Investigating the Binding Affinity of Potential Drugs and Inhibitors against MDR Klebsiella pneumoniae using Molecular Docking Simulations

 

Rashmi A Kale*, Archana N. Moon

P.G.T Department of Biochemistry, RTM, Nagpur University, Nagpur, Maharashtra, India.

 

ABSTRACT:

Background: Klebsiella pneumoniae is one of the leading causes of death a worldwide due to hypervirulent strains developing in hospitals. Also, antibiotic-resistant K. pneumoniae is progressively being involved in invasive infections with high morbidity and mortality. The current study aimed to determine antimicrobial susceptibility patterns, identify the resistance genes (genomic and plasmid) among clinical isolates of K. pneumoniae, and also to carry out the assay of beta-lactamase activity. Isolation of Genomic DNA and Plasmid DNA is thus carried out to know which gene is responsible for resistance. Molecular and Docking studies were carried out to find the presence of a mutation in the beta-lactamase gene and their docking sites with the standard inhibitors and antibiotics. Methods: Fifty clinical sputum samples of Respiratory tract infected patients were collected from different hospitals and Labs (Government Medical College, Indira Gandhi Medical College, Vishakha Lab etc) in Nagpur (Maharashtra). Out of 50, only 21 isolates were confirmed as Klebsiella pneumoniae by performing morphological and biochemical testing. An antibiotic Sensitivity test was performed. Microiodometric assay and Nitrocefin method were carried out to find out the beta-lactamase producing bacteria from clinical isolates.  These isolates were also checked for different βL genes by PCR using specific primers for ESBL genes and showed the presence SHV-1, TEM, and CTX-M genes in the results. These genes were sent for gene sequencing for confirmation, and mutational analysis was done.  After sequencing and mutational analysis were done for these isolated βL genes, Phylogenetic analysis was done to find out the closest standard protein. Automated molecular docking with all inhibitors to beta- lactamase protein was performed by using the advanced docking program Autodock 4.0. Results: The present study concluded that out of six, the five gene sequences i.e kp 13(g) CTX-M, kp 16(g) CTX-M, kp 14(p) CTX-M, kp 17(p) SHV, and kp 18(p) CTX-M contained a less number of mutations which do not show the actual change at their protein level, whereas kp 18 (g) TEM showed a maximum number of mutations and showed actual change at their protein level. The binding affinities of various inhibitors and antibiotics against TEM, SHV, and CTX-M enzymes were calculated. Durlobactam and Relebactam ligands demonstrated strong binding affinities with both wild-type and mutant TEM enzymes, indicating potent inhibitory activity Durlobactam and Ciprofloxacin exhibited the highest binding energies against the SHV enzyme, while Norfloxacin and Aztreonam showed the highest binding energies against the CTX-M enzyme. These combinations demonstrate potential as therapeutic strategies for treating resistant K. pneumoniae infections. Conclusions: Thus, this study concluded that the mutations occurring in the beta-lactamase genes may be due to variations in the environmental conditions or due to the frequent use of antibiotics makes them resistant to antibiotics. Docking helps us to know which part of the beta-lactamase protein is responsible for resistance to antibiotics. The findings of this study conclude that if we design (drug) the combination of high-binding energy Antibiotics and Inhibitors against beta-lactamase enzymes, can be useful to treat MDR K. pneumoniae.

 

 

 

 

1. INTRODUCTION:

Klebsiella pneumoniae (K. pneumoniae), one of the major Enterobacterales, has emerged as a clinically significant pathogen because of increased antibiotic resistance and its propensity to cause serious outcomes ^1,3. It has been ranked the second among Gram-negative bacteria in causing hospital-acquired infections^1,2. K. pneumoniae is recognized as the primary cause of serious nosocomial infections such as respiratory infections, urinary tract infections, soft tissue infections, bacteremia, and sepsis. By analyzing different geographic regions of India, meta-analysis reveals the prevalence of multi-drug resistant Klebsiella pneumoniae among hospital-acquired is 34.37% and the pooled prevalence of multi-drug resistant k. pneumoniae among hospital-acquired cases was found at 2%³. In the present study, we have focused on Molecular Biology and Docking work. The earlier work of this study was already published in the Research Journal of Biotechnology.

