NDM-1 Research Articles

Four genotypes of carbapenem-resistant Acinetobacter baumannii strains lacking OXA-23 production in Korea

During nationwide Fantimicrobial surveillance (Korea Global Antimicrobial Resistance Surveillance System [Kor-GLASS]), the recent emergence of non-oxacillinase (OXA)-23 production by carbapenem-resistant Acinetobacter baumannii (CRAB) isolates was noted. In this study, we evaluated resistance mechanisms other than OXA-23 production to elucidate the shift in considerable CRAB clones. The presence of OXA carbapenemase genes, such as blaOXA-23, blaOXA-24, blaOXA-58, and blaOXA-51-ISAba1, was determined by PCR. Other carbapenemase genes, such as blaIMP, blaVIM, blaNDM, blaKPC, blaGES, and blaOXA-48 , were determined using sequencing. Strains lacking carbapenemase genes were subjected to whole genome sequencing, and resistance genes were analyzed using ResFinder. Four CRAB strains were collected through a Kor-GLASS study in 2022, in which OXA-23 production was not identified. The carbapenemase genotypes of the four CRAB strains lacking blaOXA-23 were blaOXA-51-ISAba1, blaOXA-66/ACD25, blaOXA-182, and blaNDM-1. To the best of our knowledge, this is the f irst study to identify CRAB producing New Delhi metallo-β-lactamase (NDM)-1 in Korea. In conclusion, domestic CRAB resistance mechanisms may undergo subtle changes. Continuous observations are required to monitor the emergence of new clones.

Comprehensive analysis of distribution characteristics and horizontal gene transfer elements of blaNDM-1-carrying bacteria

The worldwide dissemination of New Delhi metallo-β-lactamase-1 (NDM-1), which mediates resistance to almost all clinical β-lactam antibiotics, is a major public health problem. The global distribution, species, sources, and potential transfer risk of blaNDM-1-carrying bacteria are unclear. Results of a comprehensive analysis of literature in 2010–2022 showed that a total of 6002 blaNDM-1 carrying bacteria were widely distributed around 62 countries with a high trend in the coastal areas. Opportunistic pathogens or pathogens like Klebsiella sp., Escherichia sp., Acinetobacter sp. and Pseudomonas sp. were the four main species indicating the potential microbial risk. Source analysis showed that 86.45 % of target bacteria were isolated from the source of hospital (e.g., Hospital patients and wastewater) and little from surface water (5.07 %) and farms (3.98 %). A plasmid-encoded blaNDM-1Acinetobacter sp. with the resistance mechanisms of antibiotic efflux pump, antibiotic target change and antibiotic degradation was isolated from the wastewater of a typical tertiary hospital. Insertion sequences (IS3 and IS30) located in the adjacent 5 kbp of blaNDM-1-bleMBL gene cluster indicating the transposon-mediated horizontal gene transfer risk. These results showed that the worldwide spread of blaNDM-1-carrying bacteria and its potential horizontal gene transfer risk deserve good control.

Global emergence of double and multi-carbapenemase producing organisms: epidemiology, clinical significance, and evolutionary benefits on antimicrobial resistance and virulence.

