# Efficacy of ceftazidime/avibactam in various combinations for the treatment of experimental osteomyelitis in rabbits caused by OXA-48-/ESBL-producing Escherichia coli

Benjamin Davido, Anne Claude Crémieux, Isabelle Vaugier, Pierre de Truchis, Kamel Hamami, Frédéric J. Laurent, Azzam Saleh-Mghir

► To cite this version:

Benjamin Davido, Anne Claude Crémieux, Isabelle Vaugier, Pierre de Truchis, Kamel Hamami, et al. Efficacy of ceftazidime/avibactam in various combinations for the treatment of experimental osteomyelitis in rabbits caused by OXA-48-/ESBL-producing Escherichia coli. Journal of Antimicrobial Chemotherapy, 2023, 78 (5), pp.1211-1218. 10.1093/jac/dkad070. hal-04233270

HAL Id: hal-04233270

https://hal.science/hal-04233270

Submitted on 23 Nov 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 Efficacy of ceftazidime-avibactam in various combinations for the
treatment of experimental osteomyelitis in rabbits caused by OXA-
48/ESBL-producing Escherichia coli

4

5 Running title: Ceftazidime-avibactam against OXA-48/ESBL E. coli in an
6 Experimental Osteomyelitis

7 B. Davido*¹,², A. C. Crémieux¹,³, I. Vaugier⁴, P. De Truchis¹, K. Hamami⁵, F. Laurent⁶,⁷,

8 A. Saleh-Mghir¹,²

9 ¹UMR 1173, Versailles Saint-Quentin University, Versailles, France, ²Raymond Poincaré
10 Paris Saclay University Hospital, Garches, France; ³FHU PROTHEE, St Louis-
11 Lariboisière Hospital, Paris-Cité University, Paris, France, ⁴CIC, Raymond Poincaré
12 Paris Saclay University Hospital, Garches, France, ⁵Pharmacy Unit, Raymond Poincaré
13 Paris Saclay University Hospital, Garches, France, ⁶Institut for Infectious Agents,
14 Department of Bacteriology - CNR des staphylocoques, Croix-Rousse Hospital, North
15 Biology Centre, Hospices Civils de Lyon, Lyon, France, ⁷Team “Staphylococcal
16 pathogenesis”, International Centre for Infectiology Research, INSERM U1111 - CNRS
17 UMR5308 - ENS Lyon - Lyon 1 University, Lyon, France

18

19 * Corresponding author. Mailing address: Benjamin Davido, Maladies Infectieuses,
20 Hôpital Raymond Poincaré, 104 bld Raymond Poincaré, 92380 Garches, France

21 Tel: +33 (1) 47 10 77 40; Fax: +33 (1) 47 10 77 90; benjamin.davido@aphp.fr

22
24 **Background** While the treatment of ESBL-producing Enterobacterales osteomyelitis
25 relies on carbapenems, optimal regimen for OXA48-types remains unclear. We evaluated
26 the efficacy of ceftazidime-avibactam in different combinations in an experimental model
27 of OXA-48/ESBL-producing *E. coli* osteomyelitis.

28 **Methods** *E. coli* pACYC184 is a clinical strain harbouring *bla*<sub>OXA-48</sub> and *bla*<sub>CTX-M15</sub>
29 inserts, with increased exposure susceptibility to imipenem (MIC,2 mg/L), gentamicin
30 (MIC,0.5 mg/L), colistin (MIC,0.25 mg/L), **ceftazidime-avibactam** (MIC,0.094 mg/L),
31 fosfomycin (MIC,1 mg/L), and resistant to ceftazidime (MIC,16 mg/L). Osteomyelitis
32 was induced in rabbits by tibial injection of 2x10<sup>8</sup> cfu of OXA-48/ESBL *E. coli*.
33 Treatment started 14 days later for 7 days in 6 groups: (1) control, (2) colistin 150.000
34 IU/kg SC q8h, (3) **ceftazidime-avibactam** 100/25 mg/kg SC q8h, (4) **ceftazidime-avibactam** + colistin, (5) **ceftazidime-avibactam** + fosfomycin 150 mg/kg SC q12h, (6) **ceftazidime-avibactam** + gentamicin 15 mg/kg IM q24h. Treatment was evaluated at day
35
36
37 24 according to bone cultures.

38 **Results** *In vitro*, time kill curves of **ceftazidime-avibactam** in combination showed a
39 synergistic effect. *In vivo*, compared to controls, rabbits treated with colistin alone had
40 similar bone bacterial density (p=0.50), whereas **ceftazidime-avibactam** alone or in
41 combinations significantly decreased bone bacterial densities (p=0.004 and p<0.0002,
42 respectively). Bone sterilisation was achieved using **ceftazidime-avibactam** in
43 combination with colistin (91%), or fosfomycin (100%) or gentamicin (100%)
44 (p<0.0001), whereas single therapies were not different from controls. No **ceftazidime-**
45 **avibactam**-resistant strains emerged in rabbits treated regardless of combination.
46 **Conclusions** In our model of *E. coli* OXA-48/ESBL osteomyelitis, **ceftazidime**-

