Genomic characterization and global lineage-plasmid context of a bla NDM-5-harboring Escherichia coli ST3014 isolate.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a significant public health threat, particularly as a superbug responsible for nosocomial infections. In this study, we report a novel sequence type 6758 of K. pneumoniae harboring the bla NDM-1 gene. Antimicrobial susceptibility testing was conducted according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The complete genome sequence of the strain was determined using the Illumina NovaSeq 6000 platform and long-read MinION sequencer. Genomic features and resistance mechanisms of the strain were further comprehensively analysed using various bioinformatics approaches. Antimicrobial susceptibility testing revealed that this strain exhibited resistance to multiple antimicrobials, including ceftazidime, ceftriaxone, cefazolin, cefepime, imipenem, meropenem, ampicillin/sulbactam, and sulfamethoxazole/trimethoprim. The genome analysis identified sixteen resistance genes. The bla NDM-1 carbapenemase gene is located on a 47,823 bp IncX3-type plasmid (pNDM-CRKP331). A total of 41 K. pneumoniae strains carrying similar IncX3-type plasmids were retrieved from the NCBI database, representing 20 sequence types (STs) across 11 countries. The most common resistance gene carried by these IncX3-type plasmids is bla NDM, and all these plasmids contain only the bla NDM gene. The bla NDM-carrying IncX3-type plasmids are widely prevalent in K. pneumoniae in China, spanning 15 STs. In summary, our study reports the first genome sequence of an ST 6758 K. pneumoniae strain containing the class B β-lactamase bla NDM-1 isolated from a clinical sample. Given the global emergence of bla NDM, measures should be taken to prevent the spread of these bla NDM-carrying IncX3-type plasmids. Our findings contribute to the understanding of the transmission mechanisms of bla NDM in K. pneumoniae.

Introduction. The New Delhi metallo-β-lactamase (NDM) variant NDM-5 was first described in 2011 in an isolate of Escherichia coli. We noted that a high proportion of isolates of E. coli positive for bla NDM carbapenemase genes submitted to the UK Health Security Agency (formerly Public Health England) between 2019 and mid-2021 carried the bla NDM-5 allele, with many co-harbouring rmtB, rendering them highly resistant to aminoglycosides as well as to most β-lactams.Hypothesis/Gap Statement. This observation suggested that a common plasmid may be circulating.Aim. To compare these isolates and describe the plasmids carrying these resistance elements.Methodology. All isolates were sequenced on an Illumina platform, with five also subjected to long-read nanopore sequencing to provide complete assemblies. The locations of bla NDM-5, rmtB and other associated genetic elements were identified. Susceptibility testing to a wide range of antibiotics was carried out on representative isolates.Results. The 34 isolates co-harbouring bla NDM-5 and rmtB were from 14 hospital groups and six different regions across England and consisted of 11 distinct sequence types. All carried IncF plasmids. Assembly of the NDM plasmids in five isolates revealed that they carried rmtB and bla NDM-5 in an IncF conjugative plasmid ranging in size from 85.5 to 161 kb. All carried a highly conserved region, previously described in E. coli plasmid pHC105-NDM, that included bla TEM-1B and rmtB followed by sequence bounded by two IS26 elements containing ΔISAba125, bla NDM-5, ble, trpF and tat followed by ISCR1 and an integron with sul1, aadA2 and dfrA12 cassettes. This arrangement has been described in isolates from other countries and continents, suggesting that such plasmids are widely distributed, at least in E. coli, with similar plasmids also found in Klebsiella pneumoniae. Tested isolates were resistant to most antibiotics except colistin, fosfomycin and tigecycline.Conclusion. These observations suggest that conjugative plasmids carrying a highly conserved resistance gene segment have become widespread in England and elsewhere. This study highlights the value of routine whole-genome sequencing in identifying genetic elements responsible for resistance dissemination.

