Spread Of Plasmids Research Articles

PIPdb: a comprehensive plasmid sequence resource for tracking the horizontal transfer of pathogenic factors and antimicrobial resistance genes.

Plasmids, as independent genetic elements, carrying resistance or virulence genes and transfer them among different pathogens, posing a significant threat to human health. Under the 'One Health' approach, it is crucial to control the spread of plasmids carrying such genes. To achieve this, a comprehensive characterization of plasmids in pathogens is essential. Here we present the Plasmids in Pathogens Database (PIPdb), a pioneering resource that includes 792964 plasmid segment clusters (PSCs) derived from 1 009571 assembled genomes across 450 pathogenic species from 110 genera. To our knowledge, PIPdb is the first database specifically dedicated to plasmids in pathogenic bacteria, offering detailed multi-dimensional metadata such as collection date, geographical origin, ecosystem, host taxonomy, and habitat. PIPdb also provides extensive functional annotations, including plasmid type, insertion sequences, integron, oriT, relaxase, T4CP, virulence factors genes, heavy metal resistance genesand antibiotic resistance genes. The database features a user-friendly interface that facilitates studies on plasmids across diverse host taxa, habitats, and ecosystems, with a focus on those carrying antimicrobial resistance genes (ARGs). We have integrated online tools for plasmid identification and annotation from assembled genomes. Additionally, PIPdb includes a risk-scoring system for identifying potentially high-risk plasmids. The PIPdb web interface is accessible at https://nmdc.cn/pipdb.

Characterization of novel sequence type 12531 and O8:H7 serotype carbapenem-resistant Escherichia coli with strong swimming and intestinal epithelial cell barrier migration abilities.

Carbapenem-resistant Enterobacteriaceae have become widely prevalent globally because of antibiotic misuse and the spread of drug-resistant plasmids, where carbapenem-resistant Escherichia coli (CREC) is one of the most common and prevalent pathogens. Furthermore, E. coli has been identified as a member of normal gut flora and does not cause disease under normal circumstances. However, certain strains of E. coli, due to the expression of virulence genes, can cause severe intestinal and extra-intestinal infections. Therefore, clinically, drug resistance and pathogenic E. coli strains are significantly challenging to treat. In this study, a novel CREC strain DC8855 was isolated from the ascites of a patient with intestinal perforation, identified as a novel sequence type 12531 (ST12531) and an unreported serotype O8:H7. It was revealed that the resistance of ST12531 CREC was predominantly conferred by an IncFII(K) plasmid carrying blaNDM-4. Furthermore, phylogenetic analysis indicated that this is the first discovery of such plasmids in China and the first identification in E. coli. Moreover, regarding virulence, the swimming assays, qRT-PCR, and in vitro intestinal barrier model indicated that DC8855 had significantly higher motility, flagella gene expression, and intestinal epithelial cell barrier migration ability than the other sequence types CREC strains (ST167 and ST410). In conclusion, this study identified novel CREC which was multidrug resistant as well as enteropathogenic and therefore requires continuous monitoring.

LuxS/AI-2 system facilitate the dissemination of antibiotic-resistant plasmids in Klebsiella pneumoniae

Plasmid conjugation is a central mechanism driving the dissemination of antibiotic resistance in Klebsiella pneumoniae. However, the conjugative operon requires specific stimuli for activation. Identifying signals and elucidating the underlying mechanisms is crucial in combating plasmid spread. This study uncovers a key mechanism promoting the dissemination of high-risk plasmids, including IncFII, IncX3, and IncX4 types, in K. pneumoniae. We observed that increased donor density significantly enhanced conjugation, and transcript levels of both conjugation and AI-2 quorum sensing genes were markedly upregulated. By mutating the luxS and lsrR genes in K. pneumoniae 1678, we found that plasmid conjugation efficiency decreased in the 1678ΔluxS mutant, while it significantly increased in the 1678ΔlsrR mutant. RT-qPCR and β-galactosidase assays demonstrated that LsrR represses transcription of relaxosome and T4CP genes, while AI-2 (synthesized by LuxS) activates their expression. AlphaFold3 docking models suggest LsrR may bind directly to IncX plasmid relaxase promoters, inhibiting their expression. Adding external AI-2 signals revealed no effect on plasmid conjugation when LsrR was absent, confirming the dependence of AI-2 signals on LsrR repression. In conclusion, AI-2-mediated signaling enhances donor density effects on plasmid conjugation by de-repressing LsrR-mediated suppression.

