Understanding blaNDM-1 gene regulation in CRKP infections: toward novel antimicrobial strategies for hospital-acquired pneumonia.

BACKGROUND: The escalating challenge of Carbapenem-resistant Klebsiella pneumoniae (CRKP) in hospital-acquired pneumonia (HAP) is closely linked to the blaNDM-1 gene. This study explores the regulatory mechanisms of blaNDM-1 expression and aims to enhance antibacterial tactics to counteract the spread and infection of resistant bacteria. METHODS: KP and CRKP strains were isolated from HAP patients' blood samples. Transcriptomic sequencing (RNA-seq) identified significant upregulation of blaNDM-1 gene expression in CRKP strains. Bioinformatics analysis revealed blaNDM-1 gene involvement in beta-lactam resistance pathways. CRISPR-Cas9 was used to delete the blaNDM-1 gene, restoring sensitivity. In vitro and in vivo experiments demonstrated enhanced efficacy with Imipenem and Thanatin or Subatan combination therapy. RESULTS: KP and CRKP strains were isolated with significant upregulation of blaNDM-1 in CRKP strains identified by RNA-seq. The Beta-lactam resistance pathway was implicated in bioinformatics analysis. Knockout of blaNDM-1 reinstated sensitivity in CRKP strains. Further, co-treatment with Imipenem, Thanatin, or Subactam markedly improved antimicrobial effectiveness. CONCLUSION: Silencing blaNDM-1 in CRKP strains from HAP patients weakens their Carbapenem resistance and optimizes antibacterial strategies. These results provide new theoretical insights and practical methods for treating resistant bacterial infections.

A Multiplex Recombinase-Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC -2, and blaNDM -1 Genes in Klebsiella pneumoniae.

OBJECTIVE: This study aimed to establish a highly sensitive and rapid single-tube, two-stage, multiplex recombinase-aided qPCR (mRAP) assay to specifically detect the khe, blaKPC-2, and blaNDM-1 genes in Klebsiella pneumoniae. METHODS: mRAP was carried out in a qPCR instrument within 1 h. The analytical sensitivities of mRAP for khe, blaKPC-2, and blaNDM-1 genes were tested using recombinant plasmids and dilutions of reference strains. A total of 137 clinical isolates and 86 sputum samples were used to validate the clinical performance of mRAP. RESULTS: mRAP achieved the sensitivities of 10, 8, and 14 copies/reaction for khe, blaKPC-2, and blaNDM-1 genes, respectively, superior to qPCR. The Kappa value of qPCR and mRAP for detecting khe, blaKPC-2, and blaNDM-1 genes was 1, 0.855, and 1, respectively (p < 0.05). CONCLUSION: mRAP is a rapid and highly sensitive assay for potential clinical identification of khe, blaKPC-2, and blaNDM-1 genes in K. pneumoniae.

Outer Membrane Vesicles Transmitting blaNDM-1 Mediate the Emergence of Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae.

Dissemination of hypervirulent and carbapenem-resistant Klebsiella pneumoniae (CRKP) has been reported worldwide, posing a serious threat to antimicrobial therapy and public health. Outer membrane vesicles (OMVs) act as vectors for the horizontal transfer of virulence and resistance genes. However, K. pneumoniae OMVs that transfer carbapenem resistance genes into hypervirulent K. pneumoniae (hvKP) have been insufficiently investigated. Therefore, this study investigates the transmission of the blaNDM-1 gene encoding resistance via OMVs released from CRKP and the potential mechanism responsible for the carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) emergence. OMVs were isolated via ultracentrifugation from CRKP with or without meropenem selective pressure. OMVs were then used to transform classical K. pneumoniae (ckp) ATCC 10031, extended-spectrum ß-lactamase (ESBL)-producing K. pneumoniae ATCC 700603, and hvKP NTUH-K2044. Our results showed that meropenem treatment resulted in changes in the number and diameter of OMVs secreted by CRKP. OMVs derived from CRKP mediated the transfer of blaNDM-1 to ckp and hvKP, thereby increasing the carbapenem MIC of transformants. Further experiments confirmed that NTUH-K2044 transformants exhibited hypervirulence. Our study demonstrates, for the first time, that OMVs derived from CRKP can carry blaNDM-1 and deliver resistance genes to other K. pneumoniae strains, even hvKP. The transfer of carbapenem genes into hypervirulent strains may promote the emergence and dissemination of CR-hvKP. This study elucidates a new mechanism underlying the formation of CR-hvKP.