Limited information is available on whether blaKPC-containing plasmids from isolates in a hospital outbreak can be differentiated from epidemiologically unrelated blaKPC-containing plasmids based on sequence data. This study aimed to evaluate the performance of three approaches to distinguish epidemiologically related from unrelated blaKPC-containing pKpQiL-like IncFII(k2)-IncFIB(pQiL) plasmids. Epidemiologically related isolates were subjected to short- and long-read whole-genome sequencing. A hybrid assembly was performed, and plasmid sequences were extracted from the assembly graph. Epidemiologically unrelated plasmid sequences were extracted from GenBank. Pairwise comparisons of epidemiologically related and unrelated plasmids based on SNPs using snippy and of phylogenetic distance using Roary and using a similarity index that penalizes size differences between plasmids (Stoesser index) were performed. The percentage of pairwise comparisons misclassified as genetically related or as clonally unrelated was determined using different genetic thresholds for genetic relatedness. The ranges of number of SNPs, Roary phylogenetic distance, and Stoesser index overlapped between the epidemiologically related and unrelated plasmids. When a genetic similarity threshold that classified 100% of epidemiologically related plasmid pairs as genetically related was used, the percentages of plasmids misclassified as epidemiologically related ranged from 6.7% (Roary) to 20.8% (Stoesser index). Although epidemiologically related plasmids can be distinguished from unrelated plasmids based on genetic differences, blaKPC-containing pKpQiL-like IncFII(k2)-IncFIB(pQiL) plasmids show a high degree of sequence similarity. The phylogenetic distance as determined using Roary showed the highest degree of discriminatory power between the epidemiologically related and unrelated plasmids.

Global genomic epidemiology and plasmid-mediated dissemination of blaKPC and blaNDM in the Serratia marcescens complex

Intestinal colonization by multidrug-resistant Escherichia coli provides a silent reservoir for dissemination of last-line resistance. The co-occurrence of bla NDM-5 and the plasmid-mediated colistin resistance gene mcr-1.1 is particularly concerning, yet the plasmid architectures and transfer potential of colonizing isolates remain poorly characterized. Two bla NDM-5- and mcr-1.1-positive E. coli isolates were recovered from fecal samples during colonization screening were investigated (L1771 and L2386). Antimicrobial susceptibility was determined by MIC testing. Short- and long-read sequencing was used to achieve plasmid-level resolution. S1-nuclease PFGE and Southern blot hybridization were performed to localize resistance genes. Conjugation assays were conducted to evaluate plasmid transferability. Both isolates were resistant to colistin (MIC 4 mg/L) and carbapenems (L1771: imipenem 32 mg/L, meropenem 32 mg/L; L2386: imipenem 16 mg/L, meropenem 16 mg/L). L1771 contained a bla NDM-5-carrying IncX3 plasmid (~46 kb), whereas mcr-1.1 was located on the chromosome. In contrast, plasmid-resolved analysis of L2386 revealed a high-risk configuration involving two distinct resistance plasmids: a bla NDM-5-carrying IncX3 plasmid (~48 kb) and an mcr-1.1-carrying IncI2 plasmids (~65 kb). Together, these plasmids formed a multi-plasmid "last-line resistance package" linking carbapenem and colistin resistance within a single isolate. The IncI2 mcr-1.1 plasmid p2803-MCR co-carried bla CTX-M-199. Importantly, all three resistance plasmids identified above: the bla NDM-5-carrying IncX3 plasmid in L1771 and the bla NDM-5-carrying IncX3 plasmid together with the mcr-1.1-carrying IncI2 plasmid in L2386 were transferable by conjugation, supporting strong potential for horizontal spread and co-selection. This study provides plasmid-resolved and functionally validated evidence that gut-colonizing E. coli can assemble a fully conjugative, multi-plasmid resistance package linking carbapenem and colistin resistance, including an IncI2 mcr-1.1 platform with potential broad-host-range dissemination. These findings underscore the need for surveillance that resolves plasmid backbones and transferability, not only resistance gene presence.

The emergence of carbapenem-resistant Enterobacteriaceae poses a significant threat to public health worldwide. Here, we reported a multidrug-resistant Escherichia coli strain with two different blaNDM–5-carrying plasmids from China. Illumina short-read and MinION long-read whole genome sequencing were performed. Genomic analysis found that one blaNDM–5 gene together with mphA was located on a 55-kb IncX3 plasmid, while the other blaNDM–5 gene was on a novel 68-kb IncFII plasmid. Susceptibility testing and quantitative reverse transcription PCR results further indicated that the transconjugants with the IncX3 plasmid exhibited higher-level carbapenem resistance and expression of blaNDM–5 than those with both plasmids or the IncFII plasmid. Two other β-lactamase genes (blaCTX–M–15 and blaOXA–1) were also detected on another 160-kb IncF plasmid. This is the first report of coexistence of two blaNDM–5-carrying plasmids in a single bacterial isolate, highlighting the genetic complexity of NDM-5 carbapenemase circulation, and the urgent need for continued active surveillance.