Redundant carbapenemase-producing (RCP) bacteria, which carry double or multiple carbapenemases, represent a new and concerning phenomenon. The objective of this study is to conduct a comprehensive analysis of the epidemiology and genetic mechanisms of RCP strains to support targeted surveillance and control measures. A retrospective analysis was conducted using surveillance data from 277 articles. Statistical analysis was performed to determine and evaluate species prevalence, proportions of carbapenemases, antibiotic susceptibility profiles, sample information, and patient outcomes. Complete plasmid sequencing data were utilized to investigate potential antimicrobial resistance or virulence advantages that strains may gain from acquiring redundant carbapenemases. RCP bacteria are widely distributed globally, and their prevalence is increasing over time. Several countries, including China, India, Iran, Turkey, and South Korea, have reported more than 100 RCP strains. The most commonly reported RCP species are Klebsiella pneumoniae and Acinetobacter baumannii, which exhibit varying proportions of carbapenemase combinations. Certain species-carbapenemase combinations, such as K. pneumoniae carrying New Delhi metallo-β-lactamase (NDM) + oxacillinase (OXA) (56.76%) and K. pneumoniae carbapenemase (KPC) + Verona integron-encoded metallo-β-lactamase (VIM) (50.00%) carbapenemases, are associated with high mortality rates. In patients with RCP strains isolated from the bloodstream and respiratory system, the mortality rates are 58.70% and 69.23%, respectively. Analysis of plasmids from RCP strains suggests that they may acquire additional antibiotic resistance phenotypes and virulence factors. Carbapenem-resistant bacteria carrying redundant carbapenemases pose a significant global health threat. This study provides valuable insights into the epidemiology and genetic mechanisms of these bacteria, supporting the development of effective control and prevention strategies to mitigate their transmission.IMPORTANCEThis study examined the global distribution patterns of 1,780 bacteria with double or multiple carbapenemases from 277 articles and assessed their clinical impact. The presence of multiple carbapenemases increases the chances of co-resistance to other classes of antibiotics and more virulence factors, further complicating the clinical management of infections.

Unravelling the genomic characteristics of a Klebsiella quasipneumoniae clinical isolate carrying blaNDM-1

ObjectiveDespite the increasing reports of blaNDM in Enterobacterales in Brazil, comprehensive whole genome sequencing (WGS) data remain scarce. To address this knowledge gap, our study focuses on the characterization of the genome of an New Delhi Metallo-β-lactamase (NDM)-1-producing Klebsiella quasipneumoniae subsp. quasipneumoniae (KQPN) clinical strain isolated in Brazil. MethodsThe antimicrobial susceptibility profile of the A-73.113 strain was performed by agar dilution or broth microdilution following the Brazilian Antimicrobial Susceptibility Testing Committee/European Committee on Antimicrobial Susceptibility Testing recommendations. WGS was performed using the Illumina® NextSeq platform and the generated reads were assembled using the SPAdes software. The sequences obtained were submitted to the bioinformatics pipelines to determine the sequence type, resistome, plasmidome, and virulome. ResultsThe A-73.113 strain was identified as KQPN and was susceptible to polymyxins (MICs, ≤0.25 µg/mL), tigecycline (MIC, 0.5 µg/mL), ciprofloxacin (MIC, 0.5 µg/mL), and levofloxacin (MIC, 1 µg/mL). WGS analysis revealed the presence of genes conferring resistance to β-lactams (blaNDM-1, blaCTX-M-15, blaOXA-9, blaOKP-A-5, blaTEM-1), aminoglycosides [aph(3′)-VI, aadA1, aac(6′)-Ib], and fluoroquinolones (oqxAB, qnrS1, aac(6′)-Ib-cr]. Additionally, the presence of the plasmid replicons Col(pHAD28), IncFIA(HI1), IncFIB(K) (pCAV1099-114), IncFIB(pQil), and IncFII(K), as well as virulence-encoding genes fimABCDEFGHIK (type 1 fimbria), pilW (type IV pili), iutA (aerobactin), entABCDEFS/fepABCDG/fes (Ent siderophores), iroE (salmochelin), and allABCDRS (allantoin utilization) was verified. Furthermore, we found that the A-73.113 strain belongs to ST1040. ConclusionsHere we report the genomic characteristics of an NDM-1-producing KQPN ST1040 strain isolated from blood cultures in Brazil. These data will enhance our comprehension of how this species contributes to the acquisition and dissemination of blaNDM-1 in Brazilian nosocomial settings.

