Plasmid-borne Resistance Research Articles

Microbial Degradation of Pesticides in Agricultural Environments: A Comprehensive review of Mechanisms, Factors and Biodiversity

Pesticides are used frequently in modern times to prevent and manage crop pests and diseases, but their residual effects have seriously harmed both the environment and the health of humans. Within the fields of all over the world applied restoration of the environment science and technology, the study of microbial breakdown of pesticides in soil ecosystems is a significant research area. Agricultural methods depend on the use of chemicals, including pesticides and herbicides, to control pests and weeds. However, these chemicals pose risks to the environment, human, and animal well-being. Microbes have shown promise in degrading these agricultural environmental hazards, mitigating their negative impact. The organisms that exist in the natural world, the research on bacteria that break down pesticides and herbicides, and the approach for the application of these bacteria has been summarized in the paper. This literature review aims to identify the microbes responsible for degrading these chemicals and assess their effectiveness in doing so. The goal of this review is to determine the bacteria responsible for the deterioration of agricultural environmental hazard chemicals, evaluate their efficacy in degrading these compounds, explore the factors influencing microbial degradation efficiency, and identify research gaps in the field. Inclusion criteria encompass studies published in English between 2010 and 2023 that focus on the degradation of agricultural environmental hazard chemicals by microbes, specifically microbial consortia, under controlled conditions. A systematic literature review will be conducted using databases like ScienceDirect, Web of Science, and PubMed. Data extracted from selected studies will include information on pesticide types, microorganisms involved in degradation, mechanisms of microbial degradation, factors affecting microbial degradation, current trends in microbial degradation of pesticides, biodiversity of pesticide-degrading microbes, plasmid-borne pesticide resistance in bacterial communities, and strategies for pesticide degradation by microbial consortia. This research tries to present a comprehensive knowledge of microbial degradation mechanisms, highlight the potential of microbial consortia in pesticide degradation, and contribute to sustainable and eco-friendly approaches for addressing pesticide residues in agricultural environments. This study focused on the variables that affect the microbial breakdown of pesticides and the technique by which microorganisms decompose under natural conditions. In addition, the current trends of research on the microbial degradation of pesticides as well as a few visible challenges that continue to need attention were described.

Complete genome sequence of a high-risk Escherichia coli ST10 isolated from avian feces in Pakistan.

We present the complete genome sequence of Escherichia coli strain PEC1009 isolated from avian feces in Pakistan. The strain belongs to sequence type (ST) 10, a high-risk clone. The strain possesses two plasmids, and various plasmid-borne antibiotic resistance genes and chromosome-borne virulence factor genes were identified in its genome.

Characterization of azithromycin-resistant Shigella flexneri serotype 2a isolates using whole genome sequencing in Ontario from 2016 to 2018.

