ABSTRACT
Background and Aims
Respiratory and soft tissue infections remain major public health challenges both globally and in Nepal, increasingly complicated by rising antimicrobial resistance (AMR). The emergence of multidrug‐resistant (MDR) and extensively drug‐resistant (XDR) pathogens has made effective treatment more difficult, underscoring the need for localized resistance data to guide therapy. This study aimed to investigate the distribution and AMR profiles of pathogens isolated from sputum and pus samples, with particular emphasis on MDR and XDR strains.
Methods
A retrospective study was conducted from December 2024 to April 2025, analyzing 615 sputum and 45 pus samples from patients at Madan Bhandari Hospital and Trauma Center in Eastern Nepal. Bacterial identification and antimicrobial susceptibility testing were performed according to validated government‐approved guidelines (AMR Bacteriology Standard Operating Procedure, 2024). MDR and XDR strains were classified based on the criteria provided by the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC).
Results
A total of 66 sputum (10.73%) and 17 pus (37.78%) samples showed positive bacterial growth. Pseudomonas aeruginosa (40.9%) and Klebsiella pneumoniae (39.4%) were the most prevalent pathogens in sputum samples, while Staphylococcus aureus (64.7%) dominated pus samples. The resistance patterns revealed 10.6% of sputum isolates as MDR, 7.6% as XDR, and 11.7% XDR in pus isolates. High resistance rates were observed to common antibiotics such as cefixime, cefepime, and aztreonam, while meropenem demonstrated effectiveness against many resistant strains. Gentamicin and doxycycline showed good efficacy against S. aureus and other pathogens in both samples.
Conclusion
The notable prevalence of MDR and XDR pathogens, along with antibiotic resistance, underscores the need for updated treatment guidelines. Meropenem remains effective for severe infections, while gentamicin and doxycycline are suitable for targeted therapies. Strengthening antimicrobial stewardship, diagnostics, and infection control is crucial to address AMR in Eastern Nepal.
Keywords: antimicrobial resistance (AMR), extensively drug‐resistant (XDR), multidrug‐resistant (MDR), Nepal, Pseudomonas aeruginosa, Staphylococcus aureus
1. Introduction
Respiratory and soft tissue infections are significant contributors to the global burden of disease, with sputum and pus cultures being essential in diagnosing and managing these infections [1, 2]. Respiratory infections, including pneumonia, bronchitis, and tuberculosis, are among the leading causes of morbidity and mortality worldwide, particularly in low‐ and middle‐income countries (LMICs) like Nepal [3, 4]. Similarly, wound infections and abscesses, which often involve pus samples, are common causes of hospital admissions, particularly in healthcare settings where surgical procedures, trauma, and poor hygiene conditions are prevalent [5, 6]. In Nepal, the prevalence of respiratory infections and wound‐related infections has led to substantial healthcare costs, with an increasing number of cases reported each year [6, 7].
Sputum and pus cultures provide valuable insights into the microbial etiology of these infections, with common pathogens such as Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa being frequently isolated from sputum and pus samples [8, 9]. These pathogens, particularly S. aureus and P. aeruginosa, are often associated with severe, life‐threatening infections and pose significant treatment challenges due to their growing resistance to commonly used antibiotics [8, 10].
The rising threat of antimicrobial resistance (AMR) further complicates the management of respiratory and soft tissue infections. AMR has become a major global public health concern, leading to treatment failures, prolonged hospital stays, increased healthcare costs, and higher mortality rates [11]. The overuse and misuse of antibiotics, particularly broad‐spectrum agents, unauthorized sales, inadequate diagnostic facilities, poor patient education, and non‐compliance, often due to poverty and inappropriate self‐medication, have accelerated the development of multidrug‐resistant (MDR) and extensively drug‐resistant (XDR) strains of pathogens [12, 13, 14, 15]. In Nepal, AMR is a growing problem, with numerous studies reporting high rates of resistance to commonly used antibiotics such as beta‐lactams, fluoroquinolones, and aminoglycosides [16, 17, 18]. Though the government of Nepal has already endorsed and approved the National Action Plan for Antimicrobial Resistance (AMR) Nepal 2024–2028 to combat AMR and given instructions to implement it at the provincial and local levels, we still do not know about the AMR pattern of many hospitals in the eastern region [19].
In the eastern region of Nepal, AMR remains a significant concern, yet local data on the AMR patterns of sputum and pus pathogens are scarce. While studies from central and western Nepal have highlighted resistance trends in respiratory and wound infections [20, 21], as per our literature search, no research has been conducted in the eastern hospitals, where healthcare infrastructure and infection control measures are limited [22]. Furthermore, the available studies often focus on specific patient groups, such as inpatients [23], without capturing a more comprehensive picture that includes emergency and outpatient cases. This study aims to address this gap by conducting a retrospective analysis of the spectrum of pathogens and their resistance profiles in sputum and pus samples collected from patients at Madan Bhandari Hospital and Trauma Center (MBHTC), a secondary care hospital in eastern Nepal. Findings will inform targeted treatment protocols, antimicrobial stewardship programs, clinical practices, and healthcare policies to improve infection control and reduce AMR impact in Nepal.
