn, As, and Fe) significantly affected the abundances of seven ARG subtypes (tetB(P), ant(6)-Ia, tet44, aph(3′′)-Ib, mefB, tet(L), and tet(39)). In particular, florfenicol had the most positive correlations with ARGs. Our results indicated that nutrients, heavy metals, and antibiotics all contributed to the presence and persistence of plasmid-harboring ARGs. This study provides insights into the fate of plasmids and ARGs, and related factors during the swine manure treatment process, thereby facilitating the development of a new treatment technique for removing ARGs and reducing the public health risk associated with livestock production. [ABSTRACT FROM AUTHOR]

Objectives: The global success of carbapenem-resistant pathogens has been attributed to large plasmids carrying blaKPC genes circulating among high-risk clones. In this study, we sequenced the genome of a carbapenem-resistant Escherichia coli strain (Ec351) isolated from a human infection. Phylogenomic analysis based on single nucleotide polymorphisms (SNPs) as well as the comparative resistome and plasmidome of globally disseminated blaKPC-2-positive E. coli strains with identical sequence type (ST) were further investigated. Methods: Total DNA was sequenced using an Illumina NextSeq 500 platform and was assembled using Unicycler. Genomic data were evaluated through bioinformatics tools available from the Center of Genomic Epidemiology and by in silico analysis. Results: Genomic analysis revealed the convergence of a wide resistome and virulome in E. coli ST648, showing a high-level phylogenetic relationship with a KPC-2-positive ST648 cluster identified in the USA and association with international clade 2. Additionally, the emergence of an IncQ1 small plasmid (pEc351) carrying blaKPC-2 (on an NTEKPC-IId element), aph(3')-VIa, and plasmid regulatory and replication genes in the pandemic clone ST648 is reported. Conclusion: Identification of a blaKPC-2-positive IncQ1 plasmid in a high-risk E. coli clone represents rapid adaptation and expansion of these small plasmids encoding carbapenemases to novel bacterial hosts with global distribution, which deserves continued monitoring.

The emergence of Klebsiella pneumoniae (K. pneumoniae) in German healthcare is worrying. It is not well-investigated in the veterinary world and food chains. In the current study, antibiotic susceptibility profiles of 24 K. pneumoniae strains isolated from powdered milk samples produced in Germany were investigated by a microdilution test. Next-generation sequencing (NGS) was applied to identify genomic determinants for antimicrobial resistance (AMR), virulence-associated genes and plasmids replicons. All isolates were susceptible to the majority (14/18) of tested antibiotics. Resistance to colistin, fosfomycin, chloramphenicol and piperacillin was found. The ambler class A ß-lactamase, blaSHV variants were identified in all isolates, of which blaSHV-187 was most prevalent and found in 50% of isolates. Single-nucleotide-variants of oqxA and oqxB conferring resistance to phenicol/quinolone were found in all isolates, and the oqxB17 was the most prevalent found in 46% of isolates. 67% of isolates harbored fosA genes; however, only one was fosfomycin-resistant. Two isolates harbored genes conferring resistance to colistin, despite being susceptible. The majority of identified virulome genes were iron uptake siderophores. Two enterobactins (entB, fepC), six adherence-related genes belonging to E. coli common pilus (ECP) and one secretion system (ompA gene) were found in all isolates. In contrast, yersiniabactin was found in two isolates. One ST23 strain was susceptible to all tested antibiotics, and harbored determinants discriminatory for hypervirulent strains, e.g., aerobactin, salmochelin, yersiniabactin, enterobactin and regulator of mucoid phenotype A genes that are highly associated with hypervirulent K. pneumoniae. The IncF plasmid family was found in all strains, while almost half of the isolates harbored Col440I-type plasmids and nine isolates harbored various Inc-type plasmids. The presence of K. pneumoniae carrying different resistomes and major virulent specific virulomes in powdered milk samples is alarming. This could threaten public health, particularly of neonates and infants consuming dried milk. [ABSTRACT FROM AUTHOR]

