Spread of plasmids carrying antibiotic resistance genes in soil-lettuce-snail food chain.

Fertilization can change the composition of antibiotic resistance genes(ARGs) and their host bacteria in agricultural fields, while complex microbial activities help ARGs into crops and transmit them to humans through agricultural products.Therefore, this study constructed a farmland food chain with soil-lettuce-snail as a typical structure, added genetically engineered Pseudomonas fluorescens containing multidrug-resistant plasmid RP4 to track its spread in the farmland food chain, and used different fertilization methods to explore its influence on the spread and diffusion of ARGs and intl1 in the farmland food chain. It was found that exogenous Pseudomonas can enter plants from soil and pass into snails' intestines, and there is horizontal gene transfer phenomenon of RP4 plasmid in bacteria. At different interfaces of the constructed food chain, the addition of exogenous drug-resistant bacteria had different effects on the total abundance of ARGs and intl1. Fertilization, especially manure, not only promoted the spread of Pseudomonas aeruginosa and the transfer of RP4 plasmid levels, but also significantly increased the total abundance of ARGs and intl1 at all interfaces of the constructed food chain. The main ARGs host bacteria in the constructed food chain include Proteobacteria, Bacteroides, and Firmicutes, while Flavobacterium of Bacteroides is the unique potential host bacteria of RP4 plasmid. In conclusion, this study provides a reference for the risk assessment of ARGs transmitted to the human body through the food chain, and has important practical significance to reduce the antibiotic resistance contamination of agricultural products and ensure the safety of vegetable basket.

Extensive remodeling of sugar metabolism through gene loss and horizontal gene transfer in a eukaryotic lineage.

BACKGROUND: In yeasts belonging to the subphylum Saccharomycotina, genes encoding components of the main metabolic pathways, like alcoholic fermentation, are usually conserved. However, in fructophilic species belonging to the floral Wickerhamiella and Starmerella genera (W/S clade), alcoholic fermentation was uniquely shaped by events of gene loss and horizontal gene transfer (HGT). RESULTS: Because HGT and gene losses were first identified when only eight W/S-clade genomes were available, we collected publicly available genome data and sequenced the genomes of 36 additional species. A total of 63 genomes, representing most of the species described in the clade, were included in the analyses. Firstly, we inferred the phylogenomic tree of the clade and inspected the genomes for the presence of HGT-derived genes involved in fructophily and alcoholic fermentation. We predicted nine independent HGT events and several instances of secondary loss pertaining to both pathways. To investigate the possible links between gene loss and acquisition events and evolution of sugar metabolism, we conducted phenotypic characterization of 42 W/S-clade species including estimates of sugar consumption rates and fermentation byproduct formation. In some instances, the reconciliation of genotypes and phenotypes yielded unexpected results, such as the discovery of fructophily in the absence of the cornerstone gene (FFZ1) and robust alcoholic fermentation in the absence of the respective canonical pathway. CONCLUSIONS: These observations suggest that reinstatement of alcoholic fermentation in the W/S clade triggered a surge of innovation that goes beyond the utilization of xenologous enzymes, with fructose metabolism playing a key role.

Accelerated dissemination of antibiotic resistant genes via conjugative transfer driven by deficient denitrification in biochar-based biofiltration systems.

Biofiltration systems harbored and disseminated antibiotic resistance genes (ARGs), when confronting antibiotic-contained wastewater. Biochar, a widely used environmental remediation material, can mitigate antibiotic stress on adjoining microbes by lowering the availability of sorbed antibiotics, and enhance the attachment of denitrifiers. Herein, bench-scale biofiltration systems, packed with commercial biochars, were established to explore the pivotal drivers affecting ARG emergence. Results showed that biofiltration columns, achieving higher TN removal and denitrification capacity, showed a significant decrease in ARG accumulation (p < 0.05). The relative abundance of ARGs (0.014 ± 0.0008) in the attached biofilms decreased to 1/5-folds of that in the control group (0.065 ± 0.004). Functional analysis indicated ARGs' accumulation was less attributed to ARG activation or horizontal gene transfer (HGT) driven by sorbed antibiotics. Most denitrifiers, like Bradyrhizobium, Geothrix, etc., were found to be enriched and host ARGs. Nitrosative stress from deficient denitrification was demonstrated to be the dominant driver for affecting ARG accumulation and dissemination. Metagenomic and metaproteomic analysis revealed that nitrosative stress promoted the conjugative HGT of ARGs mainly via increasing the transmembrane permeability and enhancing the amino acid transport and metabolism, such as cysteine, methionine, and valine metabolism. Overall, this study highlighted the risks of deficient denitrification in promoting ARG transfer and transmission in biofiltration systems and natural ecosystems.

