https://orcid.org/0000-0003-1510-8711
Figures
Abstract
The global rise in antimicrobial resistance is largely driven by DNA-encoded, antibiotic-hydrolyzing enzymes. This study aimed to determine the prevalence of ESBL, AmpC-BL, MBL, and carbapenemase genes. A cross-sectional study was conducted from September 2018 to March 2019 using standard microbiological and molecular methods. Among 231 Gram-negative bacterial isolates, 176 (76.2%) carried beta-lactamase genes confirmed by PCR. ESBLs were detected in 117 (50.6%), MBLs in 50 (21.6%), carbapenemases in 46 (19.9%), and AmpC-BLs in 22 (9.5%). The most frequent carriers were Klebsiella pneumoniae, Escherichia coli, and Acinetobacter species. The dominant ESBL genes were blaCTX-M (85.2%) and blaTEM (79.5%), with blaBEL (17.2%) surpassing blaSHV (9.8%), indicating a shift in resistance patterns, while among AmpC genes, blaFOX (39.4%) and blaCITM (36.4%) were the most prevalent. The blaOXA-23 (40.6%) and blaKPC (14.5%) were the most common carbapenemase genes, while blaNDM (39.1%) and blaVIM (34.8%) dominated among MBLs. The high prevalence of blaOXA-23 in Ethiopia is a significant finding, which I have not found evidence of in similar African or Asian settings. A comparable trend in Taiwan suggests medical tourism may contribute to the spread of resistant bacteria. These findings highlight the critical importance of continuous molecular surveillance and indicate that factors like medical tourism may contribute to the international dissemination of resistant strains.
Citation: Beshah D, Tessema TS, Woldemichael GB, Belachew EB, Alemayehu DH, Tesfaye A, et al. (2026) Resistance gene profiling of beta-lactamase and carbapenemase in gram-negative blood isolates: A tertiary care hospital. PLoS One 21(3): e0344856. https://doi.org/10.1371/journal.pone.0344856
Editor: Nabi Jomehzadeh, Ahvaz Jondishapour University of Medical Sciences Faculty of Medicine, IRAN, ISLAMIC REPUBLIC OF
Received: September 26, 2025; Accepted: February 26, 2026; Published: March 30, 2026
Copyright: © 2026 Beshah et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This research work was funded by the Addis Ababa University vice president for research and technology transfer office thematic research funding program, with VPRTTPY-0402018 grant numbers awarded to the author Dr. Tamrat Abebe.
Competing interests: The authors declare that there is no competing interest.
Abbreviation:: AST, Antimicrobial susceptibility test; DRM, Drug resistance mechanism; ESΒL, Extended-spectrum beta-lactamase; AmpC-BL, AmpC-beta-lactamase; MBL, Metallo beta-lactamases; Carba, Carbapenemase; ICU, Intensive care unit; NICU, Neonatal ICU; SICU, Surgical ICU; MICU, Medical ICU; SWI, Surgical wound infection; RTI, Respiratory tract infection; UTI, Urinary tract infection
Introduction
Antibiotic-resistant gram-negative bacteria (GNB) due to beta-lactamases and carbapenemases are a major cause of morbidity, mortality, and significant economic costs worldwide, and their incidence and spread are on the rise [1–3]. These antibiotic resistance determinants are passed from one bacterial community to another through horizontal gene transfer mechanisms [1,4]. The beta-lactamase-mediated resistance is increasingly associated with Plasmid-Encoded Extended-spectrum beta-lactamase (ESBL) and Carbapenemase, sometimes in combination with other resistance mechanisms (e.g., porin loss, efflux pumps, target site alteration) [4,5].
Beta-lactamases, which are produced by bacteria, lead to a variety of beta-lactam antibiotic resistance, including penicillin, cephalosporin, cephamycin, monobactam, and carbapenem [6]. The major blaCTX-M, blaSHV, blaTEM, and minor blaGES, blaVEB, blaBEL, and blaPER genes that produce ESBL enzymes are capable of hydrolyzing penicillin, monobactams, and third-generation cephalosporins and are inhibited by beta-lactamase inhibitors except for blaKPC, which can hydrolyze carbapenem drugs and beta-lactamase inhibitors [7]. The blaMOX, blaCIT, blaDHA, blaACC, blaEBC, and blaFOX genes, classified as AmpC-beta-lactamases (AmpC-BL), hydrolyze penicillins, monobactams, broad and extended-spectrum cephalosporins, and cephamycin but are sensitive to Cefepime [8,9]. The major blaNDM-1, blaIMP, blaVIM, and the minor blaGIM-1, blaSIM-1, blaAIM-1, blaDIM, and blaSPM-1 genes that produce carbapenem-resistant MBL enzymes are metalloproteins and are capable of hydrolyzing all (third-generation cephalosporin, cephamycin, and carbapenem) antibiotics except monobactams [5]. The blaOXA-23-group, blaOXA-24-group, blaOXA-48, and blaOXA-58-group genes are oxacillinase derivatives, like blaKPC and blaIMI Carbapenemase, their hydrolytic activity against carbapenems and some 3rd generation cephalosporins, including monobactam, and they are not inhibited by clavulanic acid and tazobactam [5].