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.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an important pathogen causing serious nosocomial infections. We describe an outbreak of CRAb in an intensive care unit in the Netherlands in 2021. During an outbreak of non-resistant A. baumannii, while infection control measures were in place, CRAb isolates carrying highly similar bla NDM-1 - and tet(x3)-encoding plasmids were isolated from three patients over a period of several months. The chromosomal and plasmid sequences of the CRAb and non-carbapenemase-carrying A. baumannii isolates cultured from patient materials were analysed using hybrid assemblies of short-read and long-read sequences. The CRAb isolates revealed that the CRAb outbreak consisted of two different strains, carrying similar plasmids. The plasmids contained multiple antibiotic resistance genes including the tetracycline resistance gene tet(x3), and the bla NDM-1 and bla OXA-97 carbapenemase genes. We determined minimal inhibitory concentrations (MICs) for 13 antibiotics, including the newly registered tetracycline antibiotics eravacycline and omadacycline. The CRAb isolates showed high MICs for tetracycline antibiotics including eravacycline and omadacycline, except for minocycline which had a low MIC. In this study we show the value of sequencing multidrug-resistant A. baumannii for outbreak tracking and guiding outbreak mitigation measures.

Carbapenem-resistant Acinetobacter baumannii (CRAB) represents a formidable nosocomial pathogen, with healthcare environments acting as critical reservoirs for its dissemination. In this study, we investigated the prevalence, antimicrobial resistance profiles and genomic characteristics of CRAB isolates collected from hospitals in Shanghai, China, between June and December 2024, identifying ST2Pas (84.13%) and ST164Pas (15.08%) as the predominant lineages among the 126 CRAB isolates recovered from clinical (n=94), environmental (n=29) and healthcare worker (n=3) sources. Environmental CRAB accounted for the highest proportions on patient-contact surfaces (34.48%), medical devices (31.03%) and shared items (24.14%). Within the dominant ST2Pas lineage, clinical isolates exhibited higher resistance rates to ampicillin/sulbactam, cefoperazone/sulbactam and levofloxacin, with significantly higher carriage rates of bla TEM-1D compared to environmental isolates. Compared to ST164Pas, ST2Pas CRAB isolates exhibited greater resistance to amikacin, gentamicin, trimethoprim/sulfamethoxazole and minocycline and a higher prevalence of aph(3')-Ia, aph(3″)-Ib, aph(3')-VI, aph(6)-Id, armA, bla OXA-66 , bla TEM-1D, mph(E) and tet(B), but lower rates of bla CARB-16, bla NDM-1 and bla OXA-91 (P<0.05). Notably, comparative genomic analysis suggested putative adaptive differences between the two lineages. ST2Pas retained the T6SS and biofilm-associated genes (bap), descriptive genomic features that suggest a potential capacity for active colonization. Conversely, the ST164Pas clone lacked the T6SS gene cluster but was enriched with the surface adhesin ata and immune evasion-related genes. Concordantly, ST164Pas CRAB isolates exhibited significantly stronger biofilm-forming capacities than ST2Pas in vitro. We hypothesize that these genomic alterations and phenotypic traits may represent a fitness trade-off, potentially conferring a survival advantage under antibiotic pressure. Furthermore, bla OXA-23 in ST2Pas was predominantly carried on conjugative plasmids restricted to Acinetobacter species, whereas bla NDM-1 in ST164Pas was localized on a broad-host-range non-mobile plasmid, potentially facilitating cross-genus transmission. Although our ST164Pas isolates shared high homology with clinical strains from Zhejiang, China, the genomic localization of bla NDM-1 differed between the plasmid and chromosome, respectively. These descriptive genomic findings highlight the putative adaptive trajectories of the predominant ST2Pas and emerging ST164Pas clones, underscoring the critical need for comprehensive genomic surveillance, complemented by future phenotypic validation, to monitor their rapid evolution and dissemination.

