# Assessment of Activity and Resistance Mechanisms to Cefepime in Combination with the Novel b-Lactamase Inhibitors Zidebactam, Taniborbactam, and Enmetazobactam against a Multicenter Collection of Carbapenemase-Producing Enterobacterales

Juan Carlos Vázquez-Ucha, Cristina Lasarte-Monterrubio, Paula

Guijarro-Sánchez, Marina Oviaño, Laura Álvarez-Fraga, Isaac Alonso-García,

Jorge Arca-Suárez, German Bou, Alejandro Beceiro

► To cite this version:

Juan Carlos Vázquez-Ucha, Cristina Lasarte-Monterrubio, Paula Guijarro-Sánchez, Marina Oviaño, Laura Álvarez-Fraga, et al.. Assessment of Activity and Resistance Mechanisms to Cefepime in Combination with the Novel b-Lactamase Inhibitors Zidebactam, Taniborbactam, and Enmetazobactam against a Multicenter Collection of Carbapenemase-Producing Enterobacterales. Antimicrobial Agents and Chemotherapy, 2022, 66 (e01676-21), 10.1128/AAC.01676-21. hal-04958960

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1 Title:

2 Assessment of activity and resistance mechanisms to cefepime in
3 combination with the novel β-lactamase inhibitors zidebactam,
4 taniborbactam and enmetazobactam against a multicenter collection
5 of carbapenemase-producing Enterobacterales.

6

7

8 Juan Carlos Vázquez-Ucha*¹, Cristina Lasarte-Monerrubio*¹, Paula Guijarro-Sánchez*¹, Marina
9 Oviaño¹, Laura Álvarez-Fraga¹, Isaac Alonso-García², Jorge Arca-Suárez¹, German Bou#¹ and
10 Alejandro Beceiro #¥¹ on behalf of the GEMARA-SEIMC/REIPI Enterobacterales Study Group‡

11

12 ¹Servicio de Microbiología do Complejo Hospitalario Universitario da Coruña (CHUAC), Instituto
de Investigación Biomédica da Coruña (INIBIC), Xubias de Arriba, 84, 15006 A Coruña, CIBER
de Enfermedades Infecciosas, Spain.

15 ²Servicio de Microbiología, Hospital Provincial Pontevedra, Loureiro Crespo 2,
16 36002, Pontevedra, Spain.

17

18 *Juan Carlos Vázquez-Ucha, Cristina Lasarte-Monerrubio and Paula Guijarro-Sánchez,
19 contributed equally to the study

20 #German Bou and Alejandro Beceiro, contributed equally to the study

21 ‡Members are listed in the Acknowledgements section.

22

23 ¥ Author for correspondence: Dr. Alejandro Beceiro

24 e-mail: Alejandro.Beceiro.Casas@sergas.es

25 Servicio de Microbiología. Complejo Hospitalario Universitario A Coruña (CHUAC)

26 As Xubias, 15006, A Coruña, Spain. Telephone: +34 981178000 ext. 292145 Fax: +34 981176097

27

28 KEYWORDS: β-lactamase inhibitors, antimicrobial resistance, carbapenemase-producing
29 Enterobacterales, zidebactam, taniborbactam, enmetazobactam, cefepime, restoring antimicrobial
30 activity.

31

32 Running title: New cefepime/BLIs combinations against CPE
The global distribution of carbapenemases such as KPC, MBLs and OXA-48 gives cause for concern, as these enzymes are not inhibited by classical β-lactamase inhibitors (BLIs).

The current development of new inhibitors is one of the most promising highlights for the treatment of multidrug-resistant bacteria. The activity of cefepime in combination with the novel BLIs zidebactam, taniborbactam and enmetazobactam was studied in a collection of 400 carbapenemase-producing Enterobacterales (CPE). The genomes were fully sequenced and potential mechanisms of resistance to cefepime/BLI combinations were characterized.

Cefepime resistance in the whole set of isolates was 79.5% (MIC<sub>50/90</sub> 64/≥128mg/L). The cefepime/zidebactam and cefepime/taniborbactam combinations showed the highest activity (MIC<sub>50/90</sub> ≤0.5/1 and ≤0.5/2 mg/L, respectively). Cefepime/zidebactam displayed high activity, regardless of the carbapenemase or ESBL considered (99% MIC ≤2 mg/L).

Cefepime/taniborbactam displayed excellent activity against OXA-48- and KPC-producing Enterobacterales and lower activity against MBL-producing isolates (4 strains yielded MICs ≥16 mg/L:2 NDM producers with an insertion in PBP3, 1 VIM-1 producer with non-functional OmpK35 and 1 IMP-8 producer). Cefepime/enmetazobactam displayed the lowest activity (MIC<sub>50/90</sub> 1/≥128 mg/L), with MICs ≥16 mg/L for 49 MBL producers, 40 OXA-48 producers (13 with **amino acid changes** in OmpK35/36, 4 in PBPs and 11 in RamR) and 25 KPC producers (most with an insertion in OmpK36).

These results confirm the therapeutic potential of the new β-lactamase inhibitors, shedding light on the activity of cefepime and BLIs against CPE and resistance mechanisms. The cefepime/zidebactam and cefepime/taniborbactam combinations are particularly highlighted as promising alternatives to penicillin-based inhibitors for the treatment of CPE.
57 INTRODUCTION

The main strategy to restore the effectiveness of β-lactam antibiotics is the use of β-
lactamase inhibitors (BLI). The global distribution of class A carbapenemases such as
KPC, class B β-lactamases such as VIM, IMP and NDM, and class D β-lactamases such as
OXA-48, is a cause for concern, since they are not inhibited by the classical inhibitors (1).
After a period of no new significant advances in this area, several families of broad-
spectrum inhibitors have emerged in recent years in the fight against bacterial multidrug
resistance (2–4).