Infections with ESBL-producing bacteria have been successfully treated with carbapenem drugs such as Imipenem, Meropenem, Doripenem, and Ertapenem, but the massive use of this antibiotic has accelerated the dissemination of carbapenemase-producing genes [5]. The Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii are the most common drug-resistant GNB [3]. These strains are frequently overlooked by clinicians without therapeutic options [1]. Although beta-lactam antibiotics are heavily used in many developing countries, the diversity of beta-lactamase and carbapenemase [10] genes is poorly understood [11]. There are a few studies conducted in Ethiopia. Still, the diversity of these drug-resistance genes is not well explored. Therefore, this study aims to further explore the diversity of beta-lactamase and carbapenemase genes in patients admitted to tertiary care hospitals, Tikur Anbessa Specialized Referral Hospital in Addis Ababa, Ethiopia.
Materials and methods
Description of study design and research setting
A cross-sectional study was conducted from September 2018 to March 2019 in TASH, Addis Ababa, Ethiopia, which is the largest specialized hospital in the country, with over 700 beds [12]. All patients from all age groups suspected of bloodstream infection, who visited Tikur Anbessa specialized hospital during the study period, who were willing to give blood samples, and who agreed with consent to the study, were included in this study. Convenience sampling techniques were used for all age groups of BSI-suspected patients who volunteered to provide a blood sample and sign a consent form to participate in the study. Patients who refused to participate in the study or had received antibiotics during the previous ten days were excluded.
Data collection
A standardized questionnaire was used to collect clinical and demographic information, while a trained nurse reviewed the patient’s medical records. The samples were taken by trained laboratory technologists and professional nurses who have previously gathered research data and blood samples. The lead investigator gave all data and sample collectors detailed instructions on how to use pre-made, standardized questionnaires, draw blood, and transport samples to the lab. The principal investigator and expert microbiologists monitored the microbial growth in the blood culture bottle and the rest downstream processes.
Sample collection, bacterial isolation, and identification
A total of 1486 patient samples were screened, and 231 gram-negative isolates were analyzed. Sample collection, culture, and bacterial identification were initially performed, after which antimicrobial susceptibility testing was carried out. This study was followed by our previous study on phenotypic detection of extended-spectrum beta-lactamase and carbapenem resistance, where the detailed methods are described [13].
Phenotypic ESBL and AmpC detection
Drug-resistant isolates were initially screened by routine AST. Resistance or intermediate results to ceftazidime, ceftriaxone, or cefotaxime suggested ESBL, and additional resistance to cephamycin (cefotetan) indicated possible AmpC according to CLSI criteria. Breakpoints used for ESBL screening were CAZ ≤ 22 mm, CRO ≤ 25 mm, and CTX ≤ 27 mm. AmpC screening used the same cephalosporins plus cefepime and cefoxitin, with indicative values FEP ≥ 25 mm, and FOX ≤ 18 mm. All cefoxitin-resistant isolates underwent confirmatory testing. ESBL and AmpC were confirmed by E-test following CLSI and EUCAST guidelines [8,14]. E-Test: MIC test strips of ceftazidime ± clavulanic acid and cefotaxime ± clavulanic acid were used. ESBL was confirmed when CTX ≥ 0.5 with CTX/CTL ratio ≥8, or CAZ ≥ 1 with CAZ/CAL ratio ≥8. AmpC testing used cefotetan ± cloxacillin strips; CN/CNI ratio ≥8 indicated AmpC production [15].
Carbapenem-resistant detection
The modified carbapenemase inactivation method (mCIM) and EDTA-modified mCIM (eCIM) were used to test carbapenem-resistant or intermediate Enterobacteriaceae (Imipenem, Meropenem, Doripenem, Ertapenem) and non-fermenters (Imipenem, Meropenem, Doripenem), excluding Acinetobacter spp. In mCIM, a zone of inhibition of 6–15 mm is positive, 16–18 mm is considered positive with colonies, and ≥19 mm is negative, indicating resistance via non-carbapenemase mechanisms. In eCIM, an increase of ≥5 mm in zone diameter combined with mCIM positivity indicates metallo-beta-lactamase production [14,16].
DNA extraction for gene detection
Genomic DNA was extracted using a modified boiling method. A 1000 µL aliquot of cell suspension (10⁷ cells/mL) from each GNB was incubated overnight at 37 °C, then centrifuged (4500 rpm, 5 min, 4 °C). Pellets were resuspended in 50 µL nuclease-free water, boiled at 100 °C for 5 min, and centrifuged (3000 g, 10 min). The supernatant was transferred to a new tube, mixed with 0.7 volumes of cold absolute ethanol, and centrifuged (20 min) to precipitate DNA. The pellet was washed with 70% cold ethanol, air-dried, and re-suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) [17]. The final concentration of 70–100ng/μl of the template was prepared by using a Nanodrop Spectrophotometer.
