New Delhi Metallo-β-lactamase-1 Gene Research Articles

Emergence of New Delhi metallo-β-lactamase-1 (NDM-1) producing Enterobacterales from water sources: An impending public health challenge in Adamawa-north senatorial zone, Nigeria

Background: The emergence of New Delhi metallo-beta-lactamase-1 (NDM-1) among Enterobacterales in water sources has raised a major public health concern and constitute critical threat to human health as these organisms exhibits high level of resistance to available potent antibiotics. The aim of this study is to detect the presence of NDM-1 gene among carbapenem resistant Enterobacterales (CRE) isolates from water sources.
 Methodology: A total of 256 water samples were collected from randomly selected hand-dug wells (128 samples) and river/stream (128 samples) for each of dry and rainy seasons in four out of the five local government areas (LGAs) of Adamawa-north senatorial zone, Nigeria. The water samples were filtered using membrane filtration technique and the filters introduced into appropriate bacteriologic media for bacterial growth. The bacterial isolates recovered were identified by both phenotypic and molecular protocols. Phenotypic carbapenem (imipenem) resistance was determined by disc diffusion test, blaNDM-1 gene was detected by specific polymerase chain reaction (PCR) test, and plasmid DNA was extracted and electrophoresed by standard procedure.
 Results: Of the 256 water samples analyzed for bacteria growth, 300 bacterial isolates of the order Enterobacterales were recovered. Of these, only 45 (12.6%) isolates were phenotypically resistant to carbapenem (imipenem) antibiotic and blaNDM-1 gene was detected in 30 (66.7%) of these. While blaNDM-1 gene was detected in all the isolates of Klebsiella oxytoca, Klebsiella variicola, Enterobacter aerogenes, Enterobacter hormaechei, Enterobacter asburiae, Citrobacter freundii, and Morganella morganii that were resistant to imipenem, other isolates harbored blaNDM-1 gene in varying proportion. Most of the isolates positive for blaNDM-1 also harbored R-plasmids.
 Conclusion: Emergence of carbapenem resistance mediated by NDM-1 gene in Enterobacterales isolated from water sources constitutes an emerging public health challenge with potential transmission to humans, thereby complicating the treatment of infections caused by these resistant pathogens in man. As such, the urgent need for antimicrobial surveillance and stewardship is of utmost importance.

β-Lactam Resistance Gene NDM-1 in the Aquatic Environment: A Review.

New Delhi Metallo-β-lactamase-1 (NDM-1) offers carbapenem antibiotics resistance that creates an evolving challenge in treating bacterial infections. NDM-1-bearing strains were observed in surface waters around New Delhi in 2010 and after then identified globally. The usage of antibiotics may hasten the growth of the NDM-1-producing bacteria, which pose severe hazards to human and animal health. The emergence of the NDM-1 in the aquatic environment is turning out to be a growing concern worldwide. NDM-1 gene conferring resistance to a widespread class of antibiotics has been observed in bacteria disseminated in animal production wastewaters, hospital sewage, domestic sewage, industrial effluents, wastewater treatment plants, drinking water, surface water, and even in groundwater. This review recapitulates the currently published research studies on the prevalence and geographical distribution of the NDM-1 gene in the aquatic environment, its habitats, and healthcare risk associated with NDM-1-producing bacteria, in addition to molecular techniques employed to reveal the occurrence of the NDM-1 in the aquatic environment, including conventional polymerase chain reaction, real-time qPCR, DNA hybridization, and microarray-based methods.

