Cefoxitin as an Alternative to Carbapenems in a Murine Model of Urinary Tract Infection Due to Escherichia coli Harboring CTX-M-15-Type Extended-Spectrum -Lactamase

Raphal Lepeule,a Etienne Rupp,a,b Patrick Le,c Laurent Massias,c Franoise Chau,a Amandine Nucci,d Agns Lefort,a,e and Bruno Fantina,e
Universit Paris Diderot, Paris,a AP-HP, Hpital Bichat, CNR Associ Rsistance dans les Flores Commensales, Laboratoire de Bactriologie, Paris,b AP-HP, Hpital Bichat, Laboratoire de Toxicologie-Pharmacocintique, Service de Pharmacie, Paris,c Institut Pasteur, Unit des Agents Antibactriens, Paris,d and AP-HP, Hpital Beaujon, Service de Mdecine Interne, Clichy,e France

We investigated the efciency of the cephamycin cefoxitin as an alternative to carbapenems for the treatment of urinary tract infections (UTIs) due to Escherichia coli producing CTX-M-type extended-spectrum -lactamases. The susceptible, UTIinducing E. coli CFT073-RR strain and its transconjugant CFT073-RR Tc (pblaCTX-M-15), harboring a blaCTX-M-15 carryingplasmid, were used for all experiments. MICs of cefoxitin (FOX), ceftriaxone (CRO), imipenem (IMP), and ertapenem (ETP) for CFT073-RR and CFT073-RR Tc (pblaCTX-M-15) were 4 and 4, 0.125 and 512, 0.5 and 0.5, and 0.016 and 0.032 g/ml, respectively. Bactericidal activity was similarly achieved in vitro against the two strains after 3 h of exposure to concentrations of FOX, IMI, and ETP that were 2 times the MIC, whereas CRO was not bactericidal against CFT073-RR Tc (pblaCTX-M-15). The frequencies of spontaneous mutants of the 2 strains were not higher for FOX than for IMP or ETP. In the murine model of UTIs, mice infected for 5 days were treated over 24 h. Therapeutic regimens in mice (200 mg/kg of body weight every 3 h or 4 h for FOX, 70 mg/kg every 6 h for CRO, 100 mg/kg every 2 h for IMP, and 100 mg/kg every 4 h for ETP) were chosen in order to reproduce the percentage of time that free-drug concentrations above the MIC are obtained in humans with standard regimens. All antibiotic regimens produced a signicant reduction in bacterial counts (greater than 2 log10 CFU) in kidneys and bladders for both strains (P < 0.001) without selecting resistant mutants in vivo, but the reduction obtained with CRO against CFT073-RR Tc (pblaCTX-M-15) in kidneys was signicantly lower than that obtained with FOX. In conclusion, FOX appears to be an effective therapeutic alternative to carbapenems for the treatment of UTIs due to CTX-M-producing E. coli.

ver the last decade, resistance to -lactams among Enterobacteriaceae has emerged as a major public health threat. This is mostly due to proliferation of extended-spectrum -lactamases (ESBLs), especially of the CTX-M-type, which have spread worldwide both in hospitals and the community (9). Escherichia coli isolates that produce CTX-M (especially CTX-M-15 in Western countries) are currently a serious cause of urinary tract infections (UTIs) in the community (36). Mortality in the most severe infections, particularly those evolving into bacteremia, is as high as 60% (27). In addition, associated resistance to other classes of antimicrobial agents is often observed in CTX-M producers, limiting the availability of therapeutic options. Carbapenems are widely regarded as the drug of choice for parenchymal infections due to ESBL-producing Enterobacteriaceae (35). However, several reports have described the emergence of resistance to carbapenems in Enterobacteriaceae by two mechanisms: (i) in vivo selection of porin-decient mutants under therapy with carbapenems (11, 20) and (ii) emergence of carbapenemases, reported extensively in Klebsiella pneumoniae (18, 30) and more recently in E. coli (18, 29). The rise of ESBLs has led to an increased consumption of carbapenems, which facilitated the emergence and spread of carbapenemases. Emergence of carbapenem resistance in E. coli is worrying from a public health point of view and compels exploration of alternative therapeutic options. In vitro, cephamycins demonstrate consistent activity against ESBL-producing strains of Enterobacteriaceae. There are very few published reports on the use of cephamycins for the treatment of infection due to ESBL producers. Pangon et al. reported the emergence of porin-

