Comparative Analysis of Environmental Versus Clinical Isolates of Klebsiella pneumoniae: A Key Trafficker of Antibiotic Resistance Genes (AMR)

Alakh Narayan Singh¹, Gopal Nath^1*
1Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India

*Correspondence author: Gopal Nath, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India; Email: [email protected]

Citation: Singh AN, et al. Comparative Analysis of Environmental Versus Clinical Isolates of Klebsiella Pneumoniae: A Key Trafficker of Antibiotic Resistance Genes (AMR). J Clin Immunol Microbiol. 2025;6(2):1-12.

Copyright^© 2025 by Singh AN, et al. All rights reserved. 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.

Table 1: Resistance pattern of clinical and environmental isolates of K. pneumoniae using the disc diffusion method.

Table 2: Overall resistance rate of K. pneumoniae to various antimicrobials in Asian countries (date 2005-2019).

Figure 1: Antibiotics susceptibility testing (disk diffusion method) of clinical isolate of K. pneumoniae.

Figure 2: The bar diagram shows antibiotics drug resistance pattern of clinical isolates of K. pneumoniae.

Figure 3: Dendrogram of clinical isolates of K. pneumoniae based on antibiotics susceptibility testing.

Figure 4: Antibiotics susceptibility test (disk diffusion method) of environmental isolate of K. pneumoniae.

Figure 5: Bar diagram showing antibiotics drug resistance pattern of environmental isolates of K. pneumoniae.

Figure 6: Dendrogram of environmental isolates of K. pneumoniae based on antibiotics susceptibility testing.

Figure 7: Dendrogram of K. pneumoniae based on ERIC-PCR of thirty randomly selected isolates of from clinical and environmental.

Discussion

The emergence and rapid spread of hypermucoviscosity carbapenem-colistin-resistant K. pneumoniae in healthcare settings is a serious concern worldwide. K. pneumoniae belongs to the ESKAPE group of microorganisms, especially known for the acquisition of antimicrobial resistance genes from environmental sources and also reflects the ability of ESKAPE microorganisms to escape from killing by antimicrobial substances. Thus, it defies eradication by conventional antibiotic therapy, which accounts for extensive morbidity and mortality worldwide.

 The clinical isolates were resistant at a very high level as compared to that of environmental, while 11 out of 16 antibiotics tested >90% of the clinical isolates expressed resistance while only two antibiotics, i.e., Cefuroxime and Cefazolin/Cephalexin were found to resist >90% of the environmental isolates. It is quite likely that the resistance mechanism in the environmental bacteria might be located on a chromosome, leading to intrinsic resistance [21].

On comparing the resistance pattern of clinical versus environmental isolates, it could be observed that all of the 129 (100%) clinical isolates were MDR, with 14.7% of them having PDR status. Contrary to this, only 10.8% of the environmental isolates could be designated MDR with nil PDR. Interestingly, 66.7% of the isolates were resistant to only the β-lactam group of antibiotics. This difference could be explained based on high selection pressure in the hospital environment leading to the accumulation of a variety of resistance genes in plasmids borne by the sturdy K. pneumoniae bacteria, which is incessantly between the natural environment and the hospital setup. Although not unexpected, the present observation made in this study is shown for the first time in the literature.

Interestingly, a study reported from North India during the same period, i.e., 2021-22, had a similar pattern and rate of prevalence against different antibiotics. Interestingly, there has been a rising pattern of drug resistance in K. pneumoniae from 2005 to 2019 (Table 2). It has almost reached a peak and Colistin remains the only ray of hope. The resistance against Colistin also has risen from 2.9% during 2005-2019 to 15.5% in the present study [22-31].

A review article entitled “Klebsiella pneumoniae: an increasing threat to Public Health” by Effah, et al., analyzed the 20 relevant articles published between 2005-2019 from different countries of Asia with a special focus on China [32]. Since there is a duration of 14 years, there are a lot of variations in drug resistance. When we compare the drug resistance of the present study, where the clinical samples were collected during 2021-2022, K. pneumoniae shows unusually high resistance rates against different antibiotics. These resistance rates indicate that before and during the year 2021-22, our hospital might have practiced indiscriminate use/misuse of antibiotics, except colistin, which was resisted by 15.5% of the isolates in the present study. However, previous studies from different countries also showed that the resistance rates vary from 1.8-4.4% (Table 2). The high heterogeneity index shows the huge variation in the different reports, which might have occurred due to different selection pressures over the 14 years and different antibiotics policies, if any, in different hospitals. The three Indian studies included in this review were also conducted in the years 2015 (33), 2012-14 (34) and 2014-2016 (35). Intriguingly, the study conducted recently had a similar resistance rate from North India (Amritsar) (21), whereas all the other antibiotics tested have a resistance rate ranging between 60-100% [21,33-35]. Interestingly, the resistance rate for colistin was 16%, while in the present study, it was 15.5%.

