# Activity of aztreonam in combination with novel $\beta$-lactamase inhibitors against metallo-$\beta$-lactamase-producing Enterobacterales from Spain

Juan Carlos Vázquez-Ucha, Isaac Alonso-Garcia, Paula Guijarro-Sánchez, Cristina Lasarte-Monterrubio, Laura Alvarez-Fraga, Arnau Cendón-Esteve, Michelle Outeda, Romina Maceiras, Andrea Peña-Escholano, Marta Martínez-Guitián, et al.

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Juan Carlos Vázquez-Ucha, Isaac Alonso-Garcia, Paula Guijarro-Sánchez, Cristina Lasarte-Monterrubio, Laura Alvarez-Fraga, et al. Activity of aztreonam in combination with novel $\beta$-lactamase inhibitors against metallo-$\beta$-lactamase-producing Enterobacterales from Spain. International Journal of Antimicrobial Agents, 2023, 61, 10.1016/j.ijantimicag.2023.106738. hal-04959390

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1 Activity of aztreonam in combination with novel β-lactamase inhibitors

2 against metallo-β-lactamase-producing Enterobacterales from Spain

3

4 Authors: Juan Carlos Vázquez-Ucha¹¥*, Isaac Alonso-Garcia¹*,

5 Paula Guijarro-Sánchez¹, Cristina Lasarte-Monterrubio¹, Laura Álvarez-Fraga¹,

6 Arnau Cendón-Esteve¹, Michelle Outeda¹, Romina Maceiras¹, Andrea Peña-Escolano¹,

7 Marta Martínez-Guitián¹,², Jorge Arca-Suárez¹, Germán Bou¹† and Alejandro Beceiro¹†
8 on behalf of the GEMARA-SEIMC/REIPI Enterobacterales Study Group‡.

9

10 ¹Microbiology department of the University Hospital A Coruña (CHUAC), Institute of

11 Biomedical Research of A Coruña (INIBIC), A Coruña. CIBER de Enfermedades

12 Infecciosas (CIBERINFEC), Spain.

13 ²NANOBIOFAR, Center for Research in Molecular Medicine and Chronic Diseases

14 (CiMUS), Universidad de Santiago de Compostela, 15782, Santiago de Compostela,

15 Spain.

16

17

18 * Juan Carlos Vázquez-Ucha and Isaac Alonso-García contributed equally to the study.

19 † Alejandro Beceiro and Germán Bou contributed equally to the study.

20 ¥ Author for correspondence: Dr. Juan Carlos Vázquez-Ucha

21 e-mail: Juan.Carlos.Vazquez.Ucha@sergas.es

22 Microbiology department of the University Hospital A Coruña (CHUAC), Institute of

23 Biomedical Research of A Coruña (INIBIC). As Xubias, 15006, A Coruña, Spain.

24 Telephone: +34 981178000 ext. 292145

25
26 **ABSTRACT**

27 Metallo-β-lactamase (MBL)-producing Enterobacterales are of particular concern
28 because they are widely disseminated and difficult to treat, being resistant to almost all
29 β-lactam antibiotics. Aztreonam is not hydrolysed by MBLs but is labile to serine β-
30 lactamases (SBLs), which are usually co-produced by MBL-producing Enterobacterales.
31 Here we report the activity of aztreonam in combination with novel β-lactamase inhibitors
32 (BLIs) against a national multicentre study collection of strains co-producing MBLs and
33 SBLs. Fifty-five clinical isolates co-producing MBLs (41 VIM-producers, 10 NDM-
34 producers and 4 IMP-producers) and SBLs were selected, and whole-
35 genome sequencing (WGS) was performed. Aztreonam, aztreonam/avibactam,
36 aztreonam/relebactam, aztreonam/zidebactam, aztreonam/taniborbactam,
37 aztreonam/vaborbactam and aztreonam/enmetazobactam MICs were determined. β-
38 lactam/BLI resistance mechanisms were analysed by WGS. All BLIs decreased the
39 aztreonam MICs for strains not susceptible to aztreonam. Aztreonam/zidebactam (MIC
40 ≤1 mg/L for 96.4% of isolates), aztreonam/avibactam (MIC ≤1 mg/L for 92.7% of
41 isolates) and aztreonam/taniborbactam (MIC ≤1 mg/L for 87.3% of isolates) were the
42 most active combinations. For other aztreonam/BLI combinations, 50-70% of the isolates
43 yielded MICs ≤1 mg/L. WGS data revealed that mutations in PBP3, defective
44 OmpE35/OmpK35 porins and the presence of ESBLs and class C β-lactamases were
45 some of the resistance mechanisms involved in reduced susceptibility to aztreonam/BLIs.
46 Combinations of aztreonam with new BLIs show promising activity against
47 Enterobacterales co-producing MBLs and SBLs, particularly the aztreonam/zidebactam,
48 aztreonam/avibactam and aztreonam/taniborbactam combinations. Our results show that
49 these novel drugs may represent innovative therapeutic strategies by their use in yet
50 unexplored combinations as solutions for difficult-to-treat infections.

51 **Keywords:** β-lactamase inhibitors, aztreonam, Enterobacterales, metallo-β-lactamase, CPE, WGS.

