Plasmid Sequences Research Articles

Antimicrobial Susceptibility and Resistance Mechanisms in Mannheimia haemolytica Isolates from Sheep at Slaughter.

Mannheimia haemolytica is the main pathogen contributing to pneumonic pasteurellosis in sheep. The aim of this study was to investigate the antimicrobial resistance levels in M. haemolytica isolates from the lungs of slaughtered sheep and to examine the genetic resistance mechanisms involved. A total of 256 M. haemolytica isolates, 169 from lungs with pneumonic lesions and 87 from lungs without lesions, were analyzed by the disk diffusion method for 12 antimicrobials, and the whole genome of 14 isolates was sequenced to identify antimicrobial resistance determinants. Levels of phenotypic resistance ranged from <2% for 10 antimicrobials (amoxicillin, amoxicillin-clavulanic, ceftiofur, cefquinome, lincomycin/spectinomycin, gentamicin, erythromycin, florfenicol, enrofloxacin, and doxycycline) to 4.3% for tetracycline and 89.1% for tylosin. Six isolates carried tetH genes and four isolates carried, in addition, the strA and sul2 genes in putative plasmid sequences. No mutations associated with macrolide resistance were identified in 23 rDNA sequences, suggesting that the M. haemolytica phenotypic results for tylosin should be interpreted with care in the absence of well-established epidemiological and clinical breakpoints. The identification of strains phenotypically resistant to tetracycline and of several resistance genes, some of which were present in plasmids, highlights the need for continuous monitoring of susceptibility patterns in Pasteurellaceae isolates from livestock.

Characterizing conjugative plasmids from an antibiotic-resistant dataset for use as broad-host delivery vectors.

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.

Global transmission of broad-host-range plasmids derived from the human gut microbiome.

Broad-host-range (BHR) plasmids in human gut bacteria are of considerable interest for their ability to mediate horizontal gene transfer (HGT) across large phylogenetic distance. However, the human gut plasmids, especially the BHR plasmids, remain largely unknown. Here, we identified the plasmids in the draft genomes of gut bacterial isolates from Chinese and American donors, resulting in 5372 plasmid-like clusters (PLCs), of which, 820 PLCs (comPLCs) were estimated with>60% completeness genomes and only 155 (18.9%) were classified to known replicon types (n=37). We observed that 175 comPLCs had a broad host range across distinct bacterial genera, of which, 71 were detected in at least two human populations of Chinese, American, Spanish, and Danish, and 13 were highly prevalent (>10%) in at least one human population. Haplotype analyses of two widespread PLCs demonstrated their spreading and evolutionary trajectory, suggesting frequent and recent exchanges of the BHR plasmids in environments. In conclusion, we obtained a large collection of plasmid sequences in human gut bacteria and demonstrated that a subset of the BHR plasmids can be transmitted globally, thus facilitating extensive HGT (e.g. antibiotic resistance genes) events. This study highlights the potential implications of the plasmids for global human health.

Long read genome assemblers struggle with small plasmids.

Whole-genome sequencing has become a preferred method for studying bacterial plasmids, as it is generally assumed to capture the entire genome. However, long-read genome assemblers have been shown to sometimes miss plasmid sequences - an issue that has been associated with plasmid size. The purpose of this study was to investigate the relationship between plasmid size and plasmid recovery by the long-read-only assemblers, Flye, Raven, Miniasm, and Canu. This was accomplished by determining the number of times each assembler successfully recovered 33 plasmids, ranging from 1919 to 194 062 bp in size and belonging to 14 bacterial isolates from six bacterial genera, using Oxford Nanopore long reads. These results were additionally compared to plasmid recovery rates by the short-read-first assembler, Unicycler, using both Oxford Nanopore long reads and Illumina short reads. Results from this study indicate that Canu, Flye, Miniasm, and Raven are prone to missing plasmid sequences, whereas Unicycler was successful at recovering 100 % of plasmid sequences. Excluding Canu, most plasmid loss by long-read-only assemblers was due to failure to recover plasmids smaller than 10 kb. As such, it is recommended that Unicycler be used to increase the likelihood of plasmid recovery during bacterial genome assembly.

