Carbapenem-resistant Enterobacteriaceae (CRE) isolates have been found to display high resistance to broad-spectrum antimicrobials than non-CRE, implying carbapenem resistance might be linked with resistance to several other antibiotics as well. Since carbapenem are considered to be the last resort drugs of choice, the highly resistant CRE carrying the New Delhi metallo-β-lactamase-1 gene (NDM-1) can limit the therapeutic options12. Excessive antibiotic usage in health care, agriculture and veterinary care, has led to a selection pressure favouring the survival and spread of such resistant organisms, resulting in increased hospital stay, morbidity and mortality34. NDM-1 producers belonging to Enterobacteriaceae are mainly associated with urinary tract infections (UTI), peritonitis, septicaemia, pulmonary infections, tissue and other device–associated infections5. There are reports of the presence of NDM-1 in Cedecea lapagei isolated from the neonatal intensive care unit in Aligarh67. Studies have revealed widespread dissemination of blaNDM variants through horizontal gene transfer among the Gram-negative bacilli (GNB) in developing countries co-existing with other resistance markers8. As observed for other gut-colonizing multidrug-resistant (MDR) bacteria, which precede infections by NDM producers, their transmission mainly takes place via the oro-faecal route9. The most abundant carbapenemase producer globally is the Klebsiella pneumoniae carbapenemase (KPC) (blaKPC gene), followed by the Verona integron-encoded metallo-ß-lactamase (VIM), NDM, Imipenemase (IMP) metallo-β-lactamase, and oxacillinase-48-type (OXA-48). These are all harboured by GNBs, viz. K. pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumanii and Enterobacter cloacae1011. Aggressive acquirement of antimicrobial resistance (AMR) and harbouring of mobile genetic elements by members of Enterobacteriaceae and non-Enterobacteriaceae have greatly affected the community1213.
AMR menace prevails worldwide, especially among the carbapenem group of drugs. The Ministry of Health and Family Welfare, Government of India has been actively addressing AMR issues by launching several programs spanning the length and breadth of the country towards its containment. India is a member of the 10-Global Health Security Agenda (GHSA) steering group and the largest of 17 GHSA phase 1 countries supported by several international organizations to fight against AMR14. The Indian Council of Medical Research (ICMR) and National Centre for Disease Control (NCDC) have been working together on the five-year plans, implementing AMR surveillance, health-care associated infection control and reinforcing various laboratories. With the onset of the COVID-19 pandemic, treating potentially fatal secondary bacterial infections in COVID-19 patients became imperative. In such a situation the increased levels of antimicrobials released in wastewater from hospitals may have affected levels of antimicrobials in the environment at large. Thus, in such challenging times, antibiotic stewardship plays a crucial role15.
The background of blaNDM-1 transmission
NDM-1-producing bacteria was first detected in India from a patient with UTI caused by carbapenem-resistant K. pneumonia16. What was alarming was that, the latter was found to be resistant to most of the antimicrobials16. Subsequently, reports of NDM-1 spread rapidly westward17.
It was found that most of the isolates carrying the blaNDM-1 gene were on plasmids along with several other antibiotic resistance determinants, capable of transferring such massive resistance to the associated bacteria18. These code for resistance to all aminoglycosides, macrolides and sulphamethoxazole, thus converting resident bacterial isolates to multidrug-resistant ones18. Subsequently, blaNDM-1 and blaVIM-2 genes were found together in a strain of P. aeruginosa in West Bengal, displaying co-existence of different carbapenem resistance gene19. However, others have reported different reasons behind the background of NDM-1 dissemination, stating plasmids of different sizes with genetic signatures on either side of blaNDM-1 gene being responsible for its mobility and pathogenesis. Similar gene cassette carrying resistant traits were also found in a tertiary care hospital among northern India among other Gram-negative bacteria from the same patient2021. These genes colonize together, thus spreading extensive resistance which is alarming as these bugs are usually related to causing life-threatening diseases222324.
Acquiring the NDM-1 gene and its proliferation: A multifaceted problem
Although studies related to NDM-1 have mostly been associated to the Indian subcontinent1, the Balkan countries are considered as a secondary reservoir where NDM-producing bacteria have been isolated from patients following return after seeking medical benefits from abroad9. Karthikeyan et al25 have reported detection of NDM-positive bacteria from the Middle East and North or Central Africa including Afghanisthan, Algeria, Cameroon, Egypt, Iraq, Israel, Kuwait, Lebanon, Morocco, the Sultanate of Oman and the United Arab Emirates25. The National Reference Laboratory of the Health Protection Agency, United Kingdom (UK) had investigated the rise in the unusual carbapenem-resistant isolates from UK patients26. These isolates of enterobacteriaceae showed NDM-1, but were negative for other known carbapenemase genes. All these cases had a history of travel to India or Pakistan within a brief span in common26. This calls for a routine detection and accurate reporting of NDM-1 in diagnostic laboratories, especially those from India. With the rampant dissemination of the NDM gene variants (NDM-2 to NDM-8) with varying capacity to cleave carbapenem drugs, molecular epidemiological investigations of these isolates especially from faecal specimen. The hospital sewage is a potential source of blaNDM variants outbreaks which is of major concern27. Genotyping techniques are of paramount importance for a better understanding of their rampant spread and infection control28.
