Nepal Journal of Biotechnology. D e c . 2 0 1 9 Vol. 7, No. 1: 74-81 DOI: https://doi.org/10.3126/njb.v7i1.26954 ©NJB, Biotechnology Society of Nepal 74 Nepjol.info/index.php/njb. ORIGINAL RESEARCH ARTICLE Phenotypic Characterization of Beta-lactamases Producing Gram-Negative Bacteria in a Tertiary Hospital, Nepal Elina Maharjan1,2, Pooja Shakya1, Balkrishna Bhattachan3, Bharat Prasad Baral4, Dhiraj Shrestha5,6* 1Department of Microbiology, Kathmandu College of Science & Technology (KCST), Nepal. 2Research Center for Applied Science and Technology (RECAST), Tribhuvan University, Nepal. 3Siddhi Memorial Hospital, Bhaktapur, Nepal. 4Annapurna Child and Women Hospital, Pokhara, Nepal. 5Department of Microbiology, Tri-Chandra Multiple Campus, Kathmandu, Nepal. 6Department of Microbiology, Shi-Gan International College of Science and Technology (SICOST), Kathmandu, Nepal. Abstract Infections caused by beta-lactamases producing Gram-negative bacteria are increasing, thus posing a challenge to the management of such infections. The surveillance data of such bacteria is limited in Nepal so this study aimed to detect the beta-lactamase producing Gram-negative bacteria in a tertiary setting. A total of 604 clinical samples, including urine, blood, sputum and body fluids, were cultured and identified by the routine standard laboratory protocols. Antibiotic susceptibility testing was done by Kirby Bauer disc diffusion method following Clinical and Laboratory Standard Institute guidelines (2014). Extended-spectrum beta- lactamases (ESBL) producers were identified by combined disk method and metallo-beta- lactamases (MBL) producers were identified by Imipenem- EDTA combined disk method. Out of 604 samples, 282 (46.7%) samples showed significant growth, of which 229 (81.2%) were Gram- negative bacteria. Of 229 Gram-negative bacteria, 200 (87.3%) were multidrug resistant, 67 (29.3%) were ESBL producers and 16 (7.0%) were MBL producers. Klebsiella pneumoniae were among higher ESBL producers and Pseudomonas aeruginosa were among higher MBL producers. The findings suggest higher antibacterial resistance among Gram-negative bacteria with the added burden of beta-lactamase production. Imipenem was effective against 125 of 229 Gram- negative bacteria tested. Thus, imipenem can be the drug of choice for empirical management. The higher multidrug resistance and higher beta-lactamases production among Gram-negative bacteria warrant the continuous monitoring, surveillance, early detection, and infection control practices of such bacteria. Keywords: Antibacterial resistance, extended-spectrum beta-lactamases, ESBL, Gram-negative, metallo-beta-lactamase, MBL *Corresponding Author Email: hiraj.diamond@gmail.com Introduction Antibacterial resistance (ABR) is an increasingly serious threat to human health, challenging the effective management of infections. ABR increase the health care cost as a result of prolonged illness, additional tests and pricier drugs [1]. Annually, more than 750,000 deaths are caused by resistant bacteria. The median overall increased cost to treat a resistant bacterial infection is around 700 USD [2]. ABR is a natural genetic change, thus newer mechanisms are emerging and spreading, raising multidrug-resistant (MDR) bacteria [1]. One of the most worrisome resistance mechanisms is the production of beta- lactamases, of which extended-spectrum beta- lactamases (ESBLs) and metallo-beta- lactamases (MBLs) are the most impacting ones. ESBLs can hydrolyze all beta-lactam antibiotics including cephalosporins except cephamycins or carbapenems [3]. ESBLs are often plasmid-mediated. Since it was first reported from Germany in 1983, it has then spread worldwide [4]. In Nepal, ESBLs were first reported in 2006 [5]. Similarly, MBLs can hydrolyze all broad-spectrum beta-lactams including cephalosporins, and carbapenems, except monobactams [6]. Since its first report from Japan in 1991, it has also been reported Nepal Journal of Biotechnology. D e c . 