53
## 1. Introduction

Infections caused by metallo-β-lactamase (MBL)-producing Enterobacterales are increasing globally at an alarming rate [1]. These infections present an elevated risk of mortality due to inadequate therapeutic options [2]. MBL producers are usually resistant to the vast majority of β-lactams, including carbapenems and β-lactam/β-lactamase inhibitor (BLI) combinations, therefore exist few therapeutic alternatives and optimal treatments are not well established [2,3].

New recently approved inhibitors such as avibactam (in combination with ceftazidime), relebactam (with imipenem) and vaborbactam (with meropenem) are unfortunately not able to inhibit MBLs [4]. The diazacyclooctane (DBO) avibactam is a BLI with a wide spectrum of activity against the most clinically relevant class A, C and D β-lactamases of Enterobacterales, such as KPC, GES, CTX-M, SHV and OXA-48. The second DBO approved for clinical use, relebactam (which is closely related to avibactam), displays similar activity against class A and C β-lactamases, although its activity against OXA-48 is much lower. Similarly, the boronic acid derivative vaborbactam exhibits strong activity against class A and C β-lactamases, including KPC carbapenemases, but it is not effective against class D β-lactamases [4].

Aztreonam is the only β-lactam not hydrolysed by MBLs. However, serine β-lactamases (SBLs), such as many AmpC and ESBLs, present in most MBL-producing strains, can inactivate aztreonam. Thus, combining aztreonam with an SBL inhibitor is therefore a promising therapeutic option for Enterobacterales that co-produce SBLs and MBLs. The
75 aztreonam/avibactam combination is currently undergoing clinical trials and awaiting
76 approval by regulatory agencies (NCT03580044). Combining ceftazidime/avibactam
77 with aztreonam, recommended by Infectious Diseases Society of America for treating
78 carbapenem-resistant MBL-producing Enterobacterales [5], is currently being applied in
79 clinical settings, with promising results [3]. These attractive combinations including
80 aztreonam could avoid the use of less desirable options such as colistin for combating
81 MBL infections.

82 We are now fortunately witnessing the development of other new β-lactamase inhibitors
83 (e.g. zidebactam, taniborbactam and enmetazobactam), which are currently under clinical
84 development in combination with different β-lactams. Combining these inhibitors with
85 aztreonam may represent an innovative therapeutic strategy against MBLs. However, the
86 potential activity against MBL producers remains unexplored.

87 We aimed to evaluate the activity of aztreonam in combination with the novel β-lactamase
88 inhibitors avibactam, relebactam, zidebactam, taniborbactam, vaborbactam and
89 enmetazobactam, in a multicentre collection of MBL-producing Enterobacterales, fully
90 sequenced by hybrid whole-genome sequencing (WGS), in order to identify therapeutic
91 candidates against this currently uncovered therapeutic niche.

## 2. Material and Methods

### 2.1. Bacterial isolates and susceptibility testing
The bacterial isolates under study were recovered in a Spanish multicentre survey (involving 24 hospitals) conducted during a two-month period in 2018, in order to evaluate the epidemiology and resistance mechanisms of carbapenemase-producing Enterobacterales (CPEs) [6]. From this collection of 400 non-duplicate CPE strains, all MBL-producing strains (55) were selected for this study.

Aztreonam, aztreonam/avibactam, aztreonam/relebactam, aztreonam/zidebactam, aztreonam/taniborbactam, aztreonam/vaborbactam and aztreonam/enmetazobactam MICs were determined by reference broth microdilution. MICs were also determined to imipenem, meropenem and ertapenem [7,8]. Aztreonam was tested at concentrations between 1 and 128 mg/L, alone and in all combinations. The BLIs were tested at fixed concentrations: 4 mg/L for avibactam, relebactam and taniborbactam, and 8 mg/L for vaborbactam and enmetazobactam. As both zidebactam and aztreonam exhibit antimicrobial activity, a 1:1 ratio was used in the tests to eliminate any bias in the activity of either component [9]. *Escherichia coli* ATCC 25922 and *Pseudomonas aeruginosa* ATCC 27853 reference strains were used as controls [9]. The antimicrobial activity of each inhibitor was also tested at concentrations between 1 and 128 mg/L. EUCAST v12.0 MIC breakpoints for aztreonam (≤1 mg/L for susceptibility and >4 mg/L for resistance) were adopted in this study for all aztreonam/BLI combinations (as breakpoints for these combinations have not yet been determined) [10]. EUCAST v12.0 MIC breakpoints for imipenem, meropenem and ertapenem were also used [10].

115
## 2.2. WGS and antimicrobial resistance genotypes

All isolates were sequenced in parallel using both short- (Illumina MiSeq benchtop, Illumina) and long-read (MinION, Oxford Nanopore Technologies) approaches, as previously described [6].

Antimicrobial resistance gene content, including all β-lactamase genes, was evaluated *in silico* using the Resfinder [11], AMRFinderPlus [12] and CARD [13] tools. SNPs were identified from bacterial sequencing data by variant calling with Snippy v4.6.0 [14], to evaluate the mechanisms involved in β-lactam/BLI resistance, as previously described [15].

## 3. Results

Fifty-five completely sequenced clinical isolates of Enterobacterales co-producing MBLs and SBLs were included in the study: 41 VIM-producers, 10 NDM-producers and 4 IMP-producers. The presence of MBLs is reflected in the carbapenem resistance profiles of the isolates, i.e. resistance to imipenem (29.1%), meropenem (18.2%) and ertapenem (90.9%) (Table S1).