Azithromycin-resistant shigellosis is increasing globally. This retrospective analysis of Shigella flexneri serotype 2a isolates from 2016 to 2018 in Ontario found nearly half were azithromycin (47.7%, 72/151) and ciprofloxacin (50.7%, 77/152) resistant. Moreover, 34.7% (25/72) of azithromycin-resistant isolates were also ciprofloxacin-resistant. Four isolates were ceftriaxone-resistant, although all azithromycin-resistant isolates were ceftriaxone-susceptible. Overall, 83.6% (127/152) of all S. flexneri 2a isolates were recovered from males and 97.2% (70/72) of the azithromycin-resistant cases were males. Among the azithromycin-resistant cases, some (8/72) reported international travel. Phylogenetic analysis of azithromycin-resistant isolates revealed two large male-dominated clusters, and one cluster may have been due to importation of resistant strain. Comparison of plasmids isolated from the clusters in Ontario revealed the presence of incFII plasmid with high percentage of similarity to plasmids present in global outbreaks affecting mostly males including men who have sex with men (MSM). These two large azithromycin-resistant clusters are suggestive of an outbreak among MSM, though disease exposure or sexual orientation of patients was unknown. The presence of plasmid-borne azithromycin resistance in ciprofloxacin-resistant isolates is a public health concern. Antimicrobial surveillance is important for patient management, understanding the spread of novel resistance types in local communities which sometimes is introduced by travel. We found ongoing multidrug-resistant outbreaks spanning multiple years affecting males. Reduction of future outbreaks in high-risk communities like MSM requires consorted information flow between laboratory, public health, and physicians. We impart genomic and antimicrobial characteristics of multidrug S. flexneri 2a which may serve as reference by clinicians and public health.IMPORTANCEOral ciprofloxacin and azithromycin are generally considered as the first-line therapy of shigellosis. Here, we report the emergence and transmission of azithromycin and ciprofloxacin-resistant S. flexneri serotype 2a among male adults in Ontario during 2016-2018. The percentage of azithromycin and ciprofloxacin resistance among S. flexneri 2a is higher compared to previous reports from Canada and United States. Here, we show the genetic basis of the antimicrobial resistance among these unique groups of S. flexneri 2a isolates. We describe a domestically acquired azithromycin-resistant and ciprofloxacin-resistant S. flexneri 2a lineage in Ontario. Combining whole-genome sequencing (WGS) data with travel-associated data helped in understanding dissemination and transmission. We employed WGS, which not only helped us in understanding the genetic-relationship between isolates but also mine information regarding plasmids. In the future, linking WGS, travel-related data, and clinical data can provide enhanced contact tracing and improve public-health management.

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.

Curing of a field strain of Salmonella enterica serovar Infantis isolated from poultry from its highly stable pESI like plasmid

Salmonella enterica serovar Infantis (S. infantis) is an important emerging pathogen, associated with poultry and poultry products and related to an increasing number of human infections in many countries. A concerning trend among S. infantis isolates is the presence of plasmid-mediated multidrug resistance. In many instances, the genes responsible for this resistance are carried on a megaplasmid known as the plasmid of emerging S. infantis (pESI) or pESI like plasmids. Plasmids can be remarkably stable due to the presence of multiple replicons and post-segregational killing systems (PSKs), which contribute to their maintenance within bacterial populations. To enhance our understanding of S. infantis and its multidrug resistance determinants toward the development of new vaccination strategies, we have devised a new method for targeted plasmid curing. This approach effectively overcomes plasmid addiction by leveraging the temporal overproduction of specific antitoxins coupled with the deletion of the partition region. By employing this strategy, we successfully generated a plasmid-free strain from a field isolate derived from S. infantis 119,944.This method provides valuable tools for studying S. infantis and its plasmid-borne multidrug resistance mechanisms and can be easily adopted for plasmid curing from other related bacteria.

Interphylum dissemination of NDM-5-positive plasmids in hospital wastewater from Fuzhou, China: a single-centre, culture-independent, plasmid transmission study