Conventional PCR for gene detection
The PCR was performed using hot start ready-made Multiplex PCR (5x HOT FIREPol Multiplex Master Mix) containing HOT FIREPol DNA polymerase, 5x multiplex buffer, 10 mM MgCl2, dNTPs, and BSA compounds that increase sample density for direct loading were imported from Solis Biodyne company, Estonia, according to the manufacturer’s instructions [18]. All primers were obtained from IDT except AmpC-BL and blaVIM and blaIMP primers, which were imported from LIGO Macrogen Europe. Oligonucleotide yield or lyophilized primer was reconstituted by nuclease-free TE buffer (10 mM Tris, pH 8.0; 0.1 mM EDTA, pH 8.0) using the integrated DNA technology (IDT) protocol (Tables 1 and 2). The PCR amplification was done by using the T3000 Biometra thermocycler, which is the product of Analytik Jena, a company based in Germany.
Extended-spectrum beta-lactamase gene detection
The isolates that were screened positive for ESBL were subjected to ESBL multiplex 1 and 2 PCR tests using specific primers (Table 1). In multiplex 1, ESBL, blaSHV, blaCTX-M, and blaTEM genes were amplified adopting Trung et al.‘s protocol [19]. Multiplex PCR reactions were performed in a final volume of 20 μl containing 4 μl Multiplex master mix, 0.6 μl reverse and 0.6 μl forward primers each containing (10 ng/μl and 0.2 μl), 13.8 μl nuclease-free water, and 1 μl of 70–100 ng/μl DNA template. The thermal cycling conditions were: initial denaturation at 95°C for 240 seconds, followed by 35 cycles of denaturation at 94°C for 25 s, annealing at 58°C for 45 s, extension at 72°C for 1 minute, and final extension at 72°C for 5 min. The multiplex 2 PCR was performed for ESBL, blaPER, blaGES, blaBEL, and blaVEB, using the protocols of Bogaerts et al. [20] (Table 1). The reactions were performed in a final volume of 20 μl containing 4 μl Multiplex master mix, 0.8 μl reverse and 0.8 μl forward primers each containing (10 ng/μl), 13.4 nucleases-free water, and 1 μl of 70–100 ng/μl DNA template. The thermal cycling conditions were: initial denaturation at 95°C for 300 seconds, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 90 seconds, extension at 72°C for 90 seconds, and final extension at 72°C for 5 minutes.
AmpC Beta-Lactamase genes detection
The isolates that were screened positive for AmpC-BLs were subjected to ESBL multiplex 1–2 AmpC-BLs multiplex 3 PCR tests (Table 1). A multiplex 3 PCR was set for AmpC-BLs family blaMOXM, blaCITM, blaDHAM, blaACCM, blaEBCM, and blaFOXM [21] (Table 1) in a final volume of 20 μl containing 4 μl Multiplex master mix, 1.0 μl reverse, and 1.0 μl forward primers each containing (0.6μM for blaMOXM, blaCITM, blaDHAM; 0.5 μM for blaACCM, blaEBCM, and 0.4 μM for blaFOXM), 13.0 μl nuclease-free water, and 1 μl of 70–100 ng/μl DNA template. The thermal cycling conditions were: initial denaturation at 95°C for 240 seconds, followed by 25 cycles of denaturation at 94°C for 30 seconds, annealing at 64°C for 60 seconds, extension at 72°C for 1 minute, and final extension at 72°C for 7 minutes.
Detection of carbapenemase, oxacillinase, and MBL genes
The carbapenem drug-resistant isolates were subjected to ESBL multiplex PCR 1−3 (Table 2) and carbapenemase multiplex PCR 1−3 (Table 2). Multiplex CARBA PCR 1 was employed in the initial screening test for carbapenemase blaKPC, blaOXA-48, and metallo-beta-lactamase genes of blaNDM, blaVIM, and blaIMP [20] (Table 2) in a final volume of 20 μl containing 4 μl Multiplex master mix, 0.6 μl reverse and 0.6 μl forward primers each containing (10 ng/μl), 13.8 μl nuclease-free water, and 1 μl of 70−100 ng/μl DNA template. The thermal cycling conditions were: initial denaturation at 95°C for 300 seconds, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 90 seconds, extension at 72°C for 90 seconds, and final extension at 72°C for 300 seconds. Multiplex PCR 2 for OXA family Carbapenemases producing genes of the blaOXA-23 group, blaOXA-24/143 group, and blaOXA-58 group was carried out using a similar procedure as above [20].
Minor metallo-beta-lactamase genes
The CARBA multiplex 3 was used for the detection of the minor Metallo-beta-lactamase producing genes, blaAIM, blaGIM, blaSIM, and blaDIM [22] for carbapenem-resistant strains (Table 2). It was performed in a final volume of 20 μl containing 4 μl Multiplex master mix, 0.8 μl reverse and 0.8 μl forward primers each containing (10 ng/μl), 13.4 μl nuclease-free water, and 1 μl of 70–100 ng/μl DNA template. The thermal cycling conditions were: initial denaturation at 94°C for 300 seconds, followed by 36 cycles of denaturation at 94°C for 30 seconds, annealing at 52°C for 40 seconds, extension at 72°C for 50 seconds, and final extension at 72°C for 350 seconds.
