Assessing the Synergistic Potential of Ceftazidime–avibactam and Aztreonam against Metallo-beta-lactamase Producing Enterobacterales by Disc Elution and Disc E Strip – Synergy Method

Abstract

Introduction:

Emergence of carbapenem-resistant Gram-negative pathogens poses a significant challenge due to limited treatment options and higher mortality rates associated with infections caused by these pathogens. A combination of aztreonam (AT) and ceftazidime/avibactam (CZA-AVI) is an effective combination with synergistic activity against metallo-beta-lactamase (MBL) producing carbapenem resistant Enterobacterales (CRE). This study aimed to compare the disc elution and disc-E strip method for assessing the synergistic effect of aztreonam and ceftazidime/avibactam against MBL-producing CRE, with the potential to inform effective treatment strategies.

Methodology:

A total of 55 samples were isolated from clinical samples, in which 65.45% Klebsiella pneumoniae and 27.27% Escherichia coli were resistant to the tested carbapenem (meropenem) by the Vitek-2 compact system. Molecular detection of the New Delhi MBL, Verona integron-encoded MBL, or imipenemase gene was performed by conventional multiplex polymerase chain reaction. The synergy between aztreonam (30 µg) and ceftazidime–avibactam (30/20 µg) against MBL-producing CRE was assessed using both the disc elution and the disc-E strip synergy method.

Results:

Out of the 55 carbapenem-resistant isolates, K. pneumoniae (65.4%) being the most common isolate, and urinary tract and bloodstream infections were the primary sources. Neutropenia was a significant risk factor, and high antibiotic resistance was observed, where 63.4% isolates were extensively drug-resistant, whereas 21% were pan-drug resistant, and 15.4% multidrug-resistant. With increasing awareness among clinicians, CZA-AVI + AT is being used. Comparison between disc elution and disc E-test showed 96.4% concordance.

Conclusion:

This study highlights the need to evaluate ATM and CAZ/AVI as potential therapeutic alternative for the treatment of infections caused by MBL-producing Enterobacterales. Hence, synergy testing should be incorporated into routine laboratory practises to save time and give appropriate targeted therapy.

INTRODUCTION

The global increase in antimicrobial resistance among Gram-negative bacteria has emerged as a major public health concern, particularly due to resistance to carbapenems, which are often reserved as last-line agents for severe infections. Infections caused by carbapenem-resistant organisms are associated with prolonged hospital stays, limited therapeutic options, and increased mortality. Among Gram-negative pathogens, Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii are the most frequently implicated organisms, giving rise to the clinically significant entities known as carbapenem resistant Enterobacterales (CRE), carbapenem-resistant P. aeruginosa, and carbapenem-resistant A. baumannii, respectively.[1,2]

A key mechanism contributing to carbapenem resistance in Enterobacterales is the production of carbapenem-hydrolyzing enzymes, particularly metallo-beta-lactamases (MBLs). These enzymes, classified under Ambler class B, include New Delhi metallo-beta-lactamase (NDM), Verona integron-encoded metallo-beta-lactamase (VIM), and imipenemase (IMP). MBLs are capable of hydrolyzing nearly all beta-lactam antibiotics, with the notable exception of monobactams such as aztreonam, and are not inhibited by currently available beta-lactamase inhibitors. The plasmid-mediated transmission of MBL genes further accelerates their dissemination within healthcare settings.[2,3]

Surveillance data from India have consistently demonstrated a high burden of carbapenem resistance. Reports from the Indian Council of Medical Research indicate substantial resistance rates among Klebsiella pneumoniae and Escherichia coli, with NDM being the predominant carbapenemase, either alone or in combination with other enzymes such as OXA-48.[4] This alarming trend significantly restricts effective antimicrobial choices and complicates patient management.

