Distribution of carbapenemase-encoding genes (blaIMP, blaNDM, blaKPC, blaVIM and blaOXA-48) in carbapenem-resistant Klebsiella pneumoniae from Yemen

Distribution of carbapenemase-encoding genes (blaIMP, blaNDM, blaKPC, blaVIM and blaOXA-48) in carbapenem-resistant Klebsiella pneumoniae from Yemen | 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 Distribution of carbapenemase-encoding genes (blaIMP, blaNDM, blaKPC, blaVIM and blaOXA-48) in carbapenem-resistant Klebsiella pneumoniae from Yemen Aref N A Alafif, Saleh S Bahaj, Ahmed Y Al-Jaufy, waleed Y Alkassar, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9067881/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Background The emergence of carbapenem-resistant Klebsiella pneumoniae (CR-KP) represents a critical threat to global public health, severely limiting therapeutic options. Surveillance of these resistance determinants is essential to guide infection control and antimicrobial stewardship. This study aimed to identify the prevalence and distribution of specific carbapenemase-encoding genes ( bla KPC, bla NDM, bla IMP, bla VIM, and bla OXA-48) among clinical isolates of K. pneumoniae collected from various hospitals in Sana’a, Yemen. Methods A cross-sectional study was conducted in Sana’a, Yemen from October 2023 to December 2024. One hundred eighteen non-duplicate clinical isolates of Klebsiella pneumoniae (family Enterobacteriaceae) showing resistance to carbapenems (imipenem and meropenem) were recovered from wound, urine, sputum, blood and pus specimens collected at two private hospitals and the National Center of Public Health Laboratories. Antimicrobial susceptibility testing was performed by Kirby-Bauer disk diffusion and colistin MIC by broth disk elution according to CLSI 2024. Carbapenemase production was screened by reduced carbapenem susceptibility and confirmed phenotypically with the combined imipenem/EDTA disk test for metallo-β-lactamases. Genotypic detection of bla KPC, bla NDM, bla IMP, bla VIM and bla OXA-48 was performed by real-time PCR using DNA extracted by boiling. Descriptive statistics were analyzed with SPSS v26. Results Among the 118 isolates, 84 (71.2%) were positive for at least one carbapenemase-encoding gene. The most predominant gene was bla OXA-48 (59.3%), followed by bla NDM (38.9%), bla VIM (33.9%), bla IMP (6.8%), and bla KPC (1.7%). Regarding specimen distribution, the highest positivity rate for these genes was found in pus (85.7%) and wound (77.8%) samples. Co-occurrence of multiple genes was frequent: 22 isolates (18.6%) harbored two genes, 24 (20.3%) harbored three genes, and 4 (3.4%) harbored four genes ( bla NDM, bla OXA-48, bla VIM, and either bla KPC or bla IMP). Antimicrobial susceptibility testing revealed 100% resistance to most β-lactams, including meropenem, imipenem, and ceftazidime. High resistance rates were also observed for ciprofloxacin (95.7%) and gentamicin (93.2%). Colistin remained the most effective agent, with a susceptibility rate of 97.5%. Conclusions Carbapenemase genes—particularly bla OXA-48, bla NDM and bla VIM—are widespread among carbapenem-resistant K. pneumoniae in Sana’a, Yemen, with frequent co-occurrence and extensive multidrug resistance. These findings underscore the urgent need for strengthened antimicrobial stewardship, routine molecular surveillance for carbapenemase genes, and rigorous infection-control measures to limit dissemination. Carbapenemase blaOXA-48 blaNDM blaVIM blaKPC blaIMP Klebsiella pneumoniae Enterobacteriaceae Yemen antimicrobial resistance Introduction Antimicrobial resistance (AMR) represents one of the most pressing global health challenges of the 21st century, threatening the effectiveness of modern medicine and jeopardizing the treatment of common infectious diseases. Among the various mechanisms of bacterial resistance, the production of β-lactamases—enzymes that hydrolyze β-lactam antibiotics—has emerged as a particularly concerning threat to public health systems worldwide [ 1 ]. These enzymes compromise the efficacy of a broad spectrum of antibiotics, including penicillins, cephalosporins, and critically, carbapenems, which are often considered the last line of defense against multidrug-resistant Gram-negative bacteria [ 2 ]. Klebsiella pneumoniae , a Gram-negative bacterium belonging to the family Enterobacteriaceae, has become a major nosocomial pathogen responsible for a wide range of healthcare-associated infections, including pneumonia, bloodstream infections, urinary tract infections, and surgical site infections [ 3 ]. The organism’s ability to acquire and disseminate resistance determinants through mobile genetic elements has facilitated the rapid global spread of carbapenem-resistant strains [ 4 ]. Carbapenem resistance in K. pneumoniae is primarily mediated by the production of carbapenemases, which are β-lactamases capable of hydrolyzing carbapenems and most other β-lactam antibiotics [ 5 ]. Carbapenemases are classified into different molecular classes according to the Ambler classification system. Class A carbapenemases include Klebsiella pneumoniae carbapenemase (KPC), which has become endemic in many regions worldwide [ 6 ]. Class B metallo-β-lactamases (MBLs), such as New Delhi metallo-β-lactamase (NDM), Verona integron-encoded metallo-β-lactamase (VIM), and imipenemase (IMP), require zinc ions for their catalytic activity and are not inhibited by conventional β-lactamase inhibitors [ 7 ]. Class D carbapenemases, particularly OXA-48 and its variants, have emerged as significant contributors to carbapenem resistance, especially in the Middle East and North Africa region [ 8 ]. The epidemiology of carbapenemase-producing K. pneumoniae varies significantly across different geographical regions. In the Middle East, several studies have documented the predominance of OXA-48 and NDM carbapenemases [ 9 ], [ 10 ], [ 11 ], [ 12 ]. The Arabian Peninsula has witnessed a concerning increase in carbapenem-resistant Enterobacteriaceae (CRE), with multiple carbapenemase types co-circulating in healthcare settings [ 13 ], [ 14 ]. In Saudi Arabia, molecular characterization studies have revealed the presence of multiple carbapenemase genes, including bla NDM and bla OXA-48, often co-harbored in the same isolates [ 15 ], [ 16 ], [ 17 ]. Similar patterns have been observed in other Gulf Cooperation Council (GCC) countries, where OXA-48 and NDM producers dominate among CRE isolates [ 18 ], [ 19 ], [ 20 ]. Yemen, located in the southern Arabian Peninsula, faces unique challenges in addressing antimicrobial resistance due to ongoing conflict, limited healthcare infrastructure, and disrupted surveillance systems. Previous studies from Yemen have documented the presence of carbapenemase-producing Enterobacteriaceae, including NDM-producing isolates [ 6 ], [ 9 ]. However, comprehensive data on the distribution and molecular epidemiology of carbapenemase genes in K. pneumoniae from Yemen remain limited. Understanding the local epidemiology of carbapenemase-producing organisms is crucial for implementing effective infection control measures and guiding empirical antimicrobial therapy. The detection and characterization of carbapenemase genes are essential for surveillance, infection control, and antimicrobial stewardship programs. Phenotypic methods, such as the modified carbapenem inactivation method (mCIM) and EDTA-based tests for metallo-β-lactamases, provide initial screening but may lack specificity [ 10 ], [ 16 ]. Molecular methods, particularly real-time polymerase chain reaction (PCR), offer rapid and accurate identification of specific carbapenemase genes, enabling timely implementation of infection control measures [ 21 ]. The co-occurrence of multiple carbapenemase genes in a single isolate represents a particularly concerning phenomenon, as it may confer resistance to a broader range of antimicrobial agents and complicate treatment options [ 22 ], [ 23 ]. Studies from neighboring countries have reported varying rates of co-harboring multiple carbapenemase genes, with some isolates carrying three or more different carbapenemase genes simultaneously [ 3 ], [ 15 ], [ 35 ]. Given the limited data on carbapenemase-producing K. pneumoniae in Yemen and the critical need for local surveillance data to inform clinical practice and public health policy, this study was conducted to determine the prevalence and distribution of five major carbapenemase-encoding genes ( bla KPC, bla NDM, bla IMP, bla VIM, and bla OXA-48) among carbapenem-resistant K. pneumoniae isolates collected from clinical specimens in Sana’a, Yemen. Additionally, we aimed to characterize the antimicrobial resistance profiles of these isolates and investigate the co-occurrence patterns of multiple carbapenemase genes. Methods Study Design and Setting A cross-sectional study was conducted in Sana’a, Yemen, from October 2023 to December 2024. Clinical isolates were collected from two private hospitals and the National Center of Public Health Laboratories in Sana’a. The study was approved by the institutional ethics committee, and all procedures were performed in accordance with relevant guidelines and regulations. Inclusion criteria All positive culture isolates of K. pneumoniae obtained from various clinical specimens that showed resistance to all carbapenems (imipenem, and meropenem) were included and characterized. Sample size The sample size was 118 of Klebsiella Pneumoniae positive culture, based on the population size = 1,000,000 and, the prevalence was 8.1% in the study in Pakistan by [ 28 ], the power of study was 80% at confidence interval 95%. (calculated by EPI info version7.1). Bacterial Isolates A total of 118 non-duplicate clinical isolates of Klebsiella pneumoniae showing resistance to carbapenems (imipenem and/or meropenem) were included in the study. Isolates were recovered from various clinical specimens, including wound swabs, urine, sputum, blood, and pus samples. Species identification was performed using standard biochemical tests and confirmed by automated identification systems where available. Antimicrobial Susceptibility Testing Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) 2024 guidelines. The following antimicrobial agents were tested: meropenem (MR, 10 µg), imipenem (IPM, 10 µg), ceftazidime (CAZ, 30 µg), cefepime (FEP, 30 µg), ceftriaxone (CTR, 30 µg), cefuroxime (CXM, 30 µg), cefoperazone (CS, 75 µg), amoxicillin-clavulanate (AMC, 20/10 µg), piperacillin-tazobactam (PIT, 100/10 µg), aztreonam (AZT, 30 µg), ciprofloxacin (CIP, 5 µg), levofloxacin (LE, 5 µg), gentamicin (GEN, 10 µg), amikacin (AK, 30 µg), tobramycin (TOB, 10 µg), trimethoprim-sulfamethoxazole (COT, 1.25/23.75 µg), and tigecycline (TI, 15 µg). For colistin (CT), minimum inhibitory