Quality control
The bacterial identification, AST, and phenotyping characterization of beta-lactamase production test quality controlling procedure were mentioned in the previous paper. DNA samples from reference blaTEM, blaSHV, and blaCTX-M positive strains were utilized as positive controls for ESBL detection. During PCR analysis, the known laboratory reference blaKPC, blaOXA-48, and blaNDM genes were utilized as positive controls, with Escherichia coli ATCC® 25922 serving as the negative control. Each primer pair was tested in monoplex PCR before multiplexing. Before multiplexing the ESBL and carbapenemase primers, each primer was evaluated in a monoplex PCR test.
Agarose gel electrophoresis
The amplified PCR products from all PCR reactions were loaded in a 1.5% agarose gel containing ethidium bromide and run using 120V, 400mA, for 60 minutes using a Bio-Rad gel electrophoresis machine. DNA bands were visualized using a gel doc Imaging System (Bio-Rad) and results were taken through the internet.
Statistical analysis and interpretation
Data were collected by trained data collectors, and data quality was ensured through the use of standardized data collection formats and materials. Data captured in EPI INFO were cleaned and analyzed by using SPSS version 24 software for further processing. The quantitative data collected using different techniques were analyzed using simple descriptive statistics. The association was also assessed using the χ2-test. In SPSS, sensitivity and specificity are calculated by creating a crosstab between test results and true condition variables, then interpreting the column percentages of true positives and true negatives from the output.
Ethical clearance
The research received approval from the College of Natural and Computational Sciences Institutional Review Board (CNS-IRB) on March 30, 2018, under minute no. IRB/032/2018 at Addis Ababa University. The College of Health Sciences then accepts this approval letter and gives permission to access their facilities for sample collection. The Helsinki [23] declaration’s requirements were met by this research process. We have obtained written assent (12–18) and written approval from the patient’s legal guardians for participants under the age of 18, but we have obtained patient written consent for those above 18. The study’s objectives and methodologies were communicated to the parents or guardians of the participants during the study period. Patients who provided informed consent were chosen and recruited as study subjects, and their results were disclosed to the attending physicians.
Results
Culture results
Among 1486 samples, 417 culture-positive samples were identified, and 224 (53.7%) of these samples were GNB. Of these, GNB 7 (1.68%) had polymicrobial growth, and a total of 231 GNB were identified. However, among 231 GNB, 195 of them had one or more DRM of these, 189 (81.8%) had good DNA extraction quality, and 6 (2.6%) did not; the remaining 42(18.2%) did not have DRM.
Phenotypic screening of ESBL, AmpC, Carbapenem, and MBL-resistant bacteria
A total of 188 (81.4%) ESBL and 82 (35.5%) AmpC-BLs were identified phenotypically. Phenotyping confirmatory tests done by E-test reduced from the primary screening results of 188 (81.4%) and 82(35.5%) to 122 (52.8%) and 33 (14.3%) for ESBL and AmpC, respectively. The Klebsiella pneumoniae, Acinetobacter spp, and Escherichia coli GNB showed a higher number of extended-spectrum cephalosporin and cephamycin drug resistance. The number of drug-resistant strains to carbapenem and MBL resistance strains was 74 (32.0%). But during the confirmatory test, it reduced to 69 (29.9%) carbapenem and metallo-beta-lactam drug-resistant strains. The highest carbapenem drug resistance was in Acinetobacter species [13].
Molecular characterization of drug-resistance genes in GNB from BSI
Among the 231 GNB isolates, 189 had phenotypically confirmed DRM, and 176 (76.2%) were PCR-confirmed drug-resistance enzyme-producing strains. In total, 415 drug-resistance–encoding genes were identified in this study. The highest prevalence of resistance was observed in ESBL-producing isolates, followed by MBL, Carbapenemase, and AmpC β-lactamase–producing isolates, each accounting for 117 (50.6%), 50 (21.6%), 46 (19.9%), and 22 (9.5%), respectively. The total carbapenem resistance that produces Carbapenemase and Metalo-beta-lactamase accounted for 63 (27.3%) (Table 3).
The number of AST-confirmed DRM was reduced by PCR-confirmed DRM encoding gene from ESBL 132(57.1%) to 123 (53.2%), AmpC 36(15.6%) to 22(9.5%), and Carbapenemase with MBL producer 69(29.9%) was reduced to 63(27.3%) (Table 4).
The chi-square analysis indicates a strong association between phenotypic and genotypic characterization of DRM. For all comparisons between phenotypic and genotypic ESBL, AmpC, carbapenemase, and MBLs DRM p-value was < 0.0001 (Table 5).