Sporadic clone Escherichia coli ST615 as a vector and reservoir for dissemination of crucial antimicrobial resistance genes.

There is scarce information concerning the role of sporadic clones in the dissemination of antimicrobial resistance genes (ARGs) within the nosocomial niche. We confirmed that the clinical Escherichia coli M19736 ST615 strain, one of the first isolates of Latin America that harbors a plasmid with an mcr-1 gene, could receive crucial ARG by transformation and conjugation using as donors critical plasmids that harbor bla CTX-M-15, bla KPC-2, bla NDM-5, bla NDM-1, or aadB genes. Escherichia coli M19736 acquired bla CTX-M-15, bla KPC-2, bla NDM-5, bla NDM-1, and aadB genes, being only blaNDM-1 maintained at 100% on the 10th day of subculture. In addition, when the evolved MDR-E. coli M19736 acquired sequentially bla CTX-M-15 and bla NDM-1 genes, the maintenance pattern of the plasmids changed. In addition, when the evolved XDR-E. coli M19736 acquired in an ulterior step the paadB plasmid, a different pattern of the plasmid's maintenance was found. Interestingly, the evolved E. coli M19736 strains disseminated simultaneously the acquired conjugative plasmids in different combinations though selection was ceftazidime in all cases. Finally, we isolated and characterized the extracellular vesicles (EVs) from the native and evolved XDR-E. coli M19736 strains. Interestingly, EVs from the evolved XDR-E. coli M19736 harbored bla CTX-M-15 though the pDCAG1-CTX-M-15 was previously lost as shown by WGS and experiments, suggesting that EV could be a relevant reservoir of ARG for susceptible bacteria. These results evidenced the genetic plasticity of a sporadic clone of E. coli such as ST615 that could play a relevant transitional link in the clinical dynamics and evolution to multidrug/extensively/pandrug-resistant phenotypes of superbugs within the nosocomial niche by acting simultaneously as a vector and reservoir of multiple ARGs which later could be disseminated.

Specificities and commonalities of the Planctomycetes plasmidome.

Plasmids, despite their critical role in antibiotic resistance and modern biotechnology, are understood in only a few bacterial groups in terms of their natural ecological dynamics. The bacterial phylum Planctomycetes, known for its unique molecular and cellular biology, has a largely unexplored plasmidome. This study offers a thorough exploration of the diversity of natural plasmids within Planctomycetes, which could serve as a foundation for developing various genetic research tools for this phylum. Planctomycetes plasmids encode a broad range of biological functions and appear to have coevolved significantly with their host chromosomes, sharing many homologues. Recent transfer events of insertion sequences between cohabiting chromosomes and plasmids were also observed. Interestingly, 64% of plasmid genes are distantly related to either chromosomally encoded genes or have homologues in plasmids from other bacterial groups. The planctomycetal plasmidome is composed of 36% exclusive proteins. Most planctomycetal plasmids encode a replication initiation protein from the Replication Protein A family near a putative iteron-containing replication origin, as well as active type I partition systems. The identification of one conjugative and three mobilizable plasmids suggests the occurrence of horizontal gene transfer via conjugation within this phylum. This comprehensive description enhances our understanding of the plasmidome of Planctomycetes and its potential implications in antibiotic resistance and biotechnology.

A novel approach to exploring the dark genome and its application to mapping of the vertebrate virus fossil record.