concentration (MIC) was determined using the broth disk elution method according to CLSI guidelines. Isolates with colistin MIC ≤ 2 µg/mL were considered susceptible, while those with MIC > 2 µg/mL were considered resistant. Phenotypic Detection of Carbapenemase Production Carbapenemase production was initially screened based on reduced susceptibility to carbapenems (imipenem and/or meropenem). Phenotypic confirmation of metallo-β-lactamase (MBL) production was performed using the combined disk test with imipenem and EDTA [ 24 ]. Briefly, imipenem disks (10 µg) with and without EDTA (750 µg) were placed on Mueller-Hinton agar plates inoculated with the test organism. An increase in the inhibition zone diameter of ≥ 5 mm around the imipenem-EDTA disk compared to the imipenem disk alone was interpreted as positive for MBL production. DNA Extraction Bacterial DNA was extracted using the boiling method [ 25 ]. Briefly, 2–3 bacterial colonies were suspended in 200 µL of sterile distilled water, heated at 100°C for 10 minutes, and then immediately cooled on ice. The suspension was centrifuged at 12,000 rpm for 5 minutes, and the supernatant containing the DNA was used as a template for PCR. Molecular Detection of Carbapenemase Genes Real-time PCR was performed to detect five major carbapenemase-encoding genes: bla KPC, bla NDM, bla IMP, bla VIM, and bla OXA-48. Gene-specific primers and probes were used according to published protocols. PCR reactions were performed using a real-time PCR system with appropriate positive and negative controls. Amplification conditions included an initial denaturation step followed by 40 cycles of denaturation, annealing, and extension. Results were interpreted based on the cycle threshold (Ct) values, with Ct < 35 considered positive for the target gene. Data Analysis Data were entered and analyzed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics were used to summarize the prevalence of carbapenemase genes, antimicrobial resistance patterns, and specimen distribution. Categorical variables were expressed as frequencies and percentages. The distribution of carbapenemase genes across different specimen types and the co-occurrence patterns of multiple genes were analyzed and presented in tables. Ethical consideration The study was be conducted after obtaining ethical clearance from the research and ethical review committee of the department of Medical Microbiology, Faculty of Medicine and Health Sciences, Sana'a University. (Reference no.). Additionally, an official permission letter was be obtained from the study site and from the hospital and laboratory authorities. Results Among the 118 carbapenem-resistant K. pneumoniae isolates tested, 84 (71.2%) were positive for at least one of the five carbapenemase-encoding genes investigated, while 34 isolates (28.8%) tested negative for all five genes (Table 2 ). The most predominant carbapenemase gene detected was bla OXA-48, present in 70 isolates (59.3%), followed by bla NDM in 46 isolates (38.9%), bla VIM in 40 isolates (33.9%), bla IMP in 8 isolates (6.8%), and bla KPC in 2 isolates (1.7%) (Table 1 ). Table 1 Distribution of carbapenemase-encoding genes ( bla IMP, bla NDM, bla KPC, bla VIM and bla OXA-48) in carbapenem-resistant Klebsiella pneumoniae Organism No. bla NDM bla VIM bla OXA-48 bla KPC bla IMP Klebsiella pneumoniae 118 46 (38.9%) 40 (33.9%) 70 (59.3%) 2 (1.7%) 8 (6.8%) 84 out of 118 (71.2%) K. pneumoniae isolates tested positive for carbapenemase-encoding genes, while 34 isolates (28.8%) tested negative (Table 2 ). Table 2 Distribution of all carbapenemase-encoding genes in Klebsiella pneumoniae Encoding genes Yes No No. % No. % Carbapenemase genes 84 71.2% 34 28.8% Among the 84 carbapenemase-positive isolates, 34 (28.8%) harbored a single carbapenemase gene, while 50 (42.3%) harbored multiple genes. Specifically, 22 isolates (18.6%) harbored two genes, 24 isolates (20.3%) harbored three genes, and 4 isolates (3.4%) harbored four different carbapenemase genes simultaneously (Table 3 ). Table 3 Distribution of Klebsiella pneumoniae carbapenemase-encoding genes number in clinical isolates Genes (N = 84) No. % One 34 28.8 Two 22 18.6 Three 24 20.3 Four 4 3.4 The distribution and co-occurrence patterns of carbapenemase genes among the 84 positive isolates are detailed in Table 8 . Among single-gene isolates (n = 34), bla OXA-48 was predominant (82.4%), with bla NDM present in 17.6%, while bla VIM, bla KPC, and bla IMP were absent. In two-gene isolates (n = 22), bla VIM (72.7%) and bla OXA-48 (63.6%) were most common, followed by bla NDM (54.5%) and bla IMP (9.1%). In three-gene isolates (n = 24), bla NDM and bla OXA-48 were both present in 100% of isolates, bla VIM in 83.3%, and bla IMP in 16.7%. All four-gene isolates (n = 4) carried bla NDM, bla OXA-48, and bla VIM (100%), with bla KPC and bla IMP each present in 50%.(Table 4 ). Table 4 Distribution and co-occurrence of Klebsiella pneumoniae carbapenemase-encoding genes among carbapenemase-positive isolates Genes (N = 84) One (N = 34) Two (N = 22) Three (N = 24) Four (N = 4) bla NDM (N = 46) 6 (17.6%) 12 (54.5%) 24 (100.0%) 4 (100.0%) bla VIM (N = 40) 0 (0%) 16 (72.7%) 20 (83.3%) 4 (100.0%) bla OXA-48 (N = 70) 28 (82.4%) 14 (63.6%) 24 (100.0%) 4 (100.0%) bla KPC (N = 2) 0 (0%) 0 (0%) 0 (0%) 2 (50.0%) bla IMP (N = 8) 0 (0%) 2 (9.1%) 4 (16.7%) 2 (50.0%) K. pneumoniae isolates were recovered from various clinical specimens: sputum (n = 52, 44.1%), urine (n = 20, 16.8%), wound (n = 18, 15.3%), blood (n = 14, 11.9%), and pus (n = 14, 11.9%) (Table 5 ). Table 5 Distribution of Klebsiella pneumoniae isolated from different clinical specimens Specimens No. % Wound 18 15.3 Urine 20 16.8 Sputum 52 44.1 Blood 14 11.9 Pus 14 11.9 The positivity rate for carbapenemase genes varied across specimen types. The highest positivity rate was observed in pus samples (85.7%), followed by wound (77.8%), urine (70.0%), sputum (69.2%), and blood (57.1%) (Table 6 ). Table 6 Distribution of Klebsiella pneumoniae carbapenemase-encoding genes in clinical specimens Gene Specimens (N = 118) Wound (N = 18) Urine (N = 20) Sputum (N = 52) Blood (N = 14) Pus (N = 14) Positive (N = 84) 14 (77.8%) 14 (70.0%) 36 (69.2%) 8 (57.1%) 12 (85.7%) Negative (N = 34) 4 (22.2%) 6 (30.0%) 16 (30.8%) 6 (42.9%) 2 (14.3%) The distribution of specific carbapenemase genes varied across specimen types. bla OXA-48 was detected at high rates across all specimens: pus (71.4%), urine (60.0%), sputum (57.7%), blood (57.1%), and wound (55.6%). bla NDM showed variable distribution: pus (57.1%), wound (55.6%), urine (40.0%), sputum (30.8%), and blood (28.6%). bla VIM was found in: pus (57.1%), blood (42.9%), wound (33.3%), sputum (30.8%), and urine (20.0%). bla KPC was rare, detected only in blood (14.3%). bla IMP was found in: urine (20.0%), pus (14.3%), and sputum (3.8%).(Table 7 ). Table 7 Distribution of Klebsiella pneumoniae carbapenemase-encoding genes in clinical specimens Genes (N = 84) Specimens (N = 118) Wound (N = 18) Urine (N = 20) Sputum (N = 52) Blood (N = 14) Pus (N = 14) bla NDM (N = 46) 10 (55.6%) 8 (40.0%) 16 (30.8%) 4 (28.6%) 8 (57.1%) bla VIM (N = 40) 6 (33.3%) 4 (20.0%) 16 (30.8%) 6 (42.9%) 8 (57.1%) bla OXA-48 (N = 70) 10 (55.6%) 12 (60.0%) 30 (57.7%) 8 (57.1%) 10 (71.4%) bla KPC (N = 2) 0 (0%) 0 (0%) 0 (0%) 2 (14.3%) 0 (0%) bla IMP (N = 8) 0 (0%) 4 (20.0%) 2 (3.8%) 0 (0%) 2 (14.3%) All 118 carbapenem-resistant K. pneumoniae isolates demonstrated 100% resistance to multiple β-lactam antibiotics, including meropenem, imipenem, ceftazidime, cefepime, ceftriaxone, cefuroxime, cefoperazone, amoxicillin-clavulanate, piperacillin-tazobactam, and aztreonam. High resistance rates were also observed for non-β-lactam antibiotics: ciprofloxacin (95.7%), trimethoprim-sulfamethoxazole (96.6%), gentamicin (93.2%), tobramycin (94.1%), amikacin (91.5%), and levofloxacin (89.8%). Colistin remained the most effective antimicrobial agent, with 97.5% of isolates showing susceptibility and only 2.5% demonstrating resistance (Table 8 ). Table 8 Antimicrobial resistance profiling of carbapenem-resistant Klebsiella pneumoniae Antibiotics Klebsiella pneumoniae Susceptible Resistant Colistin (CT) 115 (97.5%) 3 (2.5%) Ciprofloxacin (CIP) 5 (4.3%) 113 (95.7%) Meropenem (MR) 0 (0%) 118 (100.0%) Cefepime (FEP) 0 (0%) 118 (100.0%) Trimethoprim-sulfamethoxazole (COT) 4 (3.4%) 114 (96.6%) Levofloxacin (LE) 12 (10.2%) 106 (89.8%) Ceftazidime (CAZ) 0 (0%) 118 (100.0%) Gentamicin (GEN) 8 (6.8%) 110 (93.2%) Amoxicillin-clavulanate (AMC) 0 (0%) 118 (100.0%) Ceftriaxone (CTR) 0 (0%) 118 (100.0%) Piperacillin-tazobactam (PIT) 0 (0%) 118 (100.0%) Cefuroxime (CXM) 0 (0%) 118 (100.0%) Cefoperazone (CS) 0 (0%) 118 (100.0%) Amikacin (AK) 10 (8.5%) 108 (91.5%) Tobramycin (TOB) 7 (5.9%) 111 (94.1%) Imipenem (IPM) 0 (0%) 118 (100.0%) Aztreonam (AZT) 0 (0%) 118 (100.0%) Discussion This study provides comprehensive data on the molecular epidemiology of carbapenemase-producing K. pneumoniae in Sana’a, Yemen, revealing a high prevalence of carbapenemase genes with frequent co-occurrence of multiple genes and extensive multidrug resistance. Our findings have important implications for infection control, antimicrobial stewardship, and public health policy in Yemen and the broader Middle East region. The overall prevalence of carbapenemase genes in our study was 71.2%, indicating that the majority of carbapenem-resistant K. pneumoniae isolates in Sana’a harbor at least one carbapenemase-encoding gene. This high prevalence is consistent with reports from other Middle Eastern countries and underscores the significant burden of carbapenemase-mediated resistance in the region [ 1 ], [ 2 ], [ 3 ]. The predominance of bla OXA-48 (59.3%) in our study aligns with the epidemiological pattern observed in many Middle Eastern and North African countries. Similarly high rates of bla OXA-48 have been reported in studies from Saudi Arabia (87.0%) [ 22 ], Tunisia (86.72%) [ 23 ], Egypt (91.6%) [ 26 ], and Turkey (79.8%) [ 24 ]. The widespread dissemination of bla OXA-48 in the region has been attributed to the successful spread of specific plasmids carrying this gene, particularly the IncL/M-type plasmid [ 20 ]. However, our findings contrast with some studies from the Gulf region that reported lower bla OXA-48 prevalence, such as Qatar (20%) [ 37 ] and UAE (23.1%) [ 38 ], suggesting significant geographical variation even within the Arabian Peninsula. The prevalence of bla NDM (38.9%) in our study is comparable to reports from Qatar (39%) [ 37 ], Saudi Arabia (41.3%) [ 40 ], and Pakistan (44.8%) [ 28 ], but lower than rates reported in some studies from Egypt (90.9%, 95.8%) [ 30 ], [ 26 ], Tunisia (86.8%) [ 23 ], and Iraq (70.4%) [ 32 ]. The NDM enzyme family has emerged as a major