In Vitro Evaluation of the activity of Ceftazidime-avibactam and Aztreonam-Avibactam against 76 Stenotrophomonas maltophilia isolates from a teaching hospital in Chongqing

Background: Treatment options for Stenotrophomonas maltophilia (S. maltophilia) infections were limited. We assessed the ecacy of ceftazidime-avibactam (CAZ-AVI) and aztreonam-avibactam (ATM-AVI) against a selection of 76 S. maltophilia out of the 1179 strains isolated from the First Aliated Hospital of Chongqing Medical University during 2011-2018. Methods: We investigated the antimicrobial resistance proles of the 1179 S. maltophilia clinical isolates from the rst aliated hospital of Chongqing Medical University during 2011-2018, a collection of 76 isolates of which were available for further study of microbiological characterization. Minimum inhibitory concentrations (MICs) of ceftazidime (CAZ), ceftazidime-avibactam (CAZ-AVI), aztreonam (ATM) and aztreonam-avibactam (ATM-AVI) were determined via the broth microdilution method. We deemed that CAZ-AVI or ATM-AVI was more effective in vitro than CAZ or ATM alone when CAZ-AVI or ATM-AVI led to a category change from “Resistant” with CAZ or ATM alone to “Susceptible” or “Intermediate” with CAZ-AVI or ATM-AVI, or if the MIC of CAZ-AVI or ATM-AVI was at least 2-fold lower than the MIC of CAZ or ATM alone. Results: For the 76 clinical isolates included in the study, MICs of CAZ, ATM, CAZ-AVI and ATM-AVI ranged from 0.03-64, 1-1024, 0.016-64, and 0.06-64 μg/mL, respectively. In combined therapy, AVI was effective at restoring the susceptibility of 48.48% (16/33) and 89.71% (61/68) of S. maltophilia to CAZ and ATM, respectively. Furthermore, CAZ-AVI showed better results in terms of the proportion of susceptible isolates (77.63% vs.56.58%, P<0.001), MIC50 (2μg/mL vs. 8μg/mL, P<0.05), and MIC distribution (P<0.001) when compared to CAZ. According to our denition, CAZ-AVI was more effective in vitro than CAZ alone for 84.21% of the isolates. Similarly, ATM-AVI also showed better results in terms of the proportion of susceptible isolates (90.79%vs. 10.53%, P<0.001), MIC50 (2μg/mL vs. 64μg/mL, P<0.001), and MIC distribution (P<0.001) when compared to ATM. According to our denition, ATM-AVI was also more effective in vitro than ATM alone for 97.37% of the isolates. Conclusions: AVI potentiated the activity of both CAZ and ATM against S. maltophilia clinical isolates in vitro. We demonstrated that CAZ-AVI and ATM-AVI are both useful therapeutic options to treat infections caused by S. maltophilia. 32- and 8-fold respectively. The poor activity of CAZ-AVI against MDR S. maltophilia isolates was in accordance with a previous study by Lindsay J. Caverly et al., who demonstrated that the activity of CAZ-AVI was poor against most MDR/XDR S. maltophilia strains. ecacy of CAZ-AVI i.v. in combination with i.v. patient with to ATM and CAZ-AVI, against S. maltophilia isolates. The excellent in vitro activity of CAZ-AVI or ATM-AVI against S. maltophilia isolates in our hospital supports further evaluation of CAZ-AVI or ATM-AVI in clinical studies against S. maltophilia infections. CAZ-AVI or ATM-AVI might turn out to be useful therapeutic options to treat infections caused by S. maltophilia.


