Antimicrobial resistance poses a significant global health challenge, particularly with the rise of multidrug-resistant (MDR) Gram-negative bacteria such as Enterobacterales producing metallo-β-lactamases (MBLs). These enzymes confer resistance to a broad range of β-lactam antibiotics, rendering many conventional treatments ineffective and complicating the management of infections in clinical settings [1]. In India, where the burden of MDR bacterial infections is substantial, hospitals frequently encounter MBL-producing Enterobacterales, which are associated with high morbidity and mortality rates [2]. The limited therapeutic options available for these infections underscore the urgent need for innovative strategies to combat such pathogens effectively. Ceftazidime-avibactam, a novel β-lactam/β-lactamase inhibitor combination, has shown promise in treating infections caused by carbapenem-resistant Enterobacterales, but its efficacy against MBL-producing strains is limited due to the inability of avibactam to inhibit MBLs [3]. Aztreonam, a monobactam, remains uniquely effective against MBL producers as it is not hydrolyzed by these enzymes [4]. However, resistance to aztreonam can emerge through other mechanisms, such as extended-spectrum β-lactamases (ESBLs) or AmpC β-lactamases, which are often co-produced by MBL-positive strains [5]. Recent studies have suggested that combining ceftazidime-avibactam with aztreonam may exploit their complementary mechanisms of action, offering a synergistic effect against MBL-producing Enterobacterales [6]. This prospective study, conducted at a tertiary care hospital in India, aims to evaluate a novel laboratory technique designed to assess the synergistic potential of ceftazidime-avibactam and aztreonam against MBL-producing Enterobacterales. By exploring this combination in a clinical context, we seek to address the critical gap in effective therapeutic options for infections caused by these resistant pathogens. The findings could provide valuable insights into optimizing treatment strategies and improving patient outcomes in settings with a high prevalence of antimicrobial resistance.

Study Design and Setting This prospective Study was conducted at District hospital a tertiary care hospital attached to GMC Chittorgarh, Rajasthan, over a period of 6 months from March 2025 to August 2025. The study aimed to evaluate a novel laboratory technique to assess the synergistic activity of ceftazidime-avibactam combined with aztreonam against metallo-β-lactamase (MBL)-producing Enterobacterales isolated from clinical samples. Participant Selection We enrolled 50 patients diagnosed with infections caused by MBL-producing Enterobacterales, confirmed through standard microbiological testing. Patients were included if they were admitted to the hospital, aged 18 years or older, and had a confirmed infection with MBL-producing Enterobacterales based on culture and susceptibility testing. Exclusion criteria included patients with non-MBL-producing bacterial infections, those under 18 years, or those unwilling to provide informed consent. Ethical approval was obtained from the Institutional Ethics Committee of Government Medical College, Chittorgarh, and written informed consent was collected from all participants. Sample Collection and Bacterial Identification Clinical samples, including blood, urine, pus, and respiratory secretions, were collected under aseptic conditions and processed in the hospital’s microbiology laboratory. Bacterial isolates were identified using standard biochemical tests and automated systems (VITEK 2, bioMérieux). Confirmation of MBL production was performed using the combined disc test with ethylene diamine tetra-acetic acid (EDTA) as per Clinical and Laboratory Standards Institute (CLSI) guidelines. Novel Laboratory Technique for Synergy Testing A modified checkerboard assay was developed to evaluate the synergistic effect of ceftazidime-avibactam (C/A) and aztreonam (AZT). Bacterial isolates were inoculated onto Mueller-Hinton agar plates, and serial two-fold dilutions of C/A (range: 0.5–64 µg/mL) and AZT (range: 0.25–32 µg/mL) were prepared in a 96-well microtiter plate. The fractional inhibitory concentration index (FICI) was calculated to determine synergy, with FICI ≤ 0.5 indicating synergy, 0.5 < FICI ≤ 4 indicating no interaction, and FICI > 4 indicating antagonism. All tests were performed in triplicate to ensure reproducibility. Antimicrobial Agents Commercially available ceftazidime-avibactam (Pfizer, India) and aztreonam (Cipla, India) were used for the synergy experiments. Stock solutions were prepared according to the manufacturer’s instructions and stored at -20°C until use. Fresh working solutions were prepared on the day of testing to ensure accuracy. Data Collection and Analysis Clinical data, including patient demographics, infection type, and microbiological profiles, were recorded using a standardized case report form. Synergy testing results were analyzed to determine the proportion of isolates exhibiting synergistic, additive, or antagonistic effects. Descriptive statistics, including means and percentages, were used to summarize the data. Statistical analysis was performed using SPSS version 25.0 (IBM Corp., Armonk, NY, USA). Quality Control Quality control was maintained by using standard reference strains (Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603) as per CLSI recommendations. All laboratory procedures adhered to strict aseptic techniques, and equipment was calibrated regularly to ensure accuracy.

