Exploring the role of phage plasmids in gene transfers.

Bacteriophages and plasmids drive horizontal gene transfer (HGT) in bacteria. Phage-plasmids (P-Ps) are hybrids of plasmid and phages. Pfeifer and Rocha recently demonstrated that P-Ps can serve as intermediates in gene exchanges between these two types of elements, identified categories of preferentially transferred genes, and reconstructed gene flows involving phage P1-like P-Ps.

Host-specific plasmid evolution explains the variable spread of clinical antibiotic-resistance plasmids.

Antibiotic resistance encoded on plasmids is a pressing global health problem. Predicting which plasmids spread in the long term remains very challenging, even though some key parameters influencing plasmid stability have been identified, such as plasmid growth costs and horizontal transfer rates. Here, we show these parameters evolve in a strain-specific way among clinical plasmids and bacteria, and this occurs rapidly enough to alter the relative likelihoods of different bacterium-plasmid combinations spreading. We used experiments with Escherichia coli and antibiotic-resistance plasmids isolated from patients, paired with a mathematical model, to track long-term plasmid stability (beyond antibiotic exposure). Explaining variable stability across six bacterium-plasmid combinations required accounting for evolutionary changes in plasmid stability traits, whereas initial variation of these parameters was a relatively poor predictor of long-term outcomes. Evolutionary trajectories were specific to particular bacterium-plasmid combinations, as evidenced by genome sequencing and genetic manipulation. This revealed epistatic (here, strain-dependent) effects of key genetic changes affecting horizontal plasmid transfer. Several genetic changes involved mobile elements and pathogenicity islands. Rapid strain-specific evolution can thus outweigh ancestral phenotypes as a predictor of plasmid stability. Accounting for strain-specific plasmid evolution in natural populations could improve our ability to anticipate and manage successful bacterium-plasmid combinations.

In vivo assembly of bacterial partition condensates on circular supercoiled and linear DNA.

In bacteria, faithful DNA segregation of chromosomes and plasmids is mainly mediated by ParABS systems. These systems, consisting of a ParA ATPase, a DNA binding ParB CTPase, and centromere sites parS, orchestrate the separation of newly replicated DNA copies and their intracellular positioning. Accurate segregation relies on the assembly of a high-molecular-weight complex, comprising a few hundreds of ParB dimers nucleated from parS sites. This complex assembles in a multi-step process and exhibits dynamic liquid-droplet properties. Despite various proposed models, the complete mechanism for partition complex assembly remains elusive. This study investigates the impact of DNA supercoiling on ParB DNA binding profiles in vivo, using the ParABS system of the plasmid F. We found that variations in DNA supercoiling does not significantly affect any steps in the assembly of the partition complex. Furthermore, physical modeling, leveraging ChIP-seq data from linear plasmids F, suggests that ParB sliding is restricted to approximately 2 Kbp from parS, highlighting the necessity for additional mechanisms beyond ParB sliding over DNA for concentrating ParB into condensates nucleated at parS. Finally, explicit simulations of a polymer coated with bound ParB suggest a dominant role for ParB-ParB interactions in DNA compaction within ParB condensates.

Pdif-mediated antibiotic resistance genes transfer in bacteria identified by pdifFinder.

Modules consisting of antibiotic resistance genes (ARGs) flanked by inverted repeat Xer-specific recombination sites were thought to be mobile genetic elements that promote horizontal transmission. Less frequently, the presence of mobile modules in plasmids, which facilitate a pdif-mediated ARGs transfer, has been reported. Here, numerous ARGs and toxin-antitoxin genes have been found in pdif site pairs. However, the mechanisms underlying this apparent genetic mobility is currently not understood, and the studies relating to pdif-mediated ARGs transfer onto most bacterial genera are lacking. We developed the web server pdifFinder based on an algorithm called PdifSM that allows the prediction of diverse pdif-ARGs modules in bacterial genomes. Using test set consisting of almost 32 thousand plasmids from 717 species, PdifSM identified 481 plasmids from various bacteria containing pdif sites with ARGs. We found 28-bp-long elements from different genera with clear base preferences. The data we obtained indicate that XerCD-dif site-specific recombination mechanism may have evolutionary adapted to facilitate the pdif-mediated ARGs transfer. Through multiple sequence alignment and evolutionary analyses of duplicated pdif-ARGs modules, we discovered that pdif sites allow an interspecies transfer of ARGs but also across different genera. Mutations in pdif sites generate diverse arrays of modules which mediate multidrug-resistance, as these contain variable numbers of diverse ARGs, insertion sequences and other functional genes. The identification of pdif-ARGs modules and studies focused on the mechanism of ARGs co-transfer will help us to understand and possibly allow controlling the spread of MDR bacteria in clinical settings. The pdifFinder code, standalone software package and description with tutorials are available at https://github.com/mjshao06/pdifFinder.

