Carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter baumannii clinical isolates from tertiary care in Nepal

Carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter baumannii clinical isolates from tertiary care in Nepal | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter baumannii clinical isolates from tertiary care in Nepal Gopiram Syangtan, Laxmi Kant Khanal, Srijana Bista, Arun Bahadur Chand, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9231883/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Background: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) were listed on the WHO bacterial priority pathogens list in 2017. These gram-negative bacteria often cause healthcare-associated infections (HAIs), which pose an increasing threat to public health. This study aimed to isolate and identify CRPAs and CRABs from a tertiary care hospital in central Nepal and characterize carbapenem resistance genes at the molecular level. Materials and method s: A total of 59 Pseudomonas aeruginosa ( n =27) and Acinetobacter baumannii ( n =32) isolates were collected from different clinical samples from April 2021 to December 2022. Antibiotic susceptibility was determined via the Kirby disc diffusion method, and the carbapenem inactivation (CIM) method was used for phenotypic confirmation of carbapenem resistance. Conventional polymerase chain reaction (PCR) was used to detect carbapenem resistance genes. Results: Among the 59 isolates, 76.3% were multidrug resistant (MDR), 63.0% were extended-spectrum beta-lactamase (ESBL), and 62.7% were carbapenem resistant. Among the carbapenem-resistant isolates ( n = 37), 75.7% of metallo-beta-lactamase (MBL) producers and 70.3% carried at least one carbapenem resistance gene. The bla CTX-M gene was predominant among the ESBL-positive isolates, present in 58.8% of the P. aeruginosa isolates and 87.5% of the A. baumannii isolates, whereas 29% of both isolates harbored the bla TEM gene. Among the CR-PA isolates, 58.3% carried bla VIM-2 , 16.7% carried bla NDM-1 , and 8.33% carried bla OXA-23 . Among the CR-AB isolates, 56% harbored bla OXA-23 , 32% carried bla NDM-1 , 16% had both bla NDM-1 and bla OXA-23 , and 8% contained both bla OXA-58 and bla OXA-23 . Conclusion: This study highlights a significant burden of MDR and carbapenem-resistant P. aeruginosa and A. baumannii , with the co-occurrence of carbapenem resistance genes, in a clinical setting in Nepal. Our findings suggest that active surveillance of antibiotic resistance in pathogens could inform clinicians and healthcare providers about the treatment of infections. Carbapenem resistance ESBLs MBL P. aeruginosa A. baumannii co-occurrence Nepal Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Pseudomonas aeruginosa and Acinetobacter baumannii are included in the ESKAPE ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , A. baumannii , P. aeruginosa , and Enterobacter species ) group of pathogens identified by the World Health Organization (WHO) as critical-priority pathogens ( 1 ), indicating urgent interventions with novel antibiotics. These bacteria are epidemiologically significant opportunistic gram-negative bacteria ( 2 ), which often cause hospital-acquired infections (HAIs), such as catheter-associated urinary tract infections, surgical site infections, ventilator-associated pneumonia, central line-associated bacteremia, septicemia, meningitis, malignant external otitis, intra-abdominal infection, and wound infections ( 3 ). The ability of A. baumannii and P. aeruginosa in a hospital environment enhances antimicrobial resistance, posing a notable mortality threat, especially in developing countries such as Nepal ( 4 ). Infections caused by these bacteria pose a treatment challenge because of their emerging intrinsic and acquired resistance to different classes of antibiotics worldwide( 5 ). Carbapenem, a broad-spectrum beta-lactam antibiotic, is frequently used as a "last-line" drug of choice for the empirical treatment of critically ill patients infected with these bacteria ( 6 ). Multiple antibiotic resistance mechanisms in A. baumannii and P. aeruginosa include the overexpression of efflux pumps, alterations in porin expression, and enzymatic inactivation ( 2 ). Beta-lactam hydrolyzing enzymes such as Ambler class D (oxacillinase-23, 24, 58, and 143), Ambler class B (metallo-beta-lactamases (MBLs): IMP, VIM, NDM, SPM, GIM, and SIM), and Ambler class A (extended-spectrum beta-lactamases (ESBLs): TEM, SHV, and CTX) are responsible for antibiotic resistance in bacterial isolates ( 2 ). Over the past two decades, the burden of antibiotic resistance related to MDR bacteremia has increased substantially in Nepal due to the widespread use of antibiotics ( 7 , 8 ). Previous reports have shown that last-resort antibiotics such as carbapenems and polymyxin are used excessively in low- to middle-income countries (LMICs) in healthcare settings ( 9 ). The cause of such infection is a greater risk of treatment failure, especially in critically ill patients, those with longer hospital stays, and those with increased healthcare costs. Recent studies have shown high carbapenem resistance among E. coli , K. pneumoniae , A. baumannii , and P. aeruginosa in Southeast Asia, including Nepal ( 6 , 10 , 11 ). A recent study in Nepal reported that more than 75% of P. aeruginosa and A. baumannii are phenotypically carbapenem resistant ( 12 , 13 ). Additionally, these pathogens are highly resistant to multiple antibiotics due to intrinsic and acquired resistance mechanisms, including biofilm formation, enzymatic inactivation, and efflux pump formation. Coinfection by these pathogens leads to severe disease, leading to deadly outcomes ( 4 ). Moreover, the prevalence of these two pathogens and their antibiotic resistance is greater in Nepal ( 12 , 13 ). This highlights the importance of focusing on P. aeruginosa and A. baumannii from the ESKAPE group because of their high resistance, nosocomial prevalence, and clinical severity. However, few studies have reported carbapenemase-associated genes in CR-PA and CR-AB isolates ( 14 , 15 ). Extensive information on the distribution of carbapenemase enzymes and associated genes in CR-PA and CR-AB within clinical settings remains limited. Our study aimed to identify phenotypes and genotypes to characterize carbapenemase-producing A. baumannii and P. aeruginosa from a tertiary care hospital in central Nepal. Genotypic testing is essential for accurate antibiotic susceptibility results, as it reveals the prevalence and types of carbapenemase-encoding genes carried by phenotypically antibiotic-resistant bacteria in clinical environments. ( 9 ). The findings of this study might aid in understanding antibiotic resistance patterns and genetic epidemiological data for antibiotic stewardship programs and in developing a new strategic action plan for the treatment of MDR and carbapenem-resistant isolates for clinicians or healthcare providers in Nepal. Materials and methods Ethical considerations: Ethical approval (IRC no. 077/78/54) was obtained from the Institutional Ethical Review Committee of the KIST Medical College and Teaching Hospital, Tribhuvan University. Informed consent was obtained from each participant after clearly mentioning the benefits and possible outcomes of this study. After providing consent, demographic information was recorded, and the samples were tracked and identified via a laboratory information system (LIS-Midas Technology) in the hospital. The personal details of the participants were kept confidential and archived. Study design, site, and period A hospital-based cross-sectional study was conducted from April 2021 to December 2022 at the KIST Medical College and Teaching Hospital (KMCTH), a 900-bed superspecialty tertiary care hospital, and the Central Department of Microbiology, Tribhuvan University, Nepal. Sample collection and processing We included all samples from the culture/susceptibility (C/S) inquiry tests requested by clinicians for both inpatients and outpatients during our study period. A total of 1207 clinical samples, such as body fluids, swabs, sputum, urine, pus, blood, and tracheal aspirates, were randomly collected and processed onto culture media (blood agar, MacConkey agar, and chocolate agar (Himedia Laboratories, India)) and incubated at appropriate temperatures and times according to standard protocols at the clinical service laboratory (KMCTH) (16,17). (Describe below). Among the culture-positive isolates, A. baumannii and P. aeruginosa recovered from the various samples during the study period were included. AST was performed following the Kirby–Bauer disk diffusion method as per CLSI 2020 (18). Bacterial isolation and identification Blood agar, MacConkey agar, and chocolate agar were used to culture each sample, which was subsequently incubated for 24 hours at 37 °C. After incubation, colony morphology characterization, Gram staining, and different biochemical tests were performed to identify the isolates. Furthermore, P. aeruginosa and A. baumannii must be confirmed via special characteristic tests, which are explained below. P. aeruginosa isolates were identified as nonlactose fermenting, gram-negative slender rods that exhibited motility, thrived at 42 °C, were pigmented on cetrimide agar, and grew aerobically on MacConkey agar (19). The 27 phenotypically confirmed P. aeruginosa isolates were subsequently obtained and preserved in a tryptic soy broth tube with 40% glycerol until further experiments. Acinetobacter spp. were initially identified as nonlactose-fermenting gram-negative cocobacilli isolates with aerobic growth on MacConkey agar at 44 °C(20). The phenol: chloroform method of DNA extraction was subsequently employed (21), and genotypic identification of A. baumannii was subsequently performed by detecting the bla OXA-51 -like gene (20). The 32 confirmed isolates of A. baumannii were preserved until further experiments. Antimicrobial susceptibility testing To gauge the antimicrobial susceptibility patterns of the isolates, the Kirby–Bauer disk diffusion method was applied via Muller–Hinton agar (Hi-media Laboratories, India), and the results were interpreted according to CLSI (2020) (18). The following antimicrobial discs were used for breakpoint determination: piperacillin (PI; 100 µg), piperacillin/tazobactam (PIT; 100/10 μg), ceftriaxone (CTR; 30 μg), ceftazidime (CAZ; 30 μg), cefotaxime (CTX; 30 μg), cefepime (CPM; 30 μg), aztreonam (AT; 30 μg), imipenem (IMP; 10 μg), meropenem (MRP; 10 μg), amikacin (AK; 30 μg), gentamicin (GEN; 10 μg), and ciprofloxacin (CIP; 5 μg). (Himedia Laboratories, India). We considered MDR isolates that were resistant to two or more antibiotics from different classes, such as beta-lactam, β-lactam combination, aminoglycoside, carbapenem, and fluoroquinolone antibiotic classes (14). Phenotypic detection of ESBLs The isolates that were resistant to cefotaxime (30 µg) and/or ceftazidime (30 µg) were tested for ESBL production via combined disc methods involving the use of ceftazidime (30 µg) and ceftazidime/clavulanic acid (30 µg/10 µg) discs and cefotaxime (30 µg) and cefotaxime/clavulanic acid (30 µg/10 µg) discs. A disc was placed 20 mm apart, and a zone of ≥ 5 mm in the cefotaxime or ceftazidime with clavulanic acid disc compared with the cefotaxime or ceftazidime disc alone was considered positive for ESBL production after overnight culture on MHA plates (18). Phenotypic detection of carbapenem resistance enzymes (MBLs) Bacterial isolates that showed intermediate resistance or resistance to imipenem or meropenem were suspected to be carbapenemase producers (22). An ethylenediaminetetraacetic acid (EDTA) synergy test was used to confirm the metallo-β-lactamase production phenotype. In this method, ethylenediaminetetraacetic acid (EDTA) was used for synergistic effects with imipenem discs [imipenem and imipenem + EDTA (10 µg/750 µg)], and the zone of inhibition was compared with that of imipenem alone (23). MIC test for carbapenem The minimal inhibitory concentration (MIC) of a carbapenem (meropenem) for P. aeruginosa and A. baumannii was determined via the agar dilution method (18). The positive controls used E. coli ATCC 25922 as a reference strain and inoculated the organism, whereas the negative control was considered the only culture medium used to ensure sterility. After 24 hours of incubation at 37 °C, the MIC readings were recorded and interpreted according to the CLSI M100 (18). Isolates with carbapenem MICs of ≥8 μg/ml were considered resistant, those with MICs of 4 μg/ml were considered intermediate, and those with MICs of ≤2 μg/ml were considered susceptible (18). Molecular detection of ESBL- and carbapenemase - encoding genes The extraction of plasmid DNA from bacterial cultures in the logarithmic growth phase was conducted via the phenol:chloroform method (21). The quality and purity of the extracted DNA template were measured via NanoDrop measurements (Nanodrop, Thermo Scientific, USA). The single-plex PCR assay was carried out via a thermal cycler (Bio-Rad, USA) with primer pairs (Macrogen, Korea) for blaCTX-M, blaTEM, blaNDM-1, blaVIM-2, blaOXA-23, blaOXA-51, and blaOXA-58 to detect beta-lactamase genes (Supplementary information, S1 Table). The reaction was carried out in a total volume of 25 µl (12.5 μl of 1X FIREpol Master Mix (Solis Biodyne, Estonia), 3 μl of template genomic DNA, 3.0 μl of 10 picomole primers (1.5 μl of forward primer and 1.5 μl of reverse primer), and 6.5 μl of nuclease-free water). The PCR products were visualized by agarose gel electrophoresis (1.5%) with DNA-stained safe dye (Sigma–Aldrich, Germany) at 70 V for 50 minutes. The band sizes were compared along a 100 bp ladder. The amplified