The aztreonam susceptibility rate for the 55 MBL-producing strains was 18.2% ($MIC_{50}/90$ 64/≥128 mg/L). Aztreonam/zidebactam ($MIC \le 1$ mg/L for 96.4% of isolates, $MIC_{50}/90$ $\le 1/\le 1$ mg/L) and aztreonam/avibactam ($MIC \le 1$ mg/L for 92.7% of isolates, $MIC_{50}/90$ $\le 1/\le 1$ mg/L) were the most active combinations (Tables 1 and 2).
137 Aztreonam/vaborbactam (MIC ≤1 mg/L for 52.7% of isolates, MIC$_{50/90}$ ≤1/32 mg/L) and
138 aztreonam/enmetazobactam (MIC ≤1 mg/L for 54.5% of isolates, MIC$_{50/90}$ ≤1/32 mg/L)
139 combinations showed the lowest activity, although both inhibitors significantly reduced
140 the aztreonam MIC to ≤1 mg/L in more than 30% of isolates. The aztreonam/relebactam
141 and aztreonam/taniborbactam MICs were ≤1 mg/L in 69.1% and 87.3% of isolates,
142 respectively. The MICs determined by assessing only those isolates not susceptible to
143 aztreonam (n=45) are shown in Table S2. The activity of each BLI was also evaluated,
144 among these, only zidebactam showed antimicrobial activity by itself (MIC$_{50/90}$ ≤1/≥128
145 mg/L), while the other BLIs yielded MICs ≥128 mg/L with all isolates.

146 As the various types of MBLs have different biochemical and genetic characteristics, it
147 is of interest to examine the activity of the BLIs according to each MBL type. In the
148 subgroup of 41 VIM-producing isolates, 24.4% of strains were susceptible to aztreonam
149 alone. Aztreonam/avibactam, aztreonam/zidebactam and aztreonam/taniborbactam
150 showed high activity, with 95.1%, 95.1% and 87.8% of strains yielding MICs ≤1 mg/L,
151 respectively, whereas the percentages were lower for the other combinations (50-75%)
152 (Figure S1). Those isolates yielding MICs >4 mg/L for aztreonam/BLIs and the possible
153 resistance mechanisms are indicated in Table 2.

154 The 10 NDM-producing strains (also CTX-M-15 co-producers) displayed high resistance
155 to aztreonam (100% with MIC ≥64 mg/L). These strains showed high susceptibility to
156 aztreonam/zidebactam, aztreonam/avibactam, aztreonam/taniborbactam and
157 aztreonam/enmetazobactam, with MICs ≤1 mg/L in 100%, 90%, 80% and 80% of
158 isolates, respectively. Only one strain was resistant to aztreonam/avibactam and two
159 strains were resistant to aztreonam/relebactam, aztreonam/vaborbactam, 160 aztreonam/taniborbactam and aztreonam/enmetazobactam (Table 1 and Figure S1). 161 These were E. coli NDM-5 and NDM-7 producers and both possessed the I532L mutation 162 and the Y334_N337insYRIN insertion in PBP3 (Table 2), previously implicated in β- 163 lactam resistance [16].

164 None of the 4 IMP-producing strains were susceptible to aztreonam. The 165 aztreonam/zidebactam, aztreonam/relebactam and aztreonam/taniborbactam 166 combinations showed the highest activity (100% of strains yielding MICs ≤1 mg/L). Only 167 one strain yielded MIC >4 mg/L for aztreonam/enmetazobactam (Table 2 and Figure 168 S1).

169 In all 3 groups of MBL-producing isolates, zidebactam alone displayed good 170 antimicrobial activity (73.1-90.0% of strains yielded MICs ≤1 mg/L), with even higher 171 efficacy than aztreonam/vaborbactam and aztreonam/enmetazobactam combinations.

173 **4. Discussion**

174 In this study we explored for the first time the activity of aztreonam in combination with 175 new BLIs against a WGS-characterized collection of MBL-producing Enterobacterales. 176 Excellent overall activity of the different combinations was observed, with 177 aztreonam/zidebactam and aztreonam/avibactam being the most active (>90% of strains 178 yielding MICs ≤1 mg/L). The activity of aztreonam/avibactam against MBL- and SBL- 179 producing strains has already been extensively analysed, showing excellent activity
(currently in clinical phase III) [17]. In a previous study, we evaluated the activity of enmetazobactam, zidebactam and taniborbactam in combination with cefepime against a collection of 400 CPEs [15]. Comparison of the results obtained in both studies with the 55 MBL-producing strains showed that while the percentage of strains yielding MICs ≤1 mg/L was similar for aztreonam/zidebactam and cefepime/zidebactam (96.4% and 89.3%, respectively), important differences in activity were observed between aztreonam/taniborbactam and cefepime/taniborbactam (87.3% and 58.9%, respectively) and between aztreonam/enmetazobactam and cefepime/enmetazobactam (54.5% and 0%, respectively). Similar results were found on comparing the data obtained for the same collection of isolates for aztreonam/relebactam in this study (69.1% of strains yielding MICs ≤1 mg/L) and for imipenem/relebactam in a previous study (44.6% of strains yielding MICs ≤1 mg/L) [6]. These results show the importance of these promising combinations of new β-lactamase inhibitors and aztreonam, which are capable of improving the activity shown by approved or more developed combinations. The excellent activity shown by the aztreonam/zidebactam combination is also due to the antimicrobial activity of zidebactam itself (Table 1) [18]. The antimicrobial activity of zidebactam, identified as a β-lactam-enhancer, is due to its ability to bind to PBP2 of Enterobacterales or *P. aeruginosa* [4]. The aztreonam/zidebactam combination was therefore tested at a ratio 1:1 (unlike the other combinations, which were tested at a fixed inhibitor concentration) to eliminate the potential bias in activity due to either of the compounds.
Although scarce, some strains yielding MIC >4 mg/L for aztreonam/BLI were identified.

