Resistance Plasmid Research Articles

Klebsiella pneumoniae clinical isolates with features of both multidrug-resistance and hypervirulence have unexpectedly low virulence

Klebsiella pneumoniae has been classified into two types, classical K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). cKP isolates are highly diverse and important causes of nosocomial infections; they include globally disseminated antibiotic-resistant clones. hvKP isolates are sensitive to most antibiotics but are highly virulent, causing community-acquired infections in healthy individuals. The virulence phenotype of hvKP is associated with pathogenicity loci responsible for siderophore and hypermucoid capsule production. Recently, convergent strains of K. pneumoniae, which possess features of both cKP and hvKP, have emerged and are cause of much concern. Here, we screen the genomes of 2,608 multidrug-resistant K. pneumoniae isolates from the United States and identify 47 convergent isolates. We perform phenotypic and genomic characterization of 12 representative isolates. These 12 convergent isolates contain a variety of antimicrobial resistance plasmids and virulence plasmids. Most convergent isolates contain aerobactin biosynthesis genes and produce more siderophores than cKP isolates but not more capsule. Unexpectedly, only 1 of the 12 tested convergent isolates has a level of virulence consistent with hvKP isolates in a murine pneumonia model. These findings suggest that additional studies should be performed to clarify whether convergent strains are indeed more virulent than cKP in mouse and human infections.

Corynebacterium oculi-related bacterium may act as a pathogen and carrier of antimicrobial resistance genes in dogs: a case report

BackgroundThe genus Corynebacterium comprises well-known animal and human pathogens as well as commensals of skin and mucous membranes. Species formerly regarded as contaminants are increasingly being recognized as opportunistic pathogens. Corynebacterium oculi has recently been described as a human ocular pathogen but has so far not been reported in dogs.Case presentationHere we present two cases of infection with a novel Corynebacterium sp., a corneal ulcer and a case of bacteriuria. The two bacterial isolates could not be identified by MALDI-TOF MS. While 16 S rRNA gene (99.3% similarity) and rpoB (96.6% identity) sequencing led to the preliminary identification of the isolates as Corynebacterium (C.) oculi, whole genome sequencing revealed the strains to be closely related to, but in a separate cluster from C. oculi. Antimicrobial susceptibility testing showed high minimal inhibitory concentrations of lincosamides, macrolides, tetracycline, and fluoroquinolones for one of the isolates, which also contained an erm(X) and tet-carrying plasmid as well as a nonsynonymous mutation leading to an S84I substitution in the quinolone resistance determining region of GyrA.ConclusionsWhile the clinical signs of both dogs were alleviated by antimicrobial treatment, the clinical significance of these isolates remains to be proven. However, considering its close relation with C. oculi, a known pathogen in humans, pathogenic potential of this species is not unlikely. Furthermore, these bacteria may act as reservoir for antimicrobial resistance genes also in a One Health context since one strain carried a multidrug resistance plasmid related to pNG3 of C. diphtheriae.

Effect of Zinc Oxide and Copper Sulfate on Antibiotic Resistance Plasmid Transfer in Escherichia coli.

Heavy metals such as zinc (Zn) and copper (Cu) may be associated with antibiotic resistance dissemination. Our aim was to investigate whether sub-lethal dosage of Zn and Cu may enhance plasmid transfer and subsequently resistance genes dissemination. Plasmid conjugation frequencies (PCF) were performed with Escherichia coli strains bearing IncL-blaOXA-48, IncA/C-blaCMY-2, IncI1-blaCTX-M-1, IncF-blaCTX-M-1, and IncX3-blaNDM-5 as donors. Mating-out assays were performed with sub-dosages of zinc oxide (ZnO) and Cu sulfate (CuSO4). Quantification of the SOS response-associated gene expression levels and of the production of reactive oxygen species were determined. Increased PCF was observed for IncL, IncA/C, and IncX3 when treated with ZnO. PCF was only increased for IncL when treated with CuSO4. The ROS production presented an overall positive correlation with PCF after treatment with ZnO for IncL, IncA/C, and IncX3. For CuSO4 treatment, the same was observed only for IncL. No increase was observed for expression of SOS response-associated genes under CuSO4 treatment, and under ZnO treatment, we observed an increase in SOS response-associated genes only for IncX3. Our data showed that sub-dosages of ZnO and CuSO4 could significantly enhance PCF in E. coli, with a more marked effect observed with IncL, IncA/C, and IncX3 scaffolds. Our study suggested that use of certain heavy metals is not the panacea for avoiding use of antibiotics in order to prevent the dissemination of antibiotic resistance.

