In Vitro Susceptibility of Aztreonam-Vaborbactam, Aztreonam-Relebactam and Aztreonam-Avibactam Associations against Metallo-β-Lactamase-Producing Gram-Negative Bacteria

Background: Despite the availability of new options (ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam and cefiderocol), it is still very difficult to treat infections caused by metallo-β-lactamase (MBLs)-producers resistant to aztreonam. The in vitro efficacy of aztreonam in association with avibactam, vaborbactam or relebactam was evaluated on a collection of MBL-producing Enterobacterales, MBL-producing P. aeruginosa and highly drug-resistant S. maltophilia. Methods: A total of fifty-two non-duplicate MBL-producing Enterobacterales, five MBL-producing P. aeruginosa and five multidrug-resistant S. maltophila isolates were used in this study. The minimum inhibitory concentrations (MICs) of aztreonam, meropenem-vaborbactam and imipenem-relebactam were determined by Etest® (bioMérieux, La Balme-les-Grottes) according to EUCAST recommendations. For aztreonam-avibactam, aztreonam-vaborbactam and aztreonam-relebactam associations, the MICs were determined using Etest® on Mueller-Hinton (MH) agar supplemented with 8 mg/L of avibactam, 8 mg/L of vaborbactam and 4 mg/L of relebactam. The MICs were interpreted according to EUCAST guidelines. Results: The susceptibility rates of aztreonam-avibactam, aztreonam-vaborbactam and aztreonam-relebactam with a standard exposure of aztreonam (1g × 3, IV) were 84.6% (44/52), 55.8% and 34.6% for Enterobacterales and 0% for all combinations for P. aeruginosa and S. maltophila. The susceptibility rates of aztreonam-avibactam, aztreonam-vaborbactam and aztreonam-relebactam with a high exposure of aztreonam (2g × 4, IV) were 92.3%, 78.9% and 57.7% for Enterobacterales, 75%, 60% and 60% for P. aeruginosa and 100%, 100% and 40% for S. maltophila. Conclusions: As previously demonstrated for an aztreonam/ceftazidime-avibactam combination, aztreonam plus imipenem-relebactam and aztreonam plus meropenem-vaborbactam might be useful options, but with potentially lower efficiency, to treat infections caused by aztreonam-non-susceptible MBL-producing Gram-negative strains.


Introduction
The extensive dissemination of carbapenemase-producing Gram-negative bacteria poses a global threat to public health.It is crucial to implement new therapeutic strategies to treat infections caused by highly resistant pathogens.Carbapenemase belong to three of four classes of the Ambler classification: class A carbapenemases (mostly KPC types) [1], class B carbapenemases or metallo-β-lactamases (MBLs) (mostly NDM, VIM, or IMP types) [2], and class D carbapenemases (mostly OXA-48-like types in Enterobacterales) [3].Recently, new therapeutic alternatives have been marked for the treatment of carbapenemase producers.These new drugs include ceftazidime-avibactam, meropenemvaborbactam, imipenem-relebactam and cefiderocol.Despite the availability of these new options, it is still very difficult to treat infections caused by MBL producers resistant to aztreonam.Indeed, these isolates are resistant to ceftazidime-avibactam, meropenemvaborbactam and imipenem-relebactam through the MBL-hydrolyzing activity, which is not inhibited by the added β-lactamase inhibitor.Regarding cefiderocol, it might be active on MBLs, but NDM producers have cefiderocol's MIC 50 close to the clinical breakpoint of 2 mg/L, resulting in only 70% susceptibility [4].Since current MBLs, such as VIM, NDM or IMP, are not able to hydrolyze aztreonam, resistance to this antimicrobial in MBL producers is due to the co-production of an ESBL or an AmpC.Accordingly, it has been demonstrated that the concomitant use of aztreonam plus ceftazidime-avibactam or amoxicillin-clavulanate or ceftolozane-tazobactam are accurate options to treat infections caused by MBL + ESBL or MBL + AmpC producers, with aztreonam plus ceftazidimeavibactam being the most powerful strategy [5].Unfortunately, is has been recently shown that aztreonam-avibactam resistance emerged in NDM-producing E. coli through the association of modified penicillin-binding protein 3 (PBP3) and the production of CMY-42 cephalosporinase [6].Thus, it is crucial to consider other therapeutic alternatives.In this study, we evaluated the susceptibility of aztreonam-resistant MBL-producing isolates to aztreonam in association with two other marketed β-lactamase inhibitors: vaborbactam and relebactam.Vaborbactam, a cyclic boronic β-lactamase inhibitor and relebactam, a diazabicyclooctane inhibitor, are very potent against KPC enzymes, but are also active on diverse class A (including ESBL) and class C β-lactamases (AmpC) [7].As previously demonstrated for aztreonam-avibactam [5], aztreonam-vaborbactam and aztreonam-relebactam seemed to be effective on NDM-producing K. pneumoniae [8] and few multidrug-resistant S. maltophila [9].Here, we assessed the susceptibility of aztreonam in combination with vaborbactam or relebactam or avibactam on a collection of MBL producers including Enterobacterales (including species other than K. pneumoniae), P. aeruginosa and S. maltophilia.

Results
A total of fifty-two non-duplicate MBL-producing Enterobacterales, five MBL-producing P. aeruginosa and five multidrug-resistant S. maltophila isolates were used in this study.These isolates have already been tested for aztreonam-avibactam in a previous study [5].All strains were resistant to aztreonam due to the co-production of an ESBL and/or a cephalosporinase.The MBL-producing Enterobacterales included 30 NDM-producers with 11 isolates co-producing one OXA-48-like carbapenemase, fourteen VIM-producers, six IMPproducers, one GIM-1-producer and one TMB-1-producer.The MBL-producing P. aeruginosa were one VIM-2-, three IMP-1-and one IMP-2-producers.S. maltophila isolates were resistant to all β-lactams including ticarcillin-clavulanate, and to all other antimicrobials including fluoroquinolones and trimethoprim-sulfamethoxazole [5].