BACKGROUND: Genomic regions that remain poorly understood, often referred to as the dark genome, contain a variety of functionally relevant and biologically informative features. These include endogenous viral elements (EVEs)-virus-derived sequences that can dramatically impact host biology and serve as a virus fossil record. In this study, we introduce a database-integrated genome screening (DIGS) approach to investigate the dark genome in silico, focusing on EVEs found within vertebrate genomes. RESULTS: Using DIGS on 874 vertebrate genomes, we uncover approximately 1.1 million EVE sequences, with over 99% originating from endogenous retroviruses or transposable elements that contain EVE DNA. We show that the remaining 6038 sequences represent over a thousand distinct horizontal gene transfer events across 10 virus families, including some that have not previously been reported as EVEs. We explore the genomic and phylogenetic characteristics of non-retroviral EVEs and determine their rates of acquisition during vertebrate evolution. Our study uncovers novel virus diversity, broadens knowledge of virus distribution among vertebrate hosts, and provides new insights into the ecology and evolution of vertebrate viruses. CONCLUSIONS: We comprehensively catalog and analyze EVEs within 874 vertebrate genomes, shedding light on the distribution, diversity, and long-term evolution of viruses and reveal their extensive impact on vertebrate genome evolution. Our results demonstrate the power of linking a relational database management system to a similarity search-based screening pipeline for in silico exploration of the dark genome.

Mariculture waters as yet another hotbed for the creation and transfer of new antibiotic-resistant pathogenome.

With the rapid growth of aquaculture globally, large amounts of antibiotics have been used to treat aquatic disease, which may accelerate induction and spread of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in aquaculture environments. Herein, metagenomic and 16S rRNA analyses were used to analyze the potentials and co-occurrence patterns of pathogenome (culturable and unculturable pathogens), antibiotic resistome (ARGs), and mobilome (mobile genetic elements (MGEs)) from mariculture waters near 5000 km coast of South China. Total 207 species of pathogens were identified, with only 10 culturable species. Furthermore, more pathogen species were detected in mariculture waters than those in coastal waters, and mariculture waters were prone to become reservoirs of unculturable pathogens. In addition, 913 subtypes of 21 ARG types were also identified, with multidrug resistance genes as the majority. MGEs including plasmids, integrons, transposons, and insertion sequences were abundantly present in mariculture waters. The co-occurrence network pattern between pathogenome, antibiotic resistome, and mobilome suggested that most of pathogens may be potential multidrug resistant hosts, possibly due to high frequency of horizontal gene transfer. These findings increase our understanding of mariculture waters as reservoirs of antibiotic resistome and mobilome, and as yet another hotbed for creation and transfer of new antibiotic-resistant pathogenome.

Priority establishment of soil bacteria in rhizosphere limited the spread of tetracycline resistance genes from pig manure to soil-plant systems based on synthetic communities approach.

The spread of antibiotic resistance genes (ARGs) in agroecosystems through the application of animal manure is a global threat to human and environmental health. However, the adaptability and colonization ability of animal manure-derived bacteria determine the spread pathways of ARG in agroecosystems, which have rarely been studied. Here, we performed an invasion experiment by creating a synthetic communities (SynCom) with ten isolates from pig manure and followed its assembly during gnotobiotic cultivation of a soil-Arabidopsis thaliana (A. thaliana) system. We found that Firmicutes in the SynCom were efficiently filtered out in the rhizosphere, thereby limiting the entry of tetracycline resistance genes (TRGs) into the plant. However, Proteobacteria and Actinobacteria in the SynCom were able to establish in all compartments of the soil-plant system thereby spreading TRGs from manure to soil and plant. The presence of native soil bacteria prevented the establishment of manure-borne bacteria and effectively reduced the spread of TRGs. Achromobacter mucicolens and Pantoea septica were the main vectors for the entry of tetA into plants. Furthermore, doxycycline stress promoted the horizontal gene transfer (HGT) of the conjugative resistance plasmid RP4 within the SynCom in A. thaliana by upregulating the expression of HGT-related mRNAs. Therefore, this study provides evidence for the dissemination pathways of ARGs in agricultural systems through the invasion of manure-derived bacteria and HGT by conjugative resistance plasmids and demonstrates that the priority establishment of soil bacteria in the rhizosphere limited the spread of TRGs from pig manure to soil-plant systems.

