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Convergence of multidrug resistance with biofilm formation and hypermucoviscosity in Klebsiella pneumoniae from tertiary-care hospitals in Northwestern Pakistan

Published online by Cambridge University Press:  13 May 2026

Aiman Waheed,
Taj Ali Khan,
Sajjad Ahmad,
Fahad Ahmed and
Zahoor KhanOpen the ORCID record for Zahoor Khan[Opens in a new window]
Aiman Waheed
Affiliation:
Center of Biotechnology and Microbiology, University of Peshawar, Peshawar, Pakistan
Taj Ali Khan*
Affiliation:
Institute of Pathology and Diagnostic Medicines (IP&DM), Khyber Medical University, Peshawar, PakistanPublic Health Reference Laboratory, Khyber Medical University, Peshawar, Pakistan
Sajjad Ahmad
Affiliation:
Institute of Pathology and Diagnostic Medicines (IP&DM), Khyber Medical University, Peshawar, Pakistan
Fahad Ahmed
Affiliation:
Nutrition Innovation Centre for Food and Health (NICHE), School of Biomedical Sciences, Ulster University, Coleraine, N. Ireland, UK
Zahoor Khan*
Affiliation:
Nutrition Innovation Centre for Food and Health (NICHE), School of Biomedical Sciences, Ulster University, Coleraine, N. Ireland, UK
*
Corresponding authors: Taj Ali Khan; Email: tajalikhan.ibms@kmu.edu.pk; Zahoor Khan; Email: z.khan@ulster.ac.uk
Corresponding authors: Taj Ali Khan; Email: tajalikhan.ibms@kmu.edu.pk; Zahoor Khan; Email: z.khan@ulster.ac.uk

Abstract

This multicenter study describes the convergence of multidrug resistance, biofilm formation, and hypermucoviscosity in Klebsiella pneumoniae clinical isolates from tertiary-care hospitals in Pakistan. The high prevalence of these phenotypes highlights significant therapeutic challenges and underscores the need for strengthened surveillance, infection control, and antimicrobial stewardship.

Information

Type
Concise Communication
Information
Antimicrobial Stewardship & Healthcare Epidemiology , Volume 6 , Issue 1 , 2026, e143
Creative Commons
Creative Common License - CCCreative Common License - BY
This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited.
Copyright
© The Author(s), 2026. Published by Cambridge University Press on behalf of The Society for Healthcare Epidemiology of America

Introduction

Klebsiella pneumoniae is a Gram-negative member of the Enterobacterales and a major cause of healthcare-associated and community-acquired infections, including pneumonia, bloodstream infections, urinary tract infections, and device-associated infections. Reference Filev, Lyubomirova and Bogov1 Its clinical significance has increased globally due to the rapid emergence of multidrug-resistant (MDR) and carbapenem-resistant strains, largely mediated by mobile genetic elements encoding β-lactamases. Reference Li, Shi, Song, Yin and Liu2

In addition to antimicrobial resistance, virulence-associated phenotypes of K. pneumoniae such as biofilm formation and hypermucoviscosity contribute to persistence, immune evasion, and infection chronicity, particularly in device-associated settings. These traits may complicate treatment outcomes but are often evaluated independently of resistance patterns. Reference Chen, Chuang and Lin3 In Pakistan, available data primarily focus on antimicrobial susceptibility patterns of K. pneumoniae, with limited integration of phenotypic virulence characteristics. Reference Zada, Mubaraki and Qasim4 This study therefore aimed to characterize antimicrobial resistance patterns and selected virulence-associated phenotypes among K. pneumoniae isolates from major tertiary care hospitals in Peshawar that serve as major referral centers for all nearby districts of Khyber Pakhtunkhwa (KP), thereby providing region-specific data from a high-burden setting.

