European Journal of Cardiovascular Medicine

Multidrug-resistant (MDR) Gram-negative bacilli (GNB) have emerged as a major global health threat, particularly in critical care settings such as intensive care units (ICUs), where patients are more vulnerable to severe infections due to invasive procedures, prolonged hospital stays, and prior antibiotic exposure [1,2]. Infections caused by these organisms are associated with high morbidity, mortality, and healthcare costs [3].

Among the clinically important GNB, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii are frequently encountered in ICU patients and are often implicated in bloodstream infections, ventilator-associated pneumonia, urinary tract infections, and wound infections [4,5]. These pathogens exhibit increasing resistance to β-lactams, carbapenems, aminoglycosides, and fluoroquinolones, leaving limited therapeutic options such as colistin and tigecycline [6].

A key mechanism underlying resistance in GNB is the production of β-lactamases, including extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, and carbapenemases. ESBLs, mainly encoded by blaTEM, blaSHV, and blaCTX-M, hydrolyze third-generation cephalosporins and are often plasmid-mediated, facilitating rapid dissemination [7]. Carbapenem resistance is largely due to carbapenemase enzymes such as New Delhi metallo-β-lactamase (blaNDM), Klebsiella pneumoniae carbapenemase (blaKPC), and oxacillinase-48-like enzymes (blaOXA-48) [8,9]. The coexistence of ESBL and carbapenemase genes in a single isolate further complicates treatment and infection control [10].

India has been recognized as one of the epicenters of antimicrobial resistance (AMR), with high rates of ESBL- and carbapenemase-producing GNB reported from tertiary care hospitals [11,12]. ICU patients are disproportionately affected due to frequent exposure to broad-spectrum antibiotics and invasive interventions [13]. Molecular characterization of resistance genes is essential to understand local epidemiology, guide antimicrobial stewardship, and implement effective infection control measures [14].

Given this background, the present study was undertaken to investigate the molecular characterization and antimicrobial resistance patterns of multidrug-resistant Gram-negative bacilli isolated from ICU patients at a tertiary care hospital in Kolkata, India. This work aims to provide insights into the prevalence of resistance genes and their clinical implications, thereby contributing to better patient management and containment of antimicrobial resistance.

Study Design and Setting

This was a prospective, observational study conducted in the Department of Microbiology, Calcutta National Medical College and Hospital, Kolkata, West Bengal. The study was carried out over one year, from January 2024 to December 2024. A total of 100 clinical samples were obtained from ICU patients with suspected bacterial infections and included in the study.

Inclusion and Exclusion Criteria

• Inclusion criteria:
• ICU patients of all age groups and both sexes.
• Clinical specimens yielding Gram-negative bacilli (GNB) on culture.
• Exclusion criteria:
• Duplicate isolates from the same patient.
• Mixed growths with contaminants.
• Growth of Gram-positive bacteria or fungi.

Sample Collection and Transport

Clinical specimens, including blood, urine, sputum, endotracheal aspirates, bronchoalveolar lavage (BAL), wound swabs, pus, and catheter tips, were collected from ICU patients by treating clinicians using aseptic precautions.

• Blood samples were inoculated into automated blood culture bottles (BACTEC system) and incubated.
• Other samples were collected in sterile containers and transported to the microbiology laboratory within 1 hour of collection, following biosafety protocols.

Culture and Isolation of Bacteria

• Specimens were inoculated onto Blood agar, MacConkey agar, and Chocolate agar and incubated at 37°C for 18–24 hours under aerobic conditions.
• Preliminary identification of Gram-negative bacilli was based on Gram staining, colony morphology, and biochemical reactions including oxidase, triple sugar iron (TSI), indole, methyl red, Voges–Proskauer, citrate utilization, urease, and motility tests.
• Final species-level identification was confirmed using the VITEK-2 Compact (bioMérieux, France) automated system.