2 0 1 9 Vol. 7, No. 1: 74-81 Maharjan et al. 2019 ©NJB, Biotechnology Society of Nepal 75 Nepjol.info/index.php/njb. worldwide [7]. MBLs were first reported in Nepal in 2009 [8]. The acquisition, expression, and dissemination of beta-lactamase producing genes in pathogens have posed the major public health concern today [9]. Limited data are available on surveilling beta-lactamase producing clinical isolates in Nepal. ABR surveillance data can guide the physician in choosing appropriate therapy for effective management of infectious disease without extensive testing. Thus, this study aimed to produce updated data on surveilling beta-lactamase producing clinical isolates in a tertiary healthcare setting. This would help to formulate antimicrobial stewardship policy to circumvent the rising threat of ABR. Materials and Methods Study setting, design and study population A prospective hospital-based study was conducted in the Department of Microbiology at Annapurna Neurological Institute and Applied Science, Kathmandu, Nepal from March to November 2014. A total of 604 clinical samples from patients of all ages and both sexes, visiting the hospital and requesting a routine investigation, was included in the study. The samples include 263 urines (clean catch urine and catheter tip), 140 sputum samples, 73 blood samples and 128 body fluids (pus, pus swab, CSF, bile fluid, pleural fluid, peritoneal fluid, synovial fluid, and tracheal secretion). Contaminated samples and repeated samples from the same patient were excluded to avoid selection bias. Laboratory processing of the samples The samples were subjected to the standard microbiological procedures for isolation and identification of bacteria. In short, the samples were inoculated onto MacConkey agar (HiMedia, India) and blood agar (HiMedia, India) plate by streaking, followed by incubation at 37℃. Growths of bacteria were observed after 18-48hrs. The identification of Gram-negative bacteria was done by Gram’s stain morphology, cultural characteristics, and conventional biochemical tests. Biochemical tests employed were catalase test, oxidase test, indole test, citrate utilization test, methyl red test, Voges-Proskauer test, Christensen’s urease test, triple sugar iron test, decarboxylase test, and phenylalanine deaminase test. Antimicrobial susceptibility testing The antimicrobial susceptibility test of all identified bacteria against antibiotics of various classes was done in vitro by Kirby-Bauer disc diffusion method [10]. A zone of inhibitions (ZOI) for each disc was measured and the results were interpreted as per CLSI guideline M100-S24 [11]. Control strains Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used for the quality control of the test. Detection of extended-spectrum beta- lactamase producing strain ESBL producing isolates were identified by a phenotypic method as described in CLSI guideline M100-S24 [11]. Screening test for ESBL: Isolates exhibiting resistance against ceftriaxone (30µg) (ZOI ≤25mm) and/or ceftazidime (30µg) (ZOI ≤22mm) and/or cefpodoxime (10µg) (ZOI ≤17mm) and/or cefotaxime (30µg) (ZOI ≤27mm) were screened for ESBL production. Combined disc (CD) method as a confirmatory test for ESBL: ZOIs of isolates against ceftazidime disc (30µg) and cefotaxime (30µg) were compared against ZOIs of isolates against ceftazidime disc (30µg) containing clavulanic acid (10µg) and cefotaxime (30µg) containing clavulanic acid (10µg) when placed 25mm apart (center to center). The isolates showing the difference of 5mm or more between either of the two ZOIs of the disc and clavulanate added disc was confirmed positive for ESBL production. Klebsiella pneumoniae ATCC 700603 (ESBL positive) was used as control strains. Nepal Journal of Biotechnology. D e c . 