Resistance Plasmid Research Articles

The Effects of Cupriavidus Metallidurians on Plant Growth and Development in Areas with Heavy Metal Runoff

This project measures the effects of C. metallidurians on agricultural plant growth in environments with heavy metal runoff. Heavy metals have been known to negatively impact the environment specifically in the form of runoff. Heavy metals usually accumulate plant roots and inhibit nutrient and water transportation within plants. lIn this project, heavy metals being tested include Zinc, which prevents nutrients from being transported to the leaves; Nickel, which inhibits transpiration and photosynthesis and impairs metabolism; and Copper, which can cause protein denaturation and corrupt soil viability. C. metallidurians are a type of bacteria known to have heavy metal resistant plasmids and plasmid determinants specifically directed towards a different heavy metal each, with the cnr being resistant to copper and nickel, chr being resistant to copper and zinc, and cop being resistant to specifically copper. C. metallidurans have previously been known to detoxify high concentrations of copper and gold. They more famously have been known to pump out gold nuggets from exposure to copper metals. Since the effects of other heavy metals on C. metallidurans are not as well studied, this project aims to study the effects of those other heavy metals such as Zinc, Nickel, and Copper and apply this interaction to a real-life scenario of agriculture to see if this bacteria may be able to minimize the damage of Heavy Metal Runoff pollution.

AadT, a new weapon in Acinetobacter's fight against antibiotics.

Genes encoding a novel multidrug efflux pump, AadT, from the Drug:H+ antiporter 2 family, were discovered in Acinetobacter multidrug resistance plasmids. Here, we profiled the antimicrobial resistance potential, and examined the distribution of these genes. aadT homologs were found in many Acinetobacter and other Gram-negative species and were typically adjacent to novel variants of adeAB(C), which encodes a major tripartite efflux pump in Acinetobacter. The AadT pump decreased bacterial susceptibility to at least eight diverse antimicrobials, including antibiotics (erythromycin and tetracycline), biocides (chlorhexidine), and dyes (ethidium bromide and DAPI) and was able to mediate ethidium transport. These results show that AadT is a multidrug efflux pump in the Acinetobacter resistance arsenal and may cooperate with variants of AdeAB(C).

Genomic surveillance of multidrug-resistant Klebsiella in Wales reveals persistent spread of Klebsiella pneumoniae ST307 and adaptive evolution of pOXA-48-like plasmids.

Rising rates of multidrug-resistant Klebsiella infections necessitate a comprehensive understanding of the major strains and plasmids driving spread of resistance elements. Here, we analysed 540 clinical, screen and environmental Klebsiella isolates recovered from across Wales between 2007 and 2020 using combined short- and long-read sequencing approaches. We identified resistant clones that have spread within and between hospitals including the high-risk strain sequence type (ST)307, which acquired the bla OXA-244 carbapenemase gene on a pOXA-48-like plasmid. We found evidence that this strain, which caused an acute outbreak largely centred on a single hospital in 2019, had been circulating undetected across South Wales for several years prior to the outbreak. In addition to clonal transmission, our analyses revealed evidence for substantial plasmid spread, mostly notably involving bla KPC-2 and bla OXA-48-like (including bla OXA-244) carbapenemase genes that were found among many species and strain backgrounds. Two thirds (20/30) of the bla KPC-2 genes were carried on the Tn4401a transposon and associated with IncF plasmids. These were mostly recovered from patients in North Wales, reflecting an outward expansion of the plasmid-driven outbreak of bla KPC-2-producing Enterobacteriaceae in North-West England. A total of 92.1 % (105/114) of isolates with a bla OXA-48-like carbapenemase carried the gene on a pOXA-48-like plasmid. While this plasmid family is highly conserved, our analyses revealed novel accessory variation including integrations of additional resistance genes. We also identified multiple independent deletions involving the tra gene cluster among pOXA-48-like plasmids in the ST307 outbreak lineage. These resulted in loss of conjugative ability and signal adaptation of the plasmids to carriage by the host strain. Altogether, our study provides, to our knowledge, the first high resolution view of the diversity, transmission and evolutionary dynamics of major resistant clones and plasmids of Klebsiella in Wales, and forms an important basis for ongoing surveillance efforts. This article contains data hosted by Microreact.

