PLSDB: advancing a comprehensive database of bacterial plasmids.

Plasmids are known to contain genes encoding for virulence factors and antibiotic resistance mechanisms. Their relevance in metagenomic data processing is steadily growing. However, with the increasing popularity and scale of metagenomics experiments, the number of reported plasmids is rapidly growing as well, amassing a considerable number of false positives due to undetected misassembles. Here, our previously published database PLSDB provides a reliable resource for researchers to quickly compare their sequences against selected and annotated previous findings. Within two years, the size of this resource has more than doubled from the initial 13,789 to now 34,513 entries over the course of eight regular data updates. For this update, we aggregated community feedback for major changes to the database featuring new analysis functionality as well as performance, quality, and accessibility improvements. New filtering steps, annotations, and preprocessing of existing records improve the quality of the provided data. Additionally, new features implemented in the web-server ease user interaction and allow for a deeper understanding of custom uploaded sequences, by visualizing similarity information. Lastly, an application programming interface was implemented along with a python library, to allow remote database queries in automated workflows. The latest release of PLSDB is freely accessible under https://www.ccb.uni-saarland.de/plsdb.

Evolving origin-of-transfer sequences on staphylococcal conjugative and mobilizable plasmids-who's mimicking whom?

In Staphylococcus aureus, most multiresistance plasmids lack conjugation or mobilization genes for horizontal transfer. However, most are mobilizable due to carriage of origin-of-transfer (oriT) sequences mimicking those of conjugative plasmids related to pWBG749. pWBG749-family plasmids have diverged to carry five distinct oriT subtypes and non-conjugative plasmids have been identified that contain mimics of each. The relaxasome accessory factor SmpO, encoded by each conjugative plasmid, determines specificity for its cognate oriT. Here we characterized the binding of SmpO proteins to each oriT. SmpO proteins predominantly formed tetramers in solution and bound 5'-GNNNNC-3' sites within each oriT. Four of the five SmpO proteins specifically bound their cognate oriT. An F7K substitution in pWBG749 SmpO switched oriT-binding specificity in vitro. In vivo, the F7K substitution reduced but did not abolish self-transfer of pWBG749. Notably, the substitution broadened the oriT subtypes that were mobilized. Thus, this substitution represents a potential evolutionary intermediate with promiscuous DNA-binding specificity that could facilitate a switch between oriT specificities. Phylogenetic analysis suggests pWBG749-family plasmids have switched oriT specificity more than once during evolution. We hypothesize the convergent evolution of oriT specificity in distinct branches of the pWBG749-family phylogeny reflects indirect selection pressure to mobilize plasmids carrying non-cognate oriT-mimics.

Antibiotic resistance plasmid composition and architecture in Escherichia coli isolates from meat.

Resistance plasmids play a crucial role in the transfer of antimicrobial resistance from the veterinary sector to human healthcare. In this study plasmids from foodborne Escherichia coli isolates with a known (ES)BL or tetracycline resistance were sequenced entirely with short- and long-read technologies to obtain insight into their composition and to identify driving factors for spreading. Resistant foodborne E. coli isolates often contained several plasmids coding for resistance to various antimicrobials. Most plasmids were large and contained multiple resistance genes in addition to the selected resistance gene. The majority of plasmids belonged to the IncI, IncF and IncX incompatibility groups. Conserved and variable regions could be distinguished in each of the plasmid groups. Clusters containing resistance genes were located in the variable regions. Tetracycline and (extended spectrum) beta-lactamase resistance genes were each situated in separate clusters, but sulphonamide, macrolide and aminoglycoside formed one cluster and lincosamide and aminoglycoside another. In most plasmids, addiction systems were found to maintain presence in the cell.

Universal whole-sequence-based plasmid typing and its utility to prediction of host range and epidemiological surveillance.

