European Journal of Cardiovascular Medicine

Enterobacterales, (including Escherichia coli, Klebsiella spp, Enterobacter spp.) along with Pseudomonas spp. and Acinetobacter spp. are common cause of infection in both in-patient and out-patient settings. Given their frequency in commonly encountered infections, continuous rising rates of antimicrobial resistance among Enterobacterales are emerging as a serious threat to both the community and Hospital.  An increasing prevalence of extended-spectrum β-lactamases, which cause resistance to most β-lactams except carbapenems, limits the therapeutic benefit of β-lactam antibiotics.(1) Carbapenems have long been employed as the mainstay for managing complicated infections caused by ESBL-producing Enterobacterales. However, the widespread use of carbapenems has resulted in the emergence of carbapenem-resistant organisms (CRO). (2) In recent years, there is a growing emphasis on developing effective carbapenem-sparing alternative antibiotics to preserve the efficacy of carbapenems and to control the development of resistance. (3) Various beta-lactam/beta-lactamase inhibitor (BL/BLI) combinations such as piperacillin-tazobactam, cefoperazone-sulbactam, ceftolozane-tazobactam have been employed as carbapenem-sparing agent in treatment of ESBL-producing pathogens.(3) Cefepime is a 4th-generation cephalosporin and has broad spectrum bactericidal activity against gram-negative and gram-positive pathogens and used in injectable form. It is approved for the treatment of a variety of severe infections since 1996 at the US FDA and since 1993 in Europe. It is an oxyimino-cephalosporin targeting penicillin binding protein (PBP)-3 in gram-negative bacteria with enhanced stability to degradation by chromosomal and plasmid mediated extended spectrum AmpC lactamases as well as OXA-48 like enzymes. Cefepime’s greater activity against gram-negative bacteria that produce ESBL versus other commercially available oxyimino-cephalosporins is mainly due to its more rapid penetration into bacteria, the targeting of specific PBPs and lower affinity for several β-lactamases (4,5). Enmetazobactam is a novel penicillanic acid sulfone β-lactamase inhibitor (BLI) that is similar in structure to tazobactam but has a strategically placed methyl group that imparts a net neutral charge (zwitterionic) and enhances bacterial cell penetration. (Fig.1)

Fig.1 Chemical structures of enmetazobactam and tazobactam. The zwitterionicity of enmetazobactam is highlighted in color. (Source - Morrissey et al.)

This methyl group on the triazole ring of enmetazobactam allows for many classical and nonclassical hydrogen bonding interactions not observed for tazobactam in the active site of CTX-M-15 (Ambler class A serine beta -lactamases).(6) Therefore, Cefepime-enmetazobactam combination is a novel BL/BLI. It has shown promising activity against a wide range of ESBL-producing and multidrug-resistant Gram-negative bacilli. Currently Cefepime-enmetazobactam combination is recommended for treatment of complicated UTI and hospital-acquired as well as ventilator-associated pneumonia in United States and Europe. Therefore, the potential role of this combination as an effective alternative in treating multi-drug-resistant strains need to be evaluated in Indian context. (7) Current study was undertaken to evaluate the efficacy of cefepime-enmetazobactam as a carbapenem-sparing agent in comparison with other antibiotics, like piperacillin-tazobactam and also its effectiveness in carbapenem-resistant organisms (CRO).

This prospective cross-sectional study was conducted at Jagannath Gupta Institute of Medical Sciences and Hospital, North-Kolkata, from March 2026 to October 2026 for a period of eight months. Before beginning the study Institutional Ethical Committee (IEC) clearance was obtained. The study was conducted to evaluate the in-vitro antibiotic susceptibility (AST) of cefepime-enmetazobactam and compare these with piperacillin-tazobactam, imipenem, meropenem and ertapenem against multi-drug-resistant Gram-negative bacteria.

Inclusion criteria: Urine, sputum, ET Tube, pus, tissue, BAL fluid and wound swab samples with significant growth of Gram-negative bacteria resistant to third generation cephalosporins.

Exclusion criteria: 1. Duplicate isolates from the same patient.