Dissemination of extensively drug-resistant NDM-producing Providencia stuartii in Europe linked to patients transferred from Ukraine, March 2022 to March 2023

Background The war in Ukraine led to migration of Ukrainian people. Early 2022, several European national surveillance systems detected multidrug-resistant (MDR) bacteria related to Ukrainian patients. Aim To investigate the genomic epidemiology of New Delhi metallo-β-lactamase (NDM)-producing Providencia stuartii from Ukrainian patients among European countries. Methods Whole-genome sequencing of 66 isolates sampled in 2022–2023 in 10 European countries enabled whole-genome multilocus sequence typing (wgMLST), identification of resistance genes, replicons, and plasmid reconstructions. Five bla NDM-1-carrying-P. stuartii isolates underwent antimicrobial susceptibility testing (AST). Transferability to Escherichia coli of a bla NDM-1-carrying plasmid from a patient strain was assessed. Epidemiological characteristics of patients with NDM-producing P. stuartii were gathered by questionnaire. Results wgMLST of the 66 isolates revealed two genetic clusters unrelated to Ukraine and three linked to Ukrainian patients. Of these three, two comprised bla NDM-1-carrying-P. stuartii and the third bla NDM-5-carrying-P. stuartii. The bla NDM-1 clusters (PstCluster-001, n = 22 isolates; PstCluster-002, n = 8 isolates) comprised strains from seven and four countries, respectively. The bla NDM-5 cluster (PstCluster-003) included 13 isolates from six countries. PstCluster-001 and PstCluster-002 isolates carried an MDR plasmid harbouring bla NDM-1, bla OXA-10, bla CMY-16, rmtC and armA, which was transferrable in vitro and, for some Ukrainian patients, shared by other Enterobacterales. AST revealed PstCluster-001 isolates to be extensively drug-resistant (XDR), but susceptible to cefiderocol and aztreonam–avibactam. Patients with data on age (n = 41) were 19–74 years old; of 49 with information on sex, 38 were male. Conclusion XDR P. stuartii were introduced into European countries, requiring increased awareness and precautions when treating patients from conflict-affected areas.

The New Delhi metallo-β-lactamase-1 biosensor rapidly and accurately detected antibiotic plasma concentrations in cefuroxime-treated patients

ObjectivesTherapeutic drug monitoring (TDM) of β-lactam antibiotics in critically ill patients may benefit dose optimisation, thus improving therapeutic outcomes. However, rapidly and accurately detecting these antibiotics in blood remains a challenge. This research group recently developed a thermometric biosensor called the New Delhi metallo-β-lactamase-1 (NDM-1) biosensor, which detects multiple classes of β-lactam antibiotics in spiked plasma samples. MethodsThis study assessed the NDM-1 biosensor's effectiveness in detecting plasma concentrations of β-lactam antibiotics in treated patients. Seven patients receiving cefuroxime were studied. Plasma samples collected pre- and post-antibiotic treatment were analysed using the NDM-1 biosensor and compared with liquid chromatography coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). ResultsThe biosensor detected plasma samples without dilution, and a brief pre-treatment using a polyvinylidene fluoride filter significantly lowered matrix effects, reducing the running time to 5–8 minutes per sample. The assay's linear range for cefuroxime (6.25–200 mg/L) covered target concentrations during the trough phase of pharmacokinetics in critically ill patients. The pharmacokinetic properties of cefuroxime in treated patients determined by the NDM-1 biosensor and the UPLC-MS/MS were comparable, and the cefuroxime plasma concentrations measured by the two methods showed statistically good consistency. ConclusionThese data demonstrate that the NDM-1 biosensor assay is a fast, sensitive, and accurate method for detecting cefuroxime plasma concentrations in treated patients and highlights the NDM-1 biosensor as a promising tool for on-site TDM of β-lactam antibiotics in critically ill patients.

Risk Factors for Carbapenemase-Producing Enterobacterales Colonization upon Admission to Tertiary Hospital Hematology Units: A Retrospective Case-control Study