contributor to carbapenem resistance globally, with NDM-1 being the most common variant [ 11 ], [ 12 ]. The presence of NDM-producing organisms is particularly concerning because metallo-β-lactamases confer resistance to nearly all β-lactams except aztreonam and are not inhibited by currently available β-lactamase inhibitors [ 7 ]. The detection of bla VIM (33.9%) in our study represents a significant finding, as VIM-type carbapenemases have been less commonly reported in the Arabian Peninsula compared to other regions. Our prevalence is higher than that reported in some neighboring countries but consistent with the increasing recognition of VIM enzymes in the Middle East [ 11 ], [ 42 ]. The presence of VIM alongside other metallo-β-lactamases (NDM and IMP) suggests diverse mechanisms of metallo-β-lactamase-mediated resistance in Yemen. The low prevalence of bla KPC (1.7%) in our study is consistent with the general epidemiological pattern in the Middle East, where KPC enzymes are less common compared to OXA-48 and NDM [ 13 ], [ 14 ], [ 20 ]. However, the detection of KPC, even at low levels, is noteworthy given its association with high-risk clones and its potential for rapid dissemination [ 15 ]. Studies from Palestine [ 15 ] and Egypt [ 44 ] have documented the emergence of KPC-producing K. pneumoniae in the region, suggesting that continued surveillance is warranted. Similarly, bla IMP (6.8%) was detected at relatively low frequency in our study. IMP-type metallo-β-lactamases were among the first carbapenemases to be described and have been reported primarily from Asia and the Pacific region [ 42 ]. The presence of IMP in Yemen, albeit at low levels, indicates the diversity of carbapenemase types circulating in the country. One of the most striking findings of our study is the high frequency of co-occurrence of multiple carbapenemase genes, with 42.3% of carbapenemase-positive isolates harboring two or more genes. This phenomenon has been increasingly recognized in the Middle East and North Africa region [ 3 ], [ 15 ], [ 22 ], [ 35 ]. The co-harboring of multiple carbapenemase genes is particularly concerning for several reasons. First, it may confer resistance to a broader spectrum of β-lactam antibiotics and potentially other antimicrobial classes if the resistance genes are located on the same mobile genetic elements. Second, it complicates the detection and characterization of carbapenemase-producing organisms, as phenotypic tests may not distinguish between different carbapenemase types. Third, it suggests the presence of highly successful plasmids or clones capable of accumulating multiple resistance determinants. In our study, 24 isolates (20.3%) harbored three carbapenemase genes, and 4 isolates (3.4%) harbored four genes simultaneously. The most common combination in four-gene isolates included bla NDM, bla OXA-48, bla VIM, and either bla KPC or bla IMP. Similar patterns of multiple carbapenemase gene co-occurrence have been reported in studies from Morocco [ 35 ], Saudi Arabia [ 3 ], [ 15 ], and Egypt [ 44 ]. The co-existence of class A, B, and D carbapenemases in the same isolate represents a particularly challenging scenario for antimicrobial therapy, as it eliminates most β-lactam options and necessitates reliance on last-resort agents such as colistin. The mechanisms underlying the co-occurrence of multiple carbapenemase genes likely involve the acquisition of multiple plasmids or the presence of complex genetic structures harboring multiple resistance genes. Studies have shown that carbapenemase genes are often located on conjugative plasmids belonging to various incompatibility groups, which can be transferred horizontally between bacterial species [ 4 ], [ 22 ]. The accumulation of multiple carbapenemase genes may be facilitated by selective pressure from antimicrobial use and by the genetic plasticity of K. pneumoniae , which readily acquires and maintains mobile genetic elements. Our study revealed that carbapenemase-producing K. pneumoniae was isolated from diverse clinical specimens, with the highest proportion from sputum (44.1%), reflecting the importance of respiratory tract infections in hospitalized patients. The high positivity rate for carbapenemase genes in pus (85.7%) and wound (77.8%) samples is particularly concerning, as these infections are often associated with surgical procedures and may indicate nosocomial transmission. The detection of carbapenemase-producing organisms in blood cultures (57.1% positivity rate) is especially alarming, as bloodstream infections caused by carbapenem-resistant organisms are associated with high mortality rates and limited treatment options [ 5 ], [ 26 ]. The distribution of specific carbapenemase genes varied across specimen types, with bla OXA-48 being consistently prevalent across all specimen types. This ubiquitous distribution suggests that OXA-48-producing strains are well-established in the healthcare facilities studied and may be transmitted through various routes. The higher prevalence of bla NDM and bla VIM in pus and wound samples compared to other specimen types may reflect specific epidemiological patterns or clonal spread within surgical wards. The antimicrobial resistance profiles of the carbapenem-resistant K. pneumoniae isolates in our study demonstrate extensive multidrug resistance, with 100% resistance to all β-lactam antibiotics tested. This pan-β-lactam resistance is expected given the presence of carbapenemases, which hydrolyze virtually all β-lactam antibiotics. The high resistance rates to fluoroquinolones (ciprofloxacin 95.7%, levofloxacin 89.8%) and aminoglycosides (gentamicin 93.2%, amikacin 91.5%, tobramycin 94.1%) indicate that these isolates have acquired multiple resistance mechanisms beyond carbapenemases, likely through the accumulation of additional resistance genes on plasmids or chromosomal mutations [ 4 ], [ 31 ]. The near-universal resistance to trimethoprim-sulfamethoxazole (96.6%) further limits therapeutic options for urinary tract infections, which are commonly caused by K. pneumoniae . The extensive multidrug resistance observed in our study is consistent with reports from other countries in the region [ 18 ], [ 26 ], [ 30 ], [ 31 ], [ 33 ] and reflects the serious challenge posed by carbapenem-resistant K. pneumoniae to clinical management. Colistin remained the most effective antimicrobial agent in our study, with 97.5% susceptibility. This finding is encouraging and consistent with other studies from the Middle East that have reported high colistin susceptibility rates among carbapenem-resistant Enterobacteriaceae [ 5 ], [ 16 ], [ 18 ]. However, the detection of colistin resistance in 2.5% of isolates is concerning, as colistin is often considered the last-resort treatment for infections caused by carbapenem-resistant Gram-negative bacteria. The emergence of colistin resistance, particularly in the context of carbapenem resistance, creates a scenario of pan-drug resistance with extremely limited or no treatment options. The mechanisms of colistin resistance in Enterobacteriaceae include chromosomal mutations in genes regulating lipopolysaccharide modification and, more recently, plasmid-mediated resistance through mcr genes [ 29 ]. The findings of this study have several important implications for clinical practice and public health policy in Yemen. First, the high prevalence of carbapenemase-producing K. pneumoniae necessitates the implementation of robust infection prevention and control measures in healthcare facilities. These measures should include strict adherence to hand hygiene protocols, contact precautions for patients colonized or infected with carbapenem-resistant organisms, environmental cleaning and disinfection, and surveillance cultures to identify asymptomatic carriers [ 17 ], [ 20 ]. Second, the extensive multidrug resistance observed in our study highlights the urgent need for antimicrobial stewardship programs to optimize antimicrobial use and reduce selective pressure for resistance. Antimicrobial stewardship interventions should include guidelines for empirical therapy based on local resistance patterns, de-escalation strategies when culture results become available, and education programs for healthcare providers [ 2 ], [ 5 ]. Third, the molecular characterization of carbapenemase genes should be integrated into routine microbiology laboratory practices to enable rapid detection and appropriate infection control responses. While phenotypic methods can provide initial screening, molecular methods such as real-time PCR offer rapid and accurate identification of specific carbapenemase genes [ 16 ], [ 21 ]. The implementation of molecular diagnostics may be challenging in resource-limited settings like Yemen, but it is essential for effective surveillance and outbreak investigation. Fourth, the co-occurrence of multiple carbapenemase genes in a substantial proportion of isolates suggests that treatment decisions should not rely solely on the detection of a single carbapenemase type. Combination therapy may be necessary for serious infections, and the choice of antimicrobial agents should be guided by comprehensive susceptibility testing, including testing for colistin resistance [ 7 ], [ 26 ]. Conclusions This study demonstrates a high prevalence of carbapenemase-encoding genes among carbapenem-resistant K. pneumoniae isolates in Sana’a, Yemen, with bla OXA-48, bla NDM, and bla VIM being the most common. The frequent co-occurrence of multiple carbapenemase genes and extensive multidrug resistance, with only colistin retaining significant activity, represent serious challenges for clinical management and infection control. These findings underscore the urgent need for strengthened antimicrobial stewardship programs, routine molecular surveillance for carbapenemase genes, enhanced infection prevention and control measures, and the development of new therapeutic strategies. The implementation of these interventions is critical to prevent further dissemination of carbapenem-resistant organisms and to preserve the effectiveness of available antimicrobial agents in Yemen and the broader region. Abbreviations AMR Antimicrobial resistance CR-KP Carbapenem-resistant Klebsiella pneumoniae CRE Carbapenem-resistant Enterobacteriaceae CLSI Clinical and Laboratory Standards Institute EDTA Ethylenediaminetetraacetic acid GCC Gulf Cooperation Council IMP Imipenemase KPC Klebsiella pneumoniae carbapenemase MBL Metallo-β-lactamase mCIM Modified carbapenem inactivation method MIC Minimum inhibitory concentration NDM New Delhi metallo-β-lactamase OXA Oxacillinase PCR Polymerase chain reaction VIM Verona integron-encoded metallo-β-lactamase WHO World Health Organization Declarations Ethics approval and consent to participate This study was approved by the institutional ethics committee of the participating hospitals and the National Center of Public Health Laboratories in Sana’a, Yemen. The study involved only bacterial isolates collected as part of routine clinical care, and no additional patient interventions were performed. Patient consent was waived by the ethics committee as the study used de-identified bacterial isolates without access to patient identifiable information. Consent for publication Not applicable. