Carbapenemase-encoding Plasmid Research Articles

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

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

Whole-genome sequence analysis of carbapenem-resistant Enterobacteriaceae recovered from hospitalized patients

Carbapenems are among the few effective antibiotics against multidrug-resistant Enterobacteriaceae. This study aimed at characterizing the plasmid content and resistome of clinical carbapenem-resistant Enterobacteriaceae (CRE) recovered between 2016-2019 from hospitalized patients in Lebanon. Plasmid typing and whole-genome sequencing were used to study the genomic characteristics of 65 clinical CREs including 27 Escherichia coli, 24 Klebsiella pneumoniae, one Klebsiella quasipneumoniae, three Morganella morganii, three Citrobacter freundii, five Enterobacter hormaechei, and two Serratia marcescens. blaOXA-48 (33.8%; n=22) and blaOXA-48-like genes were among the detected resistance determinants, with two isolates co-harboring blaNDM-5. Various blaNDM variants, blaNDM-1 (16.9%; n=11), blaNDM-5 (9.2%; n=6), blaNDM-7 (9.2%; n=6), and blaNDM-19 (4.6%; n=3), different ESBLs and AmpC β-lactamases were detected. Carbapenem resistance determinants were linked a variety of incompatibility groups with IncFIB(K) (43.1%; n=28) being the most prevalent, followed by IncFIA (40.0%), IncL (35.4%), IncX3 (32.3%), IncI1 (32.3%), and IncFIIK (29.2%). We analyzed the clonality and resistance determinants of 65 multi-drug resistant (MDR) Enterobacteriaceae recovered in the period between 2016-2019 from a large tertiary hospital in Lebanon. NDM variants, OXA-48 and OXA-181were the most prevalent detected carbapenemases and were mostly linked to the dissemination of IncL, IncX3, and IncF. This study reinforces the need to track the spread and dominance of clinically relevant carbapenemase-encoding plasmids in healthcare settings.

Isolation of Hv-CRKP with co-production of three carbapenemases (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and a virulence plasmid: a study from a Chinese tertiary hospital.

The worldwide dissemination of K. pneumoniae isolates is a significant public health concern, as these organisms possess a unique capacity to acquire genetic elements encoding both resistance and hypervirulence. This study aims to investigate the epidemiological, resistance, and virulence characteristics of K. pneumoniae isolates that carry both virulence plasmids and blaOXA-48-like genes in a tertiary hospital in China. A total of 217 clinical isolates of carbapenem-resistant K. pneumoniae (CRKP) were collected between April 2020 and March 2022. The antimicrobial susceptibility test was conducted to evaluate the drug resistance profile. All isolates were screened for the presence of genes encoding carbapenemases (blaKPC, blaNDM, blaIMP, blaVIM, and blaOXA-48-like), ESBLs genes (blaCTX-M, blaSHV, blaTEM), and virulence plasmid pLVPK-borne genes (rmpA, rmpA2, iucA, iroB, and peg344) using polymerase chain reaction (PCR) amplification. Clonal lineages were assigned using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The plasmid incompatibility groups were identified using PCR-based replicon typing (PBRT). The transferability of carbapenemase-encoding plasmids and pLVPK-like virulence plasmids was assessed via conjugation. The plasmid location of rmpA2 was determined using S1-Pulsed Field Gel Electrophoresis (S1-PFGE) and southern blotting hybridization. The virulence potential of the isolates was assessed using the string test, capsular serotyping, serum killing assay and a Galleria mellonella larval infection model. Of the 217 CRKP clinical isolates collected, 23% were identified as carrying blaOXA-48-like genes. All blaOXA-48-like isolates exhibited resistance to commonly used clinical antimicrobial agents, except for ceftazidime/avibactam, colistin, tigecycline, trimethoprim-sulfamethOXAzole, polymyxin B, and nitrofurantoin. The main common OXA-48-like carbapenemase enzymes were found to be blaOXA-181 and blaOXA-232. MLST and PFGE fingerprinting analysis revealed clonal transmission and plasmid transmission. OXA-48-like producing CRKP isolates mainly clustered in K64 ST11 and K47 ST15. Results of the string Test, serum killing assay (in vitro) and Galleria mellonella infection model (in vivo) indicated hypervirulence. PBRT showed that the blaOXA-181 and blaOXA-232 producing hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP) were mainly carried on ColE-type, IncF, and IncX3. Eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1). Moreover, Southern blotting hybridization revealed that all eight isolates had a pLVPK-like virulent plasmid (138.9-216.9 kb) with an uneven number and size of plasmid. In our investigation, we have observed the emergence of hv-CRKP carrying blaOXA-48-like genes, which identified two genetic relationships: clonal transmission and plasmid transmission. PBRT analysis showed that these genes were mainly carried on ColE-type, IncF, and IncX3 plasmids. These isolates have been shown to be hypervirulent in vitro and in vivo. Additionally, eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and carrying a pLVPK-like virulent plasmid. Hence, our findings highlight the need for further investigation and active surveillance of hypervirulent OXA-48-like producing Hv-CRKP isolates to control their transmission.