47 **avibactam** in combination was more effective than any single therapy whatever the

48 companion drug used (gentamicin or colistin or fosfomycin).
49 INTRODUCTION

There are numerous mechanisms of antimicrobial resistance in Gram-negative bacteria that require appropriate antimicrobial therapies, posing a challenge that needs to be addressed. The dramatic worldwide spread of carbapenemase-producing Enterobacterales (CPE) is a constant threat and has been identified by the WHO as a top priority ¹ as the development of new effective drugs is urgently needed, as recently emphasized by the Infectious Diseases Society of America (IDSA) in 2019. ²

Historically, the main reservoirs of OXA-48-producing Enterobacterales were located in North Africa and in Turkey, where the first case was described in 2001. ³ During the 2000s, Klebsiella pneumonia was the primary host for resistance. ⁴ Since then, we have observed a widespread occurrence of the *bla* OXA-48 gene in many European countries, including France, which has been involved in numerous hospital-acquired outbreaks. ⁵ Moreover, as evidenced by the situation in France, the incidence of OXA-48-type isolates in *E. coli* has risen dramatically and, by 2017, had equaled that of Klebsiella. It is noteworthy to mention that nearly 78% of OXA-48-type strains were also found to produce extended-spectrum beta-lactamases. ⁶ One concern about these superbugs is their spread in the community, as previously described in the Mediterranean region, with a high prevalence of OXA-48 producers at admission (13%) associated with ESBL-producers (42.5%, mainly CTX-M15). ⁷ Moreover, these ESBL-producing Enterobacterales are very likely to be imported and subsequently spread during international travel, especially from southern Asia and sub-Saharan Africa. ⁸,⁹

It should be noted that OXA-48 enzymes partially hydrolyze carbapenems but spare broad-spectrum cephalosporins, and are mainly found in *K. pneumoniae* rather than *E.*
coli isolates. ¹⁰ Nevertheless, physicians should be aware that *bla* OXA-48 genes spread extremely easily and quickly among Enterobacterales within the microbiota due to a high transfer efficiency of its plasmid, rather than the expansion of a single clone or gain of virulence. ¹¹

Furthermore, while Gram-negative bacteria are rarely involved in osteomyelitis (around 10% of cases), ¹² cases of such complex infections, especially those caused by CPE, have been increasingly described ¹³⁻¹⁵ and do require expert advice to find the most appropriate combination. In addition, we recently showed in an experimental model of KPC K. pneumoniae osteomyelitis that the combination of ceftazidime-avibactam with colistin or gentamicin, but not fosfomycin, allowed significant bone sterilisation in infected rabbits. ¹⁶ As ceftazidime-avibactam restores the activity of ceftazidime against ESBL-producing Enterobacterales, but is also effective against OXA-48 producers, ¹⁷ we decided to study its effectiveness for the treatment of osteomyelitis due to OXA-48/ESBL E. coli, a deemed difficult-to-treat infection.

86

## MATERIALS AND METHODS

**Bacterial strain.** E. coli pACYC184-OXA-48-CTX-M-15 is an isogenic derivative of a urinary clinical strain of E. coli provided by The National Reference Center for Emerging Antibiotic Resistance (NARA), Fribourg, Switzerland. This isolate is a double mutant that co-expresses 2 genes of resistance including *bla* OXA-48 an OXA-48-type beta-lactamase, and *bla* CTX-M15 responsible for the ESBL activity.

This strain has increased exposure susceptibility to imipenem (MIC, 2 mg/L), and full susceptibility to gentamicin (MIC, 0.5 mg/L), colistin (MIC, 0.25 mg/L), ceftazidime-
95

avibactam (MIC, 0.094 mg/L) and fosfomycin (MIC, 1 mg/L), but is resistant to
ceftazidime (MIC, 16 mg/L).

96

97

Before being inoculated to the animal, the double mutant strain was cultured in casein
hydrolysate-yeast extract-containing (CCY) broth medium at 37°C during 18 h. After
centrifugation, the pellets were washed twice and resuspended in phosphate-buffered
saline. All inocula were assessed by plating serial dilutions on tryptic soy agar
(bioMérieux, La Balme-les-Grottes, France).

100

101

102

103

**Time-kill curve studies.** Cultures were diluted in 10 mL of fresh Mueller-Hinton broth to yield an inoculum of $10^5$ cfu/mL. The bactericidal activities of ceftazidime-avibactam, colistin, gentamicin, and fosfomycin, alone or in combination, were determined in triplicate. Antibiotic concentrations used were equivalent to 4 times the MIC to more closely reflect the optimal dosage of antibiotics. After 0, 1, 3, 6 and 24 h of incubation in a shaking water bath at 37°C, serial dilutions of 0.1 mL samples were cultured on Mueller-Hinton agar plates (Bio-Rad, Marnes la Coquettes, France) before determining the number of viable microorganisms using **cfu**. Synergistic effect was defined as a decrease of $\ge 2 \log_{10}$ **cfu**/mL for the combination compared to that of its most active component. A bactericidal activity was defined by a reduction of $\ge 3 \log_{10}$ **cfu**/mL compared with the initial inoculum.