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Authors’ contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by all authors. The first draft of the manuscript was written by the authors and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. 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Among the various mechanisms of bacterial resistance, the production of β-lactamases\u0026mdash;enzymes that hydrolyze β-lactam antibiotics\u0026mdash;has emerged as a particularly concerning threat to public health systems worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These enzymes compromise the efficacy of a broad spectrum of antibiotics, including penicillins, cephalosporins, and critically, carbapenems, which are often considered the last line of defense against multidrug-resistant Gram-negative bacteria [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, a Gram-negative bacterium belonging to the family Enterobacteriaceae, has become a major nosocomial pathogen responsible for a wide range of healthcare-associated infections, including pneumonia, bloodstream infections, urinary tract infections, and surgical site infections [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The organism\u0026rsquo;s ability to acquire and disseminate resistance determinants through mobile genetic elements has facilitated the rapid global spread of carbapenem-resistant strains [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Carbapenem resistance in \u003cem\u003eK. pneumoniae\u003c/em\u003e is primarily mediated by the production of carbapenemases, which are β-lactamases capable of hydrolyzing carbapenems and most other β-lactam antibiotics [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCarbapenemases are classified into different molecular classes according to the Ambler classification system. Class A carbapenemases include \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e carbapenemase (KPC), which has become endemic in many regions worldwide [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Class B metallo-β-lactamases (MBLs), such as New Delhi metallo-β-lactamase (NDM), Verona integron-encoded metallo-β-lactamase (VIM), and imipenemase (IMP), require zinc ions for their catalytic activity and are not inhibited by conventional β-lactamase inhibitors [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Class D carbapenemases, particularly OXA-48 and its variants, have emerged as significant contributors to carbapenem resistance, especially in the Middle East and North Africa region [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe epidemiology of carbapenemase-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e varies significantly across different geographical regions. In the Middle East, several studies have documented the predominance of OXA-48 and NDM carbapenemases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The Arabian Peninsula has witnessed a concerning increase in carbapenem-resistant Enterobacteriaceae (CRE), with multiple carbapenemase types co-circulating in healthcare settings [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In Saudi Arabia, molecular characterization studies have revealed the presence of multiple carbapenemase genes, including \u003cem\u003ebla\u003c/em\u003eNDM and \u003cem\u003ebla\u003c/em\u003eOXA-48, often co-harbored in the same isolates [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Similar patterns have been observed in other Gulf Cooperation Council (GCC) countries, where OXA-48 and NDM producers dominate among CRE isolates [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eYemen, located in the southern Arabian Peninsula, faces unique challenges in addressing antimicrobial resistance due to ongoing conflict, limited healthcare infrastructure, and disrupted surveillance systems. Previous studies from Yemen have documented the presence of carbapenemase-producing Enterobacteriaceae, including NDM-producing isolates [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, comprehensive data on the distribution and molecular epidemiology of carbapenemase genes in \u003cem\u003eK. pneumoniae\u003c/em\u003e from Yemen remain limited. Understanding the local epidemiology of carbapenemase-producing organisms is crucial for implementing effective infection control measures and guiding empirical antimicrobial therapy.\u003c/p\u003e \u003cp\u003eThe detection and characterization of carbapenemase genes are essential for surveillance, infection control, and antimicrobial stewardship programs. Phenotypic methods, such as the modified carbapenem inactivation method (mCIM) and EDTA-based tests for metallo-β-lactamases, provide initial screening but may lack specificity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Molecular methods, particularly real-time polymerase chain reaction (PCR), offer rapid and accurate identification of specific carbapenemase genes, enabling timely implementation of infection control measures [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe co-occurrence of multiple carbapenemase genes in a single isolate represents a particularly concerning phenomenon, as it may confer resistance to a broader range of antimicrobial agents and complicate treatment options [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Studies from neighboring countries have reported varying rates of co-harboring multiple carbapenemase genes, with some isolates carrying three or more different carbapenemase genes simultaneously [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the limited data on carbapenemase-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e in Yemen and the critical need for local surveillance data to inform clinical practice and public health policy, this study was conducted to determine the prevalence and distribution of five major carbapenemase-encoding genes (\u003cem\u003ebla\u003c/em\u003eKPC, \u003cem\u003ebla\u003c/em\u003eNDM, \u003cem\u003ebla\u003c/em\u003eIMP, \u003cem\u003ebla\u003c/em\u003eVIM, and \u003cem\u003ebla\u003c/em\u003eOXA-48) among carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates collected from clinical specimens in Sana\u0026rsquo;a, Yemen. Additionally, we aimed to characterize the antimicrobial resistance profiles of these isolates and investigate the co-occurrence patterns of multiple carbapenemase genes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design and Setting\u003c/h2\u003e \u003cp\u003eA cross-sectional study was conducted in Sana\u0026rsquo;a, Yemen, from October 2023 to December 2024. Clinical isolates were collected from two private hospitals and the National Center of Public Health Laboratories in Sana\u0026rsquo;a. The study was approved by the institutional ethics committee, and all procedures were performed in accordance with relevant guidelines and regulations.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInclusion criteria\u003c/h3\u003e\n\u003cp\u003eAll positive culture isolates of K. pneumoniae obtained from various clinical specimens that showed resistance to all carbapenems (imipenem, and meropenem) were included and characterized.\u003c/p\u003e\n\u003ch3\u003eSample size\u003c/h3\u003e\n\u003cp\u003eThe sample size was 118 of Klebsiella Pneumoniae positive culture, based on the population size\u0026thinsp;=\u0026thinsp;1,000,000 and, the prevalence was 8.1% in the study in Pakistan by [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], the power of study was 80% at confidence interval 95%. (calculated by EPI info version7.1).\u003c/p\u003e\n\u003ch3\u003eBacterial Isolates\u003c/h3\u003e\n\u003cp\u003eA total of 118 non-duplicate clinical isolates of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e showing resistance to carbapenems (imipenem and/or meropenem) were included in the study. Isolates were recovered from various clinical specimens, including wound swabs, urine, sputum, blood, and pus samples. Species identification was performed using standard biochemical tests and confirmed by automated identification systems where available.\u003c/p\u003e\n\u003ch3\u003eAntimicrobial Susceptibility Testing\u003c/h3\u003e\n\u003cp\u003eAntimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) 2024 guidelines. The following antimicrobial agents were tested: meropenem (MR, 10 \u0026micro;g), imipenem (IPM, 10 \u0026micro;g), ceftazidime (CAZ, 30 \u0026micro;g), cefepime (FEP, 30 \u0026micro;g), ceftriaxone (CTR, 30 \u0026micro;g), cefuroxime (CXM, 30 \u0026micro;g), cefoperazone (CS, 75 \u0026micro;g), amoxicillin-clavulanate (AMC, 20/10 \u0026micro;g), piperacillin-tazobactam (PIT, 100/10 \u0026micro;g), aztreonam (AZT, 30 \u0026micro;g), ciprofloxacin (CIP, 5 \u0026micro;g), levofloxacin (LE, 5 \u0026micro;g), gentamicin (GEN, 10 \u0026micro;g), amikacin (AK, 30 \u0026micro;g), tobramycin (TOB, 10 \u0026micro;g), trimethoprim-sulfamethoxazole (COT, 1.25/23.75 \u0026micro;g), and tigecycline (TI, 15 \u0026micro;g).\u003c/p\u003e \u003cp\u003eFor colistin (CT), minimum inhibitory concentration (MIC) was determined using the broth disk elution method according to CLSI guidelines. Isolates with colistin MIC\u0026thinsp;\u0026le;\u0026thinsp;2 \u0026micro;g/mL were considered susceptible, while those with MIC\u0026thinsp;\u0026gt;\u0026thinsp;2 \u0026micro;g/mL were considered resistant.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic Detection of Carbapenemase Production\u003c/h2\u003e \u003cp\u003eCarbapenemase production was initially screened based on reduced susceptibility to carbapenems (imipenem and/or meropenem). Phenotypic confirmation of metallo-β-lactamase (MBL) production was performed using the combined disk test with imipenem and EDTA [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, imipenem disks (10 \u0026micro;g) with and without EDTA (750 \u0026micro;g) were placed on Mueller-Hinton agar plates inoculated with the test organism. An increase in the inhibition zone diameter of \u0026ge;\u0026thinsp;5 mm around the imipenem-EDTA disk compared to the imipenem disk alone was interpreted as positive for MBL production.