resistant K. pneumoniae mutants under treatment with cefoxitin and the subsequent relapse of infection (33). Combined cephamycin and carbapenem resistances due to acquisition of the plasmidmediated blaDHA-1 and the concomitant depletion of porin (OmpK36) expression have been observed in K. pneumoniae after omoxef or cefotetan exposure (8, 22). Cefoxitin-resistant E. coli mediated by the loss of a porin has also been described in clinical isolates (3, 32). Therefore, the aim of our study was to evaluate the efciency of cefoxitin as an alternative to carbapenems for the treatment of UTIs due to E. coli producing CTX-M-type ESBLs in terms of bactericidal activity and selection of resistant mutants in vitro and in an ascending UTI murine model. (This work was partially presented at the 50th Interscience Conference on Antimicrobial Agents and Chemotherapy, Boston, MA, September 2010.)
MATERIALS AND METHODS
Bacterial strains, conjugation assays, and plasmid stability. Experiments were performed with a susceptible E. coli CFT073 strain, previously

Received 2 December 2011 Returned for modication 11 December 2011 Accepted 24 December 2011 Published ahead of print 3 January 2012 Address correspondence to Bruno Fantin, bruno.fantin@bjn.aphp.fr. Copyright 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.06233-11

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TABLE 1 MICs ( g/ml) of FOX, CTX, CRO, IMP, and ETP and proportions of resistant mutants for the E. coli strains used in the study
MIC ( g/ml) of: E. coli strain CFT073-RR CFT073-RR Tc (blaCTX-M-15)
a b

PRM (10 8)a CRO 0.125 512 IMP 0.5 0.5 ETP 0.016 0.032 FOX 0.06 0.12 0.6 1.77c IMP 8.16 18.2 13.8b 26.6b,c ETP 4.35 12.3 13.6b 15.5a,c

FOX 4 4

CTX 0.06 512

Selection by antibiotics at 4 times the MIC. Each value is the mean standard deviation of six independent experiments. PRM, proportion of resistant mutants. P 0.05 when compared with the PRM for the same strain treated with FOX. cP 0.2 when compared with the PRM for the E. coli CFT073-RR strain.