Background: Our interconnected world and the ability of bacteria to quickly swap antibiotic resistance genes (ARGs) make it particularly important to establish the epidemiological links of multidrug resistance (MDR) transfer between wastewater treatment plant (WWTP)- and human/animal-associated bacteria, under the One Health framework. However, evidence of ARGs exchange and potential factors that contribute to this transfer remain limited. Results: Here, by combining culture-based population genomics and genetic comparisons with publicly available datasets, we reconstructed the complete genomes of 82 multidrug-resistant isolates from WWTPs and found that most WWTP-associated isolates were genetically distinct from their closest human/animal-associated relatives currently available in the public database. Even in the minority of lineages that were closely related, WWTP-associated isolates were characterized by quite different plasmid compositions. We identified a high diversity of circular plasmids (264 in total, of which 141 were potentially novel), which served as the main source of resistance, and showed potential horizontal transfer of ARG-bearing plasmids between WWTP- and humans/animal-associated bacteria. Notably, the potentially transferred ARGs and virulence factors (VFs) with different genetic backgrounds were closely associated with flanking insertion sequences (ISs), suggesting the importance of synergy between plasmids and ISs in mediating a multilayered hierarchical transfer of MDR and potentiating the emergence of MDR-hypervirulent clones. Conclusion: Our findings advance the current efforts to establish potential epidemiological links of MDR transmission between WWTP- and human/animal-associated bacteria. Plasmids play an important role in mediating the transfer of ARGs and the IS-associated ARGs that are carried by conjugative plasmids should be prioritized to tackle the spread of resistance. FH5Vi-SGsgm43KEHZNerU2 Video Abstract [ABSTRACT FROM AUTHOR]

The emergence and rapid spread of carbapenemase‐producing Enterobacterales represents a serious public health concern. Critically, these global priority bacteria have begun to be reported in companion animals, implying a potential risk of cross‐transmission between humans and pets. Using long‐read (MinION) and short‐read (Illumina) sequencing technologies, we have identified and characterized a hypermucoviscous KPC‐2‐producing Klebsiella pneumoniae strain belonging to the high‐risk international clone ST11/CG258, in a dog with urinary tract infection. Strikingly, the blaKPC‐2 gene was carried by a 54‐kb IncN plasmid assignated to ST15, which shared 99.8 and 96.8% pairwise identity with IncN‐pST15 plasmids from human and environmental K. pneumoniae strains, respectively; all come from an area with high endemicity of KPC‐2. Our findings suggest that IncN‐pST15 plasmids conferring carbapenem resistance can play as important a role as clonal transmission of K. pneumoniae, representing another major challenge for One Health. [ABSTRACT FROM AUTHOR]

The European epidemic monophasic variant of Salmonella enterica serovar Typhimurium (S. 1,4,[5],12:i:-) characterized by the multi locus sequence type ST34 and the antimicrobial resistance ASSuT profile has become one of the most common serovars in Europe (EU) and the United States (US). In this study, we reconstructed the time-scaled phylogeny and evolution of this Salmonella in Europe. The epidemic S. 1,4,[5],12:i:- ST34 emerged in the 1980s by an acquisition of the Salmonella Genomic Island (SGI)-4 at the 3′ end of the phenylalanine phe tRNA locus conferring resistance to copper and arsenic toxicity. Subsequent integration of the Tn21 transposon into the fljAB locus gave resistance to mercury toxicity and several classes of antibiotics used in food-producing animals (ASSuT profile). The second step of the evolution occurred in the 1990s, with the integration of mTmV and mTmV-like prophages carrying the perC and/or sopE genes involved in the ability to reduce nitrates in intestinal contents and facilitate the disruption of the junctions of the host intestinal epithelial cells. Heavy metals are largely used as food supplements or pesticide for cultivation of seeds intended for animal feed so the expansion of the epidemic S. 1,4,[5],12:i:- ST34 was strongly related to the multiple-heavy metal resistance acquired by transposons, integrative and conjugative elements and facilitated by the escape until 2011 from the regulatory actions applied in the control of S. Typhimurium in Europe. The genomic plasticity of the epidemic S. 1,4,[5],12:i:- was demonstrated in our study by the analysis of the plasmidome. We were able to identify plasmids harboring genes mediating resistance to phenicols, colistin, and fluoroquinolone and also describe for the first time in six of the analyzed genomes the presence of two plasmids (pERR1744967-1 and pERR2174855-2) previously described only in strains of enterotoxigenic Escherichia coli and E. fergusonii.