Molecular identification of ESBL coding genes
Overall, 52.8% (122/231) of isolates were phenotypically ESBL positive for the E-test, and 95.9% (117/122) of these were confirmed positive by PCR, with 100% sensitivity and 86.4% specificity (Table 6). Major ESBL detection was used to identify blaSHV, blaCTXM, and blaTEM genes, whereas minor ESBL was used to detect blaPER, blaGES, blaBEL, and blaVEB genes (Fig 1). The blaCTX-M, detected in 85.2% (104/122) of isolates, was the most prevalent ESBL gene, followed by blaTEM 79.5% (97/122) and blaBEL 17.2% (21/122). The genes blaSHV, blaGES, blaVEB, and blaPER were detected at lower frequencies in this study. Statistical analysis demonstrated significant associations for blaTEM (p < 0.0001), blaCTX-M (p < 0.0001), blaSHV (p = 0.002), blaVEB (p = 0.005), and total PCR ESBL (p = 0.010*), indicating these genes are significantly distributed among the ESBL producers. Genes blaGES and blaBEL did not show statistically significant differences (p > 0.05) (Table 6).
a). ESBL multiplex PCR 1, Major ESBL for the detection of blaSHV, blaCTXM, and blaTEM genes from 3 patient samples with TNC-negative and TPC-positive control. b). ESBL multiplex PCR 2, Minor ESBL for the detection of blaPER, blaGES, blaBEL, and blaVEB genes from 4 patient samples with NC-negative and PC-positive control.
Klebsiella species accounted for 47% (109/231) of all gram-negative bacterial isolates. Of these isolates, 73.3% (80/109) were E-test confirmed ESBL producers, while PCR confirmed were 97.5% (78/80). In this study, 77.3% (58/75) of Klebsiella pneumoniae isolates were identified as ESBL producers using the E-test, and 96.6% (56/58) of these were confirmed to carry ESBL genes by PCR. Among the Klebsiella pneumoniae isolates, the most commonly detected ESBL genes were blaTEM and blaCTX-M, each found in 51 of 58 isolates (87.9%), followed by blaSHV, identified in 8 of 58 isolates (13.8%). Out of 27 Klebsiella oxytoca isolates, 21 were confirmed as ESBL producers by E-test, and all of which tested positive by PCR. Both blaTEM and blaCTX-M genes were detected in all PCR-positive isolates. Other Klebsiella species, such as Klebsiella ozaenae and Klebsiella rhinoscleromatis, were less frequent (Table 6).
A total of 49 Escherichia spp isolates were found, of which 24 (49%) were phenotypically confirmed as ESBL producers using the E-test, and all were confirmed positive by PCR. The most frequently detected gene was blaCTX-M 95.8% (23/24), followed by blaTEM 75% (18/24) (P < 0.0001) and blaBEL 29.2% (7/24). In this study, 47 Acinetobacter spp. Isolates were identified, of which 21.3% (10/47) were confirmed as ESBL producers by E-test. Among these, 80% (8/10) were PCR-positive, with blaBEL being the most commonly detected gene, identified in 50% (5/10) of the ESBL-producing isolates. Other gram-negative bacteria made up 9% (n = 21) of isolates, with 33.3% (7/21) ESBL producers phenotypically and 85.7% (6/7) PCR-positive for ESBL genes (Table 6).
Molecular identification of AmpC-BLs encoding genes
Among the 231 Gram-negative bacterial isolates, 14.3% (33/231) were phenotypically identified as AmpC producers. Of these, 66.7% (22/33) were confirmed positive for AmpC genes by PCR. The presence of AmpC β-lactamase genes predicted phenotypic resistance with 100% sensitivity and 92% specificity. AmpC-BL gene detection was done for the detection of blaMOXM, blaCITM, blaDHAM, blaACCM, blaEBCM, and blaFOXM genes (Fig 2). The most frequently detected gene was blaFOXM, found in 39.4% (13/33) of the isolates, followed by blaCITM in 36.4% (12/33). blaMOXM and blaEBCM were each detected in 15.2% (5/33) of the isolates, with blaEBCM showing a statistically significant association (p = 0.02), and overall PCR AmpC positivity (p = 0.045), suggesting these findings are unlikely due to chance (Table 7).
ESBL Multiplex PCR 3, AmpC-BL gene detection of blaMOXM, blaCITM, blaDHAM, blaACCM, blaEBCM, and blaFOXM genes of 6 patient samples with NC-negative and PC-positive control.
Among 109 Klebsiella isolates, 10.1% were phenotypically positive for AmpC β-lactamase production, all of which were confirmed by PCR. The most frequently detected gene was blaFOX-M, present in 54.5% (6/11) of the phenotypically positive isolates, followed by blaCIT-M in 36.4% (4/11), and blaEBC-M and blaMOXM in 27.3% (3/11) (Table 7).