104

105

106

107

108

109

110

111

112

113

114

115

**Selection of antibiotic doses in rabbits.** Plasma antibiotic concentrations were measured in uninfected animals to select doses leading to plasma concentrations equivalent to those observed in humans as previously described in our previous experiment using the same

116

117
118 drugs. ¹⁶ Of note in the present experiment gentamicin dose was halved, and
119 administrations route of fosfomycin and colistin were switched from intramuscular to
120 subcutaneous in accordance with the animal welfare committee and supported by
121 pharmacokinetics data in veterinary medicine. ¹⁸⁻²⁰

122

123 **Rabbit osteomyelitis model.** Norden's method was used to induce osteomyelitis in
124 female New Zealand white rabbits, each weighing 2 to 3 kg, as previously described. ²¹
125 The rabbits were housed in individual cages and received food and water ad libitum
126 according to the French legislation on animal experimentation. Study was approved by
127 the Animal Use Committee of Maisons-Alfort Veterinary School. The animals were
128 anaesthetized by intramuscular (im) injection of ketamine, 25 mg/kg (Vibrac, Carros,
129 France) and 2% xylazine, 25 mg/kg (Elanco, Cuxhaven, Germany). To ensure post
130 anaesthesia analgesia, a subcutaneous (sc) injection of buprenorphine, 30µg/kg (Indivior,
131 France) was carried out. Infection was induced by tibial intra-medullary injection of a
132 sclerosing agent, 0.1 ml of 3% sodium tetradecyl sulfate (Kreussler Pharma, La
133 Chaussée-Saint-Victor, France), followed by 0.2 mL of the OXA-48/ESBL E. coli
134 inoculum (10⁹ cfu/mL) and 0.1 mL of saline. A high inoculum was preferred based on
135 our previous experiments using a **Gram-negative** bacteria to achieve a persistent
136 osteomyelitis in the infected animals ²². Analgesia using a fentanyl patch of 12.5µg/h
137 (Mylan, Lyon, France) was applied and pethidine hydrochloride (Renaudin, Itxassou,
138 France), 100 mg in 500mL of water was given in the rabbit bottles during 7 days
139 following the procedure.

140
**Treatment and its evaluation.** Rabbits were randomly assigned to one of the following groups of rabbits (n=12): (1) control, (2) colistin 150 000 IU/kg q8h sc, (3) **ceftazidime-avibactam** 100/25 mg/kg sc q8h, (4) **ceftazidime-avibactam** + gentamicin 15 mg/kg im q24h, (5) **ceftazidime-avibactam** + colistin 150 000 IU/kg q8h sc, (6) **ceftazidime-avibactam** + fosfomycin 150 mg/kg q12h sc. Each regimen was initiated 14 days after bacterial inoculation on animals, and administered for a total duration of 7 days (until day 21 of the procedure).

Rabbits (infected and controls) were euthanized by intravenous (iv) injection of pentobarbital 3 days after the end of antimicrobial therapy (day 24) to avoid a carryover effect of the antibiotics and allow regrowth of any potential mutants after a wash-out period. During animal autopsy, the upper third of the tibia (3 cm long), including compact bone and marrow, was isolated, weighted and split with a bone crush in an autopulverizer with 10 mL of sterile saline (Spex 6700; Freezer/Mill Industries Inc., Metuchen, USA). Thereafter, serial dilutions were performed and plated on tryptic soy agar for incubation at 37°C for 24 h before cfu were enumerated. The results were expressed as the median [IQR] log₁₀ cfu/g of bone and as the percentage of rabbits with sterile bones as previously defined.²²

**In vivo selection of mutants.** Each undiluted bone homogenate (100 µL) of untreated and treated rabbits was plated onto Mueller-Hinton II agar and onto Mueller-Hinton II agar supplemented with **ceftazidime-avibactam** (0.125, 0.25 or 4 µg/mL) to detect resistant mutants after 24 h of incubation at 37°C. When bacterial growth was observed, E. coli identification was performed by MALDI-TOF mass spectrometry (MALDI
Biotyper, Bruker Daltonique, Wissenbourg, France), and specific enzymes of resistance were confirmed using beta-lacta test for ESBL production (Laboratoire Biorad, 92430, Marnes-La-Coquette, France) and NG-test Carba-5 for OXA-48 (Laboratoire NG-Biotech, 35480, Guipry, France). The MIC values of ceftazidime-avibactam were determined by Etest method (BioMerieux, 69280, Marcy l'Etoile, France). Resistant mutants were defined by an MIC value ≥ 4 µg/mL according to EUCAST.²³

170

**Data analysis.** Numeric variables were represented by the median and IQR, whereas categorical variables were represented by numbers and percentages. Because of the relatively limited sample size, comparisons between groups were performed using Fisher's exact test for categorical variables and the Kruskal-Wallis non-parametric method for numeric variables. The effect of the antibiotics and their 95% CIs on Log₁₀ cfu criteria were calculated by the Hodges-Lehmann estimator and the Bauer algorithm. The effects of antibiotics on the sterility criteria were estimated by the difference in the proportion of sterile bones and the 95% exact CI. A p-value <0.05 was considered as statistically significant. All analyses were realised with R software, using coin and exact2x2 packages.