Plasmer: an Accurate and Sensitive Bacterial Plasmid Prediction Tool Based on Machine Learning of Shared k-mers and Genomic Features.

Identification of plasmids in bacterial genomes is critical for many factors, including horizontal gene transfer, antibiotic resistance genes, host-microbe interactions, cloning vectors, and industrial production. There are several in silico methods to predict plasmid sequences in assembled genomes. However, existing methods have evident shortcomings, such as unbalance in sensitivity and specificity, dependency on species-specific models, and performance reduction in sequences shorter than 10 kb, which has limited their scope of applicability. In this work, we proposed Plasmer, a novel plasmid predictor based on machine-learning of shared k-mers and genomic features. Unlike existing k-mer or genomic-feature based methods, Plasmer employs the random forest algorithm to make predictions using the percent of shared k-mers with plasmid and chromosome databases combined with other genomic features, including alignment E value and replicon distribution scores (RDS). Plasmer can predict on multiple species and has achieved an average the area under the curve (AUC) of 0.996 with accuracy of 98.4%. Compared to existing methods, tests of both sliding sequences and simulated and de novo assemblies have consistently shown that Plasmer has outperforming accuracy and stable performance across long and short contigs above 500 bp, demonstrating its applicability for fragmented assemblies. Plasmer also has excellent and balanced performance on both sensitivity and specificity (both >0.95 above 500 bp) with the highest F1-score, which has eliminated the bias on sensitivity or specificity that was common in existing methods. Plasmer also provides taxonomy classification to help identify the origin of plasmids. IMPORTANCE In this study, we proposed a novel plasmid prediction tool named Plasmer. Technically, unlike existing k-mer or genomic features-based methods, Plasmer is the first tool to combine the advantages of the percent of shared k-mers and the alignment score of genomic features. This has given Plasmer (i) evident improvement in performance compared to other methods, with the best F1-score and accuracy on sliding sequences, simulated contigs, and de novo assemblies; (ii) applicability for contigs above 500 bp with highest accuracy, enabling plasmid prediction in fragmented short-read assemblies; (iii) excellent and balanced performance between sensitivity and specificity (both >0.95 above 500 bp) with the highest F1-score, which eliminated the bias on sensitivity or specificity that commonly existed in other methods; and (iv) no dependency of species-specific training models. We believe that Plasmer provides a more reliable alternative for plasmid prediction in bacterial genome assemblies.

Anthropogenic and natural disturbances along a river and its estuary alter the diversity of pathogens and antibiotic resistance mechanisms

Antibiotic resistance (AMR) in pathogens threatens human health worldwide, and antibiotic-resistant bacteria (ARB) are widespread in the environment. In particular, anthropogenically-disturbed rivers became reservoirs of ARBs and hotspots of antibiotic resistance gene (ARG) transmission. However, the diversity and sources of ARB, and the mechanisms of ARG transmission are not fully known. Here, we used deep metagenomic sequencing to study the dynamics of pathogens and their antibiotic resistance mechanisms along the Alexander River (Israel), affected by sewage and animal farm runoffs. Putative pathogens such as Aeromicrobium marinum and Mycobacterium massilipolynesiensis were enriched in western stations, following the inputs of polluted Nablus River. Aeromonas veronii was dominant in eastern stations in Spring. Several AMR mechanisms showed distinct patterns in Summer-Spring (dry season) and Winter (rainy season). We found low abundance beta-lactamases conferring carbapenem resistance: e.g., OXA-912 was linked to A. veronii in Spring; OXA-119 and OXA-205 to Xanthomonadaceae in Winter. We classified 33 % of ARG-containing contigs as putative plasmid sequences, indicating the high potential for resistome transmission. A limited number of ARGs were linked to putative phages. Our results suggest that this model river is a hotspot for AMR activity and transmission, and highlight the merit of deep sequencing for AMR discovery.

Emergence of a Hybrid IncI1-Iα Plasmid-Encoded blaCTX-M-101 Conferring Resistance to Cephalosporins in Salmonella enterica Serovar Enteritidis.