2. Methods
2.1. Study Design, Period, and Setting
This retrospective study was conducted at MBHTC, a secondary healthcare facility located in Urlabari, Morang, Nepal (geographical map shown in Figure 1). The study period spanned from December 16, 2024, to April 13, 2025. Data for this investigation were obtained from the hospital's microbiology laboratory section. MBHTC is a 50‐bed hospital under the provincial government, located in Urlabari Municipality, and serves as a key healthcare institution in the eastern region, providing comprehensive diagnostic and treatment services [24]. The hospital serves a diverse population from different districts, making it an ideal setting for examining respiratory and soft tissue infections, as well as the AMR patterns of sputum and pus pathogens. The reporting of this study conforms to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement [25].
Figure 1.
A shaded map illustrating the geographical area of the study site.
2.2. Study Population and Eligibility Criteria
The study population consisted of patients who had sputum or pus cultures performed at MBHTC between December 16, 2024, and April 13, 2025.
Inclusion criteria included both male and female patients of all age groups who presented with clinical symptoms suggestive of respiratory infections (such as cough, sputum production, shortness of breath, or fever) or soft tissue infections (such as wound infections, abscesses, or cellulitis) and had sputum or pus cultures ordered by their attending physician. Patients from the emergency, outpatient, and inpatient departments were included.
Exclusion criteria included samples that were not collected in sterile containers as per protocol, were contaminated, had billing issues, repetitive isolates from the same patients with identical culture sensitivity profiles, or samples with no bacterial growth.
2.3. Data Collection and Processing
For data collection, the research team visited the MBHTC laboratory. Data were retrieved from the hospital's electronic medical records system (MEDAS Software V3.2) for sputum and pus cultures performed between December 16, 2024, and April 13, 2025. The research team filtered reports with bacterial growth, identified bacteria, and performed antimicrobial susceptibility testing. Duplicate reports were removed, and data were exported. Key variables extracted included patient demographics, sample collection dates, microbial identification, and antimicrobial susceptibility profiles. A total of 620 sputum samples and 45 pus samples were initially received for analysis. After applying the exclusion criteria and removing five sputum samples due to billing issues, 615 sputum samples and all 45 pus samples were included in the final analysis.
2.4. Laboratory Processing and Testing
2.4.1. Sputum and Pus Specimen Collection and Processing
Sputum specimens were collected from patients using the spontaneous expectoration method, where patients were instructed to cough deeply and expectorate into sterile containers. In cases where patients were unable to expectorate, sputum was collected using a nasopharyngeal suction method. Pus samples were collected aseptically from abscesses or infected wounds using sterile swabs or aspiration techniques.
Specimens were transported promptly to the laboratory for processing. Upon arrival, specimens were processed by microscopy and cultured on media as per the AMR Bacteriology SOP: Version 2.0 (2024) [26], specifically on blood agar (BA), chocolate (heated blood) agar (CA), and MacConkey agar (MA). These media were chosen to support the growth of both aerobic and anaerobic pathogens while isolating key pathogens such as S. pneumoniae, H. influenzae, K. pneumoniae, S. aureus, and P. aeruginosa [26]. The procedures adhered to the protocols set forth in the National Public Health Laboratory's AMR Bacteriology SOP to ensure consistent and reliable bacteriological results [26].
2.4.2. Bacterial Isolation and Identification
For bacterial isolation, sputum and pus specimens were plated onto BA, CA, and MA plates using a calibrated loop. Plates were incubated aerobically at 35°C–37°C for 18–24 h. After incubation, colonies were examined for growth and morphology. Gram staining was performed to differentiate bacterial species, followed by biochemical testing to confirm identification. The identification process followed standard microbiological procedures outlined in the AMR Bacteriology SOP: Version 2.0 (2024) [26], including tests for oxidase, catalase, indole, urease production, and sugar fermentation patterns.
2.4.3. Antibiotic Susceptibility Testing (AST)
AST of bacterial isolates from sputum and pus specimens was performed using the modified Kirby‐Bauer disc diffusion method on Mueller–Hinton Agar (MHA) (HiMedia, India), as per AMR Bacteriology SOP: Version 2.0 (2024) [26]. A sterile cotton swab was used to inoculate standardized bacterial suspension equivalent to 0.5 McFarland standard onto MHA plates. After allowing the surface to dry, appropriate HiMedia antibiotic discs were placed aseptically. Each isolate was tested against a panel of antibiotics appropriate for the identified pathogens, including commonly used antibiotics for respiratory and soft tissue infections.
