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Transcriptional heterogeneity in isogenic bacterial populations can play various roles in bacterial evolution, but its detection remains technically challenging. Here, we use microbial split-pool ligation transcriptomics to study the relationship between bacterial subpopulation formation and plasmid-host interactions at the single-cell level. We find that single-cell transcript abundances are influenced by bacterial growth state and plasmid carriage. Moreover, plasmid carriage constrains the formation of bacterial subpopulations. Plasmid genes, including those with core functions such as replication and maintenance, exhibit transcriptional heterogeneity associated with cell activity. Notably, we identify a cell subpopulation that does not transcribe conjugal plasmid transfer genes, which may help reduce plasmid burden on a subset of cells. Our study advances the understanding of plasmid-mediated subpopulation dynamics and provides insights into the plasmid-bacteria interplay. Transcriptional heterogeneity in isogenic bacterial populations can play various roles in bacterial evolution, but its detection remains technically challenging. Here, Cyriaque et al. use microbial split-pool ligation transcriptomics to study the relationship between bacterial subpopulation formation and plasmid-host interactions at the single-cell level, providing insights into plasmid-bacteria dynamics. [ABSTRACT FROM AUTHOR]

Objectives To determine the frequencies and clonal distributions of putative genetic determinants of resistance to antimicrobials applied for treatment of Clostridioides difficile infection (CDI), as documented in the genomic record. Methods We scanned 26 557 C. difficile genome sequences publicly available from the EnteroBase platform for plasmids, point mutations and gene truncations previously reported to reduce susceptibility to vancomycin, fidaxomicin or metronidazole, respectively. We measured the antimicrobial susceptibility of 143 selected C. difficile isolates. Results The frequency of mutations causing reduced susceptibility to vancomycin and metronidazole, respectively, increased strongly after 2000, peaking at up to 52% of all sequenced C. difficile genomes. However, both mutations declined sharply more recently, reflecting major changes in CDI epidemiology. We detected mutations associated with fidaxomicin resistance in several major genotypes, but found no evidence of international spread of resistant clones. The pCD-METRO plasmid, conferring metronidazole resistance, was detected in a single previously unreported C. difficile isolate, recovered from a hospital patient in Germany in 2008. The pX18-498 plasmid, putatively associated with decreased vancomycin susceptibility, was confined to related, recent isolates from the USA. Phenotype measurements confirmed that most of those genetic features were useful predictors of antibiotic susceptibility, even though ranges of MICs typically overlapped among isolates with and without specific mutations. Conclusions Genomic data suggested that resistance to therapeutic antimicrobial drugs is rare in C. difficile. Public antimicrobial resistance marker databases were not equipped to detect most of the genetic determinants relevant to antibiotic therapy of CDI. [ABSTRACT FROM AUTHOR]

We analyzed antimicrobial resistance and virulence traits in multidrug-resistant (MDR) E. coli isolates obtained from imported shrimp using whole-genome sequences (WGSs). Antibiotic resistance profiles were determined phenotypically. WGSs identified key characteristics, including their multilocus sequence type (MLST), serotype, virulence factors, antibiotic resistance genes, and mobile elements. Most of the isolates exhibited resistance to gentamicin, streptomycin, ampicillin, chloramphenicol, nalidixic acid, ciprofloxacin, tetracycline, and trimethoprim/sulfamethoxazole. Multilocus sequence type (MLST), serotype, average nucleotide identity (ANI), and pangenome analysis showed high genomic similarity among isolates, except for EC15 and ECV01. The EC119 plasmid contained a variety of efflux pump genes, including those encoding the acid resistance transcriptional activators (gadE, gadW, and gadX), resistance-nodulation-division-type efflux pumps (mdtE and mdtF), and a metabolite, H1 symporter (MHS) family major facilitator superfamily transporter (MNZ41_23075). Virulence genes displayed diversity, particularly EC15, whose plasmids carried genes for adherence (faeA and faeC-I), invasion (ipaH and virB), and capsule (caf1A and caf1M). This comprehensive analysis illuminates antimicrobial resistance, virulence, and plasmid dynamics in E. coli from imported shrimp and has profound implications for public health, emphasizing the need for continued surveillance and research into the evolution of these important bacterial pathogens. [ABSTRACT FROM AUTHOR]

Sternal bursitis, a common inflammatory condition in poultry, poses significant challenges to both animal welfare and public health. This study aimed to investigate the prevalence, antimicrobial resistance, and genetic characteristics of Staphylococcus aureus isolates associated with sternal bursitis in chickens. Ninety-eight samples were collected from affected chickens, and 24 S. aureus isolates were identified. Antimicrobial susceptibility testing revealed resistance to multiple agents, with a notable prevalence of aminoglycoside resistance genes. Whole genome sequencing elucidated the genetic diversity and virulence profiles of the isolates, highlighting the predominance of clonal complex 5 (CC5) strains. Additionally, biofilm formation assays demonstrated moderate biofilm production capacity among the isolates. These findings underscore the importance of vigilant monitoring and targeted interventions to mitigate the impact of sternal bursitis in poultry production systems. [ABSTRACT FROM AUTHOR]

The global spread of plasmid-mediated mobile colistin resistance (mcr) genes threatens the vital role of colistin as a drug of last resort. We investigated whether the recurrent occurrence of specific E. coli pathotypes and plasmids in individual pig farms resulted from the continued presence or repeated reintroduction of distinct E. coli strains. E. coli isolates (n = 154) obtained from three pig farms with at least four consecutive years of mcr detection positive for virulence-associated genes (VAGs) predicting an intestinal pathogenic pathotype via polymerase chain reaction were analyzed. Detailed investigation of VAGs, antimicrobial resistance genes and plasmid Inc types was conducted using whole genome sequencing for 87 selected isolates. Sixty-one E. coli isolates harbored mcr-1, and one isolate carried mcr-4. On Farm 1, mcr-positive isolates were either edema disease E. coli (EDEC; 77.3%) or enterotoxigenic E. coli (ETEC; 22.7%). On Farm 2, all mcr-positive strains were ETEC, while mcr-positive isolates from Farm 3 showed a wider range of pathotypes. The mcr-1.1 gene was located on IncHI2 (Farm 1), IncX4 (Farm 2) or IncX4 and IncI2 plasmids (Farm 3). These findings suggest that various pathogenic E. coli strains play an important role in maintaining plasmid-encoded colistin resistance genes in the pig environment over time. [ABSTRACT FROM AUTHOR]

Raoultella planticola is an emerging bacterial pathogen responsible for causing infections in both humans and animals. Unfortunately, sporadic reports of carbapenem-resistant R. planticola (CRRP) have been documented worldwide. Here we first reported the complete genome sequence of a CRRP isolate RP_3045 co-carrying blaIMP-4 and blaSHV-12, recovered from a patient in China, and its genetic relatedness to 82 R. planticola strains deposited in the NCBI GenBank database, sourced from humans, animals, and the environment. Whole-genome sequencing was performed using the Illumina NovaSeq 6000 and Oxford Nanopore MinION platforms. Phylogenetic analysis was also performed and visualized using a single nucleotide polymorphism (SNP)-based strategy. The complete genome of R. planticola strain RP_3045 was determined to be 6,312,961 bp in length, comprising five contigs that included one chromosome and four plasmids. RP_3045 was found to be multidrug-resistant and harbored several antimicrobial resistance genes, including both blaIMP-4 and blaSHV-12 genes located on a single plasmid. The most closely related strain was hkcpe63, recovered from humans in Hong Kong, China, in 2014, with 506 SNP differences. R. planticola strains were distributed globally and exhibited strong associations among isolates obtained from different sectors. This study provides evidence for the potential of R. planticola to disseminate carbapenem resistance across different sectors, highlighting the critical need for active and continuous surveillance of CRRP. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Escherichia coli (CREC) poses a severe global public health risk. This study reveals the worldwide geographic spreading patterns and spatiotemporal distribution characteristics of resistance genes in 7918 CREC isolates belonging to 497 sequence types (ST) and originating from 75 countries. In the last decade, there has been a transition in the prevailing STs from highly virulent ST131 and ST38 to higher antibiotic-resistant ST410 and ST167. The rise of multi-drug resistant strains of CREC carrying plasmids with extended-spectrum beta-lactamase (ESBL) resistance genes could be attributed to three important instances of host-switching events. The spread of CREC was associated with the changing trends in blaNDM-5, blaKPC-2, and blaOXA-48, as well as the plasmids IncFI, IncFII, and IncI. There were intercontinental geographic transfers of major CREC strains. Various crucial transmission hubs and patterns have been identified for ST131 in the United Kingdom, Italy, the United States, and China, ST167 in India, France, Egypt, and the United States, and ST410 in Thailand, Israel, the United Kingdom, France, and the United States. This work is valuable in managing CREC infections and preventing CREC occurrence and transmission inside healthcare settings and among diverse hosts. Analysis of 7918 global carbapenem-resistant E. coli genomes reveals the transitions of diverse sequence types (STs), three host-switching events leading to the spread of resistance plasmids, and the crucial transmission country hubs and patterns of major STs. [ABSTRACT FROM AUTHOR]