Based on the constructed antibiotic susceptibility pattern dendrogram, it was interesting to observe that 30 randomly selected clinical and environment isolates had different non-identical cluster patterns; however, 5 strains had similar sensitivity patterns.

When the dendrogram was constructed based on the antibiotic susceptibility pattern and phage typing of the clinical isolates, the antibiotic susceptibility pattern had 8 small clusters comprising 2, 54, 10, 6,14, 19, 5 and 3 isolates having identical pairs. In contrast, the phage typing had only 3 identical pairs comprising 2 phages each. It means there is no correlation between the antibiotic and phage typing pattern. The lack of correlation between antibiotic typing and phage typing is due to the different mechanisms of inheritance of resistance genes, mainly plasmids, rather than the presence of various phage receptors on the bacterial surfaces.

High selection pressure may be appreciated by the dendrogram based on the resistance of clinical isolate, where 4 major clusters of identical strains could be seen. Interestingly, one cluster had 54 identical strains. On the contrary, the dendrogram prepared for environmental isolates on the same basis hardly shows the 3 or more identical strains in different branches.

The present study compares the resistance pattern in the isolates of both the origin, i.e., clinical and environmental. It shows that environment strains are only multi-drug resistant to a few antibiotics. However, high resistance rates in a few of the environmental isolates also depicted against β-lactam groups of antibiotics, especially Ampicillin, Cefotaxime, Cefpirome, Ciprofloxacin and Piperacillin, might be due to inherited chromosomal genes. However, the resistance rate of 71.4% in the environmental strains needs explanation as, despite Tazobactam, the resistance is seen in these isolates. Do these isolates have mechanisms other than ESBL?

Based on ERIC PCR, the dendrogram prepared indicates that the resistance markers are primarily not located on chromosomes but on plasmids since whole genome analysis by ERIC PCR of both types of isolates shows rare identical strains, which is contrary to the dendrogram prepared based on drug resistance. Wyres and Kathryn, 2018 in a review article published in Current Opinion in Microbiology, emphasize that K. pneumoniae are intrinsically resistant to ampicillin due to penicillinase (SHV-1) in their chromosome [5,36-38]. They further suggested that additional drug resistance occasionally may arise through mutation in chromosomal genes and most AMR in K. pneumoniae results from the equisition of large and small consignations plasmids via horizontal gene transfer [7,39-41].

It is suggested that under the selection pressure of antibiotics in a clinical environment, the accumulation of AMR genes in a single strain results in pan-resistant strains [42]. Wyres and Holt have mentioned that clinical strains of K. pneumoniae can accumulate up to 400 AMR genes, followed by A. baumannii 278 and E. cloacae 277. They have depicted that K. pneumoniae can accumulate many plasmids ranging from 2-5 per strain. It has been shown that among the Enterobacteriaceae family, K. pneumoniae has the highest number (≈5500 genes) of genes per genome. Further, GC content per gene in K. pneumoniae is highly variable. All these inherent quality of K. pneumoniae enables it to accumulate the highest number of resistance genes in a single bacterial cell; added to this, unlike E. coli, K. pneumoniae can thrive well in environmental condition and also in the gut of human, pet animals, insects and aquatic animals. As the AMR genes primarily originate from the water and soil bacteria, which produce a spectrum of antibiotics for their protection, K. pneumoniae acquires these genes and brings them to the human gut as a key trafficker to disseminate in various clinically important bacteria. This is evident in the present study as many environmental strains were detected with resistance against many of the penicillin and cephalosporin groups of drugs, probably mediated intrinsically on chromosomes [37]. We could detect MDR strains of K. pneumoniae from the environment, while most of the clinical isolates were MDR and PDR [43].

onclusion

It has been suggested that the gut microbiota of human beings only occasionally have K. pneumoniae as a commensal flora (6-10%). Therefore, K. pneumoniae may not be an essential component of the gut microbiota. When we combine all the above factors, we analyzed the K. pneumoniae position; it may be suggested that it is a key amplifier and spreader of AMR genes in the clinical environment. It may also be suggested that eradicating K. pneumoniae from the gut of human beings and from the clinical environment through the agency of bacteriophages can help us reduce the menace of antibiotic resistance.

Conflict of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This research received no specific grant from public, commercial or not-for-profit funding agencies.

Data Availability Statement

The original contributions in the present study are included in the article/supplementary material; further inquiries can be directed to the corresponding author.

Authors’ Contribution

ANS: Conceptualization, Data curation, Formal analysis, Investigation, Validation, Visualization, Writing – original draft, Writing – review and editing. GN: Conceptualization, Data curation, Formal analysis, Investigation, Validation, Visualization, Writing – review and editing, Supervision, Project administration.

Acknowledgements

We are also grateful to Viral Research and Diagnostic Laboratory, DHR, Govt. of India, for providing the infrastructure for the current research work and the financial help as an incentive grant from IoE, Banaras Hindu University, given to GN.

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