Bacterial plasmids play a large role in allowing bacteria to adapt to changing environments and can pose a significant risk to human health if they confer virulence and antimicrobial resistance (AMR). Plasmids differ significantly in the taxonomic breadth of host bacteria in which they can successfully replicate, this is commonly referred to as 'host range' and is usually described in qualitative terms of 'narrow' or 'broad'. Understanding the host range potential of plasmids is of great interest due to their ability to disseminate traits such as AMR through bacterial populations and into human pathogens. We developed the MOB-suite to facilitate characterization of plasmids and introduced a whole-sequence-based classification system based on clustering complete plasmid sequences using Mash distances (https://github.com/phac-nml/mob-suite). We updated the MOB-suite database from 12 091 to 23 671 complete sequences, representing 17 779 unique plasmids. With advances in new algorithms for rapidly calculating average nucleotide identity (ANI), we compared clustering characteristics using two different distance measures - Mash and ANI - and three clustering algorithms on the unique set of plasmids. The plasmid nomenclature is designed to group highly similar plasmids together that are unlikely to have multiple representatives within a single cell. Based on our results, we determined that clusters generated using Mash and complete-linkage clustering at a Mash distance of 0.06 resulted in highly homogeneous clusters while maintaining cluster size. The taxonomic distribution of plasmid biomarker sequences for replication and relaxase typing, in combination with MOB-suite whole-sequence-based clusters have been examined in detail for all high-quality publicly available plasmid sequences. We have incorporated prediction of plasmid replication host range into the MOB-suite based on observed distributions of these sequence features in combination with known plasmid hosts from the literature. Host range is reported as the highest taxonomic rank that covers all of the plasmids which share replicon or relaxase biomarkers or belong to the same MOB-suite cluster code. Reporting host range based on these criteria allows for comparisons of host range between studies and provides information for plasmid surveillance.

Diversity in Chlamydial plasmids.

BACKGROUND: Evolutionary studies have been conducted that have investigated the chromosomal variance in the genus of Chlamydia. However, no all-encompassing genus-wide comparison has been performed on the plasmid. Therefore, there is a gap in the current knowledge on Chlamydia plasmid diversity. AIMS: This project is aimed to investigate and establish the nature and extent of diversity across the entire genus of Chlamydia, by comparing the sequences of all currently available plasmid carrying strains. METHODS: The PUBMED database was used to identify plasmid sequences from all available strains that met the set quality criteria for their inclusion in the study. Alignments were performed on the 51 strains that fulfilled the criteria using MEGA X software. Following that Maximum Likelihood estimation was used to construct 11 phylogenetic trees of the whole plasmid sequence, the individual 8 coding sequences, the iteron and a chromosomal gene ompA as a comparator. RESULTS: The genus-wide plasmid phylogeny produced three distinct lineages labelled as alpha, beta and gamma. Nineteen genotypes were found in the initial whole plasmid analysis. Their distribution was allocated as six C. pecorum, two C. pneumoniae, one C. gallinacea, one C. avium, one C. caviae, one C. felis, two C. psittaci, one C. trachomatis, one C. muridarum, and two C. suis. The chromosomal comparative gene ompA supported this distribution, with the same number of primary clades with the same species distribution. However, ompA sequence comparison resulted in fewer genotypes due to a reduced amount of available sequences (33 out of 51). All results were statistically significant. CONCLUSION: The results of this study indicate that the common bacterial ancestor of all the species had a plasmid, which has diverged over time. Moreover, it suggests that there is a strong evolutionary selection towards these species retaining their plasmids due to its high level of conservation across the genus, with the notable exception of C. pneumoniae. Furthermore, the evolutionary analysis showed that the plasmid and the chromosome have co-evolved.

Carriage of Distinct mcr-1-Harboring Plasmids by Unusual Serotypes of Salmonella.