Global epistasis in plasmid-mediated antimicrobial resistance.

Antimicrobial resistance (AMR) in bacteria is a major public health threat and conjugative plasmids play a key role in the dissemination of AMR genes among bacterial pathogens. Interestingly, the association between AMR plasmids and pathogens is not random and certain associations spread successfully at a global scale. The burst of genome sequencing has increased the resolution of epidemiological programs, broadening our understanding of plasmid distribution in bacterial populations. Despite the immense value of these studies, our ability to predict future plasmid-bacteria associations remains limited. Numerous empirical studies have recently reported systematic patterns in genetic interactions that enable predictability, in a phenomenon known as global epistasis. In this perspective, we argue that global epistasis patterns hold the potential to predict interactions between plasmids and bacterial genomes, thereby facilitating the prediction of future successful associations. To assess the validity of this idea, we use previously published data to identify global epistasis patterns in clinically relevant plasmid-bacteria associations. Furthermore, using simple mechanistic models of antibiotic resistance, we illustrate how global epistasis patterns may allow us to generate new hypotheses on the mechanisms associated with successful plasmid-bacteria associations. Collectively, we aim at illustrating the relevance of exploring global epistasis in the context of plasmid biology.

Fitness effects of plasmids shape the structure of bacteria-plasmid interaction networks.

Antimicrobial resistance (AMR) genes are often carried on broad host range plasmids, and the spread of AMR within microbial communities will therefore depend on the structure of bacteria­plasmid networks. Empirical and theoretical studies of ecological interaction networks suggest that network structure differs between communities that are predominantly mutualistic versus antagonistic, with the former showing more generalized interactions (i.e., species interact with many others to a similar extent). This suggests that mutualistic bacteria­plasmid networks­where antibiotics are present and plasmids carry AMR genes­will be more generalized than antagonistic interactions, where plasmids do not confer benefits to their hosts. We first develop a simple theory to explain this link: fitness benefits of harboring a mutualistic symbiont promote the spread of the symbiont to other species. We find support for this theory using an experimental bacteria­symbiont (plasmid) community, where the same plasmid can be mutualistic or antagonistic depending on the presence of antibiotics. This short-term and parsimonious mechanism complements a longer-term mechanism (coevolution and stability) explaining the link between mutualistic and antagonistic interactions and network structure.

Large-Scale Identification of Known and Novel RRNPP Quorum-Sensing Systems by RRNPP_Detector Captures Novel Features of Bacterial, Plasmidic, and Viral Coevolution.

Gram-positive Firmicutes bacteria and their mobile genetic elements (plasmids and bacteriophages) encode peptide-based quorum-sensing systems (QSSs) that orchestrate behavioral transitions as a function of population densities. In their simplest form, termed "RRNPP", these QSSs are composed of two adjacent genes: a communication propeptide and its cognate intracellular receptor. RRNPP QSSs notably regulate social/competitive behaviors such as virulence or biofilm formation in bacteria, conjugation in plasmids, or lysogeny in temperate bacteriophages. However, the genetic diversity and the prevalence of these communication systems, together with the breadth of behaviors they control, remain largely underappreciated. To better assess the impact of density dependency on microbial community dynamics and evolution, we developed the RRNPP_detector software, which predicts known and novel RRNPP QSSs in chromosomes, plasmids, and bacteriophages of Firmicutes. Applying RRNPP_detector against available complete genomes of viruses and Firmicutes, we identified a rich repertoire of RRNPP QSSs from 11 already known subfamilies and 21 novel high-confidence candidate subfamilies distributed across a vast diversity of taxa. The analysis of high-confidence RRNPP subfamilies notably revealed 14 subfamilies shared between chromosomes/plasmids/phages, 181 plasmids and 82 phages encoding multiple communication systems, phage-encoded QSSs predicted to dynamically modulate bacterial behaviors, and 196 candidate biosynthetic gene clusters under density-dependent regulation. Overall, our work enhances the field of quorum-sensing research and reveals novel insights into the coevolution of gram-positive bacteria and their mobile genetic elements.