Genomic characterization of a carbapenem-resistant Citrobacter freundii clinical isolate from China carrying blaNDM-5 on a novel IncC-IncFIB-IncX3 plasmid

Citrobacter freundii is one of the important pathogens that can cause nosocomial infections. The advent of carbapenem-resistant C. freundii complicates clinical treatment. Here, we reported the genome sequence of a carbapenem-resistant C. freundii strain carrying a novel IncC-IncFIB-IncX3 plasmid in China. The genome sequence of C. freundii CRNMS1 was obtained using the Illumina NovaSeq 6000 platform and the long-read Nanopore sequencer. Multilocus sequence typing was identified using MLST (v.2.23.0). The identification of antimicrobial resistance genes (ARGs) and plasmid replicons was performed using the resfinder and plasmidfinder of ABRicate (v.1.0.1). Circular comparisons of plasmids were performed using the BLAST Ring Image Generator (BRIG). CRNMS1 belongs to ST116 in the C. freundii MLST scheme. Thirteen ARGs were predicted in all, including blaNDM-5, which was located in a plasmid. The plasmid pblaNDM5-S1, which carried the blaNDM-5 gene, was discovered to be a novel plasmid including three plasmid replicons (IncC, IncFIB, and IncX3) as well as seven ARGs (sul1, sul2, floR, dfrA17, aadA5, qnrA1, and blaNDM-5). A total of 38 blaNDM-5-bearing C. freundii strains can be retrieved from the NCBI database. Phylogenetic analysis revealed a worldwide distribution of C. freundii strains carrying the blaNDM-5 gene, with China having the highest prevalence (39%, 15/38). However, they were distantly related to CRNMS1 with SNP differences >2545. In summary, we reported a novel IncC-IncFIB-IncX3 plasmid carrying blaNDM-5 in a carbapenem-resistant C. freundii strain in China. The development of such hybrid plasmids facilitates the transmission of ARGs.

Involvement of Tn3 transposon in formation and transmission of hypervirulent and carbapenem-resistant Klebsiella pneumoniae

ABSTRACT Hypervirulent and carbapenem-resistant Klebsiella pneumoniae poses a severe threat to public health for its high pathogenicity, transmissibility, and drug resistance. This study aims to explore the evolutionary path and the role of Tn3 transposon in the virulence and carbapenem resistance transmission of K. pneumoniae . Bioinformatics analysis and some experimental tests such as plasmid conjugation experiments, antimicrobial susceptibility testing, and virulence-associated tests were performed to explore the virulence and drug resistance transmission. The complete genome sequencing, S1 nuclease pulsed-field gel electrophoresis, and bioinformatics analysis were used to investigate their transmission mechanism mediated by Tn3 transposons. Three hypervirulent and carbapenem-resistant K. pneumoniae isolates, which were obtained from different patients at different time points in the same ward, harbored a virulence plasmid and a carbapenem resistance plasmid. They were confirmed to have first evolved from hypervirulent isolates and then acquired a bla KPC -positive plasmid (CR-hvKp evolutionary pattern). The non-conjugative virulence plasmid pVir could be transferred to other bacterial strains via mobilization by the conjugative IncN/U-type plasmid pKPC, as well as by fusing with the conjugative pKPC plasmid (mediated by Tn3-based homologous recombination) to be self-transmissible, thus transferring drug resistance and virulence. The cointegration, pVir/KPC fusion plasmid, was further resolved into pVir and pKPC between the duplicated copies of the Tn3 transposon resolution site mediated by site-specific recombination. Therefore, Tn3 transposons can mediate hypervirulent and carbapenem-resistant K. pneumoniae strains transferring drug resistance and virulence to other bacteria. We must be vigilant to emerging transposon-mediated hypervirulent and carbapenem-resistant pathogens. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) is resistant to most common antibiotics, becoming the most important and prevalent nosocomial opportunity pathogen. Besides, K. pneumoniae can also cause severe community-acquired infections, such as primary liver abscess and endophthalmitis. These pathogens are commonly referred to as hvKp. CRKP and hvKp have evolved separately, each occupying its own clonal lineage and exhibiting a variety of properties. Our study provides important insights into the evolutionary events related to the arousal of virulence and drug resistance in K. pneumoniae through plasmid transmission, mediated by Tn3 transposon. Our study also provides evidence that multiple mechanisms contribute to the successful transfer of non-conjugative virulence plasmid, and the involvement of transposons enhances the efficiency. A good knowledge of its transmission mechanisms is fundamental to finding effective strategies to combat these threatening pathogens. Transposons are widely present in bacteria, spreading resistance and virulence genes between the environment and humans. Therefore, emerging transposon-mediated hypervirulent and carbapenem-resistant pathogens should be highly valued.