used in a murine model of pyelonephritis (2, 19), and its transconjugant, harboring a blaCTX-M-15-carrying plasmid. A rifampin-resistant mutant, E. coli CFT073-RR, was selected in vitro from E. coli CFT073 by plating 109 bacteria onto Mueller-Hinton (MH) agar containing a rifampin concentration of 250 g/ml. The transconjugant was obtained by conjugating E. coli CFT073-RR and a clinical E. coli CEC89 isolate harboring the blaCTX-M-15 gene (37). A plasmid transfer assay was attempted by bacterial mating in liquid broth. After incubation, transconjugants were selected by plating the conjugation mixture on Drigalski agar supplemented with cefotaxime (10 g/ml) plus rifampin (250 g/ml). Genetic backgrounds for CFT073-RR and the transconjugant were conrmed to be identical by random amplication of polymorphic DNA (RAPD) experiments using 2 primers (1254 and 1281) (31). For the clinical strain and the transconjugant, a multidrug resistance (MDR) region, similar to that described for plasmid pC15-1a (7), was investigated by PCR, using E. coli strain 666T as a positive control (21). The genes blaCTX-M-15 (with insertion sequence ISEcp1 immediately upstream), blaOXA-1, and aac(6=)-1b were present in both strains, and blaTEM-1 was not detected. Plasmid stability of the transconjugant E. coli CFT073-RR Tc (pblaCTX-M-15) was measured in vitro by subculturing every 48 h for 4 weeks in antibiotic-free tryptic soy (TS) broth and subculturing daily for 5 days in minimal medium. Cultures were plated onto MH agar with or without 10 g/ml of cefotaxime. No plasmid loss was observed in minimal medium, and after 4 weeks in TS broth, the median weekly plasmid loss was 0.5% (range, 0.4% to 2%). Antibiotic activities in vitro. MICs of cefoxitin (FOX), cefotaxime (CTX), ceftriaxone (CRO), imipenem (IMP), and ertapenem (ETP) were determined by the agar dilution method in accordance with CLSI guidelines (10). Time-kill curve kinetics were performed with FOX, CRO, IMP, and ETP for each strain in 10 ml TS broth with antibiotic concentrations equal to 2 times the MIC value for the tested strain or with no antibiotic. Viable counts were enumerated by plating 100 l of serial dilutions of cultures onto TS agar plates after 0, 1, 3, 6, and 24 h of incubation at 37C. Antibiotic carryover did not interfere with bacterial counts at the antibiotic concentrations used (34). The frequency of spontaneous resistant mutants was determined for each strain at an antibiotic concentration of 4 times the MIC, as described previously (2, 16). Briey, strains were grown at 37C overnight in 20 ml of antibiotic-free TS broth and centrifuged, and the pellet was suspended in 2 ml of sterile broth to obtain an inoculum of 1010 CFU/ml. MH agar plates containing antibiotics were inoculated with 100 l of cell suspension and incubated at 37C for 48 h. The proportion of spontaneous resistant mutants was calculated by dividing the number of CFU growing on MH agar plates with antibiotics by the number of CFU growing on antibiotic-free MH agar. Each experiment was performed at least three (time-kill curves) or six (MICs, selection of mutants) times. Geometric means were used to express the results for MICs, and means and standard deviations were calculated for rates of spontaneous mutants. UTI mouse model. Animal experiments were performed in accordance with prevailing regulations regarding the care and use of laboratory animals from the European Commission (14). The experimental protocol was approved by the Departmental Direction of Veterinary Services from Paris, France. The ascending, unobstructed UTI mouse model was used as previously described (2, 19). Eight-week-old immunocompetent CBA female mice (weight, 20 to 23 g) were used. Inocula were obtained by over-