The global spread of plasmid-borne carbapenem resistance is an ongoing public health challenge; however, the nature of such horizontal gene transfer events among complex bacterial communities remains poorly understood. We examined the in-situ transfer of the globally dominant New Delhi metallo-β-lactamase (NDM)-5-positive IncX3 plasmid (denoted pX3_NDM-5) in hospital wastewater to simulate a real-world, One Health antimicrobial resistance context. For this transmission study, we tagged pX3_NDM-5 with the green fluorescent protein gene, gfp, using a CRISPR-based method and transferred the plasmid to a donor Escherichia coli strain. Bacteria were extracted from a hospital wastewater treatment plant (Fujian Provincial Maternity and Children's Hospital, Fuzhou, China) as the bacterial recipient community. We mixed this recipient community with the E coli donor strain carrying the gfp-tagged plasmid, both with and without sodium hypochlorite (NaClO) as an environmental stressor, and conducted several culture-based and culture-independent conjugation assays. The conjugation events were observed microscopically and quantified by fluorescence-activated cell sorting. We analysed the taxonomic composition of the sorted transconjugal pool by 16S rRNA gene amplicon sequencing and assessed the stability of the plasmid in the isolated transconjugants and its ability to transfer back to E coli. We show that the plasmid pX3_NDM-5 has a broad host range and can transfer across various bacterial phyla, including between Gram-negative and Gram-positive bacteria. Although environmental stress with NaClO did not affect the overall plasmid transfer frequency, it reduced the breadth of the transconjugant pool. The taxonomic composition of the transconjugal pool was distinct from that of the recipient communities, and environmental stress modulated the permissiveness of some operational taxonomic units towards the acquisition of pX3_NDM-5. Notably, pX3_NDM-5 transconjugants included the Gram-positive pathogen Enterococcus faecalis, and the plasmid could subsequently be reconjugated back to E coli. These findings suggest that E faecalis could act as a natural shuttle vector for the wide dissemination of pX3_NDM-5 plasmids. Our culture-independent conjugation model simulates natural environmental conditions and challenges the established theory that Gram-negative and Gram-positive bacteria rarely exchange clinically important plasmids. The data show that plasmids disseminate more widely across genera and phyla than previously thought. These findings have substantial implications when considering the spread of antimicrobial resistance across One Health sectors. The Laboratory of Lingnan Modern Agriculture Project, the National Natural Science Foundation of China, the Natural Science Foundation of Fujian Province of China, and the Outstanding Young Research Talents Program of Fujian Agriculture and Forestry University.

Study of mercury resistance and Fourier transform infrared spectroscopy-based metabolic profiling of a potent Bacillus tropicus strain from forest soil.

Mercury (Hg) is a highly toxic heavy metal and Hg-resistant indigenous bacterial isolates may offer a green and cost-effective bioremediation strategy to counter Hg contamination. In this study, a potent Hg-resistant bacterium was isolated from the forest soil of a bird sanctuary. Identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry depicted the isolate as a strain of Bacillus tropicus, validated by morphological, biochemical, and molecular studies. The isolate demonstrated biological Hg removal efficiency and capacity of 50.67% and 19.76 mg g-1 , respectively. The plasmid borne resistance determinant, merA, encoding mercuric reductase, was detected in the bacterium endowing it with effective Hg volatilization and resistance capability. A Fourier-transform infrared spectroscopic comparative metabolic profiling revealed the involvement of various functional groups like -COOH, -CH2 , -OH, PO4 - and so on, resulting in differential spectral patterns of the bacterium both in control and Hg-exposed situations. A temporal variance in metabolic signature was also observed during the early and mid-log phase of growth in the presence of Hg. The bacterium described in this study is the first indigenous Hg-resistant strain isolated from the Uttar Dinajpur region, which could be further explored and exploited as a potent bioresource for Hg remediation.

Molecular Characterisation of Antimicrobial Resistance in E. coli Isolates from Piglets in the West Region of Romania.