Genome-Based Retrospective Analysis of a Providencia stuartii Outbreak in Rome, Italy: Broad Spectrum IncC Plasmids Spread the NDM Carbapenemase within the Hospital.

Providencia stuartii is a member of the Morganellaceae family, notorious for its intrinsic resistance to several antibiotics, including last-resort drugs such as colistin and tigecycline. Between February and March 2022, a four-patient outbreak sustained by P. stuartii occurred in a hospital in Rome. Phenotypic analyses defined these strains as eXtensively Drug-Resistant (XDR). Whole-genome sequencing was performed on the representative P. stuartii strains and resulted in fully closed genomes and plasmids. The genomes were highly related phylogenetically and encoded various virulence factors, including fimbrial clusters. The XDR phenotype was primarily driven by the presence of the blaNDM-1 metallo-β-lactamase alongside the rmtC 16S rRNA methyltransferase, conferring resistance to most β-lactams and every aminoglycoside, respectively. These genes were found on an IncC plasmid that was highly related to an NDM-IncC plasmid retrieved from a ST15 Klebsiella pneumoniae strain circulating in the same hospital two years earlier. Given its ability to acquire resistance plasmids and its intrinsic resistance mechanisms, P. stuartii is a formidable pathogen. The emergence of XDR P. stuartii strains poses a significant public health threat. It is essential to monitor the spread of these strains and develop new strategies for their control and treatment.

The role of plasmids in carbapenem resistant E. coli in Alameda County, California

BackgroundAntimicrobial resistant infections continue to be a leading global public health crisis. Mobile genetic elements, such as plasmids, have been shown to play a major role in the dissemination of antimicrobial resistance (AMR) genes. Despite its ongoing threat to human health, surveillance of AMR in the United States is often limited to phenotypic resistance. Genomic analyses are important to better understand the underlying resistance mechanisms, assess risk, and implement appropriate prevention strategies. This study aimed to investigate the extent of plasmid mediated antimicrobial resistance that can be inferred from short read sequences of carbapenem resistant E. coli (CR-Ec) in Alameda County, California. E. coli isolates from healthcare locations in Alameda County were sequenced using an Illumina MiSeq and assembled with Unicycler. Genomes were categorized according to predefined multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) schemes. Resistance genes were identified and corresponding contigs were predicted to be plasmid-borne or chromosome-borne using two bioinformatic tools (MOB-suite and mlplasmids).ResultsAmong 82 of CR-Ec identified between 2017 and 2019, twenty-five sequence types (STs) were detected. ST131 was the most prominent (n = 17) followed closely by ST405 (n = 12). blaCTX−M were the most common ESBL genes and just over half (18/30) of these genes were predicted to be plasmid-borne by both MOB-suite and mlplasmids. Three genetically related groups of E. coli isolates were identified with cgMLST. One of the groups contained an isolate with a chromosome-borne blaCTX−M−15 gene and an isolate with a plasmid-borne blaCTX−M−15 gene.ConclusionsThis study provides insights into the dominant clonal groups driving carbapenem resistant E. coli infections in Alameda County, CA, USA clinical sites and highlights the relevance of whole-genome sequencing in routine local genomic surveillance. The finding of multi-drug resistant plasmids harboring high-risk resistance genes is of concern as it indicates a risk of dissemination to previously susceptible clonal groups, potentially complicating clinical and public health intervention.

Characterizations of blaCTX-M-14 and blaCTX-M-64 in a clinical isolate of Escherichia coli from China.

Extended-spectrum beta-lactamase-producing Gram-negative bacteria are common in the community and hospitals. To monitor ESBLs mediated by the CTX-M genotype, we collected clinical ESBL pathogenic strains from a hospital in central China and observed a strain of Escherichia coli, namely Ec15103 carrying blaCTX-M-14, blaCTX-M-64 and blaTEM-1, isolated from the blood of a 7-day-old infant in 2015. Strain Ec15103 contains two drug resistance plasmids: pEc15103A, an IncFI-type plasmid that cannot be conjugatively transferred and carries the drug resistance genes blaTEM-1, aacC2, aadA5, sul1, mph(A), sul2, strAB, and tetA(A); and pEc15103B, an IncK2/Z-type plasmid that carries the conjugation transfer gene and blaCTX-M-14. In addition, blaCTX-M-64 is located on the chromosome of Ec15103, and it is the first report of pathogen with blaCTX-M-64 located on its chromosome (the search terms used "blaCTX-M-64" and "chromosome"). blaCTX-M-14 and blaCTX-M-64 are carried by ISEcp1-mediated transposon Tn6503a and Tn6502, respectively. The conjugation transfer ability of pEc15103B was significantly inhibited by zidovudine (AZT) and linoleic acid (LA) and that expression of blaCTX-M-14, blaCTX-M-64 and blaTEM-1 at the mRNA level did not change based on the concentration of cefotaxime or ampicillin. Co-occurrence of blaCTX-M-14 and blaCTX-M-64 in a single isolate will enhance the drug resistance of bacteria, and the presence of blaCTX-M-64 in the chromosome may make the resistance more maintain. This fact will facilitate its dissemination and persistence under different antimicrobial selection pressures. It is essential to prevent these strains from further spreading in a hospital environment.