2. Samples showing contamination, mixed growth or insignificant growth.

Demographic and clinical data including age, sex, occupation, address, phone number, underlying systemic diseases were also collected and recorded. The urine, sputum, ET Tube, pus, tissue, BAL fluid and wound swab samples were collected maintaining all the aseptic precautions and immediately sent to the microbiology laboratory for microbial culture and sensitivity testing. The isolates were subjected to antibiotic susceptibility testing using the Kirby-Bauer disc diffusion method on Mueller-Hinton agar, following Clinical and Laboratory Standards Institute (CLSI) guidelines, 2025. Cefepime-enmetazobactam (30/20 µg), piperacillin-tazobactam (100/10 µg), meropenem (10 µg), imipenem (10 µg), ertapenem (10 µg), ceftazidime (30 µg), and cefotaxime (30 µg) discs were used. The antibiotic discs were procured from commercially available sources (Microexpress, Tulip Diagnostics, Goa, India). Isolates resistant to third generation cephalosporins (ceftazidime and cefotaxime) and positive in phenotypic confirmatory test for ESBL (ceftazidime and ceftazidime-clavulanate combined disc test) were considered as ESBL producer and isolates resistant to meropenem were considered as Carbapenem-resistant organisms (CRO). The clinical breakpoints of cefepime-enmetazobactam recommended by FDA for Enterobacterales and P. aeruginosa were followed in this study. Accordingly, the zone size of ≥21 mm and ≥18 mm were considered as susceptible and zone size of ≤20 mm and ≤17 mm were considered as resistant for Enterobacterales and Pseudomonas aeruginosa respectively. ⁽⁸⁾

A total of 149 third generation cephalosporins resistant Gram-negative bacterial isolates from urine, sputum, ET tube, pus, tissue, BAL fluid and wound swab samples were included in the study. The demographic distribution of the 149 patients showed that females constituted 57.00% (n=85) and male constituted 43.00% (n=64). (Fig 2)

Age-wise, the most affected age group was 61–90 years, accounting for 45.6% (n = 68) isolates. It was followed by individuals of 41–60 years, accounting for 38.2%, (n=57), then individuals of 21–40 years, accounting for 11.4% (n=17), then 11-20 years, accounting for 2.7% ( n=4), then children of 3-10 years accounting for .7% (n=1) and below 3 years accounting for 1.34% (n=2) isolates. (Fig. 3)

The predominant isolates were Escherichia coli, consisting of 42.2% (n = 63) of the total isolates. This was followed by Klebsiella pneumoniae (35.6%, n = 53), Pseudomonas species (20.8%, n = 31), Enterobacter spp. (5%, n = 1) and Acinetobacter spp. (n=1). (Fig. 3)

Among all the isolates, resistance to piperacillin-tazobactam was 67.6% (n=96), whereas resistance to cefepime-enmetazobactam was 54.2% (n=77). If these results are analysed using a standard two-proportion Chi-square test, treating them as independent groups, the p value is 0.0346, which is less than the standard significance threshold of 0.05. Since the exact same 149 isolates were exposed to both antibiotics in a laboratory setting, a McNemar’s test is technically ideal. Number of isolates resistant to both Piperacillin-Tazobactam and Cefepime-Enmetazobactam was (n=73). Number of isolates susceptible to both Piperacillin-Tazobactam and Cefepime-Enmetazobactam was (n=36). Number of isolates resistant to Piperacillin-Tazobactam but susceptible to Cefepime-Enmetazobactam was (n=23). Number of isolates susceptible to Piperacillin-Tazobactam but resistant to Cefepime-Enmetazobactam was (n=4).

Table 1. The 2×2 Contingency Table

* Note: The provided percentages (77/149 ≈ 51.6% and 96/149 ≈ 64.4%) suggest a total sample size of 149. The sum of the contingency cells provided (73 + 36 + 23 + 4) equals 136, meaning 13 isolates had intermediate data. McNemar's test was calculated strictly using the completed paired outcomes (n=137).

The McNemar test statistic (χ²) with continuity correction was calculated and a χ² value of 12.0 with 1 degree of freedom corresponds to a p-value of approximately 0.00053 was observed. Because the p-value is well below the standard significance threshold (α = 0.05), the findings are highly statistically significant, confirming that Cefepime-Enmetazobactam (FPE) exhibits significantly better in vitro activity (lower resistance rate) than Piperacillin-Tazobactam (PIT) in this cohort.

Ertapenem resistance was observed in 46.9% (n=70), meropenem resistance in 53.0% (n=79) and imipenem resistance in 55.7% (n=83) isolates, whereas cefepime-enmetazobactam resistant isolates were 54.2% (n=77). Running a statistical comparison across these four groups yields a p-value greater than 0.05 (typically around p ≈ 0.45). Because the p-value is well above the conventional threshold of 0.05, the differences between these resistance rates are not statistically significant. Cefepime-enmetazobactam is designed to overcome extended-spectrum beta-lactamases (ESBLs). However, if these specific isolates are already highly resistant to carbapenems like ertapenem, imipenem, and meropenem, they likely carry carbapenemases or severe porin mutations. Therefore, Cefepime-enmetazobactum is not promising against carbapenem-resistant organisms (CRO).