ESBL and carbapenemase genes were visualized in a gel documentation system (Azure Biosystems, USA) and compared with a 100 bp DNA ladder (Thermo Scientific, USA). Quality control The sterility of freshly prepared culture media was confirmed by incubating 5% of the media plates at 37 °C for 24 hours to check for potential contamination. The quality of the culture media and antibiotic disks was checked by assessing the performance with E. coli ATCC 25922. Data analysis and interpretation SPSS (Student version) was used to analyze the data. Chi-square tests were used to determine the associations among the frequency of samples, antibiotic-resistant isolates, multidrug-resistant isolates, beta-lactam enzyme-producing isolates, and the MIC value of carbapenem. P values < 0.05 were considered statistically significant. The figure was created via GraphPad Prism software. Results During the study period, 1,207 samples were analyzed, and 22.5% (n = 272) were positive according to bacterial culture. Among the total positive cultures, 20.2% (n = 50/272) were gram-positive, and 79.8% (n = 217/272) were gram-negative (S-Fig. 1 ). Isolates of A. baumannii and P. aeruginosa were recovered from 32 and 27 samples, respectively. The total prevalence of nonlactose fermenter-negative bacteria (NLF-GNB) was 4.9% (n = 59/1207), 3.7% was MDR (n = 45/1207), 2.0% was ESBL (n = 24/1207), 3.1% was carbapenem resistant, and 2.3% (n = 28/1207) was an MBL producer in the total sample (Fig. 1 ). Distribution of NLF-GNB isolates and MDR, ESBL, carbapenem resistance, and MBL-producing isolates with demographic data (age and sex) and hospital settings (Table 1 ). Among the isolates, a higher prevalence of MDR, carbapenem resistance, ESBL, and MBL producers was detected in females than in males, but the difference was not statistically significant. In the age group category, a higher rate (37.8%; 17/45) of MDR was found in the 41–60-year age group, followed by the 21–40-year and older age (≥ 61 years) groups. In contrast, carbapenem-resistant, ESBL-producing, and MBL-producing isolates were more prevalent in the younger age group (21–40 years), followed by the 41–60 years and ≥ 61 years age groups. The predominant MDR and carbapenem-resistant isolates were found in inpatient populations rather than in OPD patients. However, there was no statistically significant difference between these two groups of populations (Tables 1 and 2 ). In total, most of the isolates were obtained from sputum samples (40.7%, n = 24), followed by pus/wound swabs (20.3%, n = 12) (Table S2). We found a significant difference in the distribution of NLF-GNB isolates among different types of samples ( p = 0.006). Table 1 Demographic distributions of NLF-GNB Variables Total isolate (n = 59) MDR (n = 45/59) ESBL(n = 24/59) Carbapenem Resistance(n = 37/59) MBL (n = 28/59) Demographics P. aeruginosa (n = 27) A. baumannii (n = 32) P. aeruginosa (n = 19) A. baumannii (n = 26) P. aeruginosa (n = 17) A. baumannii (n = 7) P. aeruginosa (n = 12) A. baumannii (n = 25) P. aeruginosa (n = 10) A. baumannii (n = 18) Gender (n/%) Male (n = 27) 13 (48.1) 14 (43.7) 9 (47.4) 11 (42.3)) 9 (52.9) 2 (28.6) 5 (41.7) 9 (36.0) 5 (50.0) 7 (38.9) Female (n = 32) 14 (51.9) 18 (56.3) 10 (52.6)) 15 (57.7) 8 (47.1) 5 (71.4) 7 (58.3) 16 (64.0) 5 (50.0) 11 (61.1) P value 0.7355 0.7358 0.3864 0.7394 0.5692 Age Group (n/%) (Years) ≤ 20 (n = 7) 2 (7.4) 5 (15.6) 1 (5.3) 5 (19.2) 1 (5.9) 1 (14.3) 0 (0) 4 (16.0) 0 (0) 4 (22.2) 21–40 (n = 19) 6 (22.2) 13 (40.7) 5 (26.3) 10 (38.5) 5 (29.4) 4 (57.1) 3 (25.0) 10 (40.0) 3 (30.0) 8 (44.4) 41–60 (n = 18) 9 (33.3) 9 (28.1) 7 (36.8) 8 (30.8) 6 (35.3) 2 (28.6) 4 (33.3) 8 (32.0) 4 (40.0) 3 (16.7) ≥ 61 (n = 15) 10 (37.1) 5 (15.6) 6 (31.6) 3 (11.5) 5 (29.4) 0 (0) 5 (41.7) 3 (12.0) 3 (30.0) 3 (16.7) P value 0.1615 0.2197 0.3127 0.1247 0.2126 Hospital Setting (n/%) Inpatients (n = 36) 15 (55.6) 21 (65.6) 12 (63.2) 16 (61.5) 8 (47.1) 5 (71.4) 8 (66.7) 19 (76.0) 6 (60.0) 13 (72.2) Outpatients (n = 23) 12 (44.4) 11 (34.4) 7 (38.8) 10 (38.5) 9 (52.9) 2 (28.6) 4 (33.3) 6 (24.0) 4 (40.0) 5 (27.8) P value 0.8908 0.9119 0.3864 0.6964 0.6775 Table 2 Prevalence of P. aeruginosa and A. baumannii in different samples collected from outpatients. Specimen Inpatients (n = 36) Outpatients (n = 23) P. aeruginosa (n/%) A. baumannii (n/%) P. aeruginosa (n/%) A. baumannii (n/%) Sputum (n = 24) 5 (22.8) 9 (37.5) 3 (12.5) 7 Pus/Wound swabs (n = 12) 6 (50.0) 2 (16.7) 3 (25.0) 1 (8.3) Urine (n = 9) 5 (55.6) - 2 (22.2) 2 (22.2) Blood (n = 6) 1 (16.7) 3 (50.0) - 2 (33.3) Tips (ET, Catheter & Foleys) (n = 8) 1 (12.5) 4 (50.0) 1 (12.5) 2 (25.5) Antimicrobial susceptibility test of the isolates The majority of P. aeruginosa isolates were resistant to ceftazidime (63%), followed by gentamycin (59.3%) (Table S3). In the case of A. baumannii (n = 32), the highest level of resistance was observed against ceftriaxone (96.1%), followed by ceftazidime (93.7%), cefotaxime (87.5%), piperacillin (87.5%), ciprofloxacin (87.5%), piperacillin-tazobactam and cefepime (84.4%), gentamycin (81.3%) and carbapenem (78.1%). Carbapenem-resistant (CR) isolates Among the 59 isolates, 62.7% (n = 37/59) were found to be resistant to both meropenem and imipenem (carbapenem) (Table S3). Among the 37 CR isolates, the MICs of meropenem for P. aeruginosa (n = 12) and A. baumanni (n = 25) ranged from 32 µg/mL to 512 µg/mL and from 32 µg/mL to 256 µg/mL, respectively (Fig. 2). The highest percentage of CR isolates were found in sputum samples (37.8%, n = 14/37), followed by pus/wound swabs (n = 8/37, 21.6%) and tips, including ETs, Catheter, and Foleys (6.2%, n = 6/37). The frequency of CR isolates significantly differed according to the specimen type ( p = 0.004) (Table S2). Prevalence of multidrug resistant, ESBL-producing, and carbapenemase-producing NLF-GNB strains Among the 59 isolates, 45 (76.3%) were confirmed as MDR isolates, among which 57.8% were A. baumannii and 42.2% were P. aeruginosa . Among the total isolates, 24 were confirmed to be ESBL positive, and P. aeruginosa ( n = 17) was more common than A. baumannii ( n = 7). Among the 37 carbapenemase producers, 75.7% of the isolates were metallo-β-lactamase (MBL)-positive, comprising 64.3% A. baumannii and 35.7% P. aeruginosa . The frequencies of MDR, ESBL, and carbapenemase-producing NLF-GNB (MBL) strains are summarized in Fig. 3 . Distribution and co-occurrence of carbapenemase genes All the isolates (n = 59) were examined for the presence of four carbapenemase genes, MBLs ( bla NDM−1 and bla VIM−2 ), and OXA ( bla OXA−23 and bla OXA−58 ), which are carbapenem resistance genes, via specific target primers described previously (Table S1 ). Among the CR isolates, 70.2% (n = 26/37) of the carbapenem-resistant isolates were found to harbor at least one or a combination of carbapenemase-encoding genes. Among these, 45.9% (n = 17/37) of the isolates contained metallo-β-lactamase (MBL) genes (Ambler class B), including bla NDM−1 in 10 isolates and bla VIM−2 in 7 isolates. Additionally, 48.6% (n = 18/37) of the isolates harbored OXA genes (Ambler class D), with bla OXA−23 present in 16 isolates and bla OXA−58 in 2 isolates. In the CRPA, bla VIM−2 was dominant (58.3%, n = 7/12), followed by bla NDM−1 and bla OXA−23 (16.7%, n = 2/12). Similarly, bla OXA−23 was highest (56.0%, n = 14/25) in CR-AB, followed by bla NDM−1 (32.0%, n = 8/25) and bla OXA−58 (8.0%, n = 2/25) (Table S7). Although 11 isolates (four P. aeruginosa isolates and seven A. baumannii isolates ) were phenotypically carbapenem resistant, the associated genes were undetected. Interestingly, one of the carbapenem-susceptible isolates carried the bla NDM−1 and bla OXA−23 genes (Table S7). In total, 21.6% ( n = 8/37) of the CR isolates coharbored more than one carbapenemase-encoding gene, whereas 47.4% ( n = 18/37) of the isolates harbored either a single gene (Fig. 4 ). Specifically, four CR-AB isolates carried bla NDM−1 + bla OXA−23 , two CR-AB isolates carried bla OXA−23 + bla OXA−58 , and one CR-PA isolate tested positive for bla NDM−1 + bla VIM−2 . Additionally, one CR-PA isolate was found to carry all three different carbapenemase-encoding genes ( bla NDM−1 + bla VIM−2 + bla OXA−23 ). ESBL gene cooccurrences with carbapenemase genes A total of 24 phenotypic ESBL-positive isolates were examined for the presence of ESBL-encoding genes via PCR; 66.7% (n = 16/24) and 29.2% (n = 7/24) of the isolates contained the bla CTX−M and bla TEM genes, respectively. Among them, 25% (n = 6/24) of the isolates harbored both the bla CTX−M and bla TEM genes in four of the P. aeruginosa isolates and two of the A. baumannii isolates. A higher frequency of bla CTX−M was detected in A. baumannii (85.7%), whereas bla TEM was more frequent in P. aeruginosa (29.4%). Cooccurrence of carbapenemase genes with ESBL genes Among the 37 CR isolates, 35.1% were ESBL positive. Among them, the majority of the isolates coharbored the ESBL gene. Specifically, five bla VIM−2 (all CR-PA), three bla NDM−1 (2 CR-PA and 1 CR-AB), three bla OXA−23 (3 CR-AB), and one bla OXA−58 (one CR-AB) isolate coexisted with bla CTX−M . Additionally, bla TEM cooccurred with one bla VIM−2 (in CR-PA), one bla NDM−1 (in CR-AB), and two bla OXA−23 (in CR-AB) isolates (Table S7). Discussions In this study, one-fifth of the total clinical specimens were bacterial culture positive; among them, 21.69% were A. baumannii and P. aeruginosa . CR-AB and CR-PA are the most common pathogens associated with nosocomial infections in Asian countries, including Nepal (10). A. baumannii and P. aeruginosa are frequently isolated from sputum and wound/pus samples. Carbapenem-resistant isolates were also more prevalent in those two samples, which may be due to their greater representation or could be linked to their ability to adapt to and resist specific environments where they persist. These results suggest that the dominance of antibiotic-resistant bacteria may differ in terms of survival mechanisms at different anatomical sites (29). Approximately three-fourths of the A. baumannii isolates and half of the P. aeruginosa isolates were resistant to third-generation cephalosporin and carbapenem antibiotics, which reflected the comparative differences in resistance rates among the pathogens and could be attributed to individual intrinsic resistance mechanisms. (30). Interestingly, our antimicrobial pattern was similar to several earlier observations of antibiotic resistance in Nepal. (14,31,32). Among the A. baumannii isolates, approximately 80% were carbapenem resistant, which is a relatively lower prevalence than that reported in the phenotypic research conducted by Joshi et al. (97.7%) in Kathmandu (14). In contrast, another study conducted in tertiary care hospitals in Nepal reported a lower rate of CR-AB (51%) (33) than our study did. Similarly, our neighboring countries, India (14.3%) (34) and Pakistan (22.0%) (35), also reported a low prevalence of CR-AB in clinical samples. These differences could be due to the distributions of pathogens being influenced by microbial ecology, the hospital environment, infection control practices, patient care practices, coinfections, antibiotic use, study populations, geographic areas, and sample processes (11). In our findings, approximately 45% of P. aeruginosa isolates were carbapenem resistant, which is similar to the results of a study conducted by Olalekan et al. in Nigeria. (36). However, in South Asian countries, Bangladesh reported approximately 40% carbapenem resistance, whereas Pakistan reported 65% carbapenem resistance in P. aeruginosa isolates (37). In the global scenario, approximately one-fifth of P. aeruginosa are carbapenem resistant, which is even lower than that in the Asia Pacific region (38). These results suggest that CR-PA is gradually increasing in Nepal in hospital settings. Moreover, our study reported that the MICs of meropenem were highly different for both P. aeruginosa (32--512 µg/mL) and A. baumannii (32--256 µg/mL), with a statistically significant difference that indicates an increasing rate of carbapenem resistance, which could be related to the uncontrolled consumption of carbapenem antibiotics (8). In our study, 81.3% of A. baumannii strains were MDR, which was higher than that reported in a previous study conducted in Nepal. Studies conducted in various regions of Nepal reported 30.7%-75.9% rates of MDR bacteria. (15,33,39). In addition, 70.4% of the P. aeruginosa isolates were MDR in our study, which disagrees with the findings of other studies in Nepal: the percentage ranged from 14.3% to 60.1% (15,33,39). These results show increasing trends of MDR isolates in our clinical setting, which create limited treatment options and higher mortality and morbidity rates, with respiratory tract infection, surgical site infection, and increased risk of spread in hospital environments. This study reported a substantial prevalence of ESBL production among A. baumannii and P. aeruginosa isolates, accounting for 40.7% of phenotypically identified organisms (Table S4). Notably, this study showed similarities with ESBL-producing P. aeruginosa from Kathmandu. (33). The occurrence of bla TEM and bla CTX-M genes in A. baumannii was relatively lower than that in P. aeruginosa . This study revealed that the bla CTX-M (66.7%) gene was more responsible for ESBL production than the bla TEM (29.2%) gene was and that 25% of both genes coexisted. Similar findings were reported from India and Gaza in A. baumannii and P. aeruginosa (34,40). In addition, a study conducted on Uro-pathogenic E. coli at Kathmandu reported a greater presence of bla CTX-M (87.5%) than bla TEM (53.6%) among ESBL-producing isolates (41), which indicates that the bla CTX-M genes might predominantly circulate in this area. We