Draft Genome Sequences and Genome Characterization of Three Toxigenic and Two Nontoxigenic Clostridioides difficile Clinical Isolates from Florida, USA.

Draft genome sequences of five Clostridioides difficile clinical isolates were obtained in Florida, USA. Three isolates, designated TGH29 (sequence type 1 [ST1]/clade 2), TGH79 (ST11/clade 5), and TGH91 (ST35/clade 1), contained toxin-encoding genes. The two nontoxigenic strains were classified as TGH114 (ST109/clade 4) and TGH132 (ST15/clade 1). Antimicrobial resistance determinants and plasmids were detected and putative prophages predicted in some isolates.

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.

Molecular epidemiology of enterically colonizing Escherichia coli with resistance against third-generation cephalosporins isolated from stool samples of European soldiers with concomitant diarrhea on deployment in Western African Mali.

Extended spectrum beta-lactamases (ESBL) are frequently found in Enterobacterales isolates from Western Africa. However, information on the molecular epidemiology of regional ESBL-positive Enterobacterales strains is scarce. In order to provide epidemiological information, ESBL-positive Escherichia coli isolates from stool samples of European soldiers with diarrhea deployed to a field camp in Mali were subjected to whole-genome sequencing (Illumina MiSeq and Oxford Nanopore MinION) and antimicrobial susceptibility testing. With two exemptions, sequence-based analysis suggested an absence of transmission events between soldiers as indicated by a high genetic diversity of isolates and sequence types, confirming previous rep-PCR results. Third-generation cephalosporin resistance was associated with the presence of blaCTX-M-15 genes with (n = 14) and without (n = 5) co-occurring blaTEM-1b genes. Between 0 and 6 virulence and resistance plasmids per isolate were recorded. The detected resistance plasmids could be categorized into five types, which, in turn, share different sequence-identical segments, representing particular antimicrobial resistance gene-associated mobile genetic elements (MGEs). Phenotypic resistance rates within the 19 assessed isolates that showed distinguishable colony morphologies were 94.7% (18/19) against ampicillin-sulbactam and trimethoprim/sulfamethoxazole, 68.4% (13/19) against moxifloxacin, 31.6% (6/19) against ciprofloxacin, 42.1% (8/19) against gentamicin, 31.6% (6/19) against tobramycin, and 21.1% (4/19) against piperacillin-tazobactam and fosfomycin. Virulence-associated genes mediating infectious gastroenteritis were rarely detected. The gene aggR, which is characteristic for enteroaggregative E. coli, was only detected in one single isolate. In summary, we found a variety of different strains and clonal lineages of ESBL-carrying E. coli. Transmission either between soldiers or from common contaminated sources was demonstrated in two cases and played only a minor role in this military field camp, while there were indications that resistance gene bearing MGEs had been exchanged between antimicrobial resistance gene-(ARG-)carrying plasmids.

Environmental risks of antibiotic resistance genes released from biological laboratories and its control measure.