Testing of antibiotic combinations in NDM-1-producing nosocomial carbapenem resistant Acinetobacter baumannii

Carbapenem resistant Acinetobacter baumannii (CRAB) have become a serious problem in health care settings. Its high prevalence has been associated with nosocomial transmission, high mortality rates, drug resistance and massive economic loss. The most frequent mechanisms of resistance are carbapenem-hydrolyzing class D β-lactamases (CHDL), followed by class B metallo-β-lactamases (MBL). New Delhi metallo-β-lactamase-1(NDM-1) generated much global alarm and were labeled as superbugs which documented impossible to be treated. Preceding our study, eight different antibiotic combinations were evaluated using checkerboard method. This was based on the results obtained from antimicrobial susceptibility testing and phenotypic detection of metallo-β-lactamase production of 27 CRAB selected from our previous work. The existence of NDM-1 gene was tested using two different methods. All antibiotic combinations showed synergistic significant results and no antagonism activity was found. The percentage of NDM-1 gene in the first detection procedure was 12/27 (44.44%) were positive while the other negative 15 ones, 10/15 (66.67%) poses NDM-1were found when using the second method. PCR products were then verified by DNA sequencing. The final consensus sequences were analyzed and submitted to NCBI GenBank data base, representing accession numbers: (HQ6526091), (MK682763.1), (MK682764.1), (MK682767.1), (MN251665.1), (MN251666.1) and (MN251670.1). The alignments showed similarity ranged from 94%-100% nucleotides identity. We concluded that detection of CRAB using accurate, rapid methods and supplying hospital laboratories with molecular department for typing of pathogenic bacteria are essential. Each hospital should establish its own policies according to their antibiogram, national and international guidelines. Primary caretakers should comply with the implemented policies.

Structure-guided optimization of D-captopril for discovery of potent NDM-1 inhibitors

β-lactam antibiotics have long been the mainstay for the treatment of bacterial infections. New Delhi metallo-β-lactamase 1 (NDM-1) is able to hydrolyze nearly all β-lactam antibiotics and even clinically used serine-β-lactamase inhibitors. The wide and rapid spreading of NDM-1 gene among pathogenic bacteria has attracted extensive attention, therefore high potency NDM-1 inhibitors are urgently needed. Here we report a series of structure-guided design of D-captopril derivatives that can inhibit the activity of NDM-1 in vitro and at cellular levels. Structural comparison indicates the mechanisms of inhibition enhancement and provides insights for further inhibitor optimization.

Multidrug-resistant Acinetobacter baumannii strains with NDM-1: Molecular characterization and in vitro efficacy of meropenem-based combinations.

Acinetobacter baumannii is an important cause of hospital-acquired, multidrug-resistant (MDR) infections occurring worldwide. Anti-microbial combination regimens may be the only feasible treatment option for affected patients. In the present study, the efficacy of the combined therapy of meropenem with colistin, ampicillin-sulbactam, tazobactam and vancomycin against clinical strains of MDR A. baumannii was determined. Anti-microbial susceptibility testing was performed and resistance genes were characterized by a multiplex polymerase chain reaction (PCR)-reverse line blot assay. The genetic background of New Delhi metallo-β-lactamase 1 (NDM-1) was analysed by primer walking. The presence of NDM-1 was detected using the modified Hodge test and the EDTA-combined disk test. To screen for synergistic drug effects, the fractional inhibitory concentration index was calculated using a checkerboard assay. The results of the PCR as well as the sequence analyses suggested that NDM-1 was located downstream of the ISAba125 element. In addition, a synergistic effect was determined for meropenem + vancomycin, meropenem + tazobactam and meropenem + ampicillin + sulbactam in two strains each, and in four strains for meropenem + colistin. A total of five A. baumannii strains with resistance to numerous antibiotics and carrying numerous resistance genes were identified. In the strains of A. baumannii, the NDM-1 gene was integrated in a transposon structure with a copy of the ISAba125 insertion sequence. However, the genetic background was not identical among the different species and strains. The genetic variability of NDM-1 may facilitate the rapid dissemination of this gene. In conclusion, meropenem may enhance the efficacy of antibiotics in A. baumannii strains with NDM-1-associated MDR.

Simultaneous three Enterobacteriaceae with different bla NDM-1-encoding plasmids in a patient transferred from mainland China to Taiwan.