Purpose: This study aimed to assess the prevalence and characteristics of carbapenemase-producing Enterobacterales (CPE) carriage among patients who were admitted to hematology units to identify risk factors for CPE carriage.Methods: This retrospective case-control study was carried out at a tertiary hospital, with a specific focus on adult patients (18 years of age or older) who were admitted to the hematology department. The cases comprised 73 patients with CPE, while the controls comprised 292 individuals matched randomly in a 1:4 ratio according to gender and age. A retrospective review of electronic medical records was conducted to collect data using a CPE acquisition risk factor questionnaire. Patient characteristics, drugs used, and the duration of use were compared between two groups using univariate analysis, and the risk factors associated with CPE were analyzed using multiple logistic regression analysis.Results: The research findings indicated a prevalence of CPE of 5.4%. The predominant strain identified was Klebsiella pneumoniae, and the primary genotype identified was New Delhi metallo-beta-lactamase (NDM). A history of surgery and use of penicillin-based antibiotics were identified as risk factors for CPE carriage.Conclusion: The results emphasize the importance of establishing specific criteria when proactively screening high-risk patients for CPE. Furthermore, these findings provide evidence for forecasting risk factors associated with CPE and devising efficacious preventive measures.

Machine Learning Models Identify Inhibitors of New Delhi Metallo-β-lactamase.

The worldwide spread of the metallo-β-lactamases (MBL), especially New Delhi metallo-β-lactamase-1 (NDM-1), is threatening the efficacy of β-lactams, which are the most potent and prescribed class of antibiotics in the clinic. Currently, FDA-approved MBL inhibitors are lacking in the clinic even though many strategies have been used in inhibitor development, including quantitative high-throughput screening (qHTS), fragment-based drug discovery (FBDD), and molecular docking. Herein, a machine learning-based prediction tool is described, which was generated using results from HTS of a large chemical library and previously published inhibition data. The prediction tool was then used for virtual screening of the NIH Genesis library, which was subsequently screened using qHTS. A novel MBL inhibitor was identified and shown to lower minimum inhibitory concentrations (MICs) of Meropenem for a panel of E. coli and K. pneumoniae clinical isolates expressing NDM-1. The mechanism of inhibition of this novel scaffold was probed utilizing equilibrium dialyses with metal analyses, native state electrospray ionization mass spectrometry, UV-vis spectrophotometry, and molecular docking. The uncovered inhibitor, compound 72922413, was shown to be 9-hydroxy-3-[(5-hydroxy-1-oxa-9-azaspiro[5.5]undec-9-yl)carbonyl]-4H-pyrido[1,2-a]pyrimidin-4-one.

Risk Factors and Outcomes of Patients Colonized with KPC and NDM Carbapenemase-Producing Enterobacterales.

Carbapenemase-producing enterobacterales (CPE) poses an increasing threat in hospitals worldwide. Recently, the prevalence of different carbapenemases conferring carbapenem resistance in enterobacterales changed in our country, including an increase in New Delhi Metallo-beta-lactamase (NDM)-CPE. We conducted a comparative historical study of adult patients colonized with Klebsiella pneumoniae carbapenemase (KPC)-CPE (July 2016 to June 2018, a historical cohort) vs. NDM-CPE (July 2016 to January 2023). We identified patients retrospectively through the microbiology laboratory and reviewed their files, extracting demographics, underlying diseases, Charlson Comorbidity Index (CCI) scores, treatments, and outcomes. This study included 228 consecutive patients from whom a CPE rectal swab screening was obtained: 136 NDM-CPE positive and 92 KPC-CPE positive. NDM-CPE-colonized patients had a shorter hospitalization length and a significantly lower 30-day post-discharge mortality rate (p = 0.002) than KPC-CPE-colonized patients. Based on multivariate regression, independent risk factors predicting CPE-NDM colonization included admission from home and CCI < 4 (p < 0.001, p = 0.037, respectively). The increase in NDM-CPE prevalence necessitates a modified CPE screening strategy upon hospital admission tailored to the changing local CPE epidemiology. In our region, the screening of younger patients residing at home with fewer comorbidities should be considered, regardless of a prior community healthcare contact or hospital admission.

Predominance of OXA-48 carbapenemase-producingKlebsiella pneumoniaestrains in tertiary hospital in Sarajevo, Bosnia and Herzegovina.

