Avibactam potentiated the activity of both ceftazidime and aztreonam against S. maltophilia clinical isolates in vitro

Background Treatment options for Stenotrophomonas maltophilia (S. maltophilia) infections were limited. We assessed the efficacy of ceftazidime (CAZ), ceftazidime-avibactam (CAZ-AVI), aztreonam (ATM), and aztreonam-avibactam (ATM-AVI) against a selection of 76 S. maltophilia out of the 1179 strains isolated from the First Affiliated Hospital of Chongqing Medical University during 2011–2018. Methods We investigated the antimicrobial resistance profiles of the 1179 S. maltophilia clinical isolates from the first affiliated hospital of Chongqing Medical University during 2011–2018, a collection of 76 isolates were selected for further study of microbiological characterization. Minimum inhibitory concentrations (MICs) of CAZ, CAZ-AVI, ATM and ATM-AVI were determined via the broth microdilution method. We deemed that CAZ-AVI or ATM-AVI was more active in vitro than CAZ or ATM alone when CAZ-AVI or ATM-AVI led to a category change from “Resistant” or “Intermediate” with CAZ or ATM alone to “Susceptible” with CAZ-AVI or ATM-AVI, or if the MIC of CAZ-AVI or ATM-AVI was at least 4-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 active at restoring the activity 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), and MIC50 (2 μg/mL vs. 8 μg/mL, P < 0.05) when compared to CAZ. According to our definition, CAZ-AVI was more active in vitro than CAZ alone for 81.58% (62/76) 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) and MIC50 (2 μg/mL vs. 64 μg/mL, P < 0.001) when compared to ATM. According to our definition, ATM-AVI was also more active in vitro than ATM alone for 94.74% (72/76) 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.


Background
Stenotrophomonas maltophilia (S. maltophilia) has emerged as an important cause of nosocomial infections in immunocompromised hosts. In patients with cystic fibrosis (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, 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 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 efflux 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 difficulty in effectively managing S. maltophilia infections.
S. maltophilia isolates naturally produce two βlactamases (L1 and L2). L1 is a class B3 metallo-βlactamase (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-lactamase inhibitors [1,8,9]. L2 is a class A cephalosporinase that confers resistance to extendedspectrum cephalosporins and ATM but can be inhibited by commercially available serine-β-lactamase inhibitors such as clavulanic acid and avibactam (AVI) [1,2,11,12]. AVI is a non-β-lactam, β-lactamase inhibitor without intrinsic antibacterial activity, but offers a broader βlactamase inhibition profile compared with any other recently used serine β-lactamase inhibitors [13]. Although S. maltophilia isolates naturally produce two βlactamases (L1 and L2), the inhibitory activities of CAZ and ATM against L2-producing S. maltophilia isolates might theoretically be re-established by AVI combination; furthermore, given the potential bactericidal activity of ATM against L1-producing S. maltophilia isolates, ATM-AVI might be useful to treat infections with both L1-and L2-producers. On the other hand, two recent studies from France 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 μg/ mL) [16], highlighting the potential benefit 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 in urgent need. 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 about half (16/33, 48.48%) of the CAZ-non-susceptible (CAZ-NS) S. maltophilia isolates, while ATM-AVI combination inhibited most (61/68, 89.71%) of the ATM-non-susceptible (ATM-NS) S. maltophilia strains.

Microbiological characteristics and antimicrobial susceptibility profiles of S. maltophilia isolates
In total, 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 from various body sites such as secretions from surgical incisions, burns, or conjunctival sacs (7.0%) and urine (5.0%). Department distribution analysis showed that ICUs (39.0%), medical wards (36.0%), and surgical wards (25.0%) contributed the majority of S. maltophilia isolates. With regard to the antimicrobial susceptibility profiles of the S. maltophilia isolates, its non-susceptible rates to LVX, MH, and SXT were respectively 8.23, 3.14, and 6.28% (Table 1).