Molecular characteristics of carbapenem-resistant Raoultella ornithinolytica.

Aim of this study was to explore molecular characteristics and resistance mechanisms of carbapenem-resistant Raoultella ornithinolytica (CR-ROR) isolated from patients in a hospital in China. Three CR-ROR strains were collected and bacterial identification was done by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) Vitek-MS and by digital DDH analysis. VITEK 2 compact system and Kirby-Bauer (K-B) disk diffusion were used for antimicrobial susceptibility testing. Whole genome sequencing was carried out using the Illumina platform NovaSeq sequencer. Abricate software was used for the prediction of antibiotic resistance genes of three CR-ROR strains. The phylogenetic tree was constructed through genome SNPs to investigate the genetic relationship of three CR-ROR strains. Three CR-ROR (WF1357, WF2441, and WF3367) strains were collected in this study. Two strains were isolated from neurosurgery (WF1357 and WF2441), and one was isolated from pulmonology department (WF3367). All strains harboured multiple antibiotic resistance genes. Two strains (WF1357, WF2441) carried the blaNDM-1 gene, one of the strains (WF3367) carried the blaKPC-2 gene. Three CR-RORs were resistant to different antimicrobial agents including carbapenems. The three CR-ROR strains collected in this study and 51 CR-ROR strain genomes downloaded from NCBI, were divided into six evolutionary groups (A-F). In this study, three CR-ROR strains were found to have a higher level of resistance to antibacterial agents and carried multiple antibiotic resistance genes. The CR-ROR strains carrying multiple antibacterial resistant genes require the stringent monitoring to avoid the spread of multidrug-resistant bacterial strains.

First Detection of blaNDM-1-Haboring IncHI2 Plasmid in Escherichia coli Strain Isolated from Goose in China.

Carbapenem-resistant Enterobacteriaceae infections are among the most serious threats to human and animal health worldwide. Of the 1013 strains of Escherichia coli isolated and identified in 14 regions of China from 2007 to 2018, seven strains were resistant to meropenem and all were positive for blaNDM. The seven New Delhi metallo-ß-lactamase (NDM)-positive strains belonged to five different sequence types, indicating that most of the NDM-positive strains were nonclonal. An IncHI2 plasmid carrying the blaNDM-1 element was identified in the C1147 strain from a goose source and reported for the first time, showing a specific structure. Conjugation experiments revealed that the IncHI2 plasmid was conjugatable, and the horizontal propagation of the plasmid led to the rapid propagation of NDM in the same and different strains. This study revealed that waterfowl, as a potential transmission factor for carbapenem-resistant blaNDM-1, poses a threat to human health.

Co-existence of blaIMP, blaNDM-1, and blaSHV, genes of Pseudomonas aeruginosa isolated from Quetta: Antimicrobial resistance and clinical significance.

Objective: Molecular detection and co-presence of carbapenem-resistant genes in the isolates of Pseudomonas aeruginosa are less commonly reported from Quetta. In the present study, we determined to highlight the antibiotic sensitivity profile and genetic mechanism of carbapenem resistance. Methods: The cross-sectional study was conducted from May to September 2018 at the Hi-tech laboratory, Centre for Advance Studies in Vaccinology and Biotechnology, University of Baluchistan, Quetta. Biochemical and molecular methods were ascertained for the recognition of the isolates and minimum inhibitory concentration was performed using E-test and broth microdilution methods. The molecular basis of carbapenemase activity was determined by identifying carbapenemase genes in the isolates. Results: Of the (n=23) P. aeruginosa isolated from pus aspirates obtained from surgical/burn units, we have detected blaIMP (n=7/8) 87.5%, blaNDM-1 (n=5/8) 62.5%, and blaSHV (n=4/8) 50%. The co-existence of multiple antibiotic-resistant genes, blaIMP, blaNDM-1 and blaSHV was found in (n=2/8) 25% isolates. These isolates displayed resistance against a range of antimicrobials from ß-lactams, tetracyclines, cephalosporins, quinolones, monobactams, aminoglycosides, sulphonamides, phosphoric acid, macrolides, and polypeptide groups, suggesting extensive-drug resistance. Conclusion: The emergence of MBL and ESBL producers is an alarming threat in the region. It is of great importance to determine the resistance mechanism of bacterial bugs. The lack of new antimicrobials particularly against gram-negative bacteria is quite alarming worldwide.

