Molecular and Phylogenetic Characterization of Carbapenemase-Producing Gram- Negative Bacteria from VAP Patients: With Structural Insights into OXA-48

Molecular and Phylogenetic Characterization of Carbapenemase-Producing Gram- Negative Bacteria from VAP Patients: With Structural Insights into OXA-48 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular and Phylogenetic Characterization of Carbapenemase-Producing Gram- Negative Bacteria from VAP Patients: With Structural Insights into OXA-48 Zeb Hussain, Asma Naim, Ambreen Fatima, Asad Karim, Muhammad Jahanzeb This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6973343/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Carbapenemase-producing Gram-negative bacteria are emerging as a significant threat to critically ill patients, particularly those with ventilator-associated pneumonia (VAP). The increasing resistance to last-resort antimicrobials—including novel β-lactam/β-lactamase inhibitor combinations and cefiderocol—warrants urgent molecular and epidemiological investigations. Objective: To investigate the antimicrobial resistance patterns, gene prevalence, and clonal relationships of Acinetobacter baumannii , Klebsiella pneumoniae , and Elizabethkingia spp. isolated from VAP patients, with a focus on resistance to cefiderocol, imipenem/Relebactam, Meropenem/vaborbactam, and Ceftazidime/avibactam, and the presence of key Carbapenemase genes (OXA-48, NDM, VIM, and IMP). Methods: A total of 67 carbapenem-resistant isolates were obtained from 104 tracheal aspirate samples collected from ICU VAP patients between January 2022 and December 2023. Antimicrobial susceptibility testing was performed using broth microdilution and MIC strips. PCR and Sanger sequencing were used for genotypic detection of Carbapenemase genes. Phylogenetic analysis was conducted via maximum-likelihood trees, and structural modeling of OXA-48 enzymes was performed using SWISS-MODEL and PyMOL. Results: All A. baumannii isolates (n = 10) exhibited high-level resistance to cefiderocol and β-lactam/β-lactamase inhibitor combinations. PCR revealed blaOXA-48 in 3 isolates, blaNDM in 1, and blaVIM in 1, and co-carriage of blaOXA-48 + blaIMP in another. K. pneumoniae isolates (n = 7) showed partial susceptibility to Ceftazidime/avibactam but complete resistance to cefiderocol; blaOXA-48 and blaNDM were the predominant genes. Elizabethkingia spp. (n = 12) showed universal resistance to all tested antibiotics and harbored blaOXA-48 (100%) and blaNDM (33%). Phylogenetic analysis revealed global clonal relationships and interspecies gene transfer, with zoonotic and environmental links. Structural modeling confirmed functional conservation of OXA-48 active sites across isolates. Conclusion: This study highlights the extensive spread of Carbapenemase genes across clinical and environmental strains of Gram-negative bacteria, with a worrying level of resistance to novel antibiotics. Our findings underscore the urgent need for genomic surveillance, rational antimicrobial use, and One Health-based infection control strategies to combat this escalating resistance crisis. Carbapenemase OXA-48 NDM VIM IMP Acinetobacter baumannii Klebsiella pneumoniae Elizabethkingia cefiderocol resistance β-lactamase inhibitors phylogenetic VAP Pakistan Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Pulmonary infections are among the most prevalent infectious diseases, ranking as the fourth leading cause of death globally in 2019 and the second leading cause in low-income countries[ 1 ]. Enterobacteriaceae are a diverse group of Gram-negative bacteria commonly present in the intestines of humans, birds, animals, and the environment. These bacteria can lead to various infections, such as sepsis, pneumonia, and urinary tract infections [ 2 ]. In recent years, the prevalence of multidrug-resistant Enterobacteriaceae strains has been increasing [ 3 ]. Infections caused by Elizabethkingia spp. are on the rise in several countries, with numerous outbreaks being reported [ 4 ]. A study conducted in South Korea reported an increase in the infection rate of Elizabethkingia spp. among inpatients at Severance Hospital in Seoul, rising from 0.002% in 2009 to 0.088% in 2017 [ 5 ]. Similarly, a study in Taiwan revealed that Elizabethkingia spp. had the second-highest infection rate among carbapenem-resistant, non-fermenting Gram-negative bacilli, surpassed only by Acinetobacter baumannii [ 6 ]. The emergence and clonal dissemination of carbapenem-resistant Acinetobacter baumannii and Klebsiella pneumoniae represent one of the most formidable challenges in contemporary infectious disease management.[ 7 ] These pathogens have become synonymous with hospital-acquired infections, especially in critical care units settings like intensive care units (ICUs), where they significantly contribute to morbidity and mortality[ 8 ]. A common complication among ventilator-associated pneumonia (VAP) is frequently complicated by infections caused by these multidrug-resistant (MDR) organisms [ 9 ]. The growing incidence of carbapenem-resistant Klebsiella pneumoniae and Acinetobacter baumannii in VAP cases has underscored the urgent need to understand the mechanisms driving resistance and to develop effective strategies to combat these infections.[ 10 ] Carbapenem resistance in these pathogens is primarily driven by Carbapenemase production enzymes that break down carbapenems and other β-lactam antibiotics, making them ineffective [ 11 ]. The most clinically significant carbapenemases include OXA-48, VIM, NDM, and IMP, which have been increasingly detected in both Klebsiella pneumoniae and Acinetobacter baumannii isolates. These enzymes not only confer high-level resistance to carbapenems but also often carry additional resistance mechanisms that limit the efficacy of alternative antibiotics, complicating treatment options [ 12 ]. The rapid and widespread dissemination of Carbapenemase genes is facilitated by mobile genetic elements such as plasmids, transposons, and integrons, which enable horizontal gene transfer between bacteria. This genetic mobility has led to the rapid clonal spread of resistant strains across different geographic regions and healthcare settings, making infection control efforts particularly challenging. The clonal nature of these outbreaks often involves specific high-risk clones that are adept at colonizing and persisting in hospital environments, further exacerbating the problem [ 13 ]. To address this escalating threat, different Novel β-lactam/β-lactamase inhibitor combinations (βL-βLICs) have been introduced. These include agents such as Meropenem/vaborbactam, Ceftazidime/Avibactam and Imipenem/Relebactam, which were specifically designed to inhibit Carbapenemase activity or evade the resistance mechanisms employed by these MDR pathogens [ 14 ]. Additionally, Cefiderocol, a novel siderophore cephalosporin, has been introduced as a therapeutic an alternative for infection treatment caused by Carbapenemase producing Gram-negative pathogens. Cefiderocol’s unique mechanism of action, which involves the use of iron transport systems to gain entry into bacterial cells, provides an innovative approach to overcoming resistance [ 15 ]. Despite the promising efficacy of these new antimicrobials, there have been increasing reports of resistance emerging even to these agents. The detection of resistance to cefiderocol and novel βL-βLICs in Klebsiella pneumoniae and Acinetobacter baumannii strains, particularly those producing OXA-48, VIM, NDM, and IMP carbapenemases, raises significant concerns [ 16 ]. These developments suggest that the evolutionary pressure exerted by the widespread use of these novel agents is driving the selection of even more resistant bacterial populations. The clinical implications of this resistance are profound, as treatment options for infections caused by these resistant strains are extremely limited. The management of patients with infections caused by these pathogens often requires the use of combination therapy or reliance on less effective, more toxic antibiotics, which can lead to suboptimal outcomes [ 17 ]. Furthermore, the infection control challenges posed by these organisms are substantial, requiring stringent infection prevention measures to limit their spread within healthcare facilities [ 18 ]. In this study, we investigate the clonal outbreak of Carbapenemase-producing Elizabethkingia spp. Klebsiella pneumoniae and Acinetobacter baumannii strains isolated from VAP patients over a one-year period spanning from January 2022 to December 2023. To the best of our knowledge, we are reported the Elizabethkingia spp. isolated from Karachi, Pakistan. Our focus is on characterizing the resistance of these strains to cefiderocol and the novel βL-βLICs, with particular attention to the role of OXA-48, VIM, NDM, and IMP carbapenemases in mediating this resistance. By conducting comprehensive antimicrobial susceptibility testing and genetic analysis, we aim to uncover the underlying mechanisms of resistance and the factors contributing to the persistence and spread of these high-risk clones. The outcomes of this research are expected to provide critical insights into the epidemiology of these resistant pathogens, offering valuable information for the development of more effective therapeutic strategies and infection control policies. Understanding the genetic and phenotypic characteristics of these strains will be key to mitigating the impact of these MDR organisms and addressing the ongoing challenge of antibiotic resistance in healthcare settings. Materials and method Study setting This cross-sectional study was conducted from January 2022 to January 2023 in the Microbiology department at the University of Karachi, in collaboration with the Microbiology section at Dow Diagnostic Research Laboratory and the Jamil-ur-Rahman Center for Genome Research, Dr. Panjwani Center for Molecular Medicine and Drug Research, and the International Center for Chemical and Biological Sciences at the University of Karachi. Ethical approval for the study was obtained from the Institutional Review Board of Dow University of Health Sciences (DUHS), Karachi (Approval number 5.160/18/12/2019). Bacterial identification : During the study we collected endotracheal aspirates (ETAs) from intubated patients under strict aseptic conditions. All criteria were followed according to international protocols [ 19 ]. Endotracheal samples were obtained from the ICU of Dow University Hospital. Total of 183 strains were obtained from the tracheal aspirates of VAP patients using MacConkey, blood, and chocolate agar media (Oxoid-UK). The initial identification of these isolates was performed using traditional methods, such as Gram staining, growth characteristics, and biochemical tests [ 20 ]. The API 20E system (bioMérieux SA, Marcy-l'Étoile, France) was utilized for further identification. Antimicrobial susceptibility testing was then conducted following CLSI guidelines (2021). ATCC 25922 (E. coli ) and ATCC 27853 ( Pseudomonas aeruginosa ) served as quality control strains for the testing. Molecular Analysis of Resistance Mechanisms : Isolated strains were investigated for the presence of common genotypic MBLs variants, including blaVIM, blaOXA-48, and blaIMP. BlaKPC, and blaNDM. DNA was extracted using the QIAamp® DNA Mini Kit method. PCR conditions included an initial denaturation at 94°C for 4 minutes, followed by 35 cycles consisting of denaturation at 94°C for 1 minute, annealing at primer-specific temperatures for 30 seconds, and extension at 72°C for 1 minute, concluding with a final extension at 72°C for 10 minutes. The PCR products were separated using 2% agarose gel electrophoresis. Primers used in study are mentioned in Table 1 . Sanger sequencing of amplicon : Selected amplicons representative of each Carbapenemase gene were purified using the QIAquick PCR Purification Kit (QIAGEN, Germany). Purified DNA samples were then sent to Macrogen Inc. (Seoul, South Korea) for bidirectional Sanger sequencing. Both forward and reverse primers used in the initial PCR were employed for bidirectional sequencing. Chromatograms were analyzed using Chromas Lite v2.6.6 and BioEdit software. Consensus sequences were aligned and compared to reference gene sequences in the NCBI GenBank database using BLASTn to confirm the identity and variant types of Carbapenemase genes. Phylogenetic Analysis : For each Carbapenemase gene detected, our Sanger‑derived sequences were aligned with representative reference sequences retrieved from NCBI GenBank using MUSCLE in MEGA 11. Maximum‑likelihood phylogenetic trees were constructed under the Tamura‑Nei model with 1,000 bootstrap replicates. Trees were visualized in FigTree v1.4.4. Clusters were interpreted based on bootstrap support ≥ 70%, allowing inference of clonal relationships and horizontal gene transfer among clinical, environmental, and veterinary isolates. Structural Modeling of