The European epidemic monophasic variant of Salmonella enterica serovar Typhimurium (S. 1 ,4,[5],12:i:-) characterized by the multi locus sequence type ST34 and the antimicrobial resistance ASSuT profile has become one of the most common serovars in Europe (EU) and the United States (US). In this study, we reconstructed the time-scaled phylogeny and evolution of this Salmonella in Europe. The epidemic S. 1 ,4,[5],12:i:- ST34 emerged in the 1980s by an acquisition of the Salmonella Genomic Island (SGI)-4 at the 3′ end of the phenylalanine phe tRNA locus conferring resistance to copper and arsenic toxicity. Subsequent integration of the Tn21 transposon into the fljAB locus gave resistance to mercury toxicity and several classes of antibiotics used in food-producing animals (ASSuT profile). The second step of the evolution occurred in the 1990s, with the integration of mTmV and mTmV-like prophages carrying the perC and/or sopE genes involved in the ability to reduce nitrates in intestinal contents and facilitate the disruption of the junctions of the host intestinal epithelial cells. Heavy metals are largely used as food supplements or pesticide for cultivation of seeds intended for animal feed so the expansion of the epidemic S. 1,4,[5],12:i:- ST34 was strongly related to the multiple-heavy metal resistance acquired by transposons, integrative and conjugative elements and facilitated by the escape until 2011 from the regulatory actions applied in the control of S. Typhimurium in Europe. The genomic plasticity of the epidemic S. 1,4,[5],12:i:- was demonstrated in our study by the analysis of the plasmidome. We were able to identify plasmids harboring genes mediating resistance to phenicols, colistin, and fluoroquinolone and also describe for the first time in six of the analyzed genomes the presence of two plasmids (pERR1744967-1 and pERR2174855-2) previously described only in strains of enterotoxigenic Escherichia coli and E. fergusonii. [ABSTRACT FROM AUTHOR]

Plasmids play important roles in microbial evolution and also in the spread of antibiotic resistance. Plasmid sequences are extensively studied from clinical isolates but rarely from the environment with a metagenomic approach focused on the plasmid fraction referred to as the plasmidome. A clear challenge in this context is to define a workflow for discriminating plasmids from chromosomal contaminants existing in the plasmidome. For this purpose, we benchmarked existing tools from assembly to detection of the plasmids by reference-free methods (cBar and PlasFlow) and database-guided approaches. Our simulations took into account short-reads alone or combined with moderate long-reads like those actually generated in environmental genomics experiments. This benchmark allowed us to select the best tools for limiting false-positives associated to plasmid prediction tools and a combination of reference-guided methods based on plasmid and bacterial databases. [ABSTRACT FROM AUTHOR]

High-throughput genomic sequencing has helped to reveal the plasmidome of Aeromonas salmonicida. This literature review provides an overview of A. salmonicida's rich plasmidome by presenting all the plasmids identified so far, addressing their biological importance and the functional links between them. The plasmids of A. salmonicida, especially those bearing antibiotic resistance genes, can provide clues about interactions of this species with other pathogens (animals and humans), as is the case for pRAS3-3432 and Chlamydia suis or pSN254b and Salmonella enterica. In addition to antibiotic resistance, plasmids play an important role in the virulence of A. salmonicida, particularly for the subspecies salmonicida and the plasmid pAsa5, which carries genes for the type-three secretion system, a virulence factor essential for the bacterium. The A. salmonicida plasmidome also has many cryptic plasmidswith no known biological function, but which can be used for the acquisition of new genetic elements. Striking examples are pAsa7 and pAsaXII that provide, respectively, resistance to chloramphenicol and formaldehyde and are derivatives of cryptic pAsa2. [ABSTRACT FROM AUTHOR]

