1 Introduction

Given their pivotal role in bacterial resistance to β-lactam antibiotics, class A β-lactamases pose a serious problem in the field of antibiotic resistance research. Nowadays, β-lactams constitute a significant class of antibiotics used to treat bacterial infections.

Penicillin-binding proteins (PBP) are the enzymes involved in cross-linking the peptidoglycans in the bacterial cell wall synthesis. β-lactam antibiotics mainly target and inactivate PBPs by binding to their active sites [1]. β-lactam antibiotics have a 4-membered β-lactam ring as their core structure; the β-lactam core structure imitates the d-Ala-d-Ala peptide as the substrate for transpeptidase activity for bacterial cell wall synthesis [2]. The bacterial cell wall ruptures, leading to the death of the bacteria, as a result of these antibiotics attaching to the PBPs’ active site and inhibiting cross-linking activity. Thus, binding of these antibiotics to the active site of the PBPs prevents the cross-linking activity, which in turn causes the loosing of the bacterial cell wall resulting in the death of the bacteria. Nevertheless, the production of β-lactamases (βLs) in microorganisms frequently compromises their biological effectiveness against certain bacterial strains. One of the mechanisms of action of β-lactams is by preventing the cell-wall synthesis which eventually leads to the death of the bacteria. But with the emergence of the enzyme β-lactamase, the bacteria can survive by breaking the β-lactam ring, which continually evolves, and has eventually made the new generation of cephalosporins, carbapenems, and monobactam inactive.

Similar to most antibiotics, several molecular mechanisms can lead to β-lactam resistance. These mechanisms include the generation of efflux pumps, changes in the structure or level of production of outer membrane porins, modifications to PBPs (the molecular target of β-lactams), and the synthesis of β-lactamase, which can dissolve antibiotics [3].

Presently, extended-spectrum β-lactamases (ESBLs) are a new class of enzymes that were discovered in Gram-negative (GN) rods in the 1980s [4]. These enzymes can hydrolyze several β-lactams such as monobactams, penicillins, and broad-spectrum cephalosporins by breaking down the β-lactam ring structure of these antibiotics, rendering them ineffective against the bacteria [5].

Ambler classified beta-lactamases into four classes A, B, C, and D based on amino acid homology [6], while Bush and Jacoby [7] divided them into four major groups and subclasses based on function and inhibition patterns. Class B βLs are metallo-enzymes with one or two zinc ions, while class A, C, and D enzymes have a serine residue in their active site. Within this family, Class A βLs comprise the largest and most prevalent group that can be further classified into several types of enzymes (such as TEM-, SHV-, and CTX-M-types) [2], these extended-spectrum beta-lactamase (ESBL) enzymes confirm resistance to ampicillin, piperacillin, ticarcillin, and cephalosporins [8].

β-lactamases from TEM and SHV enzymes are often possessed by Klebsiella pneumoniae (K. pneumoniae). In the instance of K. pneumoniae infection, the presence of SHV and TEM beta-lactamases can make treatment challenging by conferring resistance to multiple classes of β-lactam antibiotics, thereby limiting the efficacy of antibiotic therapy [9].

To tackle this issue of antibiotic resistance, there this a need to investigate the complexity of the interactions between class A beta-lactamase resistance and the antibiotic drug, to know how to combat them and also develop new drug strategies. Therefore, molecular modeling tools and in silico analysis can help in this process. Thus, we aim to contribute to the development of innovative strategies to counter antibiotic resistance, ultimately aiding in the preservation of the effectiveness of crucial antibacterial agents.

2 Materials and methods

2.1 Isolation and characterization of K. pneumoniae isolates

Samples were taken from hospital wastewater drainage and stored in a sterile sample bottle container. They were then transferred to the laboratory in an ice pack and processed in less than a day. Water samples were diluted and filtered using membrane filters with a pore size of 0.45 μm (Millipore, France). The filter paper was then aseptically placed on MacConkey agar and incubated for 24 h. Typical mucoid pink colonies were isolated for further analysis as presumptive K. pneumoniae. Biochemical tests such as indole, methyl red, Voges Proskauer, motility, and sugar fermentation were used for the identification of the isolate.

