Investigation of the blaNDM-1 and armA Genes in Carbapenem-Resistant Klebsiella pneumoniae Isolated from the Clinical Samples in Kathmandu, Nepal.

Rapid detection of carbapenemase producing Enterobacteriaceae direct from blood using real-time PCR

Objectives The aims of this study were as follows: to determine the incidence of multidrug-resistant Enterobacteriaceae isolated from the urine of patients admitted in different departments at Menoufia University Hospitals; to detect the presence of extended spectrum β-lactamases (ESβLs), AmpC β-lactamases, and carbapenemases among the isolated pathogens using phenotypic methods; and to investigate the presence of bla KPC and bla NDM genes using real-time PCR. Background Urinary tract infections are the most common type of nosocomial infections. ESβLs and AmpC β-lactamases are clinically significant because they may confer resistance to multiple classes of antibiotics. Carbapenemases are diverse enzymes that vary in the ability to hydrolyze carbapenems and other β-lactams. Materials and methods This study included 260 urine samples collected from 260 patients. ESβL production was detected on the basis of resistance to third-generation cephalosporins and aztreonam and then confirmed using the combined disk test. AmpC production was detected using the cefoxitin disk test and confirmed using the AmpC disk test. Carbapenemases were detected on the basis of resistance to imipenem, meropenem, and ertapenem, and then confirmed using the modified Hodge test, the phenylboronic acid combined disk test, and the imipenem/EDTA combined disk test. Carbapenemase gene (bla KPC and bla NDM) detection was performed using real-time PCR. Results Carbapenem resistance was 42.5% to ertapenem, 40.8% to meropenem, and 45% to imipenem. Risk factors associated with Hospital aquired infection were hospital stay of 14 days or more, exposure to invasive procedures, and comorbid conditions. ESβL production occurred in 55.8% and AmpC occurred in 30.8% of Enterobacteriaceae spp. Carbapenemase production was detected in 87%, Klebsiella pneumoniae carbapenemase (KPC) was detected in 37%, metallo-β-lactamase was detected in 54%, and bla KPCgene was detected in 24% of imipenem-resistant Enterobacteriaceae spp. However, bla NDM gene was not detected in all tested isolates. Conclusion Surveys of the prevalence, antibacterial susceptibility patterns, and identification of resistance patterns of bacterial isolates are important for determining appropriate empirical therapy for infections in critically ill patients.

Carbapenemase-Producing<i>Klebsiella pneumoniae</i>in Hospital, Singapore, 2011

BackgroundMultidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Pseudomonas aeruginosa are the leading cause of healthcare-associated infections worldwide.ObjectiveThe aim was to identify the resistant phenotypes among P. aeruginosa and to characterize different aminoglycosides and carbapenem resistance genes as major mechanisms of resistance in these isolates, in Theodor Bilharz Research Institute (TBRI), a tertiary care hospital in Cairo, Egypt.MethodsDuring a period of 11 months, 42 P. aeruginosa clinical isolates were collected from the microbiology laboratory by routine culture. Antimicrobial sensitivity testing to the aminoglycosides gentamicin and amikacin, and other classes of antibiotics, was performed by a disk diffusion method. Isolates were tested for aminoglycoside resistance genes, aac(6ʹ)-lb, aac-(3)-lla, rmtB, rmtC, armA, rmtD, and rmtF, and carbapenemase resistance genes blaNDM, blaVIM, and blaIMP, using conventional PCR.ResultsThirty-three (78.5%) of the clinical P. aeruginosa isolates showed MDR and XDR phenotypes at 42.4% and 57.65%, respectively, and these were included in the study. Aminoglycoside resistance was found in 97%, whereas carbapenem resistance was found in 81% of the isolates phenotypically. Only 59.4% (19/26) of the aminoglycoside-resistant isolates harbored resistance genes; none of the amikacin-susceptible isolates harbored any of the tested aminoglycoside resistance genes. Aminoglycoside resistance genes rmtB, armA, aac(6ʹ)-lb, and rmtF were found at rates of 17/33 (51.5%), 3/33 (9%), 2/33 (6%), and 2/33 (6%), respectively, whereas rmtD, acc(3)-II, and rmtC were not detected. Only 40.7% (11/27) of the carbapenem-resistant isolates harbored resistance genes. Carbapenem resistance genes, blaNDM andblaVIM, were found at rates of 7/33 (21.2%) and 6/33 (18.1%), respectively, and blaIMP was not detected.ConclusionRates of MDR and XDR P. aeruginosa and resistance to aminoglycosides and carbapenems in our setting are high. Methyltransferases and metallo-beta-lactamases are the main mechanisms of resistance to aminoglycosides and carbapenems, respectively. The presence of blaNDM and rmtF in the strains confirms their rapid dissemination in the Egyptian environment.