Klebsiella pneumoniae, a member of the Enterobacteriaceae family, demonstrates an increasing trend of resistance to carbapenems and is a common cause of both hospital- and community-acquired infections. The current study provides insights into the genetic characterization of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates circulating during 2022 in a Sarajevo tertiary hospital. Among the 87 CRKP strains analyzed, real-time polymerase chain reaction (rtPCR) results showed that 85 (97.7%) tested positive for the carbapenem resistance gene. The oxacillinase-48 (OXA-48) gene was detected in 83 (95.4%) isolates, while the Klebsiella pneumoniae carbapenemase (KPC) and the New Delhi metallo-beta-lactamase (NDM) genes were detected in one isolate each. No Verona integron-encoded-metallo-beta-lactamase (VIM) or imipenemase-metallo-beta-lactamase 1 (IMP-1) genes were found in any of the tested isolates. The multilocus sequence typing (MLST) analysis of sequence types (STs) revealed that ST101, an emerging high-risk clone exhibiting extensive drug resistance, was the most prevalent, whereas ST307 was detected in only one isolate. Phylogenetic analysis of the ten CRKP isolates indicated the presence of three clusters that could constitute an outbreak. A comparison of the results of the utilized phenotypic test (the combined-disk test [CDT]) and rtPCR showed high concordance, suggesting that the phenotypic assay may be useful for the early detection of resistance mechanisms as part of routine susceptibility testing. With the increased affordability of next-generation sequencing (NGS), its application in hospital settings has proven highly beneficial, aiding in the implementation of infection control and prevention measures. Given the significant resistance demonstrated by the CRKP isolates to most tested antibiotics, it is imperative to establish effective methods to restrict the spread of these isolates, as well as to carefully monitor the use of carbapenems in clinical practice.

Epidemiological and molecular study of Providencia rettgeri outbreak at a university hospital during the COVID-19 reference center.

In 2014, Brazil detected New Delhi metallo-β-lactamase (NDM)-producing Enterobacterales from a Providencia rettgeri isolate obtained through surveillance swabs in the Southern region. Subsequently, various species have reported several NDM enzymes. However, comprehensive data on the current epidemiology of NDM-producing P. rettgeri in Brazil remains limited. This study, aimed to provide a detailed characterization of the phenotypic, genotypic, and epidemiological profile of clinical isolates of P. rettgeri NDM. From April 2020 to December 2022, 18 carbapenem-resistant P. rettgeri strains, previously identified using Vitek2®, were isolated at the University Hospital of Londrina. Resistance and virulence genes were assessed through genetic analysis using ERIC PCR and NextSeq (Illumina) sequencing. Statistical analysis was conducted using SPSS version 2.0. Genomic analysis confirmed the presence of β-lactamase blaNDM-1 and blaOXA-1. All isolates showed the presence of the NDM encoding gene and genetic similarity above 90% between isolates. Clinical parameters of patients infected with P. rettgeri exhibited significant association with mechanical ventilation, prior use of carbapenems, and polymyxins. We also report a significant association between P. rettgeri infection and death outcome. This study characterizes NDM-1 metallo-β-lactmases isolates, among P. rettgeri isolates from patients at the University Hospital (HU), during the COVID-19 pandemic. The emergence of this novel resistance mechanism among P. rettgeri poses a significant challenge, limiting the therapeutic options for infections in our hospital.

3-O-Substituted Quercetin: an Antibiotic-Potentiating Agent against Multidrug-Resistant Gram-Negative Enterobacteriaceae through Simultaneous Inhibition of Efflux Pump and Broad-Spectrum Carbapenemases.