Development of an antimicrobial resistance plasmid transfer gene database for enteric bacteria.

Introduction: Type IV secretion systems (T4SSs) are integral parts of the conjugation process in enteric bacteria. These secretion systems are encoded within the transfer (tra) regions of plasmids, including those that harbor antimicrobial resistance (AMR) genes. The conjugal transfer of resistance plasmids can lead to the dissemination of AMR among bacterial populations. Methods: To facilitate the analyses of the conjugation-associated genes, transfer related genes associated with key groups of AMR plasmids were identified, extracted from GenBank and used to generate a plasmid transfer gene dataset that is part of the Virulence and Plasmid Transfer Factor Database at FDA, serving as the foundation for computational tools for the comparison of the conjugal transfer genes. To assess the genetic feature of the transfer gene database, genes/proteins of the same name (e.g., traI/TraI) or predicted function (VirD4 ATPase homologs) were compared across the different plasmid types to assess sequence diversity. Two analyses tools, the Plasmid Transfer Factor Profile Assessment and Plasmid Transfer Factor Comparison tools, were developed to evaluate the transfer genes located on plasmids and to facilitate the comparison of plasmids from multiple sequence files. To assess the database and associated tools, plasmid, and whole genome sequencing (WGS) data were extracted from GenBank and previous WGS experiments in our lab and assessed using the analysis tools. Results: Overall, the plasmid transfer database and associated tools proved to be very useful for evaluating the different plasmid types, their association with T4SSs, and increased our understanding how conjugative plasmids contribute to the dissemination of AMR genes.

Prevalence and Transmission of Extended-Spectrum Cephalosporin (ESC) Resistance Genes in Escherichia coli Isolated from Poultry Production Systems and Slaughterhouses in Denmark.

The emergence of extended-spectrum cephalosporin (ESC)-resistant Escherichia coli is a global concern. This study aimed to assess the prevalence and transmission of ESC-resistant E. coli in the Danish broiler production system. Samples from two vertically integrated Production Systems (1 and 2) and two slaughterhouses (A and B) were analyzed (n = 943) for the occurrence of ESC-resistant E. coli from 2015 to 2018. ESC-resistant E. coli isolates were whole-genome sequenced (WGS) for characterization of the multi-locus sequence type (MLST), antibiotic resistance genes, virulence genes, and plasmid replicon types. An ad hoc core genome (cg) MLST based on 2513 alleles was used to examine the genetic relatedness among isolates. The prevalence of ESC-resistant E. coli in the conventional Production System 1 was 2.7%, while in Production System 2 the prevalence was 26.7% and 56.5% for samples from the conventional and organic production, respectively. The overall prevalence of ESC-resistant E. coli in broiler thigh and fecal samples ranged from 19.3% in Slaughterhouse A to 22.4% in Slaughterhouse B. In total, 162 ESC-resistant E. coli were isolated and shown to belong to 16 different sequence types (STs). The most prevalent STs were ST2040 (n = 85) and ST429 (n = 22). Seven ESC resistance genes were detected: blaCMY-2 (n = 119), blaTEM-52B (n = 16), blaCTX-M-1 (n = 5), blaTEM-52C (n = 3), blaCTX-M-14 (n = 1), blaSHV-12 (n = 1), and up-regulation of ampC (n = 16), with an unknown resistance gene in one isolate (n = 1). The carriage of blaCMY-2 in 119 isolates was primarily associated with IncI1 (n = 87), and IncK plasmids (n = 31). Highly similar blaCMY-2 carrying E. coli isolates from ST429 were found in production systems as well as in slaughterhouses. In conclusion, findings from this study indicate that ESC-resistant E. coli are transferred vertically from farms in the production systems to slaughterhouses with the potential to enter the food supply.