reported that almost half (76.7%) of our carbapenem-resistant isolates produced MBL, two-thirds were A. baumannii, and one-third were P. aeruginosa . These results are well supported by a study conducted in Iran by Namaei et al. 2021 (42). However, an earlier study described a lower prevalence of MBL-producing isolates in the eastern part of Nepal (32,43) and Kathmandu (33) than our findings did. In A. baumannii, the MBL production rate ranges from 54–66.7% (32,44), whereas in P. aeruginosa , it ranges from 8–45% of the representative isolates (31–33). According to our observations, these reports help predict the increasing trend of CR-AB and CR-PA in Nepal. In this study, P. aeruginosa and A. baumannii isolates harboring a variety of carbapenemase resistance genes, including bla NDM-1 , bla VIM-2 , bla OXA-23, and bla OXA-58 , were identified (Table S7). The cooccurrences of bla NDM-1 and bla VIM-2 in P. aeruginosa and bla NDM-1 , bla OXA-23, and bla OXA-58 in A. baumannii were reported among carbapenem-resistant isolates as widespread antibiotic resistance genes. A relatively high MIC range (reduced antibiotic susceptibility rate) against the tested antibiotics was observed in MBL carbapenemase-producing isolates, which mostly harbored the bla VIM-2 and bla NDM-1 genes (Table S7). This finding is in agreement with various studies that have successfully reported the coexistence of antibiotic resistance genes among clinical isolates (45). These multiple resistance genes found in the same isolates, as observed in our study, are indicative of the existence of multidrug-resistant pathogens that could be responsible for the difficulty in treating and curing diseases. The bla VIM-2 is the dominant gene in MBL-producing P. aeruginosa and is associated with nosocomial infection (46). In our study, bla VIM-2 was also the predominant MBL gene among the CR-PAs cooccurring with bla NDM-1 . Similarly, previous studies conducted in Nepal reported increased bla VIM-2 among MBL-producing CR-PAs (47,48) . In contrast, a study from Nigeria revealed that the bla NDM-1 gene was more predominant than the bla VIM-2 gene among MBL-producing isolates (36). In addition, more than half of the CR-AB isolates carried bla OXA-23 , which was the predominant gene in our study. Other similar studies also reported bla OXA-23 as predominant among CR-ABs (35,49). A previous study in Nepal, CR-AB, has shown that the coexistence of bla OXA-23 with bla NDM-1 is significant (14,50). This study also emphasized that bla OXA-23 and bla ONDM-1 could coexist in CR-AB more than in previous studies. Notably, 8% of the isolates had the bla OXA-58 gene, which actively coexists with bla OXA-23 . For the first time, in Nepal, bla OXA-58 was reported in clinical CR-AB isolates in 2015 (50). Moreover, very few CR-AB clinical isolates carry bla OXA-58 and are reported in the global context (50). The co-occurrence of bla OXA-23 with bla ONDM-1 or bla OXA-58 is a concern, as this will limit future therapeutic options for AB infections in a healthcare setting. Moreover, some carbapenem-susceptible isolates identified via phenotypic detection may indeed be positive for carbapenemase genes by genotyping (51). In the present study, we detected bla ONDM-1 and bla OXA-23 in one isolate and bla CTX-M in two isolates of P. aeruginosa via PCR assays, which were negative for phenotypic detection. These results suggest that molecular techniques are necessary tools for improving diagnostic approaches in healthcare settings (51). Further research is needed to detect the expression levels of resistance genes and genetic variance via mutational analysis by sequencing methods to understand the molecular mechanisms of antibiotic resistance. Conclusion Overall, this study revealed a high prevalence of MDR and carbapenem-resistant P. aeruginosa and A. baumannii isolates harboring multiple carbapenem resistance genes in the hospital setting of Nepal. This poses a significant challenge to the effective treatment and management of patients with currently available antibiotics in hospital settings, particularly in the developing countries of Nepal. Therefore, active surveillance and continuous monitoring of antibiotic resistance patterns in pathogens are essential to guide clinicians and healthcare providers in optimizing patient care and improving treatment outcomes. Abbreviations AMR Antimicrobial resistance CLSI Clinical Laboratory Standard Institute CR carbapenem resistance CR-PA carbapenem-resistant Pseudomonas aeruginosa CR-AB carbapenem-resistant Acinetobacter baumannii CPase carbapenemase CONS coagulase negative Staphylococcus EDTA ethylenediaminetetraacetic acid ESBLs extended-spectrum beta-lactamases GNB gram-negative bacilli HAI healthcare-associated infection IRC Institutional Review Committee KMCTH KIST Medical College and Teaching Hospital KPC Klebsiella pneumoniae carbapenemase LMICs low- and middle-income countries MBLs metallo-beta-lactamases MDR multidrug-resistant MIC minimum inhibitory concentration NGS next-generation sequencing NLF nonlactose fermenter PCR polymerase chain reaction SPSS Statistical Package for Social Sciences WHO World Health Organization. Declarations Acknowledgments The authors acknowledge the patients who provided consent and the clinical specimens for this study. Conflict of interest: All the authors declare that there are no conflicts of interest regarding the publication of this manuscript. The authors have no relevant financial or nonfinancial interests to disclose. Funding This study was carried out with the in-house resources of the Central Department of Microbiology, T.U., and with no specific grants from public, private, or nonprofit organizations. Data availability All the information produced during this research work is presented within the manuscript and in the Supplementary file. Any queries regarding the research will be addressed by the corresponding author upon reasonable request. Human Ethics and Consent to Participate Ethical approval for the study was obtained from the Institutional Review Committee (IRC) of KIST Medical College and Teaching Hospital (Ref. No.: 077/078/54). Written consent was taken from each patient for voluntary participation before collecting demographic data and samples. 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Supplementary Files SupplementryInformationdatafile.docx Supporting Information File: Supplementary_Information_data.docx The supporting information file includes the following data. S1 Fig. 1: Frequencies of total bacterial species isolated from total samples. CONS: Coagulase-negative Staphylococcus spp Table S1:PCR primers used for the detection of the ESBL and MBL genes in this study. Table S2:Types of samplesprocessed in this study and the numbers of GN-NLF isolates and carbapenem-resistant strains. Table S3: Results of the antibiotic resistance profile by disk diffusion among nonlactose-fermenting gram-negative isolates Table S4:Distributions of MDR, CR, ESBL, and MBL among NLF-GNB isolates Table S5:Minimum inhibitory concentrations (MICs) of meropenem in CR isolates. Table S6:Carbapenem resistancegenes in the NFL-GNB isolates from the PCR assay. Table S7: Coexistencegenes conferring resistance to the β-lactam carbapenem among the carbapenem-resistantisolates. 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resistance, and MBL-producing isolates among the total samples.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9231883/v1/d373cb772b5b14af15917e20.png"},{"id":106759271,"identity":"0537a351-27b9-4ecf-bc74-399bb37e4568","added_by":"auto","created_at":"2026-04-13 08:32:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":65943,"visible":true,"origin":"","legend":"\u003cp\u003eMIC (µg/mL) values of the CR isolates (A) and MBL-producing isolates (B)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9231883/v1/b23932817fca6cee56de8a44.png"},{"id":106759273,"identity":"c12db0bf-0d3b-4a44-bae0-a9ed5c7d625b","added_by":"auto","created_at":"2026-04-13 08:32:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":91764,"visible":true,"origin":"","legend":"\u003cp\u003eNo. of isolates, MDR isolates, ESBL-producing, carbapenem-resistant, and MBL-producing isolates.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9231883/v1/765234d4c90cfed5aa361337.png"},{"id":106960483,"identity":"28057545-8b2b-4285-a4fd-2fcb70736439","added_by":"auto","created_at":"2026-04-15 09:21:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":111737,"visible":true,"origin":"","legend":"\u003cp\u003eThe presence of single genes or the occurrence of ESBL (A) and carbapenemase (B) genes in \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eA. baumannii\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9231883/v1/8b79c047b56247833fee3090.png"},{"id":106964419,"identity":"c8849a7f-8720-4d7f-b58e-2b4437266650","added_by":"auto","created_at":"2026-04-15 09:50:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1497077,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9231883/v1/3064644b-b4c5-4401-b43f-d99d85327444.pdf"},{"id":106959711,"identity":"65868f94-c6ad-43e2-9bfc-a043e1374912","added_by":"auto","created_at":"2026-04-15 09:14:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":390574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupporting Information File: \u003c/strong\u003eSupplementary_Information_data.docx\u003c/p\u003e\n\u003cp\u003eThe supporting information file includes the following data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS1 Fig. 1: \u003c/strong\u003eFrequencies of total bacterial species isolated from total samples. CONS: Coagulase-negative \u003cem\u003eStaphylococcus \u003c/em\u003espp\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1:\u003c/strong\u003ePCR primers used for the detection of the ESBL and MBL genes in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2:\u003c/strong\u003eTypes of samplesprocessed in this study and the numbers of GN-NLF isolates and carbapenem-resistant strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S3: \u003c/strong\u003eResults of the antibiotic resistance profile by disk diffusion among nonlactose-fermenting gram-negative isolates\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S4:\u003c/strong\u003eDistributions of MDR, CR, ESBL, and MBL among NLF-GNB isolates\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S5:\u003c/strong\u003eMinimum inhibitory concentrations (MICs) of meropenem in CR isolates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S6:\u003c/strong\u003eCarbapenem resistancegenes in the NFL-GNB isolates from the PCR assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable \u003c/strong\u003eS7: Coexistencegenes conferring resistance to the β-lactam carbapenem among the carbapenem-resistantisolates.\u003c/p\u003e","description":"","filename":"SupplementryInformationdatafile.docx","url":"https://assets-eu.researchsquare.com/files/rs-9231883/v1/7e7ea2aa53c57c6c0fdab4ed.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter baumannii clinical isolates from tertiary care in Nepal","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e are included in the ESKAPE (\u003cem\u003eEnterococcus faecium\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eA. baumannii\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e, and \u003cem\u003eEnterobacter species\u003c/em\u003e) group of pathogens identified by the World Health Organization (WHO) as critical-priority pathogens (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), indicating urgent interventions with novel antibiotics. These bacteria are epidemiologically significant opportunistic gram-negative bacteria (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), which often cause hospital-acquired infections (HAIs), such as catheter-associated urinary tract infections, surgical site infections, ventilator-associated pneumonia, central line-associated bacteremia, septicemia, meningitis, malignant external otitis, intra-abdominal infection, and wound infections (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The ability of \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e in a hospital environment enhances antimicrobial resistance, posing a notable mortality threat, especially in developing countries such as Nepal (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Infections caused by these bacteria pose a treatment challenge because of their emerging intrinsic and acquired resistance to different classes of antibiotics worldwide(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Carbapenem, a broad-spectrum beta-lactam antibiotic, is frequently used as a \"last-line\" drug of choice for the empirical treatment of critically ill patients infected with these bacteria (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMultiple antibiotic resistance mechanisms in \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e include the overexpression of efflux pumps, alterations in porin expression, and enzymatic inactivation (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Beta-lactam hydrolyzing enzymes such as Ambler class D (oxacillinase-23, 24, 58, and 143), Ambler class B (metallo-beta-lactamases (MBLs): IMP, VIM, NDM, SPM, GIM, and SIM), and Ambler class A (extended-spectrum beta-lactamases (ESBLs): TEM, SHV, and CTX) are responsible for antibiotic resistance in bacterial isolates (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOver the past two decades, the burden of antibiotic resistance related to MDR bacteremia has increased substantially in Nepal due to the widespread use of antibiotics (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Previous reports have shown that last-resort antibiotics such as carbapenems and polymyxin