The main reasons for this resistance include i) the inability of the novel inhibitor to inhibit SBLs, or high hydrolysis efficiency/expression of the β-lactamase and a partially effective inhibitor and ii) the structural changes in the target PBP-3 or decreased permeability to antibiotic or inhibitors. Research on the resistance mechanisms for these new combinations is scarce, and prediction of the exact causes of the resistance is difficult.

**Table 2** summarizes the putative mechanisms leading to the elevated MICs of aztreonam/BLIs. These include the presence of some ESBLs or chromosomal class C β-lactamases variants that are not completely inhibited by the new inhibitors, the lack of porin *ompK35* (*K. pneumoniae*) or *ompE35* (*E. cloacae* complex) and important mutations in PBP3. Similar mechanisms have recently been identified in aztreonam/avibactam-resistant clinical isolates and in aztreonam/avibactam-resistant single-step mutants of Enterobacterales [19–21].

Regarding the antimicrobial activity of zidebactam, high MICs (≥64 mg/L) of this compound were only obtained with nine isolates. Three of these strains belong to the species *S. marcescens*, which was found to be intrinsically resistant to zidebactam [22].

The other six isolates (three *K. pneumoniae*, two *Klebsiella oxytoca* and one *Citrobacter freundii* isolate), one *K. oxytoca* (AI2828) and one *K. pneumoniae* (AI2867) isolates were found to carry a non-functional OmpK35, which has previously been implicated in zidebactam resistance [23]. Importantly, the *K. pneumoniae* AI2867 isolate was the only one in this collection which showed resistance to the aztreonam/zidebactam combination.

The mechanisms of zidebactam resistance are largely unknown, and the high MICs
223 observed for the remaining four isolates may be due to loss of affinity, an unfavourable
224 balance of permeation, enhanced efflux (or combination of all the three), or even other as
225 yet unresolved factors [24].

226 The data suggest that some of these aztreonam/BLI combinations, especially those
227 including avibactam, zidebactam and taniborbactam, may represent innovative
228 therapeutic strategies for treating infections caused by MBL-producing Enterobacterales
229 in critically ill or immunocompromised patients, for whom treatment options are limited.
230 In vivo data, analysis of large collections of clinical strains and clinical studies are needed
231 to confirm the potential role of the novel combinations in treating MBL-producing
232 Enterobacterales, a currently uncovered therapeutic niche.

233

234 *Access to data*

235 The raw data (Illumina and Oxford Nanopore Technologies sequence reads) and
236 annotated assemblies corresponding to the CPE genomes have been submitted to the
237 European Nucleotide Archive under BioProject accessions PRJEB42440 and
238 PRJEB39112.

239
240 **Declarations**

241 **Funding:** This study was funded by Instituto de Salud Carlos III (ISCIII) through the projects N° Exp PI20/01212 and PI18/00501, integrated in the National Plan for Scientific Research, Development and Technological Innovation 2017-2020, and Co-funded by European Union - European Regional Development Fund "A way to make Europe".

245 The work was also supported by CIBERINFEC (CIBER de Enfermedades Infecciosas).

246 The study was also funded by project IN607A 2020/05 (GAIN- Agencia Gallega de Innovación - Consellería de Economía, Emprego e Industria) awarded to GB and project IN607D 2021/12 awarded to AB. The work was also supported by Planes Nacionales de I+D+i 2017-2020 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/006) co-financed by European Development Regional Fund "A way to achieve Europe" and operative program Intelligent Growth 2014-2020. JA-S was financially supported by the Juan Rodés program (ISCIII, JR21/00026), JCV-U was financially supported by IN606B-2022/009 Grant (Xunta de Galicia), CL-M was financially supported by IN606A-2019/029 Grant (Xunta de Galicia) and PI20/01212 (ISCIII) and PG-S was financially supported by IN607A 2020/05 Grant (Xunta de Galicia). AC and MO were financially supported by IN607D 2021/12. IA-G was financially supported by the Río Hortega program (ISCIII, CM21/00076). LA-F was financially supported by the IN606B-2018/011 (Xunta de Galicia). MM-G was financially supported by the RSU.UDC.M505 (Ministerio de Universidades).

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262 **Competing Interests:** Avibactam was provided by Pfizer and vaborbactam, zidebactam, taniborbactam and enmetazobactam by MedChemExpress. Merck Sharp & Dohme provided relebactam and did not exercise any control over the conduct or reporting of the research.

