New Delhi Metallo-β-lactamase Variants Research Articles

First Emergence of NDM-5 and OqxAB Efflux Pumps Among Multidrug-Resistant Klebsiella pneumoniae Isolated from Pediatric Patients in Assiut, Egypt.

New Delhi metallo-β-lactamase (NDM)-producing K. pneumoniae poses a high risk, especially among Egyptian pediatric patients who consume carbapenems antibiotics very widely and without adequate diagnostic sources. In addition, presence of efflux pump genes such as OqxAB increases resistance against many groups of antimicrobials which exacerbates the problem faced for human health. This study aimed to determine NDM variants among K. pneumoniae strains isolated from pediatric patients in Egypt, analyze the presence of OqxAB genes, and molecular characterization ofblaNDM-5-positiveK. pneumoniae. Fifty-six K. pneumoniaeisolates were recovered from pediatric patients, and tested for carbapenemase by modified carbapenem inactivation methods (mCIM) test. Minimum inhibitory concentrations of meropenem and colistin were determined by meropenem E-test strips and broth microdilution, respectively. PCR was used for the detection of the resistant genes (ESBL gene (blaCTX-M), carbapenemase genes (blaNDM, blaKPC) colistin resistant (mcr1, mcr2)) and genes for efflux pump (oqxA and oqxB). BlaNDM was sequenced. The effect of efflux pump in NDM-5-producing isolates was assessed by measuring MIC of ciprofloxacin and meropenem before and after exposure to the carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The horizontal gene transfer ability ofblaNDM-5 was determined using liquid mating assay and PCR-based replicon typing (PBRT) was done to determine the major plasmid incompatibility group. Twenty-nine isolates were positive for blaNDM-1, nine isolates were positive for blaNDM-5, and 15 isolates were positive for blaKPC. There is a significant increase of meropenem MIC of NDM-5-positive isolates compared with NDM-1-positive isolates. In addition, 38 isolates were positive for CTX-M, and 15 isolates were positive for mcr1. Both OqxA and OqxB were detected in 26 isolates and 13 isolates were positive for OqxA while 11 isolates were positive for OqxB only. All NDM-5-producing isolates except one isolate could transfer their plasmids by conjugation to their corresponding transconjugants (E. coli J53). Plasmid replicon typing showed that FII was predominant in NDM-5-producing K. pneumoniae. Similar strains were found between the three isolates and similarity was also detected between the two isolates. The highly resistant K. pneumoniae producing blaNDM-5 type was firstly isolated from pediatric patients. The association of efflux pump genes such as OqxAB is involved in resistance to ciprofloxacin. This highlighted the severity risk ofblaNDM-5-positiveK. pneumonia as it could transfer blaNDM-5 to other bacteria and has more resistance against carbapenems. This underlines the importance of continuous monitoring of infection control guidelines, and the urgent need for a national antimicrobial stewardship plan in Egyptian hospitals.

Phylogenetic diversity and mutational analysis of New Delhi Metallo-β-lactamase (NDM) producing E. coli strains from pediatric patients in Pakistan.

The evolution of NDM genes (blaNDM) in E. coli is accounted for expansive multidrug resistance (MDR), causing severe infections and morbidities in the pediatric population. This study aimed to analyze the phylogeny and mutations in NDM variants of E. coli recovered from the pediatric population. Carbapenem-resistant clinical strains of E. coli were identified using microbiological phenotypic techniques. PCR technique used to amplify the blaNDM genes, identified on agarose gel, and analyzed by DNA sequencing. The amino acid substitutions were examined for mutations after aligning with wild types. Mutational and phylogenetic analysis was performed using Lasergene, NCBI blastn, Clustal Omega, and MEGA software, whereas PHYRE2 software was used for the protein structure predictions. PCR amplification of the blaNDM genes detected 113 clinical strains of E. coli with the contribution of blaNDM-1 (46%), blaNDM-4 (3.5%), and blaNDM-5 (50%) variants. DNA sequencing of blaNDM variants showed homology to the previously described blaNDM-1, blaNDM-4, and blaNDM-5 genes available at GenBank and NCBI database. In addition, the mutational analysis revealed in frame substitutions of Pro60Ala and Pro59Ala in blaNDM-4 and blaNDM-5, respectively. The blaNDM-1 was ortholog with related sequences of E. coli available at GenBank. The phylogenetic analysis indicated that the NDM gene variants resemble other microbes reported globally with some new mutational sites.