A critical role of outer membrane vesicles in antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae

BackgroundThis study aimed to illustrate the status of carbapenem-resistant Enterobacterales (CRE) infections in a Chinese tertiary hospital and to investigate the role of outer membrane vesicles (OMVs) in antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP).MethodsThe data of CRE infections was collected from laboratory records, and the CRE isolates from two distinct periods (2015/07 to 2017/07 and 2020/04 to 2021/04) were enrolled to detect the carbapenemase genes by polymerase chain reaction (PCR). Multilocus sequence typing (MLST) was used to analyze the molecular characterization of CRKP. The conjugation assay was performed to verify the transmission of the antibiotic resistance plasmid. The OMVs of CRKP were isolated with a method combining an electrophoretic technique with a 300 kDa cut-off dialysis bag. The protein components in CRKP OMVs were analyzed by liquid chromatography tandem-mass spectrometry (LC–MS/MS), and the meropenem-hydrolyzing bioactivity of KPC in CRKP OMVs was determined with different treatments in vitro.ResultsA total of 178 CRE isolates, including 100 isolates from 2015/07 to 2017/07 and 78 isolates from 2020/04 to 2021/04, were collected for the detection of carbapenemase genes. We found that the carbapenemase gene blaKPC was the most prevalent, followed by blaNDM. By MLST, we found that sequence type (ST) 11 CRKP (96.1%) was the leading type during 2015/07 to 2017/07 and that the ST15 CRKP increased to 46.2% in the late period of 2020/04 to 2021/04. The diameters of Klebsiella pneumoniae OMVs ranged from 100 to 200 nm, and by proteomics analysis the most proteins from OMVs belonged to the “enzyme” group. The KPC enzyme was found in the OMVs from CRKP, and the OMVs could protect inside KPC from proteinase K digestion. Moreover, the KPC enzymes within OMVs, which could be released after Triton X-100 treatment, could hydrolyze meropenem.ConclusionsCRE has increasingly caused infections in hospitals, and blaKPC-positive CRKP infections have constituted a major proportion of infections in the past decade. The OMVs play a critical role in antibiotic resistance in CRKP.

The role of R-plasmids in hospital strains of Proteus

The antibiotic sensitivity of Proteus strains isolated between 2017 and 2021 was investigated. A total of 85 Proteus strains were analyzed, revealing the widespread presence of poly-resistant forms of this pathogen. The examination of the antibiotic susceptibility spectrum in these strains demonstrated that those isolated from 2017 to 2021 exhibited similarities in their resistance profiles, with an expanding range of resistance observed towards gentamycin, kanamycin, carbenicillin, and others. The study identified that hospital-derived Proteus strains possess both conjugative and non-conjugative plasmids, distributed across different compatibility groups. It was shown that 55% of Proteus strains have conjugative R-plasmids, determining stability to streptomycin, gentamycin, carbenicillin, tetracycline, chloramphenicol, refer to T, J, M, N groups of incompatibility and have molecular masses 53.0; 33.0; 38.0; 49.0 MDa, respectively. In Proteus strains plasmids belonging to Inc-PI group of incompatibility were found. Furthermore, non-conjugative R-plasmids were identified in Proteus strains, conferring stability to tetracycline and streptomycin, with molecular masses of 7.2 and 5.8 MDa, respectively. Thus, the investigation of Proteus strains isolated in two clinics highlighted the participation of both conjugative and non-conjugative resistance plasmids in the development of hospital-associated Proteus strains.

Elimination of blaKPC−2-mediated carbapenem resistance in Escherichia coli by CRISPR-Cas9 system

ObjectiveThe purpose of this study is to re-sensitive bacteria to carbapenemases and reduce the transmission of the blaKPC−2 gene by curing the blaKPC−2-harboring plasmid of carbapenem-resistant using the CRISPR-Cas9 system.MethodsThe single guide RNA (sgRNA) specifically targeted to the blaKPC−2 gene was designed and cloned into plasmid pCas9. The recombinant plasmid pCas9-sgRNA(blaKPC−2) was transformed into Escherichia coli (E.coli) carrying pET24-blaKPC−2. The elimination efficiency in strains was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by E-test strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance.ResultsIn the present study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the blaKPC−2 gene. PCR and qPCR results indicated that prokaryotic CRISPR-Cas9 plasmid transforming drug-resistant bacteria can efficiently clear blaKPC−2-harboring plasmids. In addition, the drug susceptibility test results showed that the bacterial resistance to imipenem was significantly reduced and allowed the resistant model bacteria to restore susceptibility to antibiotics after the blaKPC−2-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system.ConclusionIn conclusion, our study demonstrated that the one plasmid-mediated CRISPR-Cas9 system can be used as a novel tool to remove resistance plasmids and re-sensitize the recipient bacteria to antibiotics. This strategy provided a great potential to counteract the ever-worsening spread of the blaKPC−2 gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.

Genomics analysis of KPC-2 and NDM-5-producing Enterobacteriaceae in migratory birds from Qinghai Lake, China.