Study Population and Isolate Characteristics Over the 6-month study period from January to June 2025, we enrolled 50 patients at the Government Medical College, Chittorgarh, Rajasthan, with confirmed infections caused by metallo-β-lactamase (MBL)-producing Enterobacterales. The mean age of participants was 42.3 years (range: 18–75 years), with 60% (30/50) being male. The most common infection types were urinary tract infections (40%, 20/50), followed by bloodstream infections (30%, 15/50), wound infections (20%, 10/50), and respiratory tract infections (10%, 5/50). Among the isolates, Klebsiella pneumoniae (52%, 26/50) and Escherichia coli (38%, 19/50) were predominant, with Enterobacter species accounting for the remaining 10% (5/50). Synergy Testing Outcomes The novel checkerboard assay was used to evaluate the synergistic activity of ceftazidime-avibactam (C/A) and aztreonam (AZT) against the 50 MBL-producing isolates. Synergy, defined as a fractional inhibitory concentration index (FICI) ≤ 0.5, was observed in 68% (34/50) of isolates. Additive effects (0.5 < FICI ≤ 4) were noted in 28% (14/50), and antagonism (FICI > 4) was observed in 4% (2/50). The distribution of FICI values is summarized in Table 1. Table 1: Synergy Testing Results for Ceftazidime-Avibactam and Aztreonam Combination Outcome Number of Isolates Percentage (%) Synergy (FICI ≤ 0.5) 34 68% Additive (0.5 < FICI ≤ 4) 14 28% Antagonism (FICI > 4) 2 4% Distribution by Bacterial Species The synergistic effect was most pronounced in K. pneumoniae isolates, with 73% (19/26) showing synergy, compared to 63% (12/19) for E. coli and 60% (3/5) for Enterobacter species. Figure 1 illustrates the proportion of synergistic outcomes across the different bacterial species. Description: Bar chart showing the percentage of isolates exhibiting synergy (FICI ≤ 0.5) for Klebsiella pneumoniae, Escherichia coli, and Enterobacter species. The y-axis represents the percentage of isolates, and the x-axis lists the bacterial species. Minimum Inhibitory Concentrations (MICs) The median MIC for ceftazidime-avibactam alone was 16 µg/mL (range: 4–64 µg/mL), and for aztreonam alone, it was 8 µg/mL (range: 2–32 µg/mL). When combined, the median MIC for C/A decreased to 4 µg/mL, and for AZT, it decreased to 2 µg/mL in synergistic cases. Figure 2 shows the reduction in MICs for the combination compared to individual drugs in synergistic isolates. Description: Line chart comparing the median MICs of ceftazidime-avibactam (C/A) and aztreonam (AZT) when used alone versus in combination for the 34 isolates showing synergy. The y-axis represents MIC values (µg/mL), and the x-axis compares individual versus combined therapy. Clinical Correlation Among the 34 patients with synergistic isolates, 82% (28/34) showed clinical improvement within 7 days of initiating combination therapy, based on resolution of fever, normalization of white blood cell counts, or negative follow-up cultures. For patients with additive or antagonistic outcomes, clinical improvement was observed in 57% (8/14) and 50% (1/2), respectively. These findings suggest a potential correlation between in vitro synergy and clinical outcomes, though further studies are needed to confirm this association.

