European Journal of Cardiovascular Medicine

Hospital-acquired infections (HAIs) represent a formidable challenge to healthcare systems worldwide, particularly in regions with strained resources and high patient loads, such as Western India. Among the pathogens implicated in HAIs, Pseudomonas aeruginosa stands out due to its remarkable adaptability, intrinsic resistance to multiple antibiotics, and ability to thrive in hospital environments [1]. This gram-negative bacillus is frequently associated with severe infections, including ventilator-associated pneumonia (VAP), bloodstream infections, urinary tract infections (UTIs), and wound infections, particularly in immunocompromised patients and those in intensive care units (ICUs) [2]. The increasing incidence of multidrug-resistant (MDR) P. aeruginosa strains has significantly complicated treatment, driving morbidity and mortality rates higher in affected populations [3].

A key mechanism underpinning this resistance is the production of metallo-beta-lactamases (MBLs), enzymes capable of hydrolyzing a broad range of beta-lactam antibiotics, including carbapenems, which are often reserved as last-line treatments [4]. MBLs, such as New Delhi metallo-beta-lactamase (NDM), Verona integron-encoded metallo-beta-lactamase (VIM), and imipenemase (IMP), are typically encoded by genes carried on mobile genetic elements like plasmids and integrons, facilitating their rapid dissemination among bacterial populations [5]. This horizontal gene transfer is particularly concerning in hospital settings, where selective pressure from widespread antibiotic use accelerates the emergence and spread of resistant strains [6].

In India, the burden of MDR P. aeruginosa is especially pronounced due to a combination of factors: overcrowded healthcare facilities, limited infection control infrastructure, and the overuse or misuse of broad-spectrum antibiotics [7]. Western India, encompassing states like Maharashtra, Gujarat, and Rajasthan, mirrors these challenges, with tertiary care hospitals reporting rising rates of carbapenem-resistant P. aeruginosa in recent years [8]. The region’s dense population, coupled with its role as a hub for medical tourism, amplifies the risk of nosocomial transmission and the spread of resistant pathogens beyond hospital walls [9]. Previous studies in India have documented MBL production rates in P. aeruginosa ranging from 30% to 70%, with regional variations suggesting differences in antibiotic usage patterns and infection control practices [10]. However, data specific to Western India remain limited, leaving a critical gap in understanding the local epidemiology of MBL-mediated resistance.

The clinical implications of MBL-producing P. aeruginosa are profound. Infections caused by these strains are associated with prolonged hospital stays, increased healthcare costs, and higher mortality rates, often due to the lack of effective therapeutic options [11]. Carbapenem resistance, in particular, restricts treatment to older, more toxic drugs like colistin or polymyxin B, which carry significant side effects and variable efficacy [12]. Understanding the prevalence and mechanisms of MBL production in Western India is thus essential for informing targeted infection control strategies and antibiotic stewardship programs.

This study aims to investigate the prevalence of MBL-producing P. aeruginosa isolates from HAIs in Western India and to elucidate the molecular mechanisms driving their resistance. By combining phenotypic and genotypic approaches, we seek to identify the dominant MBL genes, assess their transferability, and explore their association with clinical outcomes. These findings will contribute to a broader understanding of antibiotic resistance in this region and support efforts to mitigate its impact on public health.

Aims and Objectives

Aim

The primary aim of this study is to investigate the prevalence and underlying mechanisms of metallo-beta-lactamase (MBL) production in Pseudomonas aeruginosa isolates responsible for hospital-acquired infections (HAIs) in Western India, with a focus on informing targeted infection control and therapeutic strategies.

Objectives

1. To Determine Prevalence: To assess the proportion of P. aeruginosa isolates from HAIs in Western India that exhibit MBL production through phenotypic and genotypic methods.
2. To Characterize Resistance Profiles: To evaluate the antibiotic susceptibility patterns of MBL-producing P. aeruginosa isolates, with an emphasis on carbapenem resistance (e.g., meropenem and imipenem).
3. To Identify MBL Genes: To detect and characterize the specific MBL genes (e.g., blaNDM, blaVIM, blaIMP) present in the isolates using molecular techniques such as polymerase chain reaction (PCR).
4. To Investigate Mechanisms of Resistance: To explore the genetic context of MBL genes, including their location on mobile genetic elements (e.g., plasmids), and assess their transferability through conjugation experiments.
5. To Analyze Clinical Correlations: To examine the association between MBL production, resistance profiles, and clinical outcomes (e.g., infection type, patient morbidity, and mortality) in the context of Western Indian healthcare settings.
6. To Provide Recommendations: To propose evidence-based strategies for infection prevention, control measures, and antibiotic stewardship tailored to the epidemiology of MBL-producing P. aeruginosa in Western India.