are used excessively in low- to middle-income countries (LMICs) in healthcare settings (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The cause of such infection is a greater risk of treatment failure, especially in critically ill patients, those with longer hospital stays, and those with increased healthcare costs. Recent studies have shown high carbapenem resistance among \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eK. pneumoniae\u003c/em\u003e, \u003cem\u003eA. baumannii\u003c/em\u003e, and \u003cem\u003eP. aeruginosa\u003c/em\u003e in Southeast Asia, including Nepal (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA recent study in Nepal reported that more than 75% of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eA. baumannii\u003c/em\u003e are phenotypically carbapenem resistant (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Additionally, these pathogens are highly resistant to multiple antibiotics due to intrinsic and acquired resistance mechanisms, including biofilm formation, enzymatic inactivation, and efflux pump formation. Coinfection by these pathogens leads to severe disease, leading to deadly outcomes (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Moreover, the prevalence of these two pathogens and their antibiotic resistance is greater in Nepal (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). This highlights the importance of focusing on \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eA. baumannii\u003c/em\u003e from the ESKAPE group because of their high resistance, nosocomial prevalence, and clinical severity. However, few studies have reported carbapenemase-associated genes in CR-PA and CR-AB isolates (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Extensive information on the distribution of carbapenemase enzymes and associated genes in CR-PA and CR-AB within clinical settings remains limited. Our study aimed to identify phenotypes and genotypes to characterize carbapenemase-producing \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e from a tertiary care hospital in central Nepal. Genotypic testing is essential for accurate antibiotic susceptibility results, as it reveals the prevalence and types of carbapenemase-encoding genes carried by phenotypically antibiotic-resistant bacteria in clinical environments. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The findings of this study might aid in understanding antibiotic resistance patterns and genetic epidemiological data for antibiotic stewardship programs and in developing a new strategic action plan for the treatment of MDR and carbapenem-resistant isolates for clinicians or healthcare providers in Nepal.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eEthical considerations:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval (IRC no. 077/78/54) was obtained from the\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eInstitutional Ethical Review Committee of the KIST Medical College and Teaching Hospital, Tribhuvan University.\u0026nbsp;Informed consent was obtained from each participant after clearly mentioning the benefits and possible outcomes of this study. After providing\u0026nbsp;consent, demographic information was recorded, and the\u0026nbsp;samples were\u0026nbsp;tracked and identified\u0026nbsp;via\u0026nbsp;a laboratory information system (LIS-Midas Technology) in the hospital. The personal details of\u0026nbsp;the\u0026nbsp;participants were kept confidential and archived.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy design, site, and period\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA hospital-based cross-sectional study was conducted from April 2021 to December 2022 at the KIST Medical College and Teaching Hospital (KMCTH), a 900-bed superspecialty\u0026nbsp;tertiary care hospital,\u0026nbsp;and the Central Department of Microbiology, Tribhuvan University, Nepal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample collection and processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe included all samples from the culture/susceptibility (C/S) inquiry tests requested by clinicians for both inpatients and outpatients during our study period. A total of 1207 clinical samples, such as body fluids, swabs, sputum, urine, pus, blood, and tracheal aspirates, were randomly collected and processed onto culture media (blood agar, MacConkey\u0026nbsp;agar, and\u0026nbsp;chocolate agar\u0026nbsp;(Himedia Laboratories, India))\u0026nbsp;and incubated at appropriate temperatures and times according to standard protocols at the clinical service laboratory\u0026nbsp;(KMCTH)\u0026nbsp;(16,17). (Describe below). Among\u0026nbsp;the\u0026nbsp;culture-positive isolates, \u003cem\u003eA. baumannii\u0026nbsp;\u003c/em\u003eand \u003cem\u003eP. aeruginosa\u003c/em\u003e recovered from the various\u0026nbsp;samples\u0026nbsp;during the study period were included. AST was performed following the\u0026nbsp;Kirby\u0026ndash;Bauer\u0026nbsp;disk diffusion method as per CLSI\u0026nbsp;2020\u0026nbsp;(18).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial isolation and identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood agar, MacConkey agar, and chocolate agar were used to culture each sample, which was subsequently incubated for 24 hours at 37 \u0026deg;C. After incubation, colony morphology\u0026nbsp;characterization, Gram staining, and different biochemical tests were performed to identify the isolates. Furthermore, \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eA. baumannii\u003c/em\u003e must\u0026nbsp;be confirmed\u0026nbsp;via\u0026nbsp;special\u0026nbsp;characteristic\u0026nbsp;tests, which are explained below.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e isolates were identified as nonlactose fermenting, gram-negative slender rods that exhibited motility, thrived at 42 \u0026deg;C, were pigmented on cetrimide agar, and grew aerobically on MacConkey agar\u0026nbsp;(19).\u0026nbsp;The 27\u0026nbsp;phenotypically confirmed \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates were\u0026nbsp;subsequently\u0026nbsp;obtained and preserved in a tryptic\u0026nbsp;soy\u0026nbsp;broth tube with 40% glycerol until further experiments.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcinetobacter\u003c/em\u003e spp. were initially identified as nonlactose-fermenting gram-negative cocobacilli isolates with aerobic growth on MacConkey agar at 44 \u0026deg;C(20).\u0026nbsp;The phenol: chloroform\u0026nbsp;method of DNA extraction was\u0026nbsp;subsequently\u0026nbsp;employed\u0026nbsp;(21), and genotypic identification of \u003cem\u003eA. baumannii\u003c/em\u003e was\u0026nbsp;subsequently\u0026nbsp;performed by detecting the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-51\u003c/sub\u003e-like gene\u0026nbsp;(20). The 32\u0026nbsp;confirmed\u0026nbsp;isolates of \u003cem\u003eA. baumannii\u003c/em\u003e were preserved until further experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntimicrobial susceptibility testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo gauge the antimicrobial susceptibility patterns of the isolates, the Kirby\u0026ndash;Bauer disk diffusion method was applied via Muller\u0026ndash;Hinton agar (Hi-media Laboratories, India), and the results were interpreted according to CLSI (2020)\u0026nbsp;(18). The following antimicrobial discs were used\u0026nbsp;for breakpoint determination:\u0026nbsp;piperacillin (PI;\u0026nbsp;100 \u0026micro;g), piperacillin/tazobactam (PIT; 100/10 \u0026mu;g), ceftriaxone (CTR;\u0026nbsp;30 \u0026mu;g), ceftazidime (CAZ;\u0026nbsp;30 \u0026mu;g), cefotaxime (CTX;\u0026nbsp;30 \u0026mu;g), cefepime (CPM;\u0026nbsp;30 \u0026mu;g), aztreonam (AT;\u0026nbsp;30 \u0026mu;g), imipenem (IMP;\u0026nbsp;10 \u0026mu;g), meropenem (MRP;\u0026nbsp;10 \u0026mu;g), amikacin (AK;\u0026nbsp;30 \u0026mu;g), gentamicin (GEN;\u0026nbsp;10 \u0026mu;g), and ciprofloxacin (CIP;\u0026nbsp;5 \u0026mu;g). (Himedia Laboratories, India).\u003c/p\u003e\n\u003cp\u003eWe considered MDR isolates that were resistant to two or more antibiotics from different classes, such as beta-lactam, \u0026beta;-lactam combination, aminoglycoside, carbapenem, and fluoroquinolone antibiotic classes\u0026nbsp;(14).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenotypic detection of ESBLs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isolates that were resistant to cefotaxime (30 \u0026micro;g) and/or ceftazidime (30 \u0026micro;g) were tested for ESBL production via combined disc methods involving the use of ceftazidime (30 \u0026micro;g) and ceftazidime/clavulanic acid (30 \u0026micro;g/10 \u0026micro;g) discs and cefotaxime (30 \u0026micro;g) and cefotaxime/clavulanic acid (30 \u0026micro;g/10 \u0026micro;g) discs. A disc was placed 20 mm apart, and a zone of \u0026ge; 5 mm in the cefotaxime or ceftazidime with clavulanic acid disc compared with the cefotaxime or ceftazidime disc alone was considered positive for ESBL production after overnight culture on MHA plates\u0026nbsp;(18).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenotypic detection of carbapenem resistance\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eenzymes\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(MBLs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial isolates that showed intermediate resistance or resistance to imipenem or meropenem were suspected to be carbapenemase producers\u0026nbsp;(22). An ethylenediaminetetraacetic acid (EDTA) synergy test was used to confirm the\u0026nbsp;metallo-\u0026beta;-lactamase production\u0026nbsp;phenotype. In this method, ethylenediaminetetraacetic acid (EDTA) was used for\u0026nbsp;synergistic effects with\u0026nbsp;imipenem\u0026nbsp;discs\u0026nbsp;[imipenem and imipenem + EDTA (10 \u0026micro;g/750 \u0026micro;g)], and the zone of inhibition was compared with\u0026nbsp;that of\u0026nbsp;imipenem alone\u0026nbsp;(23).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMIC\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;test for carbapenem\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe minimal inhibitory concentration (MIC) of a carbapenem (meropenem) for \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eA. baumannii\u0026nbsp;\u003c/em\u003ewas determined via the agar dilution method\u0026nbsp;(18). The positive controls used \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 as a reference strain and inoculated\u0026nbsp;the\u0026nbsp;organism,\u0026nbsp;whereas\u0026nbsp;the negative control was considered the only culture\u0026nbsp;medium used\u0026nbsp;to ensure sterility. After 24 hours of incubation at 37 \u0026deg;C, the MIC readings were recorded and interpreted according to the CLSI M100 (18).\u0026nbsp;Isolates\u0026nbsp;with\u0026nbsp;carbapenem MICs of \u0026ge;8 \u0026mu;g/ml were considered resistant,\u0026nbsp;those with MICs of\u0026nbsp;4 \u0026mu;g/ml were considered intermediate, and\u0026nbsp;those with MICs of\u0026nbsp;\u0026le;2 \u0026mu;g/ml were considered susceptible\u0026nbsp;(18).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular detection of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eESBL-\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and carbapenemase\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003eencoding\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003egenes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe extraction of plasmid DNA from bacterial cultures in the logarithmic growth phase was conducted via the phenol:chloroform method\u0026nbsp;(21). The quality and purity of the extracted DNA template were measured\u0026nbsp;via NanoDrop measurements\u0026nbsp;(Nanodrop, Thermo Scientific, USA).\u003c/p\u003e\n\u003cp\u003eThe single-plex PCR assay was carried out via a thermal cycler (Bio-Rad, USA) with primer pairs (Macrogen, Korea) for blaCTX-M, blaTEM, blaNDM-1, blaVIM-2, blaOXA-23, blaOXA-51, and blaOXA-58 to detect beta-lactamase genes (Supplementary information, S1 Table). The reaction was carried out in a total volume of 25 \u0026micro;l (12.5 \u0026mu;l\u0026nbsp;of\u0026nbsp;1X FIREpol Master Mix (Solis Biodyne, Estonia), 3 \u0026mu;l of template genomic DNA, 3.0 \u0026mu;l of 10 picomole\u0026nbsp;primers\u0026nbsp;(1.5 \u0026mu;l of forward\u0026nbsp;primer\u0026nbsp;and 1.5 \u0026mu;l of reverse primer), and 6.5 \u0026mu;l\u0026nbsp;of\u0026nbsp;nuclease-free water). The PCR products were visualized by agarose gel electrophoresis (1.5%) with DNA-stained safe dye (Sigma\u0026ndash;Aldrich,\u0026nbsp;Germany) at 70 V for 50 minutes. The band\u0026nbsp;sizes were\u0026nbsp;compared along a\u0026nbsp;100 bp ladder. The amplified\u0026nbsp;ESBL\u0026nbsp;and carbapenemase genes were visualized in a gel documentation system (Azure\u0026nbsp;Biosystems, USA)\u0026nbsp;and\u0026nbsp;compared with a 100 bp DNA ladder (Thermo Scientific, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuality control\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sterility of freshly prepared culture media was confirmed by incubating 5% of the media plates at 37 \u0026deg;C for 24 hours to check for potential contamination. The quality of the culture media and antibiotic disks was checked by assessing the performance with \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis and interpretation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPSS (Student version) was used to analyze the data. Chi-square tests were used to determine the associations among the frequency of samples, antibiotic-resistant isolates, multidrug-resistant isolates, beta-lactam enzyme-producing isolates, and the MIC value of carbapenem. \u003cem\u003eP\u003c/em\u003e values \u003cu\u003e\u0026lt;\u003c/u\u003e 0.05 were considered statistically significant. The figure was created via GraphPad Prism software.