Molecular detection of the New Delhi metallo-β-lactamase clinical variant with double mutation- V88L and M154L in Escherichia coli isolates from South India

Carbapenem is usually prescribed as a last resort of antibiotic for treating drug resistant Gram-negative bacterial infections. However, the rise in carbapenem resistance due to the emergence of Zn2+ dependent New Delhi metallo-β lactamase (NDM) variants imposes a huge threat to global health. In Indian context, focusing on molecular epidemiology and underpinning the genetic insights of such continuously evolving superbug is perquisite to guide any form of control strategies or therapeutic interventions. Herein, we investigated the occurrence of NDM variants in Escherichia coli (n = 70) isolated from clinical samples. PCR assay targeting blaNDM gene detected 5 E. coli isolates as NDM positive. Sequencing analyses of blaNDM region of the plasmid DNA determined two types of variants viz., NDM-1 (n = 3) and NDM-5 (n = 2). Notably, NDM-5 variants harbored V88L and M154L mutations, which are known to increase the fitness of NDM-5 variant towards host immune system and zinc starvation. On further characterization, three NDM variants showed the ESBL and AmpC β-lactamase genes, along with 3 different plasmid replicon types (L/M, Y, FIC). Virulence profiling showed traT gene as the most predominant gene among the variants. The emergence of NDM variants especially NDM-5 like variants with critical mutations in enterobacteriaceae increases the significant menaces to the global healthcare. The fact that these NDM variants harboring other antibiotic resistance markers, along with plasmid replicons and virulence genes can be transmitted underlines the gravity of this public health issue.

Frequency distribution, genotypes and prevalent sequence types of New Delhi metallo-β-lactamase-producing Escherichia coli among clinical isolates around the world: A review.

New Delhi metallo-β-lactamase (NDM)-producing Escherichia coli is perceived as one of the primary multidrug-resistant bacteria causing infections in hospitals and clinics. In this review, we present the frequency distribution, genotypes and most prevalent sequence types (STs) of NDM-producing E. coli among clinical isolates worldwide. Several international databases, including Medline, Embase, Scopus and Web of Science, were searched from 2008 to 2018 to identify studies addressing the prevalence of NDM-producing E. coli around the world. Of 974 records identified from the databases, 110 studies fulfilled the eligibility criteria and were included in this analysis. The prevalence of NDM-producing E. coli was 82.6%, 12.9%, 1.5%, 1.0% and 2.0% in Asia, Europe, America, Africa and Oceania, respectively. The most common reported STs among NDM-producing E. coli were ST101, ST167, ST131, ST405, ST410 and ST648. The spread of NDM variants among E. coli strains is a serious threat to global public health. In addition, the most prevalent E. coli clonal groups, such as ST101 and ST167, are one of the main causes of E. coli infection in different countries.

NDM Metallo-β-Lactamases and Their Bacterial Producers in Health Care Settings.