Plasmid transfers among bacterial populations can directly influence the ecological adaptation of these populations and their interactions with host species and environment. In this study, we developed a selective multiply‐primed rolling circle amplification (smRCA) approach to enrich and characterize circular plasmid DNA from sponge microbial symbionts via high‐throughput sequencing (HTS). DNA (plasmid and total community DNA) obtained from sponge (Cinachyrella sp.) samples and a bacterial symbiont (Vibrio sp. CyArs1) isolated from the same sponge species (carrying unknown plasmids) were used to develop and validate our methodology. The smRCA was performed during 16 hr with 141 plasmid‐specific primers covering all known circular plasmid groups. The amplified products were purified and subjected to a reamplification with random hexamer primers (2 hr) and then sequenced using Illumina MiSeq. The developed method resulted in the successful amplification and characterization of the sponge plasmidome and allowed us to detect plasmids associated with the bacterial symbiont Vibrio sp. CyArs1 in the sponge host. In addition to this, a large number of small (<2 kbp) and cryptic plasmids were also amplified in sponge samples. Functional analysis identified proteins involved in the control of plasmid partitioning, maintenance and replication. However, most plasmids contained unknown genes, which could potentially serve as a resource of unknown genetic information and novel replication systems. Overall, our results indicate that the smRCA‐HTS approach developed here was able to selectively enrich and characterize plasmids from bacterial isolates and sponge host microbial communities, including plasmids larger than 20 kbp. [ABSTRACT FROM AUTHOR]

Background: The rapid and global spread of Escherichia coli carrying mcr-type genes at the human-animal-environmental interface has become a serious global public health problem., Objective: To perform a genomic investigation of a colistin-resistant E. coli strain (14005RM) causing urinary tract infection, using a hybrid de novo assembly of Illumina/Nanopore sequence data, presenting phylogenomic insights into the relationship with mcr-1-positive strains circulating at the human-animal-environmental interface, in Brazil., Methods: Genomic DNA was sequenced using both the Illumina NexSeq and Nanopore MinION platforms. De novo hybrid assembly was performed by Unicycler. Genomic data were assessed by in silico prediction and bioinformatic tools., Results: The genome assembly size was 5 333 039 bp. The mcr-1.5-positive E. coli strain 14005RM belongs to the sequence type ST354 and presented a broad resistome (antibiotics, heavy metals, disinfectants, and glyphosate) and virulome. The mcr-1.5 gene was carried by an IncI2 plasmid (p14005RM, sizing 65,458 kb). Full genome SNP-based phylogenetic analysis reveals that mcr-1.5-producing E. coli strain 14005RM is highly related (> 98% identity) to colistin-resistant mcr-1.1-positive ST354 lineages associated with urinary tract infections in Brazil since 2015., Conclusion: Mobile colistin resistance within the Brazilian One Health microbiosphere is mediated by mcr gene variants propagated by IncX4, IncHI2, and IncI2 plasmids, circulating among global clones of E. coli., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The prevalence of antimicrobial resistance in zoonotic Salmonella is a significant ongoing concern over the world. Several reports have investigated the prevalence of Salmonella infections in the farm animals in China; however, there is only limited knowledge about the Salmonella cross-contamination in the slaughterhouses. Moreover, the application of genomic approaches for understanding the cross-contamination in the food-animal slaughterhouses is still in its infancy in China. In the present study, we have isolated 105 Salmonella strains from pig carcasses and environment samples collected from four independent slaughterhouses in Jiangsu, China. All the Salmonella isolates were subjected to whole genome sequencing, bioinformatics analysis for serovar predictions, multi-locus sequence types, antimicrobial resistance genes, and plasmid types by using the in-house Galaxy platform. The antimicrobial resistance of Salmonella isolates was determined using a minimal inhibitory concentration assay with 14 antimicrobials. We found that the predominant serovar and serogroup was S. Derby and O:4(B), with a prevalence of 41.9 and 55%, respectively. All the isolates were multidrug-resistant and the highest resistance was observed against antimicrobials tetracycline (95.4%) and trimethoprim and sulfamethoxazole (90.9%). Additionally, the colistin-resistant determinant mcr-1 gene was detected in five (4.8%) strains. Our study demonstrated the prevalence of antimicrobial resistance in Salmonella strains isolated from pig slaughterhouses in China and suggested that the genomic platform can serve as routine surveillance along with the food-chain investigation.