Methods

This multicenter cross-sectional study was conducted from August 2024 to July 2025 across three tertiary care hospitals, namely Khyber Teaching Hospital, Lady Reading Hospital, and Hayatabad Medical Complex in Peshawar, Pakistan, following ethical approval and informed consent procedures. A total of 3,286 clinical specimens (urine, blood, respiratory, wound-associated, and sterile body fluids) were processed using standard microbiological techniques, yielding 248 confirmed K. pneumoniae isolates from 953 culture-positive samples. Reference Zada, Mubaraki and Qasim4

Species identification was performed using API 20E identification system (bioMérieux, Lyon, France). Reference Zada, Mubaraki and Qasim4 Antimicrobial susceptibility testing was conducted using the Kirby–Bauer disk diffusion method, with colistin susceptibility assessed by broth microdilution. Reference Pruss, Kobylińska, Fijałkowski, Masiuk and Kwiatkowski5 Results were interpreted according to Clinical and Laboratory Standards Institute (CLSI M100, 34th edition) criteria. MDR and extensively drug-resistant (XDR) phenotypes were defined using ECDC/CDC criteria. Reference Waheed, Khan and Ahmad6

Hypermucoviscosity was assessed using the string test, and biofilm formation was quantified using a microtiter plate assay with optical density-based classification. Reference Hyun, Lee and Kim7 Whole-genome sequencing and molecular typing were not performed; therefore, genetic determinants and clonal relatedness could not be assessed.

Results

Among 3,286 clinical specimens, 29.0% yielded bacterial growth, of which 68.3% were Gram-negative. K. pneumoniae accounted for 38.1% of Gram-negative isolates (n = 248). This relatively high proportion should be interpreted cautiously, as differences in diagnostic practices, sampling strategies, and patient case-mix may limit generalizability.

Isolates were evenly distributed across participating hospitals (∼32%–34% per center), suggesting no clear institutional clustering. Urine was the most frequent source of isolation (33.9%), followed by wound-associated specimens (21.4%), blood (17.3%), respiratory samples (12.9%), and other sterile sites (4.8%).

Antimicrobial susceptibility testing demonstrated high resistance to β-lactams (≥76%), cephalosporins (≥75%), and fluoroquinolones (≥65%). Carbapenem resistance ranged from 18% to 20%. Colistin (88.7% susceptible) and nitrofurantoin (63.0% susceptible among urinary isolates) retained the greatest activity (Figure 1). Overall, 60.5% of isolates were classified as MDR/XDR.

Figure 1.

Antimicrobial susceptibility profile of Klebsiella pneumoniae isolates across tested antibiotic classes (n = 248). Amoxicillin–clavulanic acid (AMC), Piperacillin–tazobactam (TZP), Cefazolin (CZ), Cefoxitin (FOX), Ceftazidime (CAZ), Cefotaxime (CTX), Cefepime (FEP), Ertapenem (ETP), Meropenem (MEM), Amikacin (AK), Gentamicin (CN), Tobramycin (TOB), Nitrofurantoin (NIT), Ciprofloxacin (CIP), Levofloxacin (LEV), Tetracycline (TET), Trimethoprim–sulfamethoxazole (SXT), Colistin (COL).* for urine isolates only.

Hypermucoviscosity was observed in 41.5% of isolates. Biofilm formation was highly prevalent, with 89.9% of isolates demonstrating measurable biofilm production, predominantly of moderate intensity (Figure 2). These findings represent descriptive phenotypic frequencies only, as no molecular characterization or clonal analysis was performed.

Figure 2.

Distribution of biofilm-forming capacity among clinical isolates stratified by specimen type, demographic characteristics, and antimicrobial resistance profile (n = 248).

Discussion

This study provides a concise phenotypic characterization of antimicrobial resistance and selected virulence-associated traits among K. pneumoniae isolates in a high-burden healthcare setting. The high prevalence of MDR/XDR isolates and resistance to multiple antibiotic classes is consistent with regional and global trends and reflects sustained antimicrobial selection pressure in hospital environments. Reference Awoke, Teka and Seman8–Reference Azra, Ul Haq and Hinthong10

The retained activity of colistin and nitrofurantoin suggests their continued utility as last-line agents, particularly for urinary infections; however, concerns regarding toxicity and emerging resistance underscore the need for cautious use within antimicrobial stewardship frameworks. Reference Awoke, Teka and Seman8,Reference Zdarska, Arcari, Kolar and Mlynarcik9

Biofilm formation was observed in the majority of isolates, consistent with its recognized role in persistence on medical devices and contribution to chronic and recurrent infections. Reference Hyun, Lee and Kim7–Reference Zdarska, Arcari, Kolar and Mlynarcik9 Hypermucoviscosity was also frequently detected, although in the absence of molecular confirmation, this phenotype should be interpreted cautiously as a surrogate marker rather than definitive evidence of hypervirulence. Reference Azra, Ul Haq and Hinthong10

Importantly, this study reports descriptive frequencies only, and no causal or statistical associations between antimicrobial resistance and virulence phenotypes can be inferred. While these findings are broadly consistent with existing literature, they provide region-specific data from a setting where integrated phenotypic surveillance remains limited. The relatively high proportion of K. pneumoniae among Gram-negative isolates warrants careful interpretation, as it may reflect local epidemiology, diagnostic practices, or patient population characteristics rather than broader trends.

