A common source of nosocomial infections in patients is the gram-negative bacterial pathogen P. aeruginosa (Reynolds and Kollef, 2021). It is estimated that microbial biofilms account for 60%–70% of hospital acquired illnesses. The pathophysiology of biofilm infection includes the immune response to biofilms, which causes collateral harm to nearby tissues (Moser et al., 2021). Dental caries, periodontitis, otitis media, chronic sinusitis, persistent wound alterations, musculoskeletal infections (osteomyelitis), biliary tract infections, bacterial prostatitis, native valve endocarditis, and infections connected to medical devices are among the infections linked to biofilms (Tuon et al., 2022). P. aeruginosa secretes virulent factors such as lipopolysaccharides and outer membrane proteins to adapt to harsh conditions. These factors help with host cell adhesion, tissue damage, and resistance to antibiotics (Qin et al., 2022). P. aeruginosa has intrinsic resistance mechanisms such as low outer membrane permeability, the presence of β-lactamases such as OXA-50, AmpC, and antibiotic efflux pumps (Langendonk et al., 2021).

Antibiotic resistance in P. aeruginosa has been linked to four primary sets of efflux pumps: MexAB-OprM, MexXY, MexCD-OprJ, and MexEF-OprN. One well-known and common intracellular mechanism of clinical aminoglycoside resistance in P. aeruginosa is the drug-inducible MexXY-OprM (Dey et al., 2020). MexA, MexD, MexE, and MexY gene overexpression has been linked to resistance to ceftazidime, imipenem, and ciprofloxacin (Abavisani et al., 2021). MexAB-OprM, the first multidrug efflux pump found in P. aeruginosa, is pointed out to be the main contributor to antibiotic resistance in the species. A rise in drug concentration near the pump causes MexB to change its shape, allowing it to eject active molecules toward the periplasmic tunnel and outer membrane that MexA and OprM have formed (Lorusso et al., 2022). Strong efflux pump expression MexAB-OprM plays a major role in P. aeruginosa’s resistance to carbapenems (Pan et al., 2016). A comprehensive phenotypic and genotypic approach has been developed to identify Mex-mediated efflux pumps, validated using reference strains, and assessed for clinical relevance (Mesaros et al., 2007). In order to help manage epidemics in crucial patient management areas, whole-genome sequencing can offer comprehensive information in a clinically appropriate time (Lauritsen et al., 2021). Antibiotic resistance genes including MexB, MexF, and MexY were found in resistant isolates of P. aeruginosa (Chettri et al., 2023). Higher homology between MdtB with MuxB and MdtC with MuxC is observed in the two-Resistance-nodulation-division (RND) subunit systems of E. coli and P. aeruginosa (Górecki and McEvoy, 2020). Single nucleotide polymorphisms in efflux pumps and porins have been linked in recent research to the emergence of MDR clones that pose a significant risk (Aguilar-Rodea et al., 2022). P. aeruginosa’s MexB gene study uses the Ramachandran plot to assess the structural stability and appropriate folding of the protein, which are essential for the protein’s role in antibiotic resistance (Sennhauser et al., 2009).

This study aimed to assess the burden of P. aeruginosa isolates in medical implants associated infections, their resistant status, abundance of MexB efflux pump genes, association between multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates with MexB genes. The nucleotide sequence of MexB genes was determined and analyzed evolutionary relationships, which is critical in epidemiology. The significance of MexB structural and functional domains in antibiotic resistance was shown by the structural and domain study of the protein in isolates. It gave important information to generate inhibitors to fight resistant P. aeruginosa infections and describes how manipulations in these domains can increase efflux activity, which contributes to MDR.

A total of 42 samples of P. aeruginosa were isolated from patients with medical implant associated infections. Samples were processed in microbiology laboratory, COMSATS University Islamabad using the standard operating procedures.