Raoultella ornithinolytica is an emerging pathogen. This study aimed to characterize the genomic and molecular features of a carbapenem-resistant R.ornithinolytica strain co-harboring bla NDM-1 and bla IMP-4. Strain he2023 was identified and subjected to antimicrobial susceptibility testing using the BD Phoenix-M50 fully automated system. The strain was verified using MALDI-TOF. Carbapenemase genes were confirmed by PCR. Whole-genome sequencing (WGS) was performed to identify resistance genes and plasmid types. Conjugation experiments were conducted to assess transferability, and plasmid stability was evaluated through serial subculturing. The BD Phoenix-M50 system misidentified strain he2023 as Klebsiella pneumoniae, whereas MALDI-TOF and WGS correctly identified it as R. ornithinolytica. The strain was resistant to most antimicrobial agents, remaining susceptible only to amikacin,Tigecycline and polymyxin B. The bla NDM-1 and bla IMP-4 genes were both located on the plasmid pNDM-IMP, which belongs to the replicon type IncHI5. The bla NDM-1 was located in the structure of ISAba125-bla NDM-1-ble MBL-trpF-dsbC, residing in a region within the remnant of transposon Tn125, flanked by two copies of ISCR1. The bla IMP-4 was located downstream of bla NDM-1 within a novel class 1 integron (In1965) carrying the structure sul1-qacE-arr-3-ltrA-bla IMP-4-IntI1. Conjugation and transformation assays showed that the plasmid pNDM-IMP was non-transferable but exhibited high structural stability. This study is the first to report an IncHI5 plasmid co-harboring bla NDM-1 and bla IMP-4 in R. ornithinolytica. Non-transferable plasmids may serve as reservoirs or initial carriers for antibiotic resistance genes. Our findings highlights the importance of mobile genetic elements in the formation of multidrug resistance.

Plasmid-borne mcr-4.3 in Acinetobacter nosocomialis recovered from imported beef cattle: Genomic characterization and one health implications

PurposeNew Delhi metallo-β-lactamase 5 (NDM-5) shows stronger resistance to carbapenems and broad-spectrum cephalosporins than NDM-1 because NDM-5 differs from NDM-1 by two amino acid substitutions. In this study, our aim was to characterize a NDM-5-producing Escherichia coli isolate KY1497 from a patient with urinary tract infection in Japan, who had no recent history of overseas travel.Patients and MethodsNDM-5-producing E. coli isolate KY1497 was detected in the urine sample of a patient hospitalized in a tertiary hospital in Japan. The complete genome sequence of isolate KY1497 was determined by short- and long-read sequencing with hybrid assembly, followed by multilocus sequence typing (MLST), core-genome phylogeny analysis, plasmid analysis, and transconjugation experiments.ResultsKY1497 was classified as ST405 by MLST, and core-genome phylogeny exhibited the closest lineage to the clinical isolates in Nepal (IOMTU605) and Canada (FDAARGOS_448). KY1497 harbors blaNDM-5 in the IncFII-IncFIB(pB171) replicon plasmid (pKY1497_1, 123,767 base pairs). Plasmid analysis suggested that the cognate plasmids of pKY1497_1 have a minor plasmid background, rather than the globally disseminated IncX3 plasmid carrying blaNDM-5. Transconjugation analysis revealed that pKY1497_1 is transmissible to the recipient E. coli J53 strain.ConclusionWe characterized a novel Inc replicon plasmid (IncFII-IncFIB[pB171]) carrying blaNDM-5 and its host E. coli strain. NDMs are associated with a high risk of infection worldwide because of their antibiotic resistance and untreatable and hard-to-treat infections. Other patients in the hospital showed negative results for carbapenem-resistant Enterobacteriaceae. As NDM-producing strains are only sporadically detected in Japan, attention should be provided to the community prevalence of NDM-producing E. coli strains to prevent nosocomial infections.