Epidemiology and Transmission of Carbapenemase-Producing Enterobacteriaceae in a Health Care Network of an Acute-Care Hospital and Its Affiliated Intermediate- and Long-Term-Care Facilities in Singapore.

Movement of patients in a health care network poses challenges for the control of carbapenemase-producing Enterobacteriaceae (CPE). We aimed to identify intra- and interfacility transmission events and facility type-specific risk factors of CPE in an acute-care hospital (ACH) and its intermediate-term and long-term-care facilities (ILTCFs). Serial cross-sectional studies were conducted in June and July of 2014 to 2016 to screen for CPE. Whole-genome sequencing was done to identify strain relatedness and CPE genes (blaIMI, blaIMP-1, blaKPC-2, blaNDM-1, and blaOXA-48). Multivariable logistic regression models, stratified by facility type, were used to determine independent risk factors. Of 5,357 patients, half (55%) were from the ACH. CPE prevalence was 1.3% in the ACH and 0.7% in ILTCFs (P = 0.029). After adjusting for sociodemographics, screening year, and facility type, the odds of CPE colonization increased significantly with a hospital stay of ≥3 weeks (adjusted odds ratio [aOR], 2.67; 95% confidence interval [CI], 1.17 to 6.05), penicillin use (aOR, 3.00; 95% CI, 1.05 to 8.56), proton pump inhibitor use (aOR, 3.20; 95% CI, 1.05 to 9.80), dementia (aOR, 3.42; 95% CI, 1.38 to 8.49), connective tissue disease (aOR, 5.10; 95% CI, 1.19 to 21.81), and prior carbapenem-resistant Enterobacteriaceae (CRE) carriage (aOR, 109.02; 95% CI, 28.47 to 417.44) in the ACH. For ILTCFs, presence of wounds (aOR, 5.30; 95% CI, 1.01 to 27.72), respiratory procedures (aOR, 4.97; 95% CI, 1.09 to 22.71), vancomycin-resistant enterococcus carriage (aOR, 16.42; 95% CI, 1.52 to 177.48), and CRE carriage (aOR, 758.30; 95% CI, 33.86 to 16,982.52) showed significant association. Genomic analysis revealed only possible intra-ACH transmission and no evidence for ACH-to-ILTCF transmission. Although CPE colonization was predominantly in the ACH, risk factors varied between facilities. Targeted screening and precautionary measures are warranted.

Detection of NDM-1 producing Klebsiella pneumoniae ST15 and ST147 in Iran during 2019-2020.

Carbapenems are employed to treat infections caused by Gram-negative bacteria including Klebsiella pneumoniae. This research is aimed to perform phenotypic detection of ß-lactamases and molecular characterization of NDM-1 positive K. pneumoniae isolates. Another objective is to investigate NDM-1 producing K. pneumoniae among children in Iran. From 2019 to 2020, altogether 60 K. pneumoniae isolates were acquired from various patients in certain Iranian hospitals. Antimicrobial susceptibility testing was performed by disk diffusion and broth microdilution methods. In addition, mCIM and eCIM were used to confirm the production of carbapenemases and metallo-beta-lactamases (MBLs), respectively. Detection of resistance genes namely, blaNDM-1, blaIMP, blaVIM, blaKPC, blaOXA-48-like, blaCTX-M, blaSHV, blaTEM, and mcr-1 was performed by PCR and confirmed by DNA sequencing. Multilocus sequence typing (MLST) was employed to determine the molecular typing of the strains. According to the findings, the highest rate of carbapenem resistance was detected against doripenem 83.3% (50). Moreover, 31.7% (19) were resistant to colistin. Further to the above, altogether 80% (48) were carbapenemase-producing isolates and among them 46.7% (28) of the isolates were MBL and 33.3% (20) isolates were serine ß-lactamase producer. According to the PCR results, 14 isolates produced blaNDM-1. Remarkably, four blaNDM-1 positive isolates were detected in children. In addition, these isolates were clonally related as determined by MLST (ST147, ST15). Altogether ten blaNDM-1 positive isolates were ST147 and four blaNDM-1 positive isolates were ST15. Based on the results, the emergence of NDM-producing K. pneumoniae among children is worrying and hence, it is necessary to develop a comprehensive program to control antibiotic resistance in the country.

Dissemination of NDM-1 in pseudomonas aeruginosa and klebsiella pneumoniae isolated from pus samples in tertiary care hospitals of Quetta, Pakistan.