The increasing resistance to cephalosporins in Salmonella poses a serious threat to public health. In our previous study, the blaCTX-M-101 gene, a new blaCTX-M variant, was first reported in Salmonella enterica serovar Enteritidis (S. Enteritidis). Here, we further analyzed the genome characterization, transferability, and resistance mechanism of one S. Enteritidis isolate (SJTUF14523) carrying blaCTX-M-101 from an outpatient in 2016 in Xinjiang, China. This strain was a multidrug resistance (MDR) isolate and exhibited resistance to ceftazidime (MIC = 64 μg/mL), cefotaxime (MIC = 256 μg/mL), and cefepime (MIC = 16 μg/mL). Phylogenetic analysis revealed that SJTUF14523 had a close relationship to another S. Enteritidis isolate from the United States. In the presence of plasmid p14523A, there were 8- and 2133-fold increases in the MICs of cephalosporins in Escherichia coli C600 in the conjugation. Gene cloning results indicated that blaCTX-M-101 was the decisive mechanism leading to ceftazidime and cefotaxime resistance that could make the MICs break through the resistance breakpoint. Plasmid sequencing revealed that the blaCTX-M-101 gene was located on an IncI1-Iα transferable plasmid (p14523A) that was 85,862 bp in length. Sequence comparison showed that p14523A was a novel hybrid plasmid that might have resulted from the interaction between a homologous region. Furthermore, we found a composite transposon unit composed of ISEcp1, blaCTX-M-101, and orf477 in p14523A. ISEcp1-mediated transposition was likely to play a key role in the horizontal transfer of blaCTX-M-101 among plasmids in S. Enteritidis. Collectively, these findings underline further challenges in the prevention and control of antibiotic resistance posed by new CTX-M-101-like variants in Salmonella.

The highly diverse plasmid population found in Escherichia coli colonizing travellers to Laos and its role in antimicrobial resistance gene carriage.

Increased colonization by antimicrobial-resistant organisms is closely associated with international travel. This study investigated the diversity of mobile genetic elements involved with antimicrobial resistance (AMR) gene carriage in extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli that colonized travellers to Laos. Long-read sequencing was used to reconstruct complete plasmid sequences from 48 isolates obtained from the daily stool samples of 23 travellers over a 3 week period. This method revealed a collection of 105 distinct plasmids, 38.1 % (n=40) of which carried AMR genes. The plasmids in this population were diverse, mostly unreported and included 38 replicon types, with F-type plasmids (n=23) the most prevalent amongst those carrying AMR genes. Fine-scale analysis of all plasmids identified numerous AMR gene contexts and emphasized the importance of IS elements, specifically members of the IS6/IS26 family, in the evolution of complex multidrug resistance regions. We found a concerning convergence of ESBL and colistin resistance determinants, with three plasmids from two different F-type lineages carrying bla CTX-M and mcr genes. The extensive diversity seen here highlights the worrying probability that stable new vehicles for AMR will evolve in E. coli populations that can disseminate internationally through travel networks.

Molecular characterisation of the Australian and New Zealand livestock Chlamydia pecorum strains confirms novel but clonal ST23 in association with ovine foetal loss

Chlamydia pecorum is a veterinary pathogen associated with abortions and perinatal mortality in sheep. Recent studies investigating foetal and perinatal lamb mortality in sheep from Australia and New Zealand identified C. pecorum clonal sequence type (ST)23 strains in aborted and stillborn lambs. Presently, there is limited genotypic information on C. pecorum strains associated with reproductive disease, although whole genome sequencing (WGS) of one abortigenic ST23 C. pecorum strain identified unique features, including a deletion in the CDS1 locus of the chlamydial plasmid. We applied WGS on two ST23 strains detected in aborted and stillborn lambs from Australia and used phylogenetic and comparative analyses to compare these to the other available C. pecorum genomes. To re-evaluate the genetic diversity of contemporary strains, we applied C. pecorum genotyping, and chlamydial plasmid sequencing to a range of C. pecorum positive samples and isolates from ewes, aborted foetuses and stillborn lambs, cattle and a goat from diverse geographical regions across Australia and New Zealand.The two new C. pecorum genomes are nearly identical to the genome of the Australian abortigenic strain including the unique deletion in the chlamydial plasmid. Genotyping revealed that these novel C. pecorum ST23 strains are widespread and associated with sheep abortions on Australian and New Zealand farms. In addition, a goat C. pecorum strain (denoted ST 304) from New Zealand was also characterised. This study expands the C. pecorum genome catalogue and describes a comprehensive molecular characterisation of the novel livestock ST23 strains associated with foetal and lamb mortality.