Antibiotics are widely used for prophylaxis and therapy, reducing morbidity and mortality produced by bacterial pathogensin pigs, including infections caused by Escherichia coli. The aim of this study was to characterise antibiotic resistance phenotypes and genotypes in E. coli isolates in pigs in West Romanian grower farms. Differential phenotypic susceptibility profiles and the contribution of resistance genes to phenotypic expression of susceptibility or resistance were evaluated. A total of 76 E. coli isolates were identified and confirmed by the MicroScan Walk Away System. The occurrence of four resistance genes, ampC, blaZ, blaTEM and tetK in strains resistant to 13 antibiotics was assessed. Of the E. coli isolates, 0% showed resistance to meropenem, 3.9% to tigecycline and 10.5% to piperacillin/tazobactam, whereas, in contrast, 100% were resistant to ampicillin and mezlocillin, 76.31% to piperacillin and 59.3% to tetracycline. The prevalence of resistance genes in resistant isolates detected by q-PCR analysis was 97.0% for ampC, 96% for blaZ, 32.9% for blaTEM and 58.8% for tetK. Penetrance (the proportion of individuals carrying a particular variant of a gene that also expresses an associated trait) was 50% for ampC (32% for amoxicillin/clavulanate, 62% for cefazolin, 32% for cefepime, 100% for cefotaxime, 56% for cefuroxime and 99% for ampicillin), 65% for blaZ (32% for amoxicillin/clavulanate and 99% for ampicillin), 51% for blaTEM (81% for piperacillin) and 44% for the tetK gene (83% for tetracycline). The result of phenotypic antibiotic resistance testing may indicate the presence of plasmid-borne resistance, with a diagnostic odds ratio of a positive phenotypic resistance for tetK being 4.52. As a management decision, the maximum penetrance admitted for using a specific antibiotic for E. coli infections in pigs is recommended to be less than 20%.

Clonal diversity, antimicrobial resistance, and genome features among nonfermenting gram-negative bacteria isolated from patients with cystic fibrosis in Russia

Nonfermenting gram-negative (NFGN) bacteria were isolated from cystic fibrosis (CF) patients and subjected to susceptibility testing and whole-genome sequencing. Among 170 enrolled CF patients, 112 (65.9%) were colonized with at least 1 key NFGN species. The species-specific infection rate was highest for Pseudomonas aeruginosa (40.6%) followed by Stenotrophomonas maltophilia (14.1%), Achromobacter spp. (9.4%), and Burkholderia cepacia complex (Bcc, 8.2%) demonstrating a significant age-dependent increase for P. aeruginosa and Achromobacter spp., but not for S. maltophilia or Bcc. P. aeruginosa sequence types (STs) related to high-risk epidemic and global CF clones were carried by 12 (7.1%) and 13 (7.6%) patients, respectively. In total, 47% NFGN isolates, predominantly P. aeruginosa, harbored at least 1 plasmid-borne resistance gene; 5 ST235 isolates carried blaVIM2. Pathogenicity island-borne virulence genes were harbored by 9% NFGN isolates. These findings in conjunction with frequent early colonization by Bcc raised serious concerns regarding infection control in Russian CF centers.

Whole genome sequencing of increased number of azithromycin-resistant Shigella flexneri 1b isolates in Ontario

Azithromycin (AZM) resistance among Shigella is a major public health concern. Here, we investigated the epidemiology of Shigella flexneri serotype 1b recovered during 2016–2018 in Ontario, to describe the prevalence and spread of AZM resistance. We found that 72.3% (47/65) of cases were AZM–resistant (AZMR), of which 95.7% (45/47) were among males (P < 0.001). Whole-genome based phylogenetic analysis showed three major clusters, and 56.9% of isolates grouped within a single closely-related cluster (0–10 ∆SNP). A single AZMR clonal population was persistent over 3 years and involved 67.9% (36/53) of all male cases, and none reported international travel. In 2018, a different AZMR cluster appeared among adult males not reporting travel. A proportion of isolates (10.7%) with reduced susceptibility to ciprofloxacin (CIP) due to S83L mutation in gyrA were AZM susceptible, and 71.4% reported international travel. Resistance to AZM was due to the acquisition of mph gene-bearing incFII plasmids having > 95% nucleotide similarity to pKSR100. Plasmid-borne resistance limiting treatment options to AZM, ceftriaxone (CRO) and CIP was noted in a single isolate. We characterized AZMR isolates circulating locally among males and found that genomic analysis can support targeted prevention and mitigation strategies against antimicrobial-resistance.

A Brief Evaluation of a Copper Resistance Mobile Genetic Island in the Bacterial Leaf Spot Pathogen Xanthomonas euvesicatoria pv. perforans.

