Small Plasmids Research Articles

Impact of Predation by Ciliate Tetrahymena borealis on Conjugation in Aeromonas salmonicida subsp. salmonicida.

Antibiotic resistance gene (ARG) spread is driven by horizontal gene transfer (HGT). Ciliated protozoa may contribute to this process, as their predation has been shown to facilitate HGT in certain bacteria. Here, this phenomenon was further investigated using A. salmonicida subsp. salmonicida. This fish pathogen bears an extensive and dynamic plasmidome, suggesting a high potential for HGT. A. salmonicida strains carrying one of three conjugative plasmids bearing ARGs (pSN254b, pRAS1b or pAsa4b) were cocultured with a recipient, either A. salmonicida, E. coli or A. hydrophila. Conjugation rates were assessed in the presence and absence of the ciliate Tetrahymena borealis. PCR genotyping confirmed the acquisition of the conjugative plasmids and was used to verify the mobilization of other plasmids. The basal rate of conjugation observed was high. Under the conditions studied, ciliate predation did not appear to influence the conjugation rate, except at higher proportions of ciliates, which typically hampered conjugation. Microscopy revealed that most bacteria were digested in these conditions. PCR screening demonstrated that small mobilizable plasmids from A. salmonicida (pAsa1, pAsa2, pAsa3, and pAsal1) were acquired by the recipients along with the conjugative plasmids, with a slight effect of the ciliates in some donor/recipient cell combination. These results highlight how A. salmonicida can conjugate efficiently with different species and how complex its relationship with ciliates is.

Genetic study of Salmonella spp. Producting Betalatimase (ESBLs)

Ten isolates were collected from different clinical sources from laboratory in medicine century . These isolates were belonging to the genus Salmonella depending on morphological and biochemical tests . The antibiotic scussptibility tests against 10 antibiotics were examined , and it was found that the 60% isolates have multiple resistant to antibiotic ,(70%) of isolates were resistant to ampicillin,(50%) were resistant to augmentin ,(40%) were resistant to ceftriaxone ,(20%) were resistant to cefotaxime and (10%) were resistant to ciprofloxacin and tetracycline while all isolates showed sensitivity to piperacillin, imipenem, amikacin and erythromycin .The ability of Salmonela isolates to produce ?-lactamase enzymes were tested using iodometric method , and the results showed that all isolates produced this enzyme.The ability of these isolates to produce Extended-Spectrum ?-lactamase (ESBLs)were also determined by double disc synergy test , only five isolates produced these enzyme. Agarose gel electrophoresis showed that Salmonella isolates ?-lactamase producer have two small plasmid bands . Transformation experiments revealed that these plasmids were capable to transform E. coli MM294, an observation which indicates the ability of these plasmids to show their expression in more than one host.

Global Distribution and Diversity of Haloarchaeal pL6-Family Plasmids.

Australian isolates of Haloquadratum walsbyi, a square-shaped haloarchaeon, often harbor small cryptic plasmids of the pL6-family, approximately 6 kb in size, and five examples have been previously described. These plasmids exhibit a highly conserved gene arrangement and encode replicases similar to those of betapleolipoviruses. To assess their global distribution and recover more examples for analysis, fifteen additional plasmids were reconstructed from the metagenomes of seven hypersaline sites across four countries: Argentina, Australia, Puerto Rico, and Spain. Including the five previously described plasmids, the average plasmid size is 6002 bp, with an average G+C content of 52.5%. The tetramers GGCC and CTAG are either absent or significantly under-represented, except in the two plasmids with the highest %G+C. All plasmids share a similar arrangement of genes organized as outwardly facing replication and ATPase modules, but variations were observed in some core genes, such as F2, and some plasmids had acquired accessory genes. Two plasmids, pCOLO-c1 and pISLA-c6, shared 92.7% nt identity despite originating from Argentina and Spain, respectively. Numerous metagenomic CRISPR spacers matched sequences in the fifteen reconstructed plasmids, indicating frequent invasion of haloarchaea. Spacers could be assigned to haloarchaeal genera by mapping their associated direct repeats (DR), with half of these matching Haloquadratum. Finally, strand-specific metatranscriptome (RNA-seq) data could be used to demonstrate the active transcription of two pL6-family plasmids, including antisense transcripts.