BACKGROUND: Carbapenemases are emerging resistance determinants in Gramnegative pathogens, including Pseudomonas aeruginosa, Acinetobacter spp and Enterobacteriaceae. Acquired carbapenem resistance is largely attributed to the production of carbapenem‐hydrolysing enzymes called carbapenemases which include metallo‐beta‐lactamases (MBLs) or Klebsiella pneumoniae carbapenemases (KPCs). Detection of these enzymes has become a diagnostic challenge for clinical microbiologists. The present study was undertaken to assess the incidence of carbapenemases including KPCs and MBLs in urinary isolates of Enterobacteriaceae. MATERIALS AND METHODS: A total of 66 consecutive, non-repeat carbapenems resistant (by disk diffusion) clinical isolates of Enterobacteriaceae obtained over a period of one year (January-December, 2011) were subjected to modified Hodge test (MHT) for the detection of carbapenemases, combined disk test using phenylboronic acid(PBA) for KPC, EDTA double disk synergy test, combined disk test using EDTA, MBL Etest for MBL, and disk enhancement test with both PBA and EDTA for detecting coproduction of KPC and MBL. RESULTS: Of the total 66 resistant clinical isolates, MHT identified 61 (92.42%) isolates as carbapenemase producers, metallo-beta-lactamases (MBLs) activity was detected in 39(59.09%) isolates, KPC in 9(13.63%) and co-existent of both KPC and MBL in 3(4.54%) isolates. CONCLUSIONS: The study demonstrates the presence of carbapenemases including KPCs and MBLs in Enterobacteriaceae in our hospital. The combined disk test and MBL Etest show equal efficacy for MBL detection, but given the cost-constraints, combined disk test can be used as a convenient detection method in the clinical microbiology laboratory. Boronic acid disk test is easy to perform, interpret and reliable method in detecting KPCs.

Objectives The aim of the study was to determine the incidence of carbapenem resistance among Klebsiella pneumoniae isolates in Menoufia University Hospitals and detect the production of Klebsiella pneumoniae carbapenemases (KPCs) and metallo- b -lactamases (M b Ls) using phenotypic and molecular methods and correlate their presence with in-vitro susceptibility to carbapenems. Background Carbapenem-hydrolyzing b -lactamases are the most powerful b -lactamases. KPCs and M b Ls are able to hydrolyze carbapenems, which cause resistance to multiple classes of antibiotics. Materials and methods The study was conducted on 128 K. pneumoniae isolates. Carbapenemase production was detected by antibiotic susceptibility testing against imipenem (IPM), meropenem, and ertapenem and by IPM minimal inhibitory concentration assays. Results were confirmed by the modified Hodge test, the phenylboronic acid combined disk (PBA-CD) test, and the IPM/EDTA combined disk (IPM/EDTA-CD) test. Detection of carbapenemase genes (blaKPC, blaVIM, and blaIMP) was performed by multiplex PCR. Results Eighty K. pneumoniae isolates were reported as IPM resistant. Class A carbapenemases (KPCs) were detected in 35% of IPM-resistant K. pneumoniae by the PBA-CD test. Class B carbapenemases (M b Ls) were detected in 48.8% of IPM-resistant K. pneumoniae by the IPM/EDTA-CD test. Age, long hospital stay, invasive procedures, and history of drug intake (carbapenem) were high-risk factors for IPM-resistant K. pneumoniae. Among IPM-resistant isolates, 17.5% were positive for the blaKPC gene and 43.75% were positive for the blaVIM and blaIMP genes. In relation to PCR, the sensitivity and specificity of the PBA-CD test were 85.7 and 75.8%, respectively, whereas for the IPM/EDTA-CD test sensitivity and specificity were 94.3 and 86.7%, respectively. Conclusion Laboratory identification of carbapenemase-harboring clinical isolates is necessary for implementing contact precautions, for outbreak detection, and for proper choice of effective therapy. Phenotypic and molecular-based techniques identify carbapenemase producers with variable efficiencies.