Mobile genetic elements, including plasmids, drive the evolution of prokaryotic genomes through the horizontal transfer of genes allowing genetic exchange between bacteria. Moreover, plasmids carry accessory genes, which encode functions that may offer an advantage to the host. Thus, it is expected that in a certain ecological niche, plasmids are enriched in accessory functions, which are important for their hosts to proliferate in that niche. Puquio de Campo Naranja is a high-altitude lake from the Andean Puna exposed to multiple extreme conditions, including high UV radiation, alkalinity, high concentrations of arsenic, heavy metals, dissolved salts, high thermal amplitude and low O2 pressure. Microorganisms living in this lake need to develop efficient mechanisms and strategies to cope under these conditions. The aim of this study was to characterize the plasmidome of microbialites from Puquio de Campo Naranja, and identify potential hosts and encoded functions using a deep-sequencing approach. The potential ecological impact of the plasmidome, including plasmids from cultivable and non-cultivable microorganisms, is described for the first time in a lake representing an extreme environment of the Puna. This study showed that the recovered genetic information for the plasmidome was novel in comparison to the metagenome derived from the same environment. The study of the total plasmid population allowed the identification of genetic features typically encoded by plasmids, such as resistance and virulence factors. The resistance genes comprised resistances to heavy metals, antibiotics and stress factors. These results highlight the key role of plasmids for their hosts and impact of extrachromosomal elements to thrive in a certain ecological niche.

ABSTRACT Enterococcus faecium is a gut commensal of humans and animals but is also listed on the WHO global priority list of multidrug-resistant pathogens. Many of its antibiotic resistance traits reside on plasmids and have the potential to be disseminated by horizontal gene transfer. Here, we present the first comprehensive population-wide analysis of the pan-plasmidome of a clinically important bacterium, by whole-genome sequence analysis of 1,644 isolates from hospital, commensal, and animal sources of E. faecium. Long-read sequencing on a selection of isolates resulted in the completion of 305 plasmids that exhibited high levels of sequence modularity. We further investigated the entirety of all plasmids of each isolate (plasmidome) using a combination of short-read sequencing and machine-learning classifiers. Clustering of the plasmid sequences unraveled different E. faecium populations with a clear association with hospitalized patient isolates, suggesting different optimal configurations of plasmids in the hospital environment. The characterization of these populations allowed us to identify common mechanisms of plasmid stabilization such as toxin-antitoxin systems and genes exclusively present in particular plasmidome populations exemplified by copper resistance, phosphotransferase systems, or bacteriocin genes potentially involved in niche adaptation. Based on the distribution of k-mer distances between isolates, we concluded that plasmidomes rather than chromosomes are most informative for source specificity of E. faecium. IMPORTANCE Enterococcus faecium is one of the most frequent nosocomial pathogens of hospital-acquired infections. E. faecium has gained resistance against most commonly available antibiotics, most notably, against ampicillin, gentamicin, and vancomycin, which renders infections difficult to treat. Many antibiotic resistance traits, in particular, vancomycin resistance, can be encoded in autonomous and extrachromosomal elements called plasmids. These sequences can be disseminated to other isolates by horizontal gene transfer and confer novel mechanisms to source specificity. In our study, we elucidated the total plasmid content, referred to as the plasmidome, of 1,644 E. faecium isolates by using short- and long-read whole-genome technologies with the combination of a machine-learning classifier. This was fundamental to investigate the full collection of plasmid sequences present in our collection (pan-plasmidome) and to observe the potential transfer of plasmid sequences between E. faecium hosts. We observed that E. faecium isolates from hospitalized patients carried a larger number of plasmid sequences compared to that from other sources, and they elucidated different configurations of plasmidome populations in the hospital environment. We assessed the contribution of different genomic components and observed that plasmid sequences have the highest contribution to source specificity. Our study suggests that E. faecium plasmids are regulated by complex ecological constraints rather than physical interaction between hosts.