Comparative genomics of IncQ1 plasmids carrying blaGES variants from clinical and environmental sources in Brazil

IncQ-type plasmids have become important vectors in the dissemination of blaGES among different bacterial genera and species from different environments around the world, and studies estimating the occurrence of Guiana extended-spectrum (GES)-type β-lactamases are gaining prominence. We analyzed the genetic aspects of two IncQ1 plasmids harboring different blaGES variants from human and environmental sources. The blaGES variants were identified using polymerase chain reaction (PCR) in Aeromonas veronii isolated from hospital effluent and Klebsiella variicola isolated from a rectal swab of a patient admitted to the cardiovascular intensive care unit in a different hospital. Antimicrobial-susceptibility testing and transformation experiments were performed for phenotypic analysis. Whole-genome sequencing was performed using Illumina and Oxford Nanopore platforms. The comparative analysis of plasmids was performed using BLASTn, and the IncQ1 plasmids showed a high identity and similar size. A. veronii harbored blaGES-7 in a class 1 integron (In2061), recently described by our group, and K. variicola carried blaGES-5 in the known class 1 integron. Both integrons showed a fused gene cassette that encodes resistance to aminoglycosides and fluoroquinolones, with an IS6100 truncating the 3′-conserved segment. The fused genes are transcribed together, although the attC site is disrupted. These gene cassettes can no longer be mobilized. This study revealed a mobilome that may contribute to the dissemination of GES-type β-lactamases in Brazil. Class 1 integrons are hot spots for bacterial evolution, and their insertion into small IncQ-like plasmids displayed successful recombination, allowing the spread of blaGES variants in various environments. Therefore, they can become prevalent across clinically relevant pathogens.

Complete genome sequence of multidrug-resistant Raoultella terrigena strain RT01-5M1 isolated from farmed salmon in Canada.

The genome sequence of multidrug-resistant Raoultella terrigena RT01-5M1 strain isolated from Canadian farmed salmon was determined using Oxford nanopore and Illumina MiSeq sequencers. The assembled chromosome was estimated at 5,699,993 bp in size, with two plasmids, 164,879 bp and 82,046 bp. The chromosome and smaller plasmid contained antimicrobial resistance genes.

Hybracter: enabling scalable, automated, complete and accurate bacterial genome assemblies.

Improvements in the accuracy and availability of long-read sequencing mean that complete bacterial genomes are now routinely reconstructed using hybrid (i.e. short- and long-reads) assembly approaches. Complete genomes allow a deeper understanding of bacterial evolution and genomic variation beyond single nucleotide variants. They are also crucial for identifying plasmids, which often carry medically significant antimicrobial resistance genes. However, small plasmids are often missed or misassembled by long-read assembly algorithms. Here, we present Hybracter which allows for the fast, automatic and scalable recovery of near-perfect complete bacterial genomes using a long-read first assembly approach. Hybracter can be run either as a hybrid assembler or as a long-read only assembler. We compared Hybracter to existing automated hybrid and long-read only assembly tools using a diverse panel of samples of varying levels of long-read accuracy with manually curated ground truth reference genomes. We demonstrate that Hybracter as a hybrid assembler is more accurate and faster than the existing gold standard automated hybrid assembler Unicycler. We also show that Hybracter with long-reads only is the most accurate long-read only assembler and is comparable to hybrid methods in accurately recovering small plasmids.

Integrative and Conjugative Elements and Prophage DNA as Carriers of Resistance Genes in Erysipelothrix rhusiopathiae Strains from Domestic Geese in Poland.