Due to the continuous use of copper containing bactericides without effective alternative bactericides, copper resistance has become more prevalent in plant pathogens, including Xanthomonas euvesicatoria pv. perforans (formerly Xanthomonas perforans), a predominant cause of bacterial leaf spot disease of tomato and pepper in the Southeastern United States. Previously, reports of copper resistance have been associated with a large conjugative plasmid. However, we have characterized a copper resistance genomic island located within the chromosome of multiple X. euvesicatoria pv. perforans strains. The island is distinct from a previously described chromosomally encoded copper resistance island in X. vesicatoria strain XVP26. Computational analysis revealed the genomic island to contain multiple genes associated with genetic mobility, including both phage-related genes and transposase. Among copper-tolerant strains of X. euvesicatoria pv. perforans isolated from Florida, the majority of strains were found to have the copper resistance chromosomally encoded rather than plasmid borne. Our results suggest that this copper resistance island may have two modes of horizontal gene transfer and that chromosomally encoded copper resistance genes may provide a fitness advantage over plasmid-borne resistance.

The sulfonamide-resistance dihydropteroate synthase gene is crucial for efficient biodegradation of sulfamethoxazole by Paenarthrobacter species.

Sulfonamide antibiotics (SAs) are serious pollutants to ecosystems and environments. Previous studies showed that microbial degradation of SAs such as sulfamethoxazole (SMX) proceeds via a sad-encoded oxidative pathway, while the sulfonamide-resistant dihydropteroate synthase gene, sul, is responsible for SA resistance. However, the co-occurrence of sad and sul genes, as well as how the sul gene affects SMX degradation, was not explored. In this study, two SMX-degrading bacterial strains, SD-1 and SD-2, were cultivated from an SMX-degrading enrichment. Both strains were Paenarthrobacter species and were phylogenetically identical; however, they showed different SMX degradation activities. Specifically, strain SD-1 utilized SMX as the sole carbon and energy source for growth and was a highly efficient SMX degrader, while SD-2 did could not use SMX as a sole carbon or energy source and showed limited SMX degradation when an additional carbon source was supplied. Genome annotation, growth, enzymatic activity tests, and metabolite detection revealed that strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation and a pathway of protocatechuate degradation. A new sulfonamide-resistant dihydropteroate synthase gene, sul918, was identified in strain SD-1, but not in SD-2. Moreover, the lack of sul918 resulted in low SMX degradation activity in strain SD-2. Genome data mining revealed the co-occurrence of sad and sul genes in efficient SMX-degrading Paenarthrobacter strains. We propose that the co-occurrence of sulfonamide-resistant dihydropteroate synthase and sad genes is crucial for efficient SMX biodegradation. KEY POINTS: • Two sulfamethoxazole-degrading strains with distinct degrading activity, Paenarthrobacter sp. SD-1 and Paenarthrobacter sp. SD-2, were isolated and identified. • Strains SD-1 and SD-2 shared a sad-encoded oxidative pathway for SMX degradation. • A new plasmid-borne SMX resistance gene (sul918) of strain SD-1 plays a crucial role in SMX degradation efficiency.

Molecular mechanism of plasmid-borne resistance to sulfonamide antibiotics

The sulfonamides (sulfas) are the oldest class of antibacterial drugs and inhibit the bacterial dihydropteroate synthase (DHPS, encoded by folP), through chemical mimicry of its co-substrate p-aminobenzoic acid (pABA). Resistance to sulfa drugs is mediated either by mutations in folP or acquisition of sul genes, which code for sulfa-insensitive, divergent DHPS enzymes. While the molecular basis of resistance through folP mutations is well understood, the mechanisms mediating sul-based resistance have not been investigated in detail. Here, we determine crystal structures of the most common Sul enzyme types (Sul1, Sul2 and Sul3) in multiple ligand-bound states, revealing a substantial reorganization of their pABA-interaction region relative to the corresponding region of DHPS. We use biochemical and biophysical assays, mutational analysis, and in trans complementation of E. coli ΔfolP to show that a Phe-Gly sequence enables the Sul enzymes to discriminate against sulfas while retaining pABA binding and is necessary for broad resistance to sulfonamides. Experimental evolution of E. coli results in a strain harboring a sulfa-resistant DHPS variant that carries a Phe-Gly insertion in its active site, recapitulating this molecular mechanism. We also show that Sul enzymes possess increased active site conformational dynamics relative to DHPS, which could contribute to substrate discrimination. Our results reveal the molecular foundation for Sul-mediated drug resistance and facilitate the potential development of new sulfas less prone to resistance.