A total of 49 Escherichia coli isolates were identified in this study, of which 18.4% (9/49) were phenotypically confirmed as AmpC producers. Among these, 88.9% (8/9) were PCR-positive for AmpC genes. The most frequently detected gene in Escherichia coli was blaCITM, found in 77.8% (7/9) of the isolates, followed by blaFOXM at 66.7% (6/9). Other genes, including blaMOXM, blaDHAM, blaACCM, and blaEBCM, were detected at lower frequencies. Additionally, 47 Acinetobacter species isolates were identified, with 19.1% (9/47) phenotypically AmpC producers. PCR confirmed AmpC genes in 22.5% of these isolates. The genes blaFOXM, blaMOXM, and blaCITM were each detected in 11.1% of the PCR-positive Acinetobacter isolates (Table 7).
Molecular identification of the carbapenemase-encoding gene
A total of 69 (29.9%) Gram-negative bacilli (GNB) were identified as carbapenemase producers using the modified carbapenem inactivation method (mCIM) and the EDTA-modified carbapenem inactivation method (eCIM). Of these, 63 isolates (91.3%) were confirmed by PCR to harbor carbapenemase genes. Among the PCR-positive isolates, 46 (66.6%) carried carbapenemase-encoding genes, and 50 (72.5%) possessed metallo-β-lactamase genes. PCR exhibited a specificity of 88% and a sensitivity of 85% in predicting phenotypic carbapenem resistance. Carbapenemase gene detection was done for blaKPC, and Oxacillinase gene detection was done for blaOXA-48, blaOXA-58, blaOXA-23, and blaOXA24/143. Major MBL gene detection was done for blaNDM, (Fig 3) blaIMP, and blaVIM, whereas Minor MBL gene detection was done for blaDIM, blaSIM, blaGIM, and blaAIM genes (Fig 4).
a). CARBA Multiplex PCR 1, Carbapenemase gene detection of blaNDM, blaKPC, and blaOXA-48, genes of 3 patient samples with NC-negative and PC-positive control. b). CARBA Multiplex PCR 2, Oxacillinase gene detection of blaOXA-58, blaOXA-23, and blaOXA24/143, genes of 3 patient samples with NC-negative and PC-positive control.
a).CARBA Multiplex PCR 3, Minor MBL gene detection of blaDIM, blaSIM, blaGIM, and blaAIM genes of 4 patient samples with NC-negative and PC-positive control. b). CARBA Multiplex PCR 1, MBL gene detection of blaIMP genes of patient samples with NC-negative and PC-positive control. c). CARBA Multiplex PCR 1, MBL gene detection of blaVIM genes of patient samples with NC-negative and PC-positive control.
The most prevalent carbapenemase genes in this study were blaOXA-23, detected in 40.6% (28/69) of isolates, followed by blaNDM in 39.1% (27/69), and blaVIM in 34.8% (26/69). Other genes, such as blaKPC and both blaOXA-58 and blaOXA-48, were present in approximately 14.5% and 10.1% of isolates, respectively. Other genes like blaIMP and blaAIM (each 7.2%), blaGIM (5.8%), blaSIM (4.3%), and blaOXA-24/143 (1.4%) were less frequently detected. Statistical analysis showed significant associations for c blaOXA-48 (p = 0.008) and blaNDM (p = 0.002), indicating these genes’ distribution among isolates is unlikely due to chance (Table 8).
Klebsiella pneumoniae (n = 75) showed 24% (18/75) carbapenemase positivity by CIM, and 94.4% (17/18) of these were positive by PCR, and metallo-beta-lactamase producing genes were 66.7% (12/18) and 72.2%(13/18), respectively. Among Klebsiella pneumoniae isolates, the most prevalent carbapenemase gene was blaKPC, detected in 33% (6/18) of isolates, followed by blaOXA-23 in 28% (5/18), and blaIMP in 16.7% (3/18).
Among Acinetobacter spp. (n = 47), 61.7%(29/47) were tested positive for capbapenemase production by CIM. Of these CIM-positive isolates, 86.2% (25/29) were confirmed by PCR to carry carbapenemase and metallo-β-lactamase encoding genes. Of these, 68.9% (20/29) isolates carried carbapenemase, and 75.9% (22/29) isolates carried metallo-beta-lactamase-producing genes. The most frequently detected genes were blaVIM, present in 62.1% (18/29) of the Acinetobacter spp isolates, followed by blaOXA-23, 55.2% (16/29), and blaNDM, 27.6% (8/29) (Table 6).
Among Escherichia coli isolates (n = 38), 15.7% (6/38) tested positive for carbapenemase production using the CIM test, all of which were confirmed by PCR. Of these PCR-positive isolates, 50% (3/6) harbored carbapenemase-encoding genes, and 83.3% (5/6) carried metallo-β-lactamase genes. The most frequently detected gene was blaNDM, found in 50% (3/6) of the isolates, followed by blaVIM and blaAIM, each detected in 33.3% (2/6) (Table 8).