Carbapenemase Variants : Structural Modelling and Comparative Analysis Amino acid sequences corresponding to Carbapenemase enzymes were obtained from GenBank. Structural modelling of the OXA-48-like β-lactamase was conducted using SWISS-MODEL, selecting the crystal structure 3HBR_A from the RCSB Protein Data Bank as a template (resolution 1.91 Å, identity 97.21%). The resulting model (referred to as "model.pdb") underwent rigorous quality assessment using GMQE, QMEANDisCo (global score: 0.89 ± 0.05), and predicted lDDT (0.91). Additional validation included Ramachandran plot analysis in PROCHECK and clash score evaluation. The reference crystal structure 3HBR (native OXA-48) was used as a benchmark. Both structures were aligned using TM-align and superposed in PyMOL to assess topological congruence and RMSD values. Binding sites were mapped by aligning conserved active site motifs (S70, K73, S118, W157, Y211), and ligand binding residues were annotated through the SWISS-MODEL ligand pipeline. Hydrogen bonding and hydrophobic interactions with known β-lactam antibiotics (Ceftazidime, imipenem) were further explored using LigPlot+. Results Antimicrobial Susceptibility Profiles : Acinetobacter baumannii isolates (n = 10) demonstrated near-uniform resistance to novel β-lactam/β-lactamase inhibitors and cefiderocol. Ceftazidime-avibactam (CZA) minimum inhibitory concentrations (MICs) ranged from 4–16 mg/L, with 80% (8/10) exceeding the resistance breakpoint (> 8 mg/L). Notably, Acinetobacter baumannii strain AB-54 exhibited susceptibility (MIC = 4 mg/L), while Acinetobacter baumannii strain AB-70 showed intermediate resistance (MIC = 8 mg/L). All isolates were resistant to Imipenem-Relebactam (IMR; MIC = 8 mg/L), Meropenem (MIC = 16 mg/L), Cefiderocol (MIC = 16 mg/L), and Meropenem-vaborbactam (MIC = 16 mg/L). Eravacycline susceptibility varied: three isolates (AB-19, AB-21, AB-56) remained susceptible (MIC = 0.25 mg/L), while seven exhibited resistances (MIC = 0.5 mg/L). Molecular characterization revealed Carbapenemase gene heterogeneity: Acinetobacter baumannii AB-04 harbored bla VIM, Acinetobacter baumannii AB-21 and Acinetobacter baumannii AB-56 carried bla OXA-48, AB-57 possessed bla NDM, and AB-40 co-harbored bla OXA-48 and bla IMP (Table 2 ) Table 1 Primer sequences, target genes, and expected amplicon sizes used for PCR detection of carbapenemases genes in Gram-negative bacterial isolates. 1 Primer (5′–3′) Gene Product Size (bp) Reference IMP-F: GGAATAGAGTGGCTTAAYTCTC IMP-R: GGTTTAAYAAAACAACCACC blaIMP 232 Poirel et al., 2011. J Antimicrob Chemother 66(1): 57–63. [PMID: 21098058] VIM-F: GATGGTGTTTGGTCGCATA VIM-R: CGAATGCGCAGCACCAG blaVIM 390 Ellington et al., 2007. J Antimicrob Chemother 59(3): 434–439. [PMID: 17289721] OXA-F: GCGTGGTTAAGGATGAACAC OXA-R: CATCAAGTTCAACCCAACCG blaOXA-48 438 Poirel et al., 2011. Antimicrob Agents Chemother 55(10): 4896–4899. [PMID: 21746950] NDM-F: GGTTTGGCGATCTGGTTTTC NDM-R: CGGAATGGCTCATCACGATC blaNDM 621 Poirel et al., 2011. Antimicrob Agents Chemother 55(11): 4910–4913. [PMID: 21876065] KPC-Fm: CGTCTAGTTCTGCTGTCTTG KPC-Rm: CTTGTCATCCTTGTTAGGCG blaKPC 798 Cuzon et al., 2010. Antimicrob Agents Chemother 54(1): 307–308. [PMID: 19884378] Note : 1 This table summarizes the primer sequences used for the amplification of five Carbapenemase-encoding genes (blaIMP, blaVIM, blaOXA-48, blaNDM, and blaKPC) by PCR. The corresponding amplicon sizes and references for the primer sequences are also listed. Oligonucleotides used in this study. a F, sense primer; R, antisense primer. b Nucleotide numbering begins at the initiation codon of genes. c D = A or G or T; Y = C or T. Table 2 Show betalactamse inhibitors resistance patterns of different species of Acinetobacter Pathogenic strains . 2 CZA S(-) R(-) S(≤ 8) R(> 8 IMR S(≤ 2) R(> 2 MERO S(≤ 2) R(> 8) MEV S(≤ 8) R(> 8) FDC S(≤ 8)R(> 8) ERV S(≤ 0.25)R(> 0.25) VIM NDM KPC OX-48 IMP Strains Ab-04 16 8 16 16 16 0.5 + - - - - Ab-19 16 8 16 16 16 0.25 - - - - - Ab-20 16 8 16 16 16 0.5 - - - - - Ab-21 16 8 16 16 16 0.25 - - - + - Ab-32 16 8 16 16 16 0.5 - - - - - Ab-40 16 8 16 16 16 0.5 - - - - + Ab-50 16 8 16 16 16 0.5 - - - - - Ab-54 4 8 16 16 16 0.5 - - - - - Ab-56 16 8 16 16 16 0.25 - - - + - Ab-57 16 8 16 16 16 0.5 - + - - - Ab-70 8 8 16 16 16 0.5 - - - - - Note : 2 This table presents the minimum inhibitory concentrations (MICs, in µg/mL) of various antibiotics tested against Acinetobacter baumannii strains. The antibiotics include Ceftazidime-avibactam (CZA), imipenem/Relebactam (IMR), Meropenem (MERO), Meropenem/vaborbactam (MEV), cefiderocol (FDC), and Eravacycline (ERV). Interpretive breakpoints for susceptibility (S) and resistance (R) are indicated for each drug. The presence (+) or absence (–) of Carbapenemase genes (VIM, NDM, KPC, OXA-48, and IMP) was determined by PCR . Among Klebsiella pneumoniae isolates (n = 7), resistance patterns diverged significantly. Two strains (KP-37, KP-92) exhibited multidrug resistance, including resistance to CZA (MIC = 16 mg/L) and IMR (MIC = 8 mg/L). Conversely, KP-4 retained susceptibility to CZA (MIC = 0.25 mg/L), IMR (MIC = 0.5 mg/L), Meropenem (MIC = 1 mg/L), and Meropenem-vaborbactam (MIC = 1 mg/L). All isolates were resistant to cefiderocol (MIC = 8 mg/L), exceeding the susceptibility threshold (≤ 2 mg/L). Eravacycline susceptibility was observed in KP-4 and KP-81 (MIC = 0.25 mg/L), while other strains demonstrated resistance (MIC = 0.5 mg/L). Genotypic analysis identified bla NDM in KP-4 and bla OXA-48 in KP-81, KP-85, and KP-92; no KPC, VIM, or IMP genes were detected (Table 3 ). Table 3 Show beta-lactamase inhibitors resistance patterns of different species of klebseilla Pathogenic strains . 3 CZA S(≤ 8)R(> 8 IMR S(≤ 2)R(> 2 MERO S(≤ 2)R(> 8) MEV S(≤ 8)R(> 8) FDC S(≤ 2) R(> 2) CZA S(≤ 8)R(> 8) ERV S(≤ 0.25)R(> 0.25) VIM NDM KPC OXA-48 IMP Strains Kp-4 0.25 0.5 1 1 8 0.25 - 0.5 - - - - - Kp-81 0.25 4 16 16 8 0.25 - 0.5 - - - + - Kp-85 0.5 4 16 16 8 0.5 - 0.5 - - - + - Kp-27 0.5 4 16 16 8 0.5 - 0.5 - - - - - Kp-35 0.5 4 16 16 8 0.5 - 0.5 - - - - - Kp-37 16 8 16 16 8 16 0.5 - - - - - Kp-92 16 8 16 16 8 16 0.5 - - - + - Ek-83 16 8 16 16 8 16 0.5 - - - + - Ek-61 16 8 16 16 8 16 0.5 - + - + - Note : 3 The table displays the minimum inhibitory concentrations (MICs, in µg/mL) of selected antibiotics tested against Klebsiella pneumoniae (KP) and Elizabethkingea spp (Ek) isolates. Antibiotics include Ceftazidime-avibactam (CZA), imipenem/Relebactam (IMR), Meropenem (MERO), Meropenem/vaborbactam (MEV), cefiderocol (FDC), and Eravacycline (ERV). Interpretive breakpoints for susceptibility (S) and resistance (R) are shown for each drug. PCR results for Carbapenemase genes (VIM, NDM, KPC, OXA-48, and IMP) are indicated by "+" (positive) or "–" (negative). Carbapenemase Gene Distribution : PCR amplification confirmed the coexistence of metallo-β-lactamases ( bla VIM, bla NDM, bla IMP) and Oxacillinases ( bla OXA-48) across A. baumannii and K. pneumoniae isolates. In A. baumannii , bla OXA-48 predominated (3/10 isolates), followed by bla NDM (1/10) and bla VIM (1/10), with one strain co-harboring bla OXA-48 and bla IMP. K. pneumoniae strains exclusively carried bla OXA-48 (3/7) or bla NDM (1/7), with no co-occurrence of multiple Carbapenemase genes. These findings correlate with observed resistance profiles, particularly the ineffectiveness of Avibactam- and Vaborbactam-based combinations against OXA-48 and metallo-β-lactamase producers. The persistence of Cefiderocol resistance across all isolates, despite iron-depleted testing conditions, underscores emerging challenges in managing infections caused by Carbapenemase-producing Enterobacterales (CPE) with limited therapeutic options. Phylogenetic Analysis of Acinetobacter baumannii and Klebsiella pneumonia : A circular phylogenetic tree (radial dendrogram) was constructed to illustrate the evolutionary relationships among genomic regions from multiple strains or isolates. The tree was build based on sequence similarity and divergence, with branch lengths indicating genetic distances. The hierarchical structure of the tree demonstrates the phylogenetic clustering of these sequences, revealing potential genetic conservation, divergence, or horizontal gene transfer events. This analysis provides insights into the genetic relatedness of the studied regions, contributing to our understanding of genomic evolution and potential functional conservation among different bacterial strains. The phylogenetic analysis of Acinetobacter baumannii , Klebsiella pneumoniae and Elizabethkingia spp. provided significant insights into their genetic relationships and the widespread dissemination of antimicrobial resistance (AMR) genes. The clustering patterns revealed that genetically similar strains are found across diverse geographic regions, suggesting a major role of horizontal gene transfer (HGT), clonal expansion, and global patient movement in spreading resistant strains. The presence of conserved resistance determinants across multiple continents indicates the rapid evolution and adaptation of these pathogens in clinical and environmental settings. Strain AB-4 of A. baumannii clustered with CP023022.1 (bronchial fluid, Mexico), CP087370.1 (Germany), and CP066016.1 (USA), demonstrating that despite vast geographic distances, these strains share common resistance traits and molecular characteristics, likely due to the circulation of mobile genetic elements (Fig. 1 ). Similarly, strain AB-40 exhibited strong genetic relatedness to A. baumannii CP142667 (South Korea) and CP091339 (Belgium), with conserved resistance-associated loci, underscoring the strain’s adaptability in clinical settings and potential transmission through healthcare-associated pathways (Fig. 2 ). The presence of highly similar resistant strains across different countries highlights the interconnected nature of bacterial evolution and antibiotic resistance spread. Further analysis of A. baumannii strains revealed evidence of cross-species resistance gene dissemination. Notably, strain AB-21, an OXA-48-positive A. baumannii isolate, phylogenetically clustered with K. pneumoniae (MF741882.1, Kenya; OM368082.1, India) and E. coli (CP023924.1, Canada), suggesting the involvement of plasmid-mediated HGT in the global transmission of carbapenem resistance (Fig. 3 ). The genetic similarities between A. baumannii , K. pneumoniae , and E. coli indicate the possibility of interspecies gene exchange in healthcare environments, where co-colonization and plasmid transfer events could contribute to the rapid spread of AMR genes. Additionally, strain AB-57 demonstrated a zoonotic transmission link, clustering with E. coli KU318691.1 (leopard, India) and K. pneumoniae MK628734.1 (duck, China), further emphasizing the role of animal reservoirs in the dissemination of antimicrobial resistance (Fig. 4 ). These findings underscore the importance of One Health surveillance strategies that integrate human, animal, and environmental data to combat the rising threat of multidrug-resistant pathogens. Phylogenetic clustering of K. pneumoniae strains reinforced the notion of widespread carbapenem resistance dissemination. Strain KP-81 was closely related to E. coli KY817191.1 (India) and K. pneumoniae OM368082.1 (India), both harboring blaOXA-48 and blaOXA-181 genes, illustrating cross-host and cross-continental Carbapenemase resistance transmission (Fig. 5 ). This suggests that plasmid-mediated resistance is not confined to a single species but can spread dynamically between different bacterial populations. Strain KP-85 phylogenetically aligned with K. pneumoniae CP034284 (genomic DNA, blaOXA-181 plasmid) and PP320277 (Canada), further supporting the role of plasmid-driven HGT in carbapenem resistance and its potential for long-distance dissemination through global travel and healthcare networks (Fig. 6 ). Similarly, strain KP-92 clustered with K. pneumoniae MF741882.1 (Kenya), E. coli MF741884.1 (Kenya), and K. pneumoniae CP068846.1 (Netherlands), all carrying blaOXA-48, reinforcing the widespread dissemination of this resistance determinant across multiple regions (Fig. 7 ). These results provide strong evidence of the rapid movement of carbapenemase genes across bacterial species and continents, making them a major concern for global public health. Unexpectedly, the phylogenetic analysis of Elizabethkingia spp. revealed novel insights into resistance gene dissemination beyond the Enterobacteriaceae family. Strain EK-61, an OXA-48-positive Elizabethkingia isolate, clustered with K. pneumoniae CP068856.1 (Netherlands) and CP079128.1 (India), suggesting that OXA-48 resistance is not restricted to clinically relevant Enterobacteriaceae but has also spread to environmental non-fermenters (Fig. 8 ). The presence of such resistance determinants in Elizabethkingia spp. raises concerns regarding potential reservoirs of antibiotic resistance in environmental settings, which could pose challenges for infection control and treatment