 

2. MATERIALS AND METHODS:

In the present study, total 100 sputum samples were collected from the suspected RTI patients from different pathology labs in Nagpur region, Maharashtra, India. Out of 100 clinical isolates, 51 isolates showed the presence of Klebsiella sp. This was confirmed by Morphological and Biochemical tests⁴. The MDR pattern of 51 K. pneumoniae (Kp) strains were studied using fourteen different antibiotics. The presence of beta lactamase enzyme in the above-mentioned strains was studied. Out of 51 only 21 K. pneumoniae strains were beta-lactamase positive. We already published this study in the Research Journal of Biotechnology. After isolation of genomic DNA and plasmid DNA from Kp strains, PCR were carried out to find out the presence of CTX-M, TEM, and SHV bla genes in both genomic and plasmid DNA^5,6.

 

2.1 PCR for amplification of Beta-lactamase genes: This was done by using specific Primers for each of the bla genes (refer Table 1). These were the Universal Primers that we used for the study.

 

Table 1: Oligonucleotide primers used for the detection of β-lactamase genes

Target gene	Primers used	Tm	Molecular weight [g/mol]	Amplicon Size
SHV	F- 5’AAGATCCACTATCGCCCAGCAG-3’ R-5’AAGATCCACTATCGCCCAGCAG-3’	F- 62.120C R-62.12 0C	6,673.36 6,673.36	~471 bp
CTX-M	F-5’ACGCTGTTGTTAGGAAGTG-3’ R-5’TTGAGGCTGGGTGAAGT-3’	F-54.510C R-52.77 0C	5,898.85 5,321.48	~724bp
TEM-1	F-5’ GAGTATCAACATTTCCGTGTC- 3’ R-5’TAATCAGTGAGGCACCTTCTC- 3’	F-55.92 0C R-57.87 0C	6,396.16 6,381.15	~841bp

 

2.2 Phylogeny of gene sequences:

After PCR, the selected genes of both genomic and plasmid DNA from 10kp strains were then sent for gene sequencing to Barcode Biosciences, Banglore Karnataka. A phylogenetic tree was prepared using the Fasta file of the provided 10 sequenced genes and the Bioinformatic tools. Clustalw and Mega 11 software were used for this.

 

2.3 Single Nucleotide Polymorphism of the selected gene sequences: To check the mutations in the isolated gene sequences, Single Nucleotide Polymorphism was done by using Bioinformatic tools and two software, 1) BioEdit app, 2) Launch DnaSP6.exe app.

 

2.4 Selection of PDB structure of β-lactamase: The 3D structure of CTX-M (PDB ID: 5T66), SHV (PDB ID: 3D4F), and TEM (PDB ID: 1ERO) β-lactamase, downloaded from the protein data bank.

 

(A) Ligand Selection and Preparation: For analyzing and comparing the binding efficiency of the ligands to the active site of wt and mt proteins (CTX-M, SHV, and TEM), fourteen antibiotics and six inhibitors were used as a ligand in the molecular docking analysis. The structure of the Ligands was directly downloaded from the PubChem online portal.

 

(B) Homology Modelling for Mutant Proteins: In the study Sanger Sequencing data of FASTA format retrieved from Applied Biosystem machine and preliminary searched for BLAST Homology. Here, 10 best homologies were retrieved and previously reported as complete genes of genes (CTX-M, TEM and SHV).

 

2.5 Molecular Docking: Autodock4.0 app is used for docking of ligands with the wild type and mutant type proteins (CTX-M, TEM and SHV). Total 20 ligands were used, out of which 14 are antibiotics and 6 are inhibitors. To start docking, we have to first prepare separate folders for each ligand with each protein, then we need to have followed files ready in each folder. These are 1) Protein in PDB format, 2) Ligand in MDL SD format, 3) Autodock4 app, 4) Autogrid4 app, and 5) cygwin1.dll.

 

3. RESULTS:

Genomic DNA and plasmid DNA have been isolated from all 21 strains of K. pneumoniae isolated from sputum samples of RTI patients. All 21 strains of K. pneumoniae showed the presence of genomic DNA. However, only 4 strains (kp14, kp15, kp17 and kp18) out of 21 K. pneumoniae strains revealed the presence of plasmid DNA.