Antibiotic resistance genes (ARGs) are a growing global threat to public health. Biological laboratory wastewater contains large amounts of free ARGs. It is important to assess the risk of free ARGs from biological laboratories and to find appropriate treatments to control their spread. The fate of plasmids in the environment and the effect of different thermal treatments on their persistence activity were tested. The results showed that untreated resistance plasmids could exist in water for more than 24h (the special 245bp fragment). Gel electrophoresis and transformation assays showed that the plasmids boiled for 20min retained 3.65% ± 0.31% transformation activity of the intact plasmids, while autoclaving for 20min at 121°C could effectively degrade the plasmids and that NaCl, bovine serum albumin, and EDTA-2Na affected the degradation efficiency of the plasmids during boiling. In the simulated aquatic system, using 106 copy/μL of plasmids after autoclaving, only 102 copies/μL of the fragment after only 1-2h could be detected. By contrast, boiled plasmids for 20min were still detectable after plunging them into water for 24h. These findings suggest that untreated and boiled plasmids can remain in the aquatic environment for a certain time resulting in the risk of disseminating ARGs. However, autoclaving is an effective way of degrading waste free resistance plasmids.

Resistance genes and plasmid profile of bacteria Isolated from selected water bodies in Owo Metropolis

Resistance genes and plasmid profile of bacteria Isolated from selected water bodies in Owo Metropolis

Resistance genes and plasmid profile of bacteria Isolated from selected water bodies in Owo Metropolis

Aquatic samples from three water bodies were collected following laid down procedures. The bacteria were isolated using, Eosin Methylene Blue agar (EMB), Mannitol salt agar (MSA), Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) Agar, Muller Hinton (MH). The isolates were subjected to colonial, morphological and biochemical characterization. Pathogenicity test was carried out on the isolates using blood agar to determine their hemolytic activities. Antibiotic resistance status was also investigated against standard antibiotics using the Kirby Bauer disk diffusion method and the multiple antibiotic resistance indices of the isolates were also determined. The plasmid profiles were determined and the resistance genes were identified using molecular means. The bacteria species isolated were Veillonella Spp, Vibrio orientalis and Micrococcus luteus. Veillonella Spp showed Beta hemolysis while Micrococcus luteus showed alpha hemolysis and Vibrio orientalis showed gamma hemolysis. Antibiotic susceptibility test showed all isolates to be antibiotic resistant to varying degrees and multiple antibiotic resistance indices (MARi) depending on their level of resistance. Veillonella. spp. had MARi of 0.3 while M. luteus and V. orietalis had MARi of 0.5. Plasmid profiling showed all isolates to have plasmids with bandwidth of 10,000 bp. Vibrio spp and Veillonella Spp were positive for beta lactamase genes (bla SHV). Only Vibrio was positive for resistance to aminoglycosides (aac(3)-IV). Vibrio orientalis and Micrococcus luteus were found to have the resistance genes for quinolones (qnrA gene) while all the isolates possess genes against the sulphonamides (sul1 gene) used. The result of this work establishes the antibiotic resistance status of bacteria isolated from aquatic habitat and suggests their important role in the spread of antimicrobial resistance AMR, and antibiotic resistance genes (ARGs)

PLASMID PROFILING OF EXTENDED SPECTRUM Β-LACTAMASES PRODUCING ESCHERICHIA COLI IN SOME HOSPITALS IN BAGHDAD

This study was aimed to determine the prevalence of ESBLs in E. coli and their relation to plasmid profiling patterns in a sample of patients in Iraqi hospitals. The total number of E. coli isolates 91 (49%) were collected out of 185 clinical samples. The antibiotic sensitivity test for isolates showed high resistance percentage to Nitrofurantion 95.7%, amoxicillin and Ceftriaxone to each of which 91.3% and to Ceftazidime 90.2%, while the highest sensitivity was observed against Imipenem 100%, Meropenem 99% then to Amikacin 93.5%. The Multi drug resistant (MDR) isolates were 53(58.2) % and ESBLs producers were 45 (49.5) %. Plasmid profile analysis showed nineteen different plasmid profile patterns were obtained based on molecular weight and number of plasmids content. Most β-lactamase producers had multiple plasmids, where’s single plasmid profile predominant in non-β-lactamase-producing isolates. The presence of common plasmid among the isolates increases the distribution of resistant plasmid in the community. In conclusion, ESBLs producing bacteria can transferred between species by plasmids leading to cross resistance which give a limited options for treatment.

Within-patient horizontal transfer of pOXA-48 from a hypervirulent Klebsiella pneumoniae SL218 to Serratia marcescens following spread of the K. pneumoniae isolate among hospitalised patients, Denmark, 2021.