Treatment of infections in a health-care setting is becoming increasingly difficult due to the emergence of beta-lactamase producing multidrug resistant strains of Gram-negative bacteria. ESBLs are a group of beta-lactamase enzymes that confer resistance to third generation cephalosporins and aztreonam. (9) The major ESBLs are CTX-M, TEM and SHV belonging to Ambler class A. Various beta lactamase inhibitors like clavulanate, tazobactam, avibactam, vaborbactam and relebactam can inhibit the major ESBLs. Therefore, β-lactam/β-lactamase inhibitor combinations are popularly used in the treatment of ESBL-producing organisms. (10)  However, Gram-negative pathogens are increasingly becoming resistant to these β-lactam/β-lactamase inhibitor combinations owing to co-production of other beta-lactamases. (11) Inducible enzymes of Ambler class C like AmpC render third generation cephalosporins resistant after few days of treatment. KPC and OXA are serine carbapenemases belonging to Ambler class A and class D respectively and; NDM, VIM and IMP, popularly termed as metallo-beta-lactamase (MBL), are Ambler class B carbapenemases which typically contain zinc ion at their active site. (12) Over the last two decades rampant use of carbapenems has resulted in widespread dissemination of KPC, OXA and MBL resistant strains. Therefore, there is a growing emphasis on developing carbapenem-sparing alternatives. Although, piperacillin-tazobactam and cefoperazone-sulbactam have already been in use as carbapenem-sparing, β-lactam/β-lactamase inhibitor, Cefepime- enmetazobactam is a recent novel addition to the list.

Piperacillin-tazobactam are commonly prescribed for the treatment of nosocomial and healthcare-associated infections, not only for their antipseudomonal effect but also for their extended-spectrum activity in empirical therapy (13). The antimicrobial activity of piperacillin is enhanced by the addition of tazobactam against Gram-positive cocci (GPC), GNB, and anaerobic bacteria; therefore, piperacillin-tazobactam appears to be useful in the treatment of mixed aerobic and anaerobic infections (14). In a randomized, double-blind, clinical trial the clinical efficacy of cefepime-enmetazobactam for the treatment of ESBL-producing Enterobacterales was already demonstrated as superior when compared with piperacillin-tazobactam. Kaye et al. demonstrated that the clinical cure and microbiological eradication of complicated UTI or acute pyelonephritis caused by gram-negative pathogens, occurred in 79.1% (273/345) of patients receiving cefepime-enmetazobactam compared with 58.9% (196/333) receiving piperacillin-tazobactam. (15) In another in-vitro study Ramakrishnan et al. demonstrated that Cefepime-enmetazobactam is highly susceptibility, with 120 isolates (86.3%) being susceptible and only 19 isolates (13.7%) being resistant in disc diffusion method. This performance was superior to cefoperazone-sulbactam and piperacillin-tazobactam. Piperacillin-tazobactam showed 82% (n=114) susceptible, 1.4% (n=2) intermediate and 16.5% (n=23) resistant isolates. [16] Morrissey et al. also observed enhanced susceptibility to cefepime-enmetazobactam, though there is a substantial lack of potency of cefepime among ESBL as well as AmpC producing Enterobacterales in Europe. (17) In our current study, cefepime-enmetazobactam was susceptible in 68 out of 149 isolates (45.6%). Cefepime-enmetazobactam was resistant in 77 out of 149 isolates (51.6%). Whereas Piperacillin-tazobactam was susceptible in 42 out of 149 isolates (28.2%) and resistant in 96 out of 149 isolates (64.4%). Therefore, our study indicates that Cefepime-enmetazobactam is superior to Piperacillin-tazobactam against multidrug-resistant (MDR) Enterobacterales harvested from sputum, pus, wound swabs and endotracheal (ET) tube samples.

In our study Ertapenem resistance was observed in 46.9% (n=70), meropenem resistance in 53.0% (n=79) and imipenem resistance in 55.7% (n=83) isolates, whereas cefepime-enmetazobactam resistant isolates were 54.2% (n=77). Running a statistical comparison, we concluded that Cefepime-enmetazobactam is not promising against carbapenem-resistant organisms (CRO). Ramakrishnan et al. also demonstrated that Cefepime-enmetazobactam demonstrated poor activity against carbapenem-resistant organisms (CRO) (16).

The result of this study is indicating that cefepime-enmetazobactam might be a valuable carbapenem-sparing agent for empirical treatment of serious Gram-negative infections in a setting with high prevalence of ESBL producing Enterobacterales. It has susceptibility rates greater than piperacillin-tazobactam and its susceptibility closely matching carbapenem. Though it is not promising against carbapenem-resistant organisms (CRO), it might be a valuable carbapenem-sparing agent, thereby reducing the rampant use of carbapenems in cases with high prevalence of ESBL producing Enterobacterales. Our study is also indicating that Cefepime-enmetazobactam is superior to Piperacillin-tazobactam against multidrug-resistant (MDR) Enterobacterales harvested from sputum, pus, wound swabs and endotracheal (ET) tube samples. But as the current study is an in vitro (laboratory) study of antibiotic susceptibility, having several significant limitations because they cannot fully replicate the complex environment of the human body, so further research is needed to determine the potential role for cefepime-enmetazobactam in the treatment of complicated, ESBL producing Gram-negative bacterial infections.

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