Background: Klebsiella species are significant pathogens responsible for various healthcare-associated infections. Despite the existence of several mechanisms of resistance against high priority antibiotics such as carbapenems, the production of carbapenemases remains the most important mechanism of resistance among Klebsiella spp. The aim of this study was to determine the prevalence of carbapenemase-production among Klebsiella spp isolated from clinical specimens at the University College Hospital (UCH) Ibadan, Nigeria. Methodology: A total of 120 clinical bacterial isolates were collected from the routine laboratories in the Department of Medical Microbiology and Parasitology, UCH, Ibadan between January and August 2023. These isolates were re-identified using conventional microbiological methods, including Gram staining, and biochemical identification tests. Eighty viable isolates were screened for carbapenem resistance using imipenem, following CLSI guidelines. Carbapenemase production was confirmed by the modified carbapenem inactivation (mCIM) and EDTA carbapenem inactivation (eCIM) methods. Conventional multiplex polymerase chain reaction (PCR) was used to amplify and detect carbapenemase resistance genes (blaNDM-1, blaVIM, blaKPC, blaOXA-48, and blaIMP) in the isolates, with the amplified products resolved by agarose gel electrophoresis. Data were entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 26.0.Results: Ninety-five of the 120 isolates were biochemically identified as Klebsiella spp while only 80 remained viable. Twenty-three (28.8%) biochemically confirmed isolates were resistant to imipenem (carbapenem resistant Klebsiella), and 20 (25.0%) were confirmed as Klebsiella spp by PCR, of which 11 (55.0%) were resistant to all tested antibiotics. Carbapenemase production was detected in 5 (6.3%) isolates using mCIM and eCIM. PCR identified blaNDM-1 gene in 3 (3.8%) of the isolates, while blaVIM, blaKPC, blaOXA-48, and blaIMP genes were not detected in any isolate.Conclusion: The detection of blaNDM-1 and high resistance levels among isolates is of great concern. The emergence of carbapenem-resistant Klebsiella, especially with blaNDM-1 gene, threatens the effectiveness of ‘last resort’ antibiotics. There is a need for antimicrobial resistance surveillance and antimicrobial stewardship to stem the tide of antimicrobial resistance in healthcare settings.

BACKGROUND: Pseudomonas aeruginosa is one of the most common causes of nosocomial infections. Carbapenems are widely used for the treatment of this microorganism, but nowadays, increasing number of carbapenem-resistant P. aeruginosa (CRPA) isolates are increasing which further lead to treatment failure due to associated multidrug resistance. AIM: The present study was done to determine the prevalence of New Delhi metallo-β-lactamase-1 (bla NDM-1) in P. aeruginosa in hospitalized patients in a tertiary care setup. SETTINGS AND DESIGN: The study was conducted in the department of microbiology with the tertiary care hospital settings. It was a prospective cross-sectional observational study conducted from June 2014 to December 2014. MATERIALS AND METHODS: Antibiotic susceptibility test was performed using Kirby–Bauer disc diffusion method for screening of the isolates. P. aeruginosa isolates with the zone diameter of carbapenems ≤18 mm were considered as carbapenemase producers. Metallo-beta-lactamases (MBL) detection was done using imipenem- ethylenediaminetetraacetic acid double-disc synergy test. NDM-1 gene in P. aeruginosa was identified using conventional polymerase chain reaction. STATISTICAL ANALYSIS USED: Chi-square test was used to analyze the data and P RESULTS: Out of 460 P. aeruginosa, 74 (16.08%) isolates were CRPA. A total of 61 (13.2%) MBL-producing P. aeruginosa were isolated, and bla NDM-1 gene was detected in 18 (3.91%) P. aeruginosa isolates. CONCLUSION: The study showed increasing multidrug resistance among CRPA compared to carbapenem-sensitive P. aeruginosa. It also determined increasing prevalence of bla NDM-1 in P. aeruginosa isolates. It is emphasized to timely identify these pathogens and treat with proper antibiotics to reduce morbidity and mortality.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major public health concern, particularly in immunocompromised and critically ill patients. Colistin and tigecycline are among the last-resort treatment options, while the primary driver of CRKP emergence is carbapenemase production, especially Klebsiella pneumoniae carbapenemase (KPC) and metallo-β-lactamase (MBL). A thorough understanding of its resistance mechanisms is essential for selecting the most effective antimicrobial therapy. This study aimed to evaluate the diagnostic accuracy of the modified Hodge test (MHT) and modified carbapenem inactivation method (mCIM) in detecting molecular resistance mechanisms in CRKP clinical isolates. This exploratory study consisted of 65 CRKP isolates, which were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik GmbH, Bremen, Germany). Antimicrobial susceptibility testing was performed using the BD Phoenix system. The test isolates were subjected to MHTand mCIMand later shipped to the Central Research Laboratory (CRL), Bengaluru, India, where they were subjected to polymerase chain reaction (PCR) and whole-genome sequencing (WGS). PCR detected blaOXA-48-like , blaNDM-1 , bla NDM-5, and blaKPC genes in 79.7%, 10.2%, 64.4%, and 1.7% CRKP isolates, respectively. The PCR results were concordant with WGS. The MHTdemonstrated an overall sensitivity of 60.3%, specificity of 100%, positive predictive value (PPV) of 100%, and negative predictive value (NPV) of 4.2% for detecting carbapenemase production. It showed the highest sensitivity and specificity for blaKPC (100%) and blaOXA-48-like (75%) genes, respectively, with the highest PPV for blaOXA-48-like (91.4%) and NPV for blaKPC (100%). However, agreement between MHT and PCR for carbapenemase detection was negligible (Kappa: 0.049, p=0.223). A minimal but statistically significant agreement was noted for blaOXA-48-like detection (Kappa: 0.314, p=0.007), while no significant agreement was observed for blaNDM-1 , blaNDM-5 , or blaKPC genes. The mCIMhad an overall sensitivity of 3.63%, specificity of 100%, PPV of 100%, and NPV of 1.8%. It exhibited the highest sensitivity (4.3%) and specificity (100%) for blaOXA-48-like genes, with the highest PPV for blaOXA-48-like (100%), and NPV for blaKPC (98.1%). No statistically significant agreement was found between mCIM and PCR for carbapenemase detection (Kappa: 0.001, p=0.850). Comprehensive assessment of the diagnostic accuracy of MHT and mCIM using WGS across a broad spectrum of multi-drug-resistant (MDR) organisms should be conducted at multiple centers to produce reliable data that can guide better clinical management of the patients.