night incubation in TS broth after centrifugation at 1,800 g for 10 min. Pellets were suspended in 1 ml sterile broth to a nal inoculum of 1010 CFU/ml. Pyelonephritis was induced after general anesthesia (with an intraperitoneal administration of 150 mg/kg of body weight of ketamine and 0.5 mg/kg of xylazine) by injecting 50 l (108 CFU) of the inoculum into the bladder through a urethral catheter. In vivo growth experiments and plasmid stability. The growth rates of individual strains were studied in groups of at least 10 infected mice in order to evaluate their potential for performing pyelonephritis and to evaluate in vivo plasmid stability for the transconjugant. Mice were sacriced 2, 10, and 20 days after inoculation. Bladder and kidney were aseptically taken out and homogenized in 1 ml of saline solution. One hundred microliters of the solution or its dilution was spread onto MH agar plates and incubated for 24 h. Numbers of CFU were counted and expressed as the number of CFU/g of tissue. Plasmid loss was assessed for the strain harboring plasmid pblaCTX-M-15 by comparing the number of CFU growing on MH plates containing cefotaxime (10 g/ml) and the number of CFU growing on MH plates without cefotaxime. Antibiotic pharmacokinetics. Pharmacokinetic (PK) studies were performed on mice in order to determine the therapeutic regimen that best reproduces the same percentage of time of free-drug concentrations above the MIC (fT MIC) and the same plasmatic peak concentrations of antibiotics as those obtained in humans with standard regimens. CRO was chosen instead of CTX for the animal study because of its longer half-life in mice. First, we determined the dose of each antibiotic that produced, after a single subcutaneous injection in mice, plasmatic peak concentrations that corresponded to human concentrations, according to literature and our pilot experiments: 200 mg/kg for FOX (1), 70 mg/kg for CRO (5), and 100 mg/kg for IMP (24) and ETP (12). Then, single-dose pharmacokinetic studies were performed after a single subcutaneous injection in mice for these selected doses. The fT MIC in mice were obtained from observed data by linear regression. Finally, dosing intervals were determined in order to achieve fT MIC similar to those observed in humans (26, 38, 39). For FOX, the human regimen of 2 g every 6 h (q6h) was chosen according to Infectious Diseases Society of America (IDSA) guidelines (40); for CRO (1 g q24h), IMP (1 g q8h), and ETP (1 g q24h), human regimens were chosen according to United States Food and Drug Administration labeling. Antibiotic assays. Blood samples of at least 800 l were obtained by intracardiac puncture from 4 anesthetized mice 15, 30, 60, 120, 240, and 360 min after antibiotic injection. After blood collection, plasma was separated by centrifugation and, for IMP and ETP, immediately stabilized with 3-morpholinopropanesulfonic acid (MOPS) (15). Concentrations were determined by high-performance liquid chromatography (HPLC) with ultraviolet detection at 237 nm. The limit of quantication was 2 g/ml for CRO, 0.5 g/ml for ETP and FOX, and 0.2 g/ml for IMP. Intraday and extraday precisions were lower than 12% for all antibiotics. Free-drug concentrations were determined after ultraltration by centrifugation in a Centrifree YM-30 (Millipore SAS, Molsheim, France) at 2,000 g and 25C for 10 min. Antimicrobial treatment. At least 90 mice were inoculated with each strain. Five days after inoculation, 15 mice were sacriced before treatment (start-of-treatment controls), 15 were left untreated (end-of-

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TABLE 2 Pharmacokinetic/pharmacodynamic parameters of the usual dosing regimen in humans and the dosing regimen used in mice for experimental urinary tract infectionsa

RESULTS

MICs and in vitro selection of spontaneous resistant mutants. MICs of each antibiotic tested and the proportions of spontaneous resistant mutants to FOX, IMP, and ETP for the two study strains are presented in Table 1. No signicant differences were observed between the two study strains regarding the MICs or the proportions of resistant mutants of FOX, IMP, and ETP. In contrast, high levels of resistance to CRO and CTX were expressed by the transconjugant CFT073-RR Tc (pblaCTX-M-15) compared to those of the parental strain. Time-kill curves. Bactericidal activity was similarly achieved for FOX, IMP, and ETP at concentrations of 2 times the MIC against CFT073-RR and CFT073-RR Tc (pblaCTX-M-15). For CRO, a bactericidal effect similar to that for the other three antibiotics was observed against CFT073-RR, whereas against CFT073-RR Tc (pblaCTX-M-15), an initial killing of 2 log10 CFU/ml at 6 h was followed by a bacterial regrowth at 24 h (data not shown). Pharmacokinetics and pharmacodynamic (PD) parameters. Peak value, protein binding, and fT MIC for dosing regimens used in mice are shown in Table 2. In mice, two therapeutic regimens of FOX were tested: 200 mg/kg q3h, which achieved an fT MIC of 44% (signicantly higher than the target of 33% in humans with the 2-g q6h regimen), and 200 mg/kg q4h, which achieved an fT MIC of 33%, similar to that for humans. Mouse model of UTIs. The UTI experiment showed that the two strains were able to induce stable pyelonephritis in mice until at least day 10. Infection was persisting until day 20 but was maximal between days 5 and 10 (data not shown). On day 10, bacterial counts in kidney and bladder (mean log10 CFU/g of tissue standard deviation) were 4.9 0.9 and 7.4 1.0, respectively, for strain CFT073-RR and 5.3 0.9 and 7.4 0.8, respectively, for strain CFT073-RR Tc (pblaCTX-M-15). At day 10, no plasmid loss was observed in either kidneys or bladders (data not shown). In mice infected with either strain, the ve therapeutic regimens produced signicant reductions in viable bacterial counts of bladders and kidneys compared to the counts for start-oftreatment control mice (Tables 3 and 4). When looking at in vivo activities in the bladders, there were no statistically signicant differences between the ve treatment groups, regardless of the infecting strain (Table 3). In the kidneys, no differences between the ve regimens were noted in mice infected with CFT073-RR (Table 4). In mice infected with CFT073-RR Tc (pblaCTX-M-15), FOX was