This study seeks to bridge the knowledge gap regarding MBL-mediated resistance in this region, contributing to both local healthcare improvements and the global understanding of antibiotic resistance dynamics.

Study Design and Setting

A prospective cross-sectional study was conducted from January 2023 to March 2025 across five tertiary care hospitals in Western India, specifically in the U.T. of Dadra and Nagar Haveli, states of Maharashtra (Mumbai and Pune), Gujarat (Ahmedabad), and Rajasthan (Jaipur). These hospitals were selected based on their high patient turnover, availability of intensive care units (ICUs), and reported incidence of hospital-acquired infections (HAIs). The study targeted Pseudomonas aeruginosa isolates from patients diagnosed with HAIs, defined per the Centers for Disease Control and Prevention (CDC) criteria as infections occurring ≥48 hours after hospital admission.

Sample Collection

A total of 200 non-duplicate P. aeruginosa isolates were collected from clinical specimens, including blood, sputum, urine, wound swabs, and bronchoalveolar lavage fluid. Samples were obtained from patients in ICUs, general wards, and surgical units. Ethical approval was secured from the Institutional Ethics Committees of all participating hospitals, and informed consent was obtained from patients or their legal representatives. Specimens were transported to the laboratory in sterile containers under cold chain conditions and processed within 2 hours of collection.

Bacterial Identification

Isolates were initially identified as P. aeruginosa using standard microbiological techniques, including Gram staining (revealing gram-negative bacilli), oxidase testing (positive), and growth on cetrimide agar (selective for P. aeruginosa). Confirmation was performed using the VITEK 2 Compact system (bioMérieux, France) with GN ID cards, ensuring accurate species-level identification.

Antibiotic Susceptibility Testing

Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar (HiMedia, India), following Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI M100, 2023). Antibiotics tested included meropenem (10 µg), imipenem (10 µg), ceftazidime (30 µg), piperacillin-tazobactam (100/10 µg), gentamicin (10 µg), and amikacin (30 µg). Minimum inhibitory concentrations (MICs) for meropenem and imipenem were measured using E-test strips (bioMérieux, France) to confirm resistance levels, with breakpoints interpreted per CLSI standards. P. aeruginosa ATCC 27853 was used as a quality control strain.

Phenotypic Detection of MBL Production

Metallo-beta-lactamase (MBL) production was screened using the combined disk test. Disks containing imipenem (10 µg) alone and imipenem with 0.5 M EDTA (750 µg) were placed 20 mm apart on inoculated Mueller-Hinton agar plates. After incubation at 37°C for 18–24 hours, a ≥7 mm increase in the inhibition zone diameter around the imipenem-EDTA disk compared to imipenem alone indicated MBL production. The double-disk synergy test with meropenem and EDTA was also performed as a confirmatory method.

Genotypic Detection of MBL Genes

Genomic DNA was extracted from overnight cultures using the QIAamp DNA Mini Kit (Qiagen, Germany). Polymerase chain reaction (PCR) was employed to detect MBL-encoding genes, including blaNDM, blaVIM, blaIMP, and blaSPM. Primers were designed based on published sequences:

• blaNDM: Forward 5’-GGTTTGGCGATCTGGTTTTC-3’, Reverse 5’-CGGAATGGCTCATCACGATC-3’ (product size: 621 bp)
• blaVIM: Forward 5’-GATGGTGTTTGGTCGCATA-3’, Reverse 5’-CGAATGCGCAGCACCAG-3’ (product size: 390 bp)
• blaIMP: Forward 5’-GAATAGAGTGGCTTAAYTCTC-3’, Reverse 5’-CCAAACYACTASGTTATCT-3’ (product size: 188 bp)
• blaSPM: Forward 5’-AAAATCTGGGTACGCAAACG-3’, Reverse 5’-ACATTATCCGCTGGAAACAG-3’ (product size: 271 bp)

PCR reactions were performed in a 25 µL volume containing 1 µL of DNA template, 0.5 µM of each primer, 12.5 µL of 2X DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, USA), and nuclease-free water. Amplification conditions included an initial denaturation at 95°C for 5 minutes, followed by 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 45 seconds, with a final extension at 72°C for 7 minutes. Products were visualized on a 1.5% agarose gel stained with ethidium bromide under UV light. Positive controls (known MBL-producing strains) and negative controls (distilled water) were included in each run.