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The purpose of this study was to determine the level of horizontal transmission of the blaCTX-M-65 gene and the role of its associated mobile genetic elements (MGEs) in the bovine-derived Escherichia coli. After PCR identification, two plasmids carrying blaCTX-M-65 were successfully transferred to the recipient E. coli J53 Azr through conjugation assays and subsequently selected for Whole-Genome sequencing (WGS) analysis. The resistance profiles of these two positive strains and their transconjugants were also determined through antimicrobial susceptibility tests. Whole genome data were acquired using both the PacBio sequencing platform and the Illumina data platform. The annotated results were then submitted to the Genbank database for accession number recording. For comparison, the genetic environment of plasmids carrying the resistance gene blaCTX-M-65 was mapped using the Easyfig software. WGS analysis revealed Tn3-like composite transposons bearing blaCTX-M-65, blaTEM-1, and blaOXA-10 in the IncHI2-type plasmids of these two E. coli ST1508 strains. A phylogenetic tree was generated from all 48 assembled E. coli isolates blaCTX-M-65, blaTEM-1, and blaOXA-10 from the NCBI Pathogen Detection database with our two isolates, showing the relationships and the contribution of SNPs to the diversity between genetic samples. This study suggests that the transmissibility of blaCTX-M-65 on Tn3-like composite transposons contributes to an increased risk of its transmission in E. coli derived from dairy cattle. [ABSTRACT FROM AUTHOR]

Objectives To determine the genomic features of IMP-4-producing Klebsiella pneumoniae isolates recovered from paediatric patients and the transmission dynamics of bla IMP-4. Methods IMP-producing K. pneumoniae isolates were collected from paediatric patients in Shanghai Children's Medical Center from 2013 to 2020. WGS was performed for all isolates, and the complete genomes of three IMP-4-producing isolates were generated. The distribution of bla IMP-4-harbouring plasmids was determined, and a conjugation assay was employed to investigate the horizontal transfer of bla IMP-4-harbouring plasmids. Results We collected 21 bla IMP-carrying K. pneumoniae isolates, with IMP-4 (16/21, 76.2%) as the predominant subtype, followed by IMP-8 (n  = 3) and IMP-26 (n  = 2). IMP-4-producing isolates displayed a diverse population structure and all bla IMP-4 genes were located on plasmids, including IncN (n  = 9), IncHI5 (n  = 5), IncFII(K) (n  = 1) and IncFII(pKP91) (n  = 1), although only IncN plasmids were conjugative. Clonal transmission of ST101 strains carrying IncHI5 bla IMP-4-harbouring plasmids was observed, and the acquisition of bla IMP-4 by the international high-risk ST101 clone constituted a novel combination of ST101 clone and carbapenemase genes. Plasmid analysis demonstrated that the conjugal transfer of the IncHI5 bla IMP-4-harbouring plasmid might be blocked by the ST101 bacterial host. Conclusions The horizontal transfer of IncN plasmids and clonal spread of the international high-risk ST101 clone facilitated the nosocomial dissemination of bla IMP-4 among K. pneumoniae. The emerging IMP-4-producing ST101 clone displays diverse combinations of carbapenemase genes, and this clone could be a continually evolving threat and warrants prospective monitoring. [ABSTRACT FROM AUTHOR]

Plasmids mediate gene exchange across taxonomic barriers through conjugation, shaping bacterial evolution for billions of years. While plasmid mobility can be harnessed for genetic engineering and drug-delivery applications, rapid plasmid-mediated spread of resistance genes has rendered most clinical antibiotics useless. To solve this urgent and growing problem, we must understand how plasmids spread across bacterial communities. Here, we applied machine-learning models to identify features that are important for extending the plasmid host range. We assembled an up-to-date dataset of more than thirty thousand bacterial plasmids, separated them into 1125 clusters, and assigned each cluster a distribution possibility score, taking into account the host distribution of each taxonomic rank and the sampling bias of the existing sequencing data. Using this score and an optimized plasmid feature pool, we built a model stack consisting of DecisionTreeRegressor, EvoTreeRegressor, and LGBMRegressor as base models and LinearRegressor as a meta-learner. Our mathematical modeling revealed that sequence brevity is the most important determinant for plasmid spread, followed by P-loop NTPases, mobility factors, and β-lactamases. Ours and other recent results suggest that small plasmids may broaden their range by evading host defenses and using alternative modes of transfer instead of autonomous conjugation. [ABSTRACT FROM AUTHOR]

Background: The emergence and wide spread of carbapenemase-producing Enterobacteriaceae (CPE) poses a growing threat to global public health. However, clinically derived carbapenemase-producing Citrobacter causing multiple infections has rarely been investigated. Here we first report the isolation and comparative genomics of two blaNDM-5 carrying Citrobacter freundii (C. freundii) isolates from a patient with bloodstream and urinary tract infections. Results: Antimicrobial susceptibility testing showed that both blaNDM-5 carrying C. freundii isolates were multidrug-resistant. Positive modified carbapenem inactivation method (mCIM) and EDTA-carbapenem inactivation method (eCIM) results suggested metallo-carbapenemase production. PCR and sequencing confirmed that both metallo-carbapenemase producers were blaNDM-5 positive. Genotyping and comparative genomics analyses revealed that both isolates exhibited a high level of genetic similarity. Plasmid analysis confirmed that the blaNDM-5 resistance gene is located on IncX3 plasmid with a length of 46,161 bp, and could successfully be transferred to the recipient Escherichia coli EC600 strain. A conserved structure sequence (ISAba125-IS5-blaNDM-5-trpF-IS26-umuD-ISKox3) was found in the upstream and downstream of the blaNDM-5 gene. Conclusions: The data presented in this study showed that the conjugative blaNDM-5 plasmid possesses a certain ability to horizontal transfer. The dissemination of NDM-5-producing C. freundii isolates should be of close concern in future clinical surveillance. To our knowledge, this is the first study to characterize C. freundii strains carrying the blaNDM-5 gene from one single patient with multiple infections. [ABSTRACT FROM AUTHOR]

Introduction: The increase in clinical Enterobacteriaceae with dual carbapenemase has become a serious healthcare concern. It is essential to characterize the transferability and potential dissemination of blaKPC-2- and blaNDM-1-coharboring carbapenem-resistant Citrobacter freundii (CRCF). Methods: Four blaKPC-2- and blaNDM-1-coharboring CRCF strains were collected from our surveillance of the prevalence of carbapenem-resistant Enterobacteriaceae. The isolates were assessed using species identification, antimicrobial susceptibility testing, conjugation assays, whole-genome sequencing, plasmid stability, and fitness costs. Clonality, genome, plasmidome, and phylogeny were analyzed to reveal potential dissemination. Results: Three ST523 blaKPC-2- and blaNDM-1-coharboring CRCF strains, collected from the same hospital within 1 month, exhibited high homology (both identity and coverage >99%), implying clonal dissemination and a small-scale outbreak. Moreover, the blaKPC-2 and blaNDM-1 genes were coharbored on an IncR plasmid, probably generated by a blaKPC-2-harboring plasmid acquiring blaNDM-1, in these three strains. Importantly, the IncR plasmid may form a transferable hybrid plasmid, mediated by IS6100 via transposition, with another IncFII plasmid included in the same C. freundii strain. Furthermore, the blaKPC-2 and blaNDM-1 of the fourth CRCF strain are located on two di blaNDM-1-harboring C. freundii were divergent, and these plasmids have high homology to plasmids of other Enterobacteriaceae. Conclusion: Clonal dissemination of ST523 blaKPC-2- and blaNDM-1- coharboring CRCF strains was detected, and we first reported blaKPC-2 and blaNDM-1 concomitantly located on one plasmid, which could be transferred with mediation by IS6100 via transposition. Continued surveillance should urgently be implemented. [ABSTRACT FROM AUTHOR]

N-glycosylation is a post-translational modification of proteins that occurs across all three domains of life. In Archaea, N-glycosylation is crucial for cell stability and motility, but importantly also has significant implications for virus–host interactions. While some archaeal viruses present glycosylated proteins or interact with glycosylated host proteins, the direct influence of N-glycosylation on archaeal virus–host interactions remains to be elucidated. In this study, we generated an N-glycosylation-deficient mutant of Halorubrum lacusprofundi, a halophilic archaeon commonly used to study cold adaptation, and examined the impact of compromised N-glycosylation on the infection dynamics of two very diverse viruses. While compromised N-glycosylation had no influence on the life cycle of the head-tailed virus HRTV-DL1, we observed a significant effect on membrane-containing virus HFPV-1. Both intracellular genome numbers and extracellular virus particle numbers of HFPV-1 were increased in the mutant strain, which we attribute to instability of the surface-layer which builds the protein envelope of the cell. When testing the impact of compromised N-glycosylation on the life cycle of plasmid vesicles, specialized membrane vesicles that transfer a plasmid between host cells, we determined that plasmid vesicle stability is strongly dependent on the host glycosylation machinery. Our study thus provides important insight into the role of N-glycosylation in virus–host interactions in Archaea, while pointing to how this influence strongly differs amongst various viruses and virus-like elements. [ABSTRACT FROM AUTHOR]