Traditional treatment options for infections caused by MBL-producing organisms have largely relied on agents such as colistin and fosfomycin. However, these therapies are limited by concerns regarding nephrotoxicity, suboptimal tissue penetration, and the emergence of resistance. In recent years, the combination of ceftazidime–avibactam (CAZ-AVI) with aztreonam (ATM) has emerged as a promising therapeutic strategy. While avibactam inhibits co-produced serine beta-lactamases, aztreonam remains stable against MBL-mediated hydrolysis, thereby restoring antimicrobial activity when used in combination.[5]

Clinical and in vitro studies have demonstrated improved outcomes with the CAZ-AVI and ATM combination, including a reduction in mortality when compared with colistin-based regimens.[5] Despite these encouraging findings, routine laboratory detection of synergistic activity remains limited. Phenotypic synergy testing methods, such as disc elution and E-test-based techniques, offer practical approaches for assessing the combined activity of CAZ-AVI and ATM, although comparative data on their performance are scarce.[6,7]

Given the growing prevalence of MBL-producing CRE and the clinical relevance of combination therapy, this study was undertaken to evaluate and compare the disc elution method and disc-E strip synergy method for detecting in vitro synergy between aztreonam and ceftazidime–avibactam against MBL-producing Enterobacterales. Incorporating reliable synergy testing into routine diagnostic workflows may aid clinicians in selecting timely and targeted antimicrobial therapy, thereby improving patient outcomes and supporting antimicrobial stewardship efforts.

METHODOLOGY

The study is undertaken in a tertiary care center from June 2024 to December 2024, with a prospective design and a duration of 6 months. The study aims to include clinically significant, nonrepetitive isolates of CRE that produce MBLs (NDM, VIM, or IMP). Isolates that are carbapenem-susceptible, non-MBL producing, or from organisms other than Enterobacterales were excluded from the study. The sample size consists of all CRE isolates producing MBLs, identified through conventional polymerase chain reaction (PCR). A purposive (convenient) sampling technique will be employed, and data were collected using a structured pro forma.

The samples, such as blood, urine, respiratory samples, skin, and soft tissue received, were processed using standard techniques, and the clinically significant isolates were identified using the MALDI-TOF MS technique. The antibiotic susceptibility pattern was determined by the Vitek 2 system. Isolates of Enterobacterales with MIC values for meropenem under the resistant category by CLSI-2024 were used for further study.

The conventional multiplex PCR was used to molecularly detect the NDM, VIM, or IMP gene. Following an overnight incubation period, the isolates grown on 5% sheep blood agar plates were subjected to the boiling method to extract their DNA.

The primer sequence is as follows:

• Primer set blaNDM-1

  • NDM1 – Forward (5’-CACCTCATGTTTGAATT CGCC-3’) 561 base pair

  • NDM1 – Reverse (5’-CTCTGTCACATCGAAAT CGC-3’) 561 base pair.

• Primer set blaVIM

  • VIM – Forward (5’-GATGGTGTTTGGTCG CATA-3’) 390 base pair

  • VIM – Reverse (5’-CGAATGCGCAGCACCAG-3’) 390 base pair.

• Primer set bla IMP.

  • IMP – Forward (5’-GGAATAGAGTGGCTTAAY TCTC-3’) 232 base pair

  • IMP – Reverse (5’-GGTTTAAYAAAACAACC ACC-3’) 232 base pair.

PCR cycle temperature and time settings were 94°C for 10 min for initial denaturation, followed by 30 cycles of 40 s at 94°C, 52°C at 30 s, 1 min at 72°C, and a final extension step of 7 min at 72°C. Post-PCR analysis of the product was done by Agarose gel electrophoresis.[8]

The synergy between aztreonam (30 µg) and ceftazidime–avibactam (30/20 µg) against MBL-producing CRE was assessed using both the disc elution method and the disc-E strip synergy method, with interpretations based on the CLSI 2024 guidelines, and the results were compared.[9]

The data were entered into Microsoft Excel and analyzed using IBM SPSS Statistics for Windows, Version 29.0 (IBM Corp., Armonk, NY, USA). Quantitative variables were summarized using the mean, whereas qualitative characteristics were summarized with numbers and proportions. To compare groups, the Chi-square test was applied, with statistical significance defined as a P < 0.05. As the outcome variables, patient information, risk factors, antibiotic course, and results were gathered from medical records. The study was conducted with Institutional ethics committee approval.