Goose erysipelas is a serious problem in waterfowl breeding in Poland. However, knowledge of the characteristics of Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, the antimicrobial susceptibility and serotypes of four E. rhusiopathiae strains from domestic geese were determined, and their whole-genome sequences (WGSs) were analyzed to detect resistance genes, integrative and conjugative elements (ICEs), and prophage DNA. Sequence type and the presence of resistance genes and transposons were compared with 363 publicly available E. rhusiopathiae strains, as well as 13 strains of other Erysipelothrix species. Four strains tested represented serotypes 2 and 5 and the MLST groups ST 4, 32, 242, and 243. Their assembled circular genomes ranged from 1.8 to 1.9 kb with a GC content of 36-37%; a small plasmid was detected in strain 1023. Strains 1023 and 267 were multidrug-resistant. The resistance genes detected in the genome of strain 1023 were erm47, tetM, and lsaE-lnuB-ant(6)-Ia-spw cluster, while strain 267 contained the tetM and ermB genes. Mutations in the gyrA gene were detected in both strains. The tetM gene was embedded in a Tn916-like transposon, which in strain 1023, together with the other resistance genes, was located on a large integrative and conjugative-like element of 130 kb designated as ICEEr1023. A minor integrative element of 74 kb was identified in strain 1012 (ICEEr1012). This work contributes to knowledge about the characteristics of E. rhusiopathiae bacteria and, for the first time, reveals the occurrence of erm47 and ermB resistance genes in strains of this species. Phage infection appears to be responsible for the introduction of the ermB gene into the genome of strain 267, while ICEs most likely play a key role in the spread of the other resistance genes identified in E. rhusiopathiae.

Complete genome sequence of the rumen bacterium Butyrivibrio fibrisolvens D1T.

Butyrivibrio are anaerobic bacteria and members of the family Lachnospiraceae with important roles in fiber digestion in both animals and humans. This report describes the complete genome of Butyrivibrio fibrisolvens type strain D1T (DSM 3071) consisting of a chromosome (CP146963), megaplasmid (pNP243), and small plasmid (pNP21).

An improved technique for direct visualization of plasmid patterns in strains of Paenibacillus larvae

American Foulbrood disease (AFB) caused by Paenibacillus larvae is the most devastating bacterial disease affecting honey bee brood worldwide. Beekeepers have been using oxytetracycline for decades to prevent and control AFB. In some countries with high disease incidence, antibiotics are still employed with the consequent risks of the appearance of tetracycline-resistant phenotypes associated with small plasmids carrying a tetracycline-resistance gene. Techniques for plasmid visualization in Gram-positive bacteria, particularly spore-forming, are not always successful. The present study aims to improve a method for direct visualization of plasmid patterns in P. larvae and other spore-forming bacteria isolated from apiarian sources. We tested 51 P. larvae strains from different geographical areas, including tetracycline-sensitive and resistant ones. The results revealed six different plasmid profiles, named I, II, III, IV, and V. Within tetracycline-resistant strains, four strains showed pattern IV with two plasmids of 5,000 and 8,000 bp, and two strains exhibited pattern V, with bands of about 5,000, 7,000, and 8,000 bp. The plasmid band of 5,000 bp present in the six resistant strains corresponds to the tetracycline-resistance mobilizable plasmid reported for P. larvae. Within the tetracycline-sensitive phenotypes, 33 strains showed no plasmids (pattern I); while two additional profiles were detected in the rest, i.e., pattern III with three plasmids in one strain and pattern VI showing two plasmids in six strains. Patterns II, III, and VI are probably related to the presence of cryptic plasmids. In conclusion, the improved technique allows us to identify plasmid patterns in P. larvae, ensuring their reproducibility and feasibility.