181

## 182 RESULTS

183 *In vitro* bactericidal effect.

Time-kill curves obtained at 4 x MIC (Figure 1) showed that ceftazidime-avibactam alone was rapidly bactericidal within 3 hours compared to fosfomycin alone within 24
186 hours. Conversely, colistin or gentamicin alone had no bactericidal activity.

187 For the combination of **ceftazidime-avibactam** and colistin or gentamicin, fast and complete bactericidal and synergistic effects were observed within 6 hours. When fosfomycin was added to **ceftazidime-avibactam**, slower bactericidal activity was observed compared to other companion drugs, with a synergistic effect after 24h.

191 Furthermore, the use of a dual antibiotic regimen prevented regrowth, in contrast to the use of ceftazidime-avibactam alone.

193

194 **Therapeutic studies.**

195 In control rabbits at day 24, the median (IQR) of bacterial counts was 5.4 (4.7, 5.7) log₁₀ cfu/g of bone.

197 Compared with those of controls (Figure 2A and Figure 3), bacterial counts in rabbits treated with colistin were similar with a median (IQR) of 4.7 (3.4, 5.4) log₁₀ cfu/g of bone (p= 0.50) with no sterile bone observed.

200 Compared with those of controls, **ceftazidime-avibactam** alone significantly decreased bacterial counts with a median (IQR) of 3.5 (2.0, 4.5) log₁₀ cfu/g of bone (p=0.004).

202 However, percentage of sterile bones (n=3/11; 27%) at day 24 was comparable to controls (p=0.21) (Figure 2B). When used in combination, **ceftazidime-avibactam + colistin or fosfomycin or gentamicin** significantly decreased bone bacterial concentrations compared to controls (p<0.0001, p=0.0001 and p=0.0002, respectively). Furthermore, those combinations also significantly decreased bone bacterial density compared to **ceftazidime-avibactam** alone (see Table 1) and promoted bone sterilization from 91% to 100% (p<0.0001) compared to controls (Figure 2B).
209 Of note in colistin and ceftazidime-avibactam + gentamicin groups, 9 rabbits were analyzed knowing that in each group 2 animals died prematurely of sepsis and one was euthanized for unbearable suffering pain.
210
211

212 In accordance with time-kill curves, there was no difference in terms of bone bacterial density between the combinations whether ceftazidime-avibactam was associated with colistin, fosfomycin or gentamicin (Table 1).
213
214
215

216 Ceftazidime-avibactam-resistant mutants in non-sterile animals

217 No ceftazidime-avibactam-resistant mutants emerged in rabbits treated with this regimen in single (n=8) or in combination therapy with colistin (n=1), with a MIC maintained ≤ 0.25 mg/L. Likewise, no ceftazidime-avibactam-resistant mutants emerged in rabbits treated with colistin alone (n=9).
218
219
220

221
222 DISCUSSION

223 Our work emphasized the high efficacy of a 7-day course of ceftazidime-avibactam in dual therapy, in a subacute model of OXA-48/ESBL-producing Escherichia coli osteomyelitis. It should be reminded that in human clinical practice, osteomyelitis usually requires 6 to 12 weeks of treatment to achieve infection control.²⁴ One strength of our experiment is its originality, aiming to address the issue of bone and joint infection by combining two mechanisms of resistance (co-expression of carbapenemase and ESBL enzymes) considered as a game-changer.
224
225
226
227
228
229

230 It is reassuring to observe that despite being a complex infection, it remains eligible for new drugs with an unexpected favourable outcome. Moreover, our findings are congruent
231
232 with our previous work regarding KPC-producing *K. pneumoniae*¹⁶ which highlighted
233 the interest of such regimen therapy based on **ceftazidime-avibactam** to achieve bone
234 sterilization.