Citrobacter freundii resistant to novel β-lactamase inhibitor combinations and cefiderocol, co-producing class A, B and D carbapenemases encoded by transferable plasmids.

To characterize a carbapenem-resistant Citrobacter freundii (Cf-Emp) co-producing class A, B and D carbapenemases, resistant to novel β-lactamase inhibitor combinations (BLICs) and cefiderocol. Carbapenemase production was tested by an immunochromatography assay. Antibiotic susceptibility testing (AST) was performed by broth microdilution. WGS was performed using short- and long-read sequencing. Transfer of carbapenemase-encoding plasmids was assessed by conjugation experiments. Cf-Emp was isolated on selective medium for carbapenem-resistant Enterobacterales from the surveillance rectal swab taken at hospital admission from a patient of Moroccan origin. Cf-Emp produced three different carbapenemases, including KPC-2, OXA-181 and VIM-1, and was resistant to all β-lactams including carbapenems, novel BLICs (ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam) and cefiderocol. MIC of aztreonam/avibactam was 0.25 mg/L. The strain belonged to ST22, one of the C. freundii lineages of global diffusion, known to be associated with carbapenemase production. Each carbapenemase gene was located aboard a different plasmid (named pCf-KPC, pCf-OXA and pCf-VIM, respectively), which also carried other clinically relevant resistance genes, such as armA (pCf-KPC), blaSHV-12 (pCf-VIM) and qnrS1 (pCf-OXA). Transferability to Escherichia coli J53 by conjugation was observed for all plasmids. The finding of enterobacterial strains carrying multiple carbapenemase genes on transferable plasmids is alarming, because similar strains could provide an important reservoir for disseminating these clinically relevant resistance determinants.

Elimination of diverse Inc type plasmids carrying carbapenemase genes within Escherichia coli of clinical origin: A single-center study from North-east India

Plasmid often harbors multiple antimicrobial resistance genes and global dissemination of multidrug resistance plasmids pose a serious threat to public health as they limit treatment options. Our study characterizes carbapenemase-encoding plasmids and investigates their horizontal transferability and stability along with the efficacy of chemical agents in eliminating these resistance plasmids from Escherichia coli of clinical origin. Plasmids were extracted from multidrug resistant E. coli isolates harboring carbapenemase-encoding genes and horizontal transferability of the carbapenem resistance determinants were checked through transformation and conjugation assay. Replicon types were investigated through PBRT followed by analysis of plasmid stability via serial passage method. The effect of different chemical agents in elimination of carbapenemase-encoding plasmids were investigated using meropenem resistance as selectable marker. Carbapenemase-encoding genes within conjugatively transferable plasmids of diverse replicon types exhibiting high stability in the absence of selective pressure was identified in the present study. In the elimination assay, sodium dodecyl sulphate successfully eliminates resistance plasmids after single treatment. In addition, sodium sulphate and magnesium sulphate were able to eliminate resistance plasmids after second and third treatment respectively. The global emergence of antimicrobial resistance among clinically significant bacteria highpoints the need to explore and develop new agents that are efficient, economical and non-toxic; and can be used as an armament to combat antimicrobial resistance and their worldwide dissemination. This study highlights chemical agents that have the potential to eliminate plasmids associated with carbapenem resistance and warrants further investigation of these compounds to thwart further advancement of antimicrobial resistance.