Comparative genome analysis of Sesbania cannabina-nodulating Rhizobium spp. revealing the symbiotic and transferrable characteristics of symbiosis plasmids.

Symbiotic nitrogen fixation between legumes and rhizobia makes a great contribution to the terrestrial ecosystem. The successful symbiosis between the partners mainly depends on the nod and nif genes in rhizobia, while the specific symbiosis is mainly determined by the structure of Nod factors and the corresponding secretion systems (type III secretion system; T3SS), etc. These symbiosis genes are usually located on symbiotic plasmids or a chromosomal symbiotic island, both could be transferred interspecies. In our previous studies, Sesbania cannabina-nodulating rhizobia across the world were classified into 16 species of four genera and all the strains, especially those of Rhizobium spp., harboured extraordinarily highly conserved symbiosis genes, suggesting that horizontal transfer of symbiosis genes might have happened among them. In order to learn the genomic basis of diversification of rhizobia under the selection of host specificity, we performed this study to compare the complete genome sequences of four Rhizobium strains associated with S. cannabina, YTUBH007, YTUZZ027, YTUHZ044 and YTUHZ045. Their complete genomes were sequenced and assembled at the replicon level. Each strain represents a different species according to the average nucleotide identity (ANI) values calculated using the whole-genome sequences; furthermore, except for YTUBH007, which was classified as Rhizobium binae, the remaining three strains were identified as new candidate species. A single symbiotic plasmid sized 345-402 kb containing complete nod, nif, fix, T3SS and conjugal transfer genes was detected in each strain. The high ANI and amino acid identity (AAI) values, as well as the close phylogenetic relationships among the entire symbiotic plasmid sequences, indicate that they have the same origin and the entire plasmid has been transferred among different Rhizobium species. These results indicate that S. cannabina stringently selects a certain symbiosis gene background of the rhizobia for nodulation, which might have forced the symbiosis genes to transfer from some introduced rhizobia to the related native or local-condition-adapted bacteria. The existence of almost complete conjugal transfer related elements, but not the gene virD, indicated that the self-transfer of the symbiotic plasmid in these rhizobial strains may be realized via a virD-independent pathway or through another unidentified gene. This study provides insight for the better understanding of high-frequency symbiotic plasmid transfer, host-specific nodulation and the host shift for rhizobia.

Investigating Possible Interspecies Communication of Plasmids Associated with Transfer of Third-Generation Cephalosporin, Quinolone, and Colistin Resistance Between Simultaneously Isolated Escherichia Coli and Klebsiella Pneumoniae.