Three main families of compounds with inhibitory ability are attracting the attention of
scientific and clinical societies: i) the diazabicyclooctanes (DBOs), approved inhibitors in
this group are relebactam and avibactam (5); ii) boronic acid derivatives, within this group,
vaborbactam has been approved (6, 7) and iii) penicillin-based sulfones, such as the
classical inhibitors (8). The next-generation β-lactamase inhibitors belonging to these
families are zidebactam (WCK 5107, DBO), taniborbactam (VNRX-5133, boronate) and
enmetazobactam (AAI101, penicillanic acid sulfone) (Figure S1). The efficacy of these
three compounds is currently being evaluated in combination with cefepime in phase III
clinical trials with very promising results (9).

Cefepime's twice-a-day dosage schedule, enhanced activity against Enterobacterales and some Gram-positive organisms, and stability against AmpC give it several advantages over other cephalosporins and penicillins and allow its widespread use by physicians (10).

However, cefepime can be hydrolyzed by ESBLs and carbapenemases (with moderate resistance to hydrolysis by OXA-48). This important limitation calls for the search and development of new cefepime/BLIs combinations for use as carbapenem-sparing alternatives and also against carbapenemase-producing Enterobacterales (CPE) (9).
81

In recent years, several studies have evaluated the activity of new inhibitors in combination
with cefepime against MDR pathogens (see review of Isler *et al*) (9), however the
experimental comparison of the *in vitro* activity of the new β-lactamase inhibitors
zidebactam, taniborbactam and enmetazobactam has not yet been performed.

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The objective of this study was to evaluate these novel combinations of cefepime with
inhibitors, in phase III clinical trials, against a collection of 400 CPEs (304 OXA-48-
producing, 44-KPC-producing and 56-MBL-producing Enterobacterales), collected in a
multicenter study of Spanish hospitals in 2018, in which treatment with carbapenems
would not be the choice. Carbapenem-resistant Enterobacterales without carbapenemases
were not included in the collection, in order to evaluate the activity of new inhibitors
against these enzymes. The genomes of whole collection have been fully sequenced and
their mechanisms of resistance to β-lactam antibiotics characterized. The results of this
study will help us to reaffirm the therapeutic potential of these new alternatives and the
activity of cefepime combinations with zidebactam, taniborbactam and enmetazobactam
against CPE.

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# RESULTS AND DISCUSSION

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## Activity of cefepime/BLI combinations against carbapenemase-producing

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Enterobacterales

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Since cefepime/BLI breakpoints have not yet been established, to facilitate comparison and
to evaluate the activity of new combinations, the cefepime breakpoints of ≤2 mg/L for
susceptibility and ≥16 mg/L for resistance were adopted in this study. The cefepime
resistance rate for the complete set of isolates evaluated in this study was 79.5%, MIC<sub>50/90</sub>
64/≥128 mg/L. Cefepime/zidebactam was the most active combination (99.0% inhibited at

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MIC ≤2 mg/L, MIC<sub>50/90</sub> ≤0.5/1 mg/L), followed by cefepime/taniborbactam and cefepime/enmetazobactam (90.0 and 61.8% inhibited at MIC ≤2 mg/L, MIC<sub>50/90</sub> ≤0.5/2 and 1/≥128 mg/L, respectively) (Table 1 and Table S1). The antimicrobial activity of the inhibitors alone also was determined. Taniborbactam and enmetazobactam alone did not show antimicrobial activity and all isolates showed MICs ≥256 mg/L (data not shown). In contrast, significant activity of zidebactam alone, which shows affinity for PBP2, was observed against most of the strains tested. 73.3% of strains displayed a zidebactam MIC ≤1 mg/L with MIC<sub>50/90</sub> of ≤0.5/≥128 mg/L (Table 1).

We observed that the activity of the cefepime/BLI antimicrobial combinations varied significantly depending on the carbapenemase produced, as well as the presence/absence of ESBLs, as shown in Table 2 and Figure S2. Cefepime/zidebactam was able to decrease MICs to levels below the cefepime resistance breakpoint against all strains, regardless of the carbapenemase involved or the presence/absence of ESBLs. Cefepime/taniborbactam showed excellent activity against OXA-48-producing Enterobacterales, however the rates of KPC- and MBL-producing isolates showing low MICs (≤2 mg/L) to cefepime/taniborbactam were lower than those obtained with cefepime/zidebactam.

Finally, cefepime/enmetazobactam showed less ability overall to increase susceptibility to cefepime; ESBL- and KPC-producing isolates and OXA-48-producing isolates without ESBLs displayed the lowest MICs to this combination.

In a more detailed analysis of the OXA-48-producing group of isolates (n=304), the resistance rate to cefepime was 74.7%, and the most active combinations were cefepime/zidebactam and cefepime/taniborbactam (99.3% and 93.1% showed a MIC ≤2 mg/L, respectively, Table 2). When the presence or absence of ESBLs in this group of OXA-48-producing strains was analyzed, we observed, as expected, that the low resistance rates to cefepime in the absence of ESBLs hardly varied in combination with BLIs. In
130 ESBL-producing strains, however, the high cefepime resistance levels (only 6.1% of
131 isolates displayed a MIC ≤2 mg/L) decreased relevantly in the presence of zidebactam,
132 taniborbactam and enmetazobactam (99.2%, 93.1% and 71.7% showed MIC ≤2 mg/L,
133 respectively).