Genomic Analysis Revealed the International and Domestic Transmission of Carbapenem-Resistant Klebsiella pneumoniae in Chinese Pediatric Patients.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a severe threat to public health worldwide. Based on the genomic analysis of 198 CRKP isolates collected at Shanghai Children's Medical Center over the last 8 years (2013 to 2021), we reported the clinical risk, genetic diversity, and prevalence of antimicrobial resistance (AMR) of CRKP in pediatric patients at the genomic level. We found that the blaNDM genes were the predominant carbapenemase genes, followed by blaKPC-2 and blaIMP. All of the carbapenemases were disseminated mainly by four main types of plasmids, among which one plasmid was associated with a higher risk of bloodstream infections. Notably, we tracked disease outbreaks caused by recent introductions of ST14 CRKP from southeast Asia or western countries, and we reported frequent, repetitive introductions of ST11 from other domestic hospitals that were associated interhospital movement of the patients. The cocirculation of K. pneumoniae and AMR plasmids in hospitals highlights the importance of genome sequencing for monitoring and controlling CRKP infections. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection in pediatric patients differs from that in adults patients in terms of both genetic and phenotypic features, which remain to be elucidated. We present a summary of prevalent CRKP isolates from Chinese pediatric patients over 8 years, demonstrating the prevalence and clinical importance of New Delhi metallo-β-lactamase genes in pediatric patients, mainly describing the genomic features of two predominant CRKP clones (ST11 and ST14) in Chinese children, and identifying four carbapenemase-encoding plasmids that contribute to the transmission of most carbapenemase genes in hospitals. Overall, our research provides valuable information about the international and domestic transmission of CRKP isolates that are prevalent in Chinese children and shows the urgent need for genome sequencing-based surveillance systems for monitoring the transmission of CRKP.

Epidemiological characteristics and molecular features of carbapenem-resistant Enterobacter strains in China: a multicenter genomic study

ABSTRACT Epidemiological characteristics and molecular features of carbapenem-resistant Enterobacter (CR-Ent) species remain unclear in China. In this study, we performed a genomic study on 92 isolates from Enterobacter-caused infections from a multicenter study in China. Whole genome sequencing (WGS) was used to determine the genome sequence of 92 non-duplicated CR-Ent strains collected from multiple tertiary health centres. The precise species of Enterobacter strains were identified by average nucleotide identity (ANI) and in silico DNA–DNA hybridization (isDDH). Molecular features of high-risk CR-Ent sequence type (ST) lineages and carbapenemase-encoding plasmids were determined. The result revealed that the most common human-source CR-Ent species in China was E. xiangfangensis (66/92, 71.93%), and the proportion of carbapenemase-producing Enterobacter (CP-Ent) in CR-Ent was high (72/92, 78.26%) in comparison to other global regions. Furthermore, ST171 and ST116 E. xiangfangensis were the major lineages of CP-Ent strains, and ST171 E. xiangfangensis was more likely to cause infections in older patients. Genomic analysis also highlighted the likelihood of intra-hospital/inter-hospital clonal transmission of ST171 and ST116 E. xiangfangensis. In addition, the bla NDM-harbouring IncX3-type plasmid was identified as the prevalent carbapenemase-encoding plasmid carried by CR-Ent strains, and was experimentally confirmed to be able to self-transfer with high frequency. This study detailed the genomic and clinical characteristics of CR-Ent in China in the form of multicenter for the first time. The high risk of carbapenemase-producing ST171 and ST116 E. xiangfangensis, and the bla NDM-harbouring IncX3-type plasmid were detected and emphasized.

Dominant Carbapenemase-Encoding Plasmids in Clinical Enterobacterales Isolates and Hypervirulent Klebsiella pneumoniae, Singapore.

Dissemination of carbapenemase-encoding plasmids by horizontal gene transfer in multidrug-resistant bacteria is the major driver of rising carbapenem-resistance, but the conjugative mechanics and evolution of clinically relevant plasmids are not yet clear. We performed whole-genome sequencing on 1,215 clinical Enterobacterales isolates collected in Singapore during 2010–2015. We identified 1,126 carbapenemase-encoding plasmids and discovered pKPC2 is becoming the dominant plasmid in Singapore, overtaking an earlier dominant plasmid, pNDM1. pKPC2 frequently conjugates with many Enterobacterales species, including hypervirulent Klebsiella pneumoniae, and maintains stability in vitro without selection pressure and minimal adaptive sequence changes. Furthermore, capsule and decreasing taxonomic relatedness between donor and recipient pairs are greater conjugation barriers for pNDM1 than pKPC2. The low fitness costs pKPC2 exerts in Enterobacterales species indicate previously undetected carriage selection in other ecological settings. The ease of conjugation and stability of pKPC2 in hypervirulent K. pneumoniae could fuel spread into the community.