A hypervirulent Klebsiella pneumoniae SL218 (ST23-KL57), phylogenetically distinct from the classical hypervirulent SL23 (ST23-KL1) lineage, was transmitted between hospitalised patients in Denmark in 2021. The isolate carried a hybrid resistance and virulence plasmid containing bla NDM-1 and a plasmid containing bla OXA-48 (pOXA-48); the latter plasmid was horizontally transferred within-patient to Serratia marcescens. The convergence of drug resistance and virulence factors in single plasmids and in different lineages of K. pneumoniae is concerning and requires surveillance.

Genomic and functional characterization of carbapenem-resistant Klebsiella pneumoniae from hospital wastewater

BackgroundThe emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) attracted extensive attention. Information on CRKP from hospital wastewater (HWW) is limited. The aims of this study were to investigate the genomic characteristics and to evaluate the survivability characteristics of 11 CRKP from HWW in a Chinese teaching hospital in Fujian province.ResultsA total of 11 CRKP from HWW were recovered in this study. All CRKP from HWW were resistant to most antibiotics. Comparative genetic analysis demonstrated that all CRKP isolates were clustered into the three distinct phylogenetic clades and clade 2 and clade 3 were mixtures of samples collected from both HWW and clinical settings. Varieties of resistance genes, virulence genes and plasmid replicon types were detected in CRKP from HWW. In vitro transfer of blaKPC-2 was successful for 3 blaKPC-2-positive CRKP from HWW with high conjugation frequency. Our study demonstrated that the genetic environments of blaKPC−2 shared core structure with ISKpn27-blaKPC−2-ISKpn6. Group analysis showed that CRKP from HWW had a lower survivability in serum compared to clinical CRKP (p < 005); and CRKP from HWW had no significant difference in survivability in HWW compared to clinical CRKP (p > 005).ConclusionsWe analyzed the genomic and survivability characteristics of CRKP from HWW in a Chinese teaching hospital. These genomes represent a significant addition of genomic data from the genus and could serve as a valuable resource for future genomic studies about CRKP from HWW.

Genomic insight of extremely drug-resistant Klebsiella pneumoniae ST5378 from a paediatric bloodstream infection

This study investigated the draft genome and phylogeny of an extremely drug-resistant and novel sequence type Klebsiella pneumoniae isolated from a paediatric bloodstream infection. An isolate from a 7-year-old child with severe respiratory infection was identified, and the whole genome was sequenced using the Illumina MiSeq platform. High-quality reads were de novo assembled via Unicycler and annotated via PROKKA. Antimicrobial resistance genes, virulence factors, and plasmid and phage sequences were identified using the resistance gene identifier, VFanalyzer, Plasmidfinder, and PHASTER, respectively. Phylogenetics of closely related strains were inferred using core-genome multi-locus sequence typing and single nucleotide polymorphism. The draft genome of carbapenem-resistant K. pneumoniae RKS87 was 5 580 330 bp in size, with a GC content of 57.73%. The final assembly resulted in 38 contigs comprising 5075 CDS, 124 pseudo genes, 83 tRNA, 25 rRNA, and 10 ncRNA. The strain was assigned to a novel sequence type, ST5378, and harboured blaSHV-11, blaCTX-M-15, blaTEM-1, blaNDM-1, APH(3')-VI, OqxA, QnrS1, and fosA. We also identified the mutations in outer membrane porin (OmpK36 and OmpK37) and two-component system genes (PmrB and EptB). Three biomarkers (iroE, iroN, and iutA) associated with hypervirulent phenotype were also present in the genome. Phylogenetics of closely related strains revealed the clonal lineage of ST2938. The genome sequence and phylogenetics of the strain offer valuable insight into the clonal lineage, resistance genes, and pathogenicity of the novel sequence type ST5378.

Transcriptional modifications enhance the persistence of resistant plasmids in the presence of tetracycline in an environmentally relevant concentration

Antibiotic resistance genes (ARGs) have captured immense attention due to their widespread existence and remarkable ability to spread across species boundaries. Plasmid carriage promotes the persistence and spread of ARGs in the environment. To investigate the prevalence of plasmids, we conducted serial passage experiments on Pseudomonas putida KT2442 with multidrug-resistant plasmid RP4::gfp in the presence of tetracycline (TC) in an environmentally relevant concentration. The results showed that TC in environmental concentration compensated the fitness cost brought by the plasmid, prolonged the persistence time of the plasmid-bearing strain, and induced the reoccurrence of plasmids after the window time of plasmid loss. Transcriptome sequencing showed that plasmid recovery was compensated by the up-regulation of glyoxylic acid shunt and the down-regulation of ribosome biosynthesis. It is therefore hypothesized that transcriptional modifications may enhance the persistence of resistant plasmids within the population in the presence of TC in an environmentally relevant concentration. This work opens up an avenue for developing a technology based on the window time of plasmid loss to prevent the spread of ARGs.