134 In the group of KPC-producing Enterobacterales (n=44) on the other hand, all strains were
135 resistant to cefepime and 56.8% were inhibited at ≥16 mg/L of cefepime/enmetazobactam
136 (resistance breakpoint for cefepime). Cefepime/zidebactam and cefepime/taniborbactam
137 displayed the highest activity, being 100% and 84.1% of isolates inhibited at MIC ≤2
138 mg/L, respectively (Table 2). In the KPC- and ESBL-producing subgroup of strains the 3
139 cefepime/BLI combinations showed high activity (>85% inhibited at MIC ≤2 mg/L). In the
140 subgroup without ESBLs, 92.6% of isolates showed a MIC ≥16 mg/L to
141 cefepime/enmetazobactam, while the cefepime/taniborbactam and cefepime/zidebactam
142 combinations were able to completely overcome resistance to cefepime.

143 Lastly, in the MBL-producing group of strains (n=56), cefepime displayed high resistance
144 rates (92.9%), while cefepime/enmetazobactam was unable to inhibit MBLs, with 87.5%
145 showing a MIC ≥16 mg/L. However, cefepime/zidebactam and cefepime/taniborbactam
146 showed high activity (96.4% and 75.0% showed a MIC ≤2 mg/L, respectively, Table 2).
147 As expected, the presence/absence of ESBLs in MBL-producing strains did not
148 substantially affect the elevated MICs to cefepime. Thus, high rates of resistance to
149 cefepime were observed in both the non-ESBL- and ESBL-producing subgroups of strains
150 (83.3% and 100%, respectively). Cefepime/enmetazobactam also showed high MICs in
151 these groups (83.3% and 90.6% inhibited at MIC ≥16 mg/L), while cefepime/zidebactam
152 and cefepime/taniborbactam were highly active against the same subgroups (<10% with a
153 MIC ≥16 mg/L).
In a previous study with the same isolates from this multicenter collection, the imipenem/relebactam and ceftazidime/avibactam combinations, recently approved, were evaluated (11). In that study, following CLSI criteria, 16.2% of strains were resistant to ceftazidime/avibactam (MIC<sub>50/90</sub> 1/≥256 mg/L) and 14.2% to imipenem/relebactam (MIC<sub>50/90</sub> 0.5/4 mg/L), thus presenting less activity than cefepime/zidebactam and cefepime/taniborbactam.

With respect to the literature assessing the activity of cefepime/zidebactam, MIC<sub>50/90</sub> values of 0.5/2, 0.5/2 and 0.5/4 mg/L, were observed in the groups of OXA-48-, KPC- and MBL-producing Enterobacterales, respectively (12), which are consistent with those obtained in our study (≤0.5/≤0.5, ≤0.5/1 and ≤0.5/1 mg/L, respectively).

Cefepime/taniborbactam has recently shown high activity against a collection of carbapenemase-producing Enterobacterales (13). Similarly, in other study, KPC-producing strains showed MIC<sub>50/90</sub> values of 16/>128 and 0.12/1 mg/L for cefepime and cefepime/taniborbactam, respectively (14), while another collection of KPC-producing Enterobacterales showed MIC<sub>50/90</sub> values of >256/>256 and 2/8 mg/L, respectively (15), highlighting the good activity of taniborbactam. In line with those results, we found cefepime and cefepime/taniborbactam MIC<sub>50/90</sub> values of ≥128/≥128 and 1/4 mg/L, respectively for KPC-producing Enterobacterales. For OXA-48-producing Enterobacterales, a previous study showed MIC<sub>50/90</sub> of 2/128 and 0.25/1 mg/L for cefepime and cefepime/taniborbactam, respectively (14), while in our study, for this group were 64/≥128 and ≤0.5/2 mg/L, respectively.

One of the most relevant aspects of taniborbactam is its activity against MBLs, so that it is of interest to examine this class of β-lactamases in detail. In this study, we determined a MIC<sub>50/90</sub> of 1/8 mg/L for all MBL-producing strains, although MIC values varied considerably according to MBL subclass, which was previously observed (14). In our
179 study, in 42 VIM-producing strains, we observed a MIC<sub>50/90</sub> for cefepime/taniborbactam of 1/4 mg/L. In the other two MBL groups, with 4 IMP- and 10 NDM-producing strains, MIC<sub>50/90</sub> values for cefepime/taniborbactam were 8/16 mg/L and 2/16 mg/L, respectively (Figure S3). Consequently, cefepime/taniborbactam shows high activity against VIM-producing Enterobacterales strains, although as previously stated, taniborbactam did not show significant activity against IMP. Lastly, the NDM-producing subgroup is of particular interest. Taniborbactam has previously shown high activity against NDM-1 and NDM-1 variants (16–18), although other NDM-like enzymes were not analyzed. However, Wang *et al.* found NDM-5-producing *Escherichia coli* strains with MICs ≥16 mg/L to cefepime/taniborbactam carrying a mutation in PBP3, which may be involved in resistance (15). In our study, 10 NDM-producing isolates were tested (only 1 NDM-5-producing *E. coli*), which displayed low MICs to cefepime/taniborbactam. For all the above reasons, the activity of cefepime/taniborbactam against NDM-producing strains needs to be investigated further.