In the present study, the P. aeruginosa isolates from medical implants exhibited highest resistance against gentamycin, amikacin, and cefepime but lowest resistance against meropenem and imipenem. The most useful drug was polymyxin B followed by tigecycline. A study from 18 European countries, regarding antimicrobial resistance, showed that P. aeruginosa strains exhibited the highest degrees of resistance against ampicillin, gentamycin, ciprofloxacin, and amikacin, whereas imipenem, meropenem, colistin, and tigecycline were the most effective medications (Oliver et al., 2024). According to the World Health Organization (WHO), P. aeruginosa is a leading cause of antibiotic resistance and was listed as a “priority pathogen” by the WHO (Balakrishnan, 2022). Because these pathogens have shown resistance to certain antimicrobials, such as imipenem, meropenem, and third-generation cephalosporins—currently, the most effective medications for fighting MDR bacteria, they have presented a significant therapeutic challenge (Mulani et al., 2019). With similar findings in New Zealand and USA, the P. aeruginosa is found mostly in healthcare environments and plays a crucial part in causing nosocomial infections and resistant to diverse range of antibiotics (Moradali et al., 2017) (Reynolds and Kollef, 2021). Another study in Ethiopia concluded that P. aeruginosa pooled prevalence of antimicrobial resistance varies depending on the antibiotic: 20.9% for amikacin, 28.64% for meropenem, and 98.72% for ceftriaxone (Asmare et al., 2024). Likewise, a study conducted in Australia and concluded that P. aeruginosa resistant to a wide range of antibiotic classes, such as aminoglycosides (amikacin, gentamicin, and tobramycin), fluoroquinolones (FQs; ciprofloxacin, ofloxacin, and norfloxacin), carbapenems, and tetracyclines, its potential to develop MDR is a compelling aspect of concern (Avakh et al., 2023). Above recent studies were related to clinical isolates from hospital settings, supporting our study analysis that an emerging antimicrobial resistance in P. aeruginosa is a critical concern in healthcare settings.

In current study, the antimicrobial susceptibility profiling of P. aeruginosa revealed that 57% of isolates were MDR, 12% were XDR and 31% were sensitive. Whereas another study in Ethiopia revealed that the level of MDR was 45.9% and the XDR rate was 9.5% (Asamenew et al., 2023), this study was different in context as our study sampling source was infectious medical implants. Another study showed that 83% of P. aeruginosa isolates were MDR isolated from clinical samples (Mekonnen et al., 2021). Similar study results in Nigeria found that 12.8% of the isolates were MDR bacteria, and the majority of MDR strains that had high multiple antibiotic resistance indexes showed resistance to a variety of antibiotics, such as aminoglycosides, third- and fourth-generation cephalosporins, β-lactams, and FQs, which is constantly increasing (Adejobi et al., 2021). According to the INFORM database, a study in Spain found that P. aeruginosa that is XDR and MDR is a common and difficult nosocomial pathogen with consistently high rates that range from 9.0% to 11.2% and 11.5% to 24.7%, respectively (Recio et al., 2020).

The MexB gene, which encodes part of the MexAB-OprM efflux pump in Pseudomonas aeruginosa, is often considered more significant than its counterparts Mex-A and OprM because of its direct role in drug transport and efflux, contributing to antimicrobial resistance. That is why we only focused on Mex-B genes. According to the results of the study by Piddock (2006) and Tsutsumi et al. (2019), MexB is the active transporter in this tripartite complex, playing the most crucial role in the efflux of antibiotics. It directly binds and extrudes drugs from the bacterial cytoplasm and periplasm to the outside environment. MexA and OprM are structural components that aid the pump’s function, but, without MexB, the efflux pump cannot transport drugs, making MexB the critical efflux driver. In our study, MexB gene was identified in 69% resistant isolates and also found that it was a major factor in antibiotic resistance, with similar findings by studies in China, Switzerland, and France that AB-OM porin M (MexAB-OprM) and MexXY-OprM are multidrug efflux proteins that have been thoroughly investigated for their significant contributions to MDR. Moreover, these efflux pumps have unique resistance mechanisms that could result in the formation of XDR or PDR phenotypes and highly resistant strains (Qin et al., 2022; Dreier and Ruggerone, 2015; Compagne et al., 2023). Another research finding in Nepal concluded that 94.4% of P. aeruginosa isolates were harboring MexB genes, but their association with antimicrobial resistance was not analyzed (Sharma et al., 2023).

In current study, we have confirmed the presence of MexB gene efflux pump by CCCP assay; likewise, the previous study by Ahmed et al. (2021) examined the efflux pump expression under the heading of “Determination of Efflux Pumps Expression in Resistant Isolates”: The MICs of ciprofloxacin, meropenem, amikacin, and ceftriaxone were detected for 25 MDR P. aeruginosa isolates by agar dilution method in the presence and absence of efflux pump inhibitor CCCP (Sigma, San Jose, CA, USA) at a final concentration of 10 μM. A four-fold reduction in MIC or more of the tested antibiotics after adding CCCP is an indication for the presence of efflux pumps. The role of efflux pump MexB genes in antimicrobial resistance was confirmed by adding CCCP with antibiotics, because there was a significant reduction observed in MIC following the addition of CCCP reagents with antibiotics. Likewise, the results in France on multiple gram-negative organisms show that MIC was evaluated with and without CCCP and a two-fold decrease of colistin MIC was calculated for each strain. There was a significant decrease observed in MIC after addition of CCCP (Baron and Rolain, 2018). Recently, similar results were examined in Iran: the expression of the Mex efflux pump in 122 clinical isolates of P. aeruginosa. This study found a substantial association between MexB expression and resistance to antipseudomonal drugs, with MexB (69%) expression emerging as the second most active efflux pump (Rahbar et al., 2021). A research study in China revealed a significant decrease in MIC, following the CCCP addition against colistin-resistant bacteria (Ding et al., 2023). The study results of the abovementioned studies showed similarities with our results in the context that MDR-to-XDR ratio in clinical isolates is increasing and that MexB had a strong relationship with antimicrobial resistance, but there is a limitation that no one analyzes the molecular and structural analysis of their clinical isolates.