For sputum isolates, the following HiMedia antibiotic discs were used: penicillin G (10 units), ampicillin/sulbactam (10/10 µg), piperacillin/tazobactam (100/10 µg), ceftriaxone (30 µg), cefixime (5 µg), ceftazidime (30 µg), cefepime (30 µg), cefoperazone/sulbactam (75/30 µg), aztreonam (30 µg), meropenem (10 µg), imipenem (10 µg), ciprofloxacin (5 µg), ofloxacin (5 µg), azithromycin (15 µg), gentamicin (10 µg), tobramycin (10 µg), erythromycin (15 µg), doxycycline (30 µg), and trimethoprim–sulfamethoxazole (SXT) (25 µg).
For pus isolates, the following HiMedia antibiotic discs were used: penicillin G (10 units), ampicillin/sulbactam (10/10 µg), piperacillin/tazobactam (100/10 µg), ceftriaxone (30 µg), cefixime (5 µg), ceftazidime (30 µg), cefepime (30 µg), cefoperazone/sulbactam (75/30 µg), meropenem (10 µg), ciprofloxacin (5 µg), ofloxacin (5 µg), azithromycin (15 µg), erythromycin (15 µg), gentamicin (10 µg), doxycycline (30 µg), tigecycline (15 µg), and trimethoprim–sulfamethoxazole (SXT) (25 µg).
Plates were incubated at 35 ± 2°C for 16–18 h. The zones of inhibition were measured in millimeters and interpreted according to Clinical and Laboratory Standards Institute (CLSI) M100, 34th edition (2024) criteria [27]. Only pure isolates were used for the test, and quality control (QC) was maintained using standard ATCC strains. The zone of inhibition was measured and interpreted according to CLSI M100 breakpoints to determine the susceptibility, intermediate resistance, or resistance of the bacteria to each antibiotic, as mentioned in Annexe 2 of AMR Bacteriology SOP: Version 2.0 (2024) [26], which has been updated as per CLSI M100, 34th edition (2024) [27].
2.4.4. Identification of MDR, XDR, and Pan‐Drug‐Resistant (PDR) Isolates
The identification of MDR, XDR, and PDR isolates was performed using criteria proposed by the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC) [28]. MDR was defined as non‐susceptibility to at least one agent from three or more different antimicrobial categories, while XDR was categorized when a bacterium was non‐susceptible to at least one antimicrobial agent from all but two categories. PDR isolates were those non‐susceptible to all available antimicrobial agents tested [28].
2.4.5. Quality Control (QC)
QC for AST was conducted according to the Quality Assurance in Microbiology section of the AMR Bacteriology SOP: Version 2.0 (2024) [26]. The laboratory adhered to internal quality control (IQC) procedures by testing known control strains of Escherichia coli ATCC 25922 and S. aureus ATCC 25923 on every testing day to validate the performance of antimicrobial agents. External quality assurance (EQA) was ensured through participation in the National Public Health Laboratory (NPHL) proficiency testing program, which included quarterly dispatch of test panels for AST and identification.
2.5. Data Analysis
Data were entered and cleaned in Microsoft Excel (Version 16.96.1) and analyzed using SPSS software, Version 27. Descriptive statistics, including frequencies and percentages, were used for categorical variables.
3. Results
During the 4‐month period from December 16, 2024, to April 13, 2025, a total of 45 pus and 620 sputum samples were received for testing. Five sputum samples were excluded due to billing issues and exclusion criteria. Of these, 17 pus (37.78%) and 66 sputum (10.73%) samples showed positive bacterial growth, each yielding a single isolate.
The majority of sputum samples were from male patients (62.1%) and those aged ≥ 60 years (66.7%). Pus samples were more common in females (58.8%) and younger patients (35.3% aged ≤ 19). Most patients were outpatients (90.9% for sputum, 76.5% for pus), primarily from the Morang district. Resistance profiles showed that 10.6% (7/66) of sputum samples were MDR, 7.6% (5/66) were XDR, while no PDR isolates were found. For pus samples, 11.7% (2/17) were XDR, but no MDR or PDR isolates were detected, as shown in Table 1.
Table 1.