193 Finally, with respect to the third combination, several studies have shown excellent 194 cefepime/enmetazobactam activity against different collections of Enterobacterales strains, 195 especially ESBL-producing Enterobacterales, with similar or better activity than other 196 approved combinations, such as ceftazidime/avibactam, ceftolozane/tazobactam, and even 197 carbapenems (8, 19, 20). Other studies have shown good cefepime/enmetazobactam 198 activity against carbapenemase-producing Enterobacterales (21, 22). Considering the low 199 activity of enmetazobactam against OXA-48 (23), and that most OXA-48-producing 200 strains also produce ESBLs, the activity of cefepime/enmetazobactam is probably due 201 more to ESBL inhibition than to carbapenemase inhibition. KPC-producing strains deserve 202 a special mention, because the activity of cefepime/enmetazobactam against KPC- 203 producing Enterobacterales is more controversial, and has not yet been clarified in the
204 literature (8, 19, 23). Although enmetazobactam showed good activity against KPC
205 enzymes (23), recent studies showed that the cefepime/enmetazobactam combination had
206 limited microbiological activity against KPC-producing strains, showing MIC<sub>50/90</sub> of 32/64
207 mg/L or higher (8, 19) and did not improve the MIC<sub>50/90</sub> of cefepime by more than 4-fold.
208 In our study, including 44 KPC-producing strains, we found high MIC<sub>50/90</sub> values (64/≥128
209 mg/L) for cefepime/enmetazobactam (Table S2). Analyzing these isolates more closely, we
210 observed a relationship between MIC and the clonality of the isolates studied; all but one
211 ST512 isolate (n=22) showed MICs ≥128 mg/L for cefepime/enmetazobactam, while
212 isolates belonging to ST307 (n=15) showed MICs ≤1 mg/L, therefore the variability of the
213 activity of cefepime/enmetazobactam should be carefully evaluated depending on the
214 predominant STs. Lastly, with respect to MBLs and, as expected,
215 cefepime/enmetazobactam did not enhance the activity of cefepime in most of the strains,
216 as previously described in literature (23) (Table S3 and Figure S3).

217 The interpretations in this study have been obtained according the cefepime CLSI
218 breakpoints (S ≤2 mg/L and R ≥16 mg/L), however the EUCAST MIC breakpoints are
219 slightly different (S ≤1 mg/L and R≥8 mg/L). Following the EUCAST recommendations,
220 cefepime/zidebactam and cefepime/taniborbactam continue to be the combinations with
221 the highest activity against the whole collection, with 96.0% and 77.8% of strains showing
222 a MIC ≤1 mg/L. EUCAST (MIC ≥8 mg/L) and CLSI (MIC ≥16 mg/L) cefepime resistance
223 breakpoints recommendations do not differ by more than 6% for any combination studied.

224

225 Resistance to cefepime in presence of BLIs and characterization of resistance
226 mechanisms
227 Focusing particularly on the analysis of isolates showing MIC ≥16 mg/L (resistance
228 breakpoint of cefepime) to the three new β-lactam/β-lactamase inhibitors, out of a total of
229 400 Enterobacterales, 4 isolates displayed MIC ≥16 mg/L to cefepime/taniborbactam
230 (Table 3), 110 to cefepime/enmetazobactam (Tables S2 and S4), while none to
231 cefepime/zidebactam.

232

233 -Clinical isolates with elevated MICs to cefepime/zidebactam.

234 MICs ≤2 mg/L to cefepime/zidebactam were observed in 99% of strains included in this
235 study, while only 4 (1%) strains displayed MIC of 4-8 mg/L (cefepime susceptible-dose
236 dependent category), making cefepime/zidebactam the most active combination evaluated.

237 The ability of zidebactam to moderately inhibit class A, C and some class D together with
238 its antimicrobial activity, through binding to PBP2, makes the cefepime/zidebactam
239 combination extremely active against carbapenemase-producing Enterobacterales.

240

241 -Clinical isolates with elevated MICs to cefepime/taniborbactam.

242 In our study, 4 strains displayed cefepime/taniborbactam MICs ≥16 mg/L and all of them
243 produced MBLs (NDM-5, NDM-7, VIM-1, and IMP-8) (Table 3). One of these strains was
244 an IMP-8-producing *Enterobacter cloacae*, carbapenemase not inhibited by taniborbactam
245 (13). Among NDM-producing strains, one strain presented a MIC of 16 mg/L for
246 cefepime/taniborbactam, and another an elevated MIC of ≥128 mg/L. Although
247 taniborbactam is able to inhibit NDM-like carbapenemases, these strains produced CTX-
248 M-15 ESBL and OXA-1 β-lactamase, among others, and also exhibited relevant amino
249 acid changes in PBP2 and/or PBP3, the main targets of cefepime (24). Both strains share a
250 4-amino acid insertion (YRIN) in PBP3, which directly affects the size of the Ω loop. This
insertion has previously been associated with decreased susceptibility to several β-lactams
including the ceftolozane/tazobactam combination (25, 26). Similarly, the V522I mutation
in PBP2, found in the NDM-producing strain with the highest MIC to
cefepime/taniborbactam also has been associated with increased resistance to β-lactams
(27). Finally, a VIM-1-producing isolate of K. pneumoniae with a MIC to
cefepime/taniborbactam of ≥128 mg/L, as well as several copies of OXA-1 and CTX-M-
15, presented a nonfunctional OmpK35 due to a stop codon. Deficiencies in OmpK35 have
been associated with increased resistance to ceftazidime/avibactam in the presence of β-
lactamases such as KPC, which presents good hydrolytic activity against ceftazidime (28).

260

261 -Clinical isolates with elevated MICs to cefepime/enmetazobactam.