235 Interestingly while **ceftazidime-avibactam** alone reduced bone bacterial density, it failed
236 to obtain significant bone sterilization, indicating that medical treatment of such complex
237 osteomyelitis should rely on combined therapy. Moreover, while all the combinations
238 based on **ceftazidime-avibactam** were comparable, it is obvious that in clinical practice
239 the use of colistin, fosfomycin or gentamicin should be balanced with the drug toxicity of
240 these last resort antibiotics, especially when prescribed for a prolonged therapy such as
241 osteomyelitis.²⁵ Indeed, in humans it is acknowledged that colistin and gentamicin can
242 cause acute kidney failure when used at high doses, while fosfomycin is an additional
243 sodium overload, often poorly tolerated in elderly patients suffering from underlying
244 heart failure. Our findings are supported by the fact that **ceftazidime-avibactam** remains
245 highly effective *in vitro* against OXA-48-producing *E. coli* (n=9/9), as recently described
246 in a large analysis of samples of CPE (n=276) in the USA.²⁶ On the other hand, there is a
247 real lack of clinical data about **ceftazidime-avibactam** for the treatment of infections
248 caused by OXA-48 carbapenemase-producing *E. coli*. Indeed, a recent publication in real
249 life conditions about **ceftazidime-avibactam** used as a salvage therapy for CPE, showed
250 that it was prescribed in association with other antibiotics in 65.8% of cases, with 73.7%
251 of clinical success and/or microbiological cure. This study concerned 12 patients infected
252 by OXA-48 producers, with only one case being an *E.coli*²⁷. Likewise, in India
253 **ceftazidime-avibactam** has been reported in clinical practice to be highly effective against
254 OXA-48-producing Enterobacterales (82.35%; n=14/17), including patients in ICU²⁸. In
255 contrast to our work, those isolates were mainly composed of *K. pneumoniae* (84.2%)
256 rather than *E. coli*. Moreover, our findings revealed no emergence of mutants within the
257 limits of our sample size, even when using **ceftazidime-avibactam** in single therapy. Yet,
258 resistance has already been described in *K. pneumoniae* isolates carrying *bla*<sub>OXA-48</sub> genes
259 after single therapy with **ceftazidime-avibactam**.²⁹ Therefore, we cannot recommend its
260 use alone, especially in difficult-to-treat infections such as osteomyelitis, which often
261 requires a new aggressive surgical procedure in case of failure, particularly on prosthetic
262 materials.

263 It is important to note that a recent alert has also been reported regarding the risk of
264 emergence of resistance in vitro for aztreonam-avibactam (another combination based on
265 avibactam), against OXA-48 carbapenemase-producing *E. coli* in Germany. ³⁰ This
266 argues for cautious use of these new drugs and encourages their use in combination with
267 other drugs where they are susceptible, as resistance may develop if they are
268 inappropriately prescribed on a larger scale.

269 There is a lack of literature regarding **OXA-48-type** osteomyelitis, therefore there is no
270 recommended regimen for this particular entity. To the best of our knowledge, there are
271 only two case series that address bone and joint infections due to OXA-48-producing
272 Enterobacterales. ¹⁵,³¹

273 Concerning bone and joint infections caused by ESBL-producing Enterobacterales, they
274 have been mentioned in four original publications to date. Davido *et al.* ³² reported the
275 largest series with exclusively ESBL infections occurring on native bone and joints
276 (n=29), with a primary control of infection in 62.1% of cases, in contrast to ESBL
277 infections on orthopedic implants where Pfang *et al.* ³¹ reported infection control in
77.3% (n=17/22) after implant removal and Martinez-Pastor et al. ³³ in 42.8% (n=3/7) when the implant was retained. Similarly, Papadopoulous et al. ¹⁴ addressed the topic of multidrug-resistant Gram-negative prosthetic joint infections in a large study including ESBL-producers (n=94), as well as multidrug-resistant Pseudomonas spp. (n=33) and some CPE (n=12) and mentioned a pooled efficacy of 66.7% of cases (n=72/108). Our findings regarding the interest of a combination therapy for the treatment of complex multidrug-resistant organisms are concordant with Papadopoulous et al. showing that colistin was useful in combination in a model of prosthetic joint infections. ¹⁴

Unexpectedly, our findings show that fosfomycin combined with ceftazidime-avibactam was as effective as other agents, whereas this combination had been shown to be comparable to ceftazidime-avibactam alone in our previous work regarding KPC-carbapenemase osteomyelitis. ¹⁶ These results support the need for repeated experiments with different microorganisms expressing different mechanisms of resistance, in order to obtain the best data applicable in clinical practice.

Overall, in a subacute model of OXA-48/ESBL E. coli osteomyelitis, our results confirm the efficacy of ceftazidime-avibactam in combination regardless of the associated agent. These data might suggest it would benefit from off-label use, if necessary, in expert centers.

Nevertheless, our study has some limitations. First, as we used 12 rabbits per treatment group and considering some animals prematurely deceased, using 9 animals in the colistin group might have underestimated the efficacy of this drug. However, it should be noted that in our previous in vivo works, colistin monotherapy had no impact on bone sterilization despite using 12 animals. ¹⁶,²² Similarly, gentamicin was eventually used in
combination to ceftazidime-avibactam on a limited number of animals due to unexpected fatal sepsis or killing according to animal wellness and welfare committee. However, the ultimate finding was unchanged considering 100% of the animals were sterile. Second, our experiments focused on a single strain of E. coli co-expressing ESBL and OXA-48. Hence, our findings may not be extrapolated to any CPE, including other microorganisms such as Klebsiella species or OXA-48-like isolates. However, these findings are concordant with our previous experiment based on a KPC-99YC strain¹⁶ showing the necessity of combining drugs based on ceftazidime-avibactam to ensure bone sterilization. Third, we did not evaluate the interest of carbapenems (imipenem had increased exposure susceptibility) which are the antibiotics of choice for treating infections caused by ESBL-producing Enterobacterales. This choice was made considering our previous work relying on high-dose carbapenems ²² which caused severe diarrhoea in animals and could have led to a fatal outcome in terms of mortality with this particular virulent strain. Moreover, the increased use of carbapenems has led to a vicious cycle with increased antibiotic resistance with the emergence of CPE and may not have been the best comparator in this experiment at a time where carbapenem-sparing is being encouraged.