First report of mobile colistin resistance gene mcr-1 in avian pathogenic Escherichia coli isolated from turkeys in the Gaza Strip, Palestine.

Colistin is used to treat avian pathogenic Escherichia coli (APEC), a microorganism that affects turkey meat production in the Gaza Strip and worldwide. However, the recent emergence of plasmid-borne mobile colistin resistance (mcr) genes in pathogenic E. coli strains is a serious antimicrobial resistance (AMR) challenge for both human and animal health. In December 2018, colistin was banned as a veterinary antimicrobial in the Gaza Strip. This study aimed to detect and track the prevalence of colistin-resistant APEC isolated from turkey flocks in the Gaza Strip. This study investigated 239 APEC isolates from turkey flocks in the Gaza Strip between October 2018 and December 2021 (at 6-month intervals). The colistin-resistant APEC strains were detected using the broth microdilution method. The mcr-1 gene was identified using a polymerase chain reaction. The overall colistin resistance among the isolated APECs was 32.2% during the study period. The average resistance in the first interval was 37.5%, which significantly decreased to 9.3% in the last interval. Among the 77 phenotypically resistant isolates, 32.4% were positive for mcr-1. The average abundance of mcr-1 in the first interval was 66.6%, which decreased to 25% in the last interval. To the best of our knowledge, this is the first study reporting the presence of the mcr-1 gene among the APEC isolates from turkeys in the Gaza Strip. Banned veterinary use of colistin significantly decreased the percentage of resistant APEC isolates from turkeys in Gaza Strip. Further studies are needed to investigate other colistin resistance genes and track the emergence of AMR.

Co-occurrence of multiple plasmid-borne linezolid resistance genes-optrA, cfr, poxtA2 and cfr(D) in an Enterococcus faecalis isolate from retail meat.

To investigate the molecular characteristics and transferability of plasmid-borne linezolid resistance genes optrA, cfr, poxtA2 and cfr(D) genes in one linezolid-resistant Enterococcus faecalis DM86 from retail meat. E. faecalis DM86 was screened for the presence of known linezolid resistance genes via PCR analysis. Conjugation experiments were used to evaluate the transferability of the resistance genes. The complete genome of E. faecalis DM86 was obtained using both the Illumina and Nanopore platforms. Analysis of the complete sequence showed that E. faecalis DM86 belonged to sequence type 116 (ST116). Four linezolid resistance genes were identified on three plasmids, designated as pDM86-2-cfr, pDM86-3-optrA and pDM86-4-poxtA [cfr(D) co-located]. IS1216 mobile elements were found to flank the cfr and optrA locus on these two plasmids. pDM86-3-optrA encoded the RDK-type OptrA protein and a common genetic array of 'IS1216-fexA-optrA-erm(A)-IS1216' was identified on this plasmid. The cfr(D) gene was closely associated with the poxtA2 gene on pDM86-4-poxtA, and similar plasmids and structures were reported recently in the E. faecalis of animal origin. The intra- and inter-species horizontal transferability of this plasmid to E. faecalis JH2-2, Enterococcus faecium BM4105RF and Staphylococcus aureus RN4220 was also proved, with a frequency of 2.8 × 10-3, 1.7 × 10-3 and 3.4 × 10-5, respectively. This was the first report of the co-existence of up to four plasmid-borne linezolid resistance genes in one E. faecalis. Thus, efficient actions should be exerted to circumvent the microbiota contamination of food and the further spread of these antimicrobial resistance reservoirs.