Introduction
Stenotrophomonas maltophilia (S. maltophilia) is a Gram-negative, nonfermentative, environmental bacillus that has emerged as an important cause of nosocomial infections in immunocompromised hosts. In patients with cystic brosis (CF), S. maltophilia is known for colonizing the airways and causing chronic infections [1,2]. Although S. maltophilia is primarily associated with respiratory tract infections, this pathogen can cause a wide range of clinical syndromes, including catheter-associated bloodstream infections, and skin and soft tissue infections [2][3][4]. Furthermore, although S. maltophilia is not a highly virulent pathogen, it has emerged as an important nosocomial pathogen associated with crude mortality rates ranging from 14 to 69% in patients with bacteremia [5,6]. S.maltophilia is recognized by the World Health Organization as one of the leading MDR organisms in hospital settings for which disease prevention and treatment strategies must be developed [7]. More frustratingly, S. maltophilia is intrinsically resistant to different classes of antibiotics used in clinical practices, which was mediated by the expression of aminoglycoside-modifying enzymes, qnrB-like quinolone-resistant determinants, multidrug e ux pumps, and two β-lactamases (L1 and L2) [1,8,9]. These characteristics, together with its ability to adapt to environmental changes, contribute to the di culty in effectively managing infections with S. maltophilia.
Ceftazidime, levo oxacin, minocycline, and trimethoprim-sulfamethoxazole have been used as treatment options for S.maltophilia infections; but unfortunately, their susceptibility against S.maltophilia is declining [10]. To assist clinicians in achieving effective individualized and precise treatment for S.maltophilia infections, more high-quality epidemiology-and antimicrobial susceptibility testing (AST) to novel antibiotics-centered studies are urgently needed. β-lactamases are enzymes that hydrolyze the β-lactam amide, inactivating them, and preventing them from reaching their target, the penicillin binding protein in bacterial cell membranes. S. maltophilia isolates naturally produce two β-lactamases (L1 and L2). L1 is a class B3 metallo-β-lactamase (MBL) that hydrolyzes carbapenems and other β-lactams, with the important exception of the monobactam aztreonam (ATM), and is resistant to all clinically available β-lactams [1,8,9]. L2 is a class A cephalosporinase that confers resistance to extended-spectrum cephalosporins and ATM but can be inhibited by commercially available serine-β-lactamase inhibitors such as clavulanic acid and avibactam [1,2,11,12]. Avibactam (AVI) is a non β lactam, βlactamase inhibitor without intrinsic antibacterial activity, but offers a broader β-lactamase inhibition pro le compared with any other recently used serine βlactamase inhibitors [13]. Although S. maltophilia isolates naturally produce two β-lactamases (L1 and L2), the bactericidal activities of CAZ and ATM against L2-producing S. maltophilia isolates might theoretically be re-established by AVI combination; On the other side, given that the potential bactericidal activity of ATM against L1-producing S. maltophilia isolates might theoretically be achieved by monobactam's activity against MBL-producers, when combined with AVI, ATM/AVI might be useful to treat infections with both L1-and L2-producers, thus, AVI might restore the activity of ATM against both L1 and L2-producing S. maltophilia clinical isolates in vitro. Nevertheless, two recent studies from Suresnes and Japan demonstrated that CAZ-AVI is not active against S. maltophilia [14,15]. However, another report from France showed that while one third of the S. maltophilia isolates studied remained resistant to CAZ-AVI, 30% showed low MICs (< 1 ug/mL) [16], highlighting the potential bene t of CAZ-AVI against S. maltophilia. Therefore, for better decision making of the clinical management of S. maltophilia infections, additional epidemiology and resistance testing of S. maltophilia isolates from other countries or regions worldwide are desperately needed. In addition, alternative therapeutic strategy for S. maltophilia is urgently needed. So far, there are few data available in China describing the in vitro activities of CAZ-AVI and ATM-AVI against S. maltophilia clinical isolates. We conducted an observational study to evaluate the in vitro antimicrobial activities of CAZ/AVI and ATM/AVI against recent S. maltophilia clinical isolates in our hospital. In this paper, we demonstrated that CAZ-AVI combination inhibited nearly half (16/33, 48.48%) CAZ-non-susceptible S. maltophilia isolates, while ATM-AVI combination inhibited most (61/68, 89.71%) of the ATM-non-susceptible S. maltophilia strains.

Bacterial strains
We collected a total of 76 non-repetitive, recent nosocomial S. maltophilia strains between 2014 and 2018 in the First A liated Hospital of Chongqing Medical University. All the isolates were identi ed at the species level by the VITEK MS (bioMérieux, MO, USA) system, and routine antimicrobial susceptibility testing was performed using the disk diffusion (for levo oxacin, minocycline, and trimethoprim-sulfamethoxazole) testing methods. All the S. maltophilia colonization and infection cases (1179)