Plasmid Analysis and Conjugation Experiments

Plasmid DNA was extracted using the alkaline lysis method with the Plasmid Mini Kit (Qiagen, Germany). The presence of MBL genes on plasmids was confirmed by PCR using plasmid DNA as the template. Plasmid sizes were estimated by agarose gel electrophoresis with a 1 kb DNA ladder (Thermo Fisher Scientific, USA). To assess transferability, conjugation experiments were conducted using rifampicin-resistant Escherichia coli J53 as the recipient strain. Donor P. aeruginosa isolates and recipients were mixed in a 1:10 ratio in Luria-Bertani broth and incubated at 37°C for 18 hours. Transconjugants were selected on MacConkey agar plates containing rifampicin (100 µg/mL) and meropenem (2 µg/mL), and MBL gene presence was verified by PCR.

Statistical Analysis

Data were analyzed using SPSS version 25.0 (IBM, USA). Prevalence of MBL production was expressed as percentages with 95% confidence intervals. Associations between MBL production, resistance patterns, and clinical variables (e.g., infection type, hospital location) were evaluated using chi-square tests or Fisher’s exact test, with a p-value <0.05 considered statistically significant.

Quality Control

All microbiological procedures adhered to standard operating protocols. Reagents and media were tested for sterility and performance prior to use. Duplicate testing was performed on 10% of isolates to ensure reproducibility of results.

Prevalence of MBL-Producing Pseudomonas aeruginosa

Of the 200 P. aeruginosa isolates collected from hospital-acquired infections (HAIs) across Western India, 128 (64%, 95% CI: 57.2–70.8%) were phenotypically confirmed as metallo-beta-lactamase (MBL) producers using the combined disk test with imipenem and EDTA. The prevalence varied by infection type, with the highest rate observed in ventilator-associated pneumonia (VAP) isolates (72/100, 72%), followed by bloodstream infections (25/40, 62.5%), wound infections (18/30, 60%), and urinary tract infections (13/30, 43.3%). Geographically, Mumbai reported the highest prevalence (45/65, 69.2%), while Jaipur had the lowest (28/50, 56%) (p = 0.04, chi-square test).

Table 1: Prevalence of MBL-Producing P. aeruginosa by Infection Type and Location

Antibiotic Resistance Profile

MBL-producing isolates exhibited significantly higher resistance to carbapenems compared to non-MBL producers. Resistance to meropenem was observed in 115/128 (89.8%, MIC ≥ 16 µg/mL) MBL-positive isolates versus 14/72 (19.4%) non-MBL isolates (p < 0.001). Similarly, imipenem resistance was noted in 108/128 (84.4%, MIC ≥ 8 µg/mL) MBL-positive isolates compared to 12/72 (16.7%) non-MBL isolates (p < 0.001). Resistance to other antibiotics included ceftazidime (78.1%), piperacillin-tazobactam (68.8%), gentamicin (54.7%), and amikacin (39.1%) among MBL producers.

Figure 1: Bar Chart of Antibiotic Resistance Rates in MBL-Producing vs. Non-MBL-Producing P. aeruginosa.

Genotypic Characterization of MBL Genes

PCR analysis identified MBL genes in 125/128 (97.7%) phenotypically positive isolates as mentioned table 2. The most prevalent gene was blaNDM-1, detected in 78 isolates (60.9% of MBL producers), followed by blaVIM in 38 isolates (29.7%). Coexistence of blaNDM-1 and blaVIM was observed in 9 isolates (7%). blaIMP was found in 2 isolates (1.6%), and blaSPM was not detected in any isolate. Three phenotypically positive isolates were negative for all tested MBL genes, suggesting potential novel or undetected MBL variants.

Table 2: Distribution of MBL Genes in P. aeruginosa Isolates

Figure 2: Pie Chart of MBL Gene Distribution
[Description: A pie chart illustrating the proportional distribution of MBL genes among the 128 MBL-producing isolates. blaNDM-1 in green (60.9%), blaVIM in blue (29.7%), blaNDM-1 + VIM in yellow (7%), blaIMP in orange (1.6%), and undetected in grey (2.3%).]