2.2 Antimicrobial sensitivity testing

Antimicrobial susceptibility testing was determined by the disk-diffusion method [10]. ceftazidime (30 µg), Amoxicillin-clavulanate (30 µg), cefuroxime (30 µg), meropenem (10 µg), gentamicin (10 µg), tetracycline (5 µg), and ciprofloxacin (5 µg) were the antibiotics tested. The antibiotic discs were aseptically placed over the culture plate previously seeded with the 0.5 McFarland turbidity culture of the test isolate and then incubated at 37 °C for 18–24 h. The isolate’s growth inhibition, which indicated the antibiotics’ diffusion into the surrounding agar media, was evaluated. The zone of growth inhibition surrounding the disc was measured in millimeters to evaluate the growth inhibitory effect. Results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) 2021 guidelines [11].

2.3 Whole genome sequencing (WGS)

2.3.1 DNA extraction

The bacterial isolate was grown overnight on nutrient agar and the whole genomic deoxyribonucleic acid (DNA) of a pure 24-h culture plate was extracted using the ZymoBIOMICS™ DNA Miniprep Kit (Zymo Research, Inqaba Biotec, South Africa) as stated by the manufacturer’s instructions.

2.3.2 Library preparation and sequencing

Library preparation was performed using the custom-built IDT 10 bp unique dual indices (UDI) with a desired insert size of 320 bp. Demultiplexing, quality control, and adapter trimming were performed with bcl-convert1 (v4.1.5). The WGS of Genomic DNA was sequenced on an Illumina NovaSeq 6000 sequencer (Illumina Inc., San Diego, CA, USA) using Nextera XT in a paired-end run with 2 × 151 bp.

2.3.3 Assembly and annotation

The gene assembly and annotation were carried out using the Read assembly, annotation pipeline RAPT server (RAPTv0.55teamcity C, NCBI) (https://github.com/ncbi/rapt). The assembled genome was annotated via PATRICK and NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [12].

2.3.4 Bioinformatics tools

The sequence ty** (ST) was identified on MLST (https://cge.cbs.dtu.dk/services/MLST/), resistome, was analyzed by ResFinder 4.1 [13], Then the antibiotic resistance mechanisms of the isolate were analyzed using CARD (Comprehensive Antibiotic Resistance Database) [14]. De novo assemblies were used for these steps.

2.3.5 Accession number

This whole genome sequencing study’s Bioproject accession number is PRJNA1021015, and the sample was deposited in a biosample with accession number JAWIZY000000000. Raw reads were deposited in the Sequence Read Archive with accession number SRR 26387539.

2.4 Optimization and molecular docking

Five β-lactam antibiotics compounds (Amoxicillin, Augmentin, Cefepime, Ceftazidime and Meropenem) were modeled using Chemdraw software which were further subjected Spartan 14 for conversion to 3D format. B3LYP and 6-31G* set were employed as basis set for the optimization of the studied compounds and a series of descriptors were obtained for further examination. The descriptors obtained were highest occupied molecular orbital energy (EHOMO), lowest unoccupied molecular orbital energy (ELUMO), energy gap, dipole moment, polar surface area, lipophilicity, hydrogen bond donor, and hydrogen bond acceptor.