The emergence of Klebsiella pneumoniae (K. pneumoniae) in German healthcare is worrying. It is not well-investigated in the veterinary world and food chains. In the current study, antibiotic susceptibility profiles of 24 K. pneumoniae strains isolated from powdered milk samples produced in Germany were investigated by a microdilution test. Next-generation sequencing (NGS) was applied to identify genomic determinants for antimicrobial resistance (AMR), virulence-associated genes and plasmids replicons. All isolates were susceptible to the majority (14/18) of tested antibiotics. Resistance to colistin, fosfomycin, chloramphenicol and piperacillin was found. The ambler class A ß-lactamase, blaSHV variants were identified in all isolates, of which blaSHV-187 was most prevalent and found in 50% of isolates. Single-nucleotide-variants of oqxA and oqxB conferring resistance to phenicol/quinolone were found in all isolates, and the oqxB17 was the most prevalent found in 46% of isolates. 67% of isolates harbored fosA genes; however, only one was fosfomycin-resistant. Two isolates harbored genes conferring resistance to colistin, despite being susceptible. The majority of identified virulome genes were iron uptake siderophores. Two enterobactins (entB, fepC), six adherence-related genes belonging to E. coli common pilus (ECP) and one secretion system (ompA gene) were found in all isolates. In contrast, yersiniabactin was found in two isolates. One ST23 strain was susceptible to all tested antibiotics, and harbored determinants discriminatory for hypervirulent strains, e.g., aerobactin, salmochelin, yersiniabactin, enterobactin and regulator of mucoid phenotype A genes that are highly associated with hypervirulent K. pneumoniae. The IncF plasmid family was found in all strains, while almost half of the isolates harbored Col440I-type plasmids and nine isolates harbored various Inc-type plasmids. The presence of K. pneumoniae carrying different resistomes and major virulent specific virulomes in powdered milk samples is alarming. This could threaten public health, particularly of neonates and infants consuming dried milk.

Aeromonas salmonicida is a global distributed Gram-negative teleost pathogen, affecting mainly salmonids in fresh and marine environments. A. salmonicida strains are classified as typical or atypical depending on their origin of isolation and phenotype. Five subspecies have been described, where A. salmonicida subsp. salmonicida is the only typical subspecies, and the subsp. achromogenes, masoucida, smithia, and pectinolytica are considered atypical. Genomic differences between A. salmonicida subsp. salmonicida isolates and their relationship with the current classification have not been explored. Here, we sequenced and compared the complete closed genomes of four virulent strains to elucidate their molecular diversity and pathogenic evolution using the more accurate genomic information so far. Phenotypes, biochemical, and enzymatic profiles were determined. PacBio and MiSeq sequencing platforms were utilized for genome sequencing. Comparative genomics showed that atypical strains belong to the subsp. salmonicida, with 99.55% ± 0.25% identity with each other, and are closely related to typical strains. The typical strain A. salmonicida J223 is closely related to typical strains, with 99.17% identity with the A. salmonicida A449. Genomic differences between atypical and typical strains are strictly related to insertion sequences (ISs) activity. The absence and presence of genes encoding for virulence factors, transcriptional regulators, and non-coding RNAs are the most significant differences between typical and atypical strains that affect their phenotypes. Plasmidome plays an important role in A. salmonicida virulence and genome plasticity. Here, we determined that typical strains harbor a larger number of plasmids and virulence-related genes that contribute to its acute virulence. In contrast, atypical strains harbor a single, large plasmid and a smaller number of virulence genes, reflected by their less acute virulence and chronic infection. The relationship between phenotype and A. salmonicida subspecies’ taxonomy is not evident. Comparative genomic analysis based on completed genomes revealed that the subspecies classification is more of a reflection of the ecological niche occupied by bacteria than their divergences at the genomic level except for their accessory genome.

Mobile genetic elements, including plasmids, drive the evolution of prokaryotic genomes through the horizontal transfer of genes allowing genetic exchange between bacteria. Moreover, plasmids carry accessory genes, which encode functions that may offer an advantage to the host. Thus, it is expected that in a certain ecological niche, plasmids are enriched in accessory functions, which are important for their hosts to proliferate in that niche. Puquio de Campo Naranja is a high-altitude lake from the Andean Puna exposed to multiple extreme conditions, including high UV radiation, alkalinity, high concentrations of arsenic, heavy metals, dissolved salts, high thermal amplitude and low O2 pressure. Microorganisms living in this lake need to develop efficient mechanisms and strategies to cope under these conditions. The aim of this study was to characterize the plasmidome of microbialites from Puquio de Campo Naranja, and identify potential hosts and encoded functions using a deep-sequencing approach. The potential ecological impact of the plasmidome, including plasmids from cultivable and non-cultivable microorganisms, is described for the first time in a lake representing an extreme environment of the Puna. This study showed that the recovered genetic information for the plasmidome was novel in comparison to the metagenome derived from the same environment. The study of the total plasmid population allowed the identification of genetic features typically encoded by plasmids, such as resistance and virulence factors. The resistance genes comprised resistances to heavy metals, antibiotics and stress factors. These results highlight the key role of plasmids for their hosts and impact of extrachromosomal elements to thrive in a certain ecological niche. [ABSTRACT FROM AUTHOR]