New-Delhi metallo-β-lactamase1 (NDM-1) Enterobacteriaceae are increasing worldwide. Herein, we describe a single patient who carried three unusual NDM-1 carbapenem-resistant Enterobacteriaceae – Enterobacter cloacae (E. cloacae) yielded from a urine specimen and Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) from stool specimens. For E. cloacae, its bla NDM-1-encoding plasmid was pKP04NDM with a size of ~54 kb replicons with an IncN backbone. For K. pneumoniae, its blaNDM-1-encoding plasmid was pNDM-BTR with a size of ~59.6 kb and belonged to IncN. For E. coli, its main blaNDM-1-encoding plasmid was pIMP-HK1500, and the NDM-1 gene was obtained from a part of pNDM-BTR (8439 bp). These three clinical strains are reported for the first time and are assumed to be imported from mainland China to Taiwan. The three different plasmids were never reported in K. pneumoniae, E. coli, and Citrobacter spp before. Owing to their associated multidrug resistance, appropriate measures of periodic, targeted surveillance, and development of new antimicrobial agents are urgently needed.

Epidemiology of NDM-1 and its Variants in Multidrug Resistant Gram-Negative Bacilli Isolated from Infection in Cancer Patients

Infectious diseases are a major cause of morbidity and mortality throughout the world. Cancer patients are immunocompromised due to the disease itself and also due to multiple factors such as chemotherapy, radiotherapy, impairment of normal leukocyte function, and use of corticosteroids. This leads to development of resistant pathogens in them of which the New Delhi metallo-β-lactamase-1 (NDM-1) is mostly resistant to the drug of last resort the class of carbapenems. The increase in extended spectrum carbapenem resistance class of antibiotic has led to a worldwide need for a development of a more efficient and novel class of drug active against NDM-1 producing gram-negative bacteria. The epidemiological study of NDM-1 gene has revealed a startling fact on the widespread prevalence of extended spectrum carbapenem resistant gram-negative bacteria in cancer patients both nationally and internationally. Pharmacovigilance and formulations of different class of antibiotics are needed to tackle the problem of this widespread resistance. The various drug delivery methods and drug designing platforms are needed for the development of a novel class of drug needed to efficiently eradicate these multidrug resistant gram-negative pathogens. In the present review, we have described the epidemiological prevalence of NDM-1 and its variants producing gram negative bacilli in cancer patients both nationally and internationally, the problem of ineffectiveness of carbapenem class of antibiotics against such pathogens, and also the future line of developments needed in the field of pharmacovigilance and pharmacotherapeutics to develop a highly efficient, target specific, and cost-effective class of antibiotics.

New Delhi metallo-β-lactamase-1: structure, inhibitors and detection of producers.

Since its discovery in 2008, New Delhi metallo-β-lactamase-1 (NDM-1)-producing Enterobacteriaceae have disseminated globally, facilitated predominantly by gut colonization and the spread of plasmids carrying the bla NDM-1 gene. With few effective antibiotics against NDM-1 producers, and resistance developing to those which remain, there is an urgent need to develop new treatments. To date, most drug design in this area has been focused on developing an NDM-1 inhibitor and has been aided by the wealth of structural and mechanistic information available from high resolution x-ray crystallography and molecular modeling. This review aims to summarize current knowledge regarding the detection of NDM-1 producers, the mechanism of action of NDM-1 and to highlight recent attempts toward the development of clinically useful inhibitors.

Screening for New Delhi metallo-β-lactamase-1 in Enterobacteriaceae: Is there a role for the modified Hodge test?