New Delhi metallo-β-lactamase (NDM) is a metallo-β-lactamase able to hydrolyze almost all β-lactams. Twenty-four NDM variants have been identified in >60 species of 11 bacterial families, and several variants have enhanced carbapenemase activity. Klebsiella pneumoniae and Escherichia coli are the predominant carriers of blaNDM, with certain sequence types (STs) (for K. pneumoniae, ST11, ST14, ST15, or ST147; for E. coli, ST167, ST410, or ST617) being the most prevalent. NDM-positive strains have been identified worldwide, with the highest prevalence in the Indian subcontinent, the Middle East, and the Balkans. Most blaNDM-carrying plasmids belong to limited replicon types (IncX3, IncFII, or IncC). Commonly used phenotypic tests cannot specifically identify NDM. Lateral flow immunoassays specifically detect NDM, and molecular approaches remain the reference methods for detecting blaNDM Polymyxins combined with other agents remain the mainstream options of antimicrobial treatment. Compounds able to inhibit NDM have been found, but none have been approved for clinical use. Outbreaks caused by NDM-positive strains have been reported worldwide, attributable to sources such as contaminated devices. Evidence-based guidelines on prevention and control of carbapenem-resistant Gram-negative bacteria are available, although none are specific for NDM-positive strains. NDM will remain a severe challenge in health care settings, and more studies on appropriate countermeasures are required.

Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity

Infections by carbapenem-resistant Enterobacteriaceae are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically used β-lactamase inhibitors to counter the activity of metallo-β-lactamases often harbored by these pathogens. Of particular importance is New Delhi metallo-β-lactamase (NDM), which requires a di-nuclear zinc ion cluster for catalytic activity. Here, we compare the structures and functions of clinical NDM variants 1-17. The impact of NDM variants on structure is probed by comparing melting temperature and refolding efficiency and also by spectroscopy (UV-visible, 1H NMR, and EPR) of di-cobalt metalloforms. The impact of NDM variants on function is probed by determining the minimum inhibitory concentrations of various antibiotics, pre-steady-state and steady-state kinetics, inhibitor binding, and zinc dependence of resistance and activity. We observed only minor differences among the fully loaded di-zinc enzymes, but most NDM variants had more distinguishable selective advantages in experiments that mimicked zinc scarcity imposed by typical host defenses. Most NDM variants exhibited improved thermostability (up to ∼10 °C increased Tm ) and improved zinc affinity (up to ∼10-fold decreased Kd, Zn2). We also provide first evidence that some NDM variants have evolved the ability to function as mono-zinc enzymes with high catalytic efficiency (NDM-15, ampicillin: kcat/Km = 5 × 106 m-1 s-1). These findings reveal the molecular mechanisms that NDM variants have evolved to overcome the combined selective pressures of β-lactam antibiotics and zinc deprivation.

Novel Variant of New Delhi Metallo-β-lactamase, NDM-20, in Escherichia coli.

The spread of carbapenem-resistant Enterobacteriaceae (CRE) mediated by New Delhi metallo-β-lactamase (NDM) poses a serious challenge to clinicians and has become a major public health concern. NDM has been evolving into variants that possess different hydrolysis activity toward antibiotics, so as to affect treatment strategy. In addition, very few studies on NDM variants have focused on animal-derived bacterial isolates. Our study reports a novel NDM variant, NDM-20, in an isolate of Escherichia coli CCD1 recovered from the food animal swine in China. The isolate that was assigned to ST1114, exhibited high level resistance to all β-lactams tested, including aztreonam and carbapenems. The gene of blaNDM-20 was located on an IncX3-type plasmid, surrounded by multiple insertion sequences. Sequencing analysis demonstrated that blaNDM-20 contained three point mutations at positions 262 (G→T), 460 (A→C), and 809 (G→A), compared with blaNDM-1, and just one point mutation at position 809 (G→A), relative to blaNDM-5. Functional analysis revealed that the blaNDM-20 transformant, DH5α+pHSG398/NDM-20, exhibited a higher resistance to ertapenem than that of blaNDM-1 transformant DH5α+pHSG398/NDM-1. Kinetic parameter analysis showed that NDM-20 had increased enzymatic activity against some penicillins and cephalosporins but decreased carbapenemase activity relative to NDM-5. The identification of NDM-20 further confirms the evolution and prevalence of NDM variants in bacteria of food-animal origin.