Genetic recombination-mediated evolutionary interactions between phages of potential industrial importance and prophages of their hosts within or across the domains of Escherichia, Listeria, Salmonella, Campylobacter, and Staphylococcus.

BACKGROUND: The in-depth understanding of the role of lateral genetic transfer (LGT) in phage-prophage interactions is essential to rationalizing phage applications for human and animal therapy, as well as for food and environmental safety. This in silico study aimed to detect LGT between phages of potential industrial importance and their hosts. METHODS: A large array of genetic recombination detection algorithms, implemented in SplitsTree and RDP4, was applied to detect LGT between various Escherichia, Listeria, Salmonella, Campylobacter, Staphylococcus, Pseudomonas, and Vibrio phages and their hosts. PHASTER and RAST were employed respectively to identify prophages across the host genome and to annotate LGT-affected genes with unknown functions. PhageAI was used to gain deeper insights into the life cycle history of recombined phages. RESULTS: The split decomposition inferences (bootstrap values: 91.3-100; fit: 91.433-100), coupled with the Phi (0.0-2.836E-12) and RDP4 (P being well below 0.05) statistics, provided strong evidence for LGT between certain Escherichia, Listeria, Salmonella, and Campylobacter virulent phages and prophages of their hosts. The LGT events entailed mainly the phage genes encoding for hypothetical proteins, while some of these genetic loci appeared to have been affected even by intergeneric recombination in specific E. coli and S. enterica virulent phages when interacting with their host prophages. Moreover, it is shown that certain L. monocytogenes virulent phages could serve at least as the donors of the gene loci, involved in encoding for the basal promoter specificity factor, for L. monocytogenes. In contrast, the large genetic clusters were determined to have been simultaneously exchanged by many S. aureus prophages and some Staphylococcus temperate phages proposed earlier as potential therapeutic candidates (in their native or modified state). The above genetic clusters were found to encompass multiple genes encoding for various proteins, such as e.g., phage tail proteins, the capsid and scaffold proteins, holins, and transcriptional terminator proteins. CONCLUSIONS: It is suggested that phage-prophage interactions, mediated by LGT (including intergeneric recombination), can have a far-reaching impact on the co-evolutionary trajectories of industrial phages and their hosts especially when excessively present across microbially rich environments.

Investigating the nature of prokaryotic genomic island locations within a genome.

Horizontal gene transfer (HGT) is a powerful evolutionary force that considerably shapes the structure of prokaryotic genomes and is associated with genomic islands (GIs). A GI is a DNA segment composed of transferred genes that can be found within a prokaryotic genome, obtained through HGT. Much research has focused on detecting GIs in genomes, but here we pursue a new course, which is identifying possible preferred locations of GIs in the prokaryotic genome. Here, we identify the locations of the GIs within prokaryotic genomes to examine patterns in those locations. Prokaryotic GIs were analyzed according to the genome structure that they are located in, whether it be a circular or a linear genome. The analytical investigations employed are: (1) studying the GI locations in relation to the origin of replication (oriC); (2) exploring the distances between GIs; and (3) determining the distribution of GIs across the genomes. For each of the investigations, the analysis was performed on all of the GIs in the data set. Moreover, to void bias caused by the distribution of the genomes represented, the GIs in one genome from each species and the GIs of the most frequent species are also analyzed. Overall, the results showed that there are preferred sites for the GIs in the genome. In the linear genomes, these sites are usually located in the oriC region and terminus region, while in the circular genomes, they are located solely in the terminus region. These results also showed that the distance distribution between the GIs is almost exponential, which proves that GIs have preferred sites within genomes. The oriC and termniuns are preferred sites for the GIs and a possible natural explanation for this could be connected to the content of the oriC region. Moreover, the content of the GIs in terms of its protein families was studied and the results demonstrated that the majority of frequent protein families are close to identical in each section.

Dynamic evolution of the mTHF gene family associated with primary metabolism across life.