Objective:The New Delhi metallo-β-lactamase-1 (NDM-1) enzyme is a plasmid-encoded enzyme that inactivates carbapenem antibiotics. This study aims to ascertain if the modified Hodge test (MHT) has a role in screening for NDM-1 in Enterobacteriaceae with reduced carbapenem susceptibility.Methods:Over a period of one year, all Enterobacteriaceae isolates from all clinical specimens with reduced susceptibility to at least one carbapenem were subjected to MHT and conventional polymerase chain reaction (PCR) detection of the NDM-1 gene.Results:A total of 13,098 Enterobacteriaceae isolates were screened and 63 (0.48%) had reduced susceptibility to at least one carbapenem. Out of the 63 isolates, 45 (71.4%) were MHT-positive. The NDM-1 gene was detected in 18 of the 63 isolates (28.6%). All 18 PCR-positive isolates were also MHT-positive. Thus, the sensitivity and specificity of the MHT in detecting NDM-1 in Enterobacteriaceae with reduced carbapenem susceptibility are 100% and 40%, respectively.Conclusion:The MHT is a useful test to screen for the presence of NDM-1 in Enterobacteriaceae with reduced carbapenem susceptibility. However, due to its rather low specificity, all MHT-positive isolates should be subjected to alternative tests (e.g. PCR) for confirmation, especially if other types of carbapenemases (e.g. KPC) are prevalent.

Epidemiological characteristics of blaNDM-1 in Enterobacteriaceae and the Acinetobacter calcoaceticus-Acinetobacter baumannii complex in China from 2011 to 2012.

ObjectivesThe study aimed to investigate the prevalence and epidemiological characteristics of bla NDM-1 (encoding New Delhi metallo-β-lactamase 1) in Enterobacteriaceae and the Acinetobacter calcoaceticus-Acinetobacter baumannii complex (ABC) in China from July 2011 to June 2012.MethodsPCR was used to screen for the presence of bla NDM-1 in all organisms studied. For bla NDM-1-positive strains, 16S rRNA analysis and Analytical Profile Index (API) strips were used to identify the bacterial genus and species. The ABCs were reconfirmed by PCR detection of bla OXA-51-like. Antibiotic susceptibilities of the bacteria were assessed by determining minimum inhibitory concentration (MIC) of them using two-fold agar dilution test, as recommended by the Clinical and Laboratory Standards Institute (CLSI). Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE) and Southern blot hybridization were conducted to ascertain the gene location of bla NDM-1. Conjugation experiments were conducted to determine the transmission of bla NDM-1-positive strains.ResultsAmong 2,170 Enterobacteriaceae and 600 ABCs, seven Enterobacteriaceae strains and two A. calcoaceticus isolates from five different cities carried the bla NDM-1 gene. The seven Enterobacteriaceae strains comprised four Klebsiella pneumoniae, one Enterobacter cloacae, one Enterobacter aerogen and one Citrobacter freundii. All seven were non-susceptible to imipenem, meropenem or ertapenem. Two A. calcoaceticus species were resistant to imipenem and meropenem. Three K. pneumoniae showed the same PFGE profiles. The bla NDM-1 genes of eight strains were localized on plasmids, while one was chromosomal.ConclusionsCompared with previous reports, the numbers and species containing the bla NDM-1 in Enterobacteriaceae have significantly increased in China. Most of them are able to disseminate the gene, which is cause for concern. Consecutive surveillance should be implemented and should also focus on the dissemination of bla NDM-1 among gram-negative clinical isolates.