Human microbiome engineering is increasingly proposed as a way to modulate health outcomes. However, one of the current limitations to engineering microbial communities in situ is delivery of a genetic payload for introducing or modifying genes. Indeed, there is a need to identify novel broad-host delivery vectors for microbiome engineering. Therefore, in this study, we characterized conjugative plasmids from a publicly available dataset of antibiotic-resistant isolate genomes in order to identify potential broad-host vectors for further applications. From the 199 closed genomes available in the CDC & FDA AR Isolate Bank, we identified 439 plasmids, of which 126 were predicted to be mobilizable and 206 conjugative. Various characteristics of the conjugative plasmids, such as size, replication origin, conjugation machinery, host defense mechanisms, and plasmid stability proteins, were analyzed to determine these plasmids' potential host-range. Following this analysis, we clustered plasmid sequences and chose 22 unique, broad-host range plasmids that would be suitable for use as delivery vectors. This novel set of plasmids will provide a valuable resource for engineering microbial communities. [ABSTRACT FROM AUTHOR]

Plasmids are major drivers of increasing antibiotic resistance, necessitating an urgent need to understand their biology. Here we describe a detailed dissection of the molecular components controlling the genetics of I-complex plasmids, a group of antibiotic resistance plasmids found frequently in pathogenic Escherichia coli and other Enterobacteriaceae that cause significant human disease. We show these plasmids cluster into four distinct subgroups, with the prototype IncI1 plasmid R64 subgroup displaying low nucleotide sequence conservation to other I-complex plasmids. Using pMS7163B, an I-complex plasmid distantly related to R64, we performed a high-resolution transposon-based genetic screen and defined genes involved in replication, stability, and conjugative transfer. We identified the replicon and a partitioning system as essential for replication/stability. Genes required for conjugation included the type IV secretion system, relaxosome, and several uncharacterised genes located in the pMS7163B leading transfer region that exhibited an upstream strand-specific transposon insertion bias. The overexpression of these genes severely impacted host cell growth or reduced fitness during mixed competitive growth, demonstrating that their expression must be controlled to avoid deleterious impacts. These genes were present in >80% of all I-complex plasmids and broadly conserved across multiple plasmid incompatibility groups, implicating an important role in plasmid dissemination. Author summary: Antimicrobial resistance is one of the greatest threats to human health. Left unchecked, we risk a rapid escalation of untreatable infections. Plasmids are one of the most important vehicles for resistance gene carriage and transmission between bacteria, and thus an understanding of plasmid biology is crucial to controlling the spread antimicrobial resistance. Here, we combine advanced bioinformatics and a state-of-the-art genetic screen to understand the molecular mechanisms involved in the maintenance and spread of a group of plasmids strongly associated with antibiotic resistance among bacteria that cause human infection. We characterised genes involved in the replication and maintenance of these plasmids, and experimentally demonstrated that plasmid spread is dependent on a well-conserved secretion system. Our genetic screen also discovered a set of broadly conserved, uncharacterised genes that adversely impact host fitness and plasmid spread under dysregulation. Taken together, these findings describe a molecular blueprint for the biology of a group of clinically relevant antibiotic resistance plasmids found frequently in Gram-negative bacterial pathogens. [ABSTRACT FROM AUTHOR]

Motivation The analysis of bacterial isolates to detect plasmids is important due to their role in the propagation of antimicrobial resistance. In short-read sequence assemblies, both plasmids and bacterial chromosomes are typically split into several contigs of various lengths, making identification of plasmids a challenging problem. In plasmid contig binning, the goal is to distinguish short-read assembly contigs based on their origin into plasmid and chromosomal contigs and subsequently sort plasmid contigs into bins, each bin corresponding to a single plasmid. Previous works on this problem consist of de novo approaches and reference-based approaches. De novo methods rely on contig features such as length, circularity, read coverage, or GC content. Reference-based approaches compare contigs to databases of known plasmids or plasmid markers from finished bacterial genomes. Results Recent developments suggest that leveraging information contained in the assembly graph improves the accuracy of plasmid binning. We present PlasBin-flow, a hybrid method that defines contig bins as subgraphs of the assembly graph. PlasBin-flow identifies such plasmid subgraphs through a mixed integer linear programming model that relies on the concept of network flow to account for sequencing coverage, while also accounting for the presence of plasmid genes and the GC content that often distinguishes plasmids from chromosomes. We demonstrate the performance of PlasBin-flow on a real dataset of bacterial samples. Availability and implementation https://github.com/cchauve/PlasBin-flow. [ABSTRACT FROM AUTHOR]

Q fever is a worldwide zoonosis caused by an obligate intra-cellular pathogen called Coxiella burnetii affecting a broad range of animal hosts including horses. Most of the isolates found carry plasmids which genetic studies of C. burnetii strains suggest a critical role in C. burnetii survival. The correlation between an isolated plasmid type and the chronic or acute nature of the disease has always been controversial. This study was conducted to investigate the prevalence of C. burnetii QpH1 and QpDG plasmids in horses and assess the potential role of these species as reservoirs of infection and transmission. Nested-polymerase chain reaction (PCR) assays were performed on 320 blood serum samples drawn from horses in West Azerbaijan province, Iran, in 2020. In total, 26 (8.13%) Q fever-positive samples based on containing the IS1111 gene were tested by nested-PCR approach to amplify QpH1 and QpDG plasmid segments. The QpH1 and QpRS plasmid-specific sequences were identified in 19 (73.07%) and none in the serum samples, respectively. According to the present study, the age of the animal can be considered as an important risk factor for the prevalence of C. burnetii; but, the season, sex, and breed of the horse had no effect on the prevalence of disease. The results indicate that nested-PCR method could be suitable for routine diagnosis, to gather new information about the shedding of C. burnetii, and to improve the knowledge of contamination routes. [ABSTRACT FROM AUTHOR]

The similarity of the Listeria innocua genome with Listeria monocytogenes and their presence in the same niche may facilitate gene transfer between them. A better understanding of the mechanisms responsible for bacterial virulence requires an indepth knowledge of the genetic characteristics of these bacteria. In this context, draft whole genome sequences were completed on five L. innocua isolated from milk and dairy products in Egypt. The assembled sequences were screened for antimicrobial resistance and virulence genes, plasmid replicons and multilocus sequence types (MLST); phylogenetic analysis of the sequenced isolates was also performed. The sequencing results revealed the presence of only one antimicrobial resistance gene, fosX, in the L. innocua isolates. However, the five isolates carried 13 virulence genes involved in adhesion, invasion, surface protein anchoring, peptidoglycan degradation, intracellular survival, and heat stress; all five lacked the Listeria Pathogenicity Island 1 (LIPI-1) genes. MLST assigned these five isolates into the same sequence type (ST), ST-1085; however, single nucleotide polymorphism (SNP)-based phylogenetic analysis revealed 422-1,091 SNP differences between our isolates and global lineages of L. innocua. The five isolates possessed an ATP-dependent protease (clpL) gene, which mediates heat resistance, on a rep25 type plasmids. Blast analysis of clpL-carrying plasmid contigs showed approximately 99% sequence similarity to the corresponding parts of plasmids of L. monocytogenes strains 2015TE24968 and N1-011A previously isolated from Italy and the United States, respectively. Although this plasmid has been linked to L. monocytogenes that was responsible for a serious outbreak, this is the first report of L. innocua containing clpL-carrying plasmids. Various genetic mechanisms of virulence transfer among Listeria species and other genera could raise the possibility of the evolution of virulent strains of L. innocua. Such strains could challenge processing and preservation protocols and pose health risks from dairy products. Ongoing genomic research is necessary to identify these alarming genetic changes and develop preventive and control measures. [ABSTRACT FROM AUTHOR]

Background: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) attracted extensive attention. Information on CRKP from hospital wastewater (HWW) is limited. The aims of this study were to investigate the genomic characteristics and to evaluate the survivability characteristics of 11 CRKP from HWW in a Chinese teaching hospital in Fujian province. Results: A total of 11 CRKP from HWW were recovered in this study. All CRKP from HWW were resistant to most antibiotics. Comparative genetic analysis demonstrated that all CRKP isolates were clustered into the three distinct phylogenetic clades and clade 2 and clade 3 were mixtures of samples collected from both HWW and clinical settings. Varieties of resistance genes, virulence genes and plasmid replicon types were detected in CRKP from HWW. In vitro transfer of blaKPC-2 was successful for 3 blaKPC-2-positive CRKP from HWW with high conjugation frequency. Our study demonstrated that the genetic environments of blaKPC−2 shared core structure with ISKpn27-blaKPC−2-ISKpn6. Group analysis showed that CRKP from HWW had a lower survivability in serum compared to clinical CRKP (p < 005); and CRKP from HWW had no significant difference in survivability in HWW compared to clinical CRKP (p > 005). Conclusions: We analyzed the genomic and survivability characteristics of CRKP from HWW in a Chinese teaching hospital. These genomes represent a significant addition of genomic data from the genus and could serve as a valuable resource for future genomic studies about CRKP from HWW. [ABSTRACT FROM AUTHOR]