Studies investigating environmental hotspots of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in Nigeria are limited. This study was designed to assess various environmental sources and commonly touched surfaces as potential carriers of ARB and ARGs with implications for public health. A total of 392 samples, including sewage (36), sludge (36), diapers (20), plastics (20), water sachet polythene bags (20), food wastes (20), soil beneath dump sites (20), and frequently touched surfaces such as restroom floors (80), corridors (24), door handles (56), and room floors and walls (60), were collected and screened for the presence of resistant bacteria carrying genes such as bla KPC, bla NDM-1, bla CMY-2, bla IMP, bla OXA66 and MecA. Additionally, we employed standard techniques to detect methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii . We also evaluated the effectiveness of routine disinfection procedures in eliminating ARB from restroom floors. Our findings revealed that sewage, sludge, diapers, food wastes and restroom floors are frequently contaminated with highly and moderately resistant strains of E. coli, K. pneumoniae, P. aeruginosa and MRSA. Notably, we identified two variants of the bla OXA51-like gene (bla OXA-66 and bla OXA-180) in A. baumannii isolated from these environmental sources. Furthermore, we detected seven ESBL- K. pneumoniae , five ESBL- A. baumannii , two ESBL- E. coli and one ESBL- P. aeruginosa , all carrying one or more ARGs (bla KPC, bla NDM-1, bla CMY-2), in isolates recovered from sewage, sludge, restroom floors and plastics. It is of note that ARB persisted on restroom floors even after disinfection procedures. In conclusion, this study highlights that environmental wastes indiscriminately discarded in residential areas and shared surfaces among individuals are heavily colonized by ARB carrying ARGs of significant public health importance.

Brucella melitensis is a key etiological agent of brucellosis and has been increasingly subject to characterization using sequencing methodologies. This study aimed to investigate and compare short-read, long-read, and hybrid assemblies of B. melitensis. Eighteen B. melitensis isolates from Southern Israel were sequenced using Illumina and the Oxford Nanopore (ONP) MinION, and hybrid assemblies were generated with ONP long reads scaffolded on Illumina short reads. Short reads were assembled with INNUca with SPADes, long reads and hybrid with dragonflye. Abricate with the virulence factor database (VFDB) and in silico PCR (for the genes BetB, BPE275, BSPB, manA, mviN, omp19, perA, PrpA, VceC, and ureI) were used for identifying virulence genes, and a total of 61 virulence genes were identified in short-read, long-read, and hybrid assemblies of all 18 isolates. The phylogenetic analysis using long-read assemblies revealed several inconsistencies in cluster assignment as compared to using hybrid and short-read assemblies. Overall, hybrid assembly provided the most comprehensive data, and stand-alone short-read sequencing provided comparable data to stand-alone long-read sequencing regarding virulence genes. For genomic epidemiology studies, stand-alone ONP sequencing may require further refinement in order to be useful in endemic settings.

Recently, new technologies, such as next-generation sequencing and third-generation sequencing, have been used in carrier screening of thalassemia. However, there is no direct comparison between the 2 methods in carrier screening of thalassemia. To compare the clinical performance of third-generation sequencing with next-generation sequencing in carrier screening of thalassemia. Next-generation sequencing and third-generation sequencing were simultaneously conducted for 1122 individuals in Hainan Province. Among 1122 genetic results, 1105 (98.48%) were concordant and 17 (1.52%) were discordant between the 2 methods. Among the 17 discordant results, 4 were common thalassemia variants, 9 were rare thalassemia variants, and 4 were variations with unknown pathogenicity. Sanger sequencing and polymerase chain reaction for discordant samples confirmed all the results of third-generation sequencing. Among the 685 individuals with common and rare thalassemia variants detected by third-generation sequencing, 512 (74.74%) were carriers of α-thalassemia, 110 (16.06%) were carriers of β-thalassemia, and 63 (9.20%) had coinheritance of α-thalassemia and β-thalassemia. Three thalassemia variants were reported for the first time in Hainan Province, including -THAI, -α2.4, and ααααanti3.7. Eleven variants with potential pathogenicity were identified in 36 patients with positive hemoglobin test results. Among 52 individuals with negative hemoglobin test results, 17 were identified with thalassemia variants. In total, third-generation sequencing and next-generation sequencing correctly detected 763 and 746 individuals with variants, respectively. Third-generation sequencing yielded a 2.28% (17 of 746) increment compared with next-generation sequencing. Third-generation sequencing was demonstrated to be a more accurate and reliable approach in carrier screening of thalassemia compared with next-generation sequencing.