Colistin acts as a last-resort antibiotic against lethal infections by carbapenem-resistant Enterobacterial pathogens. Enterobacteriaceae carrying mobile colistin resistance (MCR) genes are rapidly emerging and threatening human health and food safety. Despite mcr-1 being prevalent in Escherichia coli, its dissemination in Salmonella is not well characterized. Herein, two unusual serotypes of colistin-resistant Salmonella isolates are reported first, namely serotype Ngor (ST5399) and Goldcoast (ST2529). Using whole genome sequencing, it is shown that mcr-1 is located on the IncHI2-like plasmid pTB501 (188,527 bp) of strain SSDFZ54 and the IncX4-type plasmid pTB602 (33,303 bp) in strain SSDFZ69, respectively. Furthermore, the backbone, tra- and antimicrobial resistance genes relative variable regions in the mcr-1-bearing IncHI2 plasmids are systematically characterized. Phylogenetic analysis shows that all IncHI2-type plasmids from non-Chinese regions are clustered together, suggesting a possible Chinese origin. Taken together, these findings extend the understanding of Salmonella as a vehicle of mcr-1 carriage and distribution.

PlasClass improves plasmid sequence classification.

Many bacteria contain plasmids, but separating between contigs that originate on the plasmid and those that are part of the bacterial genome can be difficult. This is especially true in metagenomic assembly, which yields many contigs of unknown origin. Existing tools for classifying sequences of plasmid origin give less reliable results for shorter sequences, are trained using a fraction of the known plasmids, and can be difficult to use in practice. We present PlasClass, a new plasmid classifier. It uses a set of standard classifiers trained on the most current set of known plasmid sequences for different sequence lengths. We tested PlasClass sequence classification on held-out data and simulations, as well as publicly available bacterial isolates and plasmidome samples and plasmids assembled from metagenomic samples. PlasClass outperforms the state-of-the-art plasmid classification tool on shorter sequences, which constitute the majority of assembly contigs, allowing it to achieve higher F1 scores in classifying sequences from a wide range of datasets. PlasClass also uses significantly less time and memory. PlasClass can be used to easily classify plasmid and bacterial genome sequences in metagenomic or isolate assemblies. It is available under the MIT license from: https://github.com/Shamir-Lab/PlasClass.

Incompatibility and phylogenetic relationship of I-complex plasmids.

Plasmid incompatibility is the inability of two plasmids to be stably maintained in one cell, resulting in loss of one of the plasmids in daughter cells. Dislodgement is a phenotypically distinct form of incompatibility, described as an imperfect reproduction, manifesting in rapid exclusion of a resident plasmid after superinfection. The relationship between plasmids of the phenotypic incompatibility groups IncB/O and IncZ is unclear. Their inability to co-exist was initially referred to as dislodgement while other research reached the conclusion that IncB/O and IncZ plasmids are incompatible. In this manuscript we re-evaluated the relationship between IncB/O and IncZ plasmids to settle these conflicting conclusions. We performed dislodgement testing of R16Δ (IncB/O) and pSFE-059 (IncZ) plasmids by electroporation in a bacterial cell and checked their stability. Stability tests of the obtained plasmid pair showed that the IncB/O plasmid was exclusively and almost completely lost from the heteroplasmid Escherichia coli population. Other IncB/O - IncZ pairs could not form a heteroplasmid population, using conjugation or electroporation. Our data supports the previous suggestion that IncB/O and IncZ plasmids may be considered phenotypically incompatible.

A megaplasmid family driving dissemination of multidrug resistance in Pseudomonas.

Multidrug resistance (MDR) represents a global threat to health. Here, we used whole genome sequencing to characterise Pseudomonas aeruginosa MDR clinical isolates from a hospital in Thailand. Using long-read sequence data we obtained complete sequences of two closely related megaplasmids (>420 kb) carrying large arrays of antibiotic resistance genes located in discrete, complex and dynamic resistance regions, and revealing evidence of extensive duplication and recombination events. A comprehensive pangenomic and phylogenomic analysis indicates that: 1) these large plasmids comprise an emerging family present in different members of the Pseudomonas genus, and associated with multiple sources (geographical, clinical or environmental); 2) the megaplasmids encode diverse niche-adaptive accessory traits, including multidrug resistance; 3) the accessory genome of the megaplasmid family is highly flexible and diverse. The history of the megaplasmid family, inferred from our analysis of the available database, suggests that members carrying multiple resistance genes date back to at least the 1970s.