BACKGROUND: The folate cycle of one-carbon (C1) metabolism, which plays a central role in the biosynthesis of nucleotides and amino acids, demonstrates the significance of metabolic adaptation. We investigated the evolutionary history of the methylenetetrahydrofolate dehydrogenase (mTHF) gene family, one of the main drivers of the folate cycle, across life. RESULTS: Through comparative genomic and phylogenetic analyses, we found that several lineages of Archaea lacked domains vital for folate cycle function such as the mTHF catalytic and NAD(P)-binding domains of FolD. Within eukaryotes, the mTHF gene family diversified rapidly. For example, several duplications have been observed in lineages including the Amoebozoa, Opisthokonta, and Viridiplantae. In a common ancestor of Opisthokonta, FolD and FTHFS underwent fusion giving rise to the gene MTHFD1, possessing the domains of both genes. CONCLUSIONS: Our evolutionary reconstruction of the mTHF gene family associated with a primary metabolic pathway reveals dynamic evolution, including gene birth-and-death, gene fusion, and potential horizontal gene transfer events and/or amino acid convergence.

Phylogenomics reveals the evolutionary origins of lichenization in chlorophyte algae.

Mutualistic symbioses have contributed to major transitions in the evolution of life. Here, we investigate the evolutionary history and the molecular innovations at the origin of lichens, which are a symbiosis established between fungi and green algae or cyanobacteria. We de novo sequence the genomes or transcriptomes of 12 lichen algal symbiont (LAS) and closely related non-symbiotic algae (NSA) to improve the genomic coverage of Chlorophyte algae. We then perform ancestral state reconstruction and comparative phylogenomics. We identify at least three independent gains of the ability to engage in the lichen symbiosis, one in Trebouxiophyceae and two in Ulvophyceae, confirming the convergent evolution of the lichen symbioses. A carbohydrate-active enzyme from the glycoside hydrolase 8 (GH8) family was identified as a top candidate for the molecular-mechanism underlying lichen symbiosis in Trebouxiophyceae. This GH8 was acquired in lichenizing Trebouxiophyceae by horizontal gene transfer, concomitantly with the ability to associate with lichens fungal symbionts (LFS) and is able to degrade polysaccharides found in the cell wall of LFS. These findings indicate that a combination of gene family expansion and horizontal gene transfer provided the basis for lichenization to evolve in chlorophyte algae.

EDTA enables to alleviate impacts of metal ions on conjugative transfer of antibiotic resistance genes.

Various heavy metals are reported to be able to accelerate horizontal transfer of antibiotic resistance genes (ARGs). In real water environmental settings, ubiquitous complexing agents would affect the environmental behaviors of heavy metal ions due to the formation of metal-organic complexes. However, little is known whether the presence of complexing agents would change horizontal gene transfer due to heavy metal exposure. This study aimed to fill this gap by investigating the impacts of a typical complexing agent ethylenediaminetetraacetic acid (EDTA) on the conjugative transfer of plasmid-mediated ARGs induced by a range of heavy metal ions. At the environmentally relevant concentration (0.64 mg L-1) of metal ions, all the tested metal ions (Mg2+, Ca2+, Co2+, Pb2+, Ni2+, Cu2+, and Fe3+) promoted conjugative transfer of ARGs, while an inhibitory effect was observed at a relatively higher concentration (3.20 mg L-1). In contrast, EDTA (0.64 mg L-1) alleviated the effects of metal ions on ARGs conjugation transfer, evidenced by 11 %-66 % reduction in the conjugate transfer frequency. Molecular docking and dynamics simulations disclosed that this is attributed to the stronger binding of metal ions with the lipids in cell membranes. Under metal-EDTA exposure, gene expressions related to oxidative stress response, cell membrane permeability, intercellular contact, energy driving force, mobilization, and channels of plasmid transfer were suppressed compared with the metal ions exposure. This study offers insights into the alleviation mechanisms of complexing agents on ARGs transfer induced by free metal ions.