266 **Ethical Approval:** Not required

267 **Sequence Information:** Genomes have been submitted to the European Nucleotide Archive under BioProject accessions PRJEB42440 and PRJEB39112

268

269

270 **Acknowledgements**

271 This research was made possible by the helpful collaboration of the following researchers:

272 Irene Merino, Emilia Cercenado, Rosa Gómez, Tamara Soler, Irene Gracia-Ahufinger,

273 Lina Martín, Fátima Galán, Nuria Tormo, Juan Carlos Rodríguez, Silvia Capilla, Francesc

274 Marco, María Dolores Quesada, Emma Padilla, Fe Tubau, Juanjo González, Ana Isabel

275 López-Calleja, José Luis del Pozo, María Inmaculada García, Mariela Martínez, Jorge

276 Calvo, Xavier Mulet, Fernanda Peña, Ana Isabel Rodríguez, María José Gude, Ana

277 Fernández, Javier Fernández, Felipe Fernández-Cuenca and Álvaro Pascual.

278
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372
<figure><img src="image_3.png" /><figcaption>**Table 1.** Cumulative MIC distribution for aztreonam in the presence and absence of novel β-lactamase inhibitors in 55 clinical isolates of metallo-β-lactamase-producing Enterobacterales.</figcaption></figure>

<table><thead><tr><th rowspan="2">Aztreonam/BLIs combinations</th><th colspan="7">Cumulative % of isolates at MIC (mg/L)</th><th colspan="2">% isolates with MICs values (mg/L)</th><th colspan="2">MIC</th></tr><tr><th>≤1</th><th>2</th><th>4</th><th>8</th><th>16</th><th>32</th><th>64</th><th>≤1</th><th>>4</th><th>MIC<sub>50</sub></th><th>MIC<sub>90</sub></th></tr></thead><tbody><tr><td>Aztreonam</td><td>18.2</td><td>27.3</td><td>36.4</td><td>40.0</td><td>45.5</td><td>49.1</td><td>63.6</td><td>18.2</td><td>63.6</td><td>64</td><td>≥128</td></tr><tr><td>Aztreonam/avibactam</td><td>92.7</td><td>98.2</td><td>98.2</td><td>100</td><td></td><td></td><td></td><td>92.7</td><td>1.8</td><td>≤1</td><td>≤1</td></tr><tr><td>Aztreonam/relebactam</td><td>69.1</td><td>81.8</td><td>90.9</td><td>92.7</td><td>94.5</td><td>98.2</td><td>98.2</td><td>69.1</td><td>9.1</td><td>≤1</td><td>4</td></tr><tr><td>Aztreonam/zidebactam</td><td>96.4</td><td>96.4</td><td>98.2</td><td>98.2</td><td>98.2</td><td>98.2</td><td>98.2</td><td>96.4</td><td>1.8</td><td>≤1</td><td>≤1</td></tr><tr><td>Zidebactam<sup>a</sup></td><td>76.4</td><td>83.6</td><td>83.6</td><td>83.6</td><td>83.6</td><td>83.6</td><td>85.5</td><td>76.4</td><td>16.4</td><td>≤1</td><td>≥128</td></tr><tr><td>Aztreonam/taniborbactam</td><td>87.3</td><td>89.1</td><td>94.5</td><td>98.2</td><td>98.2</td><td>98.2</td><td>100</td><td>87.3</td><td>5.5</td><td>≤1</td><td>4</td></tr><tr><td>Aztreonam/vaborbactam</td><td>52.7</td><td>61.8</td><td>80.0</td><td>81.8</td><td>87.3</td><td>90.9</td><td>92.7</td><td>52.7</td><td>20.0</td><td>≤1</td><td>32</td></tr><tr><td>Aztreonam/enmetazobactam</td><td>54.5</td><td>69.1</td><td>80.0</td><td>87.3</td><td>89.1</td><td>92.7</td><td>94.5</td><td>54.5</td><td>20.0</td><td>≤1</td><td>32</td></tr></tbody></table>

<footnote><sup>a</sup> All strains yielded MICs ≥64 mg/L for avibactam, relebactam, taniborbactam, vaborbactam and enmetazobactam. BLIs were tested in combination with aztreonam at the following concentrations: avibactam (4 mg/L), relebactam (4 mg/L), zidebactam (1:1 ratio with aztreonam), taniborbactam (4 mg/L), vaborbactam (8 mg/L) and enmetazobactam (8 mg/L).</footnote>
<figure><img src="image_4.png" /><figcaption>1 **Table 2.** Antimicrobial susceptibility (MICs in mg/L) to aztreonam, 2 aztreonam/avibactam, aztreonam/relebactam, aztreonam/zidebactam, zidebactam, 3 aztreonam/taniborbactam, aztreonam/vaborbactam and aztreonam/enmetazobactam of 4 MBL-producing Enterobacteriales isolates, according to the carbapenemase present, 5 description of β-lactamases produced and possible aztreonam-BLIs resistance 6 mechanisms.</figcaption></figure>