Frequent convergence of mcr-9 and carbapenemase genes in Enterobacter cloacae complex driven by epidemic plasmids and host incompatibility

ABSTRACT Convergence of mcr and carbapenemase genes has been sporadically detected in Enterobacter cloacae complex (ECC) with an upward trend. However, the state of the epidemic and underlying mechanism of such convergence has been poorly understood. In this study, the co-occurrence of MCR and carbapenemases was systematically analyzed in 230 clinical ECC isolates collected between 2000 and 2018 together with a global dataset consisting of 3,559 ECC genomes compiled from GenBank. We identified 48 mcr-9/mcr-10-positive isolates (MCR-ECC) (20.9%) in our collection, and a comparable ratio of MCR-ECC (720/3559, 20.2%) was detected in the global dataset. A high prevalence of carbapenemase-producing MCR-ECC (MCR-CREC) was further identified in the MCR-ECC of both datasets (16/48, 33.3%; 388/720, 53.9%), demonstrating a frequent convergence of mcr-9/10 and carbapenemase genes in ECC worldwide. An epidemic IncHI2/2A plasmid with a highly conserved backbone was identified and largely contributed to the dissemination of mcr-9 in ECC worldwide. A highly conserved IncX3-type NDM-1-carrying plasmid and IncN-type IMP-4-carrying plasmid were additionally detected in MCR-CREC isolated in China. Our surveillance data showed that MCR-CREC emerged (in 2013) much later than MCR-ECC (in 2000), indicating that MCR-CREC could be derived from MCR-ECC by additional captures of carbapenemase-encoding plasmids. Tests of plasmid stability and incompatibility showed that the mcr-9/mcr-10-encoding plasmids with the NDM-1-encoding plasmids stably remained in ECC but incompatible in Escherichia coli, suggesting that the convergence was host-dependent. The findings extend our concern on the convergence of resistance to the last resort antibiotics and highlight the necessity of continued surveillance in the future.

Pervasive transmission of a carbapenem resistance plasmid in the gut microbiota of hospitalized patients.

Introductory paragraphInfections caused by carbapenemase-producing enterobacteria (CPE) are a major concern in clinical settings worldwide. Two fundamentally different processes shape the epidemiology of CPE in hospitals: the dissemination of CPE clones from patient to patient (between-patient transfer), and the transfer of carbapenemase-encoding plasmids between enterobacteria in the gut microbiota of individual patients (within-patient transfer). The relative contribution of each process to the overall dissemination of carbapenem resistance in hospitals remains poorly understood. Here, we used mechanistic models combining epidemiological data from more than 9,000 patients with whole genome sequence information from 250 enterobacteria clones to characterise the dissemination routes of a pOXA-48-like carbapenemase-encoding plasmid in a hospital setting over a two-year period. Our results revealed frequent between-patient transmission of high-risk pOXA-48-carrying clones, mostly of Klebsiella pneumoniae and sporadically Escherichia coli. The results also identified pOXA-48 dissemination hotspots within the hospital, such as specific wards and individual rooms within wards. Using high-resolution plasmid sequence analysis, we uncovered the pervasive within-patient transfer of pOXA-48, suggesting that horizontal plasmid transfer occurs in the gut of virtually every colonised patient. The complex and multifaceted epidemiological scenario exposed by this study provides insights for the development of intervention strategies to control the in-hospital spread of CPE.