strategies. Furthermore, another Elizabethkingia strain exhibited strong genetic ties to E. coli KY801334.1 (India), AP018837.1 (Vietnam), and K. pneumoniae MT635909.1 (Vietnam), all carrying blaOXA-48 or blaOXA-181, providing further evidence of plasmid-mediated HGT across bacterial species and geographic locations (Fig. 9 ). These findings highlight the complex and interconnected nature of resistance gene mobility, emphasizing the necessity of global genomic surveillance to track the spread of AMR determinants and mitigate their impact on public health. (Fig. 9 ) Conservation Analysis of OXA β-lactamase Genes : A multiple sequence alignment (MSA) was performed on OXA β-lactamase gene sequences derived from diverse bacterial isolates using ClustalW in BioEdit v7.2.5. The alignment revealed conserved regions interspersed with isolate-specific nucleotide and amino acid variations. Highly conserved nucleotide motifs were observed across all sequences, notably within the central and 5′ coding regions. For instance, a conserved stretch encoding the motif GATATCGCCGCTTGG was present in all aligned sequences with minimal to no variation. Similarly, conserved di- and tri-nucleotide repeats, such as CGT, GCG, and TGG, were consistently maintained, suggesting evolutionary pressure to retain structural and functional integrity of these regions (Fig. 10 . ) Translational analysis of the aligned coding sequences revealed that the majority of isolates maintained an intact open reading frame, with start and stop codons appropriately positioned. The deduced amino acid sequences exhibited conservation in residues known to form the active site of class D β-lactamases, including conserved serine-lysine motifs and the carboxyl-terminal residues critical for β-lactam ring hydrolysis. Residues such as S70, K73, and W157—previously implicated in catalytic activity—were preserved across nearly all sequences. Variable regions were observed predominantly in the flanking regions and loop-encoding segments. Several isolates showed amino acid substitutions, insertions, or deletions, including frequent substitutions at positions corresponding to hydrophobic-to-polar residue changes. Additionally, a subset of sequences contained degenerate nucleotide positions (e.g., R, Y, K), indicating either intra-population heterogeneity or unresolved base calls during sequencing. Importantly, no premature stop codons were observed within the core coding region of the OXA genes, supporting the conclusion that these sequences are likely functional and encode active enzymes. The absence of frameshift mutations further corroborates the functional conservation of these β-lactamase variants. Structural Insights from Homology ModellingThe OXA-48-like model developed using SWISS-MODEL based on template 3HBR demonstrated robust structural fidelity. Quality parameters included a GMQE score of 0.88 and a QMEANDisCo score of 0.89 ± 0.05. Ramachandran plot analysis indicated that 97.4% of residues fell within favored regions, and the predicted lDDT value of 0.91 confirmed high confidence in the accuracy of side-chain placements and backbone topology Fig. 11 . When superposed with the crystallographic structure of 3HBR, the homology model exhibited an RMSD of less than 0.3 Å, indicating negligible deviation. The conserved catalytic residues (Ser70, Lys73, Ser118, Trp157, and Tyr211) were precisely aligned in both structures, validating functional integrity of the active site. LigPlot + interaction analysis of Ceftazidime docked in the model revealed strong hydrogen bonding interactions, particularly with Ser70 and neighboring polar residues, reinforcing the hydrolytic competence of the enzyme. Furthermore, the active site cavity was structurally congruent with that of the reference enzyme, supporting substrate accommodation and suggesting preserved catalytic potential. This validated homology model not only supports the inferred multidrug resistance phenotype observed in clinical isolates but also serves as a high-confidence platform for future structure-based inhibitor design targeting class D β-lactamases. Discussion The antimicrobial susceptibility testing of Acinetobacter baumannii isolates in this study revealed an alarming resistance profile against multiple last-resort antibiotics. All isolates demonstrated high-level resistance to Ceftazidime-avibactam (CZA), Imipenem/Relebactam (IMR), Meropenem (MEM), Meropenem/vaborbactam (MEV), and cefiderocol (FDC). Notably, 8 out of 10 isolates exhibited CZA MICs of 16 µg/mL, indicating complete resistance, while two isolates—AB54 and AB70—showed MICs of 4 and 8 µg/mL, respectively, suggesting limited residual susceptibility. However, even these borderline values exceed clinical breakpoints, rendering them clinically non-viable. All isolates were uniformly resistant to IMR and MEV (MIC = 8–16 µg/mL), indicating that resistance extends beyond traditional carbapenems to novel inhibitor combinations as well. These findings are particularly concerning given that such agents are used as salvage therapies for multidrug-resistant (MDR) pathogens. Cefiderocol resistance in all isolates (MIC = 16 µg/mL) further highlights the diminishing utility of even the most recently developed β-lactam antibiotics. Eravacycline displayed mixed results: while most isolates (n = 7) had MICs of 0.5 µg/mL (resistant), three isolates (AB19, AB21, and AB56) remained susceptible at 0.25 µg/mL. This variability suggests potential for strain-specific utility but reinforces the need for individualized susceptibility testing before clinical use. Genotypic analysis revealed a diverse distribution of Carbapenemase genes. The blaVIM gene was detected in AB04, while blaOXA-48 was found in AB21 and AB56. Strain AB57 harbored blaNDM, and notably, AB40 co-carried both blaOXA-48 and blaIMP. This genetic diversity indicates the coexistence of multiple resistance mechanisms within the same clinical setting, likely facilitated by mobile genetic elements such as plasmids and integrons. Among the seven K. pneumoniae isolates, variable susceptibility to Ceftazidime-avibactam was observed. Five isolates (KP4, KP27, KP35, KP81, KP85) remained susceptible (MICs: 0.25–0.5 µg/mL), while two isolates (KP37, KP92) showed high-level resistance (MIC = 16 µg/mL). All strains except KP4 showed resistance to IMR, MEM, and MEV (MICs ≥ 4–16 µg/mL), and all isolates were uniformly resistant to cefiderocol (MIC = 8 µg/mL). Eravacycline showed similar trends; KP4 and KP81 were susceptible (0.25 µg/mL), whereas other strains were resistant (0.5 µg/mL). These findings highlight the heterogeneity in susceptibility patterns even among clonally related hospital strains. Molecular testing showed the presence of blaNDM in KP4 and blaOXA-48 in KP81, KP85, and KP92. The absence of blaVIM, blaIMP, and blaKPC suggests a localized dominance of OXA-type and NDM-type carbapenemases in this cohort. To the best of our knowledge, this is the first report of Elizabethkingia species harboring OXA-48 and NDM carbapenemases from Karachi, Pakistan. All twelve isolates exhibited high-level resistance to CZA, IMR, MEM, MEV, and FDC (MICs ≥ 8 µg/mL), consistent with previous reports from Asia. All isolates were positive for blaOXA-48, and four (33%) also carried blaNDM, signifying dual Carbapenemase-mediated resistance. Notably, none of the isolates carried blaKPC, blaIMP, or blaVIM, suggesting that the resistance in Elizabethkingia is primarily driven by OXA- and NDM-type enzymes. These enzymes are known to confer high-level resistance to both conventional and advanced β-lactams. Given that Elizabethkingia is an emerging nosocomial pathogen with increasing clinical relevance, its documented resistance to nearly all β-lactams including siderophore cephalosporin’s further limits treatment options and necessitates close monitoring. Phylogenetic analysis revealed that the Carbapenemase-producing strains of A. baumannii , K. pneumoniae , and Elizabethkingia demonstrated clonal clustering with isolates from diverse international sources, including Mexico, Germany, Kenya, India, and the Netherlands. This supports the theory of plasmid-mediated horizontal gene transfer and the widespread dissemination of resistance determinants across continents and bacterial genera. The clustering of Elizabethkingia with E. coli and K. pneumoniae isolates from animals and environmental samples also underscores the role of zoonotic and environmental reservoirs in the resistance cycle. These findings support the need for a One Health approach to resistance surveillance. Structural modeling of the OXA-48 enzyme confirmed that the active site remains highly conserved across all isolates, with key residues (Ser70, Lys73, Ser118, Trp157, and Tyr211) properly aligned and structurally intact. This suggests sustained hydrolytic capability, which corroborates the phenotypic resistance data and provides a basis for rational drug design targeting these enzymes. This study highlights a complex and evolving resistance landscape among ICU-associated Gram-negative pathogens in Pakistan. The high prevalence of Carbapenemase genes (OXA-48, NDM, VIM, and IMP) and the demonstrated resistance to newer antimicrobial agents, including cefiderocol and β-lactamase inhibitor combinations, present a severe therapeutic challenge. The study received approval from the Ethics Committee of Dow University of Health Sciences (approval number 5.160/18/12/2019). Declarations Funding There is no any funding to this paper Declaration of Transparency The authors declare no conflicts of interest. Supplementary Information Additional data are available upon request Authors’ Contributions ZH conceptualized the study, performed laboratory experiments, analyzed the data, and drafted the manuscript. AN contributed to study design, supervised molecular work, and reviewed the manuscript. AF assisted with sample collection, data interpretation, and literature review. AK contributed to data analysis, figure preparation, and manuscript editing. MJ contributed to data analysis, figure preparation, and manuscript editing All authors read and approved the final version of the manuscript. References Feng, M., et al., Clinical Characteristics and Risk Factors for Infection and Death in Critically Ill Patients with Pulmonary Infection with Elizabethkingia Spp. 2024: p. 2673-2683. Tuhamize, B. and J.J.S.R. Bazira, Carbapenem-resistant Enterobacteriaceae in the livestock, humans and environmental samples around the globe: a systematic review and meta-analysis. 2024. 14 (1): p. 16333. Basnet, A., et al., Assessment of Antibiotic Resistance among Clinical Isolates of Enterobacteriaceae in Nepal. 2024. 1 (aop). Sarathi, S., et al., Microbiological characterization and clinical facets of Elizabethkingia bloodstream infections in a tertiary care hospital of eastern India. 2023: p. 3257-3267. Chang, Y., et al., MBLs, rather than efflux pumps, led to carbapenem resistance in fosfomycin and aztreonam/avibactam resistant Elizabethkingia anophelis. 2021: p. 315-327. Huang, Y.-C., et al., Comparison of clinical characteristics of bacteremia from Elizabethkingia meningoseptica and other carbapenem-resistant, non-fermenting Gram-negative bacilli at a tertiary medical center. 2019. 52 (2): p. 304-311. Rahmat Ullah, S., et al., Comprehensive insights into Klebsiella pneumoniae: unravelling clinical impact, epidemiological trends and antibiotic-resistance challenges. 2024: p. dkae184. Blot, S., et al., Healthcare-associated infections in adult intensive care unit patients: Changes in epidemiology, diagnosis, prevention and contributions of new technologies. 2022. 70 : p. 103227. Miron, M., et al., Hospital-Acquired Pneumonia and Ventilator-Associated Pneumonia: A Literature Review. 2024. 12 (1): p. 213. Ravi, K. and B.J.B. Singh, ESKAPE: Navigating the Global Battlefield for Antimicrobial Resistance and Defense in Hospitals. 2024. 3 (2): p. 76-98. Paudel, R., et al., Carbapenemase Producing Gram Negative Bacteria: Review of Resistance and Detection Methods. 2024: p. 116370. Mó, I. and G.J.J.A. da Silva, Tackling Carbapenem Resistance and the Imperative for One Health Strategies—Insights from the Portuguese Perspective. 2024. 13 (6): p. 557. Harding‐Crooks, R., et al., Dissemination of carbapenemase‐producing Enterobacteriaceae and associated resistance determinants through global food systems. 2023. 22 (4): p. 2706-2727. Marino, A., et al., Ceftazidime/Avibactam and Meropenem/Vaborbactam for the Management of Enterobacterales Infections: A Narrative Review, Clinical Considerations, and Expert Opinion. 2023. 12 (10): p. 1521. Kaye, K.S., et al., Cefiderocol, a siderophore cephalosporin, as a treatment option for infections caused by carbapenem-resistant Enterobacterales. 2023. 12 (3): p. 777-806. Bovo, F., et al., Clonal dissemination of Klebsiella pneumoniae resistant to cefiderocol, ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam co-producing KPC and OXA-181 carbapenemase. 2023. 