The coinfection process producing multiple species of pathogens provides a specific ecological niche for the exchange of genetic materials between pathogens, in which plasmids play a vital role in horizontal gene transfer, especially for drug resistance, but the underlying transfer pathway remains unclear. Interspecies communication of the plasmids associated with the transfer of third-generation cephalosporins, quinolones, and colistin resistance has been observed in simultaneously isolated Escherichia coli and Klebsiella pneumoniae from abdominal drainage following surgery. The MICs of antimicrobial agents were determined by the broth microdilution method. The complete chromosome and plasmid sequences were obtained by combining Illumina paired-end short reads and MinION long reads. S1-PFGE, southern blot analysis and conjugation assay confirmed the transferability of the mcr-1-harboring plasmid. Both the E. coli isolate EC15255 and K. pneumoniae isolate KP15255 from the same specimen presented multidrug resistance. Each of them harbored one chromosome and three plasmids, and two plasmids and their mediated resistance could be transferred to the recipient by conjugation. Comparison of their genome sequences suggested that several genetic communication events occurred between species, especially among their plasmids, such as whole-plasmid transfer, insertion, deletion, amplification, or inversion. Exchange of plasmids or the genetic elements they harbor plays a critical role in antimicrobial resistance gene transmission and poses a substantial threat to nosocomial infection control, necessitating the continued surveillance of multidrug resistant pathogens, especially during coinfection. IMPORTANCE The genome sequence of bacterial pathogens commonly provides a detailed clue of genetic communication among clones or even distinct species. The intestinal microecological environment is a representative ecological niche for genetic communication. However, it is still difficult to describe the details of horizontal gene transfer or other genetic events within them because the evidence in the genome sequence is incomplete and limited. In this study, the simultaneously isolated Escherichia coli and Klebsiella pneumoniae from a coinfection process provided an excellent example for observation of interspecies communication between the two genomes and the plasmids they harbor. A complete genome sequence acquired by combining the Illumina and MinION sequencing platforms facilitated the understanding of genetic communication events, such as whole-plasmid transfer, insertion, deletion, amplification, or inversion, which contribute to antimicrobial resistance gene transmission and are a substantial threat to nosocomial infection control.

Multiplexed long-read plasmid validation and analysis using OnRamp.

Recombinant plasmid vectors are versatile tools that have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and bacterial processes used to create recombinant DNA can introduce errors, sequence validation is an essential step in plasmid assembly. Sanger sequencing is the current standard for plasmid validation; however, this method is limited by an inability to sequence through complex secondary structure and lacks scalability when applied to full-plasmid sequencing of multiple plasmids owing to read-length limits. Although high-throughput sequencing does provide full-plasmid sequencing at scale, it is impractical and costly when used outside of library-scale validation. Here, we present Oxford nanopore-based rapid analysis of multiplexed plasmids (OnRamp), an alternative method for routine plasmid validation that combines the advantages of high-throughput sequencing's full-plasmid coverage and scalability with Sanger's affordability and accessibility by leveraging nanopore's long-read sequencing technology. We include customized wet-laboratory protocols for plasmid preparation along with a pipeline designed for analysis of read data obtained using these protocols. This analysis pipeline is deployed on the OnRamp web app, which generates alignments between actual and predicted plasmid sequences, quality scores, and read-level views. OnRamp is designed to be broadly accessible regardless of programming experience to facilitate more widespread adoption of long-read sequencing for routine plasmid validation. Here we describe the OnRamp protocols and pipeline and show our ability to obtain full sequences from pooled plasmids while detecting sequence variation even in regions of high secondary structure at less than half the cost of equivalent Sanger sequencing.

Plasmid classifications

Plasmids are universally present in bacteria and play key roles in the dissemination of genes such as antibiotic resistance determinants. Major concepts in Plasmid Biology derive from the efforts to classify plasmids. Here, we review the main plasmid classification systems, starting by phenotype-based methods, such as fertility inhibition and incompatibility, followed by schemes based on a single gene (replicon type and MOB class), and finishing with recently developed approaches that use genetic distances between whole plasmid sequences. A comparison of the latter highlights significant differences between them. We further discuss the need for an operational definition of plasmid species that reveals their biological features, akin to plasmid taxonomic units (PTUs).

PlasMapper 3.0-a web server for generating, editing, annotating and visualizing publication quality plasmid maps.

PlasMapper 3.0 is a web server that allows users to generate, edit, annotate and interactively visualize publication quality plasmid maps. Plasmid maps are used to plan, design, share and publish critical information about gene cloning experiments. PlasMapper 3.0 is the successor to PlasMapper 2.0 and offers many features found only in commercial plasmid mapping/editing packages. PlasMapper 3.0 allows users to paste or upload plasmid sequences as input or to upload existing plasmid maps from its large database of>2000 pre-annotated plasmids (PlasMapDB). This database can be searched by plasmid names, sequence features, restriction sites, preferred host organisms, and sequence length. PlasMapper 3.0 also supports the annotation of new or never-before-seen plasmids using its own feature database that contains common promoters, terminators, regulatory sequences, replication origins, selectable markers and other features found in most cloning plasmids. PlasMapper 3.0 has several interactive sequence editors/viewers that allow users to select and view plasmid regions, insert genes, modify restriction sites or perform codon optimization. The graphics for PlasMapper 3.0 have also been substantially upgraded. It now offers an interactive, full-color plasmid viewer/editor that allows users to zoom, rotate, re-color, linearize, circularize, edit annotated features and modify plasmid images or labels to improve the esthetic qualities of their plasmid map and textual displays. All the plasmid images and textual displays are downloadable in multiple formats. PlasMapper 3.0 is available online at https://plasmapper.ca.