Effects of eutrophication on the horizontal transfer of antibiotic resistance genes in microalgal-bacterial symbiotic systems

Overloading of nutrients such as nitrogen causes eutrophication of freshwater bodies. The spread of antibiotic resistance genes (ARGs) poses a threat to ecosystems. However, studies on the enrichment and spread of ARGs from increased nitrogen loading in algal-bacterial symbiotic systems are limited. In this study, the transfer of extracellular kanamycin resistance (KR) genes from large (RP4) small (pEASY-T1) plasmids into the intracellular and extracellular DNA (iDNA, eDNA) of the inter-algal environment of Chlorella pyrenoidosa was investigated, along with the community structure of free-living (FL) and particle-attached (PA) bacteria under different nitrogen source concentrations (0–2.5 g/L KNO3). The results showed that KR gene abundance in the eDNA adsorbed on solid particles (D-eDNA) increased initially and then decreased with increasing nitrogen concentration, while the opposite was true for the rest of the free eDNA (E-eDNA). Medium nitrogen concentrations promoted the transfer of extracellular KR genes into the iDNA attached to algal microorganisms (A-iDNA), eDNA attached to algae (B-eDNA), and the iDNA of free microorganisms (C-iDNA); high nitrogen contributed to the transfer of KR genes into C-iDNA. The highest percentage of KR genes was found in B-eDNA with RP4 plasmid treatment (66.2%) and in C-iDNA with pEASY-T1 plasmid treatment (86.88%). In addition, dissolved oxygen (DO) significantly affected the bacterial PA and FL community compositions. Nephelometric turbidity units (NTU) reflected the abundance of ARGs in algae. Proteobacteria, Cyanobacteria, Bacteroidota, and Actinobacteriota were the main potential hosts of ARGs. These findings provide new insights into the distribution and dispersal of ARGs in the phytoplankton inter-algal environment.

A15 LEVERAGING CULTURE-DEPENDENT AND -INDEPENDENT METAGENOMICS TO IDENTIFY ENTEROBACTERIACEAE GENES ASSOCIATED WITH ACTIVE ULCERATIVE COLITIS

Abstract Background Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) that is restricted to the large intestine and is characterized by mucosal inflammation. There is evidence that pathogenic bacteria contribute to UC pathogenesis in at least some patients and the bacterial family Enterobacteriaceae are prime suspects. Some strains of Enterobacteriaceae carry virulence genes important for colonizing the gut (e.g. fimH) and disrupting the intestinal epithelium (e.g. hemolysins). We hypothesize that key virulence genes carried by strains of many Enterobacteriaceae species contribute to disease activity in some UC patients. As we predict virulence genes are strain-specific, the detection of the species alone (e.g. Escherichia coli) is insufficient to predict pathogenic potential and this has confounded previous studies to identify infectious agents in UC. Aims We aim to use culture-dependent and -independent sequencing approaches to identify Enterobacteriaceae genes in the UC gut microbiome associated with active disease. Methods UC patient stool samples collected throughout enrolment in randomized control trials of fecal microbiota transplantation (FMT) for adult UC (n=10) and microbiome studies in early-onset pediatric UC (n=25) were cultured on MacConkey (MAC) agar to enrich for Enterobacteriaceae. Strains were isolated from baseline stool samples cultured on MAC agar for whole genome sequencing and virulence gene characterization. Baseline and post-treatment stool samples cultured on MAC agar were sent for metagenomic sequencing. Using a subset of 11 pediatric patients, I developed a metagenomic pipeline to identify Enterobacteriaceae genes enriched during active UC compared to periods of remission or milder disease. Results Known virulence genes, including fimH and hemolysins, were present in some strains across multiple Enterobacteriaceae species. Using an unbiased metagenomic approach with the pediatric cohort, we identified 42 genes enriched at baseline with the criteria they must be elevated in at least three of the 11 patients. The majority of the 42 genes were distributed into six gene clusters, including a small plasmid. Conclusions Our approach allows us to identify genes enriched in active UC that have not been previously described in the literature and our analysis will be repeated with our adult cohort. Genes that are enriched in active UC in the pediatric and/or adult cohort will be validated in publicly available metagenomic datasets that consist of both IBD patients and healthy controls. Furthermore, culture-dependent approaches allow us to test mechanisms in vivo that are informed by our bioinformatics. Leveraging microbiome and clinical data provides a unique window to guide future diagnostics and treatments to improve outcomes for UC patients. Funding Agencies CIHROGS

PUdOs: Concise Plasmids for Bacterial and Mammalian Cells.