2 0 1 9 Vol. 7, No. 1: 74-81 Maharjan et al. 2019 ©NJB, Biotechnology Society of Nepal 76 Nepjol.info/index.php/njb. Table 1. Sample and significant growth pattern Total samples Culture positive Gram-negative bacteria Gram-positive bacteria Gender (M: F ratio=1.45:1) Male 357 (59.1%) 174 (61.7%) 143 (62.4%) 31 (58.5%) Female 247 (40.9%) 108 (38.3%) 86 (37.6%) 22 (41.5%) Departments of patients Inpatients 471 (78%) 180 (63.8%) 157 (68.6%) 23 (76.7%) Outpatients 133 (22%) 102 (36.2%) 72 (31.4%) 30 (56.6%) Age groups (years): Infants and children, adolescents, adults, elders ≤9 19 (3.2%) 3 (1.1%) 2 (0.9%) 1 (1.9%) 10-19 56 (9.3%) 29 (10.3%) 26 (11.4%) 3 (5.7%) 20-59 313 (51.8%) 129 (45.7%) 99 (43.2%) 30 (56.6%) ≥60 216 (35.8%) 121 (42.9%) 102 (44.5%) 19 (35.8%) Total 604 (100%) 282 (100%) 229 (100%) 53 (100%) Detection of metallo-beta-lactamase producing strain Since during the study no standard protocol was available for the detection of MBL, MBL producing isolates were identified by the commonly used phenotypic method. Screening test for MBL: Isolates exhibiting resistance against ceftazidime (30µg) (ZOI<18mm) were screened for MBL production. The resistance against imipenem (10μg) and/or meropenem (10μg) was not used as a screening tool so as to avoid missing the detection of hidden MBL in bacteria. Bacterial suspension equivalent to 1:10 dilution of 0.5 McFarland was used for lawn culture in Mueller-Hinton agar before incorporating antibiotic discs [12]. Combined disc (CD) method as a confirmatory test for MBL: ZOI of isolate against imipenem disc (10 µg) was compared against ZOI of isolates against imipenem disc (10µg) containing 292µg (10µl of 0.1M) ethylenediamine-tetraacetic acid (EDTA) when placed 25mm apart (center to center). The isolates showing difference of 4mm or more between two ZOIs was confirmed positive for MBL production [12, 13]. P. aeruginosa ATCC 27853 (MBL negative) and P. aeruginosa PA 105663 (MBL positive) was used as control strains. Results Distribution of samples and isolates Of 604 samples, only 282 (46.7%) were culture positive. Higher number of samples from males were culture positive 174 (61.7%). Similarly, a higher number of samples from the inpatient department were culture positive, 180 (63.8%). Culture positivity increased with the age of patients. Among 282 culture positive, 229 (81.2%) were Gram-negative bacteria (Table 1). Eight different species of Gram-negative bacteria were identified. Among these, E. coli were the predominant, 78 (34.1%). E. coli was also predominant in urine samples, 61(55.0%) (Table 2). Nepal Journal of Biotechnology. D e c . 2 0 1 9 Vol. 7, No. 1: 74-81 Maharjan et al. 2019 ©NJB, Biotechnology Society of Nepal 77 Nepjol.info/index.php/njb. Antibiotic susceptibility of Gram- negative bacteria Of 229 Gram-negative bacteria, E. coli showed higher resistance against ampicillin, cefpodoxime, ceftriaxone, and cefixime, while imipenem and chloramphenicol were found effective. P. aeruginosa showed higher resistance against cefixime and cefpodoxime, while imipenem was found effective. Similarly, other Gram-negative bacteria showed higher resistance against ampicillin, cefpodoxime, and ceftriaxone, while imipenem was found fairly effective (Table 3). Distribution of beta-lactamase producers Of 229 Gram-negative bacteria, 200 (87.3%) were found MDR. Of these bacteria, 67 (29.3%) were ESBL producers and 16 (7.0%) were MBL producers. ESBL production was higher among sputum isolates i.e. 35 (52.2%) and MBL production was higher among urine isolates i.e. 8 (50%) (Table 4).Distribution of beta- lactamase production among Gram-negative bacteria Of 229 Gram-negative bacteria, MDR was found higher among Klebsiella spp. isolates. ESBL production was higher among K. pneumoniae 25 (37.3%), E. coli 16 (23.9%) and P. aeruginosa 16 (23.9%). Similarly, MBL production was higher among P. aeruginosa 9 (56.3%) and E. coli 3 (18.8%) (Table 5). Discussion Rise of beta-lactamases among pathogens