Analysis of the Association Between Antimicrobial Resistance Genes and Virulence Factors in ST11 and Non-ST11 CR-KP Bloodstream Infections in the Intensive Care Unit.

This study aims to investigate the association between antimicrobial resistance genes and virulence factors in ST11 and non-ST11 types of CR-KP in bloodstream infections in the intensive care unit, providing a theoretical basis for infection control and clinical diagnosis and treatment. From January 2021 to June 2023, samples of Klebsiella pneumoniae from bloodstream infections were collected at our hospital, focusing on those resistant to carbapenems. The resistance genes, housekeeping genes, and virulence genes were identified through PCR and analyzed using the GrapeTree software to perform MLST-based minimum spanning tree typing. Among the 85 CR-KP cases, 61.18% were of the ST11 type, predominantly of the KL64 capsular type; non-ST11 types were mainly ST15, accounting for 25.88%, predominantly of the KL5 capsular type. The carriage rates of virulence genes such as rmpA2, entB, silS, kpn, iucA, peg-344, and terB were significantly higher in the ST11 group than in the non-ST11 group. The primary carbapenemase identified was class A enzyme bla KPC-2, with a higher carriage rate in the ST11 group. Drug susceptibility tests showed that the resistance rates for cefepime, ertapenem, nitrofurantoin, amikacin, and gentamicin were also higher in the ST11 group, consistent with the resistance genotype findings. The study reveals that ST11 type CR-KP in intensive care unit bloodstream infections exhibits stronger resistance and higher virulence compared to non-ST11 types, posing significant challenges to clinical treatment. Thus, strict control over the use of carbapenem antibiotics is essential to prevent the spread of resistant plasmids.

Natural products from food sources can alter the spread of antimicrobial resistance plasmids in Enterobacterales.

Antimicrobial resistance (AMR) poses a significant threat to global public health. Notably, resistance to carbapenem and extended-spectrum β-lactam antibiotics in Gram-negative bacteria is a major impediment to treating infections. Genes responsible for antibiotic resistance are frequently carried on plasmids, which can transfer between bacteria. Therefore, exploring strategies to prevent this transfer and the prevalence of AMR plasmids is timely and pertinent. Here, we show that certain natural product extracts and associated pure compounds can reduce the conjugation of AMR plasmids into new bacterial hosts. Using our established high-throughput fluorescence-based flow cytometry assay, we found that the natural products were more active in reducing transmission of the IncK extended-spectrum β-lactamase-encoding plasmid pCT in Escherichia coli EC958c, compared to Klebsiella pneumoniae Ecl8 carrying the IncFII carbapenemase-encoding plasmid pKpQIL. The exception was the natural product rottlerin, also active in K. pneumoniae. In classical conjugation assays, rottlerin also reduced the conjugation frequency of the IncFII bla NDM-1 carrying plasmid pCPE16_3 from a clinical K. pneumoniae isolate. Our data indicate that the natural products tested here, in their current molecular structure, reduced conjugation by a small amount, which is unlikely to achieve a large-scale reduction in AMR in bacterial populations. However, certain natural products like rottlerin could provide a foundation for further research into compounds with effective anti-plasmid activity.

Modelling the comparative influence of conjugation and transformation on plasmid spread in biofilms

In this work, we propose a multidimensional continuum model for plasmid dissemination in biofilms via horizontal gene transfer. The model is formulated as a system of nonlocal partial differential equations derived from mass conservation laws and reaction kinetics principles. Biofilm is modelled as a homogeneous, viscous, incompressible fluid with a velocity given by Darcy’s law. The model considers plasmid-carrying cells as distinct volume fractions and their vertical and horizontal gene transfer via conjugation and natural transformation. The model encompasses local detoxification of biofilm due to plasmid-borne resistance gene and its effect at the community scale. The equations are solved numerically and simulations are performed to investigate how transformation and conjugation regulate the dynamics and the ecology of plasmid spread in both a multidimensional and one-dimensional biofilm system. Model results are able to predict relevant experimentally observed results in plasmid spread, such as the respective intensity of different horizontal gene transfer mechanisms and the importance of selective pressure. Moreover, model results predict coexistence of plasmid-carrying and plasmid-free bacteria even in conditions when one should out-compete the other, offering a simple modelling explanation on global plasmid persistence in bacterial communities.