Phage-plasmids are extra-chromosomal elements that act both as plasmids and as phages, whose eco-evolutionary dynamics remain poorly constrained. Here, we show that segregational drift and loss-of-function mutations play key roles in the infection dynamics of a cosmopolitan phage-plasmid, allowing it to create continuous productive infections in a population of marine Roseobacter. Recurrent loss-of-function mutations in the phage repressor that controls prophage induction leads to constitutively lytic phage-plasmids that spread rapidly throughout the population. The entire phage-plasmid genome is packaged into virions, which were horizontally transferred by re-infecting lysogenized cells, leading to an increase in phage-plasmid copy number and to heterozygosity in a phage repressor locus in re-infected cells. However, the uneven distribution of phage-plasmids after cell division (i.e., segregational drift) leads to the production of offspring carrying only the constitutively lytic phage-plasmid, thus restarting the lysis-reinfection-segregation life cycle. Mathematical models and experiments show that these dynamics lead to a continuous productive infection of the bacterial population, in which lytic and lysogenic phage-plasmids coexist. Furthermore, analyses of marine bacterial genome sequences indicate that the plasmid backbone here can carry different phages and disseminates trans-continentally. Our study highlights how the interplay between phage infection and plasmid genetics provides a unique eco-evolutionary strategy for phage-plasmids. Phage-plasmids are bacterial extrachromosomal elements that act both as plasmids and as viruses. Here, Shan et al. show that segregational drift and loss-of-function mutations play key roles in the infection dynamics of a cosmopolitan phage-plasmid, allowing it to create continuous productive infections in marine bacteria. [ABSTRACT FROM AUTHOR]

• Sub-inhibitory concentrations of ampcilin, gentamycin, kanamycin, tetracycline, and naphthalene remarkably promoted plasmid conjugative transfer intra-genera. • Donors had key effects on transconjugants composition. • Stimulants induced a positive effect on conjugal transfer frequency in situ mating matrix, which may be associated with the enhanced expression of conjugative genes. • Blastocatella and Chitinimonas sp. were the main potential receptors of plasmid pND6-2 in activated sludge. The activated sludge process is characterized by high microbial density and diversity, both of which facilitate antibiotic resistance gene transfer. Many studies have suggested that antibiotic and non-antibiotic drugs at sub-inhibitory concentrations are major inducers of conjugative gene transfer. The self-transmissible plasmid pND6-2 is one of the endogenous plasmids harbored in Pseudomonas putida ND6, which can trigger the transfer of another co-occurring naphthalene-degrading plasmid pND6-1. Therefore, to illustrate the potential influence of stimulants on conjugative transfer of pND6-2, we evaluated the effects of four antibiotics (ampicillin, gentamycin, kanamycin, and tetracycline) and naphthalene, on the conjugal transfer efficiency of pND6-2 by filter-mating experiment. Our findings demonstrated that all stimulants within an optimal dose promoted conjugative transfer of pND6-2 from Pseudomonas putida GKND6 to P. putida KT2440, with tetracycline being the most effective (100 µg/L and 10 µg/L), as it enhanced pND6-2-mediated intra-genera transfer by approximately one hundred-fold. Subsequently, seven AS reactors were constructed with the addition of donors and different stimulants to further elucidate the conjugative behavior of pND6-2 in natural environment. The stimulants positively affected the conjugal process of pND6-2, while donors reshaped the host abundance in the sludge. This was likely because stimulant addition enhanced the expression levels of conjugation transfer-related genes. Furthermore, Blastocatella and Chitinimonas were identified as the potential receptors of plasmid pND6-2, which was not affected by donor types. These findings demonstrate the positive role of sub-inhibitory stimulant treatment on pND6-2 conjugal transfer and the function of donors in re-shaping the host spectrum of pND6-2. [Display omitted] [ABSTRACT FROM AUTHOR]

The characteristics of the parABS system in polyploid bacteria are barely understood. We initially analyzed the physiological functions and mechanisms of the megaplasmid parABSm system in the thermophilic polyploid bacterium Thermus thermophilus. Deletion of parABm was possible only when a plasmid-born copy of parABm was provided, indicating that these genes are conditionally essential. The cell morphology of the parABm deletion mutant (ΔparABm) was changed to some extent, and in certain extra-large or twisted cells, the nucleoids were dispersed and damaged. Compared with that of the wild type, the frequency of anucleate cells was significantly increased. Genome content analyses showed that ΔparABm had lost ∼160 kb of megaplasmid and ∼23 kb of chromosomal sequences, respectively. Genome fluorescent tagging and PFGE experiments demonstrated that the truncated megaplasmid was frequently interlinked and could not be segregated correctly; thus, certain daughter cells eventually lost the entire megaplasmid and became twisted or enlarged with damaged nucleoids. Further, we found that when the megaplasmid was lost in these cells, the toxins encoded by the megaplasmid toxin-antitoxin (TA) systems (VapBC64_65 and VapBC142_143) would exert detrimental effects, such as to fragment DNA. Thus, parABSm might ensure the existence of these TA systems, thereby preventing genomic degradation. Together, our results suggested that in T. thermophilus, the megaplasmid-encoded parABS system plays an essential role in the megaplasmid partitioning process; also it is an important determination factor for the genome integrity maintenance. [ABSTRACT FROM AUTHOR]

Plasmids are major vectors of bacterial antibiotic resistance, but understanding of factors associated with plasmid antibiotic resistance gene (ARG) carriage is limited. We curated > 14,000 publicly available plasmid genomes and associated metadata. Duplicate and replicate plasmids were excluded; where possible, sample metadata was validated externally (BacDive database). Using Generalised Additive Models (GAMs) we assessed the influence of 12 biotic/abiotic factors (e.g. plasmid genetic factors, isolation source, collection date) on ARG carriage, modelled as a binary outcome. Separate GAMs were built for 10 major ARG types. Multivariable analysis indicated that plasmid ARG carriage patterns across time (collection years), isolation sources (human/livestock) and host bacterial taxa were consistent with antibiotic selection pressure as a driver of plasmid-mediated antibiotic resistance. Only 0.42% livestock plasmids carried carbapenem resistance (compared with 12% human plasmids); conversely, tetracycline resistance was enriched in livestock vs human plasmids, reflecting known prescribing practices. Interpreting results using a timeline of ARG type acquisition (determined by literature review) yielded additional novel insights. More recently acquired ARG types (e.g. colistin and carbapenem) showed increases in plasmid carriage during the date range analysed (1994–2019), potentially reflecting recent onset of selection pressure; they also co-occurred less commonly with ARGs of other types, and virulence genes. Overall, this suggests that following acquisition, plasmid ARGs tend to accumulate under antibiotic selection pressure and co-associate with other adaptive genes (other ARG types, virulence genes), potentially re-enforcing plasmid ARG carriage through co-selection. [ABSTRACT FROM AUTHOR]

The acquisition of blaOXA genes encoding different carbapenem-hydrolyzing class-D β-lactamases (CHDL) represents a main determinant of carbapenem resistance in the nosocomial pathogen Acinetobacter baumannii. The blaOXA-58 gene, in particular, is generally embedded in similar resistance modules (RM) carried by plasmids unique to the Acinetobacter genus lacking self-transferability. The ample variations in the immediate genomic contexts in which blaOXA-58-containing RMs are inserted among these plasmids, and the almost invariable presence at their borders of non-identical 28-bp sequences potentially recognized by the host XerC and XerD tyrosine recombinases (pXerC/D-like sites), suggested an involvement of these sites in the lateral mobilization of the gene structures they encircle. However, whether and how these pXerC/D sites participate in this process is only beginning to be understood. Here, we used a series of experimental approaches to analyze the contribution of pXerC/D-mediated site-specific recombination to the generation of structural diversity between resistance plasmids carrying pXerC/D-bounded blaOXA-58- and TnaphA6-containing RM harbored by two phylogenetically- and epidemiologically-closely related A. baumannii strains of our collection, Ab242 and Ab825, during adaptation to the hospital environment. Our analysis disclosed the existence of different bona fide pairs of recombinationally-active pXerC/D sites in these plasmids, some mediating reversible intramolecular inversions and others reversible plasmid fusions/resolutions. All of the identified recombinationally-active pairs shared identical GGTGTA sequences at the cr spacer separating the XerC- and XerD-binding regions. The fusion of two Ab825 plasmids mediated by a pair of recombinationally-active pXerC/D sites displaying sequence differences at the cr spacer could be inferred on the basis of sequence comparison analysis, but no evidence of reversibility could be obtained in this case. The reversible plasmid genome rearrangements mediated by recombinationally-active pairs of pXerC/D sites reported here probably represents an ancient mechanism of generating structural diversity in the Acinetobacter plasmid pool. This recursive process could facilitate a rapid adaptation of an eventual bacterial host to changing environments, and has certainly contributed to the evolution of Acinetobacter plasmids and the capture and dissemination of blaOXA-58 genes among Acinetobacter and non-Acinetobacter populations co-residing in the hospital niche. [ABSTRACT FROM AUTHOR]