Carbapenem-resistant gram-negative bacteria are an increasingly significant clinical threat globally. This risk may be underestimated in Kenya as only four carbapenemase genes in three bacterial species have been described. The study aimed to understand the antibiotic resistance profiles, genes, sequence types, and distribution of carbapenem-resistant gram-negative bacteria from patients in six hospitals across five Kenyan counties by bacterial culture, antibiotic susceptibility testing, and whole-genome sequence analysis. Forty-eight, non-duplicate, carbapenem non-susceptible, clinical isolates were identified across the five counties (predominantly in Nairobi and Kisii): twenty-seven Acinetobacter baumannii, fourteen Pseudomonas aeruginosa, three Escherichia coli, two Enterobacter cloacae, and two Klebsiella pneumoniae. All isolates were non-susceptible to β-lactam drugs with variable susceptibility to tigecycline (66%), minocycline (52.9%), tetracycline (29.4%), and levofloxacin (22.9%). Thirteen P. aeruginosa isolates were resistant to all antibiotics tested. Eleven carbapenemase genes were identified: blaNDM-1, blaOXA-23, -58, -66, -69, and -91 in A. baumannii (STs 1, 2, 164 and a novel ST1475), blaNDM-1 in E. cloacae (STs 25,182), blaNDM-1, blaVIM-1and -6, blaOXA-50 in P. aeruginosa (STs 316, 357, 654, and1203), blaOXA-181, blaNDM-1 in K. pneumoniae (STs 147 and 219), and blaNDM-5 in E. coli (ST164). Five A. baumannii isolates had two carbapenemases, blaNDM-1, and either blaOXA-23 (4) or blaOXA-58 (1). AmpC genes were detected in A. baumannii (blaADC-25), E. cloacae (blaDHA-1 and blaACT-6, 16), and K. pneumoniae (blaCMY). Significant multiple-drug resistant genes were the pan-aminoglycoside resistance16srRNA methyltransferase armA, rmtB, rmtC, and rmtF genes. This study is the first to report blaOXA-420, -58, -181, VIM-6, and blaNDM-5 in Kenyan isolates. High-risk STs of A. baumannii (ST1475, ST2), E. cloacae ST182, K. pneumoniae ST147, P. aeruginosa (ST357, 654), and E. coli ST167, ST648 were identified which present considerable therapeutic danger. The study recommends urgent carbapenem use regulation and containment of high-risk carbapenem-resistant bacteria.