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA Extraction\u003c/h3\u003e\n\u003cp\u003eBacterial DNA was extracted using the boiling method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Briefly, 2\u0026ndash;3 bacterial colonies were suspended in 200 \u0026micro;L of sterile distilled water, heated at 100\u0026deg;C for 10 minutes, and then immediately cooled on ice. The suspension was centrifuged at 12,000 rpm for 5 minutes, and the supernatant containing the DNA was used as a template for PCR.\u003c/p\u003e\n\u003ch3\u003eMolecular Detection of Carbapenemase Genes\u003c/h3\u003e\n\u003cp\u003eReal-time PCR was performed to detect five major carbapenemase-encoding genes: \u003cem\u003ebla\u003c/em\u003eKPC, \u003cem\u003ebla\u003c/em\u003eNDM, \u003cem\u003ebla\u003c/em\u003eIMP, \u003cem\u003ebla\u003c/em\u003eVIM, and \u003cem\u003ebla\u003c/em\u003eOXA-48. Gene-specific primers and probes were used according to published protocols. PCR reactions were performed using a real-time PCR system with appropriate positive and negative controls. Amplification conditions included an initial denaturation step followed by 40 cycles of denaturation, annealing, and extension. Results were interpreted based on the cycle threshold (Ct) values, with Ct\u0026thinsp;\u0026lt;\u0026thinsp;35 considered positive for the target gene.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eData were entered and analyzed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics were used to summarize the prevalence of carbapenemase genes, antimicrobial resistance patterns, and specimen distribution. Categorical variables were expressed as frequencies and percentages. The distribution of carbapenemase genes across different specimen types and the co-occurrence patterns of multiple genes were analyzed and presented in tables.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEthical consideration\u003c/h2\u003e \u003cp\u003eThe study was be conducted after obtaining ethical clearance from the research and ethical review committee of the department of Medical Microbiology, Faculty of Medicine and Health Sciences, Sana'a University. (Reference no.). Additionally, an official permission letter was be obtained from the study site and from the hospital and laboratory authorities.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAmong the 118 carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates tested, 84 (71.2%) were positive for at least one of the five carbapenemase-encoding genes investigated, while 34 isolates (28.8%) tested negative for all five genes (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The most predominant carbapenemase gene detected was \u003cem\u003ebla\u003c/em\u003eOXA-48, present in 70 isolates (59.3%), followed by \u003cem\u003ebla\u003c/em\u003eNDM in 46 isolates (38.9%), \u003cem\u003ebla\u003c/em\u003eVIM in 40 isolates (33.9%), \u003cem\u003ebla\u003c/em\u003eIMP in 8 isolates (6.8%), and \u003cem\u003ebla\u003c/em\u003eKPC in 2 isolates (1.7%) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\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\u003eDistribution of carbapenemase-encoding genes (\u003cem\u003ebla\u003c/em\u003eIMP, \u003cem\u003ebla\u003c/em\u003eNDM, \u003cem\u003ebla\u003c/em\u003eKPC, \u003cem\u003ebla\u003c/em\u003eVIM and \u003cem\u003ebla\u003c/em\u003eOXA-48) in carbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrganism\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eNDM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eVIM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eOXA-48\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eKPC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eIMP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46 (38.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40 (33.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e70 (59.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 (1.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 (6.8%)\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\u003e84 out of 118 (71.2%) K. pneumoniae isolates tested positive for carbapenemase-encoding genes, while 34 isolates (28.8%) tested negative (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\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\u003eDistribution of all carbapenemase-encoding genes in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\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\u003eEncoding genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarbapenemase genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e71.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28.8%\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\u003eAmong the 84 carbapenemase-positive isolates, 34 (28.8%) harbored a single carbapenemase gene, while 50 (42.3%) harbored multiple genes. Specifically, 22 isolates (18.6%) harbored two genes, 24 isolates (20.3%) harbored three genes, and 4 isolates (3.4%) harbored four different carbapenemase genes simultaneously (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e carbapenemase-encoding genes number in clinical isolates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes (N\u0026thinsp;=\u0026thinsp;84)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOne\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTwo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThree\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.4\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\u003eThe distribution and co-occurrence patterns of carbapenemase genes among the 84 positive isolates are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Among single-gene isolates (n\u0026thinsp;=\u0026thinsp;34), \u003cem\u003ebla\u003c/em\u003eOXA-48 was predominant (82.4%), with \u003cem\u003ebla\u003c/em\u003eNDM present in 17.6%, while \u003cem\u003ebla\u003c/em\u003eVIM, \u003cem\u003ebla\u003c/em\u003eKPC, and \u003cem\u003ebla\u003c/em\u003eIMP were absent. In two-gene isolates (n\u0026thinsp;=\u0026thinsp;22), \u003cem\u003ebla\u003c/em\u003eVIM (72.7%) and \u003cem\u003ebla\u003c/em\u003eOXA-48 (63.6%) were most common, followed by \u003cem\u003ebla\u003c/em\u003eNDM (54.5%) and \u003cem\u003ebla\u003c/em\u003eIMP (9.1%). In three-gene isolates (n\u0026thinsp;=\u0026thinsp;24), \u003cem\u003ebla\u003c/em\u003eNDM and \u003cem\u003ebla\u003c/em\u003eOXA-48 were both present in 100% of isolates, \u003cem\u003ebla\u003c/em\u003eVIM in 83.3%, and \u003cem\u003ebla\u003c/em\u003eIMP in 16.7%. All four-gene isolates (n\u0026thinsp;=\u0026thinsp;4) carried \u003cem\u003ebla\u003c/em\u003eNDM, \u003cem\u003ebla\u003c/em\u003eOXA-48, and \u003cem\u003ebla\u003c/em\u003eVIM (100%), with \u003cem\u003ebla\u003c/em\u003eKPC and \u003cem\u003ebla\u003c/em\u003eIMP each present in 50%.(Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution and co-occurrence of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e carbapenemase-encoding genes among carbapenemase-positive isolates\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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes (N\u0026thinsp;=\u0026thinsp;84)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOne (N\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTwo (N\u0026thinsp;=\u0026thinsp;22)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThree (N\u0026thinsp;=\u0026thinsp;24)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFour (N\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eNDM (N\u0026thinsp;=\u0026thinsp;46)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (17.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (54.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eVIM (N\u0026thinsp;=\u0026thinsp;40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (72.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20 (83.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eOXA-48 (N\u0026thinsp;=\u0026thinsp;70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28 (82.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14 (63.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eKPC (N\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2 (50.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eIMP (N\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (9.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (16.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2 (50.0%)\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 \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates were recovered from various clinical specimens: sputum (n\u0026thinsp;=\u0026thinsp;52, 44.1%), urine (n\u0026thinsp;=\u0026thinsp;20, 16.8%), wound (n\u0026thinsp;=\u0026thinsp;18, 15.3%), blood (n\u0026thinsp;=\u0026thinsp;14, 11.9%), and pus (n\u0026thinsp;=\u0026thinsp;14, 11.9%) (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e isolated from different clinical specimens\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecimens\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWound\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSputum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e44.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlood\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.9\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\u003eThe positivity rate for carbapenemase genes varied across specimen types. The highest positivity rate was observed in pus samples (85.7%), followed by wound (77.8%), urine (70.0%), sputum (69.2%), and blood (57.1%) (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e carbapenemase-encoding genes in clinical specimens\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecimens (N\u0026thinsp;=\u0026thinsp;118)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWound (N\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUrine (N\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSputum (N\u0026thinsp;=\u0026thinsp;52)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBlood (N\u0026thinsp;=\u0026thinsp;14)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePus (N\u0026thinsp;=\u0026thinsp;14)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive (N\u0026thinsp;=\u0026thinsp;84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14 (77.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14 (70.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36 (69.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8 (57.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12 (85.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative (N\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4 (22.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6 (30.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16 (30.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6 (42.