Introduction: The antimicrobial resistance (AMR) mobilome plays a key role in the dissemination of resistance genes encoded by mobile genetics elements (MGEs) including plasmids, transposons (Tns), and insertion sequences (ISs). These MGEs contribute to the dissemination of multidrug resistance (MDR) in enteric bacterial pathogens which have been considered as a global public health risk. Methods: To further understand the diversity and distribution of AMR genes and MGEs across different plasmid types, we utilized multiple sequence-based computational approaches to evaluate AMR-associated plasmid genetics. A collection of 1,309 complete plasmid sequences from Gammaproteobacterial species, including 100 plasmids from each of the following 14 incompatibility (Inc) types: A/C, BO, FIA, FIB, FIC, FIIA, HI1, HI2, I1, K, M, N, P except W, where only 9 sequences were available, was extracted from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST tools. The extracted FASTA files were analyzed using the AMRFinderPlus web-based tools to detect antimicrobial, disinfectant, biocide, and heavy metal resistance genes and ISFinder to identify IS/Tn MGEs within the plasmid sequences. Results and Discussion: In silico prediction based on plasmid replicon types showed that the resistance genes were diverse among plasmids, yet multiple genes were widely distributed across the plasmids from enteric bacterial species. These findings provide insights into the diversity of resistance genes and that MGEs mediate potential transmission of these genes across multiple plasmid replicon types. This notion was supported by the observation that many IS/Tn MGEs and resistance genes known to be associated with them were common across multiple different plasmid types. Our results provide critical insights about how the diverse population of resistance genes that are carried by the different plasmid types can allow for the dissemination of AMR across enteric bacteria. The results also highlight the value of computational-based approaches and in silico analyses for the assessment of AMR and MGEs, which are important elements of molecular epidemiology and public health outcomes. [ABSTRACT FROM AUTHOR]

Plasmids are extra‐chromosomal genetic elements that encode a wide variety of phenotypes and can be maintained in bacterial populations through vertical and horizontal transmission, thus increasing bacterial adaptation to hostile environmental conditions like those imposed by antimicrobial substances. To circumvent the segregational instability resulting from randomly distributing plasmids between daughter cells upon division, nontransmissible plasmids tend to be carried in multiple copies per cell, with the added benefit of exhibiting increased gene dosage and resistance levels. But carrying multiple copies also results in a high metabolic burden to the bacterial host, therefore reducing the overall fitness of the population. This trade‐off poses an existential question for plasmids: What is the optimal plasmid copy number? In this manuscript, we address this question by postulating and analyzing a population genetics model to evaluate the interaction between selective pressure, the number of plasmid copies carried by each cell, and the metabolic burden associated with plasmid bearing in the absence of selection for plasmid‐encoded traits. Parameter values of the model were estimated experimentally using Escherichia coli K12 carrying a multicopy plasmid encoding for a fluorescent protein and blaTEM‐1, a gene conferring resistance to β‐lactam antibiotics. By numerically determining the optimal plasmid copy number for constant and fluctuating selection regimes, we show that plasmid copy number is a highly optimized evolutionary trait that depends on the rate of environmental fluctuation and balances the benefit between increased stability in the absence of selection with the burden associated with carrying multiple copies of the plasmid. [ABSTRACT FROM AUTHOR]

Several species from the Alicyclobacillus genus have received much attention from the food and beverages industries. Their presence has been co-related with spoilage events of acidic food matrices, namely fruit juices and other fruit-based products, the majority attributed to Alicyclobacillus acidoterrestris. In this work, a combination of short and long reads enabled the assembly of the complete genome of A. acidoterrestris DSM 3922T, perfecting the draft genome already available (AURB00000000), and revealing the presence of one chromosome (4,222,202bp; GC content 52.3%) as well as one plasmid (124,737 bp; GC content 46.6%). From the 4,288 genes identified, 4,004 sequences were attributed to coding sequences with proteins, with more than 80% being functionally annotated. This allowed the identification of metabolic pathways and networks and the interpretation of high-level functions with significant reliability. Furthermore, the additional genes of interest related to spore germination, off-flavor production, namely the vdc cluster, and CRISPR arrays, were identified. More importantly, this is the first complete and closed genome sequence for a taint-producing Alicyclobacillus species and thus represents a valuable reference for further comparative and functional genomic studies. [ABSTRACT FROM AUTHOR]

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A strain of Clostridium perfringens was isolated from the bile sample of a patient with emphysematous cholecystitis who underwent a laparoscopic cholecystectomy, followed by treatment with meropenem and recovery. Metagenomic analysis of the bile sample showed that 99.73% of the bile microbiota consisted of C. perfringens, indicating that C. perfringens JUM001 was the causative pathogen of acute emphysematous cholecystitis in this patient. Complete genome sequencing showed that C. perfringens JUM001 contained a circular chromosome of 3,231,023 bp and two circular plasmids, pJUM001-1 of 49,289 bp and pJUM001-2 of 47,855 bp. JUM001 was found to possess a typing toxin gene, plc, but no other typing toxin genes, indicating that its toxinotype is type A. The plasmids pJUM001-1 and pJUM001-2 belonged to the pCP13-like and pCW3-like families of plasmids, respectively, which are characteristic conjugative and archetypical plasmids of C. perfringens. Phylogenetic analysis showed that JUM001 was closely related to C. perfringens strain JXNC-DD isolated from a dog in China. To our knowledge, this is the first report of whole-genome sequences of a clinical isolate of C. perfringens causing acute emphysematous cholecystitis. [ABSTRACT FROM AUTHOR]

Avian pathogenic Escherichia coli (APEC) is recognized as a primary source of foodborne extraintestinal pathogenic E. coli (ExPEC), which poses a significant risk of extraintestinal infections in humans. The potential of human infection with ST117 lineage APEC/ExPEC from poultry is particularly concerning. However, relatively few whole‐genome studies have focused on ST117 as an emerging ExPEC lineage. In this study, the complete genomes of 11 avian ST117 isolates and the draft genomes of 20 ST117 isolates in China were sequenced to reveal the genomic islands and large plasmid composition of ST117 APEC. With reference to the extensive E. coli genomes available in public databases, large‐scale comprehensive genomic analysis of the ST117 lineage APEC/ExPEC was performed to reveal the features of the ST117 pan‐genome and population. The high variability of the accessory genome emphasized the diversity and dynamic traits of the ST117 pan‐genome. ST117 isolates recovered from different hosts and geographic sources were randomly located on a phylogeny tree, suggesting that ST117 E. coli lacked host specificity. A time‐scaled phylogeny tree showed that ST117 was a recent E. coli lineage with a relatively short evolutionary period. Further characterization of a wide diversity of ExPEC‐related virulence genes, pathogenicity islands (PAIs), and resistance genes of the ST117 pan‐genome provided insights into the virulence and resistance of ST117 APEC/ExPEC. The results suggested zoonotic potential of ST117 APEC/ExPEC between birds and humans. Moreover, genomic analysis showed that a pool of diverse plasmids drove the virulence and multidrug resistance of ST117 APEC/ExPEC. Several types of large plasmids were scattered across the ST117 isolates, but there was no strong plasmid‐clade adaptation. Combined with the pan‐genome analysis, a double polymerase chain reaction (PCR) method was designed for rapid and cost‐effective detection of ST117 isolates from various avian and human APEC/ExPEC isolates. Overall, this study addressed a gap in current knowledge about the ST117 APEC/ExPEC genome, with significant implications to understand the success and spread of ST117 APEC/ExPEC. [ABSTRACT FROM AUTHOR]

Worldwide dissemination of extended-spectrum -lactamase (ESBL)-producing Escherichia coli constitutes an emerging global health issue, with animal food products contributing as potential reservoirs. ESBL E. coli infection is associated with the high mortality and mobility rate in developing countries due to less susceptibility to antibiotics. The present study aimed to elucidate the molecular characteristics and sequence-based analysis of ESBL E. coli in the Gujarat state of India. This study included 108 E. coli strains were isolated from different poultry farms (broiler and layer) in the Banaskantha District. PCR was employed to identify genotypic ESBL-producing antimicrobial resistance genes. Overall, a high occurrence of ESBL genes was found in poultry farms due to the high usage of antimicrobials. The PCR analysis revealed that 79.62% of isolates were detected positive with one or more ESBL genes. Among them, blaTEM (63.88%) was found to be the predominant genotype, followed by blaSHV (30.55%) and blaOXA (28.70%). In the blaCTX-M group, a higher occurrence was observed in blaCTX-M-9 (23.14%), followed by blaCTX-M-2 (24.07%) and blaCTX-M-1 (22.22%). We used the whole-genome sequencing (WGS) method to evaluate the antimicrobial resistance genes, virulence factors, single nucleotide polymorphisms (SNPs), plasmid replicons, and plasmid-mediated AMR genes of one ESBL E. coli isolated. We examined the genetic relatedness of a human pathogenic E. coli strain by comparing its sequence with the broad geographical reference E. coli sequences. Escherichia coli ST 681 was determined using multi-locus sequence typing. We compared our findings to the reference sequence of Escherichia coli str. K- 12 substr. MG1655. We found 24,937 SNPs with 21,792 in the genic region, 3,145 in the intergenic region, and six InDels across the genome. The WGS analysis revealed 46 antimicrobial resistance genes and seven plasmid-mediated AMR genes viz., tetA, qnrS1, dfrA14, sul2, aph(3")-lb, aph(6)-ld, and Aph(3')-la. The ST 681 was found to have Cib, traT, and terC virulence factors and two plasmid replicons, IncFII(pHN7A8) and IncI1-I(Alpha). This study revealed a higher occurrence of ESBL E. coli detected in poultry. [ABSTRACT FROM AUTHOR]