Sociodemographic characteristics of patients and resistant type of isolates from sputum (N = 66) and pus (N = 17)samples.
| Variables | Category | Sputum samples | Pus samples | ||
|---|---|---|---|---|---|
| Frequency | Percentage (%) | Frequency | Percentage (%) | ||
| Gender | Male | 41 | 62.1 | 7 | 41.2 |
| Female | 25 | 37.9 | 10 | 58.8 | |
| Age group | ≤ 19 | 5 | 7.6 | 6 | 35.3 |
| 20–40 | 9 | 13.6 | 3 | 17.6 | |
| 40–60 | 8 | 12.1 | 2 | 11.8 | |
| ≥ 60 | 44 | 66.7 | 6 | 35.3 | |
| Admission | Emergency | 3 | 4.5 | 1 | 5.9 |
| Inpatients | 3 | 4.5 | 3 | 17.6 | |
| Outpatients | 60 | 90.9 | 13 | 76.5 | |
| Patient address (district) | Morang | 61 | 92.4 | 16 | 94.1 |
| Jhapa | 4 | 6.1 | 1 | 5.9 | |
| Khotang | 1 | 1.5 | — | — | |
| Resistant types | MDR | 7 | 10.6 | 0 | 0 |
| XDR | 5 | 7.6 | 2 | 11.7 | |
| PDR | 0 | 0 | 0 | 0 | |
In sputum samples, gram‐negative bacteria dominated (89.4%), with P. aeruginosa (40.9%) and K. pneumoniae (39.4%) being the most frequent. Gram‐positive bacteria (S. aureus) accounted for 10.6%. In pus samples, gram‐positive bacteria were more common (64.7%), with S. aureus accounting for 64.7%. Gram‐negative bacteria comprised 35.29%, with K. pneumoniae (17.65%) and P. aeruginosa (5.9%) being the most prevalent, as shown in Table 2.
Table 2.
Distributions of pathogens isolated in sputum sample (N = 66) and pus sample (N = 17).
| Bacteria type | Isolated bacteria | Sputum samples | Pus samples | ||
|---|---|---|---|---|---|
| Frequency | % | Frequency | % | ||
| Gram negative | Pseudomonas aeruginosa | 27 | 40.9 | 1 | 5.9 |
| Klebsiella pneumoniae | 26 | 39.4 | 3 | 17.65 | |
| Escherichia coli | 3 | 4.5 | 1 | 5.9 | |
| Enterobacter | 2 | 3 | — | — | |
| Acinetobacter species | 1 | 1.5 | 1 | 5.9 | |
| Total gram negative | 59 | 89.4 | 6 | 35.29 | |
| Gram positive | Staphylococcus aureus | 7 | 10.6 | 11 | 64.7 |
| Total gram positive | 7 | 10.6 | 11 | 64.7 | |
| Total | 66 | 100 | 17 | 100 | |
The resistance patterns in sputum samples reveal significant AMR across several bacterial species. P. aeruginosa showed 100% (2/2) resistance to imipenem, 90.9% (10/11) resistance to cefixime, 62.5% (5/8) resistance to aztreonam, 59.09% (13/22) resistance to cefepime, and 35.71% (5/14) resistance to ceftazidime. Similarly, K. pneumoniae exhibited 100% (2/2) resistance to imipenem, 66.67% (2/3) resistance to doxycycline, ampicillin/sulbactam 37.5% (6/16), and 16.67% (4/24) resistance to ceftriaxone. Among the gram‐positive pathogens, S. aureus demonstrated 100% (3/3) resistance to cefixime, 50% (1/2) resistance to ciprofloxacin, and 33.33% (1/3) resistance to ceftriaxone, but doxycycline, 20% (1/5), showed good efficacy against S. aureus. The resistance rates for meropenem were lower, with P. aeruginosa showing 16.67% (4/24) resistance. For most antibiotics, E. coli exhibited high resistance rates, particularly 66.67% (2/3) for cefixime, as depicted in Table 3.
Table 3.
Antimicrobial resistance pattern in Sputum samples at Madhan Bhandari Turma Center, Eastern Nepal.