Clinical Evolution of New Delhi Metallo-β-Lactamase (NDM) Optimizes Resistance under Zn(II) Deprivation.

Carbapenem-resistant Enterobacteriaceae (CRE) are rapidly spreading and taking a staggering toll on all health care systems, largely due to the dissemination of genes coding for potent carbapenemases. An important family of carbapenemases are the Zn(II)-dependent β-lactamases, known as metallo-β-lactamases (MBLs). Among them, the New Delhi metallo-β-lactamase (NDM) has experienced the fastest and widest geographical spread. While other clinically important MBLs are soluble periplasmic enzymes, NDMs are lipoproteins anchored to the outer membrane in Gram-negative bacteria. This unique cellular localization endows NDMs with enhanced stability upon the Zn(II) starvation elicited by the immune system response at the sites of infection. Since the first report of NDM-1, new allelic variants (16 in total) have been identified in clinical isolates differing by a limited number of substitutions. Here, we show that these variants have evolved by accumulating mutations that enhance their stability or the Zn(II) binding affinity in vivo, overriding the most common evolutionary pressure acting on catalytic efficiency. We identified the ubiquitous substitution M154L as responsible for improving the Zn(II) binding capabilities of the NDM variants. These results also reveal that Zn(II) deprivation imposes a strict constraint on the evolution of this MBL, overriding the most common pressures acting on catalytic performance, and shed light on possible inhibitory strategies.

Clinical Variants of New Delhi Metallo-β-Lactamase Are Evolving To Overcome Zinc Scarcity.

Use and misuse of antibiotics have driven the evolution of serine β-lactamases to better recognize new generations of β-lactam drugs, but the selective pressures driving evolution of metallo-β-lactamases are less clear. Here, we present evidence that New Delhi metallo-β-lactamase (NDM) is evolving to overcome the selective pressure of zinc(II) scarcity. Studies of NDM-1, NDM-4 (M154L), and NDM-12 (M154L, G222D) demonstrate that the point mutant M154L, contained in 50% of clinical NDM variants, selectively enhances resistance to the penam ampicillin at low zinc(II) concentrations relevant to infection sites. Each of the clinical variants is shown to be progressively more thermostable and to bind zinc(II) more tightly than NDM-1, but a selective enhancement of penam turnover at low zinc(II) concentrations indicates that most of the improvement derives from catalysis rather than stability. X-ray crystallography of NDM-4 and NDM-12, as well as bioinorganic spectroscopy of dizinc(II), zinc(II)/cobalt(II), and dicobalt(II) metalloforms probe the mechanism of enhanced resistance and reveal perturbations of the dinuclear metal cluster that underlie improved catalysis. These studies support the proposal that zinc(II) scarcity, rather than changes in antibiotic structure, is driving the evolution of new NDM variants in clinical settings.

New Delhi metallo-β-lactamase - type carbapenemases producing Escherichia coli isolates from hospitalized patients: A pilot study

Background & objectives:Resistances to carbapenem group of antimicrobials among Escherichia coli due to production of carbapenemases, especially the New Delhi metallo-β-lactamase (NDM) types, pose serious challenges in the treatment of infections in healthcare settings. This study was undertaken to detect NDM producing E. coli isolates from hospitalized patients with urinary tract infection (UTI).Methods:A total of 30 non-repetitive isolates of E. coli from hospitalized patients with clinical suspicion of UTI were subjected to antimicrobial susceptibility testing. Screening for the production of extended-spectrum β-lactamases (ESBL) was carried out by minimum inhibitory concentration (MIC) test strip ESBL followed by phenotypic confirmation by double-disc synergy test. Phenotypic confirmation of carbapenemase production was carried out by MIC test strip metallo-β-lactamases. Molecular identification of the blaNDM gene was carried out by polymerase chain reaction (PCR) and sequencing of the amplified fragment.Results:Seventeen of the 30 isolates were detected as ESBL producers, of which three were found to be carbapenemase producers. NDM genes were detected by PCR followed by gene sequencing in all three isolates positive for ESBL as well as carbapenemase. The amino acid sequence of the three isolates showed complete identity to the reference sequences of NDM-1, NDM-4 and NDM-8, respectively.Interpretation & conclusions:Our study showed the circulation of NDM variants among the clinical isolates of E. coli that were producers of ESBL as well as carbapenemase.