Characterization of the Plasmidome Encoding Carbapenemase and Mechanisms for Dissemination of Carbapenem-Resistant Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae (CRE) infections, high in morbidity and mortality, pose serious clinical challenges due to limited treatment options. A previous CRE surveillance study on 1,507 patients from 43 hospitals in Osaka, Japan, revealed that 12% of patients carried CRE and that 95% of the CRE isolates were IMP-type carbapenemase producers. Here, the mechanisms for this regional dissemination of a single carbapenemase gene were investigated. Since the dissemination of CRE is primarily due to the transmission of carbapenemase genes located on plasmids, we analyzed the plasmidome of 230 CRE isolates carrying bla IMP by whole-genome sequencing and Southern blotting. bla IMP-6 was found to be predominantly disseminated among chromosomally distinct isolates through the pKPI-6 plasmid. Underlying the vast clonal dissemination of pKPI-6, various subpopulations deriving from pKPI-6 were identified, which had acquired advantages for the dissemination of CRE isolates. A cluster exhibiting heteroresistance against meropenem by the transcriptional regulation of bla IMP-6 caused an outbreak likely through covert transmission of bla IMP-6 For stable carriage of bla IMP-6, they occasionally integrated bla IMP-6 on their chromosomes. In addition, we detected one isolate that broadened the range of antimicrobial resistance through a single point mutation in bla IMP-6 on pKPI-6. Multifaceted analysis of the plasmidome granted us more accurate perspectives on the horizontal spread of CRE isolates, which is difficult to trace only by comparing the whole genomes. This study revealed the predominant spread of a specific carbapenemase-encoding plasmid accompanying the emergence of phenotypically diverse derivatives, which may facilitate further dissemination of CRE in various environments.IMPORTANCE Global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) threatens human health by limiting the efficacy of antibiotics even against common bacterial infections. Carbapenem resistance, mainly due to carbapenemase, is generally encoded on plasmids and is spread across bacterial species by conjugation. Most CRE epidemiological studies have analyzed whole genomes or only contigs of CRE isolates. Here, plasmidome analysis on 230 CRE isolates carrying bla IMP was performed to shed light into the dissemination of a single carbapenemase gene in Osaka, Japan. The predominant dissemination of bla IMP-6 by the pKPI-6 plasmid among genetically distinct isolates was revealed, as well as the emergences of pKPI-6 derivatives that acquired advantages for further disseminations. Underlying vast clonal dissemination of a carbapenemase-encoding plasmid, heteroresistance was found in CRE offspring, which was generated by the transcriptional regulation of bla IMP-6, stabilization of bla IMP-6 through chromosomal integration, or broadened antimicrobial resistance due to a single point mutation in bla IMP-6.

Clinical and Genomic Epidemiology of Carbapenem-Nonsusceptible Citrobacter spp. at a Tertiary Health Care Center over 2 Decades.

Carbapenem-nonsusceptible Citrobacter spp. (CNSC) are increasingly recognized as health care-associated pathogens. Information regarding their clinical epidemiology, genetic diversity, and mechanisms of carbapenem resistance is lacking. We examined microbiology records of adult patients at the University of Pittsburgh Medical Center (UMPC) Presbyterian Hospital (PUH) from 2000 to 2018 for CNSC, as defined by ertapenem nonsusceptibility. Over this time frame, the proportion of CNSC increased from 4% to 10% (P = 0.03), as did daily defined carbapenem doses/1,000 patient days (6.52 to 34.5; R2 = 0.831; P < 0.001), which correlated with the observed increase in CNSC (lag = 0 years; R2 = 0.660). Twenty CNSC isolates from 19 patients at PUH and other UPMC hospitals were available for further analysis, including whole-genome short-read sequencing and additional antimicrobial susceptibility testing. Of the 19 patients, nearly all acquired CNSC in the health care setting and over half had polymicrobial cultures containing at least one other organism. Among the 20 CNSC isolates, Citrobacter freundii was the predominant species identified (60%). CNSC genomes were compared with genomes of carbapenem-susceptible Citrobacter spp. from UPMC and with other publicly available CNSC genomes. Isolates carrying genes encoding carbapenemases (blaKPC-2,blaKPC-3, and blaNDM-1) were also long-read sequenced, and their carbapenemase-encoding plasmid sequences were compared with one another and with publicly available sequences. Phylogenetic analysis of 102 UPMC Citrobacter genomes showed that CNSC from our setting did not cluster together. Similarly, a global phylogeny of 64 CNSC genomes showed a diverse population structure. Our findings suggest that both local and global CNSC populations are genetically diverse and that CNSC harbor carbapenemase-encoding plasmids found in other Enterobacterales.

Environmental Presence and Genetic Characteristics of Carbapenemase-Producing Enterobacteriaceae from Hospital Sewage and River Water in the Philippines.