The plasmids from the Université d'Ottawa (pUdOs) are 28 small plasmids each comprising one of four origins of replication and one of seven selection markers, which together afford flexible use in Escherichia coli and several related gram-negative bacteria. The promoterless multicloning site is insulated from upstream spurious promoters by strong transcription terminators and contains type IIP or IIS restriction sites for conventional or Golden Gate cloning. pUdOs can be converted into efficient expression vectors through the insertion of a promoter at the user's discretion. For example, we demonstrate the utility of pUdOs as the backbone for an improved version of a Type III Secretion System reporter in Shigella. In addition, we derive a series of pUdO-based mammalian expression vectors, affording distinct levels of expression and transfection efficiency comparable to commonly used mammalian expression plasmids. Thus, pUdOs could advantageously replace traditional plasmids in a wide variety of cell types and applications.

Genomic insights into the evolution and adaptation of secondary metabolite gene clusters in fungicolous species Cladobotryum mycophilum ATHUM6906.

Mycophilic or fungicolous fungi can be found wherever fungi exist since they are able to colonize other fungi, which occupy a diverse range of habitats. Some fungicolous species cause important diseases on Basidiomycetes, and thus, they are the main reason for the destruction of mushroom cultivations. Nonetheless, despite their ecological significance, their genomic data remain limited. Cladobotryum mycophilum is one of the most aggressive species of the genus, destroying the economically important Agaricus bisporus cultivations. The 40.7 Mb whole genome of the Greek isolate ATHUM6906 is assembled in 16 fragments, including the mitochondrial genome and 2 small circular mitochondrial plasmids, in this study. This genome includes a comprehensive set of 12,282 protein coding, 56 rRNA, and 273 tRNA genes. Transposable elements, CAZymes, and pathogenicity related genes were also examined. The genome of C. mycophilum contained a diverse arsenal of genes involved in secondary metabolism, forming 106 biosynthetic gene clusters, which renders this genome as one of the most BGC abundant among fungicolous species. Comparative analyses were performed for genomes of species of the family Hypocreaceae. Some BGCs identified in C. mycophilum genome exhibited similarities to clusters found in the family Hypocreaceae, suggesting vertical heritage. In contrast, certain BGCs showed a scattered distribution among Hypocreaceae species or were solely found in Cladobotryum genomes. This work provides evidence of extensive BGC losses, horizontal gene transfer events, and formation of novel BGCs during evolution, potentially driven by neutral or even positive selection pressures. These events may increase Cladobotryum fitness under various environmental conditions and potentially during host-fungus interaction.

Complete genome sequence of bacteriocin-producing Ligilactobacillus salivarius B4311 isolated from fecal samples of broiler chicken with anti-listeria activity.

Ligilactobacillus is a genus of Gram-positive lactobacilli commonly found in the intestinal tracts of vertebrates. It has been granted a Qualified Presumption of Safety (QPS) status from the European Food Safety Authority (EFSA). One specific strain, Ligilactobacillus salivarius B4311, was isolated from fecal samples of broiler chickens from a farm associated with Chung-Ang University (Anseong, Korea). This strain was observed to have inhibitory effects against Listeria monocytogenes. In this paper, we present the complete genome sequence of Lig. salivarius B4311. The whole genome of strain B4311 comprises 2,071,255 bp assembled into 3 contigs representing a chromosome, repA-type megaplasmid, and small plasmid. The genome contains 1,963 protein-coding sequences, 22 rRNA genes, and 78 tRNA genes, with a guanine + cytosine (GC) content of 33.1%. The megaplasmid of strain B4311 was found to contain the bacteriocin gene cluster for salivaricin P, a two-peptide bacteriocin belonging to class IIb.

Transcriptional landscape of the pMP7017 megaplasmid and its impact on the Bifidobacterium breve UCC2003 transcriptome.