Genomic insight of extremely drug-resistant Klebsiella pneumoniae ST5378 from a paediatric bloodstream infection

This study investigated the draft genome and phylogeny of an extremely drug-resistant and novel sequence type Klebsiella pneumoniae isolated from a paediatric bloodstream infection. An isolate from a 7-year-old child with severe respiratory infection was identified, and the whole genome was sequenced using the Illumina MiSeq platform. High-quality reads were de novo assembled via Unicycler and annotated via PROKKA. Antimicrobial resistance genes, virulence factors, and plasmid and phage sequences were identified using the resistance gene identifier, VFanalyzer, Plasmidfinder, and PHASTER, respectively. Phylogenetics of closely related strains were inferred using core-genome multi-locus sequence typing and single nucleotide polymorphism. The draft genome of carbapenem-resistant K. pneumoniae RKS87 was 5 580 330 bp in size, with a GC content of 57.73%. The final assembly resulted in 38 contigs comprising 5075 CDS, 124 pseudo genes, 83 tRNA, 25 rRNA, and 10 ncRNA. The strain was assigned to a novel sequence type, ST5378, and harboured blaSHV-11, blaCTX-M-15, blaTEM-1, blaNDM-1, APH(3')-VI, OqxA, QnrS1, and fosA. We also identified the mutations in outer membrane porin (OmpK36 and OmpK37) and two-component system genes (PmrB and EptB). Three biomarkers (iroE, iroN, and iutA) associated with hypervirulent phenotype were also present in the genome. Phylogenetics of closely related strains revealed the clonal lineage of ST2938. The genome sequence and phylogenetics of the strain offer valuable insight into the clonal lineage, resistance genes, and pathogenicity of the novel sequence type ST5378.

Design of high-monounsaturated fatty acid soybean seed oil using GmPDCTs knockout via a CRISPR-Cas9 system.

Design of high-monounsaturated fatty acid soybean seed oil using GmPDCTs knockout via a CRISPR-Cas9 system.

Transferable IncX3 plasmid harboring blaNDM-1, bleMBL, and aph(3’)-VI genes from Klebsiella pneumoniae conferring phenotypic carbapenem resistance in E. coli

BackgroundThe dissemination of carbapenem resistance via carbapenemases, such as the metallo-β-lactamase NDM, among Enterobacterales poses a public health threat. The aim of this study was to characterize a plasmid carrying the blaNDM-1 gene, which was extracted from a clinical Klebsiella pneumoniae uropathogen from an Egyptian patient suffering from a urinary tract infection.Methods and resultsThe recovered plasmid was transformed into competent E. coli DH5α which acquired phenotypic resistance to cefoxitin, ceftazidime, and ampicillin/sulbactam, and intermediate sensitivity to ceftriaxone and imipenem (a carbapenem). Whole plasmid sequencing was performed on the extracted plasmid using the DNBSEQ™ platform. The obtained forward and reverse reads were assembled into contigs using the PRINSEQ and PLACNETw web tools. The obtained contigs were uploaded to PlasmidFinder and ResFinder for in silico plasmid typing and detection of antimicrobial resistance genes, respectively. The final consensus sequence was obtained using the Staden Package software. The plasmid (pNDMKP37, NCBI accession OK623716.1) was typed as an IncX3 plasmid with a size of 46,160 bp and harbored the antibiotic resistance genes blaNDM-1, bleMBL, and aph(3’)-VI. The plasmid also carried mobile genetic elements involved in the dissemination of antimicrobial resistance including insertion sequences IS30, IS630, and IS26.ConclusionsThis is Egypt’s first report of a transmissible plasmid co-harboring blaNDM-1 and aph(3’)-VI genes. Moreover, the respective plasmid is of great medical concern as it has caused the horizontal transmission of multidrug-resistant phenotypes to the transformant. Therefore, new guidelines should be implemented for the rational use of broad-spectrum antibiotics, particularly carbapenems.