<table><thead><tr><th>Strains</th><th>Species</th><th>Carba-<br>penemase</th><th>ATM<sup>a</sup></th><th>ATM/<br>AVI</th><th>ATM/<br>REL</th><th>ATM/<br>ZID</th><th>ZID</th><th>ATM/<br>VAB</th><th>ATM/<br>TAN</th><th>ATM/<br>ENM</th><th>Presence of<br>ESBLs</th><th>Presence of other<br>β-lactamases</th><th>Other possible<br>resistance<br>mechanisms<br>found by WGS</th></tr></thead><tbody><tr><td>AI2860</td><td>E. coli</td><td>bla<sub>VIM-1</sub></td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td></td><td>bla<sub>EC-1588</sub>, bla<sub>TEM-1</sub></td><td>-</td></tr><tr><td>AI2660</td><td>K. pneumoniae</td><td>bla<sub>VIM-1</sub></td><td>64</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>2</td><td>≤1</td><td>2</td><td></td><td>bla<sub>SHV-11</sub>, bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI2909</td><td>K. pneumoniae</td><td>bla<sub>VIM-1</sub></td><td>≥128</td><td>≤1</td><td>16</td><td>4</td><td>≥128</td><td>32</td><td>2</td><td>2</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>SHV-11</sub>, bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI3025</td><td>K. pneumoniae</td><td>bla<sub>VIM-1</sub></td><td>2</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>bla<sub>SHV-28</sub></td><td>bla<sub>TEM-1</sub></td><td>-</td></tr><tr><td>AI2867</td><td>K. pneumoniae</td><td>bla<sub>VIM-1</sub></td><td>≥128</td><td>≤1</td><td>≥128</td><td>≥128</td><td>≥128</td><td>≥128</td><td>64</td><td>4</td><td>(2x) bla<sub>CTX-M-15</sub></td><td>bla<sub>SHV-11</sub>, (2x)<br>bla<sub>OXA-1</sub></td><td>OmpK35 NF<br>(W316*)</td></tr><tr><td>AI2885</td><td>K. pneumoniae</td><td>bla<sub>VIM-1</sub></td><td>≥128</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>bla<sub>SHV-28</sub>,<br>bla<sub>CTX-M-15</sub></td><td>bla<sub>TEM-1</sub></td><td>-</td></tr><tr><td>AI2848</td><td>K. pneumoniae</td><td>bla<sub>VIM-1</sub></td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td></td><td>bla<sub>SHV-11</sub>, bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI2797</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>2</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>2</td><td>bla<sub>CTX-M-9</sub></td><td>bla<sub>ACT-1</sub>, bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI2802</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>4</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>bla<sub>CTX-M-9</sub></td><td>bla<sub>ACT-1</sub>, bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI2615</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>≥128</td><td>≤1</td><td>≤1</td><td>≤1</td><td>2</td><td>≥128</td><td>4</td><td>≥128</td><td></td><td>bla<sub>ACT-24</sub></td><td>-</td></tr><tr><td>AI2619</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>64</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>32</td><td></td><td>bla<sub>ACT-24</sub>, bla<sub>ACC-1</sub></td><td>-</td></tr><tr><td>AI2620</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>16</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>4</td><td>≤1</td><td>4</td><td></td><td>bla<sub>ACT-24</sub>, bla<sub>DHA-1</sub></td><td>-</td></tr><tr><td>AI2659</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>32</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>2</td><td>≤1</td><td>2</td><td>bla<sub>CTX-M-9</sub></td><td>bla<sub>ACT-24</sub></td><td>-</td></tr><tr><td>AI2798</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>≥128</td><td>≤1</td><td>8</td><td>≤1</td><td>≤1</td><td>8</td><td>≤1</td><td>8</td><td>bla<sub>CTX-M-9</sub>,<br>bla<sub>SHV-12</sub></td><td>bla<sub>ACT-24</sub>, bla<sub>TEM-1</sub></td><td>-</td></tr><tr><td>AI2804</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>≥128</td><td>≤1</td><td>4</td><td>≤1</td><td>≤1</td><td>16</td><td>≤1</td><td>8</td><td>bla<sub>CTX-M-9</sub>,<br>bla<sub>SHV-12</sub></td><td>bla<sub>ACT-24</sub>, (2x)<br>bla<sub>TEM-1</sub></td><td>-</td></tr><tr><td>AI2936</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>≥128</td><td>≤1</td><td>4</td><td>≤1</td><td>2</td><td>≥128</td><td>4</td><td>≥128</td><td>bla<sub>CTX-M-9</sub></td><td>bla<sub>ACT-24</sub></td><td>-</td></tr><tr><td>AI2613</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>16</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>2</td><td>≤1</td><td>8</td><td></td><td>bla<sub>ACT-17</sub>, (2x)<br>bla<sub>ACC-1</sub>, bla<sub>OXA-1</sub><br>bla<sub>ACT-17</sub>, (2x)<br>bla<sub>ACC-1</sub>, bla<sub>OXA-1</sub></td><td>OmpE35 NF<br>(F112fs)</td></tr><tr><td>AN2360</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>32</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>4</td><td></td><td>bla<sub>ACT-17</sub>, bla<sub>OXA-1</sub></td><td>OmpE35 