262 In this study, 110 isolates displayed MICs ≥16 mg/L to cefepime/enmetazobactam,
263 including 40 OXA-48 producers, 25 KPC producers (22 KPC-3 and 3 KPC-2) and 49
264 MBL producers (Tables S2 and S4). Further analysis of the OXA-48-producing isolates
265 revealed that 13% (40/304 isolates) displayed MICs ≥16 mg/L for
266 cefepime/enmetazobactam, 3 of them also producing an MBL. A total of 37 OXA-48-
267 producing strains not producing any other carbapenemase, were either slightly or not
268 inhibited by enmetazobactam, 35/37 also produce an ESBLs, and interestingly 18/37
269 exhibiting important changes (frameshifts, insertions, deletions...) in OmpK35, OmpK36,
270 PBPs and/or AcrAB-TolC expression regulators (29). The remaining OXA-48-producing
271 strains with high MICs to cefepime/enmetazobactam (19/37) showed no alterations in the
272 β-lactam resistance genes analyzed in this study, highlighting only the presence of two or
273 more ESBLs in most of these strains (Table S4).
With respect to the KPC-producing isolates, it should be highlighted that, in this study, enmetazobactam was only able to decrease the MIC of cefepime to ≤2 mg/L in less than half the strains with the KPC carbapenemase (40.9%, Tables 2 and Table S2). This result is determined by the prevalence of ST512 (MICs ≥128 mg/L) and ST307 (MICs ≤1 mg/L), as exposed above. All ST512 isolates produced a non-functional OmpK35 and, interestingly, all the strains with high MICs showed a 2 amino acid insertion in OmpK36 (G134_D135insGD) in loop 3. This insertion has previously been involved in constriction of the porin channel and increased MICs to carbapenems, which is of greater significance when combined with deletion of OmpK35 (30). These alterations have been also associated with the increase of MICs to new combinations such as meropenem/vaborbactam (31, 32) or meropenem/QPX7728 (33). Other isolates displaying high MICs for cefepime/enmetazobactam were two strains of *Citrobacter freundii*, which also produced a non-functional OmpK35. This strongly suggests that secondary mechanisms such as porin modifications must be present in KPC-producing strains in order to show elevated MICs for this combination.

Finally, the inhibition spectrum of enmetazobactam does not cover MBLs (9), so that most of the isolates producing MBLs in this study displayed MICs ≥16 mg/L for cefepime/enmetazobactam. However, 2 of the 56 MBLs producers cefepime-resistant isolates displayed a cefepime/enmetazobactam MIC of 4 mg/L (cefepime susceptible-dose dependent), producing VIM-1 and NDM-1 (Table S3 and Figure S3).

## CONCLUDING REMARKS

Resistance to β-lactam antibiotics continues to increase and new β-lactamases with a broader spectrum and higher hydrolytic activity are constantly emerging. The current
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development and emergence of new classes of β-lactamase inhibitors such as the ones evaluated here is possibly one of the most promising aspects in the ongoing fight against bacterial resistance to antibiotics.

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In our study, the combination that showed the highest activity against carbapenemase-producing Enterobacterales was cefepime/zidebactam, followed by cefepime/taniborbactam. Cefepime/enmetazobactam enhances the activity of cefepime alone in virtually all cases tested, but is less effective than the other two combinations, particularly against MBL carbapenemases. Cefepime/zidebactam and cefepime/taniborbactam, along with the β-lactam/β-lactamase inhibitor combinations that have already been approved, such as ceftazidime/avibactam and imipenem/relebactam, present results that lead us to think, with a certain degree of optimism, that in the near future we will have a wider therapeutic arsenal against the global expansion of MDR Enterobacterales.

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# MATERIALS AND METHODS

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## Bacterial isolates

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Public hospitals in Spain participated in a nationwide survey of Enterobacterales with meropenem MICs above the screening cut-off recommended by EUCAST (>0.125 mg/L) (34) and carbapenemase production. Four hundred Enterobacterales isolates in all were prospectively recovered during a 2-month period in 2018 (November-December) from 24 hospitals, and hence most regions of Spain (11). Bacterial strains were frozen in Luria Bertani broth (Sigma) with 15% glycerol and maintained at -80°C until analysis. The microbiological laboratory of the University Hospital Complex of A Coruña (CHUAC) served as reference laboratory.

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321
323 **Antimicrobial susceptibility testing**

324 The *in vitro* antibacterial activity of cefepime (Sigma) alone and in combination with the
325 BLIs zidebactam, taniborbactam and enmetazobactam (provided by MedChemExpress)
326 against 400 Enterobacterales isolates was determined in cation-adjusted Mueller-Hinton
327 broth (Becton, Dickinson and Company) using microdilution assays and following CLSI
328 recommendations (35). To potentiate the antibacterial activity of the β-lactamase
329 inhibitors, cefepime and zidebactam were tested in a 1:1 ratio, and taniborbactam and
330 enmetazobactam at a fixed concentration of 4 and 8 mg/L, respectively. Serially diluted
331 concentrations of cefepime and cefepime/BLIs ranging from 0.5 to 128 mg/L were
332 performed. For the complete set of isolates evaluated in this study (n=400), cefepime MIC
333 values ≤2 and ≥16 mg/L were used as breakpoints to define the susceptible and resistant
334 interpretive categories, respectively, following CLSI criteria. Thus, for the purposes of
335 comparison, resistance to cefepime/BLIs was interpreted in this study as MIC values ≥16
336 mg/L (35). *E. coli* ATCC 25922 and *Pseudomonas aeruginosa* ATCC 27853 reference
337 strains were used as controls; their MICs for these cefepime/BLI combinations have
338 previously been described (35).

339

340 **Whole-genome sequencing, hybrid assemblies and resistance genomics**

341 All clinical isolates were analyzed by WGS, as previously described (11). Total genomic
342 DNA was obtained using a Genomic DNA Buffer Set with Genomic-Tip 20/G (Qiagen),
343 following the manufacturer's instructions. The DNA yield was determined using the Qubit
344 dsDNA HS Assay Kit (Thermo Fisher). Purified genomic DNA from all isolates was
345 sequenced in parallel using both short- (Illumina MiSeq, Illumina) and long-read
346 (MinION, Oxford Nanopore Technologies) approaches.