CRISPR-Cas inhibits plasmid transfer and immunizes bacteria against antibiotic resistance acquisition in manure.

The horizontal transfer of antibiotic resistance genes among bacteria is a pressing global issue. The bacterial defense system clustered regularly interspaced short palindromic repeats (CRISPR)-Cas acts as a barrier to the spread of antibiotic resistance plasmids, and CRISPR-Cas-based antimicrobials can be effective to selectively deplete antibiotic-resistant bacteria. While significant surveillance efforts monitor the spread of antibiotic-resistant bacteria in the clinical context, a major, often overlooked aspect of the issue is resistance emergence in agriculture. Farm animals are commonly treated with antibiotics, and antibiotic resistance in agriculture is on the rise. Yet, CRISPR-Cas efficacy has not been investigated in this setting. Here, we evaluate the prevalence of CRISPR-Cas in agricultural Enterococcus faecalis strains and its antiplasmid efficacy in an agricultural niche: manure. Analyzing 1,986 E. faecalis genomes from human and animal hosts, we show that the prevalence of CRISPR-Cas subtypes is similar between clinical and agricultural E. faecalis strains. Using plasmid conjugation assays, we found that CRISPR-Cas is a significant barrier against resistance plasmid transfer in manure. Finally, we used a CRISPR-based antimicrobial approach to cure resistant E. faecalis of erythromycin resistance, but this was limited by delivery efficiency of the CRISPR antimicrobial in manure. However, immunization of bacteria against resistance gene acquisition in manure was highly effective. Together, our results show that E. faecalis CRISPR-Cas is prevalent and effective in an agricultural setting and has the potential to be utilized for depleting antibiotic-resistant populations. Our work has broad implications for tackling antibiotic resistance in the increasingly relevant agricultural setting, in line with a One Health approach.IMPORTANCEAntibiotic resistance is a growing global health crisis in human and veterinary medicine. Previous work has shown technologies based on CRISPR-Cas-a bacterial defense system-to be effective in tackling antibiotic resistance. Here we test if CRISPR-Cas is present and effective in agricultural niches, specifically in the ubiquitously present bacterium, Enterococcus faecalis. We show that CRISPR-Cas is both prevalent and functional in manure and has the potential to be used to specifically kill bacteria carrying antibiotic resistance genes. This study demonstrates the utility of CRISPR-Cas-based strategies for control of antibiotic resistance in agricultural settings.

Beta-blocker drives the conjugative transfer of multidrug resistance genes in pure and complex biological systems

Drug resistance poses a high risk to human health. Extensive use of non-antibiotic drugs contributes to antibiotic resistance genes (ARGs) transfer. However, how they affect the spread of broad-host plasmids in complex biological systems remains unknown. This study investigated the effect of metoprolol on the transfer frequency and host range of ARGs in both intrageneric and intergeneric pure culture systems, as well as in anammox microbiome. The results showed that environmental concentrations of metoprolol significantly promoted the intrageneric and intergeneric conjugative transfer. Initially, metoprolol induced excessive oxidative stress, resulting in high cell membrane permeability and bacterial SOS response. Meanwhile, more pili formation increased the adhesion and contact between bacteria, and the abundance of conjugation-related genes also increased significantly. Activation of the electron transport chain provided more ATP for this energy-consuming process. The underlying mechanism was further verified in the complex anammox conjugative system. Metoprolol induced the enrichment of ARGs and mobile genetic elements. The enhanced bacterial interaction and energy generation facilitated the high conjugative transfer frequency of ARGs. In addition, plasmid-borne ARGs tended to transfer to opportunistic pathogens. This work raises public concerns about the health and ecological risks of non-antibiotic drugs.

Monitoring Multi-Drug Resistant Klebsiella pneumoniae in Kitagata Hot Spring, Southwestern Uganda: A Public Health Implication.