has now been a prime threat to global health. Beta- lactamase production has been reported in P. aeruginosa, Acinetobacter spp. and Enterobacteriaceae [14]. The evidence has shown that ABR has a significant adverse impact on clinical outcomes and increases costs due to the consumption of health care resources [15]. Of 229 Gram-negative bacteria, 29.3% were found ESBL producers. Similar results were reported by previous studies as 25% in 2013 [16], 25.8% in 2014 [17], 24% in 2015 [18], 26.9% in 2015 [19] and 34.5% in 2017 [20]. ESBL producers are increasingly disseminating in Nepal, 0.6% in 2006 to 40% in 2017 [5, 16-22]. ESBL production was higher among K. pneumoniae (37.3%), E. coli (23.9%) and P. aeruginosa (23.9%). Table 2: Distribution of Gram-negative bacteria in different clinical samples Bacteria Sample Total Urine Blood Sputum Body Fluid ACB* 2 (1.8%) 0 (0%) 4 (5.5%) 2 (4.9%) 8 (3.5%) Citrobacter spp. 1 (0.9%) 0 (0%) 0 (0.0%) 0 (0.0%) 1 (0.4%) E. coli 61 (55.0%) 0 (0%) 9 (12.3%) 8 (19.5%) 78 (34.1%) K. pneumoniae 16 (14.4%) 0 (0%) 33 (45.2%) 22 (53.7%) 71 (31.0%) K. oxytoca 15 (13.5%) 0 (0%) 6 (8.2%) 1 (2.4%) 22 (9.6%) P. aeruginosa 12 (10.8%) 2 (50%) 20 (27.4%) 7 (17.1%) 41 (17.9%) Proteus spp. 4 (3.6%) 0 (0%) 1 (1.4%) 1 (2.4%) 6 (2.6%) Salmonella spp. 0 (0.0%) 2 (50%) 0 (0.0%) 0 (0.0%) 2 (0.9%) Total 111 (100%) 4 (100%) 73 (100%) 41 (100%) 229(100%) *Acinetobacter calcoaceticus-baumannii Complex Nepal Journal of Biotechnology. D e c . 2 0 1 9 Vol. 7, No. 1: 74-81 Maharjan et al. 2019 ©NJB, Biotechnology Society of Nepal 78 Nepjol.info/index.php/njb. Similar results were reported in E. coli as 25.8% by Pokhrel et al in 2014 [17], 22.4% by Raut et al in 2015 [23], 26.9% Yadav et al in 2015 [19]. Similarly, comparable results were reported in K. pneumoniae as 22.4% by Raut et al in 2015 [23] and 43.3% Nepal et al in 2017 [22]. ESBL production was higher among sputum isolates i.e. 35 (52.2%). Of 229 Gram-negative bacteria, 7% were found MBL producers. Similar results were reported by previous studies as 7.1% Khanal et al in 2013 [16], 3.2% Pokhrel et al in 2014 [17] and 4% Nepal et al in 2017 [20]. But a lower rate of 1.3% was reported by Mishra et al in 2012 [12] and a higher rate of 15% was reported Ansari et al in 2015 [18]. MBL producers are increasingly disseminating in Nepal [12, 16-18, 20]. MBL production was higher among P. aeruginosa isolates, 56.3%. Besides P. aeruginosa and Acinetobacter spp., MBL production was also found in E. coli and K. pneumoniae as well. MBL in Enterobacteriaceae was reported by similar studies reported [16-18, 20]. However, this contrast with the result of similar previous studies [12] which reported MBL production only in P. aeruginosa and Acinetobacter spp. MBL production was higher among urine isolates i.e. 50% of total MBL producers. Genotypic methods are considered superior tools for surveillance of such ESBL and MBL producers. But the evolution of newer beta- Table 3: Antibiotic susceptibility of Gram-negative bacteria Antibiotics E. coli (n=78) (%) P. aeruginosa (n=41) (%) Other GNB (n=110) (%) S I R S I R S I R Amikacin 30.8 14.1 55.1 58.5 17.1 24.4 39.1 11.8 49.1 Ampicillin 5.1 0.0 94.9 12.2 0.0 87.8 4.5 0.9 94.5 Aztreonam 10.3 15.4 74.4 34.1 19.5 46.3 20.0 10.0 70.0 Ceftriaxone 5.1 2.6 92.3 19.5 0.0 80.5 5.5 0.9 93.6 Cefixime 2.6 5.1 92.3 2.4 0.0 97.6 8.2 0.0 91.8 Cefotaxime 3.8 7.7 88.5 17.1 2.4 80.5 10.0 0.9 89.1 Cefpodoxime 5.1 0.0 94.9 12.2 0.0 87.8 5.5 0.0 94.5 Ceftazidime 9.0 2.6 88.5 17.1 4.9 78.0 10.0 0.9 89.1 Ciprofloxacin 14.1 1.3 84.6 19.5 2.4 78.0 11.8 0.0 88.2 Chloramphenicol 38.5 9.0 52.6 58.5 17.1 24.4 30.9 18.2 50.9 Co-trimoxazole 11.5 2.6 85.9 14.6 0.0 85.4 11.8 2.7 85.5 Gentamicin 21.8 5.1 73.1 41.5 4.9 53.7 20.9 2.7 76.4 Imipenem 39.7 15.4 44.9 78.0 0.0 22.0 56.4 4.5 39.1 Nitrofurantoin 18.0 4.9 77.1 NT NT NT NT NT NT Norfloxacin 12.8 1.3 85.9 9.8 4.9 85.4 13.6 0.0 86.4 Piperacillin/ Tazobactam NT NT NT 63.4 9.8 26.8 NT NT NT *GNB=Gram-negative bacteria, S=sensitive, I=intermediate, R=resistance, NT=not tested Nepal Journal of Biotechnology. D e c . 