In conclusion, in our model ceftazidime-avibactam in combination with colistin, fosfomycin or gentamicin was more effective than any other single therapy against OXA-48/ESBL-producing E. coli.

Ceftazidime-avibactam should not be prescribed alone for the treatment of difficult-to-treat infections, due to its partial effectiveness in sterilizing deep tissues, such as bones in this case. Experimental stringent models of complex infections, including multiple
324 mechanisms of resistance, may improve our knowledge to prescribe the optimal treatment
325 in the event of a deadlock situation to ensure the best clinical outcomes.

326

327 **FUNDING**

328 This study was supported by a grant from Pfizer (#52321751).

329

330 **TRANSPARENCY DECLARATIONS**

331 Authors declare no conflicts of interest.

332

333 **ACKNOWLEDGMENTS**

334 Authors would like to thank Beatrice Gadaleta from l'Institut de Médecine et
335 d'Epidémiologie Appliquée (IMEA) in Paris for her logistic assistance, the
336 microbiological department of Raymond-Poincaré teaching hospital and the staff of the
337 Plateforme de Recherche Biomédicale (PRBM) at Maisons-Alfort veterinary school for
338 their technical support.

339

340 **REFERENCES**

341 1. Shrivastava SR, Shrivastava PS, Ramasamy J. World health organization releases
342 global priority list of antibiotic-resistant bacteria to guide research, discovery, and
343 development of new antibiotics. *JMS - J Med Soc* 2018; **32**: 76-7. Available at:
344 https://www.jmedsoc.org/article.asp?issn=0972-
345 4958;year=2018;volume=32;issue=1;spage=76;epage=77;aulast=Shrivastava.
346 2. Talbot GH, Jezek A, Murray BE, et al. The Infectious Diseases Society of America's

347 10 × '20 Initiative (10 New Systemic Antibacterial Agents US Food and Drug

348 Administration Approved by 2020): Is 20 × '20 a Possibility? *Clin Infect Dis* 2019; **69**:

349 1–11. Available at: https://pubmed.ncbi.nlm.nih.gov/30715222/.

350 3. Poirel L, Héritier C, Tolün V, et al. Emergence of oxacillinase-mediated resistance to

351 imipenem in Klebsiella pneumoniae. *Antimicrob Agents Chemother* 2004; **48**: 15–22.

352 Available at: https://pubmed.ncbi.nlm.nih.gov/14693513/.

353 4. Potron A, Poirel L, Rondinaud E, et al. Intercontinental spread of OXA-48 beta-

354 lactamase-producing enterobacteriaceae over a 11-year period, 2001 to 2011.

355 *Eurosurveillance* 2013; **18**(31):20549. Available online:

356 http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20549

357 5. Nordmann P, Poirel L. The difficult-to-control spread of carbapenemase producers

358 among Enterobacteriaceae worldwide. *Clin Microbiol Infect* 2014; **20**: 821–30. Available

359 at: https://pubmed.ncbi.nlm.nih.gov/24930781/.

360 6. Jousset A, Emeraud C, Bonnin R, et al. Caractéristiques et évolution des souches

361 d'entérobactéries productrices de carbapénémases (EPC) isolées en France, 2012-2020.

362 *Bull Epidémiol Hebd* 2021; **18–19**: 351–8. Available at:

363 http://beh.santepubliquefrance.fr/beh/2021/18-19/2021_18-19_4.html.

364 7. Girlich D, Bouihat N, Poirel L, et al. High rate of faecal carriage of extended-spectrum

365 β-lactamase and OXA-48 carbapenemase-producing Enterobacteriaceae at a university

366 hospital in Morocco. *Clin Microbiol Infect* 2014; **20**: 350–4. Available at:

367 https://pubmed.ncbi.nlm.nih.gov/23927757/.

368 8. Ruppé E, Armand-Lefèvre L, Estellat C, et al. High Rate of Acquisition but Short
369 Duration of Carriage of Multidrug-Resistant Enterobacteriaceae after Travel to the
Tropics. *Clin Infect Dis* 2015; **61**: 593–600.

371 9. Arcilla MS, van Hattem JM, Haverkate MR, *et al*. Import and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae by international travellers

372 (COMBAT study): a prospective, multicentre cohort study. *Lancet Infect Dis* 2017; **17**: 78–85.

375 10. Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: the phantom menace. *J Antimicrob Chemother* 2012; **67**: 1597–606. Available at:

377 https://pubmed.ncbi.nlm.nih.gov/22499996/.

378 11. Hamprecht A, Sommer J, Willmann M, *et al*. Pathogenicity of Clinical OXA-48

379 Isolates and Impact of the OXA-48 IncL Plasmid on Virulence and Bacterial Fitness.

380 *Front Microbiol* 2019; **10**: 2509.