The concerning frequency of K. pneumoniae in various recreational settings, is noteworthy, especially regarding multi-drug resistant (MDR) strains. This superbug is linked to the rapid spread of plasmids carrying these resistance genes. The objective of this study was to evaluate the spatiotemporal prevalence of MDR-K. pneumoniae in the Kitagata hot spring, Southwestern Uganda. A laboratory-based descriptive longitudinal study was conducted between May and July 2023. During rainy and dry seasons, we collected eighty water samples in the morning and evening from the hot spring. The temperature at each point was measured prior to sample collection, and two samples were obtained at varying depths. 5 mL of each homogenized sample were pre-enriched in brain heart infusion broth, and subsequently in both blood and violet red bile agar. The Kirby-Bauer disk diffusion method was performed, followed by the detection of carbapenemase (CR) and extended-spectrum β-lactamase (ESBL) production. Polymerase chain reaction showed resistance genes viz. bla TEM, bla CTX-M and bla KPC. Data were analyzed using SPSS-20 to obtain chi-square tests and regression analysis. K. pneumoniae accounted for 30.0% of isolates obtained from Kitagata hot springs, with all isolates classified as multi-drug resistant. All isolates were resistant to ampicillin, rifampicin, ceftazidime, and azithromycin (79.2%). Additionally, 95.8% of isolates harbored bla TEM gene alone and both bla TEM and bla CTX genes, followed by bla KPC alone (33.3%), with 25% harboring all three resistance genes. During the dry season, K. pneumoniae had a higher prevalence (35.0%) compared to the wet season (25.0%). The prevalence of MDR-K. pneumoniae significantly increased over the course of the study. The presence of the three studied resistance genes in the isolates showed a positive correlation with the second phase of sample collection and the dry season but exhibited a negative correlation with temperature, except for isolates harboring either bla TEM alone or bla TEM+KPC+CTX genes. Kitagata hot spring serves as a hotspot for continuous dissemination and acquisition of MDR-K. pneumoniae harboring resistance genes that encode for ESBL and CR production. The healthcare sector ought to implement an ongoing monitoring and surveillance system as well as robust antimicrobial resistance stewardship programs aimed at delivering health education to the community.

Genomic characterization of carbapenemase-producing Enterobacterales from Dhaka food markets unveils the spread of high-risk antimicrobial-resistant clones and plasmids co-carrying bla NDM and mcr-1.1.

The transmission of carbapenemase-producing Enterobacterales (CPE) in the external environment, especially through food, presents a significant public health risk. To investigate the prevalence and genetic characteristics of CPE in food markets of Dhaka, Bangladesh, using WGS. CPE isolates were obtained from different food and water samples collected from food markets in the southern part of Dhaka, Bangladesh. The isolates subsequently underwent molecular typing, WGS employing both short- and long-read sequencers, and plasmid analysis. This study unveiled an extensive spread of CPE, with no significant difference in contamination rates observed in samples (N = 136), including meat (n = 8), fish (n = 5), vegetables (n = 36) or various food-washed water (n = 65) from markets near hospitals or residential areas. Thirty-eight Enterobacterales from 33 samples carried carbapenemase genes (bla NDM-1, -4, -7, bla KPC-2, bla OXA-181 or bla IMI-1). Among these, the high-risk Escherichia coli ST410 clone was the most prevalent and distributed across various locations. Furthermore, the identification of IncHI2 plasmids co-harbouring resistance genes like bla NDM-5 and mcr-1.1, without discernible epidemiological connections, is a unique finding, suggesting their widespread dissemination. The analysis unveils a dynamic landscape of CPE dissemination in food markets, underscored by the proliferation of novel IncHI2 hybrid plasmids carrying both colistin- and carbapenem-resistance genes. This illuminates the ever-evolving landscape of antimicrobial resistance in Dhaka, urging us to confront its emergent challenges.

A new anti-CRISPR gene promotes the spread of drug-resistance plasmids in Klebsiella pneumoniae.

The Klebsiella pneumoniae (K. pneumoniae, Kp) populations carrying both resistance-encoding and virulence-encoding mobile genetic elements (MGEs) significantly threaten global health. In this study, we identified a new anti-CRISPR gene (acrIE10) on a conjugative plasmid with self-target sequence in K. pneumoniae with type I-E* CRISPR-Cas system. AcrIE10 interacts with the Cas7* subunit of K. pneumoniae I-E* CRISPR-Cas system. The crystal structure of the AcrIE10-KpCas7* complex suggests that AcrIE10 suppresses the I-E* CRISPR-Cas by binding directly to Cas7 to prevent its hexamerization, thereby preventing the surveillance complex assembly and crRNA loading. Bioinformatic and functional analyses revealed that AcrIE10 is functionally widespread across diverse species. Our study reports a novel anti-CRISPR and highlights its potential role in spreading resistance and virulence among pathogens.