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Cmax, mean peak value of total concentration (given with or without standard deviation). fT MIC, percentage of time of free-drug concentrations above the MIC (MIC of FOX ETP 0.032 g/ml). b Data are according to references 26, 38, 39, and 40. cn 4.

treatment controls), and 60 mice were treated over 24 h with one of the 5 different therapeutic regimens determined from the antibiotic pharmacokinetic studies: FOX at 200 mg/kg q3h, FOX at 200 mg/kg q4h, CRO at 70 mg/kg q6h, IMP at 100 mg/kg q2h, or ETP at 100 mg/kg q4h. To avoid a carryover effect, treated mice were sacriced 24 h after the last dose of antibiotic. Bladder and kidney were aseptically taken out and homogenized in 1 ml of saline solution. One hundred microliters of the solution or its dilution was spread in duplicate onto MH agar plates and incubated for 24 h. Numbers of CFU were counted and expressed as numbers of CFU/g of tissue. Kidney and bladder homogenates (100 l of each) were also spread onto MH agar plates containing antibiotic at a concentration of 4 times the MIC and incubated at 37C for 72 h in order to detect resistant mutants. Statistical analysis. Results are the means and standard deviations for continuous variables. Continuous variables were compared by nonparametric (Mann-Whitney and Kruskal-Wallis) tests. Proportions of sterile organs were compared by the Fisher exact test. A P value of less than 0.05 was considered signicant. Analysis was performed using BiostaTGV statistical software (http://www.u707.jussieu.fr/biostatgv/).

fT MIC (%)

Cmax ( g/ml)

31

fT MIC (%)

Protein binding (%)

Cmax ( g/ml)

IMP

fT MIC (%)

Regimen

Protein binding (%)

Cmax ( g/ml)

fT MIC (%)

Regimen

CRO

33 33

1 g q24h 70 mg/kg q6h

150 208

25

8396 65

100 100

1 g q8h 100 mg/kg q2h

65 87

20 34

79 95

PK/PD values for each antibiotic

Protein binding (%)

Cmax ( g/ml)

Population

Regimen

FOX

Humansb Micec

2 g q6h 200 mg/kg q4h 200 mg/kg q3h

120 158

31

77 24

44

4 g/ml, MIC of IMP

ETP

Regimen

1 g q24h 100 mg/kg q4h

155 188

0.5 g/ml, and MIC of

Protein binding (%)

9295 99

100 93

Cefoxitin in ESBL Urinary Tract Infections

TABLE 3 Effect of antibiotics on viable organisms in bladders of mice infected with the E. coli strains used in this study
Results (log CFU/g of bladder E. coli strain CFT-RR CFT-RR Tc (pblaCTX-M-15)
a b

SD [no. sterile/total no.]) for mice treated with: Cefoxitin q3h 3.65 3.77 1.15a,b (3/15) 1.28a,b (1/15)d Ceftriaxone 4.57 3.94 0.63a,b (0/15) 0.80a,b (2/15) Imipenem 3.29 3.25 0.92a,b (3/15) 0.85a,b (3/15) Ertapenem 3.75 3.72 1.07a,b (2/15) 0.79a,b (1/15)

Start-of-treatment control 7.02 6.49 1.16 (0/15) 1.53 (0/16)