347 The resulting reads from each isolate were assembled using the Unicycler v0.4.6 (36)
348 hybrid assembler and annotated using Prokka v1.13 (37). The antimicrobial resistance gene
349 content of the isolates was analyzed using Resfinder v3.2 software (38), ARMFinderPlus
350 (39) and the Comprehensive Antibiotic Resistance Database (CARD) (38).

351 For the analysis of β-lactam resistance, the main genes involved in resistance were studied:
352 the *K. pneumoniae* porin genes, *ompK35* and *ompK36*, transcriptional activators of the
353 *acrAB-tolC* efflux system (*marR*, *ramR* and *acrR*), genes *pbp2* and *pbp3*, and the possible
354 presence of other β-lactamases. In other Enterobacterales species, homologous genes to the
355 ones mentioned were evaluated. The sequences of resistance genes used as reference were
356 obtained from strains selected according to the following criteria: the most conserved
357 sequences between the strains in the multicenter study with low MICs to the
358 cefepime/BLIs combinations were selected using BLASTP. Later, sequences were
359 searched for 100% identity and coverage in both Genbank and Uniprot databases, in order
360 to demonstrate they were conserved gene sequences in each Enterobacterales species.

361

362 **Data availability**

363 The BioProject accession number for strain genomes is PRJEB3911. Data will be available
364 upon request.

365

366 **Funding**
367 This work was supported by the Fondo de Investigación Sanitaria (grant numbers
368 PI17/01482 and PI20/01212 for A.B. and PI18/00501 for G.B. integrated in the Plan
369 Nacional de I+D and funded by the Instituto de Salud Carlos III, ISCIII). CIBERINF
370 (CIBER de Enfermedades Infecciosas). The research was also funded by the Spanish
371 Network of Research in Infectious Diseases (REIPI), N° RD16/0016/0006, integrated in
372 the National Plan for Scientific Research, Development and Technological Innovation
373 2013-2016 and funded by the ISCIII- General Subdirection of Assessment and Promotion of
374 the Research – European Regional Development Fund (FEDER) "A way of making
375 Europe". The study was also funded by GAIN (Agencia Gallega de Innovación,
376 Conselleria de Economía, Emprego e Industria (IN607D2021/12, A.B. and IN607A
377 2016/22, G.B.).

378 J.C.V.-U. was financially supported by the ISCIII project, FI18/00315. J.A.-S. was
379 financially supported by the Rio Hortega program (ISCIII, CM19/00219) and C.L.-M. by
380 IN606A-2019/029. A.B. was financially supported by the Miguel Servet II program,
381 CPII18/00024.

382

383 Acknowledgments

384 This research was possible thanks to the helpful collaboration of the following researchers:
385 Bruno K. Rodiño-Janeiro, Tyler Alioto, Marta Gut, Ivo Gut, Miguel Álvarez-Tejado, Irene
386 Merino, Emilia Cercenado, Rosa Gómez, Tamara Soler, Irene Gracia-Ahufinger, Lina
387 Martín, Fátima Galán, Nuria Tormo, Juan Carlos Rodríguez, Silvia Capilla, Francesc
388 Marco, María Dolores Quesada, Emma Padilla, Fe Tubau, Juanjo González, Ana Isabel
389 López-Calleja, José Luis del Pozo, María Inmaculada García, Mariela Martinez, Jorge
390 Calvo, Xavier Mulet, Fernanda Peña, Ana Isabel Rodríguez, María José Gude, Ana