NF<br>(F112fs)</td></tr><tr><td>AI2865</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>2</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td></td><td>bla<sub>ACT-like</sub></td><td>-</td></tr><tr><td>AI2772</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>8</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>4</td><td></td><td>bla<sub>ACT-27</sub>, bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI2759</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>≥128</td><td>≤1</td><td>2</td><td>≤1</td><td>≤1</td><td>16</td><td>≤1</td><td>16</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>ACT-16</sub>, bla<sub>OXA-1</sub>,<br>bla<sub>TEM-1</sub></td><td>-</td></tr><tr><td>AI2972</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>64</td><td>≤1</td><td>2</td><td>≤1</td><td>≤1</td><td>4</td><td>≤1</td><td>4</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>ACT-16</sub>, bla<sub>OXA-1</sub>,<br>bla<sub>TEM-1</sub></td><td>-</td></tr><tr><td>AI2656</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>8</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>bla<sub>CTX-M-9</sub></td><td>bla<sub>ACT-17</sub></td><td>-</td></tr><tr><td>AI2657</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>≥128</td><td>2</td><td>4</td><td>≤1</td><td>2</td><td>≥128</td><td>4</td><td>≥128</td><td>bla<sub>CTX-M-9</sub></td><td>bla<sub>ACT-17</sub></td><td>-</td></tr><tr><td>AI2658</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>16</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>2</td><td>≤1</td><td>2</td><td>bla<sub>CTX-M-9</sub></td><td>bla<sub>ACT-17</sub></td><td>-</td></tr><tr><td>AI2859</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>2</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td></td><td>bla<sub>ACT-17</sub>, bla<sub>TEM-1</sub></td><td>-</td></tr><tr><td>AI2939</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>8</td><td>2</td><td>≤1</td><td>≤1</td><td>≤1</td><td>2</td><td>≤1</td><td>2</td><td>bla<sub>CTX-M-9</sub></td><td>bla<sub>ACT-17</sub></td><td>-</td></tr><tr><td>AI2839</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td></td><td>bla<sub>ACT-like</sub>, bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI3013</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>8</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>bla<sub>CTX-M-9</sub></td><td>bla<sub>ACT-like</sub>, bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI2799</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub>+<br>bla<sub>OXA-48</sub></td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>bla<sub>CTX-M-9</sub></td><td>bla<sub>ACT-40</sub>, bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI2941</td><td>E. cloacae complex</td><td>bla<sub>VIM-1</sub></td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td></td><td>bla<sub>MIR-like</sub>, bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI2609</td><td>C. freundii</td><td>bla<sub>VIM-1</sub></td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td></td><td>bla<sub>CMY-like</sub></td><td>-</td></tr><tr><td>AI3012</td><td>K. oxytoca</td><td>bla<sub>VIM-1</sub></td><td>4</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≥128</td><td>≤1</td><td>≤1</td><td>≤1</td><td></td><td>bla<sub>OXY-2-16</sub>, bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI2918</td><td>K. oxytoca</td><td>bla<sub>VIM-1</sub></td><td>4</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td></td><td>bla<sub>OXY-2-11</sub>, bla<sub>OXA-1</sub>, (2x)<br>bla<sub>TEM-1</sub></td><td>-</td></tr><tr><td>AI2991</td><td>K. oxytoca</td><td>bla<sub>VIM-1</sub></td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td></td><td>bla<sub>OXY-2-11</sub></td><td>-</td></tr><tr><td>AI2828</td><td>K. oxytoca</td><td>bla<sub>VIM-1</sub></td><td>64</td><td>≤1</td><td>2</td><td>≤1</td><td>≥128</td><td>4</td><td>≤1</td><td>≤1</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>OXY-1-1</sub>, bla<sub>OXA-1</sub></td><td>OmpK35 NF<br>(A181fs)</td></tr><tr><td>AI2937</td><td>K. oxytoca</td><td>bla<sub>VIM-1</sub></td><td>≥128</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>2</td><td>≤1</td><td>2</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>OXY-6-like</sub>, (2x)<br>bla<sub>OXA-1</sub>, bla<sub>TEM-1</sub>,<br>bla<sub>DHA-1</sub></td><td>-</td></tr><tr><td>AI2616</td><td>S. marcescens</td><td>bla<sub>VIM-1</sub></td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≥128</td><td>≤1</td><td>≤1</td><td>≤1</td><td></td><td>bla<sub>SRT-like</sub></td><td>-</td></tr><tr><td>AI2617</td><td>S. marcescens</td><td>bla<sub>VIM-1</sub></td><td>≥128</td><td>≤1</td><td>2</td><td>≤1</td><td>≥128</td><td>16</td><td>≤1</td><td>≤1</td><td>bla<sub>SHV-12</sub></td><td>bla<sub>SRT-like</sub></td><td>-</td></tr></tbody></table>