Co-Production of NDM-1 and OXA-232 byKlebsiella pneumoniae

Co-Production of NDM-1 and OXA-232 by<i>Klebsiella pneumoniae</i>

Emergence of NDM-producing non-baumannii Acinetobacter spp. isolated from China

One hundred and thirty-six bla OXA-51-negative strains were identified from 1,067 Acinetobacter calcoaceticus-A. baumannii complex (ACB complex) isolates, which were collected during October 2010 to March 2013 from 15 general hospitals in 10 cities throughout Zhejiang Province, China. Seven of the 136 bla OXA-51-negative ACB complex isolates were New Delhi metallo-β-lactamase-1 (NDM-1)-positive, among which three were identified as A. nosocomialis and four were identified as A. pittii strains using 16S-23S rRNA gene intergenic spacer (ITS) sequencing and partial RNA polymerase β-subunit (rpoB) sequencing. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analysis showed that the seven NDM-positive isolates belonged to three clonal strains with three novel sequence types (STs). Polymerase chain reaction (PCR) assays and DNA sequence analysis of the carbapenemase and other β-lactamase genes indicated that all the isolates harbored the bla NDM-1 gene, and that only one strain of A. nosocomialis isolates harbored both bla NDM-1 and bla OXA-23. All of them were positive for bla ADC, from which three novel bla ADC genes (designated as bla ADC-69, bla ADC-70, and bla ADC-71) were detected for the first time. The presence of ISAba125 upstream of bla NDM-1 was identified through genetic environment analysis. Carbapenem resistance can be transferred from A. nosocomialis and A. pittii to Escherichia coli EC600 by the conjugation experiment. Plasmid analysis, DNA hybridization, and extraction experiments indicated that bla NDM-1 was located on a plasmid of approximately 50 kb. In conclusion, we characterized the dissemination of NDM-1-positive A. pittii strains in Zhejiang Province, China, and reported the NDM-producing A. nosocomialis for the first time.

Screening of New Delhi metallo-β-lactamase 1 for gram negative bacilli

Objective To screen metallo-β-lactamase (MBL) and New Delhi metallo-β-lactamase 1 (NDM-1) genes among imipenem-resistant gram-negative bacilli,and to provide experimental evidence for the treatment and control of bacteria carrying NDM-1. Methods A total of 7 impenem-resistant gram-negative bacilli isolated from 7 patients in Guangzhou Women and Children′s Medical Center during 2010 were screened for NDM-1 gene retrospectively.The MBLs were examined by imipenemethylenediaminetetra acetic acid (EDTA) double-disc synergy test.NDM-1 gene-specific primers were designed for PCR amplification,and the PCR products were cloned into p MD-T plasmid and then transformed to E.coli Dh5a.Recombinant plasmid was identified by EcoRⅠ,XhoⅠ double enzyme and sequencing. Results Two MBL positive strains (Acinetobacter baumannii 1542,Acinetobacter junii 1454) were identified,and NDM-1 gene was verified in Acinetobacter junii 1454,and showed high concordance with known NDM-1 gene sequences in Genbank (99.9%).Only one nucleotide G-A mutation among 813 nucleotides (G468A) was found,and the amino acid sequences were exactly the same with known NDM-1.The accession number of the sequence of Acinetobacter junii NDM-1 under Genbank is HQ603057.Acinetobacter junii 1454 was resistant to all beta-lactams antibiotics tested except aztreonam and cefoperazone/sulbactam but sensitive to tigecycline,aminoglycosides and quinolones. Conclusion NDM-1 positive Acinetobacter junii was screened from a hospitalized child,suggesting that clinical microbiology laboratories should strengthen imipenem-resistant NDM-1 gene screening.(Chin J Lab Med,2013,36:916-919) Key words: Gram-negative bacteria; beta-Lactamases; Imipenem; Drug resistance; bacterial

Identification and characterization of the first Escherichia coli strain carrying NDM-1 gene in China.

New Delhi metallo-β-lactamase-1 (NDM-1), an acquired class B carbapenemase, is a significant clinical threat due to its extended hydrolysis of β-lactams including carbapenems. In this study, we identified the first confirmed clinical isolate of Escherichia coli BJ01 harboring bla NDM-1 in China. The isolate is highly resistant to all tested antimicrobials except polymyxin. bla NDM-1, bla CTX-M-57, and bla TEM-1 were identified in the isolate. bla NDM-1 was transferable to E. coli EC600 and DH5α in both plasmid conjugation experiments and plasmid transformation tests. BJ01 was identified as a new sequence type, ST224, by multilocus sequence typing. Analysis of genetic environment shows complex transposon-like structures surrounding the bla NDM-1 gene. Genetic analysis revealed that the region flanking bla NDM-1 was very similar to previously identified NDM-positive Acinetobacter spp. isolated in China. The findings of this study raise attention to the emergence and spread of NDM-1-carrying Enterobacteriaceae in China.