Kumarswamy et al29, have reported the prevalence of NDM-1 in enterobacteriaceae isolates from India, Pakistan and in the United Kingdom. Their study selectively identified NDM-1 isolates from Guwahati, Mumbai, Varanasi, Bengaluru, Pune, Kolkata, Hyderabad, Port Blair, and New Delhi in India, several cities in Pakistan and Dhaka in Bangladesh pointing to its rampant dissemination29. Similar cases of those having travelled to Asian countries and may have acquired infection while undergoing treatment are also available2. Distribution of AMR and colistin resistant pathogenic strains have also been isolated from Tamil Nadu, West Bengal, Punjab303132. In a study conducted in south India, MDR Enterobacteriaceae colonizing the gut of adult rural population was observed indicating faecal carriage of AMR strains with a threat of rapid spread in the community33.
Several human (improper antibiotic stewardship and infection control, human migration) and bacterial factors (nosocomial spread of blaNDM gene) have consequently led to the rapid spread of this gene3435(Figure).
Drinking water quality, defective sewage system, vector–mediated viz. house flies, uncontrolled circulation of antibiotics are other major factors promoting the spread of these resistance microbes in a healthy population. Furthermore, there is a tendency to switch to alternative medication particularly the Indian traditional medicinal system such as the Ayurveda, Siddha and Unani having yielded successful results worldwide especially in remote and rural areas36.
Routine laboratory diagnosis of NDM
Currently, the NDM-1 class of carbapenemase is of clinical concern as it shares 32.4 per cent amino acid match with VIM-1/VIM-2, on the other hand minute identity match with other metallo-β-lactamases (MBLs). NDM-1 binds strongly to most cephalosporins compared to VIM-2. NDM-1 effectively hydrolyzes broad range of β-lactams including penicillin, cephalosporin and carbapenem and just sparing monobactams like other MBL9.
Screening of carbapenem-resistant Gram-negative bacilli (GNBs) carrying NDM-1 gene
Currently, detection of NDM producers is based on preliminary screening using the antibiotic susceptibility test (AST)9. Susceptibility to NDM-1 is usually by disc diffusion method in accordance with Clinical and Laboratory Standard Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST) or the British Society for Antimicrobial Chemotherapy (BSAC) guidelines as done in practice- and/or determination of minimum inhibitory concentration (MIC) by manual or automated methods373839. It is noteworthy that there might be certain differences in MIC values for isolates depending on the reference used to interpret antibiograms. Susceptibility to carbapenems are observed for some NDM producers and additional tests for carbapenemase detection are needed to identify them accurately9. Different chromogenic plate methods are used for performing AST and identification of carbapenemase producers which are detailed as:
Chromogenic plate method: Carbapenemase producers are presumptively identified using chromogenic plates useful for direct screening of high-risk asymptomatic carriers viz. stool samples. This cost-effective, rapid and simple detection of carbapenemase producers is vital for effective infection management control interventions and also in preventing outbreaks of nosocomial infections by these organisms. Different chromogenic media for detection of NDM-1 producers are available viz. ChromID ESBL and CHROMagar KPC media alongwith acceptable specificity and sensitivities ranging from 53-100 per cent40. However, its disadvantage being chromogenic media is not reliable detection for all types of carbapenemase producing isolates and requires confirmation tests41. Screening stool specimen of patients hospitalized for carbapenemase producers is done using screening culture media, viz. CHROMagar media and ChromID ESBL which is a bit time-consuming before the actual status of the patient is known4243444546. This is of primordial significance as these NDM-1 carrying enterobacteriaceae isolates mainly inhabit the gut region.
Several other routinely and commercially available automated systems which used to identify and detect NDM-1 carbapenemase-producing isolates include the Vitek 2 automated system (France). Studies evaluating the accuracy of these platforms in the detection of carbapenemase activity have shown appropriate sensitivities but inadequate specificities, which leads to the requirement of confirmatory tests4045.
Confirmatory detection of carbapenemase activity among the screened Gram-negative bacilli (GNBs)
Different techniques are available for identification of carbapenemase producers based on several phenotypic and genotypic methods41. The phenotypic methods are based on inhibitors or breaking down of carbapenem drugs or else on spectrophotometric approach, whereas genotypic detection is much more precise based on polymerase chain reaction (PCR)/real-time PCR (RT-PCR)/DNA sequencing/micro array based101842. However, phenotypic methods are much simpler, less time consuming and cheap. Phenotypic assays are designed based on the following principles:
Inhibition of metallo-carbapenemase: These phenotypic tests rely on the combined effect of discs viz. meropenem alongwith ethylenediamine-tetraacetic acid (EDTA), phenylboronic acid (PBA)/ both EDTA and PBA for differentiation of class A and B enzymes or dipicolinic acid (DPA) to specifically inhibit metallo-carbapenemase activity4647. Considering that PBA inhibits other β-lactamases as well, AmpC β-lactamases, cloxacillin (an AmpC inhibitor without activity against KPC and other class A carbapenemases) is also used routinely to differentiate between AmpC and KPC production4849 as it was found that strains harbouring both KPC and MBL could be detected using both EDTA and PBA in a single disk. Thus, ruling out of other common mechanisms for carbapenem resistance can indirectly help the laboratory to diagnose the presence of NDM-1 by virtue of exclusion50.