ABSTRACT Prokaryotes represent an ancestral lineage in the tree of life and constitute optimal resources for investigating the evolution of genomes in unicellular organisms. Many bacterial species possess multipartite genomes offering opportunities to study functional variations among replicons, how and where new genes integrate into a genome, and how genetic information within a lineage becomes encoded and evolves. To analyze these issues, we focused on the model soil bacterium Sinorhizobium meliloti, which harbors a chromosome, a chromid (pSymB), a megaplasmid (pSymA), and, in many strains, one or more accessory plasmids. The analysis of several genomes, together with 1.4 Mb of accessory plasmid DNA that we purified and sequenced, revealed clearly different functional profiles associated with each genomic entity. pSymA, in particular, exhibited remarkable interstrain variation and a high density of singletons (unique, exclusive genes) featuring functionalities and modal codon usages that were very similar to those of the plasmidome. All this evidence reinforces the idea of a close relationship between pSymA and the plasmidome. Correspondence analyses revealed that adaptation of codon usages to the translational machinery increased from plasmidome to pSymA to pSymB to chromosome, corresponding as such to the ancestry of each replicon in the lineage. We demonstrated that chromosomal core genes gradually adapted to the translational machinery, reminiscent of observations in several bacterial taxa for genes with high expression levels. Such findings indicate a previously undiscovered codon usage adaptation associated with the chromosomal core information that likely operates to improve bacterial fitness. We present a comprehensive model illustrating the central findings described here, discussed in the context of the changes occurring during the evolution of a multipartite prokaryote genome. IMPORTANCE Bacterial genomes usually include many thousands of genes which are expressed with diverse spatial-temporal patterns and intensities. A well-known evidence is that highly expressed genes, such as the ribosomal and other translation-related proteins (RTRPs), have accommodated their codon usage to optimize translation efficiency and accuracy. Using a bioinformatic approach, we identify core-genes sets with different ancestries, and demonstrate that selection processes that optimize codon usage are not restricted to RTRPs but extended at a genome-wide scale. Such findings highlight, for the first time, a previously undiscovered adaptation strategy associated with the chromosomal-core information. Contrasted with the translationally more adapted genes, singletons (i.e., exclusive genes, including those of the plasmidome) appear as the gene pool with the less-ameliorated codon usage in the lineage. A comprehensive summary describing the inter- and intra-replicon heterogeneity of codon usages in a complex prokaryote genome is presented.

ABSTRACT Naturally occurring plasmids constitute a major category of mobile genetic elements responsible for harboring and transferring genes important in survival and fitness. A targeted evaluation of plasmidomes can reveal unique adaptations required by microbial communities. We developed a model system to optimize plasmid DNA isolation procedures targeted to groundwater samples which are typically characterized by low cell density (and likely variations in the plasmid size and copy numbers). The optimized method resulted in successful identification of several hundred circular plasmids, including some large plasmids (11 plasmids more than 50 kb in size, with the largest being 1.7 Mb in size). Several interesting observations were made from the analysis of plasmid DNA isolated in this study. The plasmid pool (plasmidome) was more conserved than the corresponding microbiome distribution (16S rRNA based). The circular plasmids were diverse as represented by the presence of seven plasmid incompatibility groups. The genes carried on these groundwater plasmids were highly enriched in metal resistance. Results from this study confirmed that traits such as metal, antibiotic, and phage resistance along with toxin-antitoxin systems are encoded on abundant circular plasmids, all of which could confer novel and advantageous traits to their hosts. This study confirms the ecological role of the plasmidome in maintaining the latent capacity of a microbiome, enabling rapid adaptation to environmental stresses. IMPORTANCE Plasmidomes have been typically studied in environments abundant in bacteria, and this is the first study to explore plasmids from an environment characterized by low cell density. We specifically target groundwater, a significant source of water for human/agriculture use. We used samples from a well-studied site and identified hundreds of circular plasmids, including one of the largest sizes reported in plasmidome studies. The striking similarity of the plasmid-borne ORFs in terms of taxonomical and functional classifications across several samples suggests a conserved plasmid pool, in contrast to the observed variability in the 16S rRNA-based microbiome distribution. Additionally, the stress response to environmental factors has stronger conservation via plasmid-borne genes as marked by abundance of metal resistance genes. Last, identification of novel and diverse plasmids enriches the existing plasmid database(s) and serves as a paradigm to increase the repertoire of biological parts that are available for modifying novel environmental strains.