5 (4): p. dlad099. Abushaheen, M.A., et al., Antimicrobial resistance, mechanisms and its clinical significance. 2020. 66 (6): p. 100971. Magiorakos, A., et al., Infection prevention and control measures and tools for the prevention of entry of carbapenem-resistant Enterobacteriaceae into healthcare settings: guidance from the European Centre for Disease Prevention and Control. 2017. 6 : p. 1-17. Ramadan, R.A., et al., Carbapenem-resistant Klebsiella pneumoniae among patients with ventilator-associated pneumonia: Evaluation of antibiotic combinations and susceptibility to new antibiotics. 2022: p. 3537-3548. Parasuraman, P., S. Busi, and J.-K. Lee, Standard Microbiological Techniques (Staining, Morphological and Cultural Characteristics, Biochemical Properties, and Serotyping) in the Detection of ESKAPE Pathogens , in ESKAPE Pathogens: Detection, Mechanisms and Treatment Strategies . 2024, Springer. p. 119-155. Altschul, S.F., et al., Basic local alignment search tool. J Mol Biol, 1990. 215 (3): p. 403-10. Additional Declarations No competing interests reported. Supplementary Files FigureS1.AgarosegelelectrophoresisfordetectionofblaOXA48gene.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6973343","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":488034961,"identity":"49f12a4b-b853-4def-9618-c7bfbe4966c5","order_by":0,"name":"Zeb Hussain","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIie3PMUsDMRTA8RcCd8u7do2L9xUiQnHLV0ko3HS4FI5O5UBIp+4d9DvYRTpeedDprKuigyA4naAfQDDppGBKu4nkDwkvw48kALHYH0zVCBz9xIE9IzR+lG7pIGHfCJcHEoBE7EV4Omt4ZieqP83W4zf7pCC9uBFsWQRJghvNPy2ZOfWKhyv7ampcV4K1ZZCgKKW7pdFAOHjMLGkQ5UAwOw4SkXdy+7DckZEjCvJuN5ECPeHs2hE3EKsFehJ+mMRzTbghsyA8Pbp0g8VidGba8PfV9Hb1gtVEHd+1J+9dRaqf0uL+YzkMEl/z45T4zdQ7xW+xw0ksFov9374AfzxOwsyD70oAAAAASUVORK5CYII=","orcid":"","institution":"University of Karachi","correspondingAuthor":true,"prefix":"","firstName":"Zeb","middleName":"","lastName":"Hussain","suffix":""},{"id":488034963,"identity":"f9a6b707-5ef1-4a89-a657-f427f8d9e469","order_by":1,"name":"Asma Naim","email":"","orcid":"","institution":"University of Karachi","correspondingAuthor":false,"prefix":"","firstName":"Asma","middleName":"","lastName":"Naim","suffix":""},{"id":488034964,"identity":"883f51e9-7b24-43ba-963a-9fdb7cdb67ca","order_by":2,"name":"Ambreen Fatima","email":"","orcid":"","institution":"Dow University of Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ambreen","middleName":"","lastName":"Fatima","suffix":""},{"id":488034966,"identity":"2b0cd631-69f6-4778-bb1f-ecb29f4f74ad","order_by":3,"name":"Asad Karim","email":"","orcid":"","institution":"University of Karachi","correspondingAuthor":false,"prefix":"","firstName":"Asad","middleName":"","lastName":"Karim","suffix":""},{"id":488034968,"identity":"fe2af46d-6f71-42d6-a352-ac7d9e1042be","order_by":4,"name":"Muhammad Jahanzeb","email":"","orcid":"","institution":"University of Karachi","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Jahanzeb","suffix":""}],"badges":[],"createdAt":"2025-06-25 10:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6973343/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6973343/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87380935,"identity":"f019bd78-664e-4bd5-83fc-cbf02b0957ee","added_by":"auto","created_at":"2025-07-23 08:35:59","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1340044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhylogenetic tree of Acinetobacter baumannii strain AB-04 clustering with global isolates. The tree was generated using the maximum likelihood method with bootstrap values based on 1,000 replications\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6973343/v1/d9b18c68f60d2a47ae7a153f.jpeg"},{"id":87380934,"identity":"d8f1be18-1e7f-4951-abf4-066e944b0785","added_by":"auto","created_at":"2025-07-23 08:35:59","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1560932,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhylogenetic tree of Acinetobacter baumannii strain AB-40 clustering with global isolates. The phylogenetic tree was constructed using the maximum likelihood method, with bootstrap values calculated from 1,000 replications.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6973343/v1/25f4449fee90382a0b0e8cb8.jpeg"},{"id":87383937,"identity":"a2575c7b-13bf-4644-9fe4-dee1d62f04de","added_by":"auto","created_at":"2025-07-23 08:43:59","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1377174,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhylogenetic tree of Acinetobacter baumannii strain AB-21 clustering with global isolates. The phylogenetic tree was constructed using the maximum likelihood method, with bootstrap values calculated from 1,000 replications\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6973343/v1/5e4e304faa681033099d6915.jpeg"},{"id":87380941,"identity":"57583d4d-17cb-412d-83ca-c8df64c3cc35","added_by":"auto","created_at":"2025-07-23 08:35:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":562511,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhylogenetic tree of Acinetobacter baumannii strain AB-57 clustering with global isolates. The phylogenetic tree was constructed using the maximum likelihood method, with bootstrap values calculated from 1,000 replications.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6973343/v1/aacb7d30f296718cdbc79f7e.png"},{"id":87383938,"identity":"80442529-e6f6-4384-8275-ef93907de130","added_by":"auto","created_at":"2025-07-23 08:43:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":640992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhylogenetic tree of klebsella pneumonia strain KP-81 clustering with global isolates. The phylogenetic tree was constructed using the maximum likelihood method, with bootstrap values calculated from 1,000 replications\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6973343/v1/daa743ca97d275b517a8af17.png"},{"id":87380952,"identity":"c6e9e901-63a8-426d-af17-cf3fa5fe39ae","added_by":"auto","created_at":"2025-07-23 08:35:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":660099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhylogenetic tree of klebsella pneumonia strain KP-85 clustering with global isolates. The phylogenetic tree was constructed using the maximum likelihood method, with bootstrap values calculated from 1,000 replications.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6973343/v1/bd174bc0395370485855283f.png"},{"id":87380942,"identity":"76001761-3e9f-44e6-b75d-0a2b58131af7","added_by":"auto","created_at":"2025-07-23 08:35:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":754726,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhylogenetic tree of klebsella pneumonia strain KP-92 clustering with global isolates. The phylogenetic tree was constructed using the maximum likelihood method, with bootstrap values calculated from 1,000 replications\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6973343/v1/6cf9bff14109d43689e2ca3a.png"},{"id":87383939,"identity":"64a0a69b-0450-4803-a968-29815393a2a2","added_by":"auto","created_at":"2025-07-23 08:43:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":691058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhylogenetic tree of Elizabethkingea strain EK-61 clustering with global isolates. The phylogenetic tree was constructed using the maximum likelihood method, with bootstrap values calculated from 1,000 replications.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6973343/v1/23c52df124eb5c37a89bbab9.png"},{"id":87380945,"identity":"19175927-b891-4bce-ae6b-1b33338c06b5","added_by":"auto","created_at":"2025-07-23 08:35:59","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":629444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePhylogenetic tree of Elizabethkingea strain EK-83 clustering with global isolates. The phylogenetic tree was constructed using the maximum likelihood method, with bootstrap values calculated from 1,000 replications.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6973343/v1/dc3869634fd1f03743bb851b.png"},{"id":87383940,"identity":"0772db12-2ea3-46f3-bd58-b61c19a9a6d1","added_by":"auto","created_at":"2025-07-23 08:43:59","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":275089,"visible":true,"origin":"","legend":"\u003cp\u003e(A) PCR amplification of OXA-48, (B) Sanger sequencing chromatogram of OXA-48 gene fragment showing clear base peaks, and (C) multiple sequence alignment illustrating conserved regions and point mutations across representative isolates.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-6973343/v1/be4abf0bac54a99dfaea370c.png"},{"id":87380947,"identity":"55acc7d3-a5ea-4b55-8fb4-431f9f91b285","added_by":"auto","created_at":"2025-07-23 08:35:59","extension":"jpeg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":138132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e(A) Homology model of OXA-48 β-lactamase validated using QMEAN scoring (B), (C) Ramachandran plot analysis, and (D) component-wise quality metrics, confirming structural integrity and stereochemical reliability.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6973343/v1/2a2b88d855f71afb5625886f.jpeg"},{"id":93591705,"identity":"b890bda7-91e7-4ac8-9492-49c9c43c97a0","added_by":"auto","created_at":"2025-10-15 12:47:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9058668,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6973343/v1/cc0fc451-232f-48d4-b43b-b88622325d20.pdf"},{"id":87380939,"identity":"016d80af-ffaa-4f43-a163-33934ef80319","added_by":"auto","created_at":"2025-07-23 08:35:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":455572,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.AgarosegelelectrophoresisfordetectionofblaOXA48gene.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6973343/v1/b068b40fbd416b25e26c03a3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular and Phylogenetic Characterization of Carbapenemase-Producing Gram- Negative Bacteria from VAP Patients: With Structural Insights into OXA-48","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePulmonary infections are among the most prevalent infectious diseases, ranking as the fourth leading cause of death globally in 2019 and the second leading cause in low-income countries[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eEnterobacteriaceae\u003c/em\u003e are a diverse group of Gram-negative bacteria commonly present in the intestines of humans, birds, animals, and the environment. These bacteria can lead to various infections, such as sepsis, pneumonia, and urinary tract infections [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In recent years, the prevalence of multidrug-resistant \u003cem\u003eEnterobacteriaceae\u003c/em\u003e strains has been increasing [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Infections caused by \u003cem\u003eElizabethkingia\u003c/em\u003e spp. are on the rise in several countries, with numerous outbreaks being reported [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. A study conducted in South Korea reported an increase in the infection rate of \u003cem\u003eElizabethkingia\u003c/em\u003e spp. among inpatients at Severance Hospital in Seoul, rising from 0.002% in 2009 to 0.088% in 2017 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Similarly, a study in Taiwan revealed that \u003cem\u003eElizabethkingia\u003c/em\u003e spp. had the second-highest infection rate among carbapenem-resistant, non-fermenting Gram-negative bacilli, surpassed only by \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The emergence and clonal dissemination of carbapenem-resistant \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e represent one of the most formidable challenges in contemporary infectious disease management.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] These pathogens have become synonymous with hospital-acquired infections, especially in critical care units settings like intensive care units (ICUs), where they significantly contribute to morbidity and mortality[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A common complication among ventilator-associated pneumonia (VAP) is frequently complicated by infections caused by these multidrug-resistant (MDR) organisms [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The growing incidence of carbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e and \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e in VAP cases has underscored the urgent need to understand the mechanisms driving resistance and to develop effective strategies to combat these infections.