The study examined the epidemiological characteristics of carbapenem-resistant Enterobacteriaceae (CRE) isolated from migratory birds and surroundings in Qinghai Lake, China. We identified 69 (15.7%) CRE isolates from a total of 439 samples including 29 (6.6%) blaNDM-5 Escherichia coli and 40 (9.1%) blaKPC-2 Klebsiella pneumoniae. WGS analysis indicated that ST746, ST48, ST1011, and ST167 were the primary sequence types (ST) for blaNDM-5 E. coli, while all blaKPC-2K. pneumoniae were ST11 and harbored numerous antibiotic resistance gene types including blaCTX-M, qnrS, and rmtB. A phylogenetic tree based on core genomes revealed that blaNDM-5 E. coli was highly heterogeneous while the blaKPC-2K. pneumoniae was highly genetically similar within the group and to human Chinese isolates. IncX3, IncHI2, and IncFIB-HI2 plasmid replicon types were associated with blaNDM-5 spread, while IncFII-R and IncFII plasmids mediated blaKPC-2 spread. We also identified IncFII-R hybrid plasmids most likely formed by IS26-mediated integration of IncFII into IncR plasmid backbones. This also facilitated the persistence of IncFII-R plasmids and antibiotic resistance genes including blaKPC-2. In addition, all of the blaKPC-2K. pneumoniae isolates harbored a pLVKP-like virulence plasmid carrying a combination of two or more hypervirulence markers that included peg-344, iroB, iucA, rmpA, and rmpA2. This is the first description of ST11 K. pneumoniae that co-carried blaKPC-2- and pLVKP-like virulence plasmids from migratory birds. The blaKPC-2K. pneumoniae carried by migratory birds displayed high genetic relatedness to human isolates highlighting a high risk of transmission of these K. pneumoniae. KEY POINTS: • Multidrug resistance plasmids (blaKPC-2, bla436NDM-5, blaCTX-M, qnrS, and rmtB). • Co-occurrence of plasmid-mediated resistance and virulence genes. • High similarity between migratory bird genomes and humans.

Characterization of a Tigecycline-, Linezolid- and Vancomycin-Resistant Clinical Enteroccoccus faecium Isolate, Carrying vanA and vanB Genes.

Increasing incidence of Enterococcus faecium resistant to key antimicrobials used in therapy of hospitalized patients is a worrisome phenomenon observed worldwide. Our aim was to characterize a tigecycline-, linezolid- and vancomycin-resistant E. faecium isolate with the vanA and vanB genes, originating from a hematoma of a patient hospitalized in an intensive care unit in Poland. Antimicrobial susceptibility (a broad panel) was tested using gradient tests with predefined antibiotic concentrations. The complete genome sequence was obtained from a mixed assembly of Illumina MiSeq and Oxford Nanopore's MinION reads. The genome was analyzed with appropriate tools available at the Center for Genomic Epidemiology, PubMLST and GenBank. Transferability of oxazolidinone, tigecycline and vancomycin resistance genes was investigated by conjugation, followed by PCR screen of transconjugants for antimicrobial resistance genes and plasmid rep genes characteristic for the donor and genomic sequencing of selected transconjugants. The isolate was resistant to most antimicrobials tested; susceptibility to daptomycin, erythromycin and chloramphenicol was significantly reduced, and only oritavancin retained the full activity. The isolate represented sequence type 18 (ST18) and carried vanA, vanB, poxtA, fexB, tet(L), tet(M), aac(6')-aph(2''), ant(6)-Ia and ant(6')-Ii. The vanA, poxtA and tet(M) genes located on ~ 40-kb plasmids were transferable by conjugation yielding transconjugants resistant to vancomycin, linezolid and tigecycline. The substitutions in LiaS, putative histidine kinase, SulP, putative sulfate transporter, RpoB and RpoC were potential determinants of an elevated daptomycin MIC. Comparative analyses of the studied isolate with E. faecium isolates from other countries revealed its similarity to ST18 isolates from Ireland and Uganda from human infections. We provide the detailed characteristics of the genomic determinants of antimicrobial resistance of a clinical E. faecium demonstrating the concomitant presence of both vanA and vanB and resistance to vancomycin, linezolid, tigecycline and several other compounds and decreased daptomycin susceptibility. This isolate is a striking example of an accumulation of resistance determinants involving various mechanisms by a single hospital strain.

Genetic background of neomycin resistance in clinical Escherichia coli isolated from Danish pig farms.