Characterization of blaCTX-M-27/F1:A2:B20 Plasmids Harbored by Escherichia coli Sequence Type 131 Sublineage C1/H30R Isolates Spreading among Elderly Japanese in Nonacute-Care Settings.

We characterized 29 blaCTX-M-27-harboring plasmids of Escherichia coli sequence type 131 (ST131) sublineage C1/H30R isolates from healthy individuals and long-term-care facility (LTCF) residents. Most (27/29) plasmids were of the FIA, FIB, and FII multireplicon type with the same plasmid multilocus sequence typing (pMLST). Several plasmids (7/23) from LTCF residents harbored only blaCTX-M-27 as the resistance gene; however, their fundamental structures were very similar to those of previously isolated blaCTX-M-27/F1:A2:B20 plasmids, suggesting their prevalence as a newly arising public health concern.

Repository-based plasmid design.

There was an explosion in the amount of commercially available DNA in sequence repositories over the last decade. The number of such plasmids increased from 12,000 to over 300,000 among three of the largest repositories: iGEM, Addgene, and DNASU. A challenge in biodesign remains how to use these and other repository-based sequences effectively, correctly, and seamlessly. This work describes an approach to plasmid design where a plasmid is specified as simply a DNA sequence or list of features. The proposed software then finds the most cost-effective combination of synthetic and PCR-prepared repository fragments to build the plasmid via Gibson assembly®. It finds existing DNA sequences in both user-specified and public DNA databases: iGEM, Addgene, and DNASU. Such a software application is introduced and characterized against all post-2005 iGEM composite parts and all Addgene vectors submitted in 2018 and found to reduce costs by 34% versus a purely synthetic plasmid design approach. The described software will improve current plasmid assembly workflows by shortening design times, improving build quality, and reducing costs.

Presence and Diversity of Extended-Spectrum Cephalosporin Resistance Among Escherichia coli from Urban Wastewater and Feedlot Cattle in Alberta, Canada.

A recent preliminary study from our group found that extended-spectrum cephalosporin-resistance determinants can be detected in the majority of composite fecal samples collected from Alberta feedlot cattle. Most notably, blaCTX-M genes were detected in 46.5% of samples. Further isolate characterization identified blaCTX-M-15 and blaCTX-M-27, which are widespread in bacteria from humans. We hypothesized that Escherichia coli of human and beef cattle origins share the same pool of blaCTX-M genes. In this study, we aimed to assess and compare the genomic profiles of a larger collection of blaCTX-M-positive E. coli recovered from fecal composite samples from Canadian beef feedlot cattle and human wastewater through whole-genome sequencing. The variants blaCTX-M-55, blaCTX-M-32, blaCTX-M-27, blaCTX-M-15, and blaCTX-M-14 were found in both urban wastewater and cattle fecal isolates. Core genome multilocus sequence typing showed little similarity between the fecal and wastewater isolates. Thus, if the dissemination of genes between urban wastewater and feedlot cattle occurs, it does not appear to be related to the expansion of specific clonal lineages. Further investigations are warranted to assemble and compare plasmids carrying these genes to better understand the modalities and directionality of transfer.

Plasmid Typing and Classification.

Plasmids identification and classification is an essential parameter in current bacterial typing. The most widely used PCR-based methods are the PCR-based replicon typing (PBRT) and the degenerate primer MOB typing (DPMT). PBRT targets the replicons on the plasmids and DPMT targets the relaxase genes. A finer resolution of phylogenetic relatedness can be obtained by plasmid multiLocus sequence typing available for the major plasmid types occurring in Enterobacteriaceae.