Combined disc test (CDT): Studies evaluating CDT in comparison to other molecular tests have shown high sensitivities and specificities (90-100%) with reference to blaNDM-1, blaKPC, blaVIM, and blaIMP in carbapenemase-producing isolates from Hyderabad4151.
Double disk synergy test (DDSTs): This method uses Mg-EDTA to detect MBL-producing strains, including NDM-1 producers4152. This method has demonstrated good sensitivities (100%) and specificity of 91.0 per cent. However, this method also reports certain shortfalls, such as test outcome interpretation is subjective requiring a qualified personnel5354.
Gradient diffusion strips: For detecting MBL and KPC various gradient diffusion strips formats have been designed, which separately detect MBL and KPC. The E-test (epsilometer test) MBL using imipenem and imipenem-EDTA was simple for their detection, except in two cases (E. cloacae D and K. pneumoniae), in which interpretation of the results was not possible because the imipenem MICs were too low40555657. The sensitivity of E-test MBL for blaNDM-1 positive isolates was 66.7 per cent and specificity was 100 per cent. However, gradient diffusion strips are quite expensive58.
Detection based on carbapenem hydrolysis
The cloverleaf method [modified Hodge test, (MHT)]: According to Aguirre-Quinonero and coworkers, MHT is still being used for carbapenemase detection following CLSI guidelines4159. However, as a result of the poor performance of the Hodge test displaying ambiguous results, modifications of the assay such as the addition of ZnSO445 or cloxacillin58 to the agar plate was done. Pasteran and co-workers have reported inclusion of Triton X-100 (a non-ionic surfactant) to improve NDM-1 carbapenemases detection, as the compound solubilizes membrane lipoproteins and consequently membrane-bound carbapenemases60. This latter version was known as the Triton Hodge Test (THT)6162. The MHT did not show good results in detecting NDM-producing isolates (merely 20 and 32.5% sensitivity for meropenem and ertapenem, respectively). On the other hand, sensitivities of the THT were 100 per cent with ertapenem and 92.5 per cent with meropenem for the latter MBL producing strains60.
Assays displaying colour change due H+ ion concentration in the media has been exploited that is, pH based detection method or colorimetric assays including pH indicators viz. phenol red for Carba NP41 or bromothymol blue for Blue-CARBA.
Carba NP: This assay was initiated by Nordmann et al63 and named thereafter as Carba NP. This assay has reported specificity of 100 per cent and sensitivity between 90 and 100 per cent in the detection of carbapenemases5860. This assay was thus used for identifying blaNDM-4, blaNDM- 5, blaNDM-7 from rectal and stool swabs from a neonatal intensive care unit in Aligarh, India64.
Blue-CARBA: The Blue-CARBA which is a modification of the Carba NP yields similar results as the Carba NP9606163. The Blue-CARBA is an inexpensive test with 93.3 per cent sensitivity and 100 per cent specific detecting any type of carbapenemase of enterobacteriaceae including NDM-1 producers4965.
Carbapenem inhibition method (CIM): Initiated by Van der Zwaluw et al66, the CIM method display high sensitivity (98-100%) and specificity of 100 per cent. This method has proved to be inexpensive and easy to interpret. Moreover, this assay has been modified and further recommended to be used for carbapenemase detection (mCIM) in enterobacteriaceae and P. aeruginosa in the 28th edition of CLSI67. If mCIM comes positive, then only eCIM (carbapenemase activity is inhibited in the presence of EDTA) is done to differentiate MBL67.
Spectrometry techniques: Mass spectrometry (MS) is more often being used for the detection of carbapenemase-producing isolates.
MS: Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS is reliable for isolate identification of bacteria and fungi and is routinely in use in laboratories. This method is effective for detection of carbapenemase-producing Gram-negative bacteria especially NDM-1. Studies have found sensitivities ranging between 77 to 100 per cent and specificities from 94 to 100 per cent6668. However, MALDI-TOF is time-consuming and costly and its operation requires trained microbiologists. This technique was based on detection of a carbapenem spectrum and of its main derivatives resulting from carbapenem hydrolysis9.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS): The LC-MS/MS methods are analytical, reliable and sensitive. The chromatographic retention time, precursor ion mass, and product combine to impart a good analytical specificity. LC-MS/MS is the gold standard for small-molecule detection and quantitation6970.
Immunochromatographic assay: Detection of NDM-1 by immunochromatographic assay, using rat monoclonal antibodies are also being studied63.