In this study, partial sequenced data were submitted to GenBank, and accession number was assigned against each submitted MDR and XDR isolates of P. aeruginosa. The phylogenetic analysis was performed to find the similarities with isolates having a strong efflux pump system, which showed that 100% similarities were found with strains isolated from Switzerland, Denmark, and Germany, whereas a research study done in India performed phylogenetic tree analysis of MexB genes and also found similarities with some Asian isolates, but they did not process these samples for further structural analysis (Chettri et al., 2023). With similar study results in Japan and Belgium, the phylogenetic analysis showed that similar isolates having RND multidrug efflux pumps exhibited critical role in the resistance of gram-negative organisms. The RND efflux pumps are the most important of the several efflux pump families found in P. aeruginosa that are connected to MDR. RND pumps’ distinct structure and function make them essential for protecting against antibiotics (Yamasaki et al., 2023; Avrain et al., 2013).

In our study, the Ramachandran plot showed values in favored regions. More than 98% of residues in favored regions correspond to high-quality models, ideally. The findings of Ramachandran outliers revealed excellent structural quality, as indicated by the 0% outliers in all analyzed samples. In another study done in Malaysia, the subunit of efflux pump, Amr-B protein, indicated that the predicted model was of high quality and had appropriate backbone geometry. Amino acid T was found in the favored residue region of the Ramachandran plot, which corresponds to no steric clashes between the side chain atoms and main chain atoms (Hussin et al., 2024). Another similar study results in India on Ramachandran plot analysis program for 3D structural analysis of isolated protein models used and analyzed that 97.5% amino acids fall in the favored region and 2.5% in the allowed region, and none in the outlier region suggested an excellent model (Swain and Padhy, 2016). These study results supported our analysis scheme in a way that, for structural analysis, Ramachandran plot was the best tool to identify and interpret the best protein models.

In our study, multiple sequence alignment of protein sequences using Clustal Omega indicated conserved regions and sequence variations in partial sequence MexB genes. In the previous study, Clustal Omega was used by Memili et al. (2022), the resistant strain sequences were aligned with Clustal Omega Multiple Sequence Alignment program, and percent identity matrices were derived. Phylogenetic analyses to elucidate evolutionary relationships between sequences were conducted with the neighbor–end joining method through the Molecular Evolutionary Genetics Analysis (MEGAX) software, developed by the Institute of Molecular Evolutionary Genetics at Pennsylvania State University. Subsequent network analyses of the resistance gene, vanB, within E. faecium were derived from ScanProsite and InterPro. Conserved regions that could be involved in substrate binding and the antibiotics’ translocation from the bacterial cell and sequence variations are indicative of evolutionary divergence, adaptation to different environments, and exposure to various antibiotics (Sanz-García et al., 2018). A study done in Spain evaluated conserved regions and sequence variations in P. aeruginosa isolates but did not evaluated the MolProbity score and Ramachandran plot analysis to give insight about structural models of isolated samples (Mosquera-Rendón et al., 2016). In our study, the translated protein sequences were analyzed by InterPro, which verified that they belonged to homologous and well-established multidrug efflux transporters families, with similar findings by the study results in Iran that concluded that protein domain analysis of efflux pump genes in gram-negative bacteria showed a relationship to homologous RND families and highly conserved domains of RND MDR gram-negative bacteria (Abadi et al., 2018), yet, again, the isolation sources of samples were other than medical implants. In abovementioned studies, researchers did not highlight the details of 3D protein models of clinical isolates for target drug delivery.

The current study provides comprehensive information and significant understanding on occurrence MDR and XDR patterns of P. aeruginosa and role of MexB genes in resistance profiling. Phylogenetic trees of the MexB gene of resistant bacterial species showed clusters built on the basis of common ancestry. 3D structural modeling of protein sequences, Ramachandran plot analysis, and protein domain analysis showed excellent protein models of MexB genes that could be useful as potential therapeutic targets in the future medicines to predict drug design models. Sequence variations were indicative of evolutionary divergence, adaptation to different environments, and exposure to various antibiotics. Highly conserved regions were found in the multiple sequence alignment analysis that highly conserved regions could be used for the diagnostic purposes, and design primers for these domains to identify MexB genes in P. aeruginosa and eliminating or manipulating these highly conserved regions by different genetic-based methods can deactivate multidrug efflux pumps in P. aeruginosa.