Fluorescent tag reveals the potential mechanism of how indigenous soil bacteria affect the transfer of the wild fecal antibiotic resistance plasmid pKANJ7 in different habitat soils

Plasmids have increasingly become a point of concern since they act as a vital medium for the dissemination of antibiotic resistance genes (ARGs). Although indigenous soil bacteria are critical hosts for these plasmids, the mechanisms driving the transfer of antibiotic resistance plasmids (ARPs) have not been well researched. In this study, we tracked and visualized the colonization of the wild fecal antibiotic resistance plasmid pKANJ7 in indigenous bacteria of different habitat soils (unfertilized soil (UFS), chemical fertilized soil (CFS), and manure fertilized soil (MFS)). The results showed that plasmid pKANJ7 mainly transferred to the dominant genera in the soil and genera that were highly related to the donor. More importantly, plasmid pKANJ7 also transferred to intermediate hosts which aid in the survival and persistence of these plasmids in soil. Nitrogen levels also raised the plasmid transfer rate (14th day: UFS: 0.09%, CFS: 1.21%, MFS: 4.57%). Lastly, our structural equation model (SEM) showed that dominant bacteria shifts caused by nitrogen and loam were the major driver shaping the difference in the transfer of plasmid pKANJ7. Overall, our findings enhance the mechanistic understanding of indigenous soil bacteria’s role in plasmid transfer and inform potential methods to prevent the transmission of plasmid-borne resistance in the environment.

Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing.

Genome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and track the transmission of plasmid-borne antimicrobial resistance genes in bacteria. However, long-read sequencing suffers from low-accuracy base calling, and short-read sequencing is often required to improve genome accuracy. This increases costs and turnaround time. In this study, a novel ONT sequencing method is described, which uses the latest ONT chemistry with improved accuracy to assemble genomes of MDR strains and plasmids from long-read sequencing data only. Three strains of Salmonella carrying MDR plasmids were sequenced using the ONT SQK-LSK114 kit with flow cell R10.4.1, and de novo genome assembly was performed with average read accuracy (Q > 10) of 98.9%. For a 5-Mb-long bacterial genome, finished genome sequences with accuracy of >99.99% could be obtained at 75× sequencing coverage depth using Flye and Medaka software. Thus, this new ONT method greatly improves base-calling accuracy, allowing for the de novo assembly of high-quality finished bacterial or plasmid genomes without the need for short-read sequencing. This saves both money and time and supports the application of ONT data in critical genome-based epidemiological analyses. The novel ONT approach described in this study can take the place of traditional combination genome assembly based on short- and long-read sequencing, enabling pangenomic analyses based on high-quality complete bacterial and plasmid genomes to monitor the spread of antibiotic-resistant bacteria and antibiotic resistance genes.

Inhibiting fatty acid synthesis overcomes colistin resistance.

Treating multidrug-resistant infections has increasingly relied on last-resort antibiotics, including polymyxins, for example colistin. As polymyxins are given routinely, the prevalence of their resistance is on the rise and increases mortality rates of sepsis patients. The global dissemination of plasmid-borne colistin resistance, driven by the emergence of mcr-1, threatens to diminish the therapeutic utility of polymyxins from an already shrinking antibiotic arsenal. Restoring sensitivity to polymyxins using combination therapy with sensitizing drugs is a promising approach to reviving its clinical utility. Here we describe the ability of the biotin biosynthesis inhibitor, MAC13772, to synergize with colistin exclusively against colistin-resistant bacteria. MAC13772 indirectly disrupts fatty acid synthesis (FAS) and restores sensitivity to the last-resort antibiotic, colistin. Accordingly, we found that combinations of colistin and other FAS inhibitors, cerulenin, triclosan and Debio1452-NH3, had broad potential against both chromosomal and plasmid-mediated colistin resistance in chequerboard and lysis assays. Furthermore, combination therapy with colistin and the clinically relevant FabI inhibitor, Debio1452-NH3, showed efficacy against mcr-1 positive Klebsiella pneumoniae and colistin-resistant Escherichia coli systemic infections in mice. Using chemical genomics, lipidomics and transcriptomics, we explored the mechanism of the interaction. We propose that inhibiting FAS restores colistin sensitivity by depleting lipid synthesis, leading to changes in phospholipid composition. In all, this work reveals a surprising link between FAS and colistin resistance.