Results
Microbiological characteristics and antimicrobial susceptibility pro les of S. maltophilia isolates As shown in Figure 1, 1179 non-repetitive S. maltophilia strains were isolated during the study period, among which the predominant sample origins were sputum (73.0%), followed by secretion (7.0%) and urine (5.0%). Department distribution analysis showed that ICU (23.0%) including the respiratory intensive care unit (10.0%), and the respiratory department (9.0%), contributed the majority of S. maltophilia (Fig 2). With regard to the antimicrobial susceptibility pro les of the S. maltophilia isolates, its non-susceptible rates to levo oxacin, minocycline, and trimethoprim-sulfamethoxazole were respectively 9.08%, 3.14%, and 6.28% (Table 1).
Bactericidal activities of CAZ/AVI and ATM/AVI against S. maltophilia isolates To assess the potential e cacy of CAZ/AVI and ATM/AVI against S. maltophilia isolates, we have tested these combinations in vitro on a recent collection of 76 non-repetitive isolates available for microbiological characterization. The majority of tested isolates (60/76, 78.95%) were resistant to at least one of the following three antibiotics, namely levo oxacin (LVX), minocycline (MH), and trimethoprim-sulfamethoxazole (SXT). While the rest part of the isolates were sensitive to LVX, MH, and SXT. The in vitro antimicrobial susceptibilities of CAZ, CAZ/AVI, ATM, and ATM/AVI against these isolates were determined using the CLSI broth microdilution method. The MIC of CAZ alone was <=8 µg/mL for 43 of 76 S. maltophilia isolates (56.58%), and the MIC of CAZ-AVI was <=8 µg/mL for 59 of 76 isolates (77.63%) ( Table 2). For the 21 out of the 33 (63.64%) S. maltophilia isolates that were CAZ-nonsusceptible, the combination of AVI restored 13 strains' susceptibility to CAZ, remaining 8 isolates resistant to CAZ-AVI (main MIC: 64/4 µg/mL). AVI addition also reduced the CAZ MIC50 of the 76 S. maltophilia isolates from 8 to 2µg/mL (Table 3). In all, the MIC of ATM alone was <=8 µg/mL for 8 of the 76 S. maltophilia isolates (10.53%), and the MIC of ATM-AVI was <=8 µg/mL for 69 of 76 isolates (90.79%) ( Table 2). Notably, for 66 out of the 68 (97.06%) S. maltophilia isolates that were ATM-nonsusceptible, the addition of AVI restored their susceptibility to ATM. AVI addition also reduced the ATM MIC50 of the 76 S. maltophilia isolates from 64 to 2 µg/mL ( Table 3).
The results of susceptibility testing comparing CAZ, CAZ-AVI, ATM and ATM-AVI, are shown in (Table 3).These agents showed a wide range of activity against S. maltophilia. In detail, the ranges of MICs of CAZ and ATM for the 76 clinical S. maltophilia isolates were 0.03 to 64 and 1 to 1024 µg/mL, respectively, while those of CAZ-AVI and ATM-ACI for the same S. maltophilia isolates were 0.016 to 64 and 0.06 to 64 µg/mL, respectively (Table 3). In this study, AVI potentiated the activity of ATM against most of the S. maltophilia clinical isolates tested in vitro. Meanwhile, AVI also enhanced the activity of CAZ against most of those isolates tested in vitro. On the one hand, for S. maltophilia isolates that were nonsusceptible to LVX, MH, and/or SXT (78.95%, 60 of 76), the addition of 4 µg/mL AVI greatly increased the activity of CAZ against most species (4-fold MIC50 reduction) and the addition of 4 µg/mL AVI also signi cantly increased the activity of ATM against most species (32-fold MIC50 reduction) ( Table 3). On the other hand, AVI did not restore the activity of CAZ against the two multidrug-resistant (MDR) S. maltophilia isolates, even though AVI reduced one MDR isolate's MIC somewhat (4-fold MIC reduction) (Table 3). However, AVI did restore the activity of ATM against the two MDR S. maltophilia isolates with obvious MIC reduction of 32-or 8-fold. (Table 3) CAZ/AVI and ATM/AVI are more effective in vitro than CAZ and ATM alone against S. maltophilia isolates.