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eDuring the study period, 1,207 samples were analyzed, and 22.5% (n\u0026thinsp;=\u0026thinsp;272) were positive according to bacterial culture. Among the total positive cultures, 20.2% (n\u0026thinsp;=\u0026thinsp;50/272) were gram-positive, and 79.8% (n\u0026thinsp;=\u0026thinsp;217/272) were gram-negative (S-Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Isolates of \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e were recovered from 32 and 27 samples, respectively. The total prevalence of nonlactose fermenter-negative bacteria (NLF-GNB) was 4.9% (n\u0026thinsp;=\u0026thinsp;59/1207), 3.7% was MDR (n\u0026thinsp;=\u0026thinsp;45/1207), 2.0% was ESBL (n\u0026thinsp;=\u0026thinsp;24/1207), 3.1% was carbapenem resistant, and 2.3% (n\u0026thinsp;=\u0026thinsp;28/1207) was an MBL producer in the total sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDistribution of NLF-GNB isolates and MDR, ESBL, carbapenem resistance, and MBL-producing isolates with demographic data (age and sex) and hospital settings (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among the isolates, a higher prevalence of MDR, carbapenem resistance, ESBL, and MBL producers was detected in females than in males, but the difference was not statistically significant. In the age group category, a higher rate (37.8%; 17/45) of MDR was found in the 41\u0026ndash;60-year age group, followed by the 21\u0026ndash;40-year and older age (\u0026ge;\u0026thinsp;61 years) groups. In contrast, carbapenem-resistant, ESBL-producing, and MBL-producing isolates were more prevalent in the younger age group (21\u0026ndash;40 years), followed by the 41\u0026ndash;60 years and \u0026ge;\u0026thinsp;61 years age groups. The predominant MDR and carbapenem-resistant isolates were found in inpatient populations rather than in OPD patients. However, there was no statistically significant difference between these two groups of populations (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn total, most of the isolates were obtained from sputum samples (40.7%, n\u0026thinsp;=\u0026thinsp;24), followed by pus/wound swabs (20.3%, n\u0026thinsp;=\u0026thinsp;12) (Table S2). We found a significant difference in the distribution of NLF-GNB isolates among different types of samples (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic distributions of NLF-GNB\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTotal isolate (n\u0026thinsp;=\u0026thinsp;59)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eMDR (n\u0026thinsp;=\u0026thinsp;45/59)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eESBL(n\u0026thinsp;=\u0026thinsp;24/59)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eCarbapenem Resistance(n\u0026thinsp;=\u0026thinsp;37/59)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eMBL (n\u0026thinsp;=\u0026thinsp;28/59)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDemographics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eA. baumannii\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eA. baumannii\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eA. baumannii\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eA. baumannii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cem\u003eA. baumannii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e (n/%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale (n\u0026thinsp;=\u0026thinsp;27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (48.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14 (43.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (47.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11 (42.3))\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9 (52.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 (28.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5 (41.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e9 (36.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5 (50.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e7 (38.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale (n\u0026thinsp;=\u0026thinsp;32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (51.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 (56.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10 (52.6))\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15 (57.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8 (47.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 (71.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7 (58.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16 (64.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5 (50.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e11 (61.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.7355\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.7358\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.3864\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.7394\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e0.5692\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge Group\u003c/b\u003e (n/%)\u003c/p\u003e \u003cp\u003e\u003cb\u003e(Years)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;20 (n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (7.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (15.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (5.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 (19.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (5.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1 (14.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4 (16.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e4 (22.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u0026ndash;40 (n\u0026thinsp;=\u0026thinsp;19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (22.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (40.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (26.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10 (38.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 (29.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4 (57.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3 (25.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e10 (40.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3 (30.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e8 (44.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e41\u0026ndash;60 (n\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 (33.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9 (28.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 (36.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (30.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6 (35.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 (28.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 (33.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8 (32.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4 (40.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3 (16.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;61 (n\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (37.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (15.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (31.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (11.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 (29.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5 (41.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3 (12.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3 (30.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e3 (16.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.1615\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.2197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.3127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.1247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e0.2126\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHospital Setting\u003c/b\u003e (n/%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInpatients (n\u0026thinsp;=\u0026thinsp;36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15 (55.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21 (65.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12 (63.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16 (61.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8 (47.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 (71.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (66.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e19 (76.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6 (60.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e13 (72.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOutpatients (n\u0026thinsp;=\u0026thinsp;23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (44.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (34.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 (38.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10 (38.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9 (52.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 (28.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 (33.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6 (24.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4 (40.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e5 (27.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.8908\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.9119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.3864\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.6964\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e0.6775\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrevalence of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eA. baumannii\u003c/em\u003e in different samples collected from outpatients.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSpecimen\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eInpatients (n\u0026thinsp;=\u0026thinsp;36)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eOutpatients (n\u0026thinsp;=\u0026thinsp;23)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(n/%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eA. baumannii\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(n/%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(n/%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eA. baumannii\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(n/%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSputum (n\u0026thinsp;=\u0026thinsp;24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (22.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9 (37.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (12.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePus/Wound swabs (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (50.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (16.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (25.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (8.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrine (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (55.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (22.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (22.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlood (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (16.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (50.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (33.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTips (ET, Catheter \u0026amp; Foleys) (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (12.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (50.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (12.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (25.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial susceptibility test of the isolates\u003c/h2\u003e \u003cp\u003eThe majority of \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates were resistant to ceftazidime (63%), followed by gentamycin (59.3%) (Table S3). In the case of \u003cem\u003eA. baumannii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;32), the highest level of resistance was observed against ceftriaxone (96.1%), followed by ceftazidime (93.7%), cefotaxime (87.5%), piperacillin (87.5%), ciprofloxacin (87.5%), piperacillin-tazobactam and cefepime (84.4%), gentamycin (81.3%) and carbapenem (78.1%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCarbapenem-resistant (CR) isolates\u003c/h2\u003e \u003cp\u003eAmong the 59 isolates, 62.7% (n\u0026thinsp;=\u0026thinsp;37/59) were found to be resistant to both meropenem and imipenem (carbapenem) (Table S3). Among the 37 CR isolates, the MICs of meropenem for \u003cem\u003eP. aeruginosa\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;12) and \u003cem\u003eA. baumanni (n\u0026thinsp;=\u003c/em\u003e\u0026thinsp;25) ranged from 32 \u0026micro;g/mL to 512 \u0026micro;g/mL and from 32 \u0026micro;g/mL to 256 \u0026micro;g/mL, respectively (Fig.