| Bacteria type | Gram negative | Gram positive | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Isolated bacteria | Pseudomonas aeruginosa | Klebsiella pneumoniae | Escherichia coli | Enterobacter species | Acinetobacter species | Staphylococcus aureus | ||||||||||||
| S% | I% | R% | S% | I% | R% | S% | I% | R% | S% | I% | R% | S% | I% | R% | S% | I% | R% | |
| Penicillin G | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | NT | NT | NT | 1/3 (33.33%) | 0/3 (0%) | 2/3 (66.67%) |
| Ampicillin/Sulbactam | IR | IR | IR | 8/16 (50%) | 2/16 (12.5%) | 6/16 (37.5%) | NT | NT | NT | IR | IR | IR | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | 3/5 (60%) | 0/5 (0%) | 2/5 (40%) |
| Piperacillin/Tazobactam | 23/25 (92%) | 0/25 (0%) | 2/25 (8%) | 10/24 (41.67%) | 5/24 (20.83%) | 9/24 (37.5%) | 3/3 (100%) | 0/3 (0%) | 0/3 (0%) | 1/2 (50%) | 1/2 (50%) | 0/2 (0%) | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | 3/5 (60%) | 1/5 (20%) | 1/5 (20%) |
| Ceftriaxone | IR | IR | IR | 20/24 (83.33%) | 0/24 (0%) | 4/24 (16.67%) | 3/3 (100%) | 0/3 (0%) | 0/3 (0%) | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) | 2/3 (66.67%) | 0/3 (0%) | 1/3 (33.33%) |
| Cefixime | 1/11 (9.09%) | 0/11 (0%) | 10/11 (90.91%) | 12/20 (60%) | 0/12 (0%) | 8/12 (40%) | 1/3 (33.33%) | 0/3 (0.00%) | 2/3 (66.67%) | 0/1 (0.00%) | 0/1 (0.00%) | 1/1 (100.00%) | 0/1 (0.00%) | 0/1 (0.00%) | 1/1 (100.00%) | 0/3 (0.00%) | 0/3 (0.00%) | 3/3 (100.00%) |
| Ceftazidime | 9/14 (64.29%) | 0/14 (0.00%) | 5/14 (35.71%) | 4/7 (57.14%) | 0/7 (0%) | 3/7 (42.86%) | NT | NT | NT | 0/1 (0%) | 1/1 (100%) | 0/1 (0.00%) | NT | NT | NT | NT | NT | NT |
| Cefepime | 6/22 (27.27%) | 3/22 (13.64%) | 13/22 (59.09%) | 12/17 (70.59%) | 0/17 (0.00%) | 5/17 (29.41%) | 2/2 (100%) | 0/2 (0%) | 0/2 (0%) | 2/2 (100%) | 0/2 (0%) | 0/2 (0%) | 0/1 (0%) | 1/1 (100%) | 0/1 (0%) | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) |
| Cefoperazone/Sulbactam | NT | NT | NT | 0/1 (0.00%) | 0/1 (0.00%) | 1/1 (100%) | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| Aztreonam | 2/8 (25%) | 1/8 (12.5%) | 5/8 (62.5%) | NT | NT | NT | NT | NT | NT | NT | NT | NT | IR | IR | IR | IR | IR | IR |
| Meropenem | 20/24 (83.33%) | 0/24 (0.0%) | 4/24 (16.67%) | 13/22 (59.09%) | 2/22 (9.09%) | 7/22 (31.82%) | 3/3 (100%) | 0/3 (0%) | 0/3 (0%) | 1/2 (50%) | 0/2 (0.00%) | 1/2 (50%) | 1/1 (100.00%) | 0/1 (0.00%) | 0/1 (0.00%) | 4/4 (100.00%) | 0/4 (0.00%) | 0/4 (0.00%) |
| Imipenem | 0/2 (0.00%) | 0/2 (0.00%) | 2/2 (100.00%) | 0/2 (0%) | 0/2 (0%) | 2/2 (100%) | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT |
| Ciprofloxacin | 9/9 (100%) | 0/9 (0.00%) | 0/9 (0%) | 2/2 (100%) | 0/2 (0%) | 0/2 (0%) | NT | NT | NT | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | NT | NT | NT | 1/2 (50.00%) | 0/2 (0.00%) | 1/2 (50.00%) |
| Ofloxacin | 13/16 (81.25%) | 1/16 (6.25%) | 2/16 (12.50%) | 21/24 (87.50%) | 2/24 (8.33%) | 1/24 (4.17%) | 2/3 (66.67%) | 0/3 (0.00%) | 1/3 (33.33%) | 1/1 (100.00%) | 0/1 (0.00%) | 0/1 (0.00%) | 0/1 (0.00%) | 0/1 (0.00%) | 1/1 (100.00%) | 3/4 (75%) | 0/4 (0.00%) | 1/4 (25%) |
| Azithromycin | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | 0/2 (0.00%) | 0/2 (0.00%) | 2/2 (100.00%) |
| Gentamicin | 2/2 (100%) | 0/2 (0%) | 0/2 (0%) | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | NT | NT | NT | NT | NT | NT | NT | NT | NT | 1/2 (50%) | 0/2 (0.00%) | 1/2 (50%) |
| Tobramycin | 9/10 (90%) | 0/10 (0%) | 1/10 (10%) | 1/1 (100%) | 0/1 (0.00%) | 0/1 (0.00%) | NT | NT | NT | 0/1 (0%) | 1/1 (100%) | 0/1 (0%) | NT | NT | NT | NT | NT | NT |
| Erythromycin | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | IR | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) |
| Doxycycline | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) | 1/3 (33.33%) | 0/3 (0%) | 2/3 (66.67%) | NT | NT | NT | NT | NT | NT | NT | NT | NT | 4/5 (80%) | 0/5 (0%) | 1/5 (20%) |
| Trimethoprim–sulfamethoxazole (SXT) | IR | IR | IR | 23/25 (92%) | 0/25 (0%) | 2/25 (8%) | 2/3 (66.67%) | 0/3 (0.00%) | 1/3 (33.33%) | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | 2/3 (66.67%) | 0/3 (0.00%) | 1/3 (33.33%) |
Abbreviations: I%, intermediate resistance percentage; IR, intrinsic resistance; NT, not tested; R%, resistant percentage; S%, sensitive percentage.