Exploring the epidemiology of mcr genes, genetic context and plasmids in Enterobacteriaceae originating from pigs and humans on farms in Thailand.

In veterinary medicine, colistin has been widely used as therapeutic and prophylactic agent, and for growth promotion. However, colistin has been re-introduced into treatment of human MDR bacterial infections. We assessed the characteristics and spread of plasmid-borne colistin resistance among healthy pigs, workers with animal-contact and their household members in Thailand. WGS and MIC data of 146 mcr-positive isolates from a cross-sectional One Health study were analysed. Long-read sequencing and conjugation were performed for selected isolates. mcr-carrying isolates were detected in 38% of pooled-pig samples and 16% of human faecal samples. Of 143 Escherichia coli and three Escherichia fergusonii, mcr-1, mcr-3, and mcr-9 variants were identified in 96 (65.8%), 61 (41.8%) and one (0.7%) isolate, respectively. Twelve E. coli co-harboured two mcr variants (mcr-1 and mcr-3). Clonal transmission was detected in five out of 164 farms. mcr-1 was mostly harboured by epidemic IncX4 and IncHI1 plasmids (89.9%). Conversely, mcr-3 was harboured by a range of different plasmids. Comparative plasmid studies suggested IncP and IncFII plasmids as possible endemic mcr-3 plasmids in Asian countries. Moreover, mcr-3 was associated with different mobile genetic elements including TnAs2, ISKpn40 and IS26/15DI. Detected genetic signatures (DRs) indicated recent mcr-3 transpositions, underlining the mobilizable nature of the mcr-3 cassette. The epidemiology of mcr and the possible evolution of successful plasmids and transposition modules should be carefully monitored. Of special concern is the growing number of different horizontal gene transferring pathways encompassing various transposable modules the mcr genes can be shared between bacteria.

First Detection of mcr-9 in a Multidrug-Resistant Escherichia coli of Animal Origin in Italy Is Not Related to Colistin Usage on a Pig Farm.

The emergence of colistin resistance raises growing concerns because of its use as a last-resort antimicrobial for the treatment of severe gram-negative bacterial infections in humans. Plasmid-borne mobile colistin resistance genes (mcr) are particularly worrisome due to their high propensity to spread. An mcr-9-positive Escherichia coli was isolated from a piglet in Italy, representing the first isolation of this gene from an E. coli of animal origin in the country. Whole genome sequencing (WGS) revealed that mcr-9 was borne by an IncHI2 plasmid carrying several other resistance genes. The strain was indeed phenotypically resistant to six different antimicrobial classes, including 3rd and 4th generation cephalosporins. Despite the presence of mcr-9, the isolate was susceptible to colistin, probably because of a genetic background unfavourable to mcr-9 expression. The lack of colistin resistance, coupled with the fact that the farm of origin had not used colistin in years, suggests that mcr-9 in such a multidrug-resistant strain can be maintained thanks to the co-selection of neighbouring resistance genes, following usage of different antimicrobials. Our findings highlight how a comprehensive approach, integrating phenotypical testing, targeted PCR, WGS-based techniques, and information on antimicrobial usage is crucial to shed light on antimicrobial resistance.