2 0 1 9 Vol. 7, No. 1: 74-81 Maharjan et al. 2019 ©NJB, Biotechnology Society of Nepal 79 Nepjol.info/index.php/njb. lactamase genes would make use of such tools tough, owing to the lack of primers. Moreover, using such expensive tools in resource lacking settings, like Nepal, would be a fairy tale scenario. A combined disc phenotypic method using imipenem and EDTA for MBL, ceftazidime/clavulanate or cefotaxime /clavulanate for ESBL offers a cheaper alternative. Also, phenotypic method proves not only the presence of beta-lactamases producing genes but also their expression. This method has been reported to have a sensitivity of 100% and specificity of 98%, thus reliable [12, 24]. Of 229 Gram-negative bacteria, E. coli showed higher resistance against ampicillin, cefpodoxime, ceftriaxone, and cefixime while imipenem and chloramphenicol were found effective. P. aeruginosa showed higher resistance against cefixime and cefpodoxime while imipenem was found effective. Similarly, other Gram-negative bacteria showed higher resistance against ampicillin, cefpodoxime, and ceftriaxone while imipenem was found fairly effective. Imipenem was found as an effective drug against most of the tested Gram-negative isolates. This concords with similar reports which reported imipenem as the most sensitive drug [25, 26]. As per the expert consensus, isolates resistant to two or more classes of antibiotics are considered MDR strains [27]. Of 229 Gram-negative bacteria, 87.3% of the isolates were found MDR strains. MDR was found higher among Klebsiella spp. isolates (93.5%). Similarly, 87.8% of P. aeruginosa and 50% of Acinetobacter spp. isolates were found MDR strains. Some studies reported higher MDR in these bacteria [16, 18, 20]. In our study, nearly all ESBL and MBL producers were MDR strains. This limits physicians with therapeutics for the management of infections. Such ESBL producing strains can be inhibited by the use of beta-lacatamase inhibitors like clavulanate, but MBL producers are resistance to these inhibitors and MBL inhibitors are yet to be trialed in human. The reserve drug for MDR Gram- negative bacteria is emptied with the evolution of newer beta-lactamases. Colistin has always been the ultimate weapon in the fight against MDR superbugs in case all other therapeutic options fail. However, resistance against colistin has been reported in recent times, rendering our drug arsenal completely empty for such superbugs [28-30]. ABR is a crisis that must be managed with the utmost urgency to contain it. Such surveillance that generates updated data is required for the implementation of sound strategies and public health actions to contain ABR. ABR requires concerted cross-sectional action by governments Table 5. Distribution of Gram-negative isolates producing ESBL and MBL Bacteria Total isolates MDR bacteria ESBL producers MBL producers ACB* 8 4 4 (6.0%) 2 (12.5%) Citrobacter spp. 1 1 0 (0.0%) 0 (0.0%) E. coli 78 65 16 (23.9%) 3 (18.8%) K. oxytoca 22 21 5 (7.5%) 0 (0.0%) K. pneumoniae 71 66 25 (37.3%) 2 (12.5%) P. aeruginosa 41 36 16 (23.9%) 9 (56.3%) Proteus spp. 6 6 0 (0.0%) 0 (0.0%) Salmonella spp. 2 1 1 (1.5%) 0 (0.0%) Total 229 200 (100%) 67 (100%) 16 (100%) *Acinetobacter calcoaceticus-baumannii Complex Nepal Journal of Biotechnology. D e c . 