Antimicrobial Susceptibility Testing (AST)

• AST was performed by the Kirby–Bauer disk diffusion method on Mueller–Hinton agar as per Clinical and Laboratory Standards Institute (CLSI) 2023 guidelines.
• The antibiotic panel included:
• β-lactams: Ampicillin, cefotaxime, ceftazidime, cefepime, piperacillin-tazobactam, imipenem, meropenem
• Aminoglycosides: Amikacin, gentamicin
• Fluoroquinolones: Ciprofloxacin, levofloxacin
• Others: Trimethoprim-sulfamethoxazole, tigecycline, colistin
• MIC determination: For carbapenems, tigecycline, and colistin, MICs were determined by broth microdilution and/or E-test strips.
• Interpretation: Isolates were classified as susceptible, intermediate, or resistant as per CLSI 2023 breakpoints.
• Definition of MDR: Isolates resistant to ≥1 antimicrobial agent in ≥3 different antibiotic classes were considered multidrug-resistant (MDR).

Phenotypic Detection of Resistance Mechanisms

• Extended-Spectrum β-Lactamase (ESBL): Confirmed by combined disk test using cefotaxime and ceftazidime with and without clavulanic acid.
• Carbapenemase production: Detected using the modified Hodge test (MHT) and carbapenem inactivation method (CIM).
• AmpC β-lactamase: Detected using the AmpC disk test.

Molecular Characterization of Resistance Genes

• DNA Extraction: Performed using the boiling lysis method. Briefly, bacterial colonies were suspended in 200 µL of sterile distilled water, boiled at 100°C for 10 minutes, and centrifuged at 12,000 rpm for 10 minutes. The supernatant containing DNA was used as a template.
• PCR Amplification: Conventional PCR was carried out for the detection of resistance genes using specific primers. The following genes were targeted:
• ESBL genes: bla_TEM, bla_SHV, bla_CTX-M
• Carbapenemase genes: bla_NDM, bla_KPC, bla_OXA-48, bla_VIM, bla_IMP
• AmpC genes: bla_CMY, bla_DHA
• PCR conditions: Initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation (95°C for 30s), annealing (55–60°C for 30s depending on primer set), and extension (72°C for 1 min), with a final extension at 72°C for 7 min.
• Agarose Gel Electrophoresis: PCR products were run on 1.5% agarose gel stained with ethidium bromide and visualized under UV transillumination.
• Sequencing: Selected representative amplicons were purified and sequenced (Sanger sequencing) to confirm gene identity.

Quality Control

• The following ATCC reference strains were used for quality control:
• Escherichia coli ATCC 25922 (antimicrobial susceptibility control)
• Klebsiella pneumoniae ATCC 700603 (ESBL-positive control)
• Pseudomonas aeruginosa ATCC 27853 (susceptibility control)

Data Collection and Statistical Analysis

• Clinical and demographic data, including age, sex, ICU stay duration, comorbidities, invasive device use, prior antibiotic exposure, and patient outcomes, were recorded in a predesigned proforma.
• Laboratory findings, including isolate type, antimicrobial resistance profile, and molecular gene detection, were entered into Microsoft Excel 2019.
• Statistical analysis was carried out using SPSS version 25.0 (IBM Corp., USA).
• Categorical variables were compared using the Chi-square test or Fisher’s exact test, while continuous variables were analyzed using Student’s t-test.
• A p-value < 0.05 was considered statistically significant.

A total of 100 non-duplicate Gram-negative bacillus isolates were obtained from ICU patients during the 1-year study period at Calcutta National Medical College and Hospital. The isolates were identified, and their antimicrobial resistance patterns and molecular characterization were studied.