Molecular CP-carbapenem resistant Enterobacteriaceae (CRE) detection methods: Varying susceptibility patterns of isolates towards various carbapenem drug depends on the efficiency with which the drugs are hydrolyzed4243. This variability in hydrolysis spectrum hampers identification. Phenotypic tests are good for screening the carbapenemases, but most of them are incapable of detecting the specific carbapenemase responsible for drug resistance. The World Health Organization (WHO) in an effort towards AMR surveillance and prevention program, through the Global Antimicrobial Resistance Surveillance System (GLASS) has emphasized on the reliability of phenotypic assays whereas, molecular testing could provide additional data regarding resistance profile and mechanisms. Molecular diagnostic assays require costly set-up, rapid, with high specificity and sensitivity for identifying the harboured resistance genes viz. blaNDM-1,blaVIM,blaIMP. Thus, depending on the laboratory capacity and prevalent AMR data, WHO has stratified molecular diagnostic testing to be performed depending on its complexity and cost effectiveness. Thus molecular diagnostics are available for laboratories with low capacity, especially belonging to the low- and middle-income countries the molecular methods recommended are either fully integrated and automated cartridge-operated PCR or the loop-mediated isothermal amplification (LAMP) devices and lateral flow assays requiring visual inspection for interpreting results. Whereas, high-capacity laboratories with good experience in molecular diagnostics might prefer complex diagnostic assays, such as microarrays technology and the whole genome sequencing (WGS) to analyses/interpret the raw data. However, WHO does not endorse nor validate any particular molecular method but relies on the phenotypic detection of resistance in isolates using antimicrobial agents by measuring the MIC which is the gold standard71.
PCR allows for rapid identification of specific carbapenemase genes using primers and probes to conserve regions in the gene target for real-time assays, can be carried out by PCR. PCR being specific to a given gene, and can be further tailored to detect specific subgroups of a gene family72. As blaNDM-1 encoded plasmids are rampantly spreading worldwide especially among members of Enterobacteriaceae faecal screening should be prioritized using PCR-based molecular screening techniques viz. multiplex PCR nucleic acid amplification test (NAAT) for efficient detection of faecal pathogens73. A multilocus sequence typing (MLST) can be performed to identify house-keeping genes viz. on plasmids in pathogens resistant to carbapenem drugs and displaying widespread resistance in the community74. Plasmid sizing is done using S1-nuclease pulsed-field gel electrophoresis (PFGE) and Southern blot is performed for identification of NDM-1 plasmid4473. The specificity and sensitivity using qPCR of blaNDM-1 has been reported as 98.4 and 100 per cent, respectively3542. The primary limitation of PCR is that the only known genes can be targeted while, those encoding novel carbapenemase will be missed with molecular approaches75.
On the other hand, the LAMP method is promising tool. It is simple, rapid, cost-effective, requiring no sophisticated instrument. It is a single tube amplification reaction requiring Bst DNA polymerase for strand displacement and DNA synthesis under isothermal conditions based on auto-cycling. The LAMP technology has been widely used in clinical diagnosis; field detection of MBL harbouring bacterial strains, qualitative and quantitative detection of epidemic bacteria, viruses, and parasites; as well as in fetal sex identification76. It is suggested that time demands requiring further research in the development of the LAMP assay as a molecular tools for the detection and confirmation of NDM-1 producers along with its variants and other MBLs. Thus, a simple and affordable detection assay at a genetic level would certainly help combat MBLs spread, with screening remote and rural areas.
On the other hand, various commercial PCR-based customised assays are also available viz. the Xpert Carba-R test and the hyperplex SuperBug ID475877. These assays are specific and sensitive but costly. The controls used for culture-based tests, phenotypic assays and PCR for NDM-1 detection is usually K. pneumoniae ATCC BAA-1705 as positive control and K. pneumoniae ATCC BAA-1706 as negative control, however, other controls can be selected from in-house confirmed positive /negative strains confirmed by sequencing78.
Microarray technology is another promising tool and can be paired with PCR-based target amplification. It utilizes probes that hybridize to DNA targets, including resistance genes. Microarrays can be used to target and extract DNA from bacterial isolates or patient specimens and can handle hundreds of DNA targets, thus multiplexing with numerous carbapenemase genes, along with distinguishing between closely related variants798081.
The WGS is versatile and comprehensive with a capacity to recognize all resistance mechanisms across the bacterial genome. Apart from targeting various carbapenemases, it can also identify other contributors to resistance, such as porin loss, efflux genes, etc. The data generated in WGS can also help to elucidate the source in outbreak investigations by extracting information regarding the type of plasmid carrying resistance genes, evolutionary lineage of the isolate and its relatedness with other isolates4356. Currently, WGS is expensive and requires qualified personnel for necessary data extraction and interpretation82.
Other promising approaches worth exploring:
Using macrophage: The versatility of macrophages in response to environmental stimuli and their engagement largely in pathogenesis of numerous human diseases makes them a desired target cells to be considered suitable for future therapy. This combined with nano-biotechnology can offer advantage in improving therapy outcome83.
Lytic phage proteins: Using lytic phages or else lytic phage proteins, a concept which originated in the beginning of the 20th century84 for combating AMR holds promise in curbing its spread.