The appearance of transferable oxazolidinone resistance genes poses a major challenge to public health and environmental safety. These genes not only lead pathogenic bacteria to become resistant to linezolid but also reduce sensitivity to florfenicol, which is widely used in the veterinary field. To verify the dissemination of oxazolidinone resistance genes in enterococcal isolates from pigs at different production stages in a swine farm in China, we collected 355 enterococcal isolates that were resistant to florfenicol from 600 (150 per stage) fresh fecal swabs collected from a swine farm. Through initial PCR screening and whole-genome sequencing, 175 isolates harboring different oxazolidinone resistance genes were identified. All isolates carried the optrA gene. A total of 161 (92%, 161/175) isolates carried only the optrA gene. Three (1.71%, 3/175) isolates carried both the optrA and poxtA genes, and 11 (3.1%, 11/175) isolates contained the optrA gene and poxtA2 and cfr(D) variants. A total of 175 isolates that harbored oxazolidinone resistance genes included 161 E. faecalis, 6 E. faecium, and 8 E. hirae. By sequencing the whole genomes, we found that the 161 isolates of E. faecalis belonged to 28 different STs, including 8 new STs, and the 6 isolates of E. faecium belonged to four different STs, including one new ST. The phylogenetic tree based on SNPs of the core genome showed that both clonal spread and horizontal transfer mediated the diffusion of oxazolidone resistance genes in enterococcal isolates at specific stages in pig farms. Moreover, enterococcal isolates carrying oxazolidone resistance genes could spread from breeding pigs to fattening pigs, while transferable oxazolidone resistance genes in enterococcal isolates could persist on a pig farm throughout all production stages. Representative enterococcal isolates with different oxazolidinone resistance genes were further studied through Nanopore sequencing. We identified a novel plasmid, pM4-80L4 (15,008bp), carrying the poxtA2 and cfr(D) genes in enterococcal isolates at different stages. We also found three different plasmids harboring the poxtA gene with high genetic variation, and all poxtA genes were flanked by two copies of IS1216E elements. In addition, four genetically distinct plasmids carrying the optrA gene were identified, and Tn554 was found to mediate chromosome-localized optrA gene transfer. Our study highlighted that transferable oxazolidinone resistance genes in enterococcal isolates could persist throughout all production stages on a pig farm, and the prevalence and dissemination of oxazolidinone resistance genes in enterococcal isolates from animal farms should be continually monitored. [ABSTRACT FROM AUTHOR]

The Splendidus clade is the largest clade in Vibrionaceae, and its members are often related to mortality of marine animals with huge economic losses. The molecular bases of their pathogenicity and virulence, however, remain largely unknown. In particular, the complete genome sequences of the Splendidus clade species are rarely registered, which is one of the obstacles to predict core and/or unique genes responsible for their adaptation and pathogenicity, and to perform a fine scale meta‐transcriptome during bacterial infection to their hosts. In this study, we obtained the complete genomes of all type strains in the Splendidus clade and revealed that (1) different genome sizes (4.4–5.9 Mb) with V. lentus the biggest and most of them had several big plasmids, likely because of the different features on mobilome elements; (2) the Splendidus clade consists of 19 species except V. cortegadensis, and 3 sub‐clades (SC) were defined with the 15 most closely related members as SC1; (3) different carbohydrate degradation preferences may be the result of environmental adaptation; and (4) a broad prediction of virulence factors (VFs) revealed core and species unique VF genes. [ABSTRACT FROM AUTHOR]

Plastic pollution and antibiotic resistance are two emerging environmental and human health crises today. Although it was revealed that microplastics can serve as vectors for the dissemination of antibiotic resistance, it is still unclear how the nanoplastics influence the horizontal transfer of antibiotic resistance genes (ARGs). Herein, we firstly compared the effect of polystyrene (PS) micro/nanoplastics on the transformation of plasmid‐borne ARG, using a transformation model consisting of plasmid pUC19 (ampR) and Escherichia coli DH5α (recipient). Due to its size effect, PS nanoplastics (10–500 mg/L) significantly enhanced the transformation efficiency (2.8–5.4 folds) and frequency (3.2–8.4 folds) of exogenous ampR into E. coli, while PS microplastics exerted no influence. The detailed mechanisms were found that nanoplastics induced reactive oxygen species (ROS) overproduction, activated SOS response, increased cell membrane permeability and changed the secretion systems, thereby facilitating the uptake of exogenous DNA by bacteria. Moreover, the co‐presences of nanoplastics with humic acid or Fe3+ relieved to some extent, but did not completely alleviate the promoting effect of nanoplastics on plasmid transformation. Our findings suggest that the risk of nanoplastics on promoting the dissemination of antibiotic resistance should not be neglected, and further studies are needed to investigate such risk in complex environments. [ABSTRACT FROM AUTHOR]

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: To characterize one OXA-232-producing wzi93-KL112-O1 carbapenem-resistant Klebsiella pneumoniae (CRKP) co-harboring chromosomal blaCTX-M-15 and one rmpA2-associated virulence plasmid. Methods: Minimum inhibitory concentrations (MICs) were measured via broth microdilution method. Conjugation, chemical transformation, string test and Galleria mellonella infection model experiments were also conducted. Wholegenome sequencing (WGS) was performed on the Illumina and Nanopore platforms. Antimicrobial resistance determinants were identified using ABRicate program with ResFinder database. Insertion sequences (ISs) were identified using ISfinder. Bacterial virulence factors were identified using virulence factor database (VFDB). Wzi, capsular polysaccharide (KL) and lipoolygosaccharide (OCL) were analyzed using Kleborate with Kaptive. Phylogenetic analysis of 109 ST15 K. pneumoniae strains was performed using core genome multilocus sequence typing (cgMLST) on the Ridom SeqSphere+ server. MLST, replicons type, SNP strategies and another cgMLST analysis for 45 OXA-232-producing K. pneumoniae strains were further conducted using BacWGSTdb server. Results: K. pneumoniae KPTCM strain belongs to ST15 with wzi93, KL112 and O1. It possessed a multidrug-resistant (MDR) profile and was resistant to carbapenems (meropenem and ertapenem), ciprofloxacin and amikacin. Virulence assays demonstrated KPTCM strain possesses a low virulence phenotype. WGS revealed it contained one circular chromosome and nine plasmids. The carbapenemase-encoding gene blaOXA-232 was located in a 6141-bp ColKP3-type non-conjugative plasmid and flanked by DISEcp1 and DlysR-DereA. Interestingly, blaCTX-M-15 was located in the chromosome mediated by ISEcp1-based transposon Tn2012. Importantly, it harbored a rmpA2-associated pLVPK-like virulence plasmid with iutA-iucABCD gene cluster and one IS26-mediated MDR fusion plasmid according to 8-bp (AGCTGCAC or GGCCTTTG) target site duplications (TSD). Based on the cgMLST and SNP analysis, data showed OXA-232-producing ST15 K. pneumoniae isolates were mainly isolated from China and have evolved in recent years. Conclusions: Early detection of CRKP strains carrying chromosomal blaCTX-M-15, OXA-232 carbapenemase and pLVPK-like virulence plasmid is recommended to avoid the extensive spread of this high-risk clone. [ABSTRACT FROM AUTHOR]

Foodborne diseases and infection caused by associated pathogens is a public health concern. Majority of the investigations focus on common foodborne pathogens like Vibrio parahaemolyticus, Escherichia coli, Listeria monocytogenes, Shigella, Salmonella and Staphylococcus aureus. Limited knowledge has been accounted on Klebsiella pneumoniae. Presence of multidrug-resistant K. pneumoniae in the food supply is disturbing. Hence, this study assessed the presence of K. pneumoniae isolates from food samples (fresh vegetables and chicken), ascertained the presence of drug-resistant phenotypes, extended spectrum beta lactamase production, antibiotic resistance determinants, genes associated with virulence and their ability to form biofilm. Resistance towards ceftazidime and tetracycline was noted among all the isolates in the study, while they exhibited sensitivity to chloramphenicol and cotrimoxazole. All the isolates were potent ES BL producers carrying at least one ES BL encoding genes. Plasmid mediated quinolone resistance gene was detected in one isolate each from onion and chicken respectively. The isolates marked the absence of tetracycline and chloramphenicol resistance genes. Multiple virulence genes (ureA, khe, fimH, mrkD, wabG, uge and elt) were possessed by each of the isolates. K. pneumoniae from chicken and cucumber were moderate biofilm formers and those from tomato exhibited weak biofilm formation. Increased expression of the mrkA gene and reduction in the expression of the biofilm forming gene fimH gene was observed among the biofilm formers. One of the moderate and non-biofilm formers exhibited increased mrkD gene expression. The results from our study stipulate, that raw vegetables and meat serve as dormant source of drug-resistant and virulent K. pneumoniae. [ABSTRACT FROM AUTHOR]