391 Fernández, Javier Fernández.

392

393 **Transparency declarations**

394 The authors declare no competing financial interests.

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<table><caption>**Table 1.** Cumulative minimum inhibitory concentration (MIC) distribution of cefepime in the presence and absence of zidebactam, taniborbactam and enmetazobactam in 400 carbapenemase-producing Enterobacterales strains.</caption><thead><tr><th rowspan="2"></th><th rowspan="2">Cefepime/BLIs combinations*</th><th colspan="10">Cumulative % of isolates at MIC (mg/L)</th><th colspan="2">% isolates with MICs values</th></tr><tr><th>≤0.5</th><th>1</th><th>2</th><th>4</th><th>8</th><th>16</th><th>32</th><th>64</th><th>≥128</th><th>≤2 mg/L</th><th>≥16 mg/L</th></tr></thead><tbody><tr><td rowspan="5">All isolates (n=400)</td><td>Cefepime</td><td>5.5</td><td>12.3</td><td>15.0</td><td>18.8</td><td>20.5</td><td>25.5</td><td>38.3</td><td>52.5</td><td>100</td><td>15.0</td><td>79.5</td></tr><tr><td>Cefepime/zidebactam</td><td>87.5</td><td>96.0</td><td>99.0</td><td>99.5</td><td>100</td><td></td><td></td><td></td><td></td><td>99.0</td><td>0</td></tr><tr><td>Zidebactam</td><td>64.3</td><td>73.3</td><td>77.5</td><td>78.0</td><td>78.0</td><td>78.0</td><td>79.3</td><td>79.3</td><td>100</td><td>77.5</td><td>22.0</td></tr><tr><td>Cefepime/taniborbactam</td><td>55.0</td><td>77.8</td><td>90.0</td><td>97.0</td><td>99.0</td><td>99.5</td><td>99.5</td><td>99.5</td><td>100</td><td>90.0</td><td>1.0</td></tr><tr><td>Cefepime/enmetazobactam</td><td>36.0</td><td>50.3</td><td>61.8</td><td>67.8</td><td>72.5</td><td>79.3</td><td>83.5</td><td>86.5</td><td>100</td><td>61.8</td><td>27.5</td></tr><tr><td rowspan="5">All isolates no</td><td>No producing ESBLs (n=106)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cefepime</td><td>15.1</td><td>34.9</td><td>41.5</td><td>47.2</td><td>49.1</td><td>53.8</td><td>59.4</td><td>66.0</td><td>100</td><td>41.5</td><td>50.9</td></tr><tr><td>Cefepime/zidebactam</td><td>76.4</td><td>95.3</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td>100</td><td>0</td></tr><tr><td>Zidebactam</td><td>63.2</td><td>78.3</td><td>84.9</td><td>84.9</td><td>84.9</td><td>84.9</td><td>85.8</td><td>85.8</td><td>100</td><td>84.9</td><td>15.1</td></tr><tr><td>Cefepime/taniborbactam</td><td>51.9</td><td>73.6</td><td>88.7</td><td>96.2</td><td>99.1</td><td>100</td><td></td><td></td><td></td><td>88.7</td><td>0.9</td></tr><tr><td></td><td>Cefepime/enmetazobactam</td><td>34.9</td><td>44.3</td><td>50.9</td><td>53.8</td><td>56.6</td><td>62.3</td><td>65.1</td><td>69.8</td><td>100</td><td>50.9</td><td>43.4</td></tr><tr><td rowspan="5">All isolates no</td><td>Producing ESBLs (n=294)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cefepime</td><td>2.0</td><td>4.1</td><td>5.4</td><td>8.5</td><td>10.2</td><td>15.3</td><td>30.6</td><td>47.6</td><td>100</td><td>5.4</td><td>89.8</td></tr><tr><td>Cefepime/zidebactam</td><td>91.5</td><td>96.3</td><td>98.6</td><td>99.3</td><td>100</td><td></td><td></td><td></td><td></td><td>98.6</td><td>0</td></tr><tr><td>Zidebactam</td><td>64.6</td><td>71.4</td><td>74.8</td><td>75.5</td><td>75.5</td><td>75.5</td><td>76.9</td><td>76.9</td><td>100</td><td>74.8</td><td>24.5</td></tr><tr><td>Cefepime/taniborbactam</td><td>56.1</td><td>79.3</td><td>90.5</td><td>97.3</td><td>99.0</td><td>99.3</td><td>99.3</td><td>99.3</td><td>100</td><td>90.5</td><td>1.0</td></tr><tr><td></td><td>Cefepime/enmetazobactam</td><td>36.4</td><td>52.4</td><td>65.6</td><td>72.8</td><td>78.2</td><td>85.4</td><td>90.1</td><td>92.5</td><td>100</td><td>65.6</td><td>21.8</td></tr></tbody></table>