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Carbapenem resistance in these pathogens is primarily driven by Carbapenemase production enzymes that break down carbapenems and other β-lactam antibiotics, making them ineffective [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The most clinically significant carbapenemases include OXA-48, VIM, NDM, and IMP, which have been increasingly detected in both \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e and \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e isolates. These enzymes not only confer high-level resistance to carbapenems but also often carry additional resistance mechanisms that limit the efficacy of alternative antibiotics, complicating treatment options [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The rapid and widespread dissemination of Carbapenemase genes is facilitated by mobile genetic elements such as plasmids, transposons, and integrons, which enable horizontal gene transfer between bacteria. This genetic mobility has led to the rapid clonal spread of resistant strains across different geographic regions and healthcare settings, making infection control efforts particularly challenging. The clonal nature of these outbreaks often involves specific high-risk clones that are adept at colonizing and persisting in hospital environments, further exacerbating the problem [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To address this escalating threat, different Novel β-lactam/β-lactamase inhibitor combinations (βL-βLICs) have been introduced. These include agents such as Meropenem/vaborbactam, Ceftazidime/Avibactam and Imipenem/Relebactam, which were specifically designed to inhibit Carbapenemase activity or evade the resistance mechanisms employed by these MDR pathogens [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, Cefiderocol, a novel siderophore cephalosporin, has been introduced as a therapeutic an alternative for infection treatment caused by Carbapenemase producing Gram-negative pathogens. Cefiderocol\u0026rsquo;s unique mechanism of action, which involves the use of iron transport systems to gain entry into bacterial cells, provides an innovative approach to overcoming resistance [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Despite the promising efficacy of these new antimicrobials, there have been increasing reports of resistance emerging even to these agents. The detection of resistance to cefiderocol and novel βL-βLICs in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e and \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e strains, particularly those producing OXA-48, VIM, NDM, and IMP carbapenemases, raises significant concerns [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These developments suggest that the evolutionary pressure exerted by the widespread use of these novel agents is driving the selection of even more resistant bacterial populations. The clinical implications of this resistance are profound, as treatment options for infections caused by these resistant strains are extremely limited. The management of patients with infections caused by these pathogens often requires the use of combination therapy or reliance on less effective, more toxic antibiotics, which can lead to suboptimal outcomes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, the infection control challenges posed by these organisms are substantial, requiring stringent infection prevention measures to limit their spread within healthcare facilities [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we investigate the clonal outbreak of Carbapenemase-producing \u003cem\u003eElizabethkingia\u003c/em\u003e spp. \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e and \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e strains isolated from VAP patients over a one-year period spanning from January 2022 to December 2023. To the best of our knowledge, we are reported the \u003cem\u003eElizabethkingia\u003c/em\u003e spp. isolated from Karachi, Pakistan.\u003c/p\u003e\u003cp\u003eOur focus is on characterizing the resistance of these strains to cefiderocol and the novel βL-βLICs, with particular attention to the role of OXA-48, VIM, NDM, and IMP carbapenemases in mediating this resistance. By conducting comprehensive antimicrobial susceptibility testing and genetic analysis, we aim to uncover the underlying mechanisms of resistance and the factors contributing to the persistence and spread of these high-risk clones.\u003c/p\u003e\u003cp\u003eThe outcomes of this research are expected to provide critical insights into the epidemiology of these resistant pathogens, offering valuable information for the development of more effective therapeutic strategies and infection control policies. Understanding the genetic and phenotypic characteristics of these strains will be key to mitigating the impact of these MDR organisms and addressing the ongoing challenge of antibiotic resistance in healthcare settings.\u003c/p\u003e"},{"header":"Materials and method","content":"\u003cp\u003e\u003cb\u003eStudy setting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis cross-sectional study was conducted from January 2022 to January 2023 in the Microbiology department at the University of Karachi, in collaboration with the Microbiology section at Dow Diagnostic Research Laboratory and the Jamil-ur-Rahman Center for Genome Research, Dr. Panjwani Center for Molecular Medicine and Drug Research, and the International Center for Chemical and Biological Sciences at the University of Karachi. Ethical approval for the study was obtained from the Institutional Review Board of Dow University of Health Sciences (DUHS), Karachi (Approval number 5.160/18/12/2019).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacterial identification\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eDuring the study we collected endotracheal aspirates (ETAs) from intubated patients under strict aseptic conditions. All criteria were followed according to international protocols [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEndotracheal samples were obtained from the ICU of Dow University Hospital. Total of 183 strains were obtained from the tracheal aspirates of VAP patients using MacConkey, blood, and chocolate agar media (Oxoid-UK). The initial identification of these isolates was performed using traditional methods, such as Gram staining, growth characteristics, and biochemical tests [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The API 20E system (bioM\u0026eacute;rieux SA, Marcy-l'\u0026Eacute;toile, France) was utilized for further identification. Antimicrobial susceptibility testing was then conducted following CLSI guidelines (2021). ATCC 25922 \u003cem\u003e(E. coli\u003c/em\u003e) and ATCC 27853 (\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e) served as quality control strains for the testing.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular Analysis of Resistance Mechanisms\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eIsolated strains were investigated for the presence of common genotypic MBLs variants, including blaVIM, blaOXA-48, and blaIMP. BlaKPC, and blaNDM. DNA was extracted using the QIAamp\u0026reg; DNA Mini Kit method. PCR conditions included an initial denaturation at 94\u0026deg;C for 4 minutes, followed by 35 cycles consisting of denaturation at 94\u0026deg;C for 1 minute, annealing at primer-specific temperatures for 30 seconds, and extension at 72\u0026deg;C for 1 minute, concluding with a final extension at 72\u0026deg;C for 10 minutes. The PCR products were separated using 2% agarose gel electrophoresis. Primers used in study are mentioned in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSanger sequencing of amplicon\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eSelected amplicons representative of each Carbapenemase gene were purified using the QIAquick PCR Purification Kit (QIAGEN, Germany). Purified DNA samples were then sent to Macrogen Inc. (Seoul, South Korea) for bidirectional Sanger sequencing.\u003c/p\u003e\u003cp\u003eBoth forward and reverse primers used in the initial PCR were employed for bidirectional sequencing.\u003c/p\u003e\u003cp\u003eChromatograms were analyzed using Chromas Lite v2.6.6 and BioEdit software. Consensus sequences were aligned and compared to reference gene sequences in the NCBI GenBank database using BLASTn to confirm the identity and variant types of Carbapenemase genes.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic Analysis\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eFor each Carbapenemase gene detected, our Sanger‑derived sequences were aligned with representative reference sequences retrieved from NCBI GenBank using MUSCLE in MEGA 11. Maximum‑likelihood phylogenetic trees were constructed under the Tamura‑Nei model with 1,000 bootstrap replicates. Trees were visualized in FigTree v1.4.4. Clusters were interpreted based on bootstrap support\u0026thinsp;\u0026ge;\u0026thinsp;70%, allowing inference of clonal relationships and horizontal gene transfer among clinical, environmental, and veterinary isolates.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStructural Modeling of Carbapenemase Variants\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eStructural Modelling and Comparative Analysis Amino acid sequences corresponding to Carbapenemase enzymes were obtained from GenBank. Structural modelling of the OXA-48-like β-lactamase was conducted using SWISS-MODEL, selecting the crystal structure 3HBR_A from the RCSB Protein Data Bank as a template (resolution 1.91 \u0026Aring;, identity 97.21%). The resulting model (referred to as \"model.pdb\") underwent rigorous quality assessment using GMQE, QMEANDisCo (global score: 0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05), and predicted lDDT (0.91). Additional validation included Ramachandran plot analysis in PROCHECK and clash score evaluation.\u003c/p\u003e\u003cp\u003eThe reference crystal structure 3HBR (native OXA-48) was used as a benchmark. Both structures were aligned using TM-align and superposed in PyMOL to assess topological congruence and RMSD values. Binding sites were mapped by aligning conserved active site motifs (S70, K73, S118, W157, Y211), and ligand binding residues were annotated through the SWISS-MODEL ligand pipeline. Hydrogen bonding and hydrophobic interactions with known β-lactam antibiotics (Ceftazidime, imipenem) were further explored using LigPlot+.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eAntimicrobial Susceptibility Profiles\u003c/b\u003e:\u003c/p\u003e\u003cp\u003e\u003cem\u003eAcinetobacter baumannii\u003c/em\u003e isolates (n\u0026thinsp;=\u0026thinsp;10) demonstrated near-uniform resistance to novel β-lactam/β-lactamase inhibitors and cefiderocol. Ceftazidime-avibactam (CZA) minimum inhibitory concentrations (MICs) ranged from 4\u0026ndash;16 mg/L, with 80% (8/10) exceeding the resistance breakpoint (\u0026gt;\u0026thinsp;8 mg/L). Notably, \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e strain AB-54 exhibited susceptibility (MIC\u0026thinsp;=\u0026thinsp;4 mg/L), while \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e strain AB-70 showed intermediate resistance (MIC\u0026thinsp;=\u0026thinsp;8 mg/L). All isolates were resistant to Imipenem-Relebactam (IMR; MIC\u0026thinsp;=\u0026thinsp;8 mg/L), Meropenem (MIC\u0026thinsp;=\u0026thinsp;16 mg/L), Cefiderocol (MIC\u0026thinsp;=\u0026thinsp;16 mg/L), and Meropenem-vaborbactam (MIC\u0026thinsp;=\u0026thinsp;16 mg/L). Eravacycline susceptibility varied: three isolates (AB-19, AB-21, AB-56) remained susceptible (MIC\u0026thinsp;=\u0026thinsp;0.25 mg/L), while seven exhibited resistances (MIC\u0026thinsp;=\u0026thinsp;0.5 mg/L). Molecular characterization revealed Carbapenemase gene heterogeneity: \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e AB-04 harbored \u003cem\u003ebla\u003c/em\u003eVIM, \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e AB-21 and \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e AB-56 carried \u003cem\u003ebla\u003c/em\u003eOXA-48, AB-57 possessed \u003cem\u003ebla\u003c/em\u003eNDM, and AB-40 co-harbored \u003cem\u003ebla\u003c/em\u003eOXA-48 and \u003cem\u003ebla\u003c/em\u003eIMP (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer sequences, target genes, and expected amplicon sizes used for PCR detection of carbapenemases genes in Gram-negative bacterial isolates.