Emergence of an IncX3 plasmid co-harbouring the carbapenemase genes blaNDM-5 and blaOXA-181.

The spread of transmissible plasmids with carbapenemase genes has contributed to a global increase in carbapenemase-producing Enterobacterales over the past two decades, with blaNDM and blaOXA among the most prevalent carbapenemase genes. To characterize an Escherichia coli isolate co-carrying blaNDM-5 and blaOXA-181 (JBEHAAB-19-0176) that was isolated in the Japan Antimicrobial Resistant Bacterial Surveillance in 2019-20, and to evaluate the functional advantage of carrying both genes as opposed to only one. The whole-genome sequence of the isolate was determined using long- and short-read sequencing. Growth assay and co-culture experiments were performed for phenotypic characterization in the presence of different β-lactam antibiotics. WGS analysis showed that blaNDM-5 and blaOXA-181 were carried by the same IncX3 plasmid, pJBEHAAB-19-0176_NDM-OXA. Genetic characterization of the plasmid suggested that the plasmid emerged through the formation of a co-integrate and resolution of two typical IncX3 plasmids harbouring blaNDM-5 and blaOXA-181, which involved two recombination events at the IS3000 and IS26 sequences. When cultured in the presence of piperacillin or cefpodoxime, the growth rate of the transformant co-harbouring blaNDM-5 and blaOXA-181 was significantly higher than the transformant with only blaNDM-5. Furthermore, in co-culture where the two blaNDM-5-harbouring transformants were allowed to compete directly, the strain additionally harbouring blaOXA-181 showed a marked growth advantage. The additional carriage of blaOXA-181 confers a selective advantage to bacteria in the presence of piperacillin and cefpodoxime. These findings may explain the current epidemiology of carbapenemase-producing Enterobacterales, in which bacteria carrying both blaNDM-5 and blaOXA-48-like genes have emerged independently worldwide.

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.

Coexistence of a nonresistance-conferring IncI1 plasmid favors persistence of the blaCTX-M-bearing IncFII plasmid in Escherichia coli.

So far, plasmid-carried blaCTX-M is still the most common extended-spectrum beta-lactamase (ESBL) genotype in clinical settings worldwide. Except for the widespread use of third-generation cephalosporins, the interaction between coexisting plasmids can also affect the long-term stable existence of the blaCTX-M gene; however, the study on that is still sparse. In the present study, we assess the interaction of coinhabitant plasmids blaCTX-M-IncFII and nonresistance-IncI1. Our results confirmed that the increased fitness advantages of strain C600FII+I1 were attributable to the cohabitant nonresistance-IncI1 plasmid, which largely reduced the intracellular ATP levels of host bacteria, thus decreasing the rep gene expression of the blaCTX-M-IncFII plasmid, its copy numbers, and mating efficiencies, while the higher fitness advantages of strain C600FII+I1 enhanced the persistence of the blaCTX-M-IncFII plasmid. The results indicate that the nonresistance-IncI1 plasmid contributes to the long-term existence of the blaCTX-M-IncFII plasmid, implying a potentially new strategy for controlling the spread of resistance plasmids in clinical settings by targeting nonresistance plasmids.

Modelling Plasmid-Mediated Horizontal Gene Transfer in Biofilms

In this study, we present a mathematical model for plasmid spread in a growing biofilm, formulated as a nonlocal system of partial differential equations in a 1-D free boundary domain. Plasmids are mobile genetic elements able to transfer to different phylotypes, posing a global health problem when they carry antibiotic resistance factors. We model gene transfer regulation influenced by nearby potential receptors to account for recipient-sensing. We also introduce a promotion function to account for trace metal effects on conjugation, based on literature data. The model qualitatively matches experimental results, showing that contaminants like toxic metals and antibiotics promote plasmid persistence by favoring plasmid carriers and stimulating conjugation. Even at higher contaminant concentrations inhibiting conjugation, plasmid spread persists by strongly inhibiting plasmid-free cells. The model also replicates higher plasmid density in biofilm’s most active regions.