ORF-based binarized structure network analysis of plasmids (OSNAp), a novel approach to core gene-independent plasmid phylogeny.

OBJECTIVES: Systematic comparison of multiple plasmids remains challenging. We aimed to develop a new method for phylogenetic analysis of plasmids, open reading frame (ORF)-based binarized structure network analysis of plasmids (OSNAp). METHODS: With the OSNAp, the genetic structures of plasmids in a given plasmid group are expressed as binary sequences based on the presence or absence of ORFs regardless of their positions or directions. As a proof-of-concept, ORFs were collected from 101 complete I1 plasmid sequences, and their corresponding binary sequences were generated. A tree was generated using the neighbor-net, an algorithm for constructing phylogenetic networks based on distance between taxa, to visualize the plasmid phylogeny drawn from binary sequences. The results were compared with those of plasmid sequence types (pSTs) defined by plasmid multilocus sequence typing (pMLST). RESULTS: All I1 plasmids were placed on the phylogenetic tree constructed from the binary sequences. Most plasmids belonging to the same pSTs had Dice indices of ≥0.95 and were placed in the same OSNAp split. On the other hand, pST12 plasmids were distributed on separate splits due to differences in ORFs not used in pMLST, suggesting improved differentiation of the plasmids with OSNAp compared with pMLST. CONCLUSION: OSNAp is a novel holistic approach to assess relatedness of a population of plasmids in a given plasmid group based on nucleotide sequence data. It provides higher discrimination than pMLST, which may prove useful in tracing bacteria that harbor plasmids of shared origins.

Occurrence and Genomic Characterization of Two MCR-1-Producing Escherichia coli Isolates from the Same Mink Farmer.

The spread of colistin resistance gene mcr-1 at the animal-human interface remains largely unknown. This work aimed to investigate the molecular characteristics of two extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli strains with mcr-1, i.e., strains H8 and H9, isolated from the same mink farmer. In this study, five mcr-positive E. coli strains were isolated from the mink farm. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) identified two genetically unrelated MCR-1 producers (H8 and H9) from the same farmer and two clonally related MCR-1-positive isolates (M5 and M6) from two different mink samples. Additionally, a mcr-1 variant, designated mcr-1.12, was identified in isolate M4. MIC determination revealed that all of the MCR-producing strains exhibited multiresistant phenotypes but showed susceptibility to imipenem, meropenem, amikacin, and tigecycline. Replicon typing showed that mcr-1 was associated with IncHI2 plasmids in 4 cases, while the gene was located on an IncI2 plasmid in 1 case. PacBio sequencing and plasmid analysis confirmed that the mcr-1 gene was located on an ∼204-kb IncHI2 plasmid in H8 and was carried by an ∼61-kb IncI2 plasmid in H9. To our knowledge, this work represents the first report of the occurrence of MCR-producing isolates from mink. Moreover, our report also describes the coexistence of two different MCR-1 producers in the same farmer. It highlights that fur farms can be reservoirs of mcr-1 genes. The identification of mcr-carrying plasmids on a fur farm is of potential public health importance, as it suggests that mcr is widespread in the animal husbandry industry.IMPORTANCE Colistin resistance is a real threat for both human and animal health. The mobile colistin resistance gene mcr has contributed to the persistence and transmission of colistin resistance at the interfaces of animals, humans, and ecosystems. Although mcr genes have usually been recovered from food animals, patients, and healthy humans, transmission of mcr genes at the animal-human interface remains largely unknown. This was the first study to isolate and characterize MCR-producing isolates from mink, as well as to report the coexistence of two different MCR-1 producers in the same farmer. The characterization and analysis of two MCR-1-producing E. coli isolates may have important implications for comprehension of the transmission dynamics of these bacteria. We emphasize the importance of improved multisectorial surveillance of colistin-resistant E. coli in this region.