Reactive oxygen therapy (ROS): Targeted use of ROS to the site of infection in various forms has also been suggested as a potential alternative as ROS has antimicrobial activity towards pathogens including biofilm breakdown85.
The extent of threat owing to the spread of pathogens producing NDM-1 belonging to Enterobacteriaceae worldwide is alarming. This is of serious concern in the Indian sub-continent, parts of Asia, Europe and America. The Indian government has taken cognizance of the situation thus creating public awareness against unhygienic practices by initiating cleanliness drive throughout the country.
The lack of a routine standardized phenotypic test for NDM variant detection, expensive molecular tests may have led to under-reporting. The efforts made by various national institutions viz. NCDC, ICMR and GHSA of India and other nations worldwide; along with other international organizations viz. the WHO can steer the world to a safer and securer environment.
Financial support & sponsorship: None.
Conflict of Interest: None.
1. Durante-Mangoni E, Andini R, Zampino R Management of carbapenem-resistant Enterobacteriaceae
infections Clin Microbiol Infect 2019 25 943 50
2. Johnson AP, Woodford N Global spread of antibiotic resistance:The example of New Delhi metallo-β-lactamase (NDM)-mediated carbapenem resistance J Med Microbiol 2013 62 499 513
3. Taneja N, Sharma M Antimicrobial resistance in the environment:The Indian scenario Indian J Med Res 2019 149 119 28
4. Das A, Guha C, Biswas U, Jana PS, Chatterjee A, Samanta I Detection of emerging antibiotic resistance in bacteria isolated from subclinical mastitis in cattle in West Bengal Vet World 2017 10 517 20
5. Peri AM, Doi Y, Potoski BA, Harris PNA, Paterson DL, Righi E Antimicrobial treatment challenges in the era of carbapenem resistance Diagn Microbiol Infect Dis 2019 94 413 25
6. Ahmad N, Ali SM, Khan AU First reported New Delhi metallo-β-lactamase-1-producing Cedecea lapagei
Int J Antimicrob Agents 2017 49 118 9
7. Ahmad N, Khalid S, Ali SM, Khan AU Occurrence of blaNDM
variants among Enterobacteriaceae
from a neonatal Intensive Care Unit in a northern India hospital Front Microbiol 2018 9 407
8. Khalid S, Ahmad N, Ali SM, Khan AU Outbreak of efficiently transferred carbapenem-resistant blaNDM
-producing gram-negative bacilli isolated from neonatal Intensive Care Unit of an Indian hospital Microb Drug Resist 2020 26 284 9
9. Dortet L, Poirel L, Nordmann P Worldwide dissemination of the NDM-type carbapenemases in Gram-negative bacteria Biomed Res Int 2014 2014 249856
10. Benmahmod AB, Said HS, Ibrahim RH Prevalence and mechanisms of carbapenem resistance among Acinetobacter baumannii
clinical isolates in Egypt Microb Drug Resist 2019 25 480 8
11. Richter SS, Marchaim D Screening for carbapenem-resistant Enterobacteriaceae
:Who, when, and how? Virulence 2017 8 417 26
12. Dunn SJ, Connor C, McNally A The evolution and transmission of multi-drug resistant Escherichia coli
and Klebsiella pneumoniae
:The complexity of clones and plasmids Curr Opin Microbiol 2019 51 51 6
13. Breijyeh Z, Jubeh B, Karaman R Resistance of gram-negative bacteria to current antibacterial agents and approaches to resolve it Molecules 2020 25 1340
15. Bengoechea JA, Bamford CG SARS-CoV-2, bacterial co-infections, and AMR:The deadly trio in COVID-19? EMBO Mol Med 2020 12 e12560
16. Walia K, Madhumathi J, Veeraraghavan B, Chakrabarti A, Kapil A, Ray P, et al. Establishing antimicrobial resistance surveillance &research network in India: Journey so far Indian J Med Res 2019 149 164 79
17. Molton JS, Tambyah PA, Ang BS, Ling ML, Fisher DA The global spread of healthcare-associated multidrug-resistant bacteria:A perspective from Asia Clin Infect Dis 2013 56 1310 8
18. Anjana SK, Jyothi EK, Ravikumar R Phenotypic identification &molecular detection of blaNDM-1 gene in multidrug resistant Gram-negative bacilli in a tertiary care centre Indian J Med Res 2014 139 625 31
19. Paul D, Dhar D, Maurya AP, Mishra S, Sharma GD, Chakravarty A, et al. Occurrence of co-existing bla VIM-2 and bla NDM-1 in clinical isolates of Pseudomonas aeruginosa
from India Ann Clin Microbiol Antimicrob 2016 15 31
20. Mishra S, Sen MR, Upadhyay S, Bhattacharjee A Genetic linkage of blaNDM among nosocomial isolates of Acinetobacter baumannii
from a tertiary referral hospital in northern India Int J Antimicrob Agents 2013 41 452 6