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2 (14.3%)\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\u003eThe distribution of specific carbapenemase genes varied across specimen types. \u003cem\u003ebla\u003c/em\u003eOXA-48 was detected at high rates across all specimens: pus (71.4%), urine (60.0%), sputum (57.7%), blood (57.1%), and wound (55.6%). \u003cem\u003ebla\u003c/em\u003eNDM showed variable distribution: pus (57.1%), wound (55.6%), urine (40.0%), sputum (30.8%), and blood (28.6%). \u003cem\u003ebla\u003c/em\u003eVIM was found in: pus (57.1%), blood (42.9%), wound (33.3%), sputum (30.8%), and urine (20.0%). \u003cem\u003ebla\u003c/em\u003eKPC was rare, detected only in blood (14.3%). \u003cem\u003ebla\u003c/em\u003eIMP was found in: urine (20.0%), pus (14.3%), and sputum (3.8%).(Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e carbapenemase-encoding genes in clinical specimens\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGenes (N\u0026thinsp;=\u0026thinsp;84)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecimens (N\u0026thinsp;=\u0026thinsp;118)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWound (N\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUrine (N\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSputum (N\u0026thinsp;=\u0026thinsp;52)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBlood (N\u0026thinsp;=\u0026thinsp;14)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePus (N\u0026thinsp;=\u0026thinsp;14)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eNDM (N\u0026thinsp;=\u0026thinsp;46)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (55.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (40.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (30.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (28.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8 (57.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eVIM (N\u0026thinsp;=\u0026thinsp;40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (20.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (30.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 (42.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8 (57.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eOXA-48 (N\u0026thinsp;=\u0026thinsp;70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (55.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (60.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30 (57.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (57.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10 (71.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eKPC (N\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (14.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003eIMP (N\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (20.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (3.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 (14.3%)\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\u003eAll 118 carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates demonstrated 100% resistance to multiple β-lactam antibiotics, including meropenem, imipenem, ceftazidime, cefepime, ceftriaxone, cefuroxime, cefoperazone, amoxicillin-clavulanate, piperacillin-tazobactam, and aztreonam. High resistance rates were also observed for non-β-lactam antibiotics: ciprofloxacin (95.7%), trimethoprim-sulfamethoxazole (96.6%), gentamicin (93.2%), tobramycin (94.1%), amikacin (91.5%), and levofloxacin (89.8%). Colistin remained the most effective antimicrobial agent, with 97.5% of isolates showing susceptibility and only 2.5% demonstrating resistance (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial resistance profiling of carbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAntibiotics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSusceptible\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResistant\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColistin (CT)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e115 (97.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCiprofloxacin (CIP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (4.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e113 (95.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMeropenem (MR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e118 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCefepime (FEP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e118 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrimethoprim-sulfamethoxazole (COT)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (3.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e114 (96.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLevofloxacin (LE)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (10.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e106 (89.8%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCeftazidime (CAZ)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e118 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGentamicin (GEN)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 (6.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e110 (93.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmoxicillin-clavulanate (AMC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e118 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCeftriaxone (CTR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e118 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePiperacillin-tazobactam (PIT)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e118 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCefuroxime (CXM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e118 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCefoperazone (CS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e118 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmikacin (AK)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (8.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e108 (91.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTobramycin (TOB)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (5.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e111 (94.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImipenem (IPM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e118 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAztreonam (AZT)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e118 (100.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides comprehensive data on the molecular epidemiology of carbapenemase-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e in Sana\u0026rsquo;a, Yemen, revealing a high prevalence of carbapenemase genes with frequent co-occurrence of multiple genes and extensive multidrug resistance. Our findings have important implications for infection control, antimicrobial stewardship, and public health policy in Yemen and the broader Middle East region.\u003c/p\u003e \u003cp\u003eThe overall prevalence of carbapenemase genes in our study was 71.2%, indicating that the majority of carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates in Sana\u0026rsquo;a harbor at least one carbapenemase-encoding gene. This high prevalence is consistent with reports from other Middle Eastern countries and underscores the significant burden of carbapenemase-mediated resistance in the region [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe predominance of \u003cem\u003ebla\u003c/em\u003eOXA-48 (59.3%) in our study aligns with the epidemiological pattern observed in many Middle Eastern and North African countries. Similarly high rates of \u003cem\u003ebla\u003c/em\u003eOXA-48 have been reported in studies from Saudi Arabia (87.0%) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], Tunisia (86.72%) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], Egypt (91.6%) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and Turkey (79.8%) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The widespread dissemination of \u003cem\u003ebla\u003c/em\u003eOXA-48 in the region has been attributed to the successful spread of specific plasmids carrying this gene, particularly the IncL/M-type plasmid [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, our findings contrast with some studies from the Gulf region that reported lower \u003cem\u003ebla\u003c/em\u003eOXA-48 prevalence, such as Qatar (20%) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and UAE (23.1%) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], suggesting significant geographical variation even within the Arabian Peninsula.\u003c/p\u003e \u003cp\u003eThe prevalence of \u003cem\u003ebla\u003c/em\u003eNDM (38.9%) in our study is comparable to reports from Qatar (39%) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], Saudi Arabia (41.3%) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and Pakistan (44.8%) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], but lower than rates reported in some studies from Egypt (90.9%, 95.8%) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], Tunisia (86.8%) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and Iraq (70.4%) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The NDM enzyme family has emerged as a major contributor to carbapenem resistance globally, with NDM-1 being the most common variant [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The presence of NDM-producing organisms is particularly concerning because metallo-β-lactamases confer resistance to nearly all β-lactams except aztreonam and are not inhibited by currently available β-lactamase inhibitors [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe detection of \u003cem\u003ebla\u003c/em\u003eVIM (33.9%) in our study represents a significant finding, as VIM-type carbapenemases have been less commonly reported in the Arabian Peninsula compared to other regions. Our prevalence is higher than that reported in some neighboring countries but consistent with the increasing recognition of VIM enzymes in the Middle East [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The presence of VIM alongside other metallo-β-lactamases (NDM and IMP) suggests diverse mechanisms of metallo-β-lactamase-mediated resistance in Yemen.\u003c/p\u003e \u003cp\u003eThe low prevalence of \u003cem\u003ebla\u003c/em\u003eKPC (1.7%) in our study is consistent with the general epidemiological pattern in the Middle East, where KPC enzymes are less common compared to OXA-48 and NDM [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the detection of KPC, even at low levels, is noteworthy given its association with high-risk clones and its potential for rapid dissemination [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Studies from Palestine [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and Egypt [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] have documented the emergence of KPC-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e in the region, suggesting that continued surveillance is warranted.