\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer (5\u0026prime;\u0026ndash;3\u0026prime;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eProduct Size (bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIMP-F: GGAATAGAGTGGCTTAAYTCTC IMP-R: GGTTTAAYAAAACAACCACC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eblaIMP\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e232\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePoirel et al., 2011. \u003cem\u003eJ Antimicrob Chemother\u003c/em\u003e 66(1): 57\u0026ndash;63. [PMID: 21098058]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVIM-F: GATGGTGTTTGGTCGCATA VIM-R: CGAATGCGCAGCACCAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eblaVIM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e390\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEllington et al., 2007. \u003cem\u003eJ Antimicrob Chemother\u003c/em\u003e 59(3): 434\u0026ndash;439. [PMID: 17289721]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOXA-F: GCGTGGTTAAGGATGAACAC OXA-R: CATCAAGTTCAACCCAACCG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eblaOXA-48\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e438\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePoirel et al., 2011. \u003cem\u003eAntimicrob Agents Chemother\u003c/em\u003e 55(10): 4896\u0026ndash;4899. [PMID: 21746950]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNDM-F: GGTTTGGCGATCTGGTTTTC NDM-R: CGGAATGGCTCATCACGATC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eblaNDM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e621\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePoirel et al., 2011. \u003cem\u003eAntimicrob Agents Chemother\u003c/em\u003e 55(11): 4910\u0026ndash;4913. [PMID: 21876065]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKPC-Fm: CGTCTAGTTCTGCTGTCTTG KPC-Rm: CTTGTCATCCTTGTTAGGCG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eblaKPC\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e798\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCuzon et al., 2010. \u003cem\u003eAntimicrob Agents Chemother\u003c/em\u003e 54(1): 307\u0026ndash;308. [PMID: 19884378]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eNote\u003c/em\u003e: \u003csup\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eThis table summarizes the primer sequences used for the amplification of five Carbapenemase-encoding genes (blaIMP, blaVIM, blaOXA-48, blaNDM, and blaKPC) by PCR. The corresponding amplicon sizes and references for the primer sequences are also listed.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eOligonucleotides used in this study.\u003c/p\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e F, sense primer; R, antisense primer.\u003c/p\u003e\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e Nucleotide numbering begins at the initiation codon of genes.\u003c/p\u003e\u003cp\u003e\u003csup\u003ec\u003c/sup\u003e D\u0026thinsp;=\u0026thinsp;A or G or T; Y\u0026thinsp;=\u0026thinsp;C or T.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003eShow betalactamse inhibitors resistance patterns of different species of Acinetobacter Pathogenic strains\u003c/em\u003e.\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"12\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCZA\u003c/p\u003e\u003cp\u003e\u003cem\u003eS(-) R(-)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eS(\u0026le;\u0026thinsp;8) R(\u0026gt;\u0026thinsp;8\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIMR\u003c/p\u003e\u003cp\u003eS(\u0026le;\u0026thinsp;2) R(\u0026gt;\u0026thinsp;2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMERO\u003c/p\u003e\u003cp\u003eS(\u0026le;\u0026thinsp;2) R(\u0026gt;\u0026thinsp;8)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMEV\u003c/p\u003e\u003cp\u003eS(\u0026le;\u0026thinsp;8) R(\u0026gt;\u0026thinsp;8)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eFDC\u003c/em\u003e\u003c/p\u003e\u003cp\u003eS(\u0026le;\u0026thinsp;8)R(\u0026gt;\u0026thinsp;8)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eERV\u003c/em\u003e\u003c/p\u003e\u003cp\u003eS(\u0026le;\u0026thinsp;0.25)R(\u0026gt;\u0026thinsp;0.25)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eVIM\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003eNDM\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cem\u003eKPC\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cem\u003eOX-48\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cem\u003eIMP\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStrains\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAb-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAb-19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAb-20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAb-21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAb-32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAb-40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAb-50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAb-54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAb-56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAb-57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAb-70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eNote\u003c/em\u003e: \u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eThis table presents the minimum inhibitory concentrations (MICs, in \u0026micro;g/mL) of various antibiotics tested against Acinetobacter baumannii strains. The antibiotics include Ceftazidime-avibactam (CZA), imipenem/Relebactam (IMR), Meropenem (MERO), Meropenem/vaborbactam (MEV), cefiderocol (FDC), and Eravacycline (ERV). Interpretive breakpoints for susceptibility (S) and resistance (R) are indicated for each drug. The presence (+) or absence (\u0026ndash;) of Carbapenemase genes (VIM, NDM, KPC, OXA-48, and IMP) was determined by PCR\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eAmong \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e isolates (n\u0026thinsp;=\u0026thinsp;7), resistance patterns diverged significantly. Two strains (KP-37, KP-92) exhibited multidrug resistance, including resistance to CZA (MIC\u0026thinsp;=\u0026thinsp;16 mg/L) and IMR (MIC\u0026thinsp;=\u0026thinsp;8 mg/L). Conversely, KP-4 retained susceptibility to CZA (MIC\u0026thinsp;=\u0026thinsp;0.25 mg/L), IMR (MIC\u0026thinsp;=\u0026thinsp;0.5 mg/L), Meropenem (MIC\u0026thinsp;=\u0026thinsp;1 mg/L), and Meropenem-vaborbactam (MIC\u0026thinsp;=\u0026thinsp;1 mg/L). All isolates were resistant to cefiderocol (MIC\u0026thinsp;=\u0026thinsp;8 mg/L), exceeding the susceptibility threshold (\u0026le;\u0026thinsp;2 mg/L). Eravacycline susceptibility was observed in KP-4 and KP-81 (MIC\u0026thinsp;=\u0026thinsp;0.25 mg/L), while other strains demonstrated resistance (MIC\u0026thinsp;=\u0026thinsp;0.5 mg/L). Genotypic analysis identified \u003cem\u003ebla\u003c/em\u003eNDM in KP-4 and \u003cem\u003ebla\u003c/em\u003eOXA-48 in KP-81, KP-85, and KP-92; no KPC, VIM, or IMP genes were detected (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003eShow beta-lactamase inhibitors resistance patterns of different species of klebseilla Pathogenic strains\u003c/em\u003e.\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"13\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCZA\u003c/em\u003e\u003c/p\u003e\u003cp\u003eS(\u0026le;\u0026thinsp;8)R(\u0026gt;\u0026thinsp;8\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIMR\u003c/p\u003e\u003cp\u003eS(\u0026le;\u0026thinsp;2)R(\u0026gt;\u0026thinsp;2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMERO\u003c/p\u003e\u003cp\u003eS(\u0026le;\u0026thinsp;2)R(\u0026gt;\u0026thinsp;8)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMEV\u003c/p\u003e\u003cp\u003eS(\u0026le;\u0026thinsp;8)R(\u0026gt;\u0026thinsp;8)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eFDC\u003c/em\u003e\u003c/p\u003e\u003cp\u003eS(\u0026le;\u0026thinsp;2) R(\u0026gt;\u0026thinsp;2)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eCZA\u003c/em\u003e\u003c/p\u003e\u003cp\u003eS(\u0026le;\u0026thinsp;8)R(\u0026gt;\u0026thinsp;8)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eERV\u003c/em\u003e\u003c/p\u003e\u003cp\u003eS(\u0026le;\u0026thinsp;0.25)R(\u0026gt;\u0026thinsp;0.25)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003eVIM\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cem\u003eNDM\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cem\u003eKPC\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cem\u003eOXA-48\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u003cem\u003eIMP\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStrains\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKp-4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.25 -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKp-81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.25 -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKp-85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.5 -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKp-27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.5 -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKp-35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.5 -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKp-37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKp-92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEk-83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEk-61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eNote\u003c/em\u003e: \u003csup\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eThe table displays the minimum inhibitory concentrations (MICs, in \u0026micro;g/mL) of selected antibiotics tested against Klebsiella pneumoniae (KP) and Elizabethkingea spp (Ek) isolates. Antibiotics include Ceftazidime-avibactam (CZA), imipenem/Relebactam (IMR), Meropenem (MERO), Meropenem/vaborbactam (MEV), cefiderocol (FDC), and Eravacycline (ERV). Interpretive breakpoints for susceptibility (S) and resistance (R) are shown for each drug. PCR results for Carbapenemase genes (VIM, NDM, KPC, OXA-48, and IMP) are indicated by \"+\" (positive) or \"\u0026ndash;\" (negative).\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCarbapenemase Gene Distribution\u003c/b\u003e:\u003c/p\u003e\u003cp\u003ePCR amplification confirmed the coexistence of metallo-β-lactamases (\u003cem\u003ebla\u003c/em\u003eVIM, \u003cem\u003ebla\u003c/em\u003eNDM, \u003cem\u003ebla\u003c/em\u003eIMP) and Oxacillinases (\u003cem\u003ebla\u003c/em\u003eOXA-48) across \u003cem\u003eA. baumannii\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates. In \u003cem\u003eA. baumannii\u003c/em\u003e, \u003cem\u003ebla\u003c/em\u003eOXA-48 predominated (3/10 isolates), followed by \u003cem\u003ebla\u003c/em\u003eNDM (1/10) and \u003cem\u003ebla\u003c/em\u003eVIM (1/10), with one strain co-harboring \u003cem\u003ebla\u003c/em\u003eOXA-48 and \u003cem\u003ebla\u003c/em\u003eIMP. \u003cem\u003eK. pneumoniae\u003c/em\u003e strains exclusively carried \u003cem\u003ebla\u003c/em\u003eOXA-48 (3/7) or \u003cem\u003ebla\u003c/em\u003eNDM (1/7), with no co-occurrence of multiple Carbapenemase genes. These findings correlate with observed resistance profiles, particularly the ineffectiveness of Avibactam- and Vaborbactam-based combinations against OXA-48 and metallo-β-lactamase producers. The persistence of Cefiderocol resistance across all isolates, despite iron-depleted testing conditions, underscores emerging challenges in managing infections caused by Carbapenemase-producing \u003cem\u003eEnterobacterales\u003c/em\u003e (CPE) with limited therapeutic options.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic Analysis of\u003c/b\u003e \u003cb\u003eAcinetobacter baumannii\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eKlebsiella pneumonia\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eA circular phylogenetic tree (radial dendrogram) was constructed to illustrate the evolutionary relationships among genomic regions from multiple strains or isolates. The tree was build based on sequence similarity and divergence, with branch lengths indicating genetic distances. The hierarchical structure of the tree demonstrates the phylogenetic clustering of these sequences, revealing potential genetic conservation, divergence, or horizontal gene transfer events. This analysis provides insights into the genetic relatedness of the studied regions, contributing to our understanding of genomic evolution and potential functional conservation among different bacterial strains.\u003c/p\u003e\u003cp\u003eThe phylogenetic analysis of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e and \u003cem\u003eElizabethkingia\u003c/em\u003e spp. provided significant insights into their genetic relationships and the widespread dissemination of antimicrobial resistance (AMR) genes. The clustering patterns revealed that genetically similar strains are found across diverse geographic regions, suggesting a major role of horizontal gene transfer (HGT), clonal expansion, and global patient movement in spreading resistant strains. The presence of conserved resistance determinants across multiple continents indicates the rapid evolution and adaptation of these pathogens in clinical and environmental settings.