Neomycin is the first-choice antibiotic for the treatment of porcine enteritis caused by enterotoxigenic Escherichia coli. Resistance to this aminoglycoside is on the rise after the increased use of neomycin due to the ban on zinc oxide. We identified the neomycin resistance determinants and plasmid contents in a historical collection of 128 neomycin-resistant clinical E. coli isolates from Danish pig farms. All isolates were characterized by whole-genome sequencing and antimicrobial susceptibility testing, followed by conjugation experiments and long-read sequencing of eight selected representative strains. We detected 35 sequence types (STs) with ST100 being the most prevalent lineage (38.3%). Neomycin resistance was associated with two resistance genes, namely aph(3')-Ia and aph(3')-Ib, which were identified in 93% and 7% of the isolates, respectively. The aph(3')-Ia was found on different large conjugative plasmids belonging to IncI1α, which was present in 67.2% of the strains, on IncHI1, IncHI2, and IncN, as well as on a multicopy ColRNAI plasmid. All these plasmids except ColRNAI carried genes encoding resistance to other antimicrobials or heavy metals, highlighting the risk of co-selection. The aph(3')-Ib gene occurred on a 19 kb chimeric, mobilizable plasmid that contained elements tracing back its origin to distantly related genera. While aph(3')-Ia was flanked by either Tn903 or Tn4352 derivatives, no clear association was observed between aph(3')-Ib and mobile genetic elements. In conclusion, the spread of neomycin resistance in porcine clinical E. coli is driven by two resistance determinants located on distinct plasmid scaffolds circulating within a highly diverse population dominated by ST100. IMPORTANCE Neomycin is the first-choice antibiotic for the management of Escherichia coli enteritis in pigs. This work shows that aph(3')-Ia and to a lesser extent aph(3')-Ib are responsible for the spread of neomycin resistance that has been recently observed among pig clinical isolates and elucidates the mechanisms of dissemination of these two resistance determinants. The aph(3')-Ia gene is located on different conjugative plasmid scaffolds and is associated with two distinct transposable elements (Tn903 and Tn4352) that contributed to its spread. The diffusion of aph(3')-Ib is mediated by a small non-conjugative, mobilizable chimeric plasmid that likely derived from distantly related members of the Pseudomonadota phylum and was not associated with any detectable mobile genetic element. Although the spread of neomycin resistance is largely attributable to horizontal transfer, both resistance determinants have been acquired by a predominant lineage (ST100) associated with enterotoxigenic E. coli, which accounted for approximately one-third of the strains.

Multidrug resistance plasmids underlie clonal expansions and international spread of Salmonella enterica serotype 1,4,[5],12:i:- ST34 in Southeast Asia

Salmonella enterica serotype 1,4,[5],12:i:- (Typhimurium monophasic variant) of sequence type (ST) 34 has emerged as the predominant pandemic genotype in recent decades. Despite increasing reports of resistance to antimicrobials in Southeast Asia, Salmonella ST34 population structure and evolution remained understudied in the region. Here we performed detailed genomic investigations on 454 ST34 genomes collected from Vietnam and diverse geographical sources to elucidate the pathogen’s epidemiology, evolution and antimicrobial resistance. We showed that ST34 has been introduced into Vietnam in at least nine occasions since 2000, forming five co-circulating major clones responsible for paediatric diarrhoea and bloodstream infection. Most expansion events were associated with acquisitions of large multidrug resistance plasmids of IncHI2 or IncA/C2. Particularly, the self-conjugative IncA/C2 pST34VN2 (co-transferring blaCTX-M-55, mcr-3.1, and qnrS1) underlies local expansion and intercontinental spread in two separate ST34 clones. At the global scale, Southeast Asia was identified as a potential hub for the emergence and dissemination of multidrug resistant Salmonella ST34, and mutation analysis suggests of selection in antimicrobial responses and key virulence factors.

SHIP: identifying antimicrobial resistance gene transfer between plasmids.

Plasmids are carriers for antimicrobial resistance (AMR) genes and can exchange genetic material with other structures, contributing to the spread of AMR. There is no reliable approach to identify the transfer of AMR genes across plasmids. This is mainly due to the absence of a method to assess the phylogenetic distance of plasmids, as they show large DNA sequence variability. Identifying and quantifying such transfer can provide novel insight into the role of small mobile elements and resistant plasmid regions in the spread of AMR. We developed SHIP, a novel method to quantify plasmid similarity based on the dynamics of plasmid evolution. This allowed us to find conserved fragments containing AMR genes in structurally different and phylogenetically distant plasmids, which is evidence for lateral transfer. Our results show that regions carrying AMR genes are highly mobilizable between plasmids through transposons, integrons, and recombination events, and contribute to the spread of AMR. Identified transferred fragments include a multi-resistant complex class 1 integron in Escherichia coli and Klebsiella pneumoniae, and a region encoding tetracycline resistance transferred through recombination in Enterococcus faecalis. The code developed in this work is available at https://github.com/AbeelLab/plasmidHGT.