\u003c/p\u003e \u003cp\u003eSimilarly, \u003cem\u003ebla\u003c/em\u003eIMP (6.8%) was detected at relatively low frequency in our study. IMP-type metallo-β-lactamases were among the first carbapenemases to be described and have been reported primarily from Asia and the Pacific region [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The presence of IMP in Yemen, albeit at low levels, indicates the diversity of carbapenemase types circulating in the country.\u003c/p\u003e \u003cp\u003eOne of the most striking findings of our study is the high frequency of co-occurrence of multiple carbapenemase genes, with 42.3% of carbapenemase-positive isolates harboring two or more genes. This phenomenon has been increasingly recognized in the Middle East and North Africa region [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The co-harboring of multiple carbapenemase genes is particularly concerning for several reasons. First, it may confer resistance to a broader spectrum of β-lactam antibiotics and potentially other antimicrobial classes if the resistance genes are located on the same mobile genetic elements. Second, it complicates the detection and characterization of carbapenemase-producing organisms, as phenotypic tests may not distinguish between different carbapenemase types. Third, it suggests the presence of highly successful plasmids or clones capable of accumulating multiple resistance determinants.\u003c/p\u003e \u003cp\u003eIn our study, 24 isolates (20.3%) harbored three carbapenemase genes, and 4 isolates (3.4%) harbored four genes simultaneously. The most common combination in four-gene isolates included \u003cem\u003ebla\u003c/em\u003eNDM, \u003cem\u003ebla\u003c/em\u003eOXA-48, \u003cem\u003ebla\u003c/em\u003eVIM, and either \u003cem\u003ebla\u003c/em\u003eKPC or \u003cem\u003ebla\u003c/em\u003eIMP. Similar patterns of multiple carbapenemase gene co-occurrence have been reported in studies from Morocco [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], Saudi Arabia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and Egypt [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The co-existence of class A, B, and D carbapenemases in the same isolate represents a particularly challenging scenario for antimicrobial therapy, as it eliminates most β-lactam options and necessitates reliance on last-resort agents such as colistin.\u003c/p\u003e \u003cp\u003eThe mechanisms underlying the co-occurrence of multiple carbapenemase genes likely involve the acquisition of multiple plasmids or the presence of complex genetic structures harboring multiple resistance genes. Studies have shown that carbapenemase genes are often located on conjugative plasmids belonging to various incompatibility groups, which can be transferred horizontally between bacterial species [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The accumulation of multiple carbapenemase genes may be facilitated by selective pressure from antimicrobial use and by the genetic plasticity of \u003cem\u003eK. pneumoniae\u003c/em\u003e, which readily acquires and maintains mobile genetic elements.\u003c/p\u003e \u003cp\u003eOur study revealed that carbapenemase-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e was isolated from diverse clinical specimens, with the highest proportion from sputum (44.1%), reflecting the importance of respiratory tract infections in hospitalized patients. The high positivity rate for carbapenemase genes in pus (85.7%) and wound (77.8%) samples is particularly concerning, as these infections are often associated with surgical procedures and may indicate nosocomial transmission. The detection of carbapenemase-producing organisms in blood cultures (57.1% positivity rate) is especially alarming, as bloodstream infections caused by carbapenem-resistant organisms are associated with high mortality rates and limited treatment options [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe distribution of specific carbapenemase genes varied across specimen types, with \u003cem\u003ebla\u003c/em\u003eOXA-48 being consistently prevalent across all specimen types. This ubiquitous distribution suggests that OXA-48-producing strains are well-established in the healthcare facilities studied and may be transmitted through various routes. The higher prevalence of \u003cem\u003ebla\u003c/em\u003eNDM and \u003cem\u003ebla\u003c/em\u003eVIM in pus and wound samples compared to other specimen types may reflect specific epidemiological patterns or clonal spread within surgical wards.\u003c/p\u003e \u003cp\u003eThe antimicrobial resistance profiles of the carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates in our study demonstrate extensive multidrug resistance, with 100% resistance to all β-lactam antibiotics tested. This pan-β-lactam resistance is expected given the presence of carbapenemases, which hydrolyze virtually all β-lactam antibiotics. The high resistance rates to fluoroquinolones (ciprofloxacin 95.7%, levofloxacin 89.8%) and aminoglycosides (gentamicin 93.2%, amikacin 91.5%, tobramycin 94.1%) indicate that these isolates have acquired multiple resistance mechanisms beyond carbapenemases, likely through the accumulation of additional resistance genes on plasmids or chromosomal mutations [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe near-universal resistance to trimethoprim-sulfamethoxazole (96.6%) further limits therapeutic options for urinary tract infections, which are commonly caused by \u003cem\u003eK. pneumoniae\u003c/em\u003e. The extensive multidrug resistance observed in our study is consistent with reports from other countries in the region [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and reflects the serious challenge posed by carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e to clinical management.\u003c/p\u003e \u003cp\u003eColistin remained the most effective antimicrobial agent in our study, with 97.5% susceptibility. This finding is encouraging and consistent with other studies from the Middle East that have reported high colistin susceptibility rates among carbapenem-resistant Enterobacteriaceae [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the detection of colistin resistance in 2.5% of isolates is concerning, as colistin is often considered the last-resort treatment for infections caused by carbapenem-resistant Gram-negative bacteria. The emergence of colistin resistance, particularly in the context of carbapenem resistance, creates a scenario of pan-drug resistance with extremely limited or no treatment options. The mechanisms of colistin resistance in Enterobacteriaceae include chromosomal mutations in genes regulating lipopolysaccharide modification and, more recently, plasmid-mediated resistance through \u003cem\u003emcr\u003c/em\u003e genes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe findings of this study have several important implications for clinical practice and public health policy in Yemen. First, the high prevalence of carbapenemase-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e necessitates the implementation of robust infection prevention and control measures in healthcare facilities. These measures should include strict adherence to hand hygiene protocols, contact precautions for patients colonized or infected with carbapenem-resistant organisms, environmental cleaning and disinfection, and surveillance cultures to identify asymptomatic carriers [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSecond, the extensive multidrug resistance observed in our study highlights the urgent need for antimicrobial stewardship programs to optimize antimicrobial use and reduce selective pressure for resistance. Antimicrobial stewardship interventions should include guidelines for empirical therapy based on local resistance patterns, de-escalation strategies when culture results become available, and education programs for healthcare providers [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThird, the molecular characterization of carbapenemase genes should be integrated into routine microbiology laboratory practices to enable rapid detection and appropriate infection control responses. While phenotypic methods can provide initial screening, molecular methods such as real-time PCR offer rapid and accurate identification of specific carbapenemase genes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The implementation of molecular diagnostics may be challenging in resource-limited settings like Yemen, but it is essential for effective surveillance and outbreak investigation.\u003c/p\u003e \u003cp\u003eFourth, the co-occurrence of multiple carbapenemase genes in a substantial proportion of isolates suggests that treatment decisions should not rely solely on the detection of a single carbapenemase type. Combination therapy may be necessary for serious infections, and the choice of antimicrobial agents should be guided by comprehensive susceptibility testing, including testing for colistin resistance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates a high prevalence of carbapenemase-encoding genes among carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates in Sana\u0026rsquo;a, Yemen, with \u003cem\u003ebla\u003c/em\u003eOXA-48, \u003cem\u003ebla\u003c/em\u003eNDM, and \u003cem\u003ebla\u003c/em\u003eVIM being the most common. The frequent co-occurrence of multiple carbapenemase genes and extensive multidrug resistance, with only colistin retaining significant activity, represent serious challenges for clinical management and infection control. These findings underscore the urgent need for strengthened antimicrobial stewardship programs, routine molecular surveillance for carbapenemase genes, enhanced infection prevention and control measures, and the development of new therapeutic strategies. The implementation of these interventions is critical to prevent further dissemination of carbapenem-resistant organisms and to preserve the effectiveness of available antimicrobial agents in Yemen and the broader region.