Several limitations should be acknowledged. The absence of whole-genome sequencing precludes identification of resistance genes, virulence determinants, and mobile genetic elements. Similarly, lack of molecular typing limits inference regarding clonal relatedness and transmission dynamics across healthcare facilities. Additionally, the study design does not allow assessment of incidence or clinical outcomes, and findings reflect laboratory-based isolate frequencies rather than population-level burden.

Conclusion

This study highlights a high burden of multidrug-resistant Klebsiella pneumoniae with frequent biofilm formation and hypermucoviscosity in tertiary-care hospitals. These findings underscore important therapeutic and infection control challenges. Strengthened surveillance and antimicrobial stewardship are needed, alongside molecular studies to better understand resistance mechanisms and transmission dynamics.

Data availability statement

The data supporting the findings of this study are available within the article.

Financial support

No external funding was received for the conduct of this research.

Competing interests

The authors declare that there are no conflicts of interest.

Final Approval of Manuscript

All authors reviewed and approved the final version of the manuscript and agreed to be accountable for all aspects of the work.

References

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Li, J, Shi, Y, Song, X, Yin, X, Liu, H. Mechanisms of antimicrobial resistance in Klebsiella: advances in detection methods and clinical implications. Infect Drug Resist 2025;18:1339–1354. doi:10.2147/IDR.S509016.CrossRefGoogle ScholarPubMed
Chen, TA, Chuang, YT, Lin, CH. A decade-long review of the virulence, resistance, and epidemiological risks of Klebsiella pneumoniae in ICUs. Microorganisms 2024;12:2548. doi:10.3390/microorganisms12122548.CrossRefGoogle ScholarPubMed
Zada, H, Mubaraki, MA, Qasim, M, et al. Phenotypic and molecular characterization of Klebsiella pneumoniae in neonates and pediatric patients: antibiotic resistance, β-lactamase genes, and sociodemographic risk factors. BMC Infect Dis 2025;25:1769. doi:10.1186/s12879-025-12157-x.CrossRefGoogle ScholarPubMed
Pruss, A, Kobylińska, D, Fijałkowski, K, Masiuk, H, Kwiatkowski, P. Evaluation of colistin susceptibility of Klebsiella pneumoniae strains exposed to rotating magnetic field. Int J Mol Sci 2025;26:8281. doi:10.3390/ijms26178281.CrossRefGoogle ScholarPubMed
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Zdarska, V, Arcari, G, Kolar, M, Mlynarcik, P Antibiotic resistance in Klebsiella pneumoniae and related Enterobacterales: molecular mechanisms, mobile elements, and therapeutic challenges. Antibiotics 2026;15:37. doi:10.3390/antibiotics15010037.CrossRefGoogle ScholarPubMed
Azra, KTA, Ul Haq, I, Hinthong, W, et al. Antibiotic susceptibility patterns and virulence profiles of classical and hypervirulent Klebsiella pneumoniae strains isolated from clinical samples in Khyber Pakhtunkhwa. Pak Patho 2025;14:79. doi:10.3390/pathogens14010079.Google ScholarPubMed
Figure 0

Figure 1.Antimicrobial susceptibility profile of Klebsiella pneumoniae isolates across tested antibiotic classes (n = 248). Amoxicillin–clavulanic acid (AMC), Piperacillin–tazobactam (TZP), Cefazolin (CZ), Cefoxitin (FOX), Ceftazidime (CAZ), Cefotaxime (CTX), Cefepime (FEP), Ertapenem (ETP), Meropenem (MEM), Amikacin (AK), Gentamicin (CN), Tobramycin (TOB), Nitrofurantoin (NIT), Ciprofloxacin (CIP), Levofloxacin (LEV), Tetracycline (TET), Trimethoprim–sulfamethoxazole (SXT), Colistin (COL).* for urine isolates only.

Figure 1

Figure 2.Distribution of biofilm-forming capacity among clinical isolates stratified by specimen type, demographic characteristics, and antimicrobial resistance profile (n = 248).