The discovery of safe and efficient inhibitors against efflux pumps as well as metallo-β-lactamases (MBL) is one of the main challenges in the development of multidrug-resistant (MDR) reversal agents which can be utilized in the treatment of carbapenem-resistant Gram-negative bacteria. In this study, we have identified that introduction of an ethylene-linked sterically demanding group at the 3-OH position of the previously reported MDR reversal agent di-F-Q endows the resulting compounds with hereto unknown multitarget inhibitory activity against both efflux pumps and broad-spectrum β-lactamases including difficult-to-inhibit MBLs. A molecular docking study of the multitarget inhibitors against efflux pump, as well as various classes of β-lactamases, revealed that the 3-O-alkyl substituents occupy the novel binding sites in efflux pumps as well as carbapenemases. Not surprisingly, the multitarget inhibitors rescued the antibiotic activity of a carbapenem antibiotic, meropenem (MEM), in NDM-1 (New Delhi Metallo-β-lactamase-1)-producing carbapenem-resistant Enterobacteriaceae (CRE), and they reduced MICs of MEM more than four-fold (synergistic effect) in 8-9 out of 14 clinical strains. The antibiotic-potentiating activity of the multitarget inhibitors was also demonstrated in CRE-infected mouse model. Taken together, these results suggest that combining inhibitory activity against two critical targets in MDR Gram-negative bacteria, efflux pumps, and β-lactamases, in one molecule is possible, and the multitarget inhibitors may provide new avenues for the discovery of safe and efficient MDR reversal agents.

Molecular Epidemiology of Carbapenem-Resistant Klebsiella aerogenes in Japan.

Information regarding Klebsiella aerogenes haboring carbapenemase in Japan is limited. A comprehensive nationwide survey was conducted from September 2014 to December 2022, and 67 non-duplicate strains of carbapenem-resistant K. aerogenes were isolated from 57 healthcare facilities in Japan. Through genetic testing and whole-genome sequencing, six strains were found to possess carbapenemases, including imipenemase (IMP)-1, IMP-6, New Delhi metallo-β-lactamase (NDM)-1, and NDM-5. The strain harboring blaNDM-5 was the novel strain ST709, which belongs to the clonal complex of the predominant ST4 in China. The novel integron containing blaIMP-1 featured the oxacillinase-101 gene, which is a previously unreported structure, with an IncN4 plasmid type. However, integrons found in the strains possessing blaIMP-6, which were the most commonly identified, matched those reported domestically in Klebsiella pneumoniae, suggesting the prevalence of identical integrons. Transposons containing blaNDM are similar or identical to the transposon structure of K. aerogenes harboring blaNDM-5 previously reported in Japan, suggesting that the same type of transposon could have been transmitted to K. aerogenes in Japan. This investigation analyzed mobile genetic elements, such as integrons and transposons, to understand the spread of carbapenemases, highlighting the growing challenge of carbapenem-resistant Enterobacterales in Japan and underscoring the critical need for ongoing surveillance to control these pathogens.

Infection with the multidrug-resistant Klebsiella pneumoniae New Delhi metallo-B-lactamase strain in patients with COVID-19: Nec Hercules contra plures?

During the coronavirus disease 2019 (COVID-19) pandemic, in patients treated for SARS-CoV-2 infection, infections with the Klebsiella pneumoniae bacteria producing New Delhi metallo-B-lactamase (NDM) carbapenemase in the USA, Brazil, Mexico, and Italy were observed, especially in intensive care units (ICUs). This study aimed to assess the impact of Klebsiella pneumoniae NDM infection and other bacterial infections on mortality in patients treated in ICUs due to COVID-19. The 160 patients who qualified for the study were hospitalized in ICUs due to COVID-19. Three groups were distinguished: patients with COVID-19 infection only (N = 72), patients with COVID-19 infection and infection caused by Klebsiella pneumoniae NDM (N = 30), and patients with COVID-19 infection and infection of bacterial etiology other than Klebsiella pneumoniae NDM (N = 58). Mortality in the groups and chosen demographic data; biochemical parameters analyzed on days 1, 3, 5, and 7; comorbidities; and ICU scores were analyzed. Bacterial infection, including with Klebsiella pneumoniae NDM type, did not elevate mortality rates. In the group of patients who survived the acute phase of COVID-19 the prolonged survival time was demonstrated: the median overall survival time was 13 days in the NDM bacterial infection group, 14 days in the other bacterial infection group, and 7 days in the COVID-19 only group. Comparing the COVID-19 with NDM infection and COVID-19 only groups, the adjusted model estimated a statistically significant hazard ratio of 0.28 (p = 0.002). Multivariate analysis revealed that age, APACHE II score, and CRP were predictors of mortality in all the patient groups. In patients treated for SARS-CoV-2 infection acquiring a bacterial infection due to prolonged hospitalization associated with the treatment of COVID-19 did not elevate mortality rates. The data suggests that in severe COVID-19 patients who survived beyond the first week of hospitalization, bacterial infections, particularly Klebsiella pneumoniae NDM, do not significantly impact mortality. Multivariate analysis revealed that age, APACHE II score, and CRP were predictors of mortality in all the patient groups.

