Background Amoxicillin is among the most frequently prescribed antibiotics globally, either as monotherapy or in combination with clavulanic acid as amoxicillin-clavulanic acid (AMC). However, the prolonged use of AMC and other antibiotics has intensified selection pressure, accelerating the emergence of AMC-resistant and multidrug-resistant (MDR) strains. Klebsiella, a member of the ESKAPE pathogens, employs diverse resistance mechanisms against multiple classes of antibiotics. This study was aimed to identify environmental Klebsiella isolates resistant to AMC with MDR phenotype and to investigate the underlying genetic determinants contributing to their resistance and virulence. Methodology Water samples were collected from 14 sites, encompassing both wastewater and natural aquatic environments, and screened for AMC resistance on AMC supplemented Klebsiella-Selective agar base media. Antibiotic profiling of AMC resistant isolates was done by Kirby-Bauer’s disc diffusion test. Phenotypically positive MDR isolates were identified by MALDI-ToF MS. Furthermore, Klebsiella pneumoniae isolates were selected for PCR based detection of antibiotic resistance and virulence factor associated genes using plasmid and genomic DNA as a template respectively. Horizontal gene transfer experiment was carried out using K. pneumoniae isolates as donor and plasmid-free and antibiotic sensitive Escherichia coli J53R strain as a recipient. Biofilm formation was detected by crystal violet assay and visualised in SEM. The hypermucoviscosity of K. pneumoniae (hmvKp) was confirmed by string test. Results Of the total 178 AMC resistant bacterial isolates, 119 displayed MDR phenotype. Among 63 putative AMC-resistant, MDR isolates exhibiting a non-metallic sheen on EMB agar, MALDI-TOF MS-based identification confirmed 33 to be Klebsiella pneumoniae. PCR based screening for resistance determinants revealed the presence of blaTEM (100%), blaSHV (75.75%), blaCTX−M (54.54%), blaNDM (27.27%), blaOXA−48 (39.39%), blaCMY (48.48%), qnrA (6.06%), qnrB (87.87%), qnrS (93.93%), tetA (81.81%), and tetB (27.27%), alongside sul1 (90.90%) and dfrA12 (9.09%) genes. Additionally, virulence-associated genes viz., fimH (33.33%), mrkD (78.78%), ecpA (54.54%), iucC (54.54%), and rmpA (6.06%) were also detected. Furthermore, biofilm formation assay demonstrated that 24 (72.72%) isolates were strong biofilm-formers, indicating their potential for pathogenicity. Conclusion Occurrence of hypervirulent, AMC resistant and MDR Klebsiella pneumoniae in aquatic environment is a concern and further studies are required to explore their potential threat in dissemination of resistance and clinical implications. Graphical abstract

Pathogenic potential of amoxicillin-clavulanic acid resistant Klebsiella pneumoniae isolated from aquatic environment: a study of multidrug resistance and virulence

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2025年11月4日

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Vikar Ahmed,
Mohammad Tahir Siddiqui,
Asghar Ali,
Syed Ahmed Rizvi,
Mohammad Saif,
Shayan Ahmed,
Qazi Mohd Rizwanul Haq

摘要

Background

Amoxicillin is among the most frequently prescribed antibiotics globally, either as monotherapy or in combination with clavulanic acid as amoxicillin-clavulanic acid (AMC). However, the prolonged use of AMC and other antibiotics has intensified selection pressure, accelerating the emergence of AMC-resistant and multidrug-resistant (MDR) strains. Klebsiella, a member of the ESKAPE pathogens, employs diverse resistance mechanisms against multiple classes of antibiotics. This study was aimed to identify environmental Klebsiella isolates resistant to AMC with MDR phenotype and to investigate the underlying genetic determinants contributing to their resistance and virulence.

Methodology

Water samples were collected from 14 sites, encompassing both wastewater and natural aquatic environments, and screened for AMC resistance on AMC supplemented Klebsiella-Selective agar base media. Antibiotic profiling of AMC resistant isolates was done by Kirby-Bauer’s disc diffusion test. Phenotypically positive MDR isolates were identified by MALDI-ToF MS. Furthermore, Klebsiella pneumoniae isolates were selected for PCR based detection of antibiotic resistance and virulence factor associated genes using plasmid and genomic DNA as a template respectively. Horizontal gene transfer experiment was carried out using K. pneumoniae isolates as donor and plasmid-free and antibiotic sensitive Escherichia coli J^53R strain as a recipient. Biofilm formation was detected by crystal violet assay and visualised in SEM. The hypermucoviscosity of K. pneumoniae (hmvKp) was confirmed by string test.

Results

Of the total 178 AMC resistant bacterial isolates, 119 displayed MDR phenotype. Among 63 putative AMC-resistant, MDR isolates exhibiting a non-metallic sheen on EMB agar, MALDI-TOF MS-based identification confirmed 33 to be Klebsiella pneumoniae. PCR based screening for resistance determinants revealed the presence of bla_TEM (100%), bla_SHV (75.75%), bla_CTX−M (54.54%), bla_NDM (27.27%), bla_OXA−48 (39.39%), bla_CMY (48.48%), qnrA (6.06%), qnrB (87.87%), qnrS (93.93%), tetA (81.81%), and tetB (27.27%), alongside sul1 (90.90%) and dfrA12 (9.09%) genes. Additionally, virulence-associated genes viz., fimH (33.33%), mrkD (78.78%), ecpA (54.54%), iucC (54.54%), and rmpA (6.06%) were also detected. Furthermore, biofilm formation assay demonstrated that 24 (72.72%) isolates were strong biofilm-formers, indicating their potential for pathogenicity.

Conclusion

Occurrence of hypervirulent, AMC resistant and MDR Klebsiella pneumoniae in aquatic environment is a concern and further studies are required to explore their potential threat in dissemination of resistance and clinical implications.

Graphical abstract