Infections due to carbapenem-resistant Gram-negative bacilli (CR-GN) have become one of the greatest threats to human health globally. Carbapenemase-mediated resistance is of particular importance, both in clinical and epidemiological context. Although the frequency of enzyme types varies significantly by geographical region, the endemic occurrence of isolates producing Klebsiella pneumoniae carbapenemase (KPC) is a subject of major concern in many countries. In this context, new combinations of β-lactams and β-lactamase inhibitors, such as ceftazidime-avibactam (CZA), are being widely used worldwide [1], [2], [3].

Driven, at least partially, by the COVID-19 pandemic, a recent and worrisome emergence of Gram-negative bacilli producing metallo-β-lactamase (MBL), mainly New Delhi Metallo β-lactamase (NDM), has been observed. Worryingly, the emergence and dissemination of isolates co-producing KPC and MBL have also increased. The latter are defiant, as new generation β-lactamase inhibitors, which includes avibactam (AVI), relebactam and vaborbactam, do not inhibit metallo enzymes, limiting significantly the use of CZA (or other combinations) as therapeutic option [4], [5], [6], [7]. MBLs inactivate all β-lactams, except aztreonam (ATM). However, MBL-producing bacteria often produce other β-lactamases, such as extended spectrum β-lactamases (ESBL) or AmpC, which may inactivate ATM. Thus, the aztreonam-avibactam (AZA) combination is an alternative to treat infections caused by MBL-producing or co-producing bacteria. However, AZA is not commercially available yet. Meanwhile, the in vivo association of ATM with CZA has been used, providing favorable and encouraging outcomes [8], [9], [10], [11].

Currently, there is no easy-to-perform and rapid methodology to assess susceptibility to the combination ATM and CZA. Checkerboard test, time-kill curves and crossing of gradient strips [12] are methodologically complex, expensive and/or demand prolonged incubation, which reduces clinical impact of their results [8,[13], [14], [15], [16]]. Recently, elution methods (broth disc elution) have been evaluated and proved to be able to discriminate between ATM/CZA resistant and susceptible isolates. The latter are based in the elution of the antibiotics (ATM, CZA and the ATM plus CZA) from paper discs to Mueller-Hinton broth. Bacterial inoculum is added to the broth with eluted antibiotics and growth (turbidity) after 16-20h of incubation indicates resistance [14,17]. Recently, Viguier and coworkers [18] proposed a resazurin-based methodology to detect resistance to AZA (in powder) in up to 4.5h which presented excellent performance. The use of resazurin facilitates the detection of growth of the bacteria in the presence of the antibiotic as resazurin is blue when it is in an oxidized state and turns pink when reduced by viable cells during bacterial growth.

Ideally, the clinical use of ATM combined with CZA should be guided by the results of the susceptibility in vitro of the isolates to the combination. Therefore, we aimed to evaluate the susceptibility of CR-GN to ATM, CZA and AZA, as well as the synergism between ATM and CZA. Moreover, we proposed a variation of disc elution methodology, named ATM/CZA microelution (mATM/CZA), to evaluate susceptibility to ATM/CZA combination, using resazurin to enable results in up to 4 h.