 

3.1 Screening of PCR products of gDNA and pDNA on agarose gel for bla genes:

After the isolation of g DNA and p DNA, PCR was done using primers for three different β lactamase genes: 1) CTX-M, 2) TEM, and 3) SHV. By observing the intensity and the concentration of each band on agarose gel; the most prominent band of a particular gene was selected for further gene sequencing. A total of 10 genes (4 pDNA and 6 gDNA) were then sent for gene sequencing. After gene sequencing, it was found that 4 gene sequences matched with the CTX-M gene molecular weight, 3 gene sequences matched with the SHV gene, and 2 gene sequences with TEM gene (refer Fig: 1 and Table 1).

 

 

Fig 1: PCR product (selected) of both g DNA and p DNA

 

Table 1: Amplicon sizes of Beta-lactamase genes isolated from K. pneumoniae (Kp) strains.

Lanes	1	2	3	4	5	6	7	8	9	10	11
Samples of Kp strains	Marker	Kp6(G)	Kp7(G)	Kp18(G)	Kp19(G)	Kp16(G)	Kp13(G)	Kp15(P)	Kp17(P)	Kp14(P)	Kp18(P)
Amplicon size in bp	100 bp ladder	380bp (S)	380bp (S)	880bp (T)	880bp (T)	790bp (C)	790bp (C)	380bp (S)	400bp (S)	720bp (C)	720bp (C)

·       S= SHV gene, T= TEM gene, C= CTX-M gene

·       G= genomic DNA, P = Plasmid DNA

 

3.2 Single Nucleotide Polymorphism Analysis of selected genes: Single Nucleotide Polymorphism analysis was carried out on these 6 gene sequences out of 10 to find out the number of mutations and also know the change in their amino acid level. The remaining 4 genes were not QC pass.

 

3.3 Molecular Docking of Selected Gene Sequences isolated from Kp strains: In the present study, 777, 840 and 341 bases of genes (CTX-M, TEM and SHV) were successfully sequenced and translated into a protein of 240, 242, and 97 amino acids. Once the protein sequence aligned with close homology by accepting PDB ID code (5T66, 1ERO and 3D4F), the same PDB ID code-based protein (wild-type) was used for comparative docking investigation where we successfully modeled the protein structure of CTX-M, SHV and TEM-1 protein (mutant-type) using the SWISS Model program ^7,8,9 (as shown in Fig 2).

 

Fig 2: 3D Structure of Model 1, 2 and 3 derived from SWISS Model

3.4 Autodock Result Analysis:

A total of 20 Ligands (14 Antibiotics and 6 inhibitors) were used to dock with each of these three Proteins (CTX-M, SHV, and TEM of bla genes) both wild type and mutant type. Comparison has been made in the Binding energy of wild-type Proteins with the Ligands and the Binding energy of mutant-type Proteins with the same Ligands. It is clear that the binding energy of the mutant proteins with ligands is less than the binding energy of the wild-type proteins.

 

For TEM, wild and mutant proteins defined a change in binding energy, which suggests that mutation does affect the binding energy for sure with the Beta-lactamases available in the pathogen. The binding energy of the mutant TEM is less as compared to the wild type with all ligands except Cefazolin, Doxycycline and Gentamicin, which means that the mutant gene has a strong attraction to the antibiotic which leads to resistance. The calculated binding affinity (-8.99 and -6.54 Kcal/mol) for Durlobactam and Relebactam ligands exhibited stronger inhibition in terms of the ability to bind well against both the wild-type (1ERO) and mutant-type TEM enzyme. Cefazolin showed high binding energy (-8.59) against wild-type TEM among all antibiotics.  The combination of these inhibitors with antibiotics can be used to treat these resistant K. pneumoniae.

 

In the case of the SHV enzyme, the calculated binding affinities for Durlobactam  (-6.42 kcal/mol) against the mutant-type SHV enzyme and for Ciprofloxacin (-7.30 kcal/mol) against wild-type (3D4F) exhibited the highest binding energies among all the tested ligands. The binding energy of the mutant SHV is less as compared to the wild type with all ligands except Co-trimoxazole, Doxycycline and Nitrofurantoin, which means that the mutant gene has a strong attraction to the antibiotic which leads to resistance. The combination of these inhibitors and antibiotics could potentially be used to treat resistant K. pneumoniae infections.