Other Gram-negative bacteria (n = 21) had 33.3% (7/21) AST positivity, with lower gene detection rates. Overall, blaOXA-23 40.6% (28/69), blaNDM 39.1% (27/69), and blaVIM 34.8% (24/69) were the most prevalent carbapenemase genes detected by PCR. In contrast, other carbapenemase genes, such as blaKPC, blaOXA-23, blaNDM, blaIMP, blaAIM, blaGIM, and blaSIM, despite some having moderate prevalence, did not show statistically significant associations (p > 0.05), suggesting a more random or less impactful distribution within this sample (Table 8).
Discussion
Antibiotic-resistant Enterobacteriaceae have a significant impact on clinical outcomes and healthcare systems in tertiary hospitals, particularly among hospitalized patients. Multidrug resistance genes, including ESBLs, AmpC beta-lactamases, carbapenemases, and MBLs, significantly reduce the number of effective antibiotic treatment options [24]. In this study, overall, 52.8% (122 out of 231) of isolates were phenotypically ESBL-positive, and among these, 95.9% (117 out of 132) were confirmed to carry ESBL genes by PCR. This result is also consistent with other studies, showing up to 86.5% agreement in Escherichia coli ESBL-positive isolates [7]. The strong phenotypic–molecular agreement shows phenotypic testing is reliable, but combined methods are needed to detect a few undetected variants or other resistance mechanisms. Such mechanisms may include efflux pump overexpression, porin loss, or reduced membrane permeability, and target site alterations, none of which are detected by the PCR targets used in this study.
Out of 189 phenotypically confirmed DRM, 76.2% had PCR-confirmed drug resistance enzyme-encoding genes. This is comparable to the study done in Ethiopia, with 75% having drug-resistance genes [25], and lower than the study done in the Asian Pacific regions, 86.2% [26]. The number of non-DRM isolates increased from 42 by phenotypic confirmation to 55 PCR-negative GNB. Thirteen strains that were drug-resistant in the phenotyping confirmatory test were PCR-negative, possibly due to genes not included in this study or other than enzyme production DRM. Among PCR-confirmed beta-lactamase producers, ESBL, MBL, carbapenemase, and AmpC accounted for 53.2%, 21.6%, 19.9%, and 9.5%, respectively. Correlation analysis indicated a strong association between phenotypic and molecular characterization for ESBL, AmpC-BL, carbapenemase, and MBL.
The most commonly detected genes in this study were blaCTX-M (85.2%), followed by blaTEM (79.5%) and blaBEL (17.2%). The rapid spread of blaCTX-M is consistent with global trends, where blaCTX-M-type variants have largely replaced past blaTEM and blaSHV variants as the dominant, and this brings a change in ESBL epidemiology [27,28]. There is also a substantial proportion of isolates in this study that showed the co-occurrence of blaCTX-M and blaTEM, which may indicate horizontal gene transfer mediated by plasmids, transposons, and integrons, contributing to multidrug resistance [29]. Among minor ESBL genes, blaBEL 17.2% was more prevalent than the commonest blaSHV 9.8%. This suggests that drug-resistance gene prevalence and distribution fluctuated over time [20]. These findings underscore the dynamic nature of ESBL gene distribution and highlight the importance of continuous molecular surveillance to track resistance evolution.
In this study, Klebsiella species, particularly Klebsiella pneumoniae, were the predominant ESBL-producing bacteria, aligning with global trends where Klebsiella pneumoniae plays a key role in disseminating AMR genes from environmental microbes to clinically important pathogens [30]. The most commonly detected ESBL genes among Klebsiella pneumoniae in this study were blaTEM and blaCTX-M, each found in 51 of the isolates (87.9%), highlighting their co-dominance and significant contribution to β-lactam resistance. The high prevalence of these genes supports findings from other studies, which report that blaTEM is also present in a substantial proportion. These blaTEM and blaCTX-M genes were prevalent in Escherichia coli, which is widely recognized as a major carrier of ESBLs and widespread dissemination of variants in Enterobacteriaceae [31]. In contrast, blaSHV was less common, detected in only 13.8% (8/58) of the isolates. However, historically, blaSHV variants were among the earliest ESBLs identified in Klebsiella species. The continued dominance of blaCTX-M and blaTEM suggests ongoing horizontal gene transfer and the evolution of multidrug-resistant Klebsiella pneumoniae strains.
In this study, blaCTX-M was the most prevalent ESBL gene detected among Escherichia species, identified in 95.8% (23/24) of the isolates, and in hospital settings. The predominance of blaCTX-M is consistent with global epidemiological trends, where blaCTX-M enzymes have largely replaced older ESBL types as the dominant resistance mechanism in Escherichia coli [28].
The AmpC genes confer resistance, and they can be easily disseminated by horizontal gene transfer [32]. In this study, 14.3% (33/231) of GNB isolates were phenotypically positive for AmpC production, reflecting the growing emergence of these resistant strains in clinical settings. Molecular confirmation via PCR revealed that 66.7% (22/33) of phenotypically positive isolates harbored AmpC genes, indicating a moderate concordance between phenotypic and genotypic methods. This discrepancy may be attributed to either non-AmpC resistance mechanisms or limitations in phenotypic assays, as supported by recent findings [32].