Tebuconazole promotes spread of a multidrug-resistant plasmid into soil bacteria to form new resistant bacterial strains

The development of antibiotic resistance threatens human and environmental health. Non-antibiotic stressors, including fungicides, may contribute to the spread of antibiotic resistance genes (ARGs). We determined the promoting effects of tebuconazole on ARG dissemination using a donor, Escherichia coli MG1655, containing a multidrug-resistant fluorescent plasmid (RP4) and a recipient (E. coli HB101). The donor was then incorporated into the soil to test whether tebuconazole could accelerate the spread of RP4 into indigenous bacteria. Tebuconazole promoted the transfer of the RP4 plasmid from the donor into the recipient via overproduction of reactive oxygen species (ROS), enhancement of cell membrane permeability and regulation of related genes. The dissemination of the RP4 plasmid from the donor to soil bacteria was significantly enhanced by tebuconazole. RP4 plasmid could be propagated into more genera of bacteria in tebuconazole-contaminated soil as the exposure time increased. These findings demonstrate that the fungicide tebuconazole promotes the spread of the RP4 plasmid into indigenous soil bacteria, revealing the potential risk of tebuconazole residues enhancing the dissemination of ARGs in soil environments.

Antibiotic resistance begets more resistance: chromosomal resistance mutations mitigate fitness costs conferred by multi-resistant clinical plasmids.

Plasmids are the primary vectors of horizontal transfer of antibiotic resistance genes among bacteria. Previous studies have shown that the spread and maintenance of plasmids among bacterial populations depend on the genetic makeup of both the plasmid and the host bacterium. Antibiotic resistance can also be acquired through mutations in the bacterial chromosome, which not only confer resistance but also result in changes in bacterial physiology and typically a reduction in fitness. However, it is unclear whether chromosomal resistance mutations affect the interaction between plasmids and the host bacteria. To address this question, we introduced 13 clinical plasmids into a susceptible Escherichia coli strain and three different congenic mutants that were resistant to nitrofurantoin (ΔnfsAB), ciprofloxacin (gyrA, S83L), and streptomycin (rpsL, K42N) and determined how the plasmids affected the exponential growth rates of the host in glucose minimal media. We find that though plasmids confer costs on the susceptible strains, those costs are fully mitigated in the three resistant mutants. In several cases, this results in a competitive advantage of the resistant strains over the susceptible strain when both carry the same plasmid and are grown in the absence of antibiotics. Our results suggest that bacteria carrying chromosomal mutations for antibiotic resistance could be a better reservoir for resistance plasmids, thereby driving the evolution of multi-drug resistance.IMPORTANCEPlasmids have led to the rampant spread of antibiotic resistance genes globally. Plasmids often carry antibiotic resistance genes and other genes needed for its maintenance and spread, which typically confer a fitness cost on the host cell observed as a reduced growth rate. Resistance is also acquired via chromosomal mutations, and similar to plasmids they also reduce bacterial fitness. However, we do not know whether resistance mutations affect the bacterial ability to carry plasmids. Here, we introduced 13 multi-resistant clinical plasmids into a susceptible and three different resistant E. coli strains and found that most of these plasmids do confer fitness cost on susceptible cells, but these costs disappear in the resistant strains which often lead to fitness advantage for the resistant strains in the absence of antibiotic selection. Our results imply that already resistant bacteria are a more favorable reservoir for multi-resistant plasmids, promoting the ascendance of multi-resistant bacteria.

Spread and evolution of blaKPC-plasmid between Serratia marcescens and Klebsiella pneumoniae