381 12. Titécat M, Senneville E, Wallet F, *et al*. Bacterial epidemiology of osteoarticular

382 infections in a referent center: 10-year study. *Orthop Traumatol Surg Res OTSR* 2013; **99**: 653–8.

384 13. de Sanctis J, Teixeira L, van Duin D, *et al*. Complex prosthetic joint infections due to carbapenemase-producing Klebsiella pneumoniae: A unique challenge in the era of untreatable infections. *Int J Infect Dis* 2014; **25**: 73–8.

386

387 14. Papadopoulos A, Ribera A, Mavrogenis AF, *et al*. Multidrug-resistant and extensively drug-resistant Gram-negative prosthetic joint infections: Role of surgery and impact of colistin administration. *Int J Antimicrob Agents* 2019; **53**: 294–301. Available at:

389 http://www.ncbi.nlm.nih.gov/pubmed/30395988.

391 15. Davido B, Noussair L, Saleh-Mghir A, *et al*. Case series of carbapenemase-producing
392 Enterobacteriaceae osteomyelitis: Feel it in your bones. *J Glob Antimicrob Resist* 2020;
393 **23**: 74–8.

394 16. Davido B, Crémieux AC, Vaugier I, et al. Efficacy of ceftazidime-avibactam in
395 various combinations for the treatment of experimental osteomyelitis due to Klebsiella
396 pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae. *Int J Antimicrob
397 Agents* 2022: 106702. Available at: http://www.ncbi.nlm.nih.gov/pubmed/36476965.

398 17. Sharma R, Eun Park T, Moy S. Ceftazidime-Avibactam: A Novel Cephalosporin/β-
399 Lactamase Inhibitor Combination for the Treatment of Resistant Gram-negative
400 Organisms. *Clin Ther* 2016; **38**: 431–44. Available at:
401 https://pubmed.ncbi.nlm.nih.gov/26948862/.

402 18. Zozaya DH, Gutiérrez OL, Ocampo CL, Sumano LH. Pharmacokinetics of a single
403 bolus intravenous, intramuscular and subcutaneous dose of disodium fosfomycin in
404 horses. *J Vet Pharmacol Ther* 2008; **31**: 321–7. Available at:
405 https://pubmed.ncbi.nlm.nih.gov/18638292/.

406 19. Tang SS, Gong LJ, He JK, Jin X, Xiao XL. Residue depletion of colistin in swine
407 after intramuscular administration. *J S Afr Vet Assoc* 2009; **80**: 41–4. Available at:
408 https://pubmed.ncbi.nlm.nih.gov/19653518/.

409 20. Tomasi L. Depletion of the residues of colistin and amoxicillin in turkeys following
410 simultaneous subcutaneous administration. *Vet Res Commun* 1996; **20**: 175–82.

411 21. Norden CW, Keleti E. Treatment of experimental staphylococcal osteomyelitis with
412 rifampin and trimethoprim, alone and in combination. *Antimicrob Agents Chemother*
413 1980; **17**: 591–4.

414 22. Crémieux A-C, Dinh A, Nordmann P, et al. Efficacy of colistin alone and in various
415 combinations for the treatment of experimental osteomyelitis due to carbapenemase-
416 producing Klebsiella pneumoniae. *J Antimicrob Chemother* 2019; **74**: 2666-75.

417 Available at: http://www.ncbi.nlm.nih.gov/pubmed/31263884.

418 23. European Committee on Antimicrobial. The European Committee on Antimicrobial
419 Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters.

420 Version 12.0, 2022. http://www.eucast.org. 2022; **12.0**: 0-110. Available at:
421 https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_1
422 2.0_Breakpoint_Tables.pdf.

423 24. Berbari EF, Kanj SS, Kowalski TJ, et al. 2015 Infectious Diseases Society of
424 America (IDSA) Clinical Practice Guidelines for the Diagnosis and Treatment of Native
425 Vertebral Osteomyelitis in Adults. *Clin Infect Dis* 2015; **61**: e26-46. Available at:
426 https://academic.oup.com/cid/article/61/6/e26/452579.

427 25. Davido B, Bouchand F, Dinh A, et al. Reinforcement of an antimicrobial stewardship
428 task force aims at a better use of antibiotics of last resort: the COLITIFOS study. *Int J
429 Antimicrob Agents* 2017; **50**(2):142-147.

430 26. Tamma PD, Bergman Y, Jacobs EB, et al. Comparing the activity of novel antibiotic
431 agents against carbapenem-resistant Enterobacterales clinical isolates. *Infect Control
432 Hosp Epidemiol* 2022: 1-6. Available at: https://pubmed.ncbi.nlm.nih.gov/35822340/.

433 27. Temkin E, Torre-Cisneros J, Beovic B, et al. Ceftazidime-avibactam as salvage
434 therapy for infections caused by carbapenem-resistant organisms. *Antimicrob Agents
435 Chemother* 2017; **61**. Available at: https://pubmed.ncbi.nlm.nih.gov/27895014/.