\u0026nbsp;2). The highest percentage of CR isolates were found in sputum samples (37.8%, n\u0026thinsp;=\u0026thinsp;14/37), followed by pus/wound swabs (n\u0026thinsp;=\u0026thinsp;8/37, 21.6%) and tips, including ETs, Catheter, and Foleys (6.2%, n\u0026thinsp;=\u0026thinsp;6/37). The frequency of CR isolates significantly differed according to the specimen type (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004) (Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePrevalence of multidrug resistant, ESBL-producing, and carbapenemase-producing NLF-GNB strains\u003c/h2\u003e \u003cp\u003eAmong the 59 isolates, 45 (76.3%) were confirmed as MDR isolates, among which 57.8% were \u003cem\u003eA. baumannii\u003c/em\u003e and 42.2% were \u003cem\u003eP. aeruginosa\u003c/em\u003e. Among the total isolates, 24 were confirmed to be ESBL positive, and \u003cem\u003eP. aeruginosa\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17) was more common than \u003cem\u003eA. baumannii\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7). Among the 37 carbapenemase producers, 75.7% of the isolates were metallo-β-lactamase (MBL)-positive, comprising 64.3% \u003cem\u003eA. baumannii\u003c/em\u003e and 35.7% \u003cem\u003eP. aeruginosa\u003c/em\u003e. The frequencies of MDR, ESBL, and carbapenemase-producing NLF-GNB (MBL) strains are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDistribution and co-occurrence of carbapenemase genes\u003c/h2\u003e \u003cp\u003eAll the isolates (n\u0026thinsp;=\u0026thinsp;59) were examined for the presence of four carbapenemase genes, MBLs (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u0026minus;2\u003c/sub\u003e), and OXA (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;23\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;58\u003c/sub\u003e), which are carbapenem resistance genes, via specific target primers described previously (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the CR isolates, 70.2% (n\u0026thinsp;=\u0026thinsp;26/37) of the carbapenem-resistant isolates were found to harbor at least one or a combination of carbapenemase-encoding genes. Among these, 45.9% (n\u0026thinsp;=\u0026thinsp;17/37) of the isolates contained metallo-β-lactamase (MBL) genes (Ambler class B), including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e in 10 isolates and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u0026minus;2\u003c/sub\u003e in 7 isolates. Additionally, 48.6% (n\u0026thinsp;=\u0026thinsp;18/37) of the isolates harbored OXA genes (Ambler class D), with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;23\u003c/sub\u003e present in 16 isolates and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;58\u003c/sub\u003e in 2 isolates.\u003c/p\u003e \u003cp\u003eIn the CRPA, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u0026minus;2\u003c/sub\u003e was dominant (58.3%, n\u0026thinsp;=\u0026thinsp;7/12), followed by \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;23\u003c/sub\u003e (16.7%, n\u0026thinsp;=\u0026thinsp;2/12). Similarly, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;23\u003c/sub\u003e was highest (56.0%, n\u0026thinsp;=\u0026thinsp;14/25) in CR-AB, followed by \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e (32.0%, n\u0026thinsp;=\u0026thinsp;8/25) and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;58\u003c/sub\u003e (8.0%, n\u0026thinsp;=\u0026thinsp;2/25) (Table S7). Although 11 isolates (four \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates and seven \u003cem\u003eA. baumannii isolates\u003c/em\u003e) were phenotypically carbapenem resistant, the associated genes were undetected. Interestingly, one of the carbapenem-susceptible isolates carried the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;23\u003c/sub\u003e genes (Table S7).\u003c/p\u003e \u003cp\u003eIn total, 21.6% (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8/37) of the CR isolates coharbored more than one carbapenemase-encoding gene, whereas 47.4% (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;18/37) of the isolates harbored either a single gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Specifically, four CR-AB isolates carried \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e + \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;23\u003c/sub\u003e, two CR-AB isolates carried \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;23\u003c/sub\u003e + \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;58\u003c/sub\u003e, and one CR-PA isolate tested positive for \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e + \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u0026minus;2\u003c/sub\u003e. Additionally, one CR-PA isolate was found to carry all three different carbapenemase-encoding genes (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e + \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u0026minus;2\u003c/sub\u003e + \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;23\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eESBL gene cooccurrences with carbapenemase genes\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eA total of 24 phenotypic ESBL-positive isolates were examined for the presence of ESBL-encoding genes via PCR; 66.7% (n\u0026thinsp;=\u0026thinsp;16/24) and 29.2% (n\u0026thinsp;=\u0026thinsp;7/24) of the isolates contained the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e genes, respectively. Among them, 25% (n\u0026thinsp;=\u0026thinsp;6/24) of the isolates harbored both the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e genes in four of the \u003cem\u003eP. aeruginosa isolates\u003c/em\u003e and two of the \u003cem\u003eA. baumannii\u003c/em\u003e isolates. A higher frequency of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u003c/sub\u003e was detected in \u003cem\u003eA. baumannii\u003c/em\u003e (85.7%), whereas \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e was more frequent in \u003cem\u003eP. aeruginosa\u003c/em\u003e (29.4%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCooccurrence of carbapenemase genes with ESBL genes\u003c/h2\u003e \u003cp\u003eAmong the 37 CR isolates, 35.1% were ESBL positive. Among them, the majority of the isolates coharbored the ESBL gene. Specifically, five \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u0026minus;2\u003c/sub\u003e (all CR-PA), three \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e (2 CR-PA and 1 CR-AB), three \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;23\u003c/sub\u003e (3 CR-AB), and one \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;58\u003c/sub\u003e (one CR-AB) isolate coexisted with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u003c/sub\u003e. Additionally, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e cooccurred with one \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u0026minus;2\u003c/sub\u003e (in CR-PA), one \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e (in CR-AB), and two \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u0026minus;23\u003c/sub\u003e (in CR-AB) isolates (Table S7).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussions","content":"\u003cp\u003eIn this study, one-fifth of the total clinical specimens were bacterial culture positive; among them, 21.69% were \u003cem\u003eA. baumannii\u0026nbsp;\u003c/em\u003eand \u003cem\u003eP. aeruginosa\u003c/em\u003e. CR-AB and CR-PA are the most common pathogens associated with nosocomial infections in Asian countries, including Nepal (10). \u003cem\u003eA. baumannii\u0026nbsp;\u003c/em\u003eand \u003cem\u003eP. aeruginosa\u003c/em\u003e are frequently isolated from sputum and wound/pus samples. Carbapenem-resistant isolates were also more prevalent in those two samples, which may be due to their greater representation or could be linked to their ability to adapt to and resist specific environments where they persist. These results suggest that the dominance of antibiotic-resistant bacteria may differ in terms of survival mechanisms at different anatomical sites (29).\u003c/p\u003e\n\u003cp\u003eApproximately three-fourths of the \u003cem\u003eA. baumannii\u0026nbsp;\u003c/em\u003e\u003cem\u003eisolates\u0026nbsp;\u003c/em\u003eand half of the \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates were resistant to third-generation cephalosporin and carbapenem antibiotics, which reflected the comparative differences in resistance rates among the pathogens and could be attributed to individual intrinsic resistance mechanisms. (30). Interestingly, our antimicrobial pattern was similar to several earlier observations of antibiotic resistance in Nepal. (14,31,32).\u003c/p\u003e\n\u003cp\u003eAmong the \u003cem\u003eA. baumannii\u003c/em\u003e isolates, approximately 80% were carbapenem resistant, which is a relatively lower prevalence than that reported in the phenotypic research conducted by Joshi et al. (97.7%) in Kathmandu (14). In contrast, another study conducted in tertiary care hospitals in Nepal reported a lower rate of CR-AB (51%) (33) than our study did. Similarly, our neighboring countries, India (14.3%) (34) and Pakistan (22.0%) (35), also reported a low prevalence of CR-AB in clinical samples. These differences could be due to the distributions of pathogens being influenced by microbial ecology, the hospital environment, infection control practices, patient care practices, coinfections, antibiotic use, study populations, geographic areas, and sample processes (11).\u003c/p\u003e\n\u003cp\u003eIn our findings, approximately 45% of \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates were carbapenem resistant, which is similar to the results of a study conducted by Olalekan et al. in Nigeria. (36). However, in South Asian countries, Bangladesh reported approximately 40% carbapenem resistance, whereas Pakistan reported 65% carbapenem resistance in \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates (37). In the global scenario, approximately one-fifth of \u003cem\u003eP. aeruginosa\u003c/em\u003e are carbapenem resistant, which is even lower than that in the Asia Pacific region (38). These results suggest that CR-PA is gradually increasing in Nepal in hospital settings.\u003c/p\u003e\n\u003cp\u003eMoreover, our study reported that the MICs of meropenem were highly different for both \u003cem\u003eP. aeruginosa\u003c/em\u003e (32--512 \u0026micro;g/mL) and \u003cem\u003eA. baumannii\u0026nbsp;\u003c/em\u003e(32--256 \u0026micro;g/mL), with a statistically significant difference that indicates an increasing rate of carbapenem resistance, which could be related to the uncontrolled consumption of carbapenem antibiotics (8).\u003c/p\u003e\n\u003cp\u003eIn our study, 81.3% of\u0026nbsp;\u003cem\u003eA. baumannii\u003c/em\u003e strains were MDR, which was higher than that reported in a previous study conducted in Nepal. Studies conducted in various regions of Nepal reported 30.7%-75.9% rates of MDR bacteria. (15,33,39). In addition, 70.4% of the \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates were MDR in our study, which disagrees with the findings of other studies in Nepal: the percentage ranged from 14.3% to 60.1% (15,33,39). These results show increasing trends of MDR isolates in our clinical setting, which create limited treatment options and higher mortality and morbidity rates, with respiratory tract infection, surgical site infection, and increased risk of spread in hospital environments.\u003c/p\u003e\n\u003cp\u003eThis study reported a substantial prevalence of ESBL production among \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates,\u0026nbsp;accounting for 40.7% of phenotypically identified organisms (Table S4). Notably, this study showed similarities with ESBL-producing \u003cem\u003eP. aeruginosa\u003c/em\u003e from Kathmandu. (33). The occurrence of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M\u0026nbsp;\u003c/sub\u003egenes in \u003cem\u003eA. baumannii\u003c/em\u003e was relatively lower than that in \u003cem\u003eP. aeruginosa\u003c/em\u003e. This study revealed that the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M\u003c/sub\u003e (66.7%) gene was more responsible for ESBL production than the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e (29.2%) gene was and that 25% of both genes coexisted. Similar findings were reported from India and Gaza in \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e (34,40). In addition, a study conducted on Uro-pathogenic \u003cem\u003eE. coli\u003c/em\u003e at Kathmandu reported a greater presence of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M\u003c/sub\u003e (87.5%) than \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e (53.6%) among ESBL-producing isolates (41), which indicates that the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M\u0026nbsp;\u003c/sub\u003egenes might predominantly circulate in this area.\u003c/p\u003e\n\u003cp\u003eWe reported that almost half (76.7%) of our\u0026nbsp;carbapenem-resistant isolates produced MBL, two-thirds were \u003cem\u003eA. baumannii,\u003c/em\u003e and one-third were \u003cem\u003eP. aeruginosa\u003c/em\u003e. These results are well supported by a study conducted in Iran by Namaei et al. 2021 (42). However, an earlier study described a lower prevalence of MBL-producing isolates in the eastern part of Nepal (32,43) and Kathmandu (33) than our findings did. In \u003cem\u003eA. baumannii,\u0026nbsp;\u003c/em\u003ethe\u003cem\u003e\u0026nbsp;\u003c/em\u003eMBL production rate ranges from 54\u0026ndash;66.7% (32,44), whereas in \u003cem\u003eP. aeruginosa\u003c/em\u003e, it ranges from 8\u0026ndash;45% of the representative isolates (31\u0026ndash;33). According to our observations, these reports help predict the increasing trend of CR-AB and CR-PA in Nepal.