The RA3 plasmid, the archetype of IncU incompatibility group, represents a mosaic-modular genome of 45.9 kb. The replication module encompasses repA and repB (initiator) surrounded by two long repetitive sequences DR1 and DR2 of unknown function. Here, we mapped the origin of replication oriV to the 3′ end of repB and showed that oriV was activated by the transcription coming from orf02revp in the adjacent stability module. Using various in vivo and in vitro methods we demonstrated that the repB expression proceeded either from repBp located in the intergenic repA-repB region or from the upstream strong repAp that was autoregulated by RepA. Additionally, the repBp activity was modulated by the transcription from the overlapping, divergently oriented repXp. Both repXmRNA (antisense for repAmRNA) and its small polypeptide product, RepX, were strong incompatibility determinants. Hence, we showed that the sophisticated RA3 copy number control combined the multivalent regulation of repB expression, RepB titration by DR1, and transcriptional activation of oriV, dependent on the RA3 global regulatory network. Similarly organized replicons have been found in diverse bacterial species confirming the significance of these mechanisms in establishing the IncU plasmids in a broad spectrum of hosts. [ABSTRACT FROM AUTHOR]

Bacterial infections with the genus Enterobacter are notoriously difficult to treat and often associated with resistance to penicillin, aminoglycosides, fluoroquinolones, and third-generation cephalosporins. Also, Enterobacter species have emerged as the third most common hosts for carbapenemases worldwide, forcing the use of colistin as a "last-resort" antibiotic for the treatment. Studies on the population structure of the genus Enterobacter repeatedly detect E. xiangfangensis as a common clinical species present worldwide. Here, we report on the characteristics of an extreme drugresistant E. xiangfangensis isolate va18651 (ST88), obtained from a cervical swab of an expectant mother. The isolate was resistant to almost all the classes of antibiotics tested, including β-lactams (viz., penicillins, carbapenems, cephalosporin, monobactams, and their combinations), quinolone, aminoglycosides, and sulfonamide/dihydrofolate reductase inhibitor, and exhibited heteroresistance towards colistin. Analysis of its complete genome sequence revealed 37 antibiotic resistance genes (ARGs), including mcr-9.1, blaKPC-2, and blaOXA-48, encoded on three of the four different plasmids (cumulative plasmidome size 604,632 bp). An unusually high number of plasmid-based heavy metal resistance gene (HRG) clusters towards silver, arsenate, cadmium, copper, mercury, and tellurite were also detected. Virulence genes (VGs) for the lipopolysaccharide and capsular polysaccharide structures, iron acquisition (iroBCDEN, ent/fep/fes, sitABCD, iut, and fur), and a type VI secretion system, together with motility genes and Type IV pili, were encoded chromosomally. Thus, a unique combination of chromosomally encoded VGs, together with plasmid-encoded ARGs and HRGs, converged to result in an extreme drug-resistant, pathogenic isolate with survival potential in environmental settings. The use of a disinfectant, octenidine, led to its eradication; however, the existence of a highly antibiotic-resistant isolate with significant virulence potential is a matter of concern in public health settings and warrants further surveillance for extreme drug-resistant Enterobacter isolates. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the prevalence and characterization of tet(X4) in Escherichia coli isolates from a pig farm in Shanghai, China, and to elucidate tet(X4) dissemination mechanism in this swine farm. Forty-nine (80.33%) E. coli strains were isolated from 61 samples from a pig farm and were screened for the presence of tet(X). Among them, six (12.24%) strains were positive for tet(X4) and exhibited resistance to tigecycline (MIC = 16 mg/L). They were further sequenced by Illumina Hiseq. Six tet(X4)-positive strains belonged to ST761 with identical resistance genes, resistance profiles, plasmid replicons, and cgMLST type except that additional ColE10 plasmid was present in isolate SH21PTE35. Isolate SH21PTE31, as a representative ST761 E. coli strain, was further sequenced using Nanopore MinION. The tet(X4) in SH21PTE31 was located on IncFIA18/IncFIB(K)/IncX1 hybrid plasmid pYUSHP31-1, highly similar to other tet(X4)-carrying IncFIA18/IncFIB(K)/IncX1 plasmids from ST761 E. coli and other E. coli lineages in China. These IncFIA18/IncFIB(K)/IncX1 plasmids shared closely related multidrug resistance regions, and could reorganize, acquire or lose resistance modules mediated by mobile elements such as ISCR2 and IS26. Phylogenetic analysis were performed including all tet(X4)-positive isolates obtained in this pig farm combined with 43 tet(X4)-positive E. coli from pigs, cow, pork, wastewater, and patients with the same ST from NCBI. The 50 tet(X4)-carrying E. coli ST761 isolates from different areas in China shared a close phylogenetic relationship (0-49 SNPs). In conclusion, clonal transmission of tet(X4)-positive E. coli ST761 has occurred in this swine farm. E. coli ST761 has the potential to become a high-risk clone for tet(X4) dissemination in China. [ABSTRACT FROM AUTHOR]

Comparative mitogenomics of Ulva species have revealed remarkable variations in genome size due to the integration of exogenous DNA fragments, the proliferation of group I/II introns, and the change of repeat sequences. The genus Ulva is a species-rich taxonomic group, containing a variety of green-tide forming algae. In this study, five complete mitogenomes of the green-tide forming macroalga, Ulva meridionalis R. Horimoto and S. Shimada, were assembled and compared with the available ulvophyceae mtDNAs. The main circular mitogenomes of U. meridionalis ranged from 82.94 to 111.49 kb in size, and its 111.49-kb mitogenome was the largest Ulva mitogenome sequenced so far. The expansion of U. meridionalis mitogenomes is mainly due to the tandem integration of a 5.36-kb mitochondrial circular plasmid (pUme), as well as the proliferation of introns. An intact DNA-directed RNA polymerase gene (rpo) was present in pUme of U. meridionalis and was then detected in two putative plasmids (pUmu1 and pUmu2) found in Ulva mutabilis. The observed integration of the circular plasmid into U. meridionalis mitogenomes seems to occur via homologous recombination, and is a more recent evolutionary event. Many highly homologous sequences of these three putative plasmids can be detected in the other Ulva mtDNAs sequenced thus far, indicating the integration of different mitochondrial plasmid DNA into the mitogenomes is a common phenomenon in the evolution of Ulva mitogenomes. The random incidence of destruction of plasmidderived rpos and open reading frames (orfs) suggests that their existence is not the original characteristic of Ulva mitogenomes and there is no selective pressure to maintain their integrity. The frequent integration and rapid divergence of plasmid-derived sequences is one of the most important evolutionary forces to shape the diversity of Ulva mitogenomes. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the prevalence and diversity of extendedspectrum β-lactamases (ESBL)-producing Escherichia coli isolates from healthy individuals in a community and to elucidate their dissemination mechanism. Cefotaxime-resistant E. coli were isolated from 95 samples of healthy persons from one community in Yangzhou, China, and were tested for minimal inhibitory concentrations of 14 antimicrobial agents. The isolates were subjected to whole genome sequencing by Illumina Hiseq or PacBio singlemolecule real-time sequencing. A total of 30 cefotaxime-resistant E. coli isolates were obtained, carrying blaCTX-M (n=29) or blaDHA (n=1), of which the blaCTX-M-55 (n=19) was the most predominant genotype. One novel blaCTX-M variant blaCTX-M-252 was identified. Thirteen CTX-M-55-producing E. coli isolates belonged to ST8369 from nasal (n=12) or faecal (n=1) samples shared the identical cgMLST type, resistance profiles, resistance genes, plasmid replicons, and a 5,053-bp blaCTX-M-55 structure DIS26-DISEcp1-blaCTX-M-55-Dorf477-DTn2. The blaCTX-M-55 gene was located on IncHI2/ST3 plasmid in E. coli ST8369. The lengths of blaCTX-M/blaDHA-carrying contigs in the remaining 17 E. coli strains ranged from 1,663 to 382,836 bp, located on chromosome (n=4) or plasmids (n=5); the location of the other eight contigs could not be determined due to incomplete assembly. The blaCTX-M was associated with ISEcp1 as previously reported. Nasal colonization of CTX-M-55-producing ST8369 E. coli strains has occurred among healthy individuals in one community. There is a potential risk of antimicrobial resistance dissemination between humans within one community through close contact or environment via aerosols or dust. Therefore, surveillance of nasal carriage of blaCTX-M in communities is warranted to further monitor the spread of the antimicrobial resistance genes in China. [ABSTRACT FROM AUTHOR]

Necrotoxigenic Escherichia coli 2 (NTEC2) are defined as E. coli producing the toxin known as cytotoxic necrotizing factor 2 (CNF2), a potent toxin primarily found in bovine but also in humans. NTEC2 are mostly associated with bovine, and cnf2 is known to be carried by pVir‐like plasmids. In this study, we looked for NTEC2 in a collection of E. coli collected between 2011 and 2018 in French bovine. Thirty‐two isolates, collected from both sick (n = 19) and healthy (n = 13) animals, were identified and characterized using whole‐genome sequencing. One F74 plasmid of this bacterial collection was long‐read sequenced: its size was 138 121 bp and it carried the cnf2, F17cA‐eG, cdtB, iutA, iucC and ompP virulence factors (VFs), but no resistance gene. A large variety of genetic backgrounds was observed, but all cnf2‐carrying plasmids belonged to the IncF family, and most of them (78·1%) were of the F74 group. Similar F74 plasmids were also reported from bovine in the United Kingdom and the United States, as identified in the publically available databases. Consequently, these F74 plasmids, which are widely disseminated among E. coli from cattle in the French territory, are vectors of virulence determinants that largely went unnoticed until now. [ABSTRACT FROM AUTHOR]