The blaFOX and blaCITM genes were the most frequently detected, accounting for 39.4% and 36.4%, respectively. The predominance of blaFOX and blaCITM is consistent with other regional and global reports that have documented its high prevalence [33,34]. The highest rate of AmpC positivity was identified in Escherichia coli (21.1%), with the blaCITM gene detected in 87.5% of these isolates, mirroring similar trends reported in other countries, where this gene has shown wide dissemination [34,35]. The Klebsiella Spp (10.1%), in which the blaFOX gene was the most frequently detected, were present in 54.5% of cases. This also accords with other studies [35,36]. The high prevalence of blaFOX and blaCITM might be due to their plasmid-mediated spread, strong resistance to cephalosporins, and association with Klebsiella spp. and Escherichia coli, respectively.
Pseudomonas spp were positive for AmpC confirmatory E-test and do not express AmpC-BLs genes. This resistance may be due to mechanisms other than hydrolytic enzyme production; for example, efflux pumps are common in non-fermenters, and alterations at antibiotic-binding sites could also contribute. In addition, ten bacterial species do not express the AmpC-BLs producer gene. Continued molecular surveillance is therefore essential to monitor resistance trends, identify emerging gene variants, and inform targeted therapeutic and preventive strategies.
In general, resistance to carbapenems may occur due to three major mechanisms, which include production of β-lactamase enzymes (carbapenemases), overexpression of efflux pumps, and porin-mediated resistance [37,38]. In this study, a total of 69 (29.9%) isolates were confirmed as carbapenemase producers by antimicrobial susceptibility and CIM testing. Carbapenemase producers were found to be higher compared with other studies conducted in Ethiopia (7.7%) [25] and 16% [39], 3.8% in Sudan [40], and 14.7% in the Asia-Pacific region [26]. Of the 69 isolates that showed positive phenotype, 63 (91.3%) were PCR confirmed to carry the gene encoding carbapenemase. The isolates that were PCR confirmed to carry the carbapenemase and Metallo-beta-lactamase encoding genes were 46 (66.6%) and 50 (72.5%), respectively. The findings highlight the aggressiveness and spreading of carbapenem resistance and the need for broader genetic screening and advanced tools like whole-genome sequencing for accurate detection.
The most common GNB with carbapenemase and MBL producing genes were Acinetobacter spp (42.6% and 46.8%) and Klebsiella pneumoniae (16.0% and 17.3%), respectively. The most predominant carbapenemase genes in Acinetobacter spp were blaOXA23 (55.2%) and blaOXA-58 (13.8%), whereas in Klebsiella pneumoniae, blaKPC (33.3%) and blaOXA23 (27.8%).
Among carbapenemase-encoding genes blaOXA-23 28(40.6%) and blaKPC 10(14.5%) was predominant. The higher blaOXA-23 prevalence in Ethiopia is a new report compared with other African and Asian countries, except Taiwan, in which a similar finding was reported [26]. The reason could be that most Ethiopian patients went to Taiwan for medical tourism. The most prevalent MBL producer genes were blaNDM (39.1%) and blaVIM (34.8%). The highest prevalence of blaNDM in our study showed agreement with the study done in Taiwan [26]. The higher blaNDM was recorded in Klebsiella pneumoniae 13(72.2%) in this study. A study done in Addis Ababa TASH suggested carbapenem resistance to Klebsiella pneumoniae may be associated with medical tourism in Taiwan.
Conclusion
The phenotyping and molecular characterization of DRM show a strong association. This study highlights a high prevalence of beta-lactamase-producing genes among Gram-negative bacteria, with ESBLs being the most dominant, particularly blaCTX-M and blaTEM. The emergence of blaBEL over blaSHV suggests evolving resistance patterns, while the detection of blaOXA-23 at high levels marks a significant and previously undocumented finding in Ethiopia. Ongoing molecular surveillance, improving laboratory capacity, updating treatment guidelines, and reinforcing antimicrobial stewardship and infection prevention measures are essential to reduce resistance and improve patient outcomes.
Limitation
The limitation of this study is that some isolates showing phenotypic resistance were PCR-negative. This may occur because PCR detects only the specific genes included in the assay, whereas phenotypic AST reflects all resistance mechanisms, including those not gene-based or not covered by the PCR panel. Phenotypic detection of MBL resistance is lower compared to genotypic detection. This is because many strains carry both carbapenemase and metallo-β-lactamase resistance genes. For eCIM testing to be applicable, the isolate must be carbapenem-resistant, and the mCIM method test must be negative. However, most strains are positive for both mCIM and eCIM, limiting the ability to distinguish MBL producers phenotypically.
Acknowledgments
The authors would like to acknowledge Addis Ababa University College of Health Science Tikur Anbessa Specialized Hospital for sponsorship and facilitation to do research in the microbiology laboratory, Addis Ababa University College of Natural and Computational Science Department of microbial cellular molecular biology for teaching coaching and mentorship, and the authors would like Acknowledge Armauer Hansen Research Institute for facility permission and Molecular Laboratory staffs for technical support.
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