Genome Sequence of a Clinical Isolate of the Human Pathogenic Strain “ Candidatus Borrelia fainii” Qtaro

We report sequences of the complete linear chromosome and five linear plasmids of the relapsing fever spirochete "Candidatus Borrelia fainii" Qtaro. The chromosome sequence of 951,861 bp and the 243,291 bp of plasmid sequences were predicted to contain 852 and 239 protein-coding genes, respectively. The predicted total GC content was 28.4%.

The Plasmidomic Landscape of Clinical Methicillin-Resistant Staphylococcus aureus Isolates from Malaysia.

Methicillin-resistant Staphylococcus aureus (MRSA) is a priority nosocomial pathogen with plasmids playing a crucial role in its genetic adaptability, particularly in the acquisition and spread of antimicrobial resistance. In this study, the genome sequences of 79 MSRA clinical isolates from Terengganu, Malaysia, (obtained between 2016 and 2020) along with an additional 15 Malaysian MRSA genomes from GenBank were analyzed for their plasmid content. The majority (90%, 85/94) of the Malaysian MRSA isolates harbored 1-4 plasmids each. In total, 189 plasmid sequences were identified ranging in size from 2.3 kb to ca. 58 kb, spanning all seven distinctive plasmid replication initiator (replicase) types. Resistance genes (either to antimicrobials, heavy metals, and/or biocides) were found in 74% (140/189) of these plasmids. Small plasmids (<5 kb) were predominant (63.5%, 120/189) with a RepL replicase plasmid harboring the ermC gene that confers resistance to macrolides, lincosamides, and streptogramin B (MLSB) identified in 63 MRSA isolates. A low carriage of conjugative plasmids was observed (n = 2), but the majority (64.5%, 122/189) of the non-conjugative plasmids have mobilizable potential. The results obtained enabled us to gain a rare view of the plasmidomic landscape of Malaysian MRSA isolates and reinforces their importance in the evolution of this pathogen.

Novel plasmids in multidrug-resistant Shigella flexneri serotypes from Pakistan

Shigellosis is the main cause of food and waterborne diarrhea and is an emerging threat to human health. The current study characterized the indigenous multidrug-resistant Shigella flexneri serotypes for their plasmid profiles and genetic diversity, to characterize the plasmid evolutionary patterns and distribution. In total, 199 identified S. flexneri isolates belonging to six different serotypes were analyzed for plasmid profiling, followed by an analysis of whole genome sequencing. All isolates of S. flexneri resistant to antibiotics harbored multiple copies of plasmids with sizes ranging from 1.25 kbp to 9.4kbp. These isolates were clustered into 22 distinct plasmid patterns, labeled as p1-p22. Among these, p1 (24%) and p10 (13%) were the predominant plasmid profiles. All S. flexneri strains were grouped into 12 clades with a 75% similarity level. Also, a significant association was observed among the plasmid patterns, p23 and p17 with the drug-resistant patterns AMC, SXT, C (19.5%) and OFX, AMC, NA, CIP (13.5%), respectively. Moreover, the most widespread plasmid patterns p4, p10, and p1 showed a significant association with the serotypes 1b (29.16%), 2b (36%), and 7a (100%), respectively. After plasmid sequence assembly and annotation analysis, a variety of small plasmids that vary in size from 973 to 6200bp were discovered. Many of these plasmids displayed high homology and coverage with plasmids from non-S. flexneri. Several novel plasmids of small size were discovered in multidrug-resistant S. flexneri. The data also showed that plasmid profile analysis is more consistent than antibiotic susceptibility pattern analysis for identifying epidemic strains of S. flexneri isolated in Pakistan.