RESULTS

During 6 months, 55 CRE isolates were collected. Most of the patients were above 60, and the average age of the patients was 63 [Figure 1].

Figure 1.

Age-wise distribution of patients with Carbapenem-resistant bacteremia

The common isolated organism was K. pneumoniae (65.45%), followed by E. coli (27.27%) [Figure 2].

Figure 2.

Organism-wise distribution of patients with Carbapenem-resistant bacteremia

The most frequent source of infection was urinary tract infection and bloodstream infections (38.1%), commonly caused by K. pneumoniae, followed by respiratory tract infections (16.4%), skin and soft tissue infections (7.3%) [Figure 3].

Figure 3.

Sample-wise distribution of patients with Carbapenem-resistant bacteremia. SSTI = Skin and soft tissue infections

The common risk factors were determined, and the majority of the patients had comorbidities (82.6%), with the most common being hypertension (56.5%), followed by type 2 diabetes mellitus (54.3%) and immunocompromised status (45.65%) [Table 1].

Table 1.

Risk factors for Carbapenem resistant Enterobacterales

Risk factors	n (%)	OR	P
DM	25 (54.3)	2.59	0.357
HTN	26 (56.5)	0.288	0.286
Immunocompromised	21 (45.65)	0.204	0.206
HAI	31 (67.39)	0.415	0.375
ICU stay	28 (60.9)	0.33	0.322
Noninvasive surgical procedures	36 (78.26)	0.607	0.463
Invasive surgical procedures	29 (63.04)	0.358	0.339
Prior antibiotic use	21 (45.6)	0.198	0.199
Use of corticosteroid	13 (28.26)	0.976	0.574
Presence of neutropenia	6 (13.04)	0.0370	0.004

DM=Diabetes mellitus, HTN=Hypertension, HAI=Healthcare-associated infection, ICU=Intensive care unit, OR=Odds ratio

The medical records of patients who tested positive for NDM were compared to identify major risk factors, and the association was evaluated using the Chi-square test. P < 0.05 were regarded as statistically significant.

Among the risk factors, the presence of neutropenia was a statistically important risk factor for CRE (P < 0.05).

The antibiotic susceptibility pattern was noted for the CRE isolates. Resistance was seen for all the drugs, 98% to cefoperazone sulbactam and piperacillin tazobactam, 94.2% to ciprofloxacin, 61.5% to ofloxacin, 63.46% to gentamicin, 63.46% to cotrimoxazole, 73.07% to ticarcillin clavulanate, 33.3% to fosfomycin (E. coli). 63.4% isolates were extensively drug-resistant (XDR), whereas 21% were pan drug resistant (PDR), and 15.4% multidrug-resistant (MDR) [Figure 4].

Figure 4.

Antibiotic susceptibility pattern of Carbapenem-resistant Enterobacterales isolates

Out of the total patients with CRE, 71% survived, whereas 29% succumbed to the infection. Patients who succumbed to infection were found to be statistically important (P < 0.05) [Figure 5].

Figure 5.

Clinical outcome of patients with Carbapenem-resistant Enterobacterales

Out of 55 isolates of MBL-producing CRE, 53 demonstrated synergy between aztreonam (30 µg) and ceftazidime–avibactam (30/20 µg) using both the disc elution and E-test synergy methods, while 2 isolates showed no synergy in the disc E-test method [Figure 6].

Figure 6.

This figure demonstrates the disk elution for detecting Aztreonam and ceftazidime-avibactam resistance. Tube 1 – Control tube – (Growth control); Tube 2 – 30 µg/mL concentration of Aztreonam; Tube 3 – 30/20 µg/mL concentration of Ceftazidime-avibactam; Tube 4 – Combination of 30 µg/mL concentration of Aztreonam and 30/20 µg/mL concentration of Ceftazidime-avibactam, which showed that the organism is sensitive to Aztreonam + Ceftazidime-avibactam combination

The synergy of ceftazidime-avibactam and aztreonam was also tested by the E-strip synergy technique. According to the findings, an inverted D sign was present in 53 out of 55 isolates. A comparison between the E-synergy test and disc elution was conducted. The results were 96.36% concordance [Figure 7].