21. Mishra S, Upadhyay S, Sen MR, Maurya AP, Choudhury D, Bhattacharjee A Genetic acquisition of NDM gene offers sustainability among clinical isolates of Pseudomonas aeruginosa
in clinical settings PLoS One 2015 10 e0116611
22. Mitra S, Mukherjee S, Naha S, Chattopadhyay P, Dutta S, Basu S Evaluation of co-transfer of plasmid-mediated fluoroquinolone resistance genes and blaNDM
gene in Enterobacteriaceae
causing neonatal septicaemia Antimicrob Resist Infect Control 2019 8 46
23. Raczynska JE, Shabalin IG, Minor W, Wlodawer A, Jaskolski M A close look onto structural models and primary ligands of metallo-β-lactamases Drug Resist Updat 2018 40 1 12
24. Poirel L, Ros A, Carricajo A, Berthelot P, Pozzetto B, Bernabeu S, et al. Extremely drug-resistant Citrobacter freundii
isolate producing NDM-1 and other Carbapenemases identified in a patient returning from India Antimicrob Agents Chemother 2011 55 447 8
25. Karthikeyan K, Thirunarayan MA, Krishnan P Coexistence of bla OXA-23 with blaNDM - 1 and arm –A in clinical isolates of Acinetobacter baumanii
from India J Antimicrob Chemother 2010 65 2253 70
26. Moellering RC Jr NDM-1 –A cause for worldwide concern N Engl J Med 2010 363 2377 9
27. Parvez S, Khan AU Hospital sewage water:A reservoir for variants of New Delhi metallo-β-lactamase (NDM)- and extended-spectrum β-lactamase (ESBL)- producing Enterobacteriaceae
Int J Antimicrob Agents 2018 51 82 8
28. Ranjan A, Shaik S, Mondal A, Nandanwar N, Hussain A, Semmler T, et al. Molecular epidemiology and genome dynamics of New Delhi metallo-β-lactamase producing extra-intestinal pathogenic Escherichia coli
strains from India Antimicrob Agents Chemother 2016 60 6795 805
29. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK:A molecular, biological, and epidemiological study Lancet Infect Dis 2010 10 597 602
30. Manohar P, Shanthini T, Ayyanar R, Bozdogan B, Wilson A, Tamhankar AJ, et al. The distribution of carbapenem- and colistin-resistance in Gram-negative bacteria from the Tamil Nadu region in India J Med Microbiol 2017 66 874 83
31. Sodhi K, Mittal V, Arya M, Kumar M, Phillips A, Kajla B Pattern of colistin resistance in Klebsiella
isolates in an Intensive Care Unit of a tertiary care hospital in India J Infect Public Health 2020 13 1018 21
32. Bardhan T, Chakraborty M, Bhattacharjee B Prevalence of colistin-resistant, carbapenem-hydrolyzing proteobacteria in hospital water bodies and out-falls of West Bengal, India Int J Environ Res Public Health 2020 17 1007
33. Antony S, Ravichandran K, Kanungo R Multidrug-resistant Enterobacteriaceae
colonising the gut of adult rural population in South India Indian J Med Microbiol 2018 36 488 93
34. Wailan AM, Paterson DL The spread and acquisition of NDM-1:A multifactorial problem Expert Rev Anti Infect Ther 2014 12 91 115
35. Rogers BA, Aminzadeh Z, Hayashi Y, Paterson DL Country-to-country transfer of patients and the risk of multi-resistant bacterial infection Clin Infect Dis 2011 53 49 56
36. Sen S, Chakraborty R Revival, modernization and integration of Indian traditional herbal medicine in clinical practice:Importance, challenges and future J Tradit Complement Med 2017 7 234 44
37. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria:An international expert proposal for interim standard definitions for acquired resistance Clin Microbiol Infect 2012 18 268 81
38. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility
tests, 10th ed. CLSI document M2-A10 Wayne, PA CLSI 2009
39. European Committee on Antimicrobial Susceptibility
Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 1.1 2010 Available from: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_breakpoints_v1.1.pdf
accessed on June 8, 2018
40. Nordmann P, Poirel L, Carrër A, Toleman MA, Walsh TR How to detect NDM-1 producers J Clin Microbiol 2011 49 718 21
41. Aguirre-Quiñonero A, Martínez-Martínez L Non-molecular detection of carbapenemases in Enterobacteriaceae
clinical isolates J Infect Chemother 2017 23 1 11
42. Naas T, Ergani A, Carrër A, Nordmann P Real-time PCR for detection of NDM-1 carbapenemase genes from spiked stool samples Antimicrob Agents Chemother 2011 55 4038 43
43. Jain M, Sharma A, Sen MK, Rani V, Gaind R, Suri JC Phenotypic and molecular characterization of Acinetobacter baumannii
isolates causing lower respiratory infections among ICU patients Microb Pathog 2019 128 75 81
44. Mohan B, Prasad A, Kaur H, Hallur V, Gautam N, Taneja N Fecal carriage of carbapenem-resistant Enterobacteriaceae