The resistance patterns in pus samples revealed significant findings for clinically important antibiotics. P. aeruginosa exhibited 100% (1/1) intermediate resistance to piperacillin/tazobactam and no resistance to meropenem, indicating full susceptibility to Meropenem. Klebsiella species showed 100% (3/3) resistance to piperacillin/tazobactam, 100% (2/2) resistance to cefixime, and 66.67% (2/3) resistance to meropenem. S. aureus demonstrated 100% (1/1) resistance to ciprofloxacin, 87.5% (7/8) to cefixime, and 20% (1/5) resistance to Meropenem. Ceftriaxone showed 33.33% (1/3) resistance in Klebsiella isolates, while cefixime exhibited 100% (2/2) resistance. Additionally, ofloxacin had 50% (1/2) resistance to Klebsiella spp., and doxycycline exhibited 100% (1/1) sensitivity to E. coli, as shown in Table 4.
Table 4.
Antimicrobial resistance pattern in pus samples at Madhan Bhandari Trauma Center, Eastern Nepal.
| Bacteria type | Gram negative | Gram positive | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Isolated bacteria | Klebsiella species | Escherichia coli | Pseudomonas aeruginosa | Acinetobacter species | Staphylococcus aureus | ||||||||||
| S% | I% | R% | S% | I% | R% | S% | I% | R% | S% | I% | R% | S% | I% | R% | |
| Penicillin G | IR | IR | IR | NT | NT | NT | IR | IR | IR | IR | IR | IR | 0/3 (0%) | 0/3 (0%) | 3/3 (100.00) |
| Ampicillin/Sulbactam | NT | NT | NT | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) | IR | IR | IR | NT | NT | NT | 3/11 (27.27%) | 0/11 (0%) | 8/11 (72.73%) |
| Piperacillin/Tazobactam | 0/3 (0%) | 0/3 (0%) | 3/3 (100%) | 0/1 (0%) | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) | 0/1 (0%) | 1/1 (100%) | 0/1 (0%) | 0/1(0%) | 2/3 (66.67%) | 0/3 (0%) | 1/3 (33.33%) |
| Ceftriaxone | 2/3 (66.67%) | 0/3 (0%) | 1/3 (33.33%) | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) | IR | IR | IR | NT | NT | NT | 4/7 (57.14%) | 0/7 (0%) | 3/7 (42.86%) |
| Cefixime | 0/2 (0%) | 0/2 (0%) | 2/2 (100%) | NT | NT | NT | NT | NT | NT | NT | NT | NT | 1/8 (12.5%) | 0/0 (0%) | 7/8 (87.50%) |
| Ceftazidime | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | NT | NT | NT | NT | NT | NT | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) |
| Cefepime | NT | NT | NT | NT | NT | NT | NT | NT | NT | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | NT | NT | NT |
| Cefoperazone/Sulbactam | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | NT | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) |
| Meropenem | 1/3 (33.33%) | 0/3 (0.0%) | 2/3 (66.67%) | 0/1 (0%) | 1/1 (100%) | 0/1 (0%) | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | 1/1 (100.00%) | 0/1 (0.00%) | 0/1 (0.00%) | 4/5 (80%) | 0/5 (0%) | 1/5 (20%) |
| Ciprofloxacin | 1/1 (100%) | 0/1 (0.00%) | 0/1 (0%) | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) | NT | NT | NT | NT | NT | NT | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) |
| Ofloxacin | 1/2 (50%) | 0/2 (0%) | 1/2 (50%) | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) | 1/1 (100%) | 0/1 (0.00%) | 0/1 (0%) | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | 3/10 (30%) | 0/10 (0%) | 7/10 (70%) |
| Azithromycin | IR | IR | IR | NT | NT | NT | IR | IR | IR | IR | IR | IR | 1/4 (25%) | 0/4 (0%) | 3/4 (75%) |
| Erythromycin | IR | IR | IR | NT | NT | NT | IR | IR | IR | IR | IR | IR | 1/7 (14.29%) | 0/7 (0%) | 6/7 (85.71%) |
| Gentamicin | NT | NT | NT | NT | NT | NT | NT | NT | NT | 1/1 (100%) | 0/1 (0%) | 0/1 (0%) | 3/3 (100%) | 0/3 (0.00%) | 0/3 (0%) |
| Doxycycline | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) | 1/1 (100%) | 0/1 (0%) | 0/(0%) | NT | NT | NT | NT | NT | NT | 7/8 (87.5%) | 0/8 (0%) | 1/8 (12.5%) |
| Tigecycline | 1/1 (100.00%) | 0/1 (0.00%) | 0/1 (0.00%) | 1/1 (100.00%) | 0/1 (0.00%) | 0/1 (0.00%) | IR | IR | IR | NT | NT | NT | NT | NT | NT |
| Trimethoprim–sulfamethoxazole (SXT) | 2/3 (66.67%) | 0/3 (0%) | 1/3 (33.33%) | 0/1 (0%) | 0/1 (0%) | 1/1 (100%) | IR | IR | IR | NT | NT | NT | 1/7 (14.29%) | 0/7 (0%) | 6/7 (85.71%) |
Abbreviations: I%, intermediate resistance percentage; IR, intrinsic resistance; NT, not tested; R%, resistant percentage; S%, sensitive percentage.