Genomic characterization of ST38 NDM-5-producing Escherichia coli isolates from an outbreak in the Czech Republic.

A 2-year national genomic screening in the Czech Republic identified a notable prevalence of the New Delhi metallo-β-lactamase 5 (NDM-5)-producing Escherichia coli sequence type 38 (ST38) in the city of Brno. Forty-two ST38 E. coli isolates harbored the blaNDM-5 gene on the chromosome. Virulence factors confirmed the persistence of these isolates through biofilm formation. Single Nucleotide Polymorphisms (SNPs)-based phylogeny and CRISPR assay typing showed minimal genomic variations, implying a clonally driven outbreak. Results suggest that this high-risk clone may impose a nationwide problem.

A CRISPR/Cas12a-Based System for Sensitive Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales.

Antimicrobial-resistant (AMR) bacteria pose a significant global health threat, and bacteria that produce New Delhi metallo-β-lactamase (NDM) are particularly concerning due to their resistance to most β-lactam antibiotics, including carbapenems. The emergence and spread of NDM-producing genes in food-producing animals highlight the need for a fast and accurate method for detecting AMR bacteria. We therefore propose a PCR-coupled CRISPR/Cas12a-based fluorescence assay that can detect NDM-producing genes (blaNDM) in bacteria. Thanks to its designed gRNA, this CRISPR/Cas12a system was able to simultaneously cleave PCR amplicons and ssDNA-FQ reporters, generating fluorescence signals. Our method was found to be highly specific when tested against other foodborne pathogens that do not carry blaNDM and also demonstrated an excellent capability to distinguish single-nucleotide polymorphism. In the case of blaNDM-1 carrying E. coli, the assay performed exceptionally well, with a detection limit of 2.7 × 100 CFU/mL: 100 times better than conventional PCR with gel electrophoresis. Moreover, the developed assay detected AMR bacteria in food samples and exhibited enhanced performance compared to previously published real-time PCR assays. Thus, this novel PCR-coupled CRISPR/Cas12a-based fluorescence assay has considerable potential to improve current approaches to AMR gene detection and thereby contribute to mitigating the global threat of AMR.

Emergence and genomic characterization of Proteus mirabilis harboring blaNDM-1 in Korean companion dogs

Proteus mirabilis is a commensal bacterium dwelling in the gastrointestinal (GI) tract of humans and animals. Although New Delhi metallo-β-lactamase 1 (NDM-1) producing P. mirabilis is emerging as a threat, its epidemiology in our society remains largely unknown. LHPm1, the first P. mirabilis isolate harboring NDM-1, was detected from a companion dog that resides with a human owner. The whole-genome study revealed 20 different antimicrobial resistance (AMR) genes against various classes of antimicrobial agents, which corresponded to the MIC results. Genomic regions, including MDR genes, were identified with multiple variations and visualized in a comparative manner. In the whole-genome epidemiological analysis, multiple phylogroups were identified, revealing the genetic relationship of LHPm1 with other P. mirabilis strains carrying various AMR genes. These genetic findings offer comprehensive insights into NDM-1-producing P. mirabilis, underscoring the need for urgent control measures and surveillance programs using a “one health approach”.

Evaluation of the in vitro susceptibility of clinical isolates of NDM-producing Klebsiella pneumoniae to new antibiotics included in a treatment regimen for infections.