OBJECTIVE: To highlight the prevalence and epidemiology of New Delhi metallo-ß-lactamase-1. producers in pus samples. METHODS: The cross-sectional study was conducted from April to August 2018 at the Biotechnology Laboratory, Balochistan University of Information Technology Engineering and Management Sciences, Quetta, Hi-tech Laboratory, Centre for Advance Studies in Vaccinology and Biotechnology, University of Balochistan, Quetta and Microbiology Laboratory, Bolan Medical Complex Hospital, Quetta, Pakistan. Biochemical and molecular approaches were used for the identification of the isolates and Modified Hodge Test for the phenotypic detection of class-A carbapenemase activity. Minimum Inhibitory Concentration was performed using E-test and broth microdilution method. Molecular basis of carbapenemase activity was ascertained by the recognition of blaNDM-1 gene in the isolates. RESULTS: Of the 300 pus samples taken from surgical/burn units, 6(2%) blaNDM-1 harbouring isolates were found; 3(50%) each being Klebsiella pneumoniae and Pseudomonas aeruginosa. Klebsiella. pneumoniae isolates were extensively drug-resistant. The Pseudomonas aeruginosa isolates displayed resistance against 21 antibiotics of tetracyclines, quinolones, b-lactams, aminoglycosides, monobactams, sulphonamides, macrolides, cephalosporins, phosphonic acid and polypeptide groups, suggesting pan-drug resistance. CONCLUSIONS: The resistance pattern of the bacterial isolates poses a significant clinical threat in the region.

Genetic diversity and molecular analysis of metallo beta lactamases among imipenem resistant clinical isolates of Pseudomonas aeruginosa from Peshawar, Pakistan.

OBJECTIVES: Pseudomonas aeruginosa is an opportunistic pathogen with remarkable adaptation ability to thrive in diverse environmental conditions. This study aimed at phenotypic and molecular analysis of metallo beta lactamases (blaIMP, blaVIM, blaNDM-1 and blaSPM-1) and genetic diversity analysis among imipenem resistant clinical isolates of Pseudomonas aeruginosa. METHODS: This study was conducted from May 2017 to June 2018. The study included 187 Pseudomonas aeruginosa isolates collected from different clinical specimens from Peshawar, Pakistan. The isolates were analyzed for resistance to imipenem. Combined disc test (CDT) was then performed for phenotypic detection of metallo beta lactamases among imipenem resistant isolates of Pseudomonas aeruginosa. Molecular detection of metallo beta lactamases genes i.e. blaIMP, blaVIM, blaNDM-1 and blaSPM-1 was analyzed through polymerase chain reaction. Genetic diversity was determined through RAPD-PCR. RESULTS: MBL production was observed in 76% (n=19) isolates. The occurrence of MBL genes blaIMP, blaNDM-1 and blaVIM was 68% (n=17), 48% (n=12), and 4% (n=1) respectively. The blaSPM-1 gene was not detected. High genetic diversity was observed in current study. Out of 182 isolates 171 isolates showed different RAPD profiles (93.95% polymorphism); 160 were unique RAPD strains and based on similarity coefficient ≥ 80%, 22 isolates were clustered into 11 distinct clones. CONCLUSION: A high prevalence of blaIMP and blaNDM-1 among imipenem resistant isolates of Pseudomonas aeruginosa is alarming that calls for proper control and prevention strategies. RAPD technique was found to be a good genotyping technique when limited resources are available.

Co-occurrence of Plasmid-Mediated Tigecycline and Carbapenem Resistance in Acinetobacter spp. from Waterfowls and Their Neighboring Environment.

Tigecycline serves as one of the antibiotics of last resort to treat multidrug-resistant (including carbapenem-resistant) pathogens. However, the recently emerged plasmid-mediated tigecycline resistance mechanism, Tet(X), challenges the clinical efficacy of this class of antibiotics. In this study, we detected 180 tet(X)-harboring Acinetobacter isolates (8.9%, n = 180) from 2,018 samples collected from avian farms and adjacent environments in China. Eighteen tet(X)-harboring isolates (10.0%) were found to cocarry the carbapenemase gene blaNDM-1, mostly from waterfowl samples (94.4%, 17/18). Interestingly, among six Acinetobacter strains, tet(X) and blaNDM-1 were found to colocalize on the same plasmids. Moreover, whole-genome sequencing (WGS) revealed a novel orthologue of tet(X) in the six isolates coharboring tet(X) and blaNDM-1 Inverse PCR suggested that the two tet(X) genes form a single transposable unit and may be cotransferred. Sequence comparison between six tet(X)- and blaNDM-1-coharboring plasmids showed that they shared a highly homologous plasmid backbone even though they were isolated from different Acinetobacter species (three from Acinetobacter indicus, two from Acinetobacter schindleri, and one from Acinetobacter lwoffii) from various sources and from different geological regions, suggesting the horizontal genetic transfer of a common tet(X)- and blaNDM-1-coharboring plasmid among Acinetobacter species in China. Emergence and spread of such plasmids and strains are of great clinical concern, and measures must be implemented to avoid their dissemination.