Inhibitory activities of CAZ-AVI and ATM-AVI against S. maltophilia isolates
To assess the potential efficacy of CAZ-AVI and ATM-AVI against S. maltophilia isolates, we have tested these combinations in vitro on a 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 LVX, MH, and SXT. While the remaining 16 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 Clinical and Laboratory Standards Institute (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 <=8/4 μg/mL for 59 of 76 isolates (77.63%) ( Table 2). According to our definition, CAZ-AVI was more active in vitro than CAZ alone for 81.58% (62/ 76) of the isolates. For the 16 out of the 33 (48.48%) S. maltophilia isolates that were CAZ-NS, the combination of AVI restored their in vitro activity to CAZ (9 isolates from resistant to sensitive, and 7 isolates from intermediate to sensitive). AVI addition also reduced the CAZ MIC50 of the 76 S. maltophilia isolates from 8 to 2 μg/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 <=8/4 μg/mL for 69 of 76 isolates (90.79%) ( Table 2). ATM-AVI was more active in vitro than ATM alone for 94.74% (72/76) of the isolates. Notably, for 61 out of the 68 (89.71%) S. maltophilia isolates that were ATM-NS, the addition of AVI restored in vitro activity to ATM (60 isolates from resistant to sensitive, and 1 isolate from intermediate to sensitive). 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-AVI 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. 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 isolates (4-fold MIC50 reduction) and the addition of 4 μg/mL AVI also significantly increased the activity of ATM against most isolates (32fold MIC50 reduction) ( Table 3). 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).

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 and MIC50. Furthermore, while ATM-AVI was more active in vitro than ATM alone for 94.74% of the isolates, CAZ-AVI was more active in vitro than CAZ alone for 81.58% of the isolates. However, it is noteworthy that CAZ-AVI resistance was found in 12 S. maltophilia strains isolated from patients with no history of previous CAZ-AVI-based treatment, moreover, 5 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. As expected, the strains that were resistant to CAZ-AVI or ATM-AVI combinations had also been resistant to CAZ or ATM alone.
Notably, although AVI did not restore the activity of CAZ against the two MDR S. maltophilia isolates, it did restore the activity of ATM against the two MDR S. maltophilia strains with significant 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.   [17], who demonstrated that the activity of CAZ-AVI was poor against most MDR/XDR S. maltophilia strains.
Recently, the efficacy of CAZ-AVI (2.5 g i.v. every 8 h) in combination with ATM (2 g 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 [12]. The synergy among CAZ, ATM and AVI is encouraging and deserves further exploration. When compared with CAZ-AVI, ATM-AVI exhibited obviously superior inhibitory activity, inhibiting the growth of 89.71% of the ATM-NS isolates (61/68) ( Table  2). Nevertheless, although the results of some prior studies [14,15] didn't show CAZ-AVI to be active against S. maltophilia, in our research, CAZ-AVI has been demonstrated to inhibit the growth of about half of the CAZ-NS isolates (48.48%, 16/33). As S. maltophilia isolates naturally produce L1 and/or L2 β-lactamases: while L2 is a class A cephalosporinase that confers resistance to CAZ but can be inhibited by AVI [1,2,11,12], L1 is an MBL that can hydrolyze CAZ but can't be inhibited by AVI. Thus, while the L1-producers can't be killed by CAZ-AVI, the growth of the L2-producers might be inhibited by CAZ-AVI. Thus the variable results from different studies may have been due to the different βlactamases that different S. maltophilia isolates from various districts produced.
In this study, we likewise proposed the possible emergence of both CAZ-AVI non-susceptible and ATM-AVI non-susceptible S. maltophilia strains isolated from patients without previous antimicrobial exposure to CAZ-AVI and ATM-AVI, which are consistent with our previous findings 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 nonsusceptibilities in our S. maltophilia isolates. Secondly, this was a single-center retrospective study with relatively small sample size conducted in Chongqing.

Conclusions
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. maltophilia.

Bacterial strains
We collected a total of 76 non-repetitive, recent nosocomial S. maltophilia strains between 2014 and 2018 in the First Affiliated Hospital of Chongqing Medical University. All the isolates were identified at the species level by the VITEK MS (bioMérieux, MO, USA) system, and routine antimicrobial susceptibility testing was performed using the disk diffusion (for LVX, MH, and SXT) testing methods. All the S. maltophilia colonization and infection cases (1179) with complete medical records during 2011-2018 were investigated for clinical and antimicrobial resistance profiles, among which 76 recent isolates during 2014-2018 were selected for further microbiological characterization. In order to systemically include a range of different resistance profiles, we collected 60 S. maltophilia strains resistant to at least one of the three clinically available drugs (LVX, MH, and SXT), and 16 isolates sensitive to all the three antibiotics as comparison.