<table><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td></td><td>AI2618</td><td>S. marcescens</td><td>bla<sub>VIM-1</sub></td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≥128</td><td>≤1</td><td>≤1</td><td>≤1</td><td>bla<sub>SRT-like</sub></td><td>-</td></tr><tr><td></td><td>AI3010</td><td>K.<br>quasipneumoniae</td><td>bla<sub>VIM-1</sub></td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>bla<sub>OKP-B-41</sub></td><td>-</td></tr><tr><td rowspan="10">NDM-producing isolates</td><td>AI2843</td><td>E. coli</td><td>bla<sub>NDM-5</sub></td><td>≥128</td><td>≤1</td><td>32</td><td>≤1</td><td>≤1</td><td>32</td><td>8</td><td>8</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>EC-1149</sub>, bla<sub>CMY-2</sub>,<br>bla<sub>OXA-1</sub>, (3x)<br>bla<sub>TEM-1</sub></td><td>PBP3 (1532L,<br>Y334_N337insY<br>RIN)</td></tr><tr><td>AI2858</td><td>E. coli</td><td>bla<sub>NDM-7</sub></td><td>≥128</td><td>8</td><td>32</td><td>≤1</td><td>≤1</td><td>64</td><td>8</td><td>32</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>EC-725</sub>, bla<sub>CMY-6</sub>,<br>bla<sub>OXA-1</sub></td><td>PBP3 (1532L,<br>Y334_N337insY<br>RIN)</td></tr><tr><td>AI2880</td><td>K. pneumoniae</td><td>bla<sub>NDM-1</sub></td><td>≥128</td><td>≤1</td><td>2</td><td>≤1</td><td>≤1</td><td>4</td><td>≤1</td><td>≤1</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>SHV-1</sub>, bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI2898</td><td>K. pneumoniae</td><td>bla<sub>NDM-1</sub></td><td>≥128</td><td>≤1</td><td>2</td><td>≤1</td><td>≤1</td><td>4</td><td>≤1</td><td>≤1</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>SHV-1</sub>, (2x)<br>bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI3007</td><td>K. pneumoniae</td><td>bla<sub>NDM-1</sub></td><td>≥128</td><td>≤1</td><td>4</td><td>≤1</td><td>≤1</td><td>4</td><td>≤1</td><td>≤1</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>SHV-1</sub>, (2x)<br>bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI3008</td><td>K. pneumoniae</td><td>bla<sub>NDM-1</sub>+<br>bla<sub>OXA-48</sub></td><td>≥128</td><td>≤1</td><td>2</td><td>≤1</td><td>≤1</td><td>4</td><td>≤1</td><td>≤1</td><td>(2x) bla<sub>CTX-M-15</sub></td><td>bla<sub>SHV-1</sub>, bla<sub>OXA-1</sub></td><td>-</td></tr><tr><td>AI2830</td><td>K. pneumoniae</td><td>bla<sub>NDM-1</sub></td><td>64</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>SHV-11</sub></td><td>-</td></tr><tr><td>AI2934</td><td>K. pneumoniae</td><td>bla<sub>NDM-1</sub></td><td>≥128</td><td>≤1</td><td>4</td><td>≤1</td><td>≥128</td><td>4</td><td>≤1</td><td>≤1</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>SHV-11</sub>, bla<sub>OXA-1</sub>,<br>bla<sub>TEM-1</sub></td><td>-</td></tr><tr><td>AI2779</td><td>K. pneumoniae</td><td>bla<sub>NDM-23</sub></td><td>64</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>SHV-like</sub>, bla<sub>OXA-1</sub>,<br>bla<sub>TEM-1</sub></td><td>-</td></tr><tr><td>AI2793</td><td>K. pneumoniae</td><td>bla<sub>NDM-1</sub></td><td>64</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>SHV-like</sub>, bla<sub>OXA-1</sub>,<br>bla<sub>TEM-1</sub></td><td>-</td></tr><tr><td rowspan="4">IMP-producing isolates</td><td>AI2992</td><td>E. cloacae complex</td><td>bla<sub>IMP-8</sub></td><td>≥128</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>4</td><td>≤1</td><td>64</td><td>bla<sub>MIR-like</sub>, bla<sub>OXA-101</sub></td><td>-</td></tr><tr><td>AI2747</td><td>E. cloacae complex</td><td>bla<sub>IMP-13</sub></td><td>64</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>4</td><td>≤1</td><td>2</td><td>bla<sub>CTX-M-15</sub></td><td>bla<sub>ACT-16</sub>, bla<sub>OXA-1</sub>,<br>(3x) bla<sub>TEM-1</sub>,<br>bla<sub>OXA-2</sub></td><td>-</td></tr><tr><td>AI2760</td><td>E. cloacae complex</td><td>bla<sub>IMP-13</sub>+<br>bla<sub>OXA-48</sub></td><td>2</td><td>2</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>≤1</td><td>bla<sub>ACT-16</sub>, bla<sub>OXA-10</sub>,<br>bla<sub>OXA-2</sub></td><td>bla<sub>CMY-like</sub>, bla<sub>OXA-2</sub>,<br>(2x) bla<sub>TEM-like</sub></td><td>-</td></tr><tr><td>AI2642</td><td>C. freundii</td><td>bla<sub>IMP-22</sub>+<br>bla<sub>KPC-2</sub></td><td>≥128</td><td>≤1</td><td>≤1</td><td>≤1</td><td>64</td><td>≤1</td><td>≤1</td><td>4</td><td>bla<sub>CMY-like</sub>, bla<sub>OXA-2</sub>,<br>(2x) bla<sub>TEM-like</sub></td><td>-</td></tr></tbody></table>

7 aATM, aztreonam; ATM/AVI, aztreonam/avibactam; ATM/REL, aztreonam/relebactam; ATM/ZID,

8 aztreonam/zidebactam, ZID, zidebactam; ATM/VAB, aztreonam/vaborbactam; ATM/TAN,

9 aztreonam/taniborbactam; ATM/ENM, aztreonam/enmetazobactam. (2x), 2 copies of the gene; (3x), 3 copies of the gene. NF, non-functional.; fs, frameshift; ins, insertion; *, stop codon. BLIs were tested in combination with aztreonam at the following concentrations: avibactam (4 mg/L), relebactam (4 mg/L), zidebactam (1:1 ratio with aztreonam), taniborbactam (4 mg/L), vaborbactam (8 mg/L) and enmetazobactam (8 mg/L).