Discussion
In conclusion, as previously demonstrated for the aztreonam/ceftazidime-avibactam combination [10][11][12][13][14][15][16][17][18], aztreonam plus imipenem-relebactam [19][20][21] and aztreonam plus meropenem-vaborbactam [21] might be useful options to treat infections caused by aztreonamnon-susceptible MBL-producing Gram-negative strains.Despite the fact that avibactam seems to be the most potent inhibitor in association with aztreonam, it should be noted that ceftazidime is strongly hydrolyzed by ESBL and MBL with MICs >256 mg/L resulting in a non-residual effect of this drug in the tripartite combination aztreonam-ceftazidimeavibactam.Contrarily, imipenem and meropenem are not hydrolyzed by ESBL or AmpC and might retain partial activity on MBL (sometimes the MICs range from 2 to 8 mg/L).Accordingly, the tripartite associations of aztreonam-imipenem-relebactam and aztreonammeropenem-vaborbactam might benefit not only the activity of the inhibitor with aztreonam, but also partly the residual activity of the carbapenem.Notably, our collection of Gram-negative bacteria included only a few isolates of P. aeruginosa and S. maltophilia.Due to this limitation, the results of the susceptibility of these two species have to be confirmed in further studies.
According to the recent guidelines of the Infectious Diseases Society of America (IDSA) [22] and the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) [23], aztreonam-avibactam association produced by injecting aztreonam and ceftazidime-avibactam simultaneously [24] is considered the first-line treatment for infections caused by MBL producers.This is particularly the case for NDM producers that often display an increased MIC to cefiderocol [4,[25][26][27], the other potential option for the treatment of infections caused by MBL producers [22,23].
In our collection, four NDM-producing E. coli displayed resistance to the aztreonamavibactam association.In E. coli, the impact of the alteration of PBP3 has been recently described to be responsible for increased resistance to aztreonam-avibactam [11] and a cross-resistance to cefiderocol [28,29].With the recent emergence of aztreonam-avibactam resistance among carbapenemase-producing Enterobacterales [11,29,30], these tripartite associations of aztreonam-imipenem-relebactam and aztreonam-meropenem-vaborbactam have to be taken into consideration as last-resort treatment options.Unfortunately, resistance to aztreonam-meropenem-vaborbactam has already been reported in a KPC variant producing K. pneumoniae [31].Accordingly, the rapid development of additional options, such as new combinations with more potent inhibitors (e.g., zidebactam and taniborbactam) are mandatory [32][33][34].However, as it was observed for aztreonam-avibactam, which is not already marketed, resistance to these very novel inhibitors already exists, as reported for cefepime-taniborbactam and NDM-9 producers [35,36].
In conclusion, aztreonam-vaborbactam and aztreonam-relebactam could be useful options for the treatment of infections caused by aztreonam-resistant MBL-producing isolates but with a potential lower efficiency compared to aztreonam-avibactam.

Materials and Methods
A total of 52 non-duplicate MBL-producing Enterobacterales, 5 MBL-producing P. aeruginosa and 5 multidrug-resistant S. maltophila isolates were used in this study.All strains were sequenced using the Illumina technique and the resistance genes were identified using Resfinder 4.1 (https://cge.food.dtu.dk/services/ResFinder/,27 September 2023).
The MICs of aztreonam, meropenem-vaborbactam and imipenem-relebactam were determined by Etest ® (bioMérieux, La Balme-les-Grottes) according to EUCAST recommendations.For aztreonam-avibactam, aztreonam-vaborbactam and aztreonam-relebactam associations, MICs were determined using Etest ® on MH agar supplemented with 8 mg/L of avibactam (provided by Pfizer, France), 8 mg/L of vaborbactam (CliniSciences, Nanterre, France) and 4 mg/L of relebactam (provided by MSD, Puteaux, France).The MICs of aztreonam-avibactam, aztreonam-vaborbactam and aztreonam-relebactam were interpreted in the same way as aztreonam alone, according to EUCAST guidelines as updated in 2022 (Table 2).Since no recommendation exists regarding these molecules for S. maltophila, interpretation criteria of Pseudomonas spp.were used (Table 3).A control of the accuracy of β-lactam inhibitor-supplemented MH agar was performed.For this purpose, the MICs of 13 strains, obtained with the imipenem and meropenem Etest ® (bioMérieux, France) on MH agar supplemented with 8 mg/L of vaborbactam and 4 mg/L of relebactam, were compared to the MICs obtained with the imipenem-relebactam and meropenem-vaborbactam Etest ® (bioMérieux, France).The difference in MICs obtained between the two methods did not exceed two dilutions, validating the methodology used in this study (Table 3).However, due to the relatively low number of tested isolates, the full validity of this method might have to be confirmed in further studies.Of note is the fact that the accuracy of determining the MIC of aztreonam-avibactam using MH agar supplemented with 8 mg/L of avibactam has already been demonstrated on this strain collection.In the previous study, this method was also demonstrated to give similar results with the Etest ® strip superposition method [5].E. coli ATCC 53126, K. pneumoniae ATCC 700,603 and two KPC-producing strains were used as quality controls.

Percentage of susceptible strains of Enterobacterales with high exposure 8.1% 92.3% 78.9% 57.7% Percentage of resistant strains of Enterobacterales 91
Black: resistant; Grey: susceptible at high exposure; Blank: susceptible at standard dosage.