<footnote>* Taniborbactam and enmetazobactam did not show antimicrobial activity, displaying MICs ≥256 mg/L for all isolates.</footnote>
<table><caption>**Table 2.** MIC<sub>50</sub>, MIC<sub>90</sub> and % isolates with MICs values ≤2 and ≥16 mg/L to cefepime in the presence and absence of zidebactam, taniborbactam and enmetazobactam in OXA-48-, KPC- and MBL-producing Enterobacterales strains.<sup>a</sup></caption><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th rowspan="2"></th><th colspan="2">Cefepime/BLIs combinations</th><th colspan="2">% isolates with MICs values</th></tr><tr><th>MIC<sub>50</sub></th><th>MIC<sub>90</sub></th><th>≤2 mg/L</th><th>≥16 mg/L</th></tr></thead><tbody><tr><td rowspan="12">OXA-48-producing isolates</td><td rowspan="4">All isolates (n=304)</td><td>Cefepime</td><td>64</td><td>≥128</td><td>18.8</td><td>74.7</td></tr><tr><td>Cefepime/zidebactam</td><td>≤0.5</td><td>≤0.5</td><td>99.3</td><td>0</td></tr><tr><td>Zidebactam</td><td>≤0.5</td><td>≥128</td><td>75.7</td><td>24.0</td></tr><tr><td>Cefepime/taniborbactam</td><td>≤0.5</td><td>2</td><td>93.1</td><td>0</td></tr><tr><td rowspan="4">No producing ESBLs (n=57)</td><td>Cefepime</td><td>1</td><td>16</td><td>73.7</td><td>15.8</td></tr><tr><td>Cefepime/zidebactam</td><td>≤0.5</td><td>≤0.5</td><td>100</td><td>0</td></tr><tr><td>Zidebactam</td><td>≤0.5</td><td>≥128</td><td>82.5</td><td>17.5</td></tr><tr><td>Cefepime/taniborbactam</td><td>≤0.5</td><td>1</td><td>93.0</td><td>0</td></tr><tr><td rowspan="4">Producing ESBLs (n=247)</td><td>Cefepime</td><td>≥128</td><td>≥128</td><td>6.1</td><td>88.3</td></tr><tr><td>Cefepime/zidebactam</td><td>≤0.5</td><td>≤0.5</td><td>99.2</td><td>0</td></tr><tr><td>Zidebactam</td><td>≤0.5</td><td>≥128</td><td>74.1</td><td>25.5</td></tr><tr><td>Cefepime/taniborbactam</td><td>≤0.5</td><td>2</td><td>93.1</td><td>0</td></tr><tr><td rowspan="12">KPC-producing isolates</td><td rowspan="4">All isolates (n=44)</td><td>Cefepime</td><td>≥128</td><td>≥128</td><td>0</td><td>100</td></tr><tr><td>Cefepime/zidebactam</td><td>≤0.5</td><td>1</td><td>100</td><td>0</td></tr><tr><td>Zidebactam</td><td>1</td><td>2</td><td>90.9</td><td>6.8</td></tr><tr><td>Cefepime/taniborbactam</td><td>1</td><td>4</td><td>84.1</td><td>0</td></tr><tr><td rowspan="4">No producing ESBLs (n=27)</td><td>Cefepime</td><td>≥128</td><td>≥128</td><td>0</td><td>100</td></tr><tr><td>Cefepime/zidebactam</td><td>1</td><td>1</td><td>100</td><td>0</td></tr><tr><td>Zidebactam</td><td>1</td><td>2</td><td>92.6</td><td>7.4</td></tr><tr><td>Cefepime/taniborbactam</td><td>2</td><td>4</td><td>81.5</td><td>0</td></tr><tr><td rowspan="4">Producing ESBLs (n=17)</td><td>Cefepime</td><td>≥128</td><td>≥128</td><td>7.4</td><td>92.6</td></tr><tr><td>Cefepime/zidebactam</td><td>32</td><td>≥128</td><td>0</td><td>100</td></tr><tr><td>Zidebactam</td><td>≤0.5</td><td>≤0.5</td><td>100</td><td>0</td></tr><tr><td>Cefepime/taniborbactam</td><td>≤0.5</td><td>2</td><td>88.2</td><td>5.9</td></tr><tr><td rowspan="4">MBL-producing isolates</td><td rowspan="4">All isolates (n=56)</td><td>Cefepime</td><td>≥128</td><td>≥128</td><td>3.6</td><td>92.9</td></tr><tr><td>Cefepime/zidebactam</td><td>≤0.5</td><td>1</td><td>96.4</td><td>0</td></tr><tr><td>Zidebactam</td><td>≤0.5</td><td>≥128</td><td>75.0</td><td>25.0</td></tr><tr><td>Cefepime/taniborbactam</td><td>1</td><td>8</td><td>75.0</td><td>7.1</td></tr><tr><td rowspan="4">No producing ESBLs (n=24)</td><td>Cefepime</td><td>64</td><td>≥128</td><td>8.3</td><td>83.3</td></tr><tr><td>Cefepime/zidebactam</td><td>≤0.5</td><td>≤0.5</td><td>100</td><td>0</td></tr><tr><td>Zidebactam</td><td>≤0.5</td><td>≥128</td><td>79.2</td><td>20.8</td></tr><tr><td>Cefepime/taniborbactam</td><td>1</td><td>4</td><td>79.2</td><td>4.2</td></tr><tr><td rowspan="4">Producing ESBLs (n=32)</td><td>Cefepime</td><td>≥128</td><td>≥128</td><td>0</td><td>100</td></tr><tr><td>Cefepime/zidebactam</td><td>≤0.5</td><td>2</td><td>93.8</td><td>0</td></tr><tr><td>Zidebactam</td><td>≤0.5</td><td>≥128</td><td>71.9</td><td>28.1</td></tr><tr><td>Cefepime/taniborbactam</td><td>1</td><td>8</td><td>71.9</td><td>9.4</td></tr><tr><td></td><td></td><td>Cefepime/enmetazobactam</td><td>64</td><td>≥128</td><td>0</td><td>90.6</td></tr></tbody></table>

<sup>a</sup>Five strains produced two carbapenemases: OXA-48 + KPC-3, OXA-48 + IMP-13, OXA-48 + NDM-1,

OXA-48 + VIM-1 and KPC-2+ IMP-22.
<table><caption>**Table 3.** Bacterial species, sequence type, susceptibility to cefepime of clinical Enterobacterales showing high MIC values for cefepime/taniborbactam (MIC ≥16mg/L).</caption><thead><tr><th rowspan="2">Genome no.</th><th rowspan="2">Species</th><th rowspan="2">ST</th><th colspan="2">MIC (mg/L)</th><th rowspan="2">Carbapenemase</th><th colspan="2">Other β-lactamases</th><th rowspan="2">Porins</th><th rowspan="2">PBPs</th><th rowspan="2">Transcription regulators AcrAB-TolC</th></tr><tr><th>Cefepime</th><th>Cefepime/Taniborbactam</th><th>ESBL β-lactamases</th><th>Non-ESBL β-lactamases</th></tr></thead><tbody><tr><td>AI2843</td><td><i>E. coli</i></td><td>410</td><td>≥128</td><td>16</td><td>NDM-5</td><td>CTX-M-15</td><td>EC-like, (2x)TEM-1, (2x)OXA-1, CMY-2</td><td>-</td><td>PBP3 (I532L, Y334_N337insYRIN)</td><td>-</td></tr><tr><td>AI2858</td><td><i>E. coli</i></td><td>648</td><td>≥128</td><td>≥128</td><td>NDM-7</td><td>CTX-M-15</td><td>EC-like, CMY-6, OXA-1</td><td>-</td><td>PBP2 (V522I)<br>PBP3 (I532L, Y334_N337insYRIN)</td><td>-</td></tr><tr><td>AI2867</td><td><i>K. pneumoniae</i></td><td>39</td><td>≥128</td><td>≥128</td><td>VIM-1</td><td>(2x)CTX-M-15</td><td>(2x)OXA-1, SHV-11</td><td>OmpK35 NF (W316*)</td><td>-</td><td>-</td></tr><tr><td>AI2992</td><td><i>E. cloacae</i><br>complex</td><td>96</td><td>16</td><td>16</td><td>IMP-8</td><td></td><td>MIR-like, OXA-101</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

(2x): indicates that the strain has 2 copies of the gene coding for the specified β-lactamase. NF, indicates non-functional; asterisk (*): indicates premature stop codon.