This study aimed to evaluate the prevalence and genetic characteristics of carbapenemase-producing Enterobacteriaceae (CPE) in hospital sewage and river water in the Philippines, which has a typical tropical maritime climate. We collected 83 water samples from 7 hospital sewage and 10 river water sites. CPE were identified using CHROMagar mSuperCARBA, and Gram-negative strains were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) or 16S rRNA gene sequencing. Resistance genes in Enterobacteriaceae strains were identified using PCR and DNA sequencing, and transferability of carbapenemase genes from the CPE was investigated with conjugation experiments. Genotyping was performed using multilocus sequence typing (MLST) for Escherichia coli and Klebsiella pneumoniae Out of 124 Enterobacteriaceae isolates, we identified 51 strains as CPE and divided these into 7 species, 11 E. coli, 14 Klebsiella spp., 15 Enterobacter spp., and 11 others, including 4 additional species. Conjugation experiments via broth mating and using E. coli J53 revealed that 24 isolates can transfer carbapenemase-encoding plasmids. MLST analysis showed that 6 of 11 E. coli isolates belonged to clonal complex 10 (CC10). Of 11 K. pneumoniae strains, 9 unique sequence types (STs) were identified, including ST147. Five types of carbapenemase genes were identified, with the most prevalent being NDM (n = 39), which is epidemic in clinical settings in the Philippines. E. coli CC10 and K. pneumoniae ST147, which are often detected in clinical settings, were the dominant strains. In summary, our results indicate that hospital sewage and river water are contaminated by CPE strains belonging to clinically important clonal groups.IMPORTANCE Carbapenemase-producing Enterobacteriaceae (CPE) cause severe health care-associated infections, and their increasing prevalence is a serious concern. Recently, natural ecosystems have been recognized as important reservoirs of antibiotic resistance genes. We investigated the prevalence and genetic characteristics of CPE isolated from the environment (hospital sewage and river water) in the Philippines and found several CPE, including Escherichia coli and other species, with different carbapenemases. The most prevalent carbapenemase gene type was NDM, which is endemic in clinical settings. This study revealed that isolates belonging to carbapenemase-producing E. coli CC10 and K. pneumoniae sequence type 147 (ST147), which are often detected in clinical settings, were dominant in the natural environment. Our work here provides a report on the presence and characteristics of CPE in the environment in the Philippines and demonstrates that both hospital sewage and river water are contaminated by CPE strains belonging to clinically important clonal groups.

Occurrence of carbapenemase-producing Enterobacteriaceae in a Portuguese river: blaNDM, blaKPC and blaGES among the detected genes

Carbapenems are used as last-resort drugs to treat infections caused by multidrug-resistant bacteria. Despite the increasing number of reports of carbapenem-resistant Enterobacteriaceae (CRE), there is still limited information on their distribution or prevalence in the environment. Our aim was to assess the occurrence of CRE in the Lis river (Portugal) and to characterize the genetic platforms linked to carbapenemase genes. We collected six water samples from sites near a wastewater treatment plant (n = 4 samples) and livestock farms (n = 2). Twenty-four CRE were characterized by BOX element-polymerase chain reaction (BOX-PCR), and thirteen representative isolates were analysed by Pulsed-Field Gel Electrophoresis (PFGE) and by sequencing the 16S rRNA gene. Antimicrobial susceptibility testing, PCR screening for carbapenemase-encoding genes, conjugation experiments and plasmid analysis were performed. Four isolates were chosen for whole-genome sequencing. All water samples contained CRE (4.0 CFU/mL on average). Representative isolates were multidrug-resistant (resistant to ciprofloxacin, trimethoprim-sulfamethoxazole and to all β-lactams tested) and were identified as K. pneumoniae, Enterobacter and Citrobacter. Isolates carried plasmids and harboured carbapenemase-encoding genes: blaKPC-3 in K. pneumoniae (n = 9), blaNDM-1 in Enterobacter (n = 3) and blaGES-5 in Citrobacter (n = 1). Conjugation experiments were successful in two Klebsiella isolates. Enterobacter PFGE profiles grouped in one cluster while Klebsiella were divided in three clusters and a singleton. Whole-genome sequencing analysis revealed blaGES-5 within a novel class 3 integron (In3-16) located on an IncQ/pQ7-like plasmid in Citrobacter freundii CR16. blaKPC-3 was present on IncFIA-FII pBK30683-like plasmids, which were subsequently confirmed in all K. pneumoniae (n = 9). Furthermore, blaKPC-3 was part of a genomic island in K. pneumoniae CR12. In E. roggenkampii CR11, blaNDM-1 was on an IncA/C2 plasmid. The carbapenemase-encoding plasmids harboured other resistance determinants and mobile genetic elements. Our results demonstrate that Lis river is contaminated with CRE, highlighting the need for monitoring antibiotic resistance in aquatic environments, especially to last-resort drugs.