The optimized compounds were subjected to Autodock tool software [15] for the purpose of conversion to .pdbqt before docking calculation using Autodock vina software [16]. The studied receptors (class A beta-lactamase (PDB id: 1n9b) [17] and SHV-1 class A beta-lactamase (PDB id: 2zd8) [18]) were retrieved from protein data bank [19] with the resolution of 0.90Å and 1.05 Å respectively. The retrieved receptors were treated using Discovery studio software [20] by removing water molecules, small molecules downloaded with the receptors and further saved in .pdb format. The active sites in the treated receptors were located via Autodock tool software before docking. The predicted Grid box for the two receptors were set as follows: center (X = 2.890877, Y= − 27.231308 and Z = 46.482723) for class A beta-lactamase (PDB id: 1n9b) (Figs. 1 and 2) while center (X = 23.809919, Y = 29.185145 and Z = 3.684532) (Figs. 3 and 4) was set for SHV-1 class A beta-lactamase (PDB id: 2zd8) and the same size (X = 40, Y = 40, and Z = 40) and the spacing which was set to be 1.00Å was the same for both targets and saved as .pdbqt format. The prepared ligands and receptors were docked using Autodock Vina so as to calculate binding affinity and observe the types of non-bonding interactions between the studied complexes.

Fig. 1
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3D structure of treated 1n9b

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Fig. 2
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3D structure of treated 1n9b with binding site

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Fig. 3
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3D structure of treated 2zd8

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Fig. 4
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3D structure of treated 2zd8 with binding site

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3 Result and discussion

The identities of the eleven isolated K. pneumoniae obtained were reconfirmed by conventional biochemical methods and the results show the isolates were non-motile, MR negative, VP positive, indole negative, glucose positive, lactose positive, and maltose positive.

The annotated result of the selected isolate for WGS analysis further confirmed the isolate to be K. pnuemoniae with sequencing ty** ST 163.

Antimicrobial resistance is a serious worldwide health concern that is growing at a rapid rate, the infections are linked to significant morbidity and mortality rates [21].

The isolates were analyzed by Kirby Bauer disk diffusion method and showed varying percentages of resistance to ciprofloxacin (73%), ceftazidime (100%), Augmentin (45%), Cefepime (73) and tetracycline (64%), but showed 91% sensitivity to gentamicin, and meropenem respectively. Figure 5 shows the percentage susceptibility of the isolates, while Fig. 6 shows the plate of zone of inhibition of bacterial growth and also area of no zone.

Fig. 5
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Percentage susceptibility of the isolates

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Fig. 6
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Antibiotic disk diffusion plate of Isolate P3

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3.1 Bioinformatics analysis

Phenotypically confirmed one resistant isolate to cefepime, ceftazidime, amoxicillin-clavulanic acid and intermediate resistant to meropenem was selected to detect the presence of blaTEM and blaSHV gene by whole genome sequencing.

Bioinformatics analysis of the isolate revealed the presence of tetracycline resistance gene (tetA), sulfonamide resistance gene (sul1), fluoroquinolone resistance gene (acrR), aminoglycoside-modifying enzyme (aadA), beta-lactam resistance gene (blaTEM215, a different variant of blaSHV, together with OMPK36 and OMPK37).

Bla genes belonging to SHV, CTX-M, OXA and TEM families are clinically relevant ESBL and K. pneumoniae positive strains harbored them most frequently. The K. pneumoniae strains that produce ESBLs pose a major threat to public health both locally and worldwide [22].

From the bioinformatics result, our isolate carries blaTEM and blaSHV genes which revealed it as an ESBL producer. This is in agreement with other researchers in Nigeria who have also reported the detection of ESBL genes from hospital wastewater [23, 24]. The phenotypic ampicillin resistance of our isolate was further confirmed by the detection of SHV-type beta-lactamase genes as revealed. According to Hennequin and Robin [25], these genes are chromosome-encoded which confers K. pneumoniae ampicillin resistance; the isolate also exhibited resistance to other beta-lactam antibiotics. This fact complies with the presence of the beta-lactamase genes blaSHV, and blaTEM215, five different SHV (40, 56, 85, 89, and 144) variants were seen in the isolate, OMPK 36 and 37 genes were also recovered. TEM and SHV β-lactamases are narrow-spectrum β-lactamases, which belong to group 2be in the Bush-Jacoby classification scheme [26, 27].