436 28. Nagvekar V, Shah A, Unadkat VP, et al. Clinical outcome of patients on ceftazidime-
437 avibactam and combination therapy in carbapenem-resistant enterobacteriaceae. *Indian J*
438 *Crit Care Med* 2021; **25**: 780–4.

439 29. Lübbert C, Lippmann N, Busch T, *et al*. Long-term carriage of Klebsiella

440 pneumoniae carbapenemase-2-producing K pneumoniae after a large single-center

441 outbreak in Germany. *Am J Infect Control* 2014; **42**: 376–80. Available at:

442 http://www.ncbi.nlm.nih.gov/pubmed/24679563.

443 30. Nordmann P, Yao Y, Falgenhauer L, Sadek M, Imirzalioglu C, Chakraborty T.

444 Recent emergence of aztreonam-avibactam resistance in NDM and OXA-48

445 carbapenemase-producing Escherichia coli in Germany. *Antimicrob Agents Chemother*

446 2021; **65**. Available at: https://journals.asm.org/doi/10.1128/AAC.01090-21.

447 31. Pfang BG, García-Cañete J, García-Lasheras J, *et al*. Orthopedic Implant-Associated

448 Infection by Multidrug Resistant Enterobacteriaceae. *J Clin Med* 2019; **8**: 220.

449 32. Davido B, Saleh-Mghir A, Rottman M, *et al*. Native bone and joint infections caused

450 by extended-spectrum β-lactamase-producing Enterobacterales: experience of a reference

451 centre in the Greater Paris area. *Int J Antimicrob Agents* 2022; **59**. Available at:

452 https://pubmed.ncbi.nlm.nih.gov/34906675/.

453 33. Martínez-Pastor JC, Vilchez F, Pitart C, Sierra JM, Soriano A. Antibiotic resistance in

454 orthopaedic surgery: acute knee prosthetic joint infections due to extended-spectrum

455 beta-lactamase (ESBL)-producing Enterobacteriaceae. *Eur J Clin Microbiol Infect Dis*

456 2010; **29**(8): 1039–41. Available at: http://link.springer.com/10.1007/s10096-010-0950-y.

457

458
459 **Table 1.** Pairwise comparisons of ceftazidime-avibactam in combination with various
460 antibiotics in carbapenemase (OXA-48) and ESBL-producing *E. coli* experimental
461 osteomyelitis
462

<table><thead><tr><th>Antibiotics</th><th>Difference in log<sub>10</sub> cfu<br>(95% CI)</th><th>P</th></tr></thead><tbody><tr><td><strong>Ceftazidime-avibactam + gentamicin compared with</strong></td><td></td><td></td></tr><tr><td>Colistin alone</td><td>-3.54 (-5.27 ; -1.16)</td><td>0.0004</td></tr><tr><td>Ceftazidime-avibactam alone</td><td>-2.32 (-3.77 ; -0.12)</td><td>0.002</td></tr><tr><td>Ceftazidime-avibactam + colistin</td><td>-0.01 (-0.13 ; 0.07)</td><td>0.51</td></tr><tr><td>Ceftazidime-avibactam + fosfomycin</td><td>-0.04 (-0.12 ; 0.08)</td><td>0.44</td></tr><tr><td><strong>Ceftazidime-avibactam + fosfomycin compared with</strong></td><td></td><td></td></tr><tr><td>Colistin alone</td><td>-3.50 (-4.38 ; -2.10)</td><td>0.0003</td></tr><tr><td>Ceftazidime-avibactam alone</td><td>-2.30 (-3.73 ; -0.18)</td><td>0.007</td></tr><tr><td>Ceftazidime-avibactam + colistin</td><td>0.01 (-0.09 ; 0.10)</td><td>0.97</td></tr><tr><td><strong>Ceftazidime-avibactam + colistin compared with</strong></td><td></td><td></td></tr><tr><td>Colistin alone</td><td>-3.51 (-4.29 ; -1.85)</td><td>0.0003</td></tr><tr><td>Ceftazidime-avibactam alone</td><td>-2.23 (-2.88 ; -0.13)</td><td>0.004</td></tr><tr><td><strong>Ceftazidime-avibactam compared with</strong></td><td></td><td></td></tr><tr><td>Colistin alone</td><td>-1.31 (-3.40 ; 0.36)</td><td>0.15</td></tr></tbody></table>
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>
469 **Figure 2:** Effect of ceftazidime-avibactam alone or in combination with various antibiotics on log₁₀ cfu/g of bone (a) and on percentage of sterile bones (b) in animals with OXA-48/ESBL-producing *E. coli* experimental osteomyelitis.

<figure><img src="image_7.png" /></figure>

473 **Figure 3:** Bacterial density (log₁₀ cfu/g of bone) of ceftazidime-avibactam alone or in combination with various antibiotics for the treatment of OXA-48/ESBL-producing *E. coli* experimental osteomyelitis. Whiskers are the minimum and the maximum values and the horizontal line in each box plot, which covers the IQR, is the median.

477