Due to the growing resistance to routinely used antibiotics, the search for new antibiotics or their combinations with effective inhibitors against multidrug-resistant microorganisms is ongoing. In our study, we assessed the in vitro drug susceptibility of Klebsiella pneumoniae strains producing New Delhi metallo-β-lactamases (NDM) to antibiotics included in the Infectious Diseases Society of America (IDSA) and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) recommendations. A total of 60 strains of NDM-producing K. pneumoniae were obtained from different patients hospitalized at the 4th Military Hospital in Wroclaw between 2019 and 2022 and subjected to drug susceptibility to selected antibiotics, including the effects of drug combinations. Among the tested antibiotics, the highest sensitivity (100%) was observed for cefiderocol, eravacycline (interpreted according to the European Committee on Antimicrobial Susceptibility Testing [EUCAST]), and tigecycline. Sensitivity to intravenous fosfomycin varied depending on the method used. Using the "strip stacking" method, determining cumulative sensitivity to ceftazidime/avibactam and aztreonam demonstrated 100% in vitro sensitivity to this combination among the tested strains. The in vitro susceptibility assessment demonstrated that, the best therapeutic option for treating infections caused by carbapenemase-producing strains seems to be a combination of ceftazidime/avibactam with aztreonam. Due to the safety of using both drugs, cost effectiveness, and the broadest indications for use among the tested antibiotics, this therapy should be the first-line treatment for carbapenemase-producing Enterobacterales infections. Nevertheless, a comprehensive evaluation of the efficacy of treating infections caused by NDM-producing K. pneumoniae strains should include not only in vitro susceptibility assessment but also an analysis of clinical cases.

Oxidized glutathione reverts carbapenem resistance in blaNDM-1-carrying Escherichia coli.

The emergence of drug-resistant Enterobacteriaceae carrying plasmid-mediated β-lactamase genes has become a significant threat to public health. Organisms in the Enterobacteriaceae family containing New Delhi metallo-β-lactamase‑1 (NDM-1) and its variants, which are capable of hydrolyzing nearly all β-lactam antibacterial agents, including carbapenems, are referred to as superbugs and distributed worldwide. Despite efforts over the past decade, the discovery of an NDM-1 inhibitor that can reach the clinic remains a challenge. Here, we identified oxidized glutathione (GSSG) as a metabolic biomarker for blaNDM-1 using a non-targeted metabolomics approach and demonstrated that GSSG supplementation could restore carbapenem susceptibility in Escherichia coli carrying blaNDM-1 in vitro and in vivo. We showed that exogenous GSSG promotes the bactericidal effects of carbapenems by interfering with intracellular redox homeostasis and inhibiting the expression of NDM-1 in drug-resistant E. coli. This study establishes a metabolomics-based strategy to potentiate metabolism-dependent antibiotic efficacy for the treatment of antibiotic-resistant bacteria.

Characterization of blaNDM-19-producing IncX3 plasmid isolated from carbapenem-resistant Escherichia coli and Klebsiellapneumoniae

The increase in the prevalence of carbapenem-producing Enterobacterales (CPE) is a major threat, with the New Delhi metallo-β-lactamase (NDM) enzyme-producing CPEs being one of the major causative agents of healthcare settings infections. In this study, we characterized an IncX3 plasmid harboring blaNDM-19 in Lebanon, recovered from three Escherichia coli belonging to ST167 and one Klebsiella pneumoniae belonging to ST16 isolated from a clinical setting. Plasmid analysis using PBRT, Plasmid Finder, and PlasmidSPAdes showed that all four isolates carried a conjugative 47-kb plasmid having blaNDM-19, and was designated as pLAU-NDM19. We constructed a sequence-based maximum likelihood phylogenetic tree and compared pLAU-NDM19 to other representative IncX3 plasmids carrying NDM-variants and showed that it was closely linked to NDM-19 positive IncX3 plasmid from K. pneumoniae reported in China. Our findings also revealed the route mediating resistance transmission, the IncX3 dissemination among Enterobacterales, and the NDM-19 genetic environment. We showed that mobile elements contributed to the variability of IncX3 genomic environment and highlighted that clonal dissemination in healthcare settings facilitated the spread of resistance determinants. Antimicrobial stewardship programs implemented in hospitals should be coupled with genomic surveillance to better understand the mechanisms mediating the mobilization of resistance determinants among nosocomial pathogens and their subsequent clonal dissemination.