\u003c/p\u003e\n\u003cp\u003eIn this study,\u003cem\u003e\u0026nbsp;P. aeruginosa\u003c/em\u003e and \u003cem\u003eA. baumannii\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eisolates harboring a variety of carbapenemase resistance genes, including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM-2\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-23,\u0026nbsp;\u003c/sub\u003eand \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-58\u003c/sub\u003e, were identified (Table S7). The cooccurrences of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM-2\u0026nbsp;\u003c/sub\u003ein \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-23,\u0026nbsp;\u003c/sub\u003eand \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-58\u0026nbsp;\u003c/sub\u003ein \u003cem\u003eA. baumannii\u0026nbsp;\u003c/em\u003ewere reported among carbapenem-resistant isolates as widespread antibiotic resistance genes. A relatively high MIC range (reduced antibiotic susceptibility rate) against the tested antibiotics was observed in MBL carbapenemase-producing isolates, which mostly harbored the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM-2\u0026nbsp;\u003c/sub\u003eand \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u0026nbsp;\u003c/sub\u003egenes (Table S7). This finding is in agreement with various studies that have successfully reported the coexistence of antibiotic resistance genes among clinical isolates (45). These multiple resistance genes found in the same isolates, as observed in our study, are indicative of the existence of multidrug-resistant pathogens that could be responsible for the difficulty in treating and curing diseases.\u003c/p\u003e\n\u003cp\u003eThe\u003cem\u003e\u0026nbsp;bla\u003c/em\u003e\u003csub\u003eVIM-2\u0026nbsp;\u003c/sub\u003eis the dominant gene in MBL-producing \u003cem\u003eP. aeruginosa\u003c/em\u003e and is associated with nosocomial infection (46). In our study, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM-2\u0026nbsp;\u003c/sub\u003ewas also the predominant MBL gene among the CR-PAs cooccurring with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e. Similarly, previous studies conducted in Nepal reported increased \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM-2\u003c/sub\u003e among MBL-producing CR-PAs (47,48)\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eIn contrast, a study from Nigeria revealed that the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e gene was more predominant than the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM-2\u0026nbsp;\u003c/sub\u003egene among MBL-producing isolates (36).\u003c/p\u003e\n\u003cp\u003eIn addition, more than half of the CR-AB isolates carried \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-23\u003c/sub\u003e,\u003csub\u003e\u0026nbsp;\u003c/sub\u003ewhich was the predominant gene in our study. Other similar studies also reported \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-23\u003c/sub\u003e as predominant among CR-ABs (35,49). A previous study in Nepal, CR-AB, has shown that the coexistence of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-23\u003c/sub\u003e with bla\u003cem\u003e\u003csub\u003eNDM-1\u003c/sub\u003e\u003c/em\u003e is significant (14,50). This study also emphasized that \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-23\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eONDM-1\u0026nbsp;\u003c/sub\u003ecould coexist in CR-AB more than in previous studies. Notably, 8% of the isolates had the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-58\u003c/sub\u003e gene, which actively coexists with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-23\u003c/sub\u003e. For the first time, in Nepal, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-58\u003c/sub\u003e was reported in clinical CR-AB isolates in 2015 (50). Moreover, very few CR-AB clinical isolates carry \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-58\u0026nbsp;\u003c/sub\u003eand are reported in the global context (50). The co-occurrence of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-23\u003c/sub\u003e with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eONDM-1\u0026nbsp;\u003c/sub\u003eor \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-58\u003c/sub\u003e is a concern, as this will limit future therapeutic options for AB infections in a healthcare setting.\u003c/p\u003e\n\u003cp\u003eMoreover, some carbapenem-susceptible isolates identified via phenotypic detection may indeed be positive for carbapenemase genes by genotyping (51). In the present study, we detected \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eONDM-1\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-23\u003c/sub\u003e in one isolate and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M\u0026nbsp;\u003c/sub\u003ein two isolates of \u003cem\u003eP. aeruginosa\u003c/em\u003e via PCR assays, which were negative for phenotypic detection. These results suggest that molecular techniques are necessary tools for improving diagnostic approaches in healthcare settings (51). Further research is needed to detect the expression levels of resistance genes and genetic variance via mutational analysis by sequencing methods to understand the molecular mechanisms of antibiotic resistance.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, this study revealed a high prevalence of MDR and carbapenem-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eA. baumannii\u003c/em\u003e isolates harboring multiple carbapenem resistance genes in the hospital setting of Nepal. This poses a significant challenge to the effective treatment and management of patients with currently available antibiotics in hospital settings, particularly in the developing countries of Nepal. Therefore, active surveillance and continuous monitoring of antibiotic resistance patterns in pathogens are essential to guide clinicians and healthcare providers in optimizing patient care and improving treatment outcomes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAntimicrobial resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLSI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClinical Laboratory Standard Institute\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarbapenem resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCR-PA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarbapenem-resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCR-AB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarbapenem-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCPase\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarbapenemase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCONS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecoagulase negative \u003cem\u003eStaphylococcus\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEDTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eethylenediaminetetraacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eESBLs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextended-spectrum beta-lactamases\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGNB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egram-negative bacilli\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHAI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehealthcare-associated infection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIRC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInstitutional Review Committee\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKMCTH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKIST Medical College and Teaching Hospital\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKPC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e carbapenemase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLMICs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elow- and middle-income countries\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMBLs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emetallo-beta-lactamases\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emultidrug-resistant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eminimum inhibitory concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enext-generation sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNLF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enonlactose fermenter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPSS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStatistical Package for Social Sciences\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWHO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWorld Health Organization.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the patients who provided consent and the clinical specimens for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eAll the authors declare that there are no conflicts of interest regarding the publication of this manuscript. The authors have no relevant financial or nonfinancial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was carried out with the in-house resources of the\u0026nbsp;Central Department of Microbiology, T.U., and with no specific grants from public, private, or nonprofit organizations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the information produced during this research work is presented within the manuscript and in the Supplementary file. Any queries regarding the research will be addressed by the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman Ethics and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for the study was obtained from the Institutional Review Committee (IRC) of KIST Medical College and Teaching Hospital (Ref. No.: 077/078/54). Written consent was taken from each patient for voluntary participation before collecting demographic data and samples. The Declaration of Helsinki and the guidelines of the Nepal Health Research Council were adhered to throughout the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Number:\u003c/strong\u003e Not applicable.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWorld Health Organization. Bacterial Priority Pathogens List, 2017. bacterial pathogens of public health importance to guide research, development, and strategies to prevent and control antimicrobial resistance. 2017. https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently needed.\u003c/li\u003e\n \u003cli\u003eOliveira DMP De, Forde BM, Kidd TJ, Harris PNA, Schembri MA, Beatson SA, et al. Antimicrobial Resistance in ESKAPE Pathogens. Clin Microbiol Rev. 2020;33(3):00181\u0026ndash;219. \u003cu\u003ehttps://doi.org/10.1128/cmr.00181-19\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eAbdeta A, Bitew A, Fentaw S, Tsige E, Assefa D, Lejisa T, et al. Phenotypic characterization of carbapenem nonsusceptible gram-negative bacilli isolated from clinical specimens. PLoS One. 2021;16 (12): e0256556. \u003cu\u003ehttps://doi.org/10.1371/journal.pone.0256556\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eTomczyk S, Zanichelli V, Grayson ML, Twyman A, Abbas M, Pires D, et al. Control of carbapenem-resistant enterobacteriaceae, acinetobacter baumannii, and pseudomonas aeruginosa in healthcare facilities: A systematic review and reanalysis of quasiexperimental studies. Clin Infect Dis. 2019;68(5):873\u0026ndash;84. doi: 10.1093/cid/ciy752\u003c/li\u003e\n \u003cli\u003eBalasubramanian R, Boeckel TP Van, Carmeli Y, Cosgrove S, Laxminarayan R. Global incidence in hospital-associated infections resistant to antibiotics : An analysis of point prevalence surveys from 99 countries. PLoS Med. 2023;20(6): e1004178. \u003cu\u003ehttps://doi.org/10.1371/journal. pmed.1004178\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eHsu L-Y, Apisarnthanarak A, Khan E, Suwantarat N, Ghafur A, Tambyah PA. Carbapenem-Resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e and Enterobacteriaceae in South and Southeast Asia. Clin Microbiol Rev. 2017;30(1):1\u0026ndash;22. doi: 10.1128/CMR.masthead.30-1\u003c/li\u003e\n \u003cli\u003eZellweger RM, Basnyat B, Shrestha P, Prajapati KG, Dongol S, Sharma PK, et al. Changing antimicrobial resistance trends in Kathmandu, Nepal: A 23-year retrospective analysis of bacteraemia. Front Med. 2018;5: 262. \u003cu\u003edoi: 10.3389/fmed.2018.00262\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eRijal KR, Banjara MR, Dhungel B, Kafle S, Gautam K, Ghimire B, et al. 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BMC Res Notes. 2020;13(1):27. \u003cu\u003ehttps://doi.org/10.1186/s13104-020-4890-z.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eAbouelfetouh A, Torky AS, Aboulmagd E. Phenotypic and genotypic characterization of carbapenem-resistant \u003cem\u003eAcinetobacter baumannii\u0026nbsp;\u003c/em\u003eisolates from Egypt. Antimicrob Resist Infect Control. 2019;20(8):185. \u003cu\u003edoi: 10.1186/s13756-019-0611-6\u0026nbsp;\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eSambrook J, Russell DW. Molecular Cloning A Laboratory Manual. 3rd ed. Argentine J, editor. New York: Cold Spring Harbor Laboratory Press. 2001\u003c/li\u003e\n \u003cli\u003eElbadawi HS, Elhag KM, Mahgoub E, Altayb HN, Ntoumi F, Elton L, et al. Detection and characterization of carbapenem resistant Gram‐negative bacilli isolates recovered from hospitalized patients at Soba University Hospital, Sudan. BMC Microbiol. 2021;21(1):136. \u003cu\u003edoi: 10.1186/s12866-021-02133-1\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eYong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem-EDTA disk method for differentiation of metallo-\u0026beta;-lactamase-producing clinical isolates of \u003cem\u003ePseudomonas\u003c/em\u003e spp. and \u003cem\u003eAcinetobacter\u003c/em\u003e spp. J Clin Microbiol. 2002;40(10):3798\u0026ndash;801. \u003cu\u003edoi: 10.1128/JCM.40.10.3798-3801.2002.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eEdelstein M, Pimkin M, Palagin I, Edelstein I, Stratchounski L. Prevalence and Molecular Epidemiology of CTX-M Extended-Spectrum \u0026beta;-Lactamase-Producing \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e in Russian Hospitals. Antimicrob Agents Chemother. 