Genome analyses of colistin-resistant high-risk blaNDM-5 producing Klebsiella pneumoniae ST147 and Pseudomonas aeruginosa ST235 and ST357 in clinical settings.

BACKGROUND: Colistin is a last-resort antibiotic used in extreme cases of multi-drug resistant (MDR) Gram-negative bacterial infections. Colistin resistance has increased in recent years and often goes undetected due to the inefficiency of predominantly used standard antibiotic susceptibility tests (AST). To address this challenge, we aimed to detect the prevalence of colistin resistance strains through both Vitek®2 and broth micro-dilution. We investigated 1748 blood, tracheal aspirate, and pleural fluid samples from the Intensive Care Unit (ICU), Neonatal Intensive Care Unit (NICU), and Tuberculosis and Respiratory Disease centre (TBRD) in an India hospital. Whole-genome sequencing (WGS) of extremely drug-resitant (XDR) and pan-drug resistant (PDR) strains revealed the resistance mechanisms through the Resistance Gene Identifier (RGI.v6.0.0) and Snippy.v4.6.0. Abricate.v1.0.1, PlasmidFinder.v2.1, MobileElementFinder.v1.0.3 etc. detected virulence factors, and mobile genetic elements associated to uncover the pathogenecity and the role of horizontal gene transfer (HGT). RESULTS: This study reveals compelling insights into colistin resistance among global high-risk clinical isolates: Klebsiella pneumoniae ST147 (16/20), Pseudomonas aeruginosa ST235 (3/20), and ST357 (1/20). Vitek®2 found 6 colistin-resistant strains (minimum inhibitory concentrations, MIC = 4 µg/mL), while broth microdilution identified 48 (MIC = 32-128 µg/mL), adhering to CLSI guidelines. Despite the absence of mobile colistin resistance (mcr) genes, mechanisms underlying colistin resistance included mgrB deletion, phosphoethanolamine transferases arnT, eptB, ompA, and mutations in pmrB (T246A, R256G) and eptA (V50L, A135P, I138V, C27F) in K. pneumoniae. P. aeruginosa harbored phosphoethanolamine transferases basS/pmrb, basR, arnA, cprR, cprS, alongside pmrB (G362S), and parS (H398R) mutations. Both strains carried diverse clinically relevant antimicrobial resistance genes (ARGs), including plasmid-mediated blaNDM-5 (K. pneumoniae ST147) and chromosomally mediated blaNDM-1 (P. aeruginosa ST357). CONCLUSION: The global surge in MDR, XDR and PDR bacteria necessitates last-resort antibiotics such as colistin. However, escalating resistance, particularly to colistin, presents a critical challenge. Inefficient colistin resistance detection methods, including Vitek2, alongside limited surveillance resources, accentuate the need for improved strategies. Whole-genome sequencing revealed alarming colistin resistance among K. pneumoniae and P. aeruginosa in an Indian hospital. The identification of XDR and PDR strains underscores urgency for enhanced surveillance and infection control. SNP analysis elucidated resistance mechanisms, highlighting the complexity of combatting resistance.

Tandem gene duplication selected by activation of horizontally transferred gene in bacteria.