and risk factor analysis in hospitalised patients:A single centre study from India Indian J Med Microbiol 2017 35 555 62
45. Fattouh R, Tijet N, McGeer A, Poutanen SM, Melano RG, Patel SN, et al. What it the appropriate meropenem MIC for screening of carbapenemase-producing Enterobacteriaceae
in low-prevalence settings? Antimicrob Agents Chemother 2016 60 1556 9
46. Tsakris A, Kristo I, Poulou A, Markou F, Ikonomidis A, Pournaras S First occurrence of KPC-2-possessing Klebsiellapneumoniae
in a Greek hospital and recommendation for detection with boronic acid disc tests J Antimicrob Chemother 2008 62 1257 60
47. Pasteran F, Mendez T, Guerriero L, Rapoport M, Corso A Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae
J Clin Microbiol 2009 47 1631 9
48. Giske CG, Gezelius L, Samuelsen O, Warner M, Sundsfjord A, Woodford N A sensitive and specific phenotypic assay for detection of metallo-ß-lactamases and KPC in Klebsiella pneumonia
with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin Clin Microbiol Infect 2011 17 552 6
49. Pournaras S, Poulou A, Tsakris A Inhibitor-based methods for the detection of KPC carbapenemase-producing Enterobacteriaceae
in clinical practice by using boronic acid compounds J Antimicrob Chemother 2010 65 1319 21
50. Miriagou V, Tzelepi E, Kotsakis SD, Daikos GL, Bou Casals J, Tzouvelekis LS Combined disc methods for the detection of KPC- and/or VIM-positive Klebsiella pneumoniae
:Improving reliability for the double carbapenemase producers Clin Microbiol Infect 2013 19 412 5
51. Solanki R, Vanjari L, Subramanian S, Aparna B, Nagapriyanka E, Lakshmi V Comparative evaluation of multiplex PCR and routine laboratory phenotypic methods for detection of carbapenemases among gram negative bacilli J Clin Diagn Res 2014 8 C23 6
52. Naim H, Rizvi M, Azam M, Gupta R, Taneja N, Shukla I, et al. Alarming emergence, molecular characterization, and outcome of blaNDM-1 in patients infected with multidrug-resistant Gram-negative bacilli in a tertiary care hospital J Lab Physicians 2017 9 170 6
53. Kali A, Srirangaraj S, Kumar S, Divya HA, Kalyani A, Umadevi S Detection of metallo-betalactamase producing Pseudomonas aeruginosa
in Intensive Care Units Australas Med J 2013 6 686 93
54. Fujisaki M, Sadamoto S, Hishinuma A Evaluation of the double-disk synergy test for New Delhi metallo-β-lactamase-1 and other metallo-β-lactamase producing gram-negative bacteria by using metal-ethylenediaminetetraacetic acid complexes Microbiol Immunol 2013 57 346 52
55. Shenoy AK, Jyothi EK, Ravikumar R Phenotypic identification &molecular detection of blaNDM-1 gene in multidrug resistant Gram-negative bacilli in a tertiary care centre Indian J Med Res 2014 139 625 31
56. Roodsari MR, Fallah F, Taherpour A, Vala MH, Hashemi A Carbapenem-resistant bacteria and laboratory detection methods Arch Pedia Infect Dis 2014 2 188 91
57. Hrabák J, ChudáčkováE, Papagiannitsis CC Detection of carbapenemases in Enterobacteriaceae
:A challenge for diagnostic microbiological laboratories Clin Microbiol Infect 2014 20 839 53
58. Baran I, Aksu N Phenotypic and genotypic characteristics of carbapenem-resistant Enterobacteriaceae
in a tertiary-level reference hospital in Turkey Ann Clin Microbiol Antimicrob 2016 15 20
59. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility
testing: 21st informational supplement. CLSI document M100-S21 Wayne, PA CLSI 2011
60. Pasteran F, Gonzalez LJ, Albornoz E, Bahr G, Vila AJ, Corso A Triton hodge test:Improved protocol for modified hodge test for enhanced detection of NDM and other carbapenemase producers J Clin Microbiol 2016 54 640 9
61. Takayama Y, Adachi Y, Nihonyanagi S, Okamoto R Modified Hodge test using Mueller-Hinton agar supplemented with cloxacillin improves screening for carbapenemase-producing clinical isolates of Enterobacteriaceae
J Med Microbiol 2015 64 774 7
62. Girlich D, Poirel L, Nordmann P Value of the modified Hodge test for detection of emerging carbapenemases in Enterobacteriaceae
J Clin Microbiol 2012 50 477 9
63. Nordmann P, Poirel L, Dortet L Rapid detection of carbapenemase-producing Enterobacteriaceae
Emerg Infect Dis 2012 18 1503 7
64. Ahmad N, Ali SM, Khan AU Detection of New Delhi metallo-β-lactamase variants NDM-4, NDM-5, and NDM-7 in Enterobacter aerogenes
isolated from a neonatal intensive care unit of a north India hospital:A first report Microb Drug Resist 2018 24 161 5