2 0 1 9 Vol. 7, No. 1: 74-81 Maharjan et al. 2019 ©NJB, Biotechnology Society of Nepal 80 Nepjol.info/index.php/njb. and society as a whole. Currently, ‘the global action plan on antimicrobial resistance-2015’, as formulated by WHO [31], must be implemented as envisioned to tackle the ABR. Conclusion The findings suggest higher MDR, ESBL, and MBL not only among P. aeruginosa and Acinetobacter spp. but also among Enterobacteriaceae family, including E. coli and K. pneumoniae. Imipenem showed promising sensitivity against most of the Gram-negative isolates, thus it can be the antibiotic of choice for management of such infections. Evolving beta- lactamases against newer generation beta- lactams have posed a serious threat to public health. Only the continuous monitoring, surveillance, early detection, and infection control practices can ensure the effective management of such resistant strains. Conflict of Interest The authors declare no conflict of interest. Acknowledgments We acknowledge Asst. Prof. Archana Katuwal and Dr. Ranga Bahadur Basnet for supervising this study and providing the methodology for the study. Consent to publish Not applicable Ethical approval and consent to the participant No patient-related data were collected and thus ethical approval was not required. The study was a laboratory-based study and a part of the study was a routine patient care investigation. Oral informed consent was taken from all patients or from a guardian on behalf of the patients. Availability of data and materials All data generated or analyzed during this study are included in the article. Raw data can be made available upon request to the corresponding author. Funding None Authors’ Contributions All authors made substantial contributions to the study. EM, DS and PS conceived and designed the study. EM and PS collected sample, investigated and recorded the laboratory findings at the hospital. DS, BB and BPB curated the data and analyzed the data. EM and DS administered the project, reviewed literature, and drafted the manuscript. DS helped with critical review and revision of the manuscript by compiling, formatting, editing and writing the final version of the manuscript. All authors read and approved the final manuscript. References 1. Antimicrobial resistance [https://www. who.int/en/news-room/fact-sheets/detail /antimicrobial-resistance ] 2. The global threat of antibiotic resistance [https://www.reactgroup.org/antibiotic- resistance/the-threat/] 3. Philippon A, Labia R, Jacoby G: Extended- spectrum beta-lactamases. Antimicrob Agents Chemother.1989 33(8):1131-1136. 4. Knothe H, Shah P, Krcmery V, Antal M, Mitsuhashi S: Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection. 1983, 11(6):315-317. 5. Pokharel BM, Koirala J, Dahal RK, Mishra SK, Khadga PK, Tuladhar NR: Multidrug- resistant and extended-spectrum beta- lactamase (ESBL)-producing Salmonella enterica (serotypes Typhi and Paratyphi A) from blood isolates in Nepal: surveillance of resistance and a search for newer alternatives. Int J Inf Dis. 2006 10(6):434-438. 6. Walsh TR: Emerging carbapenemases: a global perspective. Int J Antimicrob Agents. 2010 36:S8-S14. 7. Watanabe M, Iyobe S, Inoue M, Mitsuhashi S: Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1991 35(1):147-151. 8. Mishra SK, Acharya J, Kattel HP, Shah AS, Rijal BP, Pokhrel BM: Metallo-beta-lactamase producing bacterial isolates: First report from Nepal. In. Kathmandu: IOM souvenir 2009: 57. 9. Shah AA, Hasan F, Ahmed S, Hameed A: Characteristics, epidemiology and clinical importance of emerging strains of Gram- negative bacilli producing extended- spectrum β-lactamases. Res Microbiol. 2004 155(6):409-421. 10. Bauer AW, Kirby WMM, Sherris JC, Turck M: Antibiotic Susceptibility Testing by a Nepal Journal of Biotechnology. D e c . 2 0 1 9 Vol. 7, No. 1: 74-81 Maharjan et al. 2019 ©NJB, Biotechnology Society of Nepal 81 Nepjol.info/index.php/njb. Standardized Single Disk Method. Am J Clin Pathol 1966 45(4):493-496. 11. Clinical and Laboratory Standards Institute (CLSI): Performance standards for antimicrobial susceptibility testing, in twenty-fourth informational supplement (M100-S24). In. Wayne: Clinical and Laboratory Standards Institute; 2014. 12. Mishra SK, Acharya J, Kattel HP, Koirala J, Rijal BP, Pokhrel BM: Metallo-beta-lactamase producing gram-negative bacterial isolates. J Nepal Health Res Counc. 