 

Similarly, the calculated binding affinities for Avibactam (-8.39 kcal/mol) against the mutant-type CTX-M enzyme and for Aztreonam (-7.75 kcal/mol) against wild-type (5T66) displayed the highest binding energies among all the ligands tested. In contrast, the mutant CTX-M enzyme showed reduced binding energy compared to wild-type CTX-M with most ligands, except Avibactam, Clavulanic acid, Durlobactam, and Tazobactam. The combination of these antibiotics may offer a potential treatment for resistant K. pneumoniae infections.

 

4. DISCUSSION:

In the present study, the determination of CTX-M, TEM and SHV bla genes were carried out. By observing the intensity and concentration of each band on agarose gel, the most prominent band of a particular gene was selected for further gene sequencing. A total of 10 genes (4 pDNA and 6 gDNA) were then sent for gene sequencing. After gene sequencing, it was found that 4 gene sequences matched the CTX-M gene molecular weight, 3 gene sequences matched the SHV gene, and 2 gene sequences matched with the TEM gene. In kp16 (g) [CTX-M] gene, a total 3 mutations were found, in which the first mutation was a silent mutation where there was no change in amino acid, whereas the other two mutations were not defined. There was no actual change in their protein level. Same way in the remaining genes also.

 

In one of the study, it has been reported that CTX-M is the most prominent ESBL enzyme worldwide. They explained that more than 50 % of strains were CTX-M positive out of 97 strains of K. pneumoniae. The TEM gene has shown maximum virulence as compared to CTX-M and SHV ¹⁰. In our study also, only in the Kp18 (g) [TEM] gene, 572 mutations were found, which becomes difficult for us to process further. From this, we can assume that the TEM gene is continuously undergoing mutation which will be harmful to our society. Their pleomorphic nature will make it a new approach to work on it as a separate study further.

 

According to other study, published in (2021), three-dimensional structure of Beta-lactamases and its docking with peptide macrocycles as an inhibitor and similar study able to understand the change in binding energy due to mutations and wild-type and found to be important in the current study ¹¹.

 

In the present study, it has been observed that mutations are prevalent in the computationally modeled proteins of CTX-M, SHV, and TEM as compared to wild type and the possibility is that they may affect the binding energy of antibiotics and inhibitors once tested In silico using drug docking tools.

 

In a similar study, k. pneumoniae is treated by using beta lactam antibiotics and in counter action organism produces beta- lactamases to inactive the medicine. They used the In silico approach by using ­­1n9b and 2zd8, when docked with drug like Cefepime and Amoxcillin and others. They found the best docking value of proteins TEM and SHV as -8.23 Kcal/mol and -8.53 Kcal/mol respectively which is in agreement to the present study ^{12, 13}.

 

In another study, reported new Delhi Beta- lactamases for which a defined inhibitor has not been reported yet. Here they screened 500 natural compounds for docking and among them for promising plant compounds reported to be efficient and hence they for the first time showcased such possibility ¹⁴.

 

The use of beta-lactamase inhibitors has been steadily increasing, intensifying the competition for effective antibiotics and inhibitors. Researchers have reported several escape mutations that enable bacterial growth even in the presence of these inhibitors. Notably, these mutations are rare and specific to particular drugs ¹⁵.

 

In the present study, we conclude that mutations in the proteins certainly affect the binding affinity via a change in the energy of binding and its change in the binding site. The rising use of beta-lactamase inhibitors and the emergence of escape mutations in bacteria underscore the urgent need for responsible antibiotic use and robust investment in research for new treatments. As these mutations are rare but drug-specific, it highlights the importance of tailoring medical interventions and developing innovative strategies to stay ahead of evolving bacterial resistance. Society must prioritize awareness, judicious antibiotic usage, and support for scientific advancements to combat this growing threat and safeguard global health.

 

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Received on 06.05.2025      Revised on 19.09.2025 Accepted on 25.11.2025      Published on 01.07.2026 Available online from July 04, 2026 Research J. Pharmacy and Technology. 2026;19(7):3189-3193. DOI: 10.52711/0974-360X.2026.00453 © RJPT All right reserved
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