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Focusing on resistance trends and transmission patterns of pathogenic microorganisms is a major priority for national surveillance programs. The use of whole-genome sequencing for antimicrobial susceptibility testing (WGS-AST) is a powerful alternative to traditional microbiology laboratory methods. Yersinia enterocolitica antimicrobial resistance (AMR) in the Ningxia Hui Autonomous Region has yet to be described thoroughly in current studies. We assessed and monitored the development of Y. enterocolitica AMR in the Ningxia Hui Autonomous Region during 2007-2019 based on WGS-AST. Resistance genotypes were predicted based on WGS. Antimicrobial resistance testing using classical microbiology determined resistance to 13 antimicrobial agents in 189 Y. enterocolitica isolates from Ningxia. The highest resistance level was 97.88% for cefazolin, followed by ampicillin (AMP) (44.97%), ciprofloxacin (CIP) (25.40%), streptomycin (STR) (11.11%), and tetracycline (TET) (10.58%). Isolates emerged as chloramphenicol (CHL) and trimethoprim/sulfamethoxazole (SXT) resistant. The primary plasmid types were IncFII(Y) and ColRNAI. The TET, STR, and SXT resistance were mediated by the tetA, aph(6)-Id, aph(30 0)-Ib, and sul2 genes located on the IncQ1 plasmid. The resistant strains were predominantly biotype 4/O:3/ST429 and the hosts were pigs and patients. The number of multidrug-resistant (MDR) strains was of concern, at 27.51%. At present, the prediction of antimicrobial resistance based on WGS requires a combination of phenotypes. From 2007 to 2019, Y. enterocolitica isolates from the Ningxia Hui Autonomous Region showed a relatively high rate of resistance to cefazolin (CZO) and some resistance to AMP, CIP, STR, and TET. CIP, SXT, and TET showed a relatively clear trend of increasing resistance. Plasmids carrying multiple drug resistance genes are an important mechanism for the spread of antimicrobial resistance. Isolates with low pathogenicity were more likely to present an AMR phenotype than non-pathogenic isolates. [ABSTRACT FROM AUTHOR]

Wild birds, particularly silver gulls (Chroicocephalus novaehollandiae) that nest near anthropogenic sites, often harbour bacteria resistant to multiple antibiotics, including those considered of clinical importance. Here, we describe the whole genome sequence of Escherichia coli isolate CE1867 from a silver gull chick sampled in 2012 that hosted an I1 pST25 plasmid with blaSHV-12, a β-lactamase gene that encodes the ability to hydrolyze oxyimino β-lactams, and other antibiotic resistance genes. Isolate CE1867 is an ST297 isolate, a phylogroup B1 lineage, and clustered with a large ST297 O130:H11 clade, which carry Shiga toxin genes. The I1 plasmid belongs to plasmid sequence type 25 and is notable for its carriage of an atypical sul3-class 1 integron with mefB∆260, a structure most frequently reported in Australia from swine. This integron is a typical example of a Tn21-derived element that captured sul3 in place of the standard sul1 structure. Interestingly, the mercury resistance (mer) module of Tn21 is missing and has been replaced with Tn2-blaTEM-1 and a blaSHV-12 encoding module flanked by direct copies of IS26. Comparisons to similar plasmids, however, demonstrate a closely related family of ARG-carrying plasmids that all host variants of the sul3-associated integron with conserved Tn21 insertion points and a variable presence of both mer and mefB truncations, but predominantly mefB∆260. [ABSTRACT FROM AUTHOR]

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an important pathogen causing hospital-associated outbreaks worldwide. The spread of K. pneumoniae carbapenemase-2 (KPC-2)-producing CRKP is primarily associated with sequence type (ST) 11. Methods: A total of 152 KPC-2-producing K. pneumoniae ST11 isolates were collected from the respiratory department of a tertiary care hospital in Beijing, China between 2009 and 2018. The genome sequencing of these isolates was performed on the HiSeq X Ten sequencer. Multilocus sequence typing (MLST), capsular type, plasmid replicon types and resistance genes were identified. Fifteen isolates were selected for the subsequent single-molecule real-time (SMRT) sequencing on the PacBio RS II. Alignment of the complete sequences of the plasmids carrying bla KPC–2 and/or virulence genes was performed by using BRIG and Easyfig. Results: From 2012 to 2018, the detection rate of the bla KPC–2-carrying CRKP rose rapidly from 3.3 to 28.1%. KPC-2-producing K. pneumoniae ST11 isolates were dominant in CRKP, which emerged in 2012 and caused several outbreaks. Most isolates exhibited multidrug-resistant to commonly used antibiotics, while all the isolates remained susceptible to tigecycline and polymyxin B. The single nucleotide polymorphism (SNP) analysis showed that all these 152 KPC-2-producing K. pneumoniae ST11 isolates could be divided into three genetically distinct clades (A, B, and C) and eleven subclades (A1–A9 and B1–B2). The majority belonged to clade A with KL47 serotype (n = 117, 77.0%), while KL64 and KL16 were identified in clades B and C, respectively. The bla KPC–2-carrying plasmids exhibited diverse types, namely, IncFII (pHN7A8)/IncR(6/15), IncFII (pHN7A8)/IncpA1763–KPC (5/15), IncFII (pHN7A8) (1/15), IncR (1/15), and IncpA1763–KPC (1/15). The genetic environment of bla KPC–2 showed nine IS 26 -based composite transposons, which had a basic core structure IS Kpn27-bla KPC–2-ΔIS Kpn6. About 27.6% (42/152) isolates co-carried 2 to 4 virulence marker genes (namely, peg344 , iucA , iroB , rmpA , and rmpA2) for hvKp strains. At least three isolates were identified to harbor virulence gene-carrying plasmids. Conclusion: KPC-2-producing K. pneumoniae ST11 was highly heterogeneous in our hospital. Transmission of these strains was mainly mediated by twelve high-risk clones. The bla KPC–2-carrying plasmids and genetic environment of bla KPC–2 genes exhibited active evolution in K. pneumoniae ST11. More attention should be paid to the tendency of KPC-2-ST11 to acquire hypervirulent plasmids. [ABSTRACT FROM AUTHOR]

Background: Urinary tract infection (UTI) is one of the most common outpatient bacterial infections. In this study, we isolated and characterized an extensively-drug resistant (XDR) NDM-5-producing Escherichia coli EC1390 from a UTI patient by using whole-genome sequencing (WGS) in combination with phenotypic assays. Methods: Antimicrobial susceptibility to 23 drugs was determined by disk diffusion method. The genome sequence of EC1390 was determined by Nanopore MinION MK1C platform. Conjugation assays were performed to test the transferability of EC1390 plasmids to E. coli recipient C600. Phenotypic assays, including growth curve, biofilm formation, iron acquisition ability, and cell adhesion, were performed to characterize the function of EC1390 plasmids. Results: Our results showed that EC1390 was only susceptible to tigecycline and colistin, and thus was classified as XDR E. coli. A de novo genome assembly was generated using Nanopore 73,050 reads with an N50 value of 20,936 bp and an N90 value of 7,624 bp. WGS analysis showed that EC1390 belonged to the O101-H10 serotype and phylogenetic group A E. coli. Moreover, EC1390 contained 2 conjugative plasmids with a replicon IncFIA (pEC1390-1 with 156,286 bp) and IncFII (pEC1390-2 with 71,840 bp), respectively. No significant difference was observed in the bacterial growth rate in LB broth and iron acquisition ability between C600, C600 containing pEC1390-1, C600 containing pEC1390-2, and C600 containing pEC1390-1 and pEC1390-2. However, the bacterial growth rate in nutrition-limited M9 broth was increased in C600 containing pEC1390-2, and the cell adhesion ability was increased in C600 containing both pEC1390-1 and pEC1390-2. Moreover, these plasmids modulated the biofilm formation under different conditions. Conclusions: In summary, we characterized the genome of XDR-E. coli EC1390 and identified two plasmids contributing to the antimicrobial resistance, growth of bacteria in a nutrition-limited medium, biofilm formation, and cell adhesion. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is a fast-growing threat to global health. The genes conferring AMR to bacteria are often located on plasmids, circular extrachromosomal DNA molecules that can be transferred between bacterial strains and species. Therefore, effective methods to characterize bacterial plasmids and detect the presence of resistance genes can assist in managing AMR, for example, during outbreaks in hospitals. However, existing methods for plasmid analysis either provide limited information or are expensive and challenging to implement in low-resource settings. Herein, we present a simple assay based on CRISPR/Cas9 excision and DNA combing to detect antimicrobial resistance genes on bacterial plasmids. Cas9 recognizes the gene of interest and makes a double-stranded DNA cut, causing the circular plasmid to linearize. The change in plasmid configuration from circular to linear, and hence the presence of the AMR gene, is detected by stretching the plasmids on a glass surface and visualizing by fluorescence microscopy. This single-molecule imaging based assay is inexpensive, fast, and in addition to detecting the presence of AMR genes, it provides detailed information on the number and size of plasmids in the sample. We demonstrate the detection of several β-lactamase-encoding genes on plasmids isolated from clinical samples. Furthermore, we demonstrate that the assay can be performed using standard microbiology and clinical laboratory equipment, making it suitable for low-resource settings. [ABSTRACT FROM AUTHOR]