4. Discussion
In this study, we examined the spectrum of pathogens and their AMR profiles in sputum and pus samples from patients at MBHTC, Eastern Nepal. Our findings highlight a notable prevalence of MDR and XDR pathogens, emphasizing the growing threat of AMR in the region. The high resistance rates observed in common pathogens such as P. aeruginosa, K. pneumoniae, and S. aureus underline the need for urgent intervention to address the rising tide of AMR.
The sputum culture positivity rate in this study (10.73%) is similar to that reported from Shree Birendra Hospital, Kathmandu, Nepal, where the rate was 9.87% [29]. Likewise, the pus culture positivity rate (37.78%) aligns with findings from the Pakistan Institute of Medical Sciences (PIMS), which reported 49.5% positive cultures [30]. These comparisons suggest that the prevalence of bacterial growth in sputum and pus samples in our study is broadly consistent with previously reported data in similar hospital settings.
The distribution of pathogens in our study revealed that P. aeruginosa and K. pneumoniae were the most frequently isolated organisms from sputum samples, together accounting for nearly four‐fifths of the isolates. In contrast, S. aureus was identified as the predominant pathogen in pus samples, accounting for nearly two‐thirds of the isolates. These findings are consistent with studies from other regions, which also report P. aeruginosa and Klebsiella as common respiratory pathogens [31, 32]. In contrast, a higher proportion of gram‐positive bacteria, such as S. aureus, was found in pus samples in our study, similar to reports from South Asia and Africa [33, 34].
The resistance patterns observed in this study align with global trends, particularly the high resistance rates among gram‐negative pathogens [23, 35]. P. aeruginosa exhibited notable resistance to multiple antibiotics, including cefepime, ceftazidime, and aztreonam, reflecting the growing difficulty in treating infections caused by this pathogen, as seen in studies from Nepal and Ethiopia [32, 36, 37]. For instance, a recent study in sputum samples from Nepal revealed that 88% of P. aeruginosa were resistant to ceftazidime [38]. Likewise, an Indian study shows 75% of P. aeruginosa were resistant to amoxicillin/clavulanate in sputum samples [39]. Similarly, K. pneumoniae demonstrated considerable resistance to key antibiotics such as imipenem, ampicillin/sulbactam, and piperacillin/tazobactam, which is consistent with findings from international surveillance studies [40]. For instance, an Indian study revealed 40% resistance to piperacillin/tazobactam in K. pneumoniae [39]. In the case of gram‐positive bacteria, S. aureus also showed marked resistance to commonly used agents like cefixime, ciprofloxacin, and ceftriaxone, echoing growing concerns of resistance among gram‐positive pathogens globally [40].
Our findings reveal that although no PDR isolates were identified in this study, MDR and XDR pathogens were identified in sputum and pus samples, which is concerning, as they reflect the broader trends observed in LMICs [41]. The emergence of XDR K. pneumoniae in pus samples is particularly alarming, as these pathogens are difficult to treat with standard antibiotics and pose a significant threat to patient outcomes [42]. This is in line with studies from Ethiopia and Vietnam, where XDR pathogens have become a significant challenge in clinical settings [43, 44]. A considerable proportion of isolates exhibited intermediate resistance to key antibiotics, such as cefepime in P. aeruginosa and piperacillin/tazobactam in K. pneumoniae, adding further complexity to treatment, as such strains may progress toward full resistance over time.
The use of meropenem remains crucial for the treatment of severe infections caused by MDR pathogens. However, resistance to imipenem was alarmingly high, with many isolates showing complete resistance. Resistance to meropenem was also concerning, with K. pneumoniae and P. aeruginosa exhibiting moderate levels of resistance, reflecting a troubling trend in the efficacy of last‐resort antibiotics. These findings echo concerns in global studies, where carbapenem resistance is on the rise, particularly among hospital‐acquired infections [45]. Nevertheless, carbapenems such as meropenem still demonstrate considerable efficacy against resistant pathogens, underscoring the need for judicious use of these antibiotics to preserve their effectiveness.
Furthermore, our study highlighted the potential efficacy of gentamicin and doxycycline as treatment options for resistant gram‐positive pathogens. S. aureus isolates from pus samples showed complete sensitivity to gentamicin, while a large majority were also sensitive to doxycycline, suggesting these antibiotics may still be effective therapeutic choices in such infections. These findings align with those from South Asian studies, which have identified gentamicin and doxycycline as effective alternatives in cases of MDR [46, 47].