Culture and PCR based detection of bacteria causing urinary tract infection in urine specimen.

OBJECTIVES: Urinary tract infections are the second most common bacterial infections occurring at all ages and both sexes. The increasing rate of antibiotic resistance is a global concern. The use of routinely used antibiotics is resulting in treatment failure. The objective of this study was to diagnose the urinary tract infections by routine culture sensitivity test and by molecular methods. METHODS: This study was conducted in Microbiology laboratory, Bolan Medical Complex Hospital, Quetta, from July 1st to 31st March 2019. Isolates were identified biochemically by API20E & API20NE. Antibiogram was performed using disc diffusion Kirby Bauer technique. The 16S rDNA gene approach was used for molecular identification of bacterial isolates. The presence of the bla NDM-1 gene was identified by polymerase chain reaction (PCR). RESULTS: We isolated 146 bacterial isolates namely Escherichia coli (n=99) 67.80%, Klebsiella pneumoniae (n=33) 22.60%, Pseudomonas aeruginosa (n=11) 7.53% and Proteus mirabilis (n=3) 2.05% from 2032 urine samples. The resistance pattern was dominated by Multi Drug Resistance (MDR). Remarkably, four isolates of Escherichia coli (n=3) and Klebsiella pneumoniae (n=1) were displaying resistance against a range of antibiotics used in the study, including carbapenems but sensitive to tigecycline and polymyxins only, suggesting extensive drug resistance having bla NDM-1 gene. CONCLUSION: This is the first report on direct molecular detection of bacterial pathogens from urinary tract infected patients in Balochistan. The presence of bla NDM-1 in different bacterial species and their extensive drug resistance pattern poses a significant clinical threat. Molecular detection of bacteria and resistant gene may reduce the diagnostic time of patients.

Heavy Metal Resistance Genes Are Associated with blaNDM-1- and blaCTX-M-15-Carrying Enterobacteriaceae.

The occurrence of heavy metal resistance genes in multiresistant Enterobacteriaceae possessing blaNDM-1 or blaCTX-M-15 genes was examined by PCR and pulsed-field gel electrophoresis with S1 nuclease. Compared with clinical susceptible isolates (10.0% to 30.0%), the pcoA, merA, silC, and arsA genes occurred with higher frequencies in blaNDM-1-positive (48.8% to 71.8%) and blaCTX-M-15-positive (19.4% to 52.8%) isolates, and they were mostly located on plasmids. Given the high association of metal resistance genes with multidrug-resistant Enterobacteriaceae, increased vigilance needs to be taken with the use of heavy metals in hospitals and the environment.

Molecular detection of blaNDM-1 (New Delhi metallobetalactamase-1) in nosocomial Enterobacteriaceae isolates by nested, multiplex polymerase chain reaction.

BACKGROUND: Carbapenems are considered "drugs of last resort" in many life-threatening infections. Advent of carbapenemases like KPC, OXA-48, VIM, IMP, and NDM have greatly affected the efficacy of these drugs, posing serious threat to global health and infection control. NDM bears special significance to the India subcontinent, labeled as place of origin and reservoir. NDM tends to escape detection by routine phenotypic methods, requiring molecular confirmation. This study utilizes nested, multiplex polymerase chain reaction (PCR) for reliable detection of blaNDM-1 in nosocomial Enterobacteriaceae isolates. METHODS: This study was conducted to detect prevalence of blaNDM-1, blaIMP, blaVIMand blaKPC genes by multiplex PCR among multidrug/carbapenem-resistant nosocomial Enterobacteriaceae isolates. From March 2013 to April 2014, 100 consecutive non-repeat isolates of Enterobacteriaceae from various inpatient clinical samples were analyzed. Imipenem-resistant isolates identified by Kirby Bauer disk diffusion method with Clinical and Laboratory Standards Institute guidelines were further subjected to nested, multiplex PCR to simultaneously detect blaNDM-1, blaIMP, blaVIMand blaKPC genes. RESULTS: Out of 100 isolates, 17 (17%) were found to be imipenem-resistant. blaNDM-1 was detected in all 17 isolates by nested, multiplex PCR. blaVIM was co-carried in 4 isolates while one isolate co-harbored blaIMP with blaNDM-1. Imipenem resistance and NDM-1 carriage was predominant amongst Klebsiella isolates. Maximum NDM-1 producers were isolated from the intensive care unit (70.6%). CONCLUSION: NDM-1 prevalence in nosocomial Enterobacteriaceae isolates in our hospital was found to be 17%. A nested, multiplex PCR was used for rapid detection of various carbapenemase genes with high sensitivity and specificity which is essential not only for favorable patient outcome but also for timely implementation of appropriate infection control practices to prevent further spread of such organisms.