Uropathogenic E. coli and Hybrid Pathotypes in Mexican Women with Urinary Tract Infections: A Comprehensive Molecular and Phenotypic Overview.

Uropathogenic Escherichia coli (UPEC) is the main cause of urinary tract infections (UTIs) and carries virulence and resistance factors often found in mobilizable genetic elements, such as plasmids or pathogenicity islands (PAIs). UPEC is part of the extraintestinal pathogenic E. coli (ExPEC), but hybrid strains possessing both diarrheagenic E. coli (DEC) and ExPEC traits, termed "hypervirulent", present a significant health threat. This study assessed the prevalence of UPEC PAIs, ExPEC sequence types (ST), DEC genes, carbapenemase and extended-spectrum ß-lactamase (ESBL) phenotypes, resistance genotypes, and plasmids in 40 clinical isolates of UPEC. Results showed that 72.5% of isolates had PAIs, mainly PAI IV536 (53%). ESBL phenotypes were found in 65% of ß-lactam-resistant isolates, with 100% of carbapenem-resistant isolates producing carbapenemase. The predominant ESBL gene was blaCTX-M-2 (60%), and the most common resistance gene in fluoroquinolone and aminoglycoside-resistant isolates was aac(6')Ib (93%). Plasmids were present in 57% of isolates, and 70% belonged to the ST131 clonal group. Molecular markers for DEC pathotypes were detected in 20 isolates, with 60% classified as hybrid pathotypes. These findings indicate significant pathogenic potential and the presence of hybrid pathotypes in E. coli UTI clinical isolates in the Mexican population.

Unlocking the potential of microbiome editing: A review of conjugation-based delivery.

In recent decades, there has been a rapid increase in the prevalence of multidrug-resistant pathogens, posing a challenge to modern antibiotic-based medicine. This has highlighted the need for novel treatments that can specifically affect the target microorganism without disturbing other co-inhabiting species, thus preventing the development of dysbiosis in treated patients. Moreover, there is a pressing demand for tools to effectively manipulate complex microbial populations. One of the approaches suggested to address both issues was to use conjugation as a tool to modify the microbiome by either editing the genome of specific bacterial species and/or the removal of certain taxonomic groups. Conjugation involves the transfer of DNA from one bacterium to another, which opens up the possibility of introducing, modifying or deleting specific genes in the recipient. In response to this proposal, there has been a significant increase in the number of studies using this method for gene delivery in bacterial populations. This MicroReview aims to provide a detailed overview on the use of conjugation for microbiome engineering, and at the same time, to initiate a discussion on the potential, limitations and possible future directions of this approach.

Type I BREX system defends against antibiotic-resistant plasmids in Escherichia coli.

The Bacteriophage Exclusion (BREX) system is a novel antiphage defense system identified in Bacillus cereus in 2015. The purpose of this study was to investigate the presence of the BREX system defenses against antibiotic-resistant plasmids such as blaKPC and blaNDM invasion in Escherichia coli. The BREX system was present in 5.4% (23/424) of E. coli clinical isolates and 6.5% (84/1283) of E. coli strains with completely sequenced genomes in the GenBank database. All 23 BREX-positive E. coli clinical isolates were susceptible to carbapenems, while all five isolates carrying blaKPC and 11 carrying blaNDM were BREX-negative. For E. coli strains in the GenBank database, 37 of 38 strains carrying blaKPC and 109 of 111 strains carrying blaNDM were BREX negative. The recognition site sequence of methyltransferase PglX in a clinical E. coli 3756 was 5'-CANCATC-3' using PacBio single-molecular real-time sequencing. The transformation efficiency of plasmid psgRNA-ColAori-target with the PglX recognition site was reduced by 100% compared with the plasmid without the recognition site in E. coli DH5α-pHSG398-BREX. The BREX showed lower defense efficacy against plasmid psgRNA-15Aori-target which had the same plasmid backbone but different surrounding sequences of recognition sites with psgRNA-ColAori-target. The conjugation frequency of the KPC-2 plasmid and NDM-5 plasmid in E. coli 3756-ΔBREX was higher than that in E. coli 3756 clinical isolate (1.0 × 10-6 vs 1.3 × 10-7 and 5.5 × 10-7 vs 1.7 × 10-8, respectively). This study demonstrated that the type I BREX system defends against antibiotic-resistant plasmids in E. coli.