Background and objectives: Carbapenem resistance is an emerging problem worldwide. Detection of carbapenem resistance genes is important to institute appropriate therapy and to initiate preventive measures. This study was conducted to determine the presence of carbapenemase enzyme producing Escherichia coli and Klebsiella species in a tertiary care hospital of Bangladesh, as well as to observe the patterns of antibiotic resistance and carbapenem resistance genes among them.&#x0D; Methodology: Total 166 Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca were isolated from urine, wound swab, pus, sputum and blood samples of patients of Dhaka Medical College Hospital. Antibiotic susceptibility test was performed by disk-diffusion technique. Carbapenemase producers were detected phenotypically by Double-disk synergy (DDS) test, Modified Hodge test (MHT) and Combined disk (CD) assay. Minimum inhibitory concentration (MIC) of imipenem was done by agar dilution method among carbapenemase producing strains. Genotypically carbapenemase genes (blaNDM-1, blaVIM, blaIMP, blaKPC, blaOXA-48/blaOXA-181) among the imipenem resistant Escherichia coli and Klebsiella species were detected by polymerase chain reaction (PCR). Sequencing was done to differentiate blaOXA-181 gene from blaOXA-48 gene. Class 1 integron were also detected by PCR using specific primer among carbapenemase producers.&#x0D; Results: Thirty seven (22.29%) imipenem resistant isolates were detected during disk-diffusion technique, among them 16 (43.24%) carbapenemase producers were detected by MHT, 20 (54.05%) by DDS test, 22 (59.46%) by CD assay and 23 (62.16%) by PCR. Out of 23 carbapenemase producing strains, MIC of imipenem ranged from 4 μg/ml up to ≥128 μg/ml. NDM-1 (43.24%) was the dominant genotype in imipenem resistant strains followed by KPC (21.62%) and OXA-181 (18.92%). Class 1 integron were present in 16 (69.57%) of the genotypically identified carbapenemase producers.&#x0D; Conclusion: The results of this study showed high proportion of carbapenemase enzyme producing Escherichia coli and Klebsiella species in Bangladesh. Genes encoding carbapenemase enzymes including blaNDM-1, blaKPC, blaVIM, blaIMP and blaOXA-181 were responsible for imipenem resistance. blaNDM-1 producers are increasing and blaKPC and blaOXA-181 producers are emerging in Bangladesh. Regular surveillance of antibiotic resistance should be done in every tertiary care hospital to prevent spread of these strains.&#x0D; Bangladesh J Med Microbiol 2020; 14 (2): 25-29

BackgroundMultidrug resistant, extremely drug-resistant, pan-drug resistant, carbapenem-resistant, and carbapenemase-producing gram-negative bacteria are becoming more common in health care settings and are posing a growing threat to public health.ObjectiveThe study was aimed to detect and phenotypically characterize carbapenem no- susceptible gram-negative bacilli at the Ethiopian Public Health Institute.Materials and methodsA prospective cross-sectional study was conducted from June 30, 2019, to May 30, 2020, at the national reference laboratory of the Ethiopian Public Health Institute. Clinical samples were collected, inoculated, and incubated for each sample in accordance with standard protocol. Antimicrobial susceptibility testing was conducted using Kirby-Bauer disk diffusion method. Identification was done using the traditional biochemical method. Multidrug-resistant and extensively drug-resistant isolates were classified using a standardized definition established by the European Centre for Disease Prevention and Control and the United States Centers for Disease Prevention and Control. Gram-negative organisms with reduced susceptibility to carbapenem antibiotics were considered candidate carbapenemase producers and subjected to modified carbapenem inactivation and simplified carbapenem inactivation methods. Meropenem with EDTA was used to differentiate metallo-β-lactamase (MBL) from serine carbapenemase. Meropenem (MRP)/meropenem + phenylboronic acid (MBO) were used to differentiate Klebsiella pneumoniae carbapenemase (KPC) from other serine carbapenemase producing gram-negative organisms.ResultsA total of 1,337 clinical specimens were analyzed, of which 429 gram-negative bacterial isolates were recovered. Out of 429 isolates, 319, 74, and 36 were Enterobacterales, Acinetobacter species, and Pseudomonas aeruginosa respectively. In our study, the prevalence of multidrug-resistant, extensively drug-resistant, carbapenemase-producing, and carbapenem nonsusceptible gram-negative bacilli were 45.2%, 7.7%, 5.4%, and 15.4% respectively. Out of 429 isolates, 66 demonstrated reduced susceptibility to the antibiotics meropenem and imipenem. These isolates were tested for carbapenemase production of which 34.8% (23/66) were carbapenemase producers. Out of 23 carbapenemase positive gram-negative bacteria, ten (10) and thirteen (13) were metallo-beta-lactamase and serine carbapenemase respectively. Three of 13 serine carbapenemase positive organisms were Klebsiella pneumoniae carbapenemase.ConclusionThis study revealed an alarming level of antimicrobial resistance (AMR), with a high prevalence of multidrug-resistant (MDR) and extremely drug-resistant, carbapenemase-producing gram-negative bacteria, particularly among intensive care unit patients at the health facility level. These findings point to a scenario in which clinical management of infected patients becomes increasingly difficult and necessitates the use of “last-resort” antimicrobials likely exacerbating the magnitude of the global AMR crisis. This mandates robust AMR monitoring and an infection prevention and control program.