Prevalence and molecular characterisation of New Delhi metallo-β-lactamases NDM-1, NDM-5, NDM-6 and NDM-7 in multidrug-resistant Enterobacteriaceae from India

The growing prevalence of carbapenem resistance in Enterobacteriaceae worldwide is a major concern. New Delhi metallo-β-lactamase (NDM)-mediated carbapenem resistance has been identified in Enterobacteriaceae from numerous countries including those of the Indian subcontinent. Currently, seven NDM β-lactamase variants (NDM-1 to -7) have been identified. This study evaluated the detection and molecular characterisation of NDM variants in Enterobacteriaceae at a tertiary care hospital in India. A total of 464 isolates were tested; 57 (12.3%) were resistant or showed reduced susceptibility to imipenem and meropenem. All carbapenem-resistant isolates were blaNDM-positive by PCR, but 13 isolates bore variants that differed in sequence from blaNDM-1. NDM-5, NDM-6 and NDM-7 were identified in two, eight and three isolates, respectively. blaNDM variants were located on plasmids of >100kb with IncF, IncA/C and untypeable replicon types. Genes encoding the 16S rRNA methyltransferases RmtB, RmtC and ArmA as well as those for AmpC β-lactamases were also located on the same plasmids as blaNDM in different combinations. The prevalence of NDM-5 to -7 variants was significantly higher in Escherichia coli (P=0.015) and they were more frequently isolated from the urology ward (P=0.037) than NDM-1. The mortality rate was comparable between patients infected with isolates positive for blaNDM-1 and blaNDM variants [25% (11/44) vs. 23% (3/13)]. Expression of blaNDM variants in E. coli using the same promoter showed that NDM-7 conferred higher resistance to imipenem. The diverse genotypic features of blaNDM indicate rapid evolution of NDM resulting from their wide spread in the Indian subcontinent.

Detection of NDM-7 in Germany, a new variant of the New Delhi metallo- -lactamase with increased carbapenemase activity

This study characterized a new variant of the New Delhi metallo-β-lactamase (NDM). A multidrug-resistant Escherichia coli isolate was recovered from the wounds, throat and rectum of a Yemeni patient who presented at the Frankfurt University Hospital in Germany. The presence of β-lactamase genes was analysed by PCR and sequencing. The isolate was further characterized by susceptibility testing, conjugation and transformation assays and plasmid analysis. The E. coli isolate was resistant to all β-lactams including carbapenems. By PCR analysis, the β-lactamase genes blaCMY-2, blaCTX-M-15, blaTEM-1 and blaNDM were identified. Sequencing revealed a blaNDM gene that differed from blaNDM-1 by two point mutations at positions 388 (G→A) and 460 (A→C) corresponding to amino acid substitutions Asp130Asn and Met154Leu, respectively. This NDM variant was identified as NDM-7. The blaNDM-7 gene was located on a self-transferable IncX3 plasmid of 60 kb. E. coli TOP10 transformants harbouring NDM-7 showed higher MICs of β-lactams including carbapenems compared with transformants harbouring NDM-1. Multilocus sequence typing analysis revealed that the E. coli isolate belonged to a novel sequence type (ST599). This study identified a novel NDM variant in E. coli, NDM-7, possessing a high ability to hydrolyse β-lactam antibiotics. Given the diversity of NDM variants located on self-transferable plasmids found in different Gram-negative species and isolated in different countries, the blaNDM gene will most likely efficiently disseminate worldwide.