ObjectivesblaKPC-carrying Enterobacterales have post great challenges to global healthcare systems. In this study, we reported the evolution and spread of blaKPC between Serratia marcescens and Klebsiella pneumoniae. MethodsFour S. marcescens and one K. pneumoniae strains were isolated from the sputum samples of the patient. Antimicrobial susceptibility tests and whole genome sequencing were performed to investigate the phenotype & genotype of strains. Conjugation assays, cloning experiment and kinetic parameters measuring were performed to explore the spread and antimicrobial resistance mechanisms. ResultsThe evolution and transmission of blaKPC-2 occurred during the treatment of ceftazidime-avibactam and trimethoprim-sulfamethoxazole. Analysis of the antimicrobial susceptibility and genetic profiles of the clinical strains showed that blaKPC-2 evolved into blaKPC-71 and blaKPC-44, together with resistance to ceftazidime-avibactam and carbapenems susceptibility recovery under antimicrobial pressure. Cloning and expression of blaKPC-44 & blaKPC-71 in E. coli DH5α showed that KPC-44 and KPC-71 resulted in a 64∼128-fold increase in the MIC value for ceftazidime-avibactam. Meanwhile, the kinetic assays also showed that the enzyme activity of KPC-44 and KPC-71 towards carbapenems was destroyed and couldn't be inhibited by avibactam. Based on the conjugation assay and whole genome sequence analyses, we provided evolutionary insights into the transmission pathway trace of blaKPC-bearing plasmids between S. marcescens and K. pneumoniae. ConclusionsMixed-species co-infection is one of the risk factors leading to the spread of plasmids carrying carbapenem-resistant genes, and increased surveillance of multidrug-resistant Enterobacterales is urgently needed.

The spread of pESI-mediated extended-spectrum cephalosporin resistance in Salmonella serovars-Infantis, Senftenberg, and Alachua isolated from food animal sources in the United States.

The goal of this study is to investigate the origin, prevalence, and evolution of the pESI megaplasmid in Salmonella isolated from animals, foods, and humans. We queried 510,097 Salmonella genomes under the National Center for Biotechnology Information (NCBI) Pathogen Detection (PD) database for the presence of potential sequences containing the pESI plasmid in animal, food, and environmental sources. The presence of the pESI megaplasmid was confirmed by using seven plasmid-specific markers (rdA, pilL, SogS, TrbA, ipf, ipr2 and IncFIB(pN55391)). The plasmid and chromosome phylogeny of these isolates was inferred from single nucleotide polymorphisms (SNPs). Our search resolved six Salmonella clusters carrying the pESI plasmid. Four were emergent Salmonella Infantis clusters, and one each belonged to serovar Senftenberg and Alachua. The Infantis cluster with a pESI plasmid carrying blaCTX-M-65 gene was the biggest of the four emergent Infantis clusters, with over 10,000 isolates. This cluster was first detected in South America and has since spread widely in United States. Over time the composition of pESI in United States has changed with the average number of resistance genes showing a decrease from 9 in 2014 to 5 in 2022, resulting from changes in gene content in two integrons present in the plasmid. A recent and emerging cluster of Senftenberg, which carries the blaCTX-M-65 gene and is primarily associated with turkey sources, was the second largest in the United States. SNP analysis showed that this cluster likely originated in North Carolina with the recent acquisition of the pESI plasmid. A single Alachua isolate from turkey was also found to carry the pESI plasmid containing blaCTX-M-65 gene. The study of the pESI plasmid, its evolution and mechanism of spread can help us in developing appropriate strategies for the prevention and further spread of this multi-drug resistant plasmid in Salmonella in poultry and humans.

Host defense peptides mitigate the spread of antibiotic resistance in physiologically relevant condition.

Antibiotic resistance represents a significant challenge to public health and human safety. The primary driver behind the dissemination of antibiotic resistance is the horizontal transfer of plasmids. Current conjugative transfer assay is generally performed in a standardized manner, ignoring the effect of the host environment. Host defense peptides (HDPs) possess a wide range of biological targets and play an essential role in the innate immune system. Herein, we reveal that sub-minimum inhibitory concentrations of HDPs facilitate the conjugative transfer of RP4-7 plasmid in the Luria Broth medium, and this observation is reversed in the RPMI medium, designed to simulate the host environment. Out of these HDPs, indolicidin (Ind), a cationic tridecapeptide from bovine neutrophils, significantly inhibits the conjugation of multidrug resistance plasmids in a dose-dependent manner, including blaNDM- and tet(X4)-bearing plasmids. We demonstrate that the addition of Ind to RPMI medium as the incubation substrate downregulates the expression of conjugation-related genes. In addition, Ind weakens the tricarboxylic acid cycle, impedes the electron transport chain, and disrupts the proton motive force, consequently diminishing the synthesis of adenosine triphosphate and limiting the energy supply. Our findings highlight the importance of the host-like environments for the development of horizontal transfer inhibitors and demonstrate the potential of HDPs in preventing the spread of resistance plasmids.