The global clustering of gene families through network analysis has been demonstrated in whole genome, plasmid, and microbiome analyses. In this study, we carried out a plasmidome network analysis of all available complete bacterial plasmids to determine plasmid associations. A blastp clustering search at 100% aa identity cut-off and sharing at least one gene between plasmids, followed by a multilevel community network analysis revealed that a surprisingly large number of the plasmids were connected by one largest connected component (LCC), with dozens of community sub-groupings. The LCC consisted mainly of Bacilli and Gammaproteobacteria plasmids. Intriguingly, horizontal gene transfer (HGT) was noted between different phyla (i.e., Staphylococcus and Pasteurellaceae), suggesting that Pasteurellaceae can acquire antimicrobial resistance (AMR) genes from closely contacting Staphylococcus spp., which produce the external supplement of V-factor (NAD). Such community network analysis facilitate displaying possible recent HGTs like a class 1 integron, str and tet resistance markers between communities. Furthermore, the distribution of the Inc replicon type and AMR genes, such as the extended-spectrum ß-lactamase (ESBL) CTX-M or the carbapenemases KPC NDM-1, implies that such genes generally circulate within limited communities belonging to typical bacterial genera. Thus, plasmidome network analysis provides a remarkable discriminatory power for plasmid-related HGT and evolution.

Plasmids contribute to microbial diversity and adaptation, providing microorganisms with the ability to thrive in a wide range of conditions in extreme environments. However, while the number of marine microbiome studies is constantly increasing, very little is known about marine plasmids, and they are very poorly represented in public databases. To extend the repertoire of environmental marine plasmids, we established a pipeline for the de novo assembly of plasmids in the marine environment by analyzing available microbiome metagenomic sequencing data. By applying the pipeline to data from the Red Sea, we identified 362 plasmid candidates. We showed that the distribution of plasmids corresponds to environmental conditions, particularly, depth, temperature, and physical location. At least 7 of the 362 candidates are most probably real plasmids, based on a functional analysis of their open reading frames (ORFs). Only one of the seven has been described previously. Three plasmids were identified in other public marine metagenomic data from different locations all over the world; these plasmids contained different cassettes of functional genes at each location. Analysis of antibiotic and metal resistance genes revealed that the same positions that were enriched with genes encoding resistance to antibiotics were also enriched with resistance to metals, suggesting that plasmids contribute site-dependent phenotypic modules to their ecological niches. Finally, half of the ORFs (50.8%) could not be assigned to a function, emphasizing the untapped potential of the unique marine plasmids to provide proteins with multiple novel functions. IMPORTANCE Marine plasmids are understudied and hence underrepresented in databases. Plasmid functional annotation and characterization is complicated but, if successful, may provide a pool of novel genes and unknown functions. Newly discovered plasmids and their functional repertoire are potentially valuable tools for predicting the dissemination of antimicrobial resistance, providing vectors for molecular cloning and an understanding of plasmid-bacterial interactions in various environments., Competing Interests: The authors declare no conflict of interest.

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 bla CTX-M-15 was acquired multiple times by a high diversity of F-type mosaic plasmids, other antibiotic resistance genes (ARGs) were dispersed by IncL ( bla OXA-48 ) or IncC ( bla CMY/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., Competing Interests: The authors declare no conflict of interest.

The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognised not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture–dependent or –independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e. the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which – despite their frequent occurrence in a large number of bacteria – are largely neglected in prevalent plasmidome conceptions.