Horizontal gene transfer occurs frequently in bacteria, but the mechanism driving activation and optimization of the expression of horizontally transferred genes (HTGs) in new recipient strains is not clear. Our previous study found that spontaneous tandem DNA duplication resulted in rapid activation of HTGs. Here, we took advantage of this finding to develop a novel technique for tandem gene duplication, named tandem gene duplication selected by activation of horizontally transferred gene in bacteria (TDAH), in which tandem duplication was selected by the activation of horizontally transferred selectable marker gene. TDAH construction does not contain any reported functional elements based on homologous or site-specific recombination and DNA amplification. TDAH only contains an essential selectable marker for copy number selection and 9-bp-microhomology border sequences for precise illegitimate recombination. One transformation and 3 days were enough to produce a high-copy strain, so its procedure is simple and fast. Without subsequent knockout of the endogenous recombination system, TDAH could also generate the relatively stable high-copy tandem duplication for plasmid-carried and genome-integrated DNA. TDAH also showed an excellent capacity for increase gene expression and worked well in different industrial bacteria. We also applied TDAH to select the optimal high copy number of ribA for vitamin B2 production in E. coli; the yield was improved by 3.5 times and remained stable even after 12 subcultures. TDAH is a useful tool for recombinant protein production and expression optimization of biosynthetic pathways. KEY POINTS: • We develop a novel and efficient technique (TDAH) for tandem gene duplication in bacterium. TDAH is based on the mechanism of HTG rapid activation. TDAH does not contain any reported functional elements based on homologous recombination and DNA amplification. TDAH only contains an essential selectable marker for copy number selection, so its construction and procedure are very simple and fast. • TDAH is the first reported selected and stable tandem-gene-duplication technique in which the selected high-copy plasmid-carried and genome-integrated DNA could remain stable without the subsequent knockout of recombination system. • TDAH showed an excellent capacity for regulating gene expression and worked well in different industrial bacteria, indicating it is a useful tool for recombinant protein production and expression optimization of biosynthetic pathways. • TDAH was applied to select the optimal high copy number of ribA for vitamin B2 production in E. coli; the yield was improved by 3.5-fold and remained stable even after 12 subcultures.

Unveiling the genetic architecture and transmission dynamics of a novel multidrug-resistant plasmid harboring blaNDM-5 in E. Coli ST167: implications for antibiotic resistance management.

BACKGROUND: The emergence of multidrug-resistant (MDR) Escherichia coli strains poses significant challenges in clinical settings, particularly when these strains harbor New Delhi metallo-ß-lactamase (NDM) gene, which confer resistance to carbapenems, a critical class of last-resort antibiotics. This study investigates the genetic characteristics and implications of a novel blaNDM-5-carrying plasmid pNDM-5-0083 isolated from an E. coli strain GZ04-0083 from clinical specimen in Zhongshan, China. RESULTS: Phenotypic and genotypic evaluations confirmed that the E. coli ST167 strain GZ04-0083 is a multidrug-resistant organism, showing resistance to diverse classes of antibiotics including ß-lactams, carbapenems, fluoroquinolones, aminoglycosides, and sulfonamides, while maintaining susceptibility to monobactams. Investigations involving S1 pulsed-field gel electrophoresis, Southern blot analysis, and conjugation experiments, alongside genomic sequencing, confirmed the presence of the blaNDM-5 gene within a 146-kb IncFIB plasmid pNDM-5-0083. This evidence underscores a significant risk for the horizontal transfer of resistance genes among bacterial populations. Detailed annotations of genetic elements-such as resistance genes, transposons, and insertion sequences-and comparative BLAST analyses with other blaNDM-5-carrying plasmids, revealed a unique architectural configuration in the pNDM-5-0083. The MDR region of this plasmid shares a conserved gene arrangement (repA-IS15DIV-blaNDM-5-bleMBL-IS91-suI2-aadA2-dfrA12) with three previously reported plasmids, indicating a potential for dynamic genetic recombination and evolution within the MDR region. Additionally, the integration of virulence factors, including the iro and sit gene clusters and enolase, into its genetic architecture poses further therapeutic challenges by enhancing the strain's pathogenicity through improved host tissue colonization, immune evasion, and increased infection severity. CONCLUSIONS: The detailed identification and characterization of pNDM-5-0083 enhance our understanding of the mechanisms facilitating the spread of carbapenem resistance. This study illuminates the intricate interplay among various genetic elements within the novel blaNDM-5-carrying plasmid, which are crucial for the stability and mobility of resistance genes across bacterial populations. These insights highlight the urgent need for ongoing surveillance and the development of effective strategies to curb the proliferation of antibiotic resistance.

Genomic insights unveil the plasmid transfer mechanism and epidemiology of hypervirulent Klebsiella pneumoniae in Vietnam.