Development of a multi-locus typing scheme for an Enterobacteriaceae linear plasmid that mediates inter-species transfer of flagella.

Due to the public health importance of flagellar genes for typing, it is important to understand mechanisms that could alter their expression or presence. Phenotypic novelty in flagellar genes arise predominately through accumulation of mutations but horizontal transfer is known to occur. A linear plasmid termed pBSSB1 previously identified in Salmonella Typhi, was found to encode a flagellar operon that can mediate phase variation, which results in the rare z66 flagella phenotype. The identification and tracking of homologs of pBSSB1 is limited because it falls outside the normal replicon typing schemes for plasmids. Here we report the generation of nine new pBSSB1-family sequences using Illumina and Nanopore sequence data. Homologs of pBSSB1 were identified in 154 genomes representing 25 distinct serotypes from 67,758 Salmonella public genomes. Pangenome analysis of pBSSB1-family contigs was performed using roary and we identified three core genes amenable to a minimal pMLST scheme. Population structure analysis based on the newly developed pMLST scheme identified three major lineages representing 35 sequence types, and the distribution of these sequence types was found to span multiple serovars across the globe. This in silico pMLST scheme has shown utility in tracking and subtyping pBSSB1-family plasmids and it has been incorporated into the plasmid MLST database under the name "pBSSB1-family".

Molecular epidemiology of carbapenem-resistance plasmids using publicly available sequences.

Carbapenem-resistant bacteria have quickly become a worldwide concern in nosocomial infections. Of the seven known carbapenemases, four have been shown to be particularly problematic: KPC, NDM, IMP, and VIM. To date, many local and species- or carbapenemase-specific epidemiological studies have been performed, which often focus on the organism itself. This report attempts to perform an inclusive (encompass both species and carbapenemase) epidemiologic study using publicly available plasmid sequences from NCBI. In this report, the gene content of these various plasmids has been characterized, replicon types of the plasmids identified, and the global spread and species promiscuity of the plasmids analyzed. Additionally, support to several groups targeting plasmid maintenance and transfer mechanisms to slow the spread of resistance plasmids is given.

[Evolution and Comparative Genomics of the pSM22 Plasmid of the IncF/MOBF12 Group].

A new plasmid, pSM22, was isolated from Serratia marcescens and sequenced. Its 43 190-bp sequence with an average GC-content of 58% contains 31 open reading frames (ORFs) which form replication, conjugation, stability, and adaptive modules. The replication module includes a site of initiation of leading-strand synthesis in plasmid replication, a replication termination site (terC), the rep A (=repA1) and repA4 genes, and the copA sequence, which codes for an antisense RNA (asRNA). These structures are functionally integrated in an FII replicon (incompatibility group IncFII). Based on the significant differences between the FII replicon and the canonical sequences of the R plasmids R1 and NR1 (=R100=R222), pSM22 was assigned to a new subtype. The conjugation module includes 13 genes with a high identity to the genes responsible for conjugation of the F plasmid. A comparative genomic analysis showed that the conjugation modules of pSM22 and F are structurally similar. By the conjugation system and the presence of three conserved motifs in relaxase (TraI), pSM22 belongs to the F12 clade of the MOBF type. The stability module includes the resD and parA genes, which are responsible for the resolution of multimeric plasmid forms and their subsequent segregation between daughter cells. The adaptive module contains the microcin H47 (MccH47) secretion/processing and UV resistance genes. The mosaic structure of pSM22 and reductive evolution of its modules suggest high genomic plasticity for the genus Serratia. An analysis of the architecture of the pSM22 modules clarifies the evolutionary relationships among IncF/MOBF12 group plasmids in bacteria of the family Enterobacteriaceae and opens a novel avenue for further comparative genomic studies of Serratia plasmids.