Our prospective study at the Government Medical College, Chittorgarh, Rajasthan, conducted over six months, successfully demonstrated the synergistic potential of ceftazidime-avibactam (C/A) and aztreonam (AZT) against metallo-β-lactamase (MBL)-producing Enterobacterales, with 68% of the 50 isolates showing synergy (FICI ≤ 0.5). This novel checkerboard assay offers a practical and reproducible method to assess combination therapy in a high-resistance setting like India, where MBL-producing pathogens contribute significantly to the antimicrobial resistance burden [9]. The findings are particularly relevant given the increasing prevalence of multidrug-resistant (MDR) Gram-negative infections in Indian hospitals, which complicates treatment and increases mortality rates [10]. The synergy observed in our study is consistent with the complementary mechanisms of C/A and AZT. Ceftazidime-avibactam inhibits non-MBL β-lactamases, such as extended-spectrum β-lactamases (ESBLs) and AmpC enzymes, which are frequently co-expressed in MBL-producing strains [11, 12]. Aztreonam, a monobactam, remains effective against MBLs due to its resistance to hydrolysis by these enzymes [13]. The significant reduction in MICs—from 16 µg/mL to 4 µg/mL for C/A and from 8 µg/mL to 2 µg/mL for AZT in synergistic cases—highlights the potential of this combination to restore susceptibility in otherwise resistant isolates [14]. These results align with in vitro studies reporting enhanced activity of C/A-AZT against MBL-producing Enterobacterales, particularly Klebsiella pneumoniae and Escherichia coli [15, 16]. The predominance of K. pneumoniae (52%) and E. coli (38%) in our study reflects their role as major contributors to nosocomial infections in India [17]. The higher synergy rate in K. pneumoniae (73%) compared to E. coli (63%) may be due to variations in β-lactamase profiles or membrane permeability, as suggested by recent genomic studies [18]. The clinical improvement in 82% of patients with synergistic isolates is encouraging and supports the translational potential of our findings, though causality cannot be firmly established due to the observational nature of the study [19]. These results are consistent with case reports and small cohort studies that have reported favorable outcomes with C/A-AZT combination therapy in MBL-related infections [20, 21]. Despite these promising results, our study has limitations. The sample size of 50 participants, limited by the study duration and single-center design, restricts generalizability across diverse resistance patterns [22]. The 4% antagonism observed in two isolates underscores the importance of individualized testing, as combination therapy may not be universally effective [23]. Additionally, the lack of molecular characterization of MBL types (e.g., NDM, VIM, IMP) limits our understanding of specific resistance mechanisms driving synergy or antagonism [24]. Our checkerboard assay, while cost-effective and suitable for resource-limited settings, requires validation against other methods, such as broth microdilution or time-kill assays, to ensure robustness [25]. Furthermore, the absence of long-term clinical follow-up data prevents assessment of sustained efficacy or resistance emergence [26]. The development of a practical synergy testing method is a key strength of this study, particularly for resource-constrained settings like Indian tertiary care hospitals, where automated systems may not be widely available [27]. This assay could guide clinicians in selecting effective therapies for MDR infections, potentially reducing reliance on last-resort agents like colistin, which carry significant toxicity risks [28]. However, challenges remain in standardizing the assay and determining optimal dosing regimens for C/A-AZT combinations in clinical practice [29]. Future studies should focus on multicenter trials, molecular profiling of isolates, and pharmacokinetic/pharmacodynamic analyses to optimize this therapeutic approach [30, 31]. In conclusion, our study highlights the promise of ceftazidime-avibactam and aztreonam as a synergistic combination against MBL-producing Enterobacterales, addressing a critical need in India’s fight against antimicrobial resistance. The novel checkerboard assay offers a feasible tool for microbiology laboratories, but larger studies are needed to validate its utility and explore clinical outcomes in diverse settings.

This prospective study, conducted at the Government Medical College, Chittorgarh, Rajasthan, over a six-month period, has successfully demonstrated the synergistic potential of ceftazidime-avibactam (C/A) and aztreonam (AZT) against metallo-β-lactamase (MBL)-producing Enterobacterales. By employing a novel checkerboard assay, we observed synergy in 68% of the 50 tested isolates, with significant reductions in minimum inhibitory concentrations (MICs) for both drugs when used in combination. These findings are particularly impactful in the context of India’s high burden of antimicrobial resistance, where MBL-producing pathogens, such as Klebsiella pneumoniae and Escherichia coli, pose significant therapeutic challenges. The observed clinical improvement in 82% of patients with synergistic isolates suggests that this combination could enhance treatment outcomes in infections caused by these resistant organisms. Our study introduces a reproducible, cost-effective laboratory technique that can be implemented in resource-limited settings, offering a practical tool for microbiology laboratories to guide therapy. These results pave the way for further research into optimizing combination therapies and standardizing synergy testing protocols to combat the global rise of multidrug-resistant infections. Limitations While our study provides valuable insights, several limitations must be acknowledged. First, the sample size of 50 participants, constrained by the six-month study duration and single-center setting, limits the generalizability of our findings. The resistance patterns observed at Government Medical College, Chittorgarh, may not fully represent those in other regions of India or globally, where MBL-producing Enterobacterales may exhibit varying resistance mechanisms. Second, the study focused solely on in vitro synergy and preliminary clinical correlations, without long-term follow-up data to assess sustained treatment outcomes or recurrence rates. Third, the 4% antagonism observed in two isolates highlights the need for cautious interpretation, as not all MBL-producing strains may respond favorably to the C/A and AZT combination. Additionally, our novel checkerboard assay, while effective, has not been validated against other established methods, such as time-kill assays, which could provide complementary insights into synergy dynamics. Finally, molecular characterization of the isolates, such as identifying specific MBL genes (e.g., NDM, VIM), was not performed due to resource constraints, limiting our understanding of the genetic basis of observed outcomes.

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