\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\"\u003eCR-KP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarbapenem-resistant Enterobacteriaceae\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 and Laboratory Standards Institute\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\"\u003eGCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGulf Cooperation Council\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImipenemase\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\"\u003eMBL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMetallo-β-lactamase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emCIM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eModified carbapenem inactivation method\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\"\u003eNDM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNew Delhi metallo-β-lactamase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOXA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOxacillinase\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\"\u003eVIM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVerona integron-encoded metallo-β-lactamase\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":"\u003ch3\u003eEthics approval and consent to participate\u003c/h3\u003e\n\u003cp\u003eThis study was approved by the institutional ethics committee of the participating hospitals and the National Center of Public Health Laboratories in Sana\u0026rsquo;a, Yemen. The study involved only bacterial isolates collected as part of routine clinical care, and no additional patient interventions were performed. Patient consent was waived by the ethics committee as the study used de-identified bacterial isolates without access to patient identifiable information.\u003c/p\u003e\n\u003ch3\u003eConsent for publication\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eAvailability of data and materials\u003c/h3\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch3\u003eCompeting interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003ch3\u003eAuthors\u0026rsquo; contributions\u003c/h3\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by all authors. The first draft of the manuscript was written by the authors and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch3\u003eAcknowledgements\u003c/h3\u003e\n\u003cp\u003eThe authors would like to thank the staff of the participating hospitals and the National Center of Public Health Laboratories in Sana\u0026rsquo;a, Yemen, for their assistance in sample collection and processing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAl-Abdely, H., AlHababi, R., Dada, H. M., Roushdy, H., Alanazi, M. M., Alessa, A. A., . . . Al-Dughmani, H. (2021). Molecular characterization of carbapenem-resistant Enterobacterales in thirteen tertiary care hospitals in Saudi Arabia. \u003cem\u003eAnnals of Saudi Medicine, 41\u003c/em\u003e(2), 63-70.\u003c/li\u003e\n \u003cli\u003eAl-Tawfiq, J. A., Rabaan, A. 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Journal of Infection and Public Health, 17(3), 424-432. doi: 10.1016/j.jiph.2024.02.011\u003c/li\u003e\n \u003cli\u003eInfection and Drug Resistance (2022). Carbapenemase genes and antimicrobial resistance patterns in carbapenem-resistant Klebsiella pneumoniae isolated from ventilator-associated pneumonia in Egypt. Infection and Drug Resistance, 15, 2847-2858.\u003c/li\u003e\n \u003cli\u003eAlizadeh, H., Khodavandi, A., Alizadeh, F., et al.\u0026nbsp;(2021). Molecular Characteristics of Carbapenem-Resistant Klebsiella pneumoniae Isolates Producing blaVIM, blaNDM, and blaIMP in Clinical Centers in Isfahan, Iran. Jundishapur Journal of Microbiology, 14(2), e114473. doi: 10.5812/JJM.114473\u003c/li\u003e\n \u003cli\u003eAl Laham, N., Afifi, A.A., Mellmann, A., et al.\u0026nbsp;(2023). Characterization of carbapenem-resistant Klebsiella pneumoniae from blood cultures in Gaza Strip hospitals, Palestine. International Journal of Medical Microbiology, 313(1), 151645. doi: 10.1016/j.ijmm.2025.151645\u003c/li\u003e\n \u003cli\u003eEl-Sayed Ahmed, M.A., Yang, Y., Yang, Y., et al.\u0026nbsp;(2021). Emergence of Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae Co-harbouring a blaNDM-1-Carrying Virulent Plasmid and a blaKPC-2-Carrying Plasmid in an Egyptian Hospital. mSphere, 6(3), e00088-21. doi: 10.1128/mSphere.00088-21\u003c/li\u003e\n \u003cli\u003eGhazawi, A., Elbediwi, M., P\u0026aacute;l, T., et al.\u0026nbsp;(2025). Comparative Genomic Study of blaKPC-2-Carrying Multidrug-Resistant Klebsiella pneumoniae in the United Arab Emirates. Research Square [Preprint]. doi: 10.21203/rs.3.rs-7111183/v1\u003c/li\u003e\n \u003cli\u003eMenif, B., Worley, J., Mansour, N., et al.\u0026nbsp;(2025). Leveraging the global genomic epidemiology of carbapenemase-producing Klebsiella pneumoniae to inform infection prevention in Tunisian hospitals. medRxiv [Preprint]. doi: 10.1101/2025.09.22.25332449\u003c/li\u003e\n \u003cli\u003eLee Y-L, Chen H-M, Hii M, Hsueh P-R. Carbapenemase-producing enterobacterales infections: recent advances in diagnosis and treatment. Int J Antimicrob Agents. 2022;59:106528.\u003c/li\u003e\n \u003cli\u003eChakraborty T, Sadek M, Yao Y, Imirzalioglu C, Stephan R, Poirel L, et al.\u0026nbsp;Crossborder emergence of Escherichia coli producing the carbapenemase NDM-5 in Switzerland and Germany. J Clin Microbiol. 2021;59:10\u0026ndash;1128.\u003c/li\u003e\n \u003cli\u003eZhang Y, Chen C, Wu J, Jin J, Xu T, Zhou Y, et al. Sequence-based Genomic Analysis Reveals Transmission of Antibiotic Resistance and virulence among carbapenemase-producing Klebsiella pneumoniae strains. mSphere. 2022;7:e00143\u0026ndash;22.\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":"Carbapenemase, blaOXA-48, blaNDM, blaVIM, blaKPC, blaIMP, Klebsiella pneumoniae, Enterobacteriaceae, Yemen, antimicrobial resistance","lastPublishedDoi":"10.21203/rs.3.rs-9067881/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9067881/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch3\u003eBackground\u003c/h3\u003e\n\u003cp\u003eThe emergence of carbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (CR-KP) represents a critical threat to global public health, severely limiting therapeutic options. Surveillance of these resistance determinants is essential to guide infection control and antimicrobial stewardship. This study aimed to identify the prevalence and distribution of specific carbapenemase-encoding genes (\u003cem\u003ebla\u003c/em\u003eKPC, \u003cem\u003ebla\u003c/em\u003eNDM, \u003cem\u003ebla\u003c/em\u003eIMP, \u003cem\u003ebla\u003c/em\u003eVIM, and \u003cem\u003ebla\u003c/em\u003eOXA-48) among clinical isolates of \u003cem\u003eK. pneumoniae\u003c/em\u003e collected from various hospitals in Sana’a, Yemen.\u003c/p\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cp\u003eA cross-sectional study was conducted in Sana’a, Yemen from October 2023 to December 2024. One hundred eighteen non-duplicate clinical isolates of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (family Enterobacteriaceae) showing resistance to carbapenems (imipenem and meropenem) were recovered from wound, urine, sputum, blood and pus specimens collected at two private hospitals and the National Center of Public Health Laboratories. Antimicrobial susceptibility testing was performed by Kirby-Bauer disk diffusion and colistin MIC by broth disk elution according to CLSI 2024. Carbapenemase production was screened by reduced carbapenem susceptibility and confirmed phenotypically with the combined imipenem/EDTA disk test for metallo-β-lactamases. Genotypic detection of \u003cem\u003ebla\u003c/em\u003eKPC, \u003cem\u003ebla\u003c/em\u003eNDM, \u003cem\u003ebla\u003c/em\u003eIMP, \u003cem\u003ebla\u003c/em\u003eVIM and \u003cem\u003ebla\u003c/em\u003eOXA-48 was performed by real-time PCR using DNA extracted by boiling. Descriptive statistics were analyzed with SPSS v26.\u003c/p\u003e\n\u003ch3\u003eResults\u003c/h3\u003e\n\u003cp\u003eAmong the 118 isolates, 84 (71.2%) were positive for at least one carbapenemase-encoding gene. The most predominant gene was \u003cem\u003ebla\u003c/em\u003eOXA-48 (59.3%), followed by \u003cem\u003ebla\u003c/em\u003eNDM (38.9%), \u003cem\u003ebla\u003c/em\u003eVIM (33.9%), \u003cem\u003ebla\u003c/em\u003eIMP (6.8%), and \u003cem\u003ebla\u003c/em\u003eKPC (1.7%). Regarding specimen distribution, the highest positivity rate for these genes was found in pus (85.7%) and wound (77.8%) samples. Co-occurrence of multiple genes was frequent: 22 isolates (18.6%) harbored two genes, 24 (20.3%) harbored three genes, and 4 (3.4%) harbored four genes (\u003cem\u003ebla\u003c/em\u003eNDM, \u003cem\u003ebla\u003c/em\u003eOXA-48, \u003cem\u003ebla\u003c/em\u003eVIM, and either \u003cem\u003ebla\u003c/em\u003eKPC or \u003cem\u003ebla\u003c/em\u003eIMP). Antimicrobial susceptibility testing revealed 100% resistance to most β-lactams, including meropenem, imipenem, and ceftazidime. High resistance rates were also observed for ciprofloxacin (95.7%) and gentamicin (93.2%). Colistin remained the most effective agent, with a susceptibility rate of 97.5%.\u003c/p\u003e\n\u003ch3\u003eConclusions\u003c/h3\u003e\n\u003cp\u003eCarbapenemase genes—particularly \u003cem\u003ebla\u003c/em\u003eOXA-48, \u003cem\u003ebla\u003c/em\u003eNDM and \u003cem\u003ebla\u003c/em\u003eVIM—are widespread among carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e in Sana’a, Yemen, with frequent co-occurrence and extensive multidrug resistance. These findings underscore the urgent need for strengthened antimicrobial stewardship, routine molecular surveillance for carbapenemase genes, and rigorous infection-control measures to limit dissemination.\u003c/p\u003e","manuscriptTitle":"Distribution of carbapenemase-encoding genes (blaIMP, blaNDM, blaKPC, blaVIM and blaOXA-48) in carbapenem-resistant Klebsiella pneumoniae from Yemen","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-31 11:24:26","doi":"10.21203/rs.3.rs-9067881/v1","editorialEvents":[{"type":"communityComments","content":1},{"type":"reviewerAgreed","content":"23224067184936621278108842940831995690","date":"2026-05-19T06:49:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42554816237658053262678329385958914817","date":"2026-05-18T13:49:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-26T12:19:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-18T09:00:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-18T09:00:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2026-03-09T03:13:15+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":"50731d6e-75b0-4ef2-b6b7-b055c8bb7ffa","owner":[],"postedDate":"March 31st, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"23224067184936621278108842940831995690","date":"2026-05-19T06:49:08+00:00","index":37,"fulltext":""},{"type":"reviewerAgreed","content":"42554816237658053262678329385958914817","date":"2026-05-18T13:49:15+00:00","index":36,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-31T11:24:26+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-31 11:24:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9067881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9067881","identity":"rs-9067881","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}