IncFII Conjugative Plasmid-Mediated Transmission of blaNDM-1 Elements among Animal-Borne Escherichia coli Strains.

This study aims to investigate the prevalence and transmission dynamics of the blaNDM-1 gene in animal Escherichia coli strains. Two IncFII blaNDM-1-encoding plasmids with only minor structural variation in the MDR region, pHNEC46-NDM and pHNEC55-NDM, were found to be responsible for the transmission of blaNDM-1 in these strains. The blaNDM-1 gene can be incorporated into plasmids and stably inherited in animal-borne E. coli strains that can be maintained in animal gut microflora even without carbapenem selection pressure.

Isolation of New Delhi metallo-ß-lactamase 1-producing Vibrio cholerae non-O1, non-O139 strain carrying ctxA, st and hly genes in southern Vietnam.

Vibrio cholerae non-O1, non-O139 (VC_NAG) organisms are universally present in the aquatic environment and regarded as non-pathogenic bacteria. However, considering that they do occasionally induce gastroenteritis, a study of their virulence and antibiotic resistance genes is important. The presence of enteropathogenic genes, including ctxA, VC_NAG-specific heat-stable toxin gene (st), hemolysin (hly), and zona occludens toxin (zot) was determined by PCR in 100 VC_NAG strains isolated in southern Vietnam in 2010-2013 from 94 environmental and six human origins. These 100 VC_NAG strains were also tested phenotypically and genotypically for the presence of the New Delhi metallo-ß-lactamase (NDM-1). Of the 100 VC_NAG strains tested, six were positive for ctxA; five from the environment and one of human origin. The st gene was detected in 17 isolates, 15 and two of which were of environmental and human origins, respectively. Gene hly was detected in 19 VC_NAG strains examined, two of which were isolated from humans and 17 from environments. The zot gene was not detected in any of the strains tested. Three VC_NAG strains of environmental origin were confirmed to produce NDM-1 and the blaNDM-1 gene was detected in those strains by PCR. Of note, one of the three NDM-1-producing VC_NAG strains was confirmed to carry ctxA, st and hly genes concurrently. This is the first report of isolation of NDM-1-producing VC_NAG strains in Vietnam.

Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of bla NDM-1.

New Delhi metallo-ß-lactamase-1 gene (bla NDM-1 ) codes for New Delhi metallo-beta-lactamase-1 (NDM-1) enzyme that cleaves the amide bond of ß-lactam ring, and provides resistance against major classes of ß-lactam antibiotics. Dissemination of the plasmid borne bla NDM-1 through horizontal gene transfer is a potential threat to the society. In this study, a rapid non-culture method for detecting NDM-1 positive bacteria was developed by Loop Mediated Isothermal Amplification (LAMP) of bla NDM-1 . Sensitivity of this method was found to be one femtogram of plasmid DNA, which translates into 2.6-25.8 copies depending on the size of the plasmid DNA. This method was applied to detect NDM-1 positive bacteria in 81 water samples that were collected from environmental and drinking water sources. NDM-1 positive bacteria were detected in three drinking water samples by LAMP but not by PCR. These three samples were collected from the water sources that were treated with chlorine for decontamination before public distribution. NDM-1 positive bacteria were not detected in lake water samples or in the samples that were collected from the water sources that were purified by reverse osmosis before public distribution. Detection of NDM-1 positive bacteria using LAMP was found to be safe, sensitive and rapid for screening large number of samples from diverse sources. This method could be developed as on-field detection kit by using fluorescent dyes to visualize the amplified bla NDM-1 gene.