65. Tijet N, Boyd D, Patel SN, Mulvey MR, Melano RG Evaluation of the Carba NP test for rapid detection of carbapenemase-producing Enterobacteriaceae
and Pseudomonas aeruginosa
Antimicrob Agents Chemother 2013 57 4578 80
66. Zwaluw KV, Haan A, Pluister GN, Bootsma HJ, Neeling AJ, Schouls LM, et al. The Carbapenemase Inactivation Method (CIM), a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in Gram-negative rods PLoS One 2015 10 e0123690
67. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility
testing: 28th edition supplement. CLSI document M100 Wayne, PA CLSI 2018
68. Tijet N, Patel SN, Melano RG Detection of carbapenemase activity in Enterobacteriaceae
:Comparison of the carbapenem inactivation method versus the Carba NP test J Antimicrob Chemother 2016 71 274 6
69. Nordmann P, Cuzon G, Naas T The real threat of Klebsiella pneumonia
carbapenemase-producing bacteria Lancet Infect Dis 2009 9 228 36
70. Kulkarni MV, Zurita AN, Pyka JS, Murray TS, Hodsdon ME, Peaper DR Use of imipenem to detect KPC, NDM, OXA, IMP, and VIM carbapenemase activity from gram-negative rods in 75 minutes using liquid chromatography-tandem mass spectrometry J Clin Microbiol 2014 52 2500 5
71. World Health Organization. Molecular methods for antimicrobial resistance (AMR) diagnostics to enhance the global antimicrobial resistance surveillance system Geneva WHO 2019
72. Shanthi M, Sekar U, Kamalanathan A, Sekar B Detection of New Delhi metallo beta lactamase-1 (NDM-1) carbapenemase in Pseudomonas aeruginosa
in a single centre in southern India Indian J Med Res 2014 140 546 50
73. Miller MJ, Binnicker MJ, Campbell S, Carroll KC, Chapin KC, Gilligan PH, et al. A guide to utilization of the microbiology laboratory for diagnosis of infectious diseases:2018 update by the infectious diseases society of America and the American Society for Microbiology Clin Infectious Dis 2018 67 e1 94
74. Ahmad N, Ali SM, Khan AU Molecular characterization of novel sequence type of carbapenem-resistant New Delhi metallo-β-lactamase-1-producing Klebsiella pneumoniae
in the neonatal intensive care unit of an Indian hospital Int J Antimicrob Agents 2019 53 525 9
75. Vira HJ, Bhat VG Epidemiology of NDM-1 and its variants in multidrug resistant gram-negative bacilli isolated from infection in cancer patients Med Chem (Los Angeles) 2017 7 368 70
76. Liu W, Zou D, Li Y, Wang X, He X, Wei X, et al. Sensitive and rapid detection of the New Delhi metallo-beta lactamase gene by loop-mediated isothermal amplification J Clin Microbiol 2012 50 1580 5
77. Lai CC, Chen CC, Lu YC, Chen HJ, Su BA, Weng TC, et al. Simultaneous three Enterobacteriaceae
with different blaNDM-1-encoding plasmids in a patient transferred from mainland China to Taiwan Infect Drug Resist 2018 11 2555 60
78. Agarwal A, Srivastava J, Maheshwari U, Iftikhar M Molecular characterization and antimicrobial susceptibility
profile of New Delhi metallo-beta-lactamase-1-producing Escherichia coli
among hospitalized patients J Lab Physicians 2018 10 149 54
79. Papagiannitsis CC, Študentová V, Izdebski R, Oikonomou O, Pfeifer Y, Petinaki E, et al. Matrix-assisted laser desorption ionization-time of flight mass spectrometry meropenem hydrolysis assay with NH4HCO3, a reliable tool for direct detection of carbapenemase activity J Clin Microbiol 2015 53 1731 5
80. Huang TD, Berhin C, Bogaerts P, Glupczynski Y Comparative evaluation of two chromogenic tests for rapid detection of carbapenemase in Enterobacteriaceae
and in Pseudomonas aeruginosa
isolates J Clin Microbiol 2014 52 3060 3
81. Chandola P, Gupta RM, Lall M, Sen S, Shergill SPS, Dutta V Molecular detection of blaNDM-1
(New Delhi metallobetalactamase-1) in nosocomial Enterobacteriaceae
isolates by nested, multiplex polymerase chain reaction Med J Armed Forces India 2018 74 108 15
82. Anandan S, Damodaran S, Gopi R, Bakthavatchalam YD, Veeraraghavan B Rapid screening for carbapenem resistant organisms:Current esults and future approaches J Clin Diagn Res 2015 9 DM01 3
83. Ponzoni M, Pastorino F, Di Paolo D, Perri P, Brignole C Targeting macrophages as a potential therapeutic intervention:Impact on inflammatory diseases and cancer Int J Mol Sci 2018 19 1953
84. Waters EM, Neill DR, Kaman B, Sahota JS, Clokie MRJ, Winstanley C, et al. Phage therapy is highly effective against chronic lung infections with Pseudomonas aeruginosa
Thorax 2017 72 666 7
85. Dryden M Reactive oxygen species:A novel antimicrobial Int J Antimicrob Agents 2018 51 299 303