Cefoxitin q4h 4.42 4.06 1.49a,b (1/15) 1.37a,b (1/15)c

P P cP dP

0.001 when compared with results for the start-of-treatment control group. 0.14 when the different treatment groups are compared. 0.76 when compared with the results for the same treatment for mice infected with CFT-RR. 0.30 when compared with the results for the same treatment for mice infected with CFT-RR.

as active as IMP and ETP and signicantly more active than CRO in terms of bacterial counts and proportions of sterile kidneys. When the activity of FOX was analyzed according to the infecting strain, the activity against CFT073-RR Tc (pblaCTX-M-15) in terms of bacterial count reduction and rates of sterilization was not reduced from that against CFT073-RR (Tables 3 and 4). Thus, acquisition of the blaCTX-M-15 plasmid did not modify the activity of FOX. As expected, the activities of IMP and ETP were similar against the two strains in both bladders and kidneys. No FOX-, IMP-, or ETP-resistant mutant of CFT073-RR or CFT073-RR Tc (pblaCTX-M-15) was detected at the end of treatment with any of these antibiotics.
DISCUSSION

Despite their good in vitro activity and stability in resistance to hydrolysis by ESBLs, the cephamycins (e.g., cefoxitin) are not currently recommended as a rst-line treatment in infections with ESBL-producing Enterobacteriaceae. Indeed, several strains of ESBL-producing K. pneumoniae and E. coli resistant to cephamycins by impermeability have been reported. These strains showed two resistance mechanisms: production of ESBL and decreased expression of the outer membrane protein (32). However, this mechanism has been more often described in K. pneumoniae than in E. coli (8, 23, 33). Our objective was to compare cefoxitin and carbapenem activities. The two carbapenems chosen were imipenem and ertapenem, two reference drugs in the treatment of urinary tract infections with ESBL-producing E. coli. This comparison focused on the bactericidal activity and the selection of resistant mutants during treatment. Our in vitro and in vivo studies were based on isogenic strains of

E. coli harboring pblaCTX-M-15 and E. coli without pblaCTX-M-15. Before considering the effect of the acquisition of plasmid pblaCTX M-15, we assessed the in vitro and in vivo stability of the plasmid by conjugation in E. coli CFT073-RR. The plasmid loss rate was less than 5% overall. A possible explanation for the high stability of this plasmid could be the presence of addiction systems (28). The transconjugant CFT073-RR Tc (pblaCTX-M-15) was very comparable to CTX-M-15-producing clinical isolates in terms of antibiotic resistance and plasmid stability. In vitro, the transconjugant CFT073-RR Tc (pblaCTX-M-15) was highly resistant to expanded-spectrum cephalosporins. FOX and IMP MICs were not affected by the production of CTX-M-15. In contrast, we observed a moderate but constant and reproducible 1-dilution increase of the ETP MIC. This result is in accordance with the fact that the MIC90 of ETP may demonstrate an increase of 3 dilutions for ESBL-producing strains compared to that for wild-type strains (25). The antibiotics showed comparable rapid bactericidal activities against both strains in vitro. Thus, the presence of CTX-M-15 did not alter the bactericidal activity of FOX or that of the two carbapenems (IMP and ETP). In the experimental pyelonephritis model in mice, we aimed to reproduce the percentage of time of free-drug concentrations above the MIC (fT MIC) obtained in humans with standard regimens. Indeed, for -lactams, this PK/PD criterion is correlated with clinical efciency (4). We took into account protein binding that was measured in mice and that could signicantly differ from that in humans (Table 2). For FOX, two regimens were tested: one to achieve an fT MIC of 33%, a gure similar to the fT MIC found in humans for a dose of 2 g every 6 h, which is a regimen recommended by the IDSA for the treatment of severe infection (40), and a second to produce an fT MIC of 44% in order to

TABLE 4 Effect of antibiotics on viable organisms in kidneys of mice infected with the E. coli strains used in this study
Results (log CFU/g of kidney E. coli strain CFT-RR CFT-RR Tc (pblaCTX-M-15)
a b