Phenotypical profile and global transcriptomic profile of Hypervirulent Klebsiella pneumoniae due to carbapenemase-encoding plasmid acquisition

BackgroundPlasmids play an vital role in driving the rapid global spread of antimicrobial resistance and adaptation to changing ambient conditions. It has been suggested that the presence of plasmids can pose tremendous impacts on the host physiology. However, little is known regarding the contributions of carbapenemase-encoding plasmid carriage on the physiology and pathogenicity of hypervirulent K. pneumoniae (hvKP).ResultsHere we performed a transcriptomic analysis of hvKP with or without carbapenemase-encoding plasmid p24835-NDM5. The results had shown 683 genes with differential expression (false discovery rate, ≤0.001; > 2-fold change), of which 107 were up-regulated and 576 were down-regulated. Gene groups with functions relating to carbohydrate metabolism and multidrug efflux system were increased in genes with increased expression, and those relating to capsule biosynthesis and virulence factors were increased in the genes with decreased expression. In agreement with these changes, survival rate of TfpNDM-hvKP in the presence of normal human serum decreased, and competitive index (CI values) indicated significant fitness defects in the plasmid-carrying hvKP strain when co-cultured with its plasmid-free isogenic ancestor and the ATCC control. Moreover, the p24835-NDM5-containing hvKP strain retained its high neutrophil-mediated phagocytosis and murine lethality.ConclusionThese data indicate that hvKP responds to carbapenemase-encoding plasmid by altering the expression of genes involved in carbohydrate metabolism, antibiotic resistance, capsule biosynthesis and virulence expression. Apart from antibiotic resistance selective advantages, carbapenemase-encoding plasmid carriage may also lead to virulence change or adaption to specific habitats in hvKP strain.

Sharing of carbapenemase-encoding plasmids between Enterobacteriaceae in UK sewage uncovered by MinION sequencing.

Dissemination of carbapenem resistance among pathogenic Gram-negative bacteria is a looming medical emergency. Efficient spread of resistance within and between bacterial species is facilitated by mobile genetic elements. We hypothesized that wastewater contributes to the dissemination of carbapenemase-producing Enterobacteriaceae (CPE), and studied this through a cross-sectional observational study of wastewater in the East of England. We isolated clinically relevant species of CPE in untreated and treated wastewater, confirming that waste treatment does not prevent release of CPE into the environment. We observed that CPE-positive plants were restricted to those in direct receipt of hospital waste, suggesting that hospital effluent may play a role in disseminating carbapenem resistance. We postulated that plasmids carrying carbapenemase genes were exchanged between bacterial hosts in sewage, and used short-read (Illumina) and long-read (MinION) technologies to characterize plasmids encoding resistance to antimicrobials and heavy metals. We demonstrated that different CPE species (Enterobacter kobei and Raoultella ornithinolytica) isolated from wastewater from the same treatment plant shared two plasmids of 63 and 280 kb. The former plasmid conferred resistance to carbapenems (blaOXA-48), and the latter to numerous drug classes and heavy metals. We also report the complete genome sequence for Enterobacter kobei. Small, portable sequencing instruments such as the MinION have the potential to improve the quality of information gathered on antimicrobial resistance in the environment.

Carbapenemase-producing enterobacteriaceae recovered from a Spanish river ecosystem.

The increasing resistance to carbapenems is an alarming threat in the fight against multiresistant bacteria. The dissemination properties of antimicrobial resistance genes are supported by their detection in a diverse population of bacteria, including strains isolated from the environment. The objective of this study was to investigate the presence of carbapenemase-producing Enterobacteriaceae (CPE) collected from a river ecosystem in the Barcelona metropolitan area (Spain). Identification of β-lactamases and other resistance determinants was determined as was the antimicrobial susceptibility profile. Moreover, screening of virulence factors, plasmid addiction systems, plasmid partition systems and replicon typing was performed. The results identified 8 isolates belonging to different species (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Klebsiella oxytoca, Raoultella ornithinolytica). The most prevalent enzyme was KPC-2 (n = 6), followed by VIM-1 (n = 2) and IMI-2 (n = 1), whereas no OXA-48-type was detected. In addition, one strain was positive for both KPC-2 and VIM-1 enzymes. All the carbapenemase-encoding plasmids carried at least one plasmid addiction or partition system, being vagCD and parAB the most frequently detected, respectively. E. coli and K. pneumoniae isolates carried a low number of virulence-associated factors and none of the detected clones has previously been identified in the clinical setting. These findings support the high dissemination potential of the carbapanemase-encoding genes and reinforce the idea that the environment is another reservoir that may play an important role in the capture, selection and dissemination of carbapenem resistance genes.