The findings of this study emphasize the urgent need for updated empirical treatment guidelines for respiratory and soft tissue infections in Eastern Nepal, informed by regional AMR patterns. Given the high resistance rates to first‐line antibiotics like ciprofloxacin, ceftriaxone, and cefixime, these antibiotics should no longer be recommended for empirical therapy. Meropenem remains a viable option for severe infections, but its use must be carefully controlled to prevent further resistance, as it belongs to the “Watch” category in the WHO AWaRe Classification [48]. For uncomplicated infections, gentamicin and doxycycline show promising results, especially in treating gram‐positive pathogens like S. aureus. Strengthening antimicrobial stewardship, enhancing diagnostic capabilities, and improving infection control measures are critical steps toward combating the rising threat of AMR in Nepal's healthcare system.
4.1. Strengths and Limitations
This study has several limitations that should be considered when interpreting the findings. The short study duration (December 2024–April 2025) and single‐center design limit the generalizability of the results to other healthcare settings in Eastern Nepal. The relatively small sample size for pus isolates and the exclusion of five sputum samples due to billing issues and study criteria may have slightly affected prevalence estimates. The retrospective design restricted access to detailed clinical data, including patient comorbidities, prior antibiotic use, and infection history, which could have provided additional insights into risk factors for AMR. Laboratory testing did not differentiate between specific resistant subtypes, such as extended‐spectrum beta‐lactamase (ESBL) or methicillin‐resistant S. aureus (MRSA), limiting a more nuanced understanding of highly resistant strains. Additionally, although a range of samples was analyzed, the overall culture positivity was low, and the antibiotic panel tested was limited for some pathogens.
Despite these limitations, the study offers valuable insights into the spectrum of pathogens and their resistance profiles in sputum and pus samples. The sample size for culture and sensitivity testing, though limited by the exclusion of certain data, still provides a solid foundation for understanding local resistance patterns. The study adhered to internationally recognized guidelines for classifying isolates into MDR, XDR, and PDR categories, which strengthens the relevance of the findings in comparison to global studies. The use of multiple antibiotics across various pathogen types, along with data from a range of hospital wards (emergency, inpatient, outpatient), adds depth to the analysis and makes the findings useful for informing treatment decisions in this setting.
5. Conclusion
This study reveals a concerning prevalence of MDR and XDR pathogens in sputum and pus samples, particularly P. aeruginosa, K. pneumoniae, and S. aureus, in Eastern Nepal. Meropenem remains effective against several resistant strains, but its use must be judicious to prevent further resistance development. Gentamicin and doxycycline show promise for treating gram‐positive infections, emphasizing the need for updated empirical treatment guidelines. Strengthening antimicrobial stewardship, enhancing diagnostic facilities, and improving infection control measures are essential to combat the growing threat of AMR in the region. These results provide crucial insights for refining clinical practices and developing policies to mitigate AMR's impact on public health in Nepal.
Author Contributions
Rahi Bikram Thapa: conceptualization, investigation, methodology, data curation, formal analysis, validation, visualization, writing – original draft, writing – review and editing. Prakash Chandra Karkee: investigation, data curation, methodology, validation, project administration, visualization. Sabin Shrestha: conceptualization, methodology, validation, visualization.
Funding
The authors have nothing to report.
Ethics Statement
Ethical approval for this study was obtained from the Institutional Review Committee (IRC) of Manmohan Memorial Institute of Health Sciences, with reference number (MMIHS‐IRC Ref. No.: NEHCO‐IRC/081/109). The hospital administration also granted written permission (Ref. No. 388) to use the laboratory data for research purposes. Given the study's retrospective nature, informed consent from individual participants was waived by the IRC. All patient data were kept confidential, and the study adhered to ethical guidelines set by the Nepal Health Research Council (NHRC). Data were anonymized and used solely for the purpose of this research.
Conflicts of Interest
The authors declare no conflicts of interest.
Transparency Statement
The lead author, Rahi Bikram Thapa, affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.
Acknowledgments
The authors would like to acknowledge Miss Usha Bhattrai and Mr. Rewati Pokharel for their assistance with data collection, and Dr. Neha Yadav for her review of the methodology section. Special thanks to the laboratory team at MBHTC and to the Ministry of Health of Koshi Province for providing invaluable support in conducting this study. Furthermore, we extend our heartfelt thanks to Mr. Ravindra Khadka for his excellent creation of the study site image and for his continued support. All authors have read and approved the final version of the manuscript. Mr. Rahi Bikram Thapa had full access to all data in this study and takes full responsibility for the integrity and accuracy of the data analysis.
Data Availability Statement
Data are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data are available from the corresponding author upon reasonable request.