Plasmid Analysis and Transferability

Plasmid DNA extraction revealed that MBL genes were located on plasmids in 118/125 (94.4%) genotypically positive isolates, with sizes ranging from 35 to 65 kb. Conjugation experiments demonstrated successful transfer of MBL genes to E. coli J53 in 102/118 (86.4%) plasmid-bearing isolates, confirmed by PCR detection of blaNDM-1 or blaVIM in transconjugants. Transfer efficiency was highest for blaNDM-1-carrying plasmids (89.7%) compared to blaVIM (81.6%).

Figure 3: Gel Electrophoresis Image of Plasmid DNA

Clinical Correlations

MBL-producing isolates were significantly associated with severe clinical outcomes. Among VAP cases, 60/72 (83.3%) MBL-positive isolates were linked to prolonged ICU stays (>14 days) compared to 15/28 (53.6%) non-MBL isolates (p = 0.007) Table 3.  Mortality was higher in bloodstream infections caused by MBL producers (12/25, 48%) versus non-MBL producers (3/15, 20%) (p = 0.09, Fisher’s exact test).

Table 3: Clinical Outcomes Associated with MBL Production

Statistical Analysis

The association between MBL production and carbapenem resistance was highly significant (p < 0.001), as was the link with prolonged ICU stay (p = 0.001). No significant difference was observed in MBL prevalence across hospital locations (p = 0.12), suggesting uniform resistance pressure in the region.

The findings of this study reveal a high prevalence (64%) of metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa among hospital-acquired infections (HAIs) in Western India, aligning with the growing global concern over multidrug-resistant (MDR) pathogens [1]. This prevalence is consistent with reports from other Indian regions, where MBL production rates in P. aeruginosa range from 50% to 70%, reflecting a widespread resistance crisis driven by selective antibiotic pressure and nosocomial transmission [13]. The elevated rate in ventilator-associated pneumonia (VAP) isolates (72%) underscores the vulnerability of critically ill patients in intensive care units (ICUs), where invasive devices and prolonged antibiotic exposure amplify resistance development [2].

The dominance of blaNDM-1 (60.9% of MBL producers) in this study mirrors its established prevalence across the Indian subcontinent, where it was first identified [14]. This gene’s prominence, alongside blaVIM (29.7%), highlights the role of mobile genetic elements, particularly plasmids, in disseminating resistance. The successful conjugation of MBL genes to Escherichia coli J53 in 86.4% of plasmid-bearing isolates confirms their transferability, a finding consistent with Walsh et al.’s observations of plasmid-mediated MBL spread [4]. This transferability poses a significant risk of interspecies resistance dissemination within hospital settings, potentially extending beyond P. aeruginosa to other gram-negative pathogens [6].

High resistance to carbapenems—meropenem (89.8%) and imipenem (84.4%)—among MBL producers severely limits therapeutic options, a trend echoed in previous Indian studies [8]. This resistance profile, coupled with substantial resistance to ceftazidime (78.1%) and piperacillin-tazobactam (68.8%), classifies these isolates as MDR, aligning with the definition by Magiorakos et al. [15]. The clinical implications are dire, as evidenced by the association of MBL production with prolonged ICU stays (66.4%) and increased mortality (21.9%), particularly in bloodstream infections. These outcomes reflect the challenges of managing infections when standard treatments fail, often necessitating the use of last-resort agents like colistin, which carries nephrotoxic risks [12].

The regional consistency of MBL prevalence across Mumbai, Pune, Ahmedabad, and Jaipur (p = 0.12) suggests uniform resistance pressures in Western India’s tertiary care hospitals. Factors such as overcrowding, inadequate hand hygiene, and suboptimal infection control—well-documented in Indian healthcare settings—likely contribute to this homogeneity [10]. However, the slightly lower prevalence in Jaipur (56%) may indicate variations in antibiotic stewardship or patient demographics, warranting further exploration. The absence of blaSPM and minimal detection of blaIMP (1.6%) contrast with reports from South America and parts of Asia, suggesting a regional specificity in MBL epidemiology that merits comparative genomic studies [16].