Characterization of carbapenemase-producing Enterobacteriaceae in the West Midlands region of England: 2007–14

Carbapenemase-producing Enterobacteriaceae (CPE) have been increasingly reported in the UK since 2003. We analysed patient and isolate data for CPE confirmed by the national reference laboratory from laboratories in the West Midlands region from November 2007 to December 2014. MICs were determined by BSAC agar dilution methodology and isolates exhibiting resistance to one or more carbapenems were screened for carbapenemase genes by PCR. Plasmid analyses were performed after electro-transformation of carbapenemase-encoding plasmids. WGS was performed on both transformants and clinical isolates. Patient data provided by the sending laboratories were reviewed. During the study period, CPE ( n = 139) were submitted from 13 laboratories in the West Midlands region, originating from 108 patients and including one environmental isolate. CPE submissions increased significantly from 2009 onwards. Isolates were predominantly Klebsiella pneumoniae (89/139) obtained from inpatients. WGS was performed on all clinical isolates and transformants. After deduplication 119 isolates and 96 transformants remained for analysis. Within these, four families of carbapenemase genes were identified: bla NDM (69/119), bla KPC (26/119), bla OXA-48-like (16/119) and bla VIM (7/119); one isolate carried both bla NDM and bla OXA-48-like . Isolates represented diverse STs and plasmid replicon types. Plasmid analyses identified plasmids of different replicon types encoding bla KPC , bla NDM and bla OXA-48-like genes, found across several species and STs. CPE have been reported increasingly in the West Midlands region over a 7 year period. bla NDM , bla KPC and bla OXA-48-like were the dominant carbapenemase genes and were found in a range of diverse genomic/plasmid environments, highlighting their ability to mobilize across different plasmids, often impeding the detection of outbreaks.

Low biological cost of carbapenemase-encoding plasmids following transfer from Klebsiella pneumoniae to Escherichia coli.

The objective of this study was to determine the biological cost, stability and sequence of two carbapenemase-encoding plasmids containing blaKPC-2 (pG12-KPC-2) and blaVIM-1 (pG06-VIM-1) isolated from Klebsiella pneumoniae when newly acquired by uropathogenic Escherichia coli clinical isolates of different genetic backgrounds. The two plasmids were transferred into plasmid-free E. coli clinical isolates by transformation. The fitness effect of newly acquired plasmids on the host cell was assessed in head-to-head competitions with the corresponding isogenic strain. Plasmid stability was estimated by propagating monocultures for ∼312 generations. Plasmid nucleotide sequences were determined using next-generation sequencing technology. Assembly, gap closure, annotation and comparative analyses were performed. Both plasmids were stably maintained in three of four E. coli backgrounds and resulted in low to moderate reductions in host fitness ranging from 1.1% to 3.6%. A difference in fitness cost was observed for pG12-KPC-2 between two different genetic backgrounds, while no difference was detected for pG06-VIM-1 between three different genetic backgrounds. In addition, a difference was observed between pG12-KPC-2 and pG06-VIM-1 in the same genetic background. In general, the magnitude of biological cost of plasmid carriage was both host and plasmid dependent. The sequences of the two plasmids showed high backbone similarity to previously circulating plasmids in K. pneumoniae. The low to modest fitness cost of newly acquired and stably maintained carbapenemase-encoding plasmids in E. coli indicates a potential for establishment and further dissemination into other Enterobacteriaceae species. We also show that the fitness cost is both plasmid and host specific.

Comparative genome analysis of IncHI2 VIM-1 carbapenemase-encoding plasmids of Escherichia coli and Salmonella enterica isolated from a livestock farm in Germany

Carbapenem-resistant Enterobacteriaceae are not any more isolated only from human settings, but also from livestock. We reported for the first time the presence of VIM-1 carbapenemases in a livestock farm in Germany. The VIM-1 resistance gene found in these farms was located on IncHI2 plasmids. In order to be able to analyse these plasmids in more detail, two different plasmids from a single farm (pRH-R27 from Salmonella enterica and pRH-R178 from Escherichia coli) were completely sequenced and analysed for the presence of antibiotic and heavy metal resistances. The plasmids showed to harbour blaVIM-1, aacA4, aadA1, sul1, qacEΔ (encoded in an In110 class 1 integron), as well as blaACC-1, strA/strB, and catA1 genes together with resistance to heavy metals (ter-, mer-, sil-, ars-, rcn-, and pco). Comparison with other IncHI2 plasmid revealed that while pRH-R27 is a mosaic IncHI2 plasmid with both high homology to the plasmid pSTm-A54650 and R478, both isolated from humans, pRH-R178 is a deletion derivative of pRH-R27, presumably caused by several IS-mediated deletions indicating genetic evolution of plasmids in this environment.