2012 10(22):208-213. 13. Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y: Imipenem-EDTA disk method for differentiation of metallo-beta-lactamase- producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol. 2002 40(10):3798-3801. 14. Bonomo RA: β-Lactamases: A Focus on Current Challenges. Cold Spring Harb Perspect Mede 2017 7(1). 15. Lakshmi R, Nusrin KS, Ann GS, Sreelakshmi KS: Role of beta-lactamases in antibiotic resistance: a review. Int Res J Pharm. 2014 5(2):37-40. 16. Khanal S, Joshi DR, Bhatta DR, Devkota U, Pokhrel BM: β -Lactamase-Producing Multidrug-Resistant Bacterial Pathogens from Tracheal Aspirates of Intensive Care Unit Patients at National Institute of Neurological and Allied Sciences, Nepal. In: ISRN Microbiol. 2013 2013: 847569. 17. Pokhrel RH, Thapa B, Kafle R, Shah PK, Tribuddharat C: Co-existence of beta- lactamases in clinical isolates of Escherichia coli from Kathmandu, Nepal. BMC Res Notes. 2014 7(1):694. 18. Ansari S, Nepal HP, Gautam R, Shrestha S, Neopane P, Gurung G, et al: Community acquired multi-drug resistant clinical isolates of Escherichia coli in a tertiary care center of Nepal. Antimicrob Resist Infect Control 2015 4(1):15. 19. Yadav KK, Adhikari N, Khadka R, Pant AD, Shah B: Multidrug resistant Enterobact- eriaceae and extended spectrum β-lactamase producing Escherichia coli: a cross-sectional study in National Kidney Center, Nepal. Antimicrob Resist Infect Control. 2015 4(1):42. 20. Nepal K, Pant ND, Neupane B, Belbase A, Baidhya R, Shrestha RK, et al: Extended spectrum beta-lactamase and metallo beta- lactamase production among Escherichia coli and Klebsiella pneumoniae isolated from different clinical samples in a tertiary care hospital in Kathmandu, Nepal. Ann Clin Microbiol Antimicrob. 2017 16(1):62. 21. Poudyal S, Bhatta DR, Shakya G, Upadhyaya B, Dumre SP, Buda GC, et al: Extended- Spectrum beta-lactamase producing multidrug resistant clinical bacterial isolates at National Public Health Laboratory, Nepal. Nepal Med Coll J. 2011 13(1):34-38. 22. Nepal HP, Neopane P, Shrestha R, Gautam R, Paudel R, Ansari S, et al: Biofilm formation and antimicrobial resistance in Klebsiella pneumoniae isolated from patients visiting a tertiary care center of Nepal. Asian Pac J Trop Dis. 2017 7(6):347-351. 23. Raut S, Gokhale S, Adhikari B: Prevalence of extended-spectrum beta-lactamases among Escherichia coli and Klebsiella spp isolates in Manipal Teaching Hospital, Pokhara, Nepal. J Microbiol Infect Dis. 2015 5(2):69-75. 24. Franklin C, Liolios L, Peleg AY: Phenotypic Detection of Carbapenem-Susceptible Metallo-beta-Lactamase-Producing Gram- Negative Bacilli in the Clinical Laboratory. J Clin Microbiol. 2006 44:3139-3144. 25. Gaur A, Garg A, Prakash P, Anupurba S, Mohapatra TM: Observations on carbapenem resistance by minimum inhibitory concentration in nosocomial isolates of Acinetobacter species: an experience at a tertiary care hospital in North India. J Health Popn Nutr. 2008 26(2):183-188. 26. Lee K, Lim CH, Cho JH, Lee WG, Uh Y, Kim HJ, et al: High Prevalence of Ceftazidime- Resistant Klebsiella pneumoniae and Increase of Imipenem-Resistant Pseudomonas aeruginosa and Acinetobacter spp. in Korea: a KONSAR Program in 2004. Yonsei Med J. 2006 47(5):634-645. 27. 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(3):268-281. 28. Vaara M: Polymyxins and Their Potential Next Generation as Therapeutic Antibiotics. Front Microbiol. 2019 10:1689. 29. Petrosillo N, Taglietti F, Granata G: Treatment Options for Colistin Resistant Klebsiella pneumoniae: Present and Future. J Clin Med. 2019 8:934. 30. Karaiskos I, Lagou S, Pontikis K, Rapti V, Poulakou G: The “Old” and the “New” Antibiotics for MDR Gram-Negative Pathogens: For Whom, When, and How. Front Public Health. 2019, 7:151. 31. World Health Organization (WHO): Global Action Plan on Antimicrobial Resistance. In. Edited by (WHO) WHO. Geneva: WHO; 2015.