Genome profiling of uropathogenic E. coli from strictly defined community-acquired UTI in paediatric patients: a multicentric study

BackgroundUrinary tract infection (UTI) in children is a common bacterial infection. The emergence of extended-spectrum beta-lactamases (ESBLs) poses a major challenge against the treatment of uropathogens. We aimed to characterize the E. coli isolates recovered from children with UTI for their resistance profile and circulating sequence types (ST).MethodsChildren (> 1.5–18 years of age) from different community health centres of India with symptoms of UTI were enrolled. Isolates causing significant bacteriuria were identified by Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) and tested for antimicrobial susceptibility by the automated system, VITEK-2 (Biomeriux, Durhum, US). Nineteen E. coli isolates (15 ESBL positive and 4 ESBL negative) were sequenced in Oxford Nanopore platform followed by core-genome phylogeny, accessory genome cluster analysis, identification of sequence types, mobile genetic elements, genetic antimicrobial resistance markers. The correlation between detection of antimicrobial resistance genes with phenotypic resistance profiles was also investigated.ResultsEleven percent of children had significant bacteriuria [male:female—1:1, > 50% were 11–18 years of age group]. E. coli was predominant (86%) followed by K. pneumoniae (11%). Susceptibility of E. coli was highest against fosfomycin (100%) followed by carbapenems (90.7%) and nitrofurantoin (88.8%). ST131 (15.8%) and ST167 (10.5%) found as high-risk clones with the presence of plasmid [IncFIB (63.1%), IncFIA (52.6%)], and composite transposon [Tn2680 (46.6%)] in many isolates. Few isolates coharboured multiple beta-lactamases including blaNDM-5 (33.3%), blaOXA-1 (53.3%), blaCTX-M-15 (60%) and blaTEM-4 (60%).ConclusionsThis study highlights horizontal transmission of resistance genes and plasmids in paediatric patients at community centers across the nation harbouring multidrug-resistant genes such as blaNDM-5 and blaCTX-M-15 associated with high-risk clones ST131 and ST167. The data is alarming and emphasizes the need for rapid identification of resistance markers to reduce the spread in community. To our knowledge, this is the first multicentric study targeting paediatric UTI patients from the community setting of India.

Insertion sequences mediate clinical ST34 monophasic Salmonella enterica serovar Typhimurium plasmid polymorphism

Hybrid plasmids can combine the genetic elements of multiple plasmids, with the potential to carry a variety of antibiotic resistance genes and virulence genes, causing a great public health concern. Hybrid plasmids formed by fusion events may further exacerbate the spread of antibiotic resistance genes as well as plasmid evolution. Salmonella enterica serovar 4,[5],12:i:- is a monophasic variant of S. Typhimurium, which is one of the major causes of foodborne disease outbreaks worldwide. To assess the risk of transmission due to plasmid structure changes, we investigated the structural diversity of plasmids in two S. 4,[5],12:i:- isolates. Nanopore long-read sequencing was performed for plasmid comparison between original plasmids (donor isolates) and reorganized plasmids. We found that the IncHI2-IncHI2A multidrug resistance (MDR) plasmids in S. 4,[5],12:i:- possessed high plasticity, and could undergo recombination with other plasmids to form fusion plasmids of different sizes. Plasmid structural polymorphisms were mainly mediated by insertion sequences such as IS26 and ISPa40, and led to the rearrangement of the plasmid internal structures. To the best of our knowledge, this is the first report of the fusion of the IncHI2-IncHI2A and IncB/O/K/Z plasmids in S. 4,[5],12:i:- mediated by IS26. In addition, we also found that the mcr-1 gene was able to generate duplication during conjugation. Polymorphic changes in MDR plasmids during conjugation may further reduce the choice of clinical therapeutic agents. Therefore, continuous monitoring regarding plasmid polymorphic changes during transmission in both in vitro and in vivo is urgently needed to decipher the MDR plasmid evolution.