Genetic characterization of a Salmonella enterica serovar Typhimurium isolated from an infant with concurrent resistance to ceftriaxone, ciprofloxacin and azithromycin

To investigate the resistance mechanism of a Salmonella Typhimurium (S. Typhimurium) isolated from a faecal sample of an infant, which exhibited concurrent resistance to ceftriaxone, ciprofloxacin and azithromycin. Antimicrobial susceptibility testing was performed by broth microdilution in two kinds of drug-sensitive plates. Antimicrobial resistance (AMR) genes were identified by whole genome sequencing and bioinformatics analysis. Genotyping of the strain was performed by multilocus sequence typing (MLST). Plasmid DNA was sequenced and analysed using plasmid bioinformatics tools. The SH11G993 strain was resistant to 28 antibiotics and carried 54 AMR genes. MLST results showed that the strain belonged to a rare genotype. The plasmid profile and plasmid sequencing showed that the strain carried two resistance plasmids. The pSH11G993-1 carried 14 AMR genes (especially co-harboured blaCMY-2, mphA and ermB) and a variety of insertion sequences, belonging to the IncC. The pSH11G993-2 carried 3 AMR genes and 9 virulence genes, belonging to the IncFIB-FII, forming a novel resistance and virulence co-harbouring plasmid. Our findings highlight that continuously monitor the changes in antibiotic resistance patterns and research on the resistance mechanisms in potential human pathogens are imperative.

Genomic epidemiology reveals multidrug resistant plasmid spread between Vibrio cholerae lineages in Yemen

Since 2016, Yemen has been experiencing the largest cholera outbreak in modern history. Multidrug resistance (MDR) emerged among Vibrio cholerae isolates from cholera patients in 2018. Here, to characterize circulating genotypes, we analysed 260 isolates sampled in Yemen between 2018 and 2019. Eighty-four percent of V. cholerae isolates were serogroup O1 belonging to the seventh pandemic El Tor (7PET) lineage, sub-lineage T13, whereas 16% were non-toxigenic, from divergent non-7PET lineages. Treatment of severe cholera with macrolides between 2016 and 2019 coincided with the emergence and dominance of T13 subclones carrying an incompatibility type C (IncC) plasmid harbouring an MDR pseudo-compound transposon. MDR plasmid detection also in endemic non-7PET V. cholerae lineages suggested genetic exchange with 7PET epidemic strains. Stable co-occurrence of the IncC plasmid with the SXT family of integrative and conjugative element in the 7PET background has major implications for cholera control, highlighting the importance of genomic epidemiological surveillance to limit MDR spread.

Genomic epidemiological investigation of an outbreak of Serratia marcescens neurosurgical site infections associated with contaminated haircutting toolkits in a hospital barber shop

Nine surgical site infections caused by Serratia marcescens were diagnosed in neurosurgical patients in a 3500-bed hospital between 2nd February and 6th April 2022. To trace the source of infections caused by S.marcescens to expedite termination of the outbreak and prevent future epidemics. A review of all surgical procedures and cultures yielding S.marcescens since February 2022 was conducted. Samples were collected from patients and environmental sources. S.marcescens isolates were characterized by antibiotic susceptibility testing. Whole-genome sequencing (WGS) was used to investigate genetic relationships. Resistance genes, virulence genes and plasmid replicons were identified. S.marcescens was isolated from patients' puncture fluid, cerebrospinal fluid and other secretions, and was also cultured from the barbers' haircutting tools, including leather knives, slicker scrapers and razors. In total, 15 isolates were obtained from patients and eight isolates were obtained from haircutting tools. All isolates exhibited identical antibiotic resistance patterns. WGS revealed close clustering among the 23 isolates which differed significantly from previous strains. Three resistance genes and nine virulence-associated genes were detected in all isolates, and 19 of 23 isolates harboured an MOBP-type plasmid. The results confirmed an outbreak of S.marcescens, which was traced to contaminated haircutting tools in the hospital barber shop. The outbreak ended after extensive reinforcement of infection control procedures and re-education of the barbers. These results highlight the risk of postoperative infections related to pre-operative skin preparation, and demonstrate the value of next-generation sequencing tools to expedite outbreak investigations.