The 190 kb megaplasmid pMP7017 of Bifidobacterium breve JCM7017 represents the first conjugative and largest plasmid characterised within this genus to date. In the current study, we adopted an integrated approach combining transcriptomics, whole genome comparative analysis and metagenomic data mining to understand the biology of pMP7017 and related megaplasmids, and to assess the impact of plasmid-carriage on the host strain. The data generated revealed variations within basic features of promoter elements which correlate with a high level of transcription on the plasmid and highlight the transcriptional activity of genes encoding both offensive and defensive adaptations, including a Type IIL restriction-modification system, an anti-restriction system and four Type II toxin-antitoxin systems. Furthermore, a highly transcribed tmRNA, which likely provides translational support to the host strain, was identified, making pMP7017 the first plasmid of the Bifidobacterium genus and the smallest plasmid known to express a tmRNA. Analyses of synteny and variability among pMP7017 and related plasmids indicate substantial diversity in gene organisation and accessory gene cargo highlighting diverse (co-)evolution and potential host-specific rearrangements and adaptations. Systematic analysis of the codon usage profile of transcriptionally active pMP7017-encoded genes suggests that pMP7017 originated from (sub)species of Bifidobacterium longum. Furthermore, mining of metagenomic data suggests the presence of pMP7017-homologues in ~10% of microbiome samples, mostly infants and/or mothers from various geographical locations. Comparative transcriptome analysis of the B. breve UCC2003 chromosome in the presence or absence of pMP7017 revealed differential expression of genes representing 8% of the total gene pool. Genes involved in genetic information processing were exclusively upregulated, while altered expression of genes involved in biofilm production and polysaccharide biosynthesis was also observed.

Novel Plastid Genome Characteristics in Fugacium kawagutii and the Trend of Accelerated Evolution of Plastid Proteins in Dinoflagellates.

Typical (peridinin-containing) dinoflagellates possess plastid genomes composed of small plasmids named "minicircles". Despite the ecological importance of dinoflagellate photosynthesis in corals and marine ecosystems, the structural characteristics, replication dynamics, and evolutionary forcing of dinoflagellate plastid genomes remain poorly understood. Here, we sequenced the plastid genome of the symbiodiniacean species Fugacium kawagutii and conducted comparative analyses. We identified psbT-coding minicircles, features previously not found in Symbiodiniaceae. The copy number of F. kawagutii minicircles showed a strong diel dynamics, changing between 3.89 and 34.3 copies/cell and peaking in mid-light period. We found that F. kawagutii minicircles are the shortest among all dinoflagellates examined to date. Besides, the core regions of the minicircles are highly conserved within genus in Symbiodiniaceae. Furthermore, the codon usage bias of the plastid genomes in Heterocapsaceae, Amphidiniaceae, and Prorocentraceae species are greatly influenced by selection pressure, and in Pyrocystaceae, Symbiodiniaceae, Peridiniaceae, and Ceratiaceae species are influenced by both natural selection pressure and mutation pressure, indicating a family-level distinction in codon usage evolution in dinoflagellates. Phylogenetic analysis using 12 plastid-encoded proteins and five nucleus-encoded plastid proteins revealed accelerated evolution trend of both plastid- and nucleus-encoded plastid proteins in peridinin- and fucoxanthin-dinoflagellate plastids compared to plastid proteins of nondinoflagellate algae. These findings shed new light on the structure and evolution of plastid genomes in dinoflagellates, which will facilitate further studies on the evolutionary forcing and function of the diverse dinoflagellate plastids. The accelerated evolution documented here suggests plastid-encoded sequences are potentially useful for resolving closely related dinoflagellates.

Evaluation of Safety and Probiotic Traits from a Comprehensive Genome-Based In Silico Analysis of Ligilactobacillus salivarius P1CEA3, Isolated from Pigs and Producer of Nisin S.