A Loop-Mediated Isothermal Amplification Method for Rapid Detection of NDM-1 Gene

A loop-mediated isothermal amplification (LAMP) method was developed for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-β-lactamase-1 (NDM-1), with its specificity and sensitivity having been evaluated. Six primers, including a pair of outer primers, a pair of inner primers, and a pair of loop primers, were specially designed for recognizing eight distinct sequences on the target NDM-1 gene. The amplification reaction was performed within only 40 min under isothermal conditions at 65°C in a regular water bath. The LAMP assay showed good specificity and higher sensitivity than the conventional polymerase chain reaction (PCR), with a detection limit of 1 pg genomic DNA per tube of one NDM-1-positive reference strain. The detection result for the 345 clinical samples showed 100% consistence with the result by the PCR method, and three contaminated samples could be detected correctly by LAMP assays, while they could not be detected by PCR. The LAMP method reported here demonstrated a potential and valuable means for detection of the NDM-1 gene: easy, rapid, visual, specific, accurate, and sensitive, especially useful for on-the-spot investigation.

Identification of New Delhi metallo-β-lactamase gene (NDM-1) from a clinical isolate of Acinetobacter junii in China

New Delhi metallo-β-lactamase-1 (NDM-1) is a novel type of metallo-β-lactamase (MBL) responsible for bacterial resistance to β-lactam antibiotics. Acinetobacter junii was previously shown to possess a MBL phenotype; however, the genes responsible for this phenotype were not identified. In this study, we reported the identification of NDM-1 gene in a clinical isolate of A. junii from a child patient in China, which was resistant to all β-lactams except aztreonam but sensitive to aminoglycosides and quinolones. The cloned NDM-1 gene contained an open reading frame of 813 bp and had a nucleotide sequence 99.9% identical (812/813) to reported NDM-1 genes carried by Acinetobacter baumannii , Enterococcus faecium , Escherichia coli , and Klebsiella pneumoniae . Recombinant NDM-1 protein was successfully expressed in E.coli BL21, and antibiotic sensitivities of the NDM-1-producing E.coli were largely similar to the A.junii 1454 isolate. The findings of this study raise attention to the emergence and spread of NDM-1-carrying bacteria in China.

Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study

SummaryBackgroundGram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1 (NDM-1) are potentially a major global health problem. We investigated the prevalence of NDM-1, in multidrug-resistant Enterobacteriaceae in India, Pakistan, and the UK.MethodsEnterobacteriaceae isolates were studied from two major centres in India—Chennai (south India), Haryana (north India)—and those referred to the UK's national reference laboratory. Antibiotic susceptibilities were assessed, and the presence of the carbapenem resistance gene blaNDM-1 was established by PCR. Isolates were typed by pulsed-field gel electrophoresis of XbaI-restricted genomic DNA. Plasmids were analysed by S1 nuclease digestion and PCR typing. Case data for UK patients were reviewed for evidence of travel and recent admission to hospitals in India or Pakistan.FindingsWe identified 44 isolates with NDM-1 in Chennai, 26 in Haryana, 37 in the UK, and 73 in other sites in India and Pakistan. NDM-1 was mostly found among Escherichia coli (36) and Klebsiella pneumoniae (111), which were highly resistant to all antibiotics except to tigecycline and colistin. K pneumoniae isolates from Haryana were clonal but NDM-1 producers from the UK and Chennai were clonally diverse. Most isolates carried the NDM-1 gene on plasmids: those from UK and Chennai were readily transferable whereas those from Haryana were not conjugative. Many of the UK NDM-1 positive patients had travelled to India or Pakistan within the past year, or had links with these countries.InterpretationThe potential of NDM-1 to be a worldwide public health problem is great, and co-ordinated international surveillance is needed.FundingEuropean Union, Wellcome Trust, and Wyeth.