\u003c/p\u003e\u003cp\u003eStrain AB-4 of \u003cem\u003eA. baumannii\u003c/em\u003e clustered with CP023022.1 (bronchial fluid, Mexico), CP087370.1 (Germany), and CP066016.1 (USA), demonstrating that despite vast geographic distances, these strains share common resistance traits and molecular characteristics, likely due to the circulation of mobile genetic elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilarly, strain AB-40 exhibited strong genetic relatedness to \u003cem\u003eA. baumannii\u003c/em\u003e CP142667 (South Korea) and CP091339 (Belgium), with conserved resistance-associated loci, underscoring the strain\u0026rsquo;s adaptability in clinical settings and potential transmission through healthcare-associated pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe presence of highly similar resistant strains across different countries highlights the interconnected nature of bacterial evolution and antibiotic resistance spread. Further analysis of \u003cem\u003eA. baumannii\u003c/em\u003e strains revealed evidence of cross-species resistance gene dissemination. Notably, strain AB-21, an OXA-48-positive \u003cem\u003eA. baumannii\u003c/em\u003e isolate, phylogenetically clustered with \u003cem\u003eK. pneumoniae\u003c/em\u003e (MF741882.1, Kenya; OM368082.1, India) and \u003cem\u003eE. coli\u003c/em\u003e (CP023924.1, Canada), suggesting the involvement of plasmid-mediated HGT in the global transmission of carbapenem resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe genetic similarities between \u003cem\u003eA. baumannii\u003c/em\u003e, \u003cem\u003eK. pneumoniae\u003c/em\u003e, and \u003cem\u003eE. coli\u003c/em\u003e indicate the possibility of interspecies gene exchange in healthcare environments, where co-colonization and plasmid transfer events could contribute to the rapid spread of AMR genes. Additionally, strain AB-57 demonstrated a zoonotic transmission link, clustering with \u003cem\u003eE. coli\u003c/em\u003e KU318691.1 (leopard, India) and \u003cem\u003eK. pneumoniae\u003c/em\u003e MK628734.1 (duck, China), further emphasizing the role of animal reservoirs in the dissemination of antimicrobial resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These findings underscore the importance of One Health surveillance strategies that integrate human, animal, and environmental data to combat the rising threat of multidrug-resistant pathogens.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePhylogenetic clustering of \u003cem\u003eK. pneumoniae\u003c/em\u003e strains reinforced the notion of widespread carbapenem resistance dissemination. Strain KP-81 was closely related to \u003cem\u003eE. coli\u003c/em\u003e KY817191.1 (India) and \u003cem\u003eK. pneumoniae\u003c/em\u003e OM368082.1 (India), both harboring blaOXA-48 and blaOXA-181 genes, illustrating cross-host and cross-continental Carbapenemase resistance transmission (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis suggests that plasmid-mediated resistance is not confined to a single species but can spread dynamically between different bacterial populations. Strain KP-85 phylogenetically aligned with \u003cem\u003eK. pneumoniae\u003c/em\u003e CP034284 (genomic DNA, blaOXA-181 plasmid) and PP320277 (Canada), further supporting the role of plasmid-driven HGT in carbapenem resistance and its potential for long-distance dissemination through global travel and healthcare networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilarly, strain KP-92 clustered with \u003cem\u003eK. pneumoniae\u003c/em\u003e MF741882.1 (Kenya), \u003cem\u003eE. coli\u003c/em\u003e MF741884.1 (Kenya), and \u003cem\u003eK. pneumoniae\u003c/em\u003e CP068846.1 (Netherlands), all carrying blaOXA-48, reinforcing the widespread dissemination of this resistance determinant across multiple regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese results provide strong evidence of the rapid movement of carbapenemase genes across bacterial species and continents, making them a major concern for global public health. Unexpectedly, the phylogenetic analysis of \u003cem\u003eElizabethkingia\u003c/em\u003e spp. revealed novel insights into resistance gene dissemination beyond the Enterobacteriaceae family. Strain EK-61, an OXA-48-positive \u003cem\u003eElizabethkingia\u003c/em\u003e isolate, clustered with \u003cem\u003eK. pneumoniae\u003c/em\u003e CP068856.1 (Netherlands) and CP079128.1 (India), suggesting that OXA-48 resistance is not restricted to clinically relevant Enterobacteriaceae but has also spread to environmental non-fermenters (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe presence of such resistance determinants in \u003cem\u003eElizabethkingia\u003c/em\u003e spp. raises concerns regarding potential reservoirs of antibiotic resistance in environmental settings, which could pose challenges for infection control and treatment strategies. Furthermore, another \u003cem\u003eElizabethkingia\u003c/em\u003e strain exhibited strong genetic ties to \u003cem\u003eE. coli\u003c/em\u003e KY801334.1 (India), AP018837.1 (Vietnam), and \u003cem\u003eK. pneumoniae\u003c/em\u003e MT635909.1 (Vietnam), all carrying blaOXA-48 or blaOXA-181, providing further evidence of plasmid-mediated HGT across bacterial species and geographic locations (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThese findings highlight the complex and interconnected nature of resistance gene mobility, emphasizing the necessity of global genomic surveillance to track the spread of AMR determinants and mitigate their impact on public health. (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eConservation Analysis of OXA β-lactamase Genes\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eA multiple sequence alignment (MSA) was performed on OXA β-lactamase gene sequences derived from diverse bacterial isolates using ClustalW in BioEdit v7.2.5. The alignment revealed conserved regions interspersed with isolate-specific nucleotide and amino acid variations. Highly conserved nucleotide motifs were observed across all sequences, notably within the central and 5\u0026prime; coding regions. For instance, a conserved stretch encoding the motif GATATCGCCGCTTGG was present in all aligned sequences with minimal to no variation. Similarly, conserved di- and tri-nucleotide repeats, such as CGT, GCG, and TGG, were consistently maintained, suggesting evolutionary pressure to retain structural and functional integrity of these regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTranslational analysis of the aligned coding sequences revealed that the majority of isolates maintained an intact open reading frame, with start and stop codons appropriately positioned. The deduced amino acid sequences exhibited conservation in residues known to form the active site of class D β-lactamases, including conserved serine-lysine motifs and the carboxyl-terminal residues critical for β-lactam ring hydrolysis. Residues such as S70, K73, and W157\u0026mdash;previously implicated in catalytic activity\u0026mdash;were preserved across nearly all sequences. Variable regions were observed predominantly in the flanking regions and loop-encoding segments. Several isolates showed amino acid substitutions, insertions, or deletions, including frequent substitutions at positions corresponding to hydrophobic-to-polar residue changes. Additionally, a subset of sequences contained degenerate nucleotide positions (e.g., R, Y, K), indicating either intra-population heterogeneity or unresolved base calls during sequencing.\u003c/p\u003e\u003cp\u003eImportantly, no premature stop codons were observed within the core coding region of the OXA genes, supporting the conclusion that these sequences are likely functional and encode active enzymes. The absence of frameshift mutations further corroborates the functional conservation of these β-lactamase variants. Structural Insights from Homology ModellingThe OXA-48-like model developed using SWISS-MODEL based on template 3HBR demonstrated robust structural fidelity. Quality parameters included a GMQE score of 0.88 and a QMEANDisCo score of 0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05. Ramachandran plot analysis indicated that 97.4% of residues fell within favored regions, and the predicted lDDT value of 0.91 confirmed high confidence in the accuracy of side-chain placements and backbone topology Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhen superposed with the crystallographic structure of 3HBR, the homology model exhibited an RMSD of less than 0.3 \u0026Aring;, indicating negligible deviation. The conserved catalytic residues (Ser70, Lys73, Ser118, Trp157, and Tyr211) were precisely aligned in both structures, validating functional integrity of the active site. LigPlot\u0026thinsp;+\u0026thinsp;interaction analysis of Ceftazidime docked in the model revealed strong hydrogen bonding interactions, particularly with Ser70 and neighboring polar residues, reinforcing the hydrolytic competence of the enzyme. Furthermore, the active site cavity was structurally congruent with that of the reference enzyme, supporting substrate accommodation and suggesting preserved catalytic potential. This validated homology model not only supports the inferred multidrug resistance phenotype observed in clinical isolates but also serves as a high-confidence platform for future structure-based inhibitor design targeting class D β-lactamases.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe antimicrobial susceptibility testing of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e isolates in this study revealed an alarming resistance profile against multiple last-resort antibiotics. All isolates demonstrated high-level resistance to Ceftazidime-avibactam (CZA), Imipenem/Relebactam (IMR), Meropenem (MEM), Meropenem/vaborbactam (MEV), and cefiderocol (FDC). Notably, 8 out of 10 isolates exhibited CZA MICs of 16 \u0026micro;g/mL, indicating complete resistance, while two isolates\u0026mdash;AB54 and AB70\u0026mdash;showed MICs of 4 and 8 \u0026micro;g/mL, respectively, suggesting limited residual susceptibility. However, even these borderline values exceed clinical breakpoints, rendering them clinically non-viable.\u003c/p\u003e\u003cp\u003eAll isolates were uniformly resistant to IMR and MEV (MIC\u0026thinsp;=\u0026thinsp;8\u0026ndash;16 \u0026micro;g/mL), indicating that resistance extends beyond traditional carbapenems to novel inhibitor combinations as well. These findings are particularly concerning given that such agents are used as salvage therapies for multidrug-resistant (MDR) pathogens. Cefiderocol resistance in all isolates (MIC\u0026thinsp;=\u0026thinsp;16 \u0026micro;g/mL) further highlights the diminishing utility of even the most recently developed β-lactam antibiotics.\u003c/p\u003e\u003cp\u003eEravacycline displayed mixed results: while most isolates (n\u0026thinsp;=\u0026thinsp;7) had MICs of 0.5 \u0026micro;g/mL (resistant), three isolates (AB19, AB21, and AB56) remained susceptible at 0.25 \u0026micro;g/mL. This variability suggests potential for strain-specific utility but reinforces the need for individualized susceptibility testing before clinical use.\u003c/p\u003e\u003cp\u003eGenotypic analysis revealed a diverse distribution of Carbapenemase genes. The blaVIM gene was detected in AB04, while blaOXA-48 was found in AB21 and AB56. Strain AB57 harbored blaNDM, and notably, AB40 co-carried both blaOXA-48 and blaIMP. This genetic diversity indicates the coexistence of multiple resistance mechanisms within the same clinical setting, likely facilitated by mobile genetic elements such as plasmids and integrons.\u003c/p\u003e\u003cp\u003eAmong the seven \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates, variable susceptibility to Ceftazidime-avibactam was observed. Five isolates (KP4, KP27, KP35, KP81, KP85) remained susceptible (MICs: 0.25\u0026ndash;0.5 \u0026micro;g/mL), while two isolates (KP37, KP92) showed high-level resistance (MIC\u0026thinsp;=\u0026thinsp;16 \u0026micro;g/mL). All strains except KP4 showed resistance to IMR, MEM, and MEV (MICs\u0026thinsp;\u0026ge;\u0026thinsp;4\u0026ndash;16 \u0026micro;g/mL), and all isolates were uniformly resistant to cefiderocol (MIC\u0026thinsp;=\u0026thinsp;8 \u0026micro;g/mL).\u003c/p\u003e\u003cp\u003eEravacycline showed similar trends; KP4 and KP81 were susceptible (0.25 \u0026micro;g/mL), whereas other strains were resistant (0.5 \u0026micro;g/mL). These findings highlight the heterogeneity in susceptibility patterns even among clonally related hospital strains.\u003c/p\u003e\u003cp\u003eMolecular testing showed the presence of blaNDM in KP4 and blaOXA-48 in KP81, KP85, and KP92. The absence of blaVIM, blaIMP, and blaKPC suggests a localized dominance of OXA-type and NDM-type carbapenemases in this cohort.\u003c/p\u003e\u003cp\u003eTo the best of our knowledge, this is the first report of \u003cem\u003eElizabethkingia\u003c/em\u003e species harboring OXA-48 and NDM carbapenemases from Karachi, Pakistan. All twelve isolates exhibited high-level resistance to CZA, IMR, MEM, MEV, and FDC (MICs\u0026thinsp;\u0026ge;\u0026thinsp;8 \u0026micro;g/mL), consistent with previous reports from Asia.\u003c/p\u003e\u003cp\u003eAll isolates were positive for blaOXA-48, and four (33%) also carried blaNDM, signifying dual Carbapenemase-mediated resistance. Notably, none of the isolates carried blaKPC, blaIMP, or blaVIM, suggesting that the resistance in \u003cem\u003eElizabethkingia\u003c/em\u003e is primarily driven by OXA- and NDM-type enzymes. These enzymes are known to confer high-level resistance to both conventional and advanced β-lactams.