Ligilactobacillus salivarius is an important member of the porcine gastrointestinal tract (GIT). Some L. salivarius strains are considered to have a beneficial effect on the host by exerting different probiotic properties, including the production of antimicrobial peptides which help maintain a healthy gut microbiota. L. salivarius P1CEA3, a porcine isolated strain, was first selected and identified by its antimicrobial activity against a broad range of pathogenic bacteria due to the production of the novel bacteriocin nisin S. The assembled L. salivarius P1CEA3 genome includes a circular chromosome, a megaplasmid (pMP1CEA3) encoding the nisin S gene cluster, and two small plasmids. A comprehensive genome-based in silico analysis of the L. salivarius P1CEA3 genome reveals the presence of genes related to probiotic features such as bacteriocin synthesis, regulation and production, adhesion and aggregation, the production of lactic acid, amino acids metabolism, vitamin biosynthesis, and tolerance to temperature, acid, bile salts and osmotic and oxidative stress. Furthermore, the strain is absent of risk-related genes for acquired antibiotic resistance traits, virulence factors, toxic metabolites and detrimental metabolic or enzymatic activities. Resistance to common antibiotics and gelatinase and hemolytic activities have been discarded by in vitro experiments. This study identifies several probiotic and safety traits of L. salivarius P1CEA3 and suggests its potential as a promising probiotic in swine production.

Transposon-aided capture (TRACA) of plasmids from the human gut.

The gut microbiota consists of a vast and diverse assemblage of microorganisms that play a pivotal role in maintaining host health. Nevertheless, a significant portion of the human gut microbiota remains uncultivated. Plasmids, a type of MGE, assume a critical function in the biological evolution and adaptation of bacteria to varying environments. To investigate the plasmids present within the gut microbiota community, we used the transposon-aided capture method (TRACA) to explore plasmids derived from the gut microbiota. In this study, fecal samples were collected from two healthy human volunteers and subsequently subjected to the TRACA method for plasmid isolation. Then, the complete sequence of the plasmids was obtained using the genome walking method, and sequence identity was also analyzed. A total of 15 plasmids were isolated. At last, 13 plasmids were successfully sequenced, of which 12 plasmids were highly identical to the plasmids in the National Center for Biotechnology Information (NCBI) database and were all small plasmids. Furthermore, a putative novel plasmid, named pMRPHD, was isolated, which had mobilized elements (oriT and oriV) and a potential type II restriction-modification (R-M) system encoded by DNA cytosine methyltransferase and type II restriction enzyme (Ban I), whose specific functions and applications warrant further exploration.

Insights into the circular: The cryptic plasmidome and its derived antibiotic resistome in the urban water systems

Plasmids have been a concern in the dissemination and evolution of antibiotic resistance in the environment. In this study, we investigated the total pool of plasmids (plasmidome) and its derived antibiotic resistance genes (ARGs) in different compartments of urban water systems (UWSs) in three European countries representing different antibiotic usage regimes. We applied a direct plasmidome approach using wet-lab methods to enrich circular DNA in the samples, followed by shotgun sequencing and in silico contig circularisation. We identified 9538 novel sequences in a total of 10,942 recovered circular plasmids. Of these, 66 were identified as conjugative, 1896 mobilisable and 8970 non-mobilisable plasmids. The UWSs’ plasmidome was dominated by small plasmids (≤10 Kbp) representing a broad diversity of mobility (MOB) types and incompatibility (Inc) groups. A shared collection of plasmids from different countries was detected in all treatment compartments, and plasmids could be source-tracked in the UWSs. More than half of the ARGs-encoding plasmids carried mobility genes for mobilisation/conjugation. The richness and abundance of ARGs-encoding plasmids generally decreased with the flow, while we observed that non-mobilisable ARGs-harbouring plasmids maintained their abundance in the Spanish wastewater treatment plant. Overall, our work unravels that the UWS plasmidome is dominated by cryptic (i.e., non-mobilisable, non-typeable and previously unknown) plasmids. Considering that some of these plasmids carried ARGs, were prevalent across three countries and could persist throughout the UWSs compartments, these results should alarm and call for attention.

Machine Learning Suggests That Small Size Helps Broaden Plasmid Host Range.

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.