SD [no. sterile/total no.]) for mice treated with: Cefoxitin q3h 1.62 2.34 1.02 (3/15) 1.19a (9/15)f,g
a,b c

Start-of-treatment control 4.85 4.64 0.86 (0/15) 1.26 (0/15)

Cefoxitin q4h 2.74 1.41 1.75 (3/15) 1.52a (9/15)f,g

a,b

Ceftriaxone 1.95 3.90 0.63 (7/15) 0.80d,e (2/15)

a,b

Imipenem 2.58 2.11 1.42 (4/15) 1.76a (7/15)c

a,b

Ertapenem 1.68 1.98 1.08a,b (6/15) 0.97a (3/15)

P P cP dP eP fP gP

0.001 when compared with results for the start-of-treatment control group. 0.25 when the different treatment groups are compared. 0.01 when compared with proportions (in parentheses) for the start-of-treatment control group. 0.05 when compared with results for the start-of-treatment control group. 0.001 when compared with results for mice treated with cefoxitin q4h. 0.001 when compared with proportions (in parentheses) for the start-of-treatment control group. 0.02 when compared with proportions (in parentheses) for mice treated with ceftriaxone.

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evaluate a possible benet of such an extension of the fT MIC. In fact, the 2 FOX regimens showed comparable activities in kidneys and bladders. Despite PK/PD parameters giving advantage to carbapenems over cephamycin, FOX, IMP, and ETP similarly and signicantly reduced the bacterial density in kidneys and bladders in mice. Moreover, this efciency was found regardless of the strain. Thus, in our model, the production of CTX-M-15 was not associated with a reduced efciency of FOX, IMP, and ETP treatment, and the effectiveness of cefoxitin was comparable to that of the carbapenems. In vitro, we did not nd increased proportions of resistant mutants in the strain harboring the plasmid pblaCTX-M-15 compared to those in the parental strain. This result is similar to that observed by Girlich et al. with ertapenem (16). In vivo, we did not detect any mutants resistant after antibiotic exposure. This may be due in part to the maximum size of the bacterial inoculum in the pyelonephritis model (maximum total number of bacteria is 5 104 CFU in the kidneys and 5 105 CFU into the bladder), which is lower than the inverse of the proportion of resistant mutants detected in vitro (between 10 6 and 10 9). Moreover, the treatment might have been too short to select mutants. Regarding the activity of CRO on the ESBL-producing strain, our results show an activity comparable to that of other antibiotics in the bladders. CRO remained effective in the kidneys but was signicantly less effective than the three other antibiotics. These results are in accordance with reports of good clinical activity of expanded-spectrum cephalosporins in human urinary tract infections due to ESBL-producing Enterobacteriaceae (6, 13, 41). This activity can be explained by the high urinary concentrations observed with this antibiotic. Indeed, in humans, urinary concentrations are higher than the MIC of our ESBL-producing strain (17). In conclusion, our results suggest that cefoxitin activity is comparable to that of imipenem and ertapenem against CTX-M-15producing E. coli strains, both in vitro and in a UTI murine model and in terms of both bactericidal activity and selection of resistant mutants. Several limitations of our study should be underlined: use of a single resistant strain, a short duration of treatment, and a relatively low in vivo inoculum does not favor selection of resistant strains during therapy. However, this molecule may be regarded as a therapeutic alternative to carbapenems for the treatment of documented urinary tract infections due to CTX-M-producing E. coli.
ACKNOWLEDGMENTS
We thank Louis Garry and Sara Dion from INSERM U722, Facult de Mdecine Xavier Bichat, Universit Paris Diderot, for technical help in the experimental model, Thierry Lambert and Patrice Courvalin from Institut Pasteur for technical assistance for gel electrophoresis, and Michael Mulvey for providing strain 666T. Raphal Lepeule was supported by a grant from the Fondation Recherche Mdicale (Mastre2 DEA20090616066).

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