Figure 7.

On the Muller-–Hinton agar plate, CAZ-AVI E-test with a preset AVI concentration of 4 mg/L. An ATM disc (30 g) was positioned 15 mm from the E-Strip’s center to the disc’s center. It was positioned at the 8 mg/L CAZ-AVI breakpoint, Result: presence of an inverse-D was interpreted as synergy

A wide array of antibiotics was employed for empirical therapy in the patients, with piperacillin–tazobactam (20.8%), cefoperazone–sulbactam (16.6%), and meropenem–teicoplanin (12.5%), being the most frequently used combinations. A combination of empirical antibiotics was used in 52% of the infection. About 43.7% received a combination of definitive antibiotic therapy. With increasing awareness among clinicians, CAZ-AVI+ AT is being used.

DISCUSSION

The rapid emergence and dissemination of CRE represent a critical challenge to contemporary clinical practice. Early identification of resistance mechanisms and timely initiation of effective therapy are essential to reduce morbidity and mortality associated with these infections. In the present study, we evaluated the synergistic activity of ceftazidime–avibactam combined with aztreonam against MBL-producing CRE isolates using two phenotypic methods, and assessed their concordance.

In this study, K. pneumoniae was the predominant isolate, accounting for nearly two-thirds of CRE cases, followed by E. coli. These findings are consistent with national and international surveillance data that identify K. pneumoniae as the most common carbapenem-resistant pathogen within the Enterobacterales group.[10] The majority of affected patients were elderly, with a mean age above 60 years, highlighting advanced age as an important demographic factor associated with CRE infections, as previously reported.[10]

Urinary tract and bloodstream infections were the most frequent clinical presentations observed in our cohort. Similar patterns have been documented in earlier studies, emphasizing these sites as major reservoirs for CRE infections in hospitalized patients.[11,12] The high prevalence of comorbid conditions such as diabetes mellitus and hypertension further underscores the vulnerability of patients with chronic illnesses to MDR infections.

Among the evaluated risk factors, neutropenia showed a statistically significant association with CRE infection.[13] This finding aligns with studies conducted among patients with hematological disorders, where immunosuppression has been identified as a major contributor to colonization and infection with carbapenem-resistant Gram-negative bacteria.[14] Other factors, including intensive care unit stay, invasive procedures, and prior antibiotic exposure, were common but did not demonstrate a statistically significant association in our cohort.[15]

Antimicrobial susceptibility testing revealed extensive resistance across multiple drug classes, with a high proportion of isolates classified as XDR or PDR. Such resistance patterns severely limit therapeutic options and reinforce the need for alternative treatment strategies. In this context, the CAZ-AVI and ATM combination has gained increasing attention due to its mechanistic rationale and demonstrated clinical benefit.[5,16]

In our study, synergy between ceftazidime–avibactam and aztreonam was observed in 96.4% of MBL-producing CRE isolates using both disc elution and disc-E strip methods, with a high degree of concordance between the two techniques. These findings are comparable to previous studies reporting high rates of in vitro synergy using similar methods.[6,7,17] The disc elution method, in particular, has been highlighted as a reliable and cost-effective approach for routine laboratory use.[7]

Clinically, the overall survival rate in our cohort was 71%, although mortality remained substantial. Prior studies have demonstrated significantly lower mortality among patients treated with CAZ-AVI plus aztreonam compared with colistin-based regimens, particularly in bloodstream infections caused by MBL-producing Enterobacterales.[16] The increasing use of this combination in our institution reflects growing clinician awareness and confidence in its efficacy.

CONCLUSION

The findings of this study support the integration of synergy testing into routine diagnostic microbiology workflows, especially in regions with a high prevalence of MBL-producing organisms. Early detection of synergistic activity can facilitate the timely initiation of targeted combination therapy, potentially improving clinical outcomes while minimizing unnecessary exposure to toxic agents.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.