Table 1: Distribution of Clinical Specimens

Table 2: Distribution of Gram-Negative Bacilli Isolates

Table 3: Antimicrobial Resistance Pattern of Isolates

Table 4: Prevalence of Multidrug Resistance (MDR)

Table 5: Molecular Detection of Resistance Genes

Fig; 1 Molecular Detection of Resistance Genes

Table 6: Co-existence of Resistance Genes

Table 7: Clinical Outcomes of ICU Patients (n=100)

The emergence and dissemination of multidrug-resistant (MDR) Gram-negative bacilli (GNB) in intensive care units (ICUs) have become a major global health concern, posing significant challenges to patient management and infection control. Critically ill patients are highly vulnerable due to invasive procedures, prolonged hospital stays, and broad-spectrum antibiotic exposure, which promote colonization and infection by resistant pathogens [1]. Our study, conducted on 100 ICU patients at Calcutta National Medical College and Hospital over one year, provides valuable insights into the spectrum of MDR GNB, their antimicrobial resistance patterns, and molecular mechanisms of resistance.

In our study, Klebsiella pneumoniae (35%) was the most frequently isolated MDR GNB, followed by Acinetobacter baumannii (25%), Pseudomonas aeruginosa (20%), Escherichia coli (15%), and others (5%). This distribution is in agreement with previous Indian studies, where K. pneumoniae and A. baumannii have been reported as predominant ICU pathogens [2,3]. The high prevalence of K. pneumoniae and Acinetobacter reflects their ability to survive in hospital environments, form biofilms, and acquire resistance determinants.

Antimicrobial Resistance Patterns

We observed alarmingly high resistance rates to cephalosporins (>80%), fluoroquinolones (>70%), and carbapenems (65% overall). Similar resistance trends have been documented in national and international studies, underscoring the widespread dissemination of extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, and carbapenemases [4–6]. Carbapenem resistance was particularly notable in A. baumannii (80%) and K. pneumoniae (70%), which is concerning since carbapenems are often the last line of defense. Colistin retained good activity (susceptibility ~85%), though emerging resistance (15%) was noted, consistent with recent reports of plasmid-mediated mcr genes [7].

Molecular Characterization

Molecular assays revealed that bla_NDM-1 and bla_OXA-48 were the predominant carbapenemase genes, particularly in K. pneumoniae and A. baumannii. Bla_NDM-1 was detected in 40% of carbapenem-resistant isolates, while bla_OXA-48 was present in 30%. This correlates with earlier studies from India reporting the widespread prevalence of bla_NDM in Enterobacteriaceae and bla_OXAin Acinetobacter [8–10]. Co-existence of multiple resistance genes was observed in some isolates, indicating horizontal gene transfer and clonal dissemination.

Clinical and Epidemiological Significance

The predominance of MDR GNB in ICU patients has serious therapeutic implications. Mortality and morbidity are significantly higher in patients infected with carbapenem-resistant organisms [11]. The limited efficacy of available antibiotics necessitates the rational use of last-resort agents like colistin and tigecycline. Furthermore, the detection of bla_NDM and bla_OXA highlights the urgent need for strict infection control, antimicrobial stewardship, and routine molecular surveillance to curb the spread of these resistance genes.

Comparison with Other Studies

Our findings are consistent with multicentric Indian surveillance programs that have reported rising carbapenem resistance in K. pneumoniae (55–70%) and A. baumannii (>75%) [12]. A similar pattern was observed in global studies, including the SENTRY Antimicrobial Surveillance Program, where carbapenem resistance exceeded 50% in Asia-Pacific isolates [13]. The emergence of bla_NDM-positive strains from Indian hospitals has been well-documented, reinforcing India’s role as a hotspot for the dissemination of these resistance determinants [14].

Limitations

The study was limited by its single-center design and sample size of 100, which may not reflect the entire region’s epidemiology. Molecular characterization was limited to common carbapenemase genes; whole-genome sequencing would provide deeper insights into clonal relatedness and resistance mechanisms.

This study highlights the high prevalence of multidrug-resistant Gram-negative bacilli among ICU patients, with Klebsiella pneumoniae and Acinetobacter baumannii being the most frequent isolates. A significant proportion exhibited carbapenem resistance and production of ESBLs and MBLs, posing serious challenges to therapeutic management. The findings emphasize the urgent need for strict antimicrobial stewardship, infection control practices, and continuous surveillance to prevent the spread of resistance and improve patient outcomes.

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