When compared to CAZ, CAZ-AVI showed better results in terms of the proportion of susceptible isolates ((77.63% vs.56.58%, P<0.001), MIC50 (2μg/mL vs.8μg/mL, P<0.05), and MIC distribution (Table 4) (P<0.001). According to our de nition, CAZ-AVI was more effective in vitro than CAZ alone for 84.21% of the isolates. On the other hand, ATM-AVI likewise showed better results in terms of the proportion of susceptible isolates (90.79% vs.10.53%, P<0.001), MIC50 (2μg/mL vs.64μg/mL, P<0.001), and MIC distribution (Table 5) (P<0.001) when compared to ATM. According to our de nition, ATM-AVI was also more effective in vitro than ATM alone for 97.37% of the isolates Discussion S. maltophilia infections pose a major challenge for clinicians because of limited therapeutic options. For the 76 clinical isolates included in the present study, both CAZ-AVI and ATM-AVI exerted promising results in terms of the proportion of susceptible isolates, MIC50, and MIC distribution. Furthermore, while ATM-AVI was more effective in vitro than ATM alone for 97.37% of the isolates, CAZ-AVI was more effective in vitro than CAZ alone for 84.21% of the isolates. However, it is noteworthy that CAZ-AVI resistance was found in 17 S. maltophilia strains isolated from patients with no history of previous CAZ-AVIbased treatment, moreover, 7 S. maltophilia strains isolated from patients without previous ATM-AVI exposure demonstrated in vitro resistance to ATM-AVI, indicating that incidence of CAZ-AVI and ATM-AVI resistance could emerge in S. maltophilia strains without previous antimicrobial exposure. Notably, although AVI did not restore the activity of CAZ against the 2 MDR S. maltophilia isolates, it did restore the activity of ATM against the two MDR S. maltophilia strains with signi cant MIC reductions of 32-and 8-fold respectively. The poor activity of CAZ-AVI against MDR S. maltophilia isolates was in accordance with a previous study by Lindsay J. Caverly et al., who demonstrated that the activity of CAZ-AVI was poor against most MDR/XDR S. maltophilia strains. Recently, the e cacy of CAZ-AVI (2.5 g i.v. every 8h) in combination with ATM (2g i.v. every 8 h) for 48 days was demonstrated for a young renal transplant patient with S. maltophilia resistant to SXT, meropenem and CAZ [11]. In our research, CAZ/AVI has been demonstrated to inhibit the growth of about half of the CAZ-NS isolates (48.48%, 16/33), still showing high-level resistance (MIC: 64/4 µg/mL) in 10 isolates. Nevertheless, compared with CAZ/AVI, ATM/AVI exhibited obviously superior bactericidal activity, inhibiting the growth of 89.71% of the ATM-NS isolates (61/68) ( Table 2 and Figure   3). The synergy among CAZ, ATM and AVI is encouraging and deserves further exploration. In this study, we likewise con rmed the emergence of both CAZ-AVI-resistant and ATM-AVI-resistant S. maltophilia strains isolated from patients without previous antimicrobial exposure to CAZ-AVI and ATM-AVI, which are consistent with our previously ndings that demonstrated CAZ-AVI resistance in the carbapenem-resistant Enterobacteriaceae (CRE) bacteremia isolates from patients with no history of previous CAZ-AVI exposure [18].
The current study has several limitations. First of all, we only evaluated the activity of CAZ, ATM, CAZ-AVI and ATM-AVI without exploring the resistance mechanisms for both CAZ-AVI and ATM-AVI non-susceptibilities in our S. maltophilia isolates. Secondly, this was a single-center retrospective study with relatively small sample size conducted in Chongqing.
In summary, ATM-AVI showed the most potent in vitro activity among the other related agents, including CAZ, ATM and CAZ-AVI, against S. maltophilia isolates. The excellent in vitro activity of CAZ-AVI or ATM-AVI against S. maltophilia isolates in our hospital supports further evaluation of CAZ-AVI or ATM-AVI in clinical studies against S. maltophilia infections. CAZ-AVI or ATM-AVI might turn out to be useful therapeutic options to treat infections caused by S.

Ethics approval
The data and samples analyzed in the present study were obtained in accordance with the standards and approved by the Chongqing Medical University Institutional Review Board and Biomedical Ethics Committee. For this study, samples were collected at the microbiology laboratory of our hospital, with no contact with the patients. This study was retrospective and there was no patient identi cation performed during data collection. Therefore, the ethics committee determined that informed consent was not required.

Consent for publication
Not applicable.

Strains
Group without AVI Group with AVI Z P