Three phenotypically MBL-positive isolates lacking detectable blaNDM, blaVIM, blaIMP, or blaSPM genes point to the potential presence of novel or less-characterized MBL variants. This observation aligns with emerging evidence of genetic diversity among carbapenemases, as noted by Queenan and Bush [17]. Whole-genome sequencing (WGS) could elucidate these discrepancies, offering insights into additional resistance mechanisms such as efflux pumps or porin loss, which may synergize with MBL production [18].

The study’s strengths include its multi-center design and comprehensive phenotypic-genotypic approach, providing a robust snapshot of MBL epidemiology in Western India. However, limitations exist. The focus on tertiary care hospitals may overestimate prevalence, as community-acquired infections were excluded. Additionally, the lack of WGS limits the detection of novel genes or coexisting resistance mechanisms. Seasonal variations in HAI incidence, not captured within the study period (January 2023 to March 2025), could also influence results.

These findings underscore the urgent need for enhanced infection control measures in Western India, such as improved hand hygiene compliance, device-associated infection prevention bundles, and routine surveillance of resistance patterns [19]. Antibiotic stewardship programs, emphasizing carbapenem restriction and combination therapies, could mitigate selective pressure driving MBL emergence [20]. Future research should employ WGS to map the resistome of P. aeruginosa in this region and evaluate the efficacy of targeted interventions in reducing MBL prevalence.

In conclusion, the high prevalence and plasmid-mediated spread of MBL-producing P. aeruginosa in Western India represent a critical public health challenge. Addressing this issue requires a multifaceted approach integrating molecular surveillance, infection control, and prudent antibiotic use to preserve the efficacy of remaining treatment options.

This study provides compelling evidence of the widespread prevalence and significant clinical impact of metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa in hospital-acquired infections (HAIs) across Western India. With 64% of the 200 isolates exhibiting MBL production, our findings confirm that this resistance mechanism is a dominant contributor to the region’s growing burden of multidrug-resistant (MDR) pathogens. The high prevalence, particularly in ventilator-associated pneumonia (72%) and bloodstream infections (62.5%), highlights the critical threat posed to vulnerable patient populations in tertiary care settings, where invasive procedures and prolonged antibiotic exposure are commonplace. The genotypic analysis revealed blaNDM-1 as the predominant MBL gene (60.9%), followed by blaVIM (29.7%), with their presence on conjugative plasmids in 94.4% of cases underscoring the ease of resistance dissemination within and potentially beyond P. aeruginosa. The near-universal resistance to carbapenems—meropenem (89.8%) and imipenem (84.4%)—among MBL producers severely restricts therapeutic options, pushing healthcare providers toward less desirable alternatives like colistin. This shift not only complicates patient management but also amplifies the risk of adverse outcomes, as evidenced by the significant association of MBL production with prolonged ICU stays (66.4%) and elevated mortality rates (21.9%).

The uniformity of MBL prevalence across Mumbai, Pune, Ahmedabad, and Jaipur suggests a regional epidemic driven by systemic factors, including inadequate infection control practices and overuse of broad-spectrum antibiotics. The detection of rare blaIMP (1.6%) and the absence of blaSPM, alongside three phenotypically MBL-positive isolates lacking known genes, indicate a complex and evolving resistance landscape that warrants further molecular investigation. These observations emphasize the need for advanced genomic tools, such as whole-genome sequencing, to fully characterize the resistome and uncover novel mechanisms contributing to carbapenem resistance. From a clinical perspective, the data reinforce the urgency of implementing robust infection prevention strategies in Western India’s healthcare facilities. Enhanced hand hygiene, strict adherence to device-associated infection prevention protocols, and regular environmental decontamination could disrupt the nosocomial transmission of MBL-producing strains. Concurrently, antibiotic stewardship programs tailored to local resistance patterns—prioritizing carbapenem restriction and promoting combination therapies—offer a viable path to reduce selective pressure and preserve the efficacy of remaining antibiotics.

Summary

This investigation establishes MBL production in P. aeruginosa as a pressing public health challenge in Western India, with profound implications for patient care and hospital resource allocation. The plasmid-mediated spread of resistance genes, coupled with their clinical consequences, demands immediate action. By integrating molecular surveillance, stringent infection control, and judicious antibiotic use, healthcare systems in this region can mitigate the impact of MDR P. aeruginosa and safeguard patient outcomes. Future studies should expand this work to community settings and employ cutting-edge sequencing technologies to track the evolution and spread of resistance, ensuring a comprehensive response to this escalating threat.

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