The emergence of class A β-lactamases, like TEM and SHV found in our isolates, has been linked to the inactivation of various β-lactam drugs that are effective in treating K. pneumoniae infections, particularly cephalosporin. This can result in treatment failure and prolonged hospital stays for patients [9].

The presence of these genes (TEM and SHV) may be due to the frequent use of penicillin and cephalosporin drugs which are beta-lactam antibiotics involved in treating infection caused by K. pneumoniae in the hospital [28]. The fact that the majority of hospital wastewater contains urine and feces of patients with partially metabolized drugs, and spilled drugs may also be a contributing factor in our isolate’s status as an antibiotic-resistant strain and ESBL producer. This wastewater can be washed down through hospital sinks to the drains and occasionally contains antibiotic residues, which are likely to contain pathogens and their resistance genes. As a result, hospital wastewater serves as an ideal environment for resistant bacteria and their genes [29], which when spread to the environment can pose the risk of antibiotic resistance that can lead to high morbidity and mortality.

The docking results from Table 1 show Amoxicillin with a binding score of − 6.45598841 kcal/mol bound with three amino acids (ASP, ARG & SER), Augmentin with a binding score of − 5.41116095 kcal/mol bound with four amino acids (ARG, ASN, SER & ALA), Cefepime has the least binding score of − 8.23996162 which made it the most effective drug against SHV organisms with three amino acids (VAL, ARG & LEU) bound to it during the docking process. Ceftazidime with a binding score of − 8.0068655 bound with five amino acids (ARG, LYS, SER, ALA & GLU) while meropenem with a binding score of − 6.82272816 kcal/mol bound only with one amino acid (ALA 237).

The docking results from Table 2 show the docking scores of the antibiotic with amino acids connected to the 2zd8 inhibitor and their amino acid residues as well as non-bonding interactions. The Table also shows that Cefepime has the lowest binding score of − 8.5358305 kcal/mol which makes it the most effective β-lactams antibiotics among others that were considered.

3.2 Docking analysis

Five β-lactams antibiotic drugs were used for the docking against two enzymes (1n9b and 2zd8). According to a report by [30], the lowest binding affinity value depicts the highest ability to inhibit the studied targets; thus, Augmentin proved to have the lowest inhibiting activity against class A beta-lactamase (PDB id: 1n9b) and SHV-1 class A beta-lactamase (PDB id: 2zd8) (Tables 1 and 2). The calculated binding affinity for cefepime exhibited stronger inhibition in terms of the ability to bind well against both the TEM and SHV enzymes. This was shown in the calculated binding affinity against 1n9b and 2zd8 (− 8.23996162 kcal/mol and − 8.5358305 kcal/mol) (Figs. 7 and 8). More so, the combinations of atoms that formed cefepime were observed to have enhanced its biological activity/inhibiting activities against the studied targets. In this work, cefepime which showed a superior performance in terms of binding activity proved to have potentials as a preferred choice of drug-like compound in clinical settings for treating infections caused by Klebsiella pneumoniae. The increasing order of the predicted binding affinity via calculated scoring against TEM and SHV enzymes was observed as: Augmentin < Amoxicillin < Meropenem < Ceftazidime < Cefepime.

Table 1 Calculated scoring, amino acid residue, and types of non-bonding interactions
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Table 2 Calculated scoring with 2zd8, amino acid residue, and types of non-bonding interactions
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Fig. 7
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Cefepime with amino acid residues in the active site of 1n9b

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Fig. 8
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Cefepime with amino acid residues in the active site of 2zd8

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4 Conclusion

This study shows that hospital wastewater represents a reservoir of class A β-lactamases. K. pneumoniae isolates from hospital wastewater samples were resistant to all commonly used antibiotics such as β-lactam. The environment is at risk if this wastewater spreads to humans and animals. Thus, it can be concluded that wastewater treatment and antibiotic stewardship in hospitals is necessary for controlling antibiotic resistance.

From the docking result, it is also concluded that cefepime has a strong binding affinity to the class A enzymes and so it can be effective in treating infection caused by K. pnuemoniae.