Co-integrate Col3m bla NDM-1-harboring plasmids in clinical Providencia rettgeri isolates from Argentina.

The first cases of bla NDM in Argentina were detected in three Providencia rettgeri (Pre) recovered from two hospitals in Buenos Aires city in 2013. The isolates were genetically related, but the plasmid profile was different. Here, we characterized the bla NDM-1-harboring plasmids of the first three cases detected in Argentina. Hybrid assembly obtained from short- and long-read sequencing rendered bla NDM-1 in Col3M plasmids of ca. 320 kb (p15268A_320) in isolate PreM15268, 210 kb (p15758B_210) in PreM15758, and 225 kb (p15973A_225) in PreM15973. In addition, PreM15758 harbored a 98-kb circular plasmid (p15758C_98) flanked by a putative recombination site (hin-TnAs2), with 100% nucleotide ID and coverage with p15628A_320. Analysis of PFGE/S1-nuclease gel, Southern hybridization with bla NDM-1 probe, hybrid assembly of short and long reads suggests that pM15758C_98 can integrate by homologous recombination. The three bla NDM-1-plasmids were non-conjugative in vitro. Moreover, tra genes were incomplete, and oriT was not found in the three bla NDM-1-plasmids. In two isolates, blaNDM-1 was embedded in a partially conserved structure flanked by two ISKox2. In addition, all plasmids harbored aph(3')-Ia, aph(3')-VI, and qnrD1 genes and aac(6´)Ib-cr, bla OXA-1, catB3, and arr3 as part of a class 1 integron. Also, p15268A_320 and p15973A_225 harbored bla PER-2. To the best of our knowledge, this is the first report of clinical P. rettgeri harboring blaNDM-1 in an atypical genetic environment and located in unusual chimeric Col3M plasmids. The study and continuous surveillance of these pathogens are crucial to tracking the evolution of these resistant plasmids and finding solutions to tackle their dissemination. IMPORTANCE Infections caused by carbapenem hydrolyzing enzymes like NDM (New Delhi metallo-beta-lactamase) represent a serious problem worldwide because they restrict available treatment options and increase morbidity and mortality, and treatment failure prolongs hospital stays. The first three cases of NDM in Argentina were caused by genetically related P. rettgeri recovered in two hospitals. In this work, we studied the genetic structure of the plasmids encoding bla NDM in those index cases and revealed the enormous plasticity of these genetic elements. In particular, we found a small plasmid that was also found inserted in the larger plasmids by homologous recombination as a co-integrate element. We also found that the bla NDM plasmids were not able to transfer or move to other hosts, suggesting their role as reservoir elements for the acquisition of resistance genes. It is necessary to unravel the dissemination strategies and the evolution of these resistant plasmids to find solutions to tackle their spread.

Genomic characterization of cocirculating Corynebacterium diphtheriae and non-diphtheritic Corynebacterium species among forcibly displaced Myanmar nationals, 2017-2019.

Respiratory diphtheria is a serious infection caused by toxigenic Corynebacterium diphtheriae, and disease transmission mainly occurs through respiratory droplets. Between 2017 and 2019, a large diphtheria outbreak among forcibly displaced Myanmar nationals densely settled in Bangladesh was investigated. Here we utilized whole-genome sequencing (WGS) to characterize recovered isolates of C. diphtheriae and two co-circulating non-diphtheritic Corynebacterium (NDC) species - C. pseudodiphtheriticum and C. propinquum. C. diphtheriae isolates recovered from all 53 positive cases in this study were identified as toxigenic biovar mitis, exhibiting intermediate resistance to penicillin, and formed four phylogenetic clusters circulating among multiple refugee camps. Additional sequenced isolates collected from two patients showed co-colonization with non-toxigenic C. diphtheriae biovar gravis, one of which exhibited decreased susceptibility to the first-line antibiotics and harboured a novel 23-kb multidrug resistance plasmid. Results of phylogenetic reconstruction and virulence-related gene contents of the recovered NDC isolates indicated they were likely commensal organisms, though 80.4 %(45/56) were not susceptible to erythromycin, and most showed high minimum inhibition concentrations against azithromycin. These results demonstrate the high resolution with which WGS can aid molecular investigation of diphtheria outbreaks, through the quantification of bacterial genetic relatedness, as well as the detection of virulence factors and antibiotic resistance markers among case isolates.