2003;47(12):3724\u0026ndash;32. \u003cu\u003edoi: 10.1128/AAC.47.12.3724\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003ePandit R, Awal B, Shrestha SS, Joshi G, Rijal BP, Parajuli NP. Extended-spectrum \u0026beta; -Lactamase (ESBL) Genotypes among Multidrug-Resistant Uropathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e Clinical Isolates from a Teaching Hospital of Nepal. Interdiscip Perspect Infect Dis. 2020;2020:6525826. \u003cu\u003edoi: 10.1155/2020/6525826\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eYong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, et al. Characterization of a new metallo-\u0026beta;-lactamase gene, \u003cem\u003ebla\u003c/em\u003eNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e sequence type 14 from India. 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Metallo-\u0026beta; lactamase producing nonfermentative gram-negative bacilli from various clinical isolates in a tertiary care hospital: A descriptive cross-sectional study. J Nepal Med Assoc. 2021;59(241):875\u0026ndash;80. \u003cu\u003edoi:10.31729/jnma.6408\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003ePandey R, Mishra SK, Shrestha A. Characterization of ESKAPE pathogens with special reference to multidrug resistance and biofilm production in a Nepalese hospital. Infect Drug Resist. 2021;14:2201\u0026ndash;12. \u003cu\u003edoi: 10.2147/IDR.S306688\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eKumari N, Kumar M, Katiyar A, Kumar A, Priya P, Kumar B, et al. Genome-wide identification of carbapenem-resistant gram-negative bacterial (CR-GNB) isolates retrieved from hospitalized patients in Bihar, India. Sci Rep. 2022;12(1):8477. \u003cu\u003ehttps://doi.org/10.1038/s41598-022-12471-3.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eZahra N, Zeshan B, Qadri MMA, Ishaq M, Afzal M, Ahmed N. Phenotypic and genotypic evaluation of antibiotic resistance of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e bacteria isolated from surgical ICU patients in Pakistan. Jundishapur J Microbiol. 2021;14(4): e113008. \u003cu\u003edoi:10.5812/JJM.113008\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eOlalekan A, Bader BK, Iwalokun B, Wolf S, Lalremruata A, Dike A, et al. High incidence of carbapenemase-producing\u003cem\u003e\u0026nbsp;Pseudomonas aeruginosa\u003c/em\u003e clinical isolates from Lagos, Nigeria. JAC-Antimicrobial Resist. 2023;5(2): dlad038. \u003cu\u003edoi: 10.1093/jacamr/dlad038\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eDiorio-Toth L, Irum S, Potter RF, Wallace MA, Arslan M, Munir T, et al. Genomic Surveillance of clinical \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e isolates reveals an additive effect of carbapenemase production on carbapenem resistance. Microbiol Spectr. 2022;10(3): e0076622. \u003cu\u003edoi: 10.1128/spectrum.00766-22\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eGales AC, Stone G, Sahm DF, Wise MG, Utt E. Incidence of ESBLs and carbapenemases among Enterobacterales and carbapenemases in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e isolates collected globally: results from ATLAS 2017\u0026ndash;2019. J Antimicrob Chemother. 2023;78(7):1606\u0026ndash;15. \u003cu\u003edoi:10.1093/jac/dkad127\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eBaral S, Pokharel A, Subramanya SH, Nayak N. Clinico-epidemiological profile of \u003cem\u003eAcinetobacter\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e infections, and their antibiotic resistant pattern in a tertiary care center, Western Nepal. Nepal J Epidemiol. 2019;9(4):804\u0026ndash;11. \u003cu\u003edoi: 10.3126/nje.v9i4.26962\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eEl Aila NA, Al Laham NA, Ayesh BM. Prevalence of extended spectrum beta-lactamase and molecular detection of \u003cem\u003ebla\u003c/em\u003eTEM, \u003cem\u003ebla\u003c/em\u003eSHV and \u003cem\u003ebla\u003c/em\u003eCTX-M genotypes among gram-negative bacilli isolates from pediatric patient population in Gaza strip. BMC Infect Dis. 2023;23(1):99. \u003cu\u003edoi:10.1186/s12879-023-08017-1\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eNayaju T, Upreti MK, Ghimire A, Shrestha B, Maharjan B, Joshi RD, et al. Higher Prevalence of Extended Spectrum b-Lactamase Producing Uropathogenic \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eAmong Patients with Diabetes from a Tertiary Care Hospital of Kathmandu, Nepal. Am J Trop Med Hyg. 2021;105(5):1347\u0026ndash;55. \u003cu\u003edoi: 10.4269/ajtmh.21-0691\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eNamaei MH, Yousefi M, Askari P, Roshanravan B, Hashemi A, Rezaei Y. High prevalence of multidrug-resistant nonfermentative gram-negative bacilli harboring \u003cem\u003ebla\u003c/em\u003eIMP-1 and \u003cem\u003ebla\u003c/em\u003eVIM-1 metallo-beta-lactamase genes in Birjand, south‒east Iran. Iran J Microbiol. 2021;13(4):470\u0026ndash;9. \u003cu\u003edoi: 10.18502/ijm.v13i4.6971\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eKumari M, Bhattarai NR, Rai K, Pandit TK, Khanal B. Multidrug-Resistant \u003cem\u003eAcinetobacter\u003c/em\u003e: Detection of ESBL, MBL, \u003cem\u003ebla\u003c/em\u003eNDM-1 Genotype, and Biofilm Formation at a Tertiary Care Hospital in Eastern Nepal. Int J Microbiol. 2022;2022: 8168000.\u003cu\u003e\u0026nbsp;doi: 10.1155/2022/8168000\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eYadav SK, Bhujel R, Hamal P, Mishra SK, Sharma S, Sherchand JB. Burden of multidrug-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e infection in hospitalized patients in a tertiary care hospital of Nepal. Infect Drug Resist. 2020;13:725\u0026ndash;32. \u003cu\u003edoi: 10.2147/IDR.S239514\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003ePaul D, Dhar D, Maurya AP, Mishra S, Sharma GD, Chakravarty A, et al. Occurrence of coexisting \u003cem\u003ebla\u003c/em\u003eVIM-2 and \u003cem\u003ebla\u003c/em\u003eNDM-1 in clinical isolates of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e from India. Ann Clin Microbiol Antimicrob. 2016;15(31):1\u0026ndash;6. \u003cu\u003edoi: 10.1186/s12941-016-0146-0.\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eVan Der Bij AK, Van Mansfeld R, Peirano G, Goessens WHF, Severin JA, Pitout JDD, et al. First outbreak of VIM-2 metallo-\u0026beta;-lactamase-producing \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e in the Netherlands: Microbiology, epidemiology and clinical outcomes. Int J Antimicrob Agents. 2011;37(6):513\u0026ndash;8. \u003cu\u003edoi:10.1016/j.ijantimicag.2011.02.010\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eAcharya M, Joshi PR, Thapa K, Aryal R, Kakshapati T, Sharma S. Detection of metallo-\u0026beta;-lactamases-encoding genes among clinical isolates of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e in a tertiary care hospital, Kathmandu, Nepal. BMC Res Notes. 2017;10(1):718. \u003cu\u003edoi:10.1186/s13104-017-3068-9\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eTakahashi T, Tada T, Shrestha S, Hishinuma T, Sherchan JB, Tohya M, et al. Molecular characterization of carbapenem-resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e clinical isolates in Nepal. J Glob Antimicrob Resist. 2021;26:279\u0026ndash;84. \u003cu\u003edoi:10.1016/j.jgar.2021.07.003\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eRanjbar R, Farahani A. Study of genetic diversity, biofilm formation, and detection of Carbapenemase, MBL, ESBL, and tetracycline resistance genes in multidrug-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e isolated from burn wound infections in Iran. Antimicrob Resist Infect Control. 2019;8(1):172.\u003cu\u003e\u0026nbsp;doi:\u003c/u\u003e\u003cu\u003e\u0026nbsp;10.1186/s13756-019-0612-5\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eShrestha S, Tada T, Miyoshi-Akiyama T, Ohara H, Shimada K, Satou K, et al. Molecular epidemiology of multidrug-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e isolates in a university hospital in Nepal reveals the emergence of a novel epidemic clonal lineage. Int J Antimicrob Agents. 2015;46(5):526\u0026ndash;31. \u003cu\u003edoi:10.1016/j.ijantimicag.2015.07.012\u003c/u\u003e\u003c/li\u003e\n \u003cli\u003eTawfick MM, Alshareef WA, Bendary HA, Elmahalawy H, Abdulall AK. The emergence of carbapenemase \u003cem\u003ebla\u0026nbsp;\u003c/em\u003eNDM genotype among carbapenem-resistant Enterobacteriaceae isolates from Egyptian cancer patients. Eur J Clin Microbiol Infect Dis. 2020;39(7):1251\u0026ndash;59. \u003cu\u003edoi:\u003c/u\u003e\u003cu\u003e\u0026nbsp;10.1007/s10096-020-03839-2\u003c/u\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Carbapenem resistance, ESBLs, MBL, P. aeruginosa, A. baumannii, co-occurrence, Nepal","lastPublishedDoi":"10.21203/rs.3.rs-9231883/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9231883/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003cem\u003e \u003c/em\u003eCarbapenem-resistant \u003cem\u003ePseudomonas aeruginosa \u003c/em\u003e(CRPA)\u003cem\u003e \u003c/em\u003eand \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e (CRAB) were listed on the WHO bacterial priority pathogens list in 2017. These gram-negative bacteria often cause healthcare-associated infections (HAIs), which pose an increasing threat to public health. This study aimed to isolate and identify CRPAs and CRABs from a tertiary care hospital in central Nepal and characterize carbapenem resistance genes at the molecular level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and method\u003c/strong\u003es: A total of\u0026nbsp;59 \u003cem\u003ePseudomonas aeruginosa \u003c/em\u003e(\u003cem\u003en\u003c/em\u003e=27)\u003cem\u003e \u003c/em\u003eand \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e=32) isolates were collected from different clinical samples from April 2021 to December 2022. Antibiotic susceptibility was determined via the Kirby disc diffusion method, and the carbapenem inactivation (CIM) method was used for phenotypic confirmation of carbapenem resistance. Conventional polymerase chain reaction (PCR) was used to detect carbapenem resistance genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Among the 59 isolates, 76.3% were multidrug resistant (MDR), 63.0% were extended-spectrum beta-lactamase (ESBL), and 62.7% were carbapenem resistant. Among the carbapenem-resistant isolates (\u003cem\u003en \u003c/em\u003e= 37), 75.7% of metallo-beta-lactamase (MBL) producers and 70.3% carried at least one carbapenem resistance gene. The \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX-M \u003c/sub\u003egene was predominant among the ESBL-positive isolates, present in 58.8% of the \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates and 87.5% of the \u003cem\u003eA. baumannii\u003c/em\u003e isolates, whereas 29% of both isolates harbored the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e gene. Among the CR-PA isolates, 58.3% carried \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM-2\u003c/sub\u003e, 16.7% carried \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e, and 8.33% carried \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-23\u003c/sub\u003e. Among the CR-AB isolates, 56% harbored \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-23\u003c/sub\u003e, 32% carried \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e, 16% had both \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1 \u003c/sub\u003eand \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-23\u003c/sub\u003e, and 8% contained both \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-58 \u003c/sub\u003eand \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA-23\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThis study highlights a significant burden of MDR and carbapenem-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eA. baumannii\u003c/em\u003e, with the co-occurrence of carbapenem resistance genes, in a clinical setting in Nepal. Our findings suggest that active surveillance of antibiotic resistance in pathogens could inform clinicians and healthcare providers about the treatment of infections.\u003c/p\u003e","manuscriptTitle":"Carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter baumannii clinical isolates from tertiary care in Nepal","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 08:32:19","doi":"10.21203/rs.3.rs-9231883/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-05T07:40:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T20:54:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-17T18:51:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235294893374236893179356820576431321595","date":"2026-04-11T14:57:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204768562449377793362822119479699727817","date":"2026-04-11T08:13:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329604566851901257347387075300676017909","date":"2026-04-10T09:14:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200785319470322834277889091296620562969","date":"2026-04-07T03:33:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-06T16:55:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-06T04:44:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-03T15:37:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-03T09:08:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2026-04-03T08:56:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a008f9b0-f4bc-4f24-91c8-1216850a0ca3","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-05T07:40:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T20:54:18+00:00","index":96,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-05T07:54:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 08:32:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9231883","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9231883","identity":"rs-9231883","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}