Hypervirulent Klebsiella pneumoniae (hvKp) is a significant cause of severe invasive infections in Vietnam, yet data on its epidemiology, population structure and dynamics are scarce. We screened hvKp isolates from patients with bloodstream infections (BSIs) at a tertiary infectious diseases hospital in Vietnam and healthy individuals, followed by whole genome sequencing and plasmid analysis. Among 700 BSI-causing Kp strains, 100 (14.3%) were hvKp. Thirteen hvKp isolates were identified from 350 rectal swabs of healthy adults; none from 500 rectal swabs of healthy children. The hvKp isolates were genetically diverse, encompassing 17 sequence types (STs), predominantly ST23, ST86 and ST65. Among the 113 hvKp isolates, 14 (12.6%) carried at least one antimicrobial resistance (AMR) gene, largely mediated by IncFII, IncR, and IncA/C plasmids. Notably, the acquisition of AMR conjugative plasmids facilitated horizontal transfer of the non-conjugative virulence plasmid between K. pneumoniae strains. Phylogenetic analysis demonstrated hvKp isolates from BSIs and human carriage clustered together, suggesting a significant role of intestinal carriage in hvKp transmission. Enhanced surveillance is crucial to understand the factors driving intestinal carriage and hvKp transmission dynamics for informing preventive measures. Furthermore, we advocate the clinical use of our molecular assay for diagnosing hvKp infections to guide effective management.

Plasmid-mediated horizontal gene mobilisation: Insights from two lactococcal conjugative plasmids.

The distinct conjugation machineries encoded by plasmids pNP40 and pUC11B represent the most prevalent plasmid transfer systems among lactococcal strains. In the current study, we identified genetic determinants that underpin pNP40- and pUC11B-mediated, high-frequency mobilisation of other, non-conjugative plasmids. The mobilisation frequencies of the smaller, non-conjugative plasmids and the minimal sequences required for their mobilisation were determined, owing to the determination of the oriT sequences of both pNP40 and pUC11B, which allowed the identification of similar sequences in some of the non-conjugative plasmids that were shown to promote their mobilisation. Furthermore, the auxiliary gene mobC, two distinct functional homologues of which are present in several plasmids harboured by the pNP40- and pUC11B-carrying host strains, was observed to confer a high-frequency mobilisation phenotype. These findings provide mechanistic insights into how lactococcal conjugative plasmids achieve conjugation and promote mobilisation of non-conjugative plasmids. Ultimately, these insights would be harnessed to optimise conjugation and mobilisation strategies for the rapid and predictable development of robust and technologically improved strains.

Integrative and conjugative elements of Pasteurella multocida: Prevalence and signatures in population evolution.

Pasteurella multocida (P. multocida) is a bacterial pathogen responsible for a range of infections in humans and various animal hosts, causing significant economic losses in farming. Integrative and conjugative elements (ICEs) are important horizontal gene transfer elements, potentially enabling host bacteria to enhance adaptability by acquiring multiple functional genes. However, the understanding of ICEs in P. multocida and their impact on the transmission of this pathogen remains limited. In this study, 42 poultry-sourced P. multocida genomes obtained by high-throughput sequencing together with 393 publicly available P. multocida genomes were used to analyse the horizontal transfer of ICEs. Eighty-two ICEs were identified in P. multocida, including SXT/R391 and Tn916 subtypes, as well as three subtypes of ICEHin1056 family, with the latter being widely prevalent in P. multocida and carrying multiple resistance genes. The correlations between insertion sequences and resistant genes in ICEs were also identified, and some ICEs introduced the carbapenem gene blaOXA-2 and the bleomycin gene bleO to P. multocida. Phylogenetic and collinearity analyses of these bioinformatics found that ICEs in P. multocida were transmitted vertically and horizontally and have evolved with host specialization. These findings provide insight into the transmission and evolution mode of ICEs in P. multocida and highlight the importance of understanding these elements for controlling the spread of antibiotic resistance.

PrgE: an OB-fold protein from plasmid pCF10 with striking differences to prototypical bacterial SSBs.

A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded type IV secretion systems (T4SSs). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many, their function remains unclear. Here, we characterize PrgE, a proposed SSB from the Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very unusual DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string supported by its N-terminal tail. In vitro studies highlight the plasticity of PrgE oligomerization and confirm the importance of the N-terminus. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from eukaryotic SSBs.