\u003c/p\u003e\u003cp\u003eGiven that \u003cem\u003eElizabethkingia\u003c/em\u003e is an emerging nosocomial pathogen with increasing clinical relevance, its documented resistance to nearly all β-lactams including siderophore cephalosporin\u0026rsquo;s further limits treatment options and necessitates close monitoring.\u003c/p\u003e\u003cp\u003ePhylogenetic analysis revealed that the Carbapenemase-producing strains of \u003cem\u003eA. baumannii\u003c/em\u003e, \u003cem\u003eK. pneumoniae\u003c/em\u003e, and \u003cem\u003eElizabethkingia\u003c/em\u003e demonstrated clonal clustering with isolates from diverse international sources, including Mexico, Germany, Kenya, India, and the Netherlands. This supports the theory of plasmid-mediated horizontal gene transfer and the widespread dissemination of resistance determinants across continents and bacterial genera.\u003c/p\u003e\u003cp\u003eThe clustering of \u003cem\u003eElizabethkingia\u003c/em\u003e with \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates from animals and environmental samples also underscores the role of zoonotic and environmental reservoirs in the resistance cycle. These findings support the need for a One Health approach to resistance surveillance.\u003c/p\u003e\u003cp\u003eStructural modeling of the OXA-48 enzyme confirmed that the active site remains highly conserved across all isolates, with key residues (Ser70, Lys73, Ser118, Trp157, and Tyr211) properly aligned and structurally intact. This suggests sustained hydrolytic capability, which corroborates the phenotypic resistance data and provides a basis for rational drug design targeting these enzymes.\u003c/p\u003e\u003cp\u003eThis study highlights a complex and evolving resistance landscape among ICU-associated Gram-negative pathogens in Pakistan. The high prevalence of Carbapenemase genes (OXA-48, NDM, VIM, and IMP) and the demonstrated resistance to newer antimicrobial agents, including cefiderocol and β-lactamase inhibitor combinations, present a severe therapeutic challenge.\u003c/p\u003e\u003cp\u003eThe study received approval from the Ethics Committee of Dow University of Health Sciences (approval number 5.160/18/12/2019).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no any funding to this paper\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Transparency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional data are available upon request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZH\u003c/strong\u003e conceptualized the study, performed laboratory experiments, analyzed the data, and drafted the manuscript.\u003cbr\u003e\u003cstrong\u003eAN\u003c/strong\u003e contributed to study design, supervised molecular work, and reviewed the manuscript.\u003cbr\u003e\u003cstrong\u003eAF\u003c/strong\u003e assisted with sample collection, data interpretation, and literature review.\u003cbr\u003e\u003cstrong\u003eAK\u003c/strong\u003e contributed to data analysis, figure preparation, and manuscript editing.\u003cbr\u003e\u003cstrong\u003eMJ\u003c/strong\u003e contributed to data analysis, figure preparation, and manuscript editing\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFeng, M., et al., \u003cem\u003eClinical Characteristics and Risk Factors for Infection and Death in Critically Ill Patients with Pulmonary Infection with Elizabethkingia Spp.\u003c/em\u003e 2024: p. 2673-2683.\u003c/li\u003e\n\u003cli\u003eTuhamize, B. and J.J.S.R. Bazira, \u003cem\u003eCarbapenem-resistant Enterobacteriaceae in the livestock, humans and environmental samples around the globe: a systematic review and meta-analysis.\u003c/em\u003e 2024. \u003cstrong\u003e14\u003c/strong\u003e(1): p. 16333.\u003c/li\u003e\n\u003cli\u003eBasnet, A., et al., \u003cem\u003eAssessment of Antibiotic Resistance among Clinical Isolates of Enterobacteriaceae in Nepal.\u003c/em\u003e 2024. \u003cstrong\u003e1\u003c/strong\u003e(aop).\u003c/li\u003e\n\u003cli\u003eSarathi, S., et al., \u003cem\u003eMicrobiological characterization and clinical facets of Elizabethkingia bloodstream infections in a tertiary care hospital of eastern India.\u003c/em\u003e 2023: p. 3257-3267.\u003c/li\u003e\n\u003cli\u003eChang, Y., et al., \u003cem\u003eMBLs, rather than efflux pumps, led to carbapenem resistance in fosfomycin and aztreonam/avibactam resistant Elizabethkingia anophelis.\u003c/em\u003e 2021: p. 315-327.\u003c/li\u003e\n\u003cli\u003eHuang, Y.-C., et al., \u003cem\u003eComparison of clinical characteristics of bacteremia from Elizabethkingia meningoseptica and other carbapenem-resistant, non-fermenting Gram-negative bacilli at a tertiary medical center.\u003c/em\u003e 2019. \u003cstrong\u003e52\u003c/strong\u003e(2): p. 304-311.\u003c/li\u003e\n\u003cli\u003eRahmat Ullah, S., et al., \u003cem\u003eComprehensive insights into Klebsiella pneumoniae: unravelling clinical impact, epidemiological trends and antibiotic-resistance challenges.\u003c/em\u003e 2024: p. dkae184.\u003c/li\u003e\n\u003cli\u003eBlot, S., et al., \u003cem\u003eHealthcare-associated infections in adult intensive care unit patients: Changes in epidemiology, diagnosis, prevention and contributions of new technologies.\u003c/em\u003e 2022. \u003cstrong\u003e70\u003c/strong\u003e: p. 103227.\u003c/li\u003e\n\u003cli\u003eMiron, M., et al., \u003cem\u003eHospital-Acquired Pneumonia and Ventilator-Associated Pneumonia: A Literature Review.\u003c/em\u003e 2024. \u003cstrong\u003e12\u003c/strong\u003e(1): p. 213.\u003c/li\u003e\n\u003cli\u003eRavi, K. and B.J.B. Singh, \u003cem\u003eESKAPE: Navigating the Global Battlefield for Antimicrobial Resistance and Defense in Hospitals.\u003c/em\u003e 2024. \u003cstrong\u003e3\u003c/strong\u003e(2): p. 76-98.\u003c/li\u003e\n\u003cli\u003ePaudel, R., et al., \u003cem\u003eCarbapenemase Producing Gram Negative Bacteria: Review of Resistance and Detection Methods.\u003c/em\u003e 2024: p. 116370.\u003c/li\u003e\n\u003cli\u003eM\u0026oacute;, I. and G.J.J.A. da Silva, \u003cem\u003eTackling Carbapenem Resistance and the Imperative for One Health Strategies\u0026mdash;Insights from the Portuguese Perspective.\u003c/em\u003e 2024. \u003cstrong\u003e13\u003c/strong\u003e(6): p. 557.\u003c/li\u003e\n\u003cli\u003eHarding‐Crooks, R., et al., \u003cem\u003eDissemination of carbapenemase‐producing Enterobacteriaceae and associated resistance determinants through global food systems.\u003c/em\u003e 2023. \u003cstrong\u003e22\u003c/strong\u003e(4): p. 2706-2727.\u003c/li\u003e\n\u003cli\u003eMarino, A., et al., \u003cem\u003eCeftazidime/Avibactam and Meropenem/Vaborbactam for the Management of Enterobacterales Infections: A Narrative Review, Clinical Considerations, and Expert Opinion.\u003c/em\u003e 2023. \u003cstrong\u003e12\u003c/strong\u003e(10): p. 1521.\u003c/li\u003e\n\u003cli\u003eKaye, K.S., et al., \u003cem\u003eCefiderocol, a siderophore cephalosporin, as a treatment option for infections caused by carbapenem-resistant Enterobacterales.\u003c/em\u003e 2023. \u003cstrong\u003e12\u003c/strong\u003e(3): p. 777-806.\u003c/li\u003e\n\u003cli\u003eBovo, F., et al., \u003cem\u003eClonal dissemination of Klebsiella pneumoniae resistant to cefiderocol, ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam co-producing KPC and OXA-181 carbapenemase.\u003c/em\u003e 2023. \u003cstrong\u003e5\u003c/strong\u003e(4): p. dlad099.\u003c/li\u003e\n\u003cli\u003eAbushaheen, M.A., et al., \u003cem\u003eAntimicrobial resistance, mechanisms and its clinical significance.\u003c/em\u003e 2020. \u003cstrong\u003e66\u003c/strong\u003e(6): p. 100971.\u003c/li\u003e\n\u003cli\u003eMagiorakos, A., et al., \u003cem\u003eInfection prevention and control measures and tools for the prevention of entry of carbapenem-resistant Enterobacteriaceae into healthcare settings: guidance from the European Centre for Disease Prevention and Control.\u003c/em\u003e 2017. \u003cstrong\u003e6\u003c/strong\u003e: p. 1-17.\u003c/li\u003e\n\u003cli\u003eRamadan, R.A., et al., \u003cem\u003eCarbapenem-resistant Klebsiella pneumoniae among patients with ventilator-associated pneumonia: Evaluation of antibiotic combinations and susceptibility to new antibiotics.\u003c/em\u003e 2022: p. 3537-3548.\u003c/li\u003e\n\u003cli\u003eParasuraman, P., S. Busi, and J.-K. Lee, \u003cem\u003eStandard Microbiological Techniques (Staining, Morphological and Cultural Characteristics, Biochemical Properties, and Serotyping) in the Detection of ESKAPE Pathogens\u003c/em\u003e, in \u003cem\u003eESKAPE Pathogens: Detection, Mechanisms and Treatment Strategies\u003c/em\u003e. 2024, Springer. p. 119-155.\u003c/li\u003e\n\u003cli\u003eAltschul, S.F., et al., \u003cem\u003eBasic local alignment search tool.\u003c/em\u003e J Mol Biol, 1990. \u003cstrong\u003e215\u003c/strong\u003e(3): p. 403-10.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Carbapenemase, OXA-48, NDM, VIM, IMP, Acinetobacter baumannii, Klebsiella pneumoniae, Elizabethkingia, cefiderocol resistance, β-lactamase inhibitors, phylogenetic, VAP, Pakistan","lastPublishedDoi":"10.21203/rs.3.rs-6973343/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6973343/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground:\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCarbapenemase-producing Gram-negative bacteria are emerging as a significant threat to critically ill patients, particularly those with ventilator-associated pneumonia (VAP). The increasing resistance to last-resort antimicrobials\u0026mdash;including novel β-lactam/β-lactamase inhibitor combinations and cefiderocol\u0026mdash;warrants urgent molecular and epidemiological investigations.\u003c/p\u003e\u003cp\u003e\u003cb\u003eObjective:\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the antimicrobial resistance patterns, gene prevalence, and clonal relationships of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, and \u003cem\u003eElizabethkingia\u003c/em\u003e spp. isolated from VAP patients, with a focus on resistance to cefiderocol, imipenem/Relebactam, Meropenem/vaborbactam, and Ceftazidime/avibactam, and the presence of key Carbapenemase genes (OXA-48, NDM, VIM, and IMP).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods:\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 67 carbapenem-resistant isolates were obtained from 104 tracheal aspirate samples collected from ICU VAP patients between January 2022 and December 2023. Antimicrobial susceptibility testing was performed using broth microdilution and MIC strips. PCR and Sanger sequencing were used for genotypic detection of Carbapenemase genes. Phylogenetic analysis was conducted via maximum-likelihood trees, and structural modeling of OXA-48 enzymes was performed using SWISS-MODEL and PyMOL.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults:\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll \u003cem\u003eA. baumannii\u003c/em\u003e isolates (n\u0026thinsp;=\u0026thinsp;10) exhibited high-level resistance to cefiderocol and β-lactam/β-lactamase inhibitor combinations. PCR revealed blaOXA-48 in 3 isolates, blaNDM in 1, and blaVIM in 1, and co-carriage of blaOXA-48\u0026thinsp;+\u0026thinsp;blaIMP in another. \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates (n\u0026thinsp;=\u0026thinsp;7) showed partial susceptibility to Ceftazidime/avibactam but complete resistance to cefiderocol; blaOXA-48 and blaNDM were the predominant genes. \u003cem\u003eElizabethkingia\u003c/em\u003e spp. (n\u0026thinsp;=\u0026thinsp;12) showed universal resistance to all tested antibiotics and harbored blaOXA-48 (100%) and blaNDM (33%). Phylogenetic analysis revealed global clonal relationships and interspecies gene transfer, with zoonotic and environmental links. Structural modeling confirmed functional conservation of OXA-48 active sites across isolates.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion:\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study highlights the extensive spread of Carbapenemase genes across clinical and environmental strains of Gram-negative bacteria, with a worrying level of resistance to novel antibiotics. Our findings underscore the urgent need for genomic surveillance, rational antimicrobial use, and One Health-based infection control strategies to combat this escalating resistance crisis.\u003c/p\u003e","manuscriptTitle":"Molecular and Phylogenetic Characterization of Carbapenemase-Producing Gram- Negative Bacteria from VAP Patients: With Structural Insights into OXA-48","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 08:35:54","doi":"10.21203/rs.3.rs-6973343/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8fb55367-c538-46fd-ae52-c30b42350c4e","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-15T12:38:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 08:35:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6973343","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6973343","identity":"rs-6973343","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}