Introduction: Carbapenems belong to the Beta Lactam group of antimicrobial agents. They are often used as “lastline agents” or “antibiotics of last resort” in critically ill patients. Carbapenem resistance in Enterobacteriaceae may be due to various reasons but Klebsiella pneumoniae Carbapenemase (KPC) enzyme production is the commonest. Phenotypic as well as genotypic methods can be used to detect Carbapenemases. Among the phenotypic tests, Modified Hodge Test (MHT) is relatively easy to perform. Aims and Objectives: This study aimed to determine the prevalence of carbapenem resistance among Enterobacteriaceae isolates and calculate the sensitivity of MHT as an indicator of KPC production. Materials and Methods: All Enterobacteriaceae isolates from clinical samples were included in this study and were screened for Carbapenem resistance. 45 randomly chosen Carbapenem Resistant Enterobacteriaceae isolates were subjected to MHT and blaKPC gene detection by PCR. Results: 2035 Enterobacteriaceae isolates were tested and 5.2% were found to be resistant to Imipenem, 22.9 % were resistant to Meropenem and 4.42 % were resistant to both Imipenem and Meropenem. The sensitivity of MHT was calculated to be 90% and specificity was calculated to be 60%

Hospitals, as a source of transmission and spread of bacteria carrying antibiotic-resistant genes (ARGs) as well as a high source of antibiotic compounds, are a significant reservoir of antibiotic-resistant gene transfer that causes bacteria to develop multidrug resistance (MDR). Klebsiella pneumonia is a bacterium often found in wastewater with a high level of Extended Spectrum β-lactamase (ESBL) and carbapenemase resistance. This study aimed to determine Klebsiella pneumonia and resistance genes encoding ESBL and carbapenemase in hospital wastewater to obtain primary Antimicrobial Resistance (AMR) data in the environment, which was first conducted in Indonesia. The resistance gene detection method was developed using High Throughput Real-Time and conventional Real-Time (HT-RT PCR). The data obtained were relative abundance and copy number. The results showed that blaTEM and blaNDM were the highest ARGs. Conventional Real-Time PCR results showed better sensitivity in detecting K. pneumoniae and ARGs than HT-RT PCR. The detection of K. pneumoniae and ARGs in RSCM wastewater in Indonesia indicates the need to improve the handling of RSCM WWTP to monitor the number of microbial resistances to antibiotics.

The latest threat of multidrug-resistant Gram-negative bacteria corresponds to the emergence of carbapenemase New Delhi metallo-β-lactamase (NDM) and Klebsiella pneumoniae carbapenemase (KPC) producers. Rapid molecular detection is essential to limit their spread. In this study, a duplex real-time polymerase chain reaction (PCR) that was specific for the detection of blaNDM and blaKPC with the same limit of detection of ten plasmid copies was developed. The assay was linear over eight log dilutions for blaNDM (R2 = 0.971; slope, -3.273) and blaKPC (R2 = 0.992; slope, -2.997) with efficiencies of 102% and 115%, respectively. The assay was validated with 157 clinical isolates and showed 100% concordance with conventional PCR. The excellent performance of the duplex PCR assay makes it a powerful tool for surveillance of the carbapenemases NDM and KPC.