Temocillin against Enterobacteriaceae isolates from community-acquired urinary tract infections: low rate of resistance and good accuracy of routine susceptibility testing methods

Temocillin against Enterobacteriaceae isolates from community-acquired urinary tract infections:... Abstract Background Temocillin is an old ‘revived’ antibiotic that may play an important role in the treatment of febrile urinary tract infection (UTI). Data regarding its activity against current Enterobacteriaceae isolates as well as the performance of routine susceptibility testing methods are, however, scarce. Objectives To determine the MICs of temocillin for Enterobacteriaceae strains reflecting the current epidemiology and to analyse the accuracy of three commercial methods. Methods Enterobacteriaceae isolates causing community-acquired UTI were prospectively collected from September 2015 to January 2017 in two French centres. Temocillin MIC was determined by agar dilution (AD) as the reference method and then compared with: (i) susceptibility testing by disc diffusion; (ii) MIC determination by Etest; and (iii) MIC estimation by the Vitek 2 automated system. Results A total of 762 Enterobacteriaceae were analysed comprising 658 (86.4%) Escherichia coli and 37 (4.9%) ESBL-producing isolates. Susceptibility rate assessed by AD was 99.6% according to the 8 mg/L clinical breakpoint and was significantly lower against the ESBL-producing isolates than the non-ESBL-producing isolates (94.6% versus 99.9%, P < 0.01). The MIC50 and MIC90 for the total set were 3 and 6 mg/L, respectively. According to the 8 mg/L clinical breakpoint, the major error rate was <1% for disc diffusion and Etest, and significantly higher for Vitek 2 (4.3%, P < 0.01), but still low. No very major error was noticed. Conclusions Temocillin showed a high level of activity against Enterobacteriaceae from community-acquired UTI and good to excellent reliability of routine methods for susceptibility testing in such a setting. Introduction Temocillin (6-α-methoxy derivative of ticarcillin) is an old ‘revived’ antibiotic from the penicillin family currently available in several European countries (Belgium, France, Luxembourg and the UK) that has very attractive characteristics: narrow spectrum mainly restricted to Enterobacteriaceae, high resistance to hydrolysis by numerous β-lactamases including ESBL and hyperproduced AmpC, and minimal risk of Clostridium difficile infection. Thus, it may act as an alternative to carbapenems for the treatment of serious Enterobacteriaceae infections such as systemic urinary tract infections (UTIs).1 As for other forgotten antibiotics, some microbiological data are, however, scarce. Studies are still in progress to better define the clinical breakpoints of temocillin, while Enterobacteriaceae are currently categorized susceptible for MIC values ≤8, ≤16 or ≤32 mg/L depending on the country (Table 1).1 If the in vitro activity of temocillin against ESBL-producing isolates has largely been described,2–4 data are paradoxically lacking for non-ESBL-producing isolates still commonly encountered in community-acquired UTIs. Table 1. Clinical breakpoints of temocillin currently in use in different countries where this antibiotic is available Country MIC (mg/L) susceptible resistant Belgiuma ≤16 ≥32 Franceb ≤8 >8 UK, systemic infectionc ≤8 >8 UK, uncomplicated UTIc ≤32 >32 Country MIC (mg/L) susceptible resistant Belgiuma ≤16 ≥32 Franceb ≤8 >8 UK, systemic infectionc ≤8 >8 UK, uncomplicated UTIc ≤32 >32 a See Fuchs et al.11 b See Comité de l’Antibiogramme de la Société Française de Microbiologie.13 c See BSAC.12 Table 1. Clinical breakpoints of temocillin currently in use in different countries where this antibiotic is available Country MIC (mg/L) susceptible resistant Belgiuma ≤16 ≥32 Franceb ≤8 >8 UK, systemic infectionc ≤8 >8 UK, uncomplicated UTIc ≤32 >32 Country MIC (mg/L) susceptible resistant Belgiuma ≤16 ≥32 Franceb ≤8 >8 UK, systemic infectionc ≤8 >8 UK, uncomplicated UTIc ≤32 >32 a See Fuchs et al.11 b See Comité de l’Antibiogramme de la Société Française de Microbiologie.13 c See BSAC.12 Moreover, the performance of the three commercial methods commonly used to determine susceptibility (disc diffusion, Etest and the Vitek 2 automated instrument system) are not well described for the temocillin/Enterobacteriaceae pairs. Two previous studies concerning temocillin have reported marked discrepancies (up to 36%) between routine tests and a reference method,5,6 but these studies were biased by an overrepresentation of strains from healthcare-associated infections. In this context, we aimed to precisely determine the MIC of temocillin for a large set of Enterobacteriaceae strains reflecting the current epidemiology of community-acquired UTIs and to assess the accuracy of disc diffusion, Etest and the Vitek 2 automated instrument system. Materials and methods Bacterial set Enterobacteriaceae isolates causing community-acquired UTIs were prospectively collected from September 2015 to January 2017 in two French centres—a private practice laboratory (analysing ∼4000 urine samples per year) and a teaching hospital—as part of an ongoing epidemiological study on community-acquired UTIs. Identification to the species level was performed routinely using either Vitek 2 or MALDI-TOF technology. Reliable clinical data were prospectively collected for patients by a dedicated staff, to ensure that no strain was hospital acquired or healthcare related. Duplicate specimens defined as the same species from the same patient within 3 months from the first positive culture were excluded. Escherichia coli ATCC 25922 was used as the reference strain for quality control in each series. Ethics Participating patients received an information letter and provided written informed consent. This study was approved by the Medical Research Ethics Committee of the Rouen University Hospital (ClinicalTrials.gov identifier: NCT02292160). Susceptibility testing Each strain was first tested against first-line antibiotics by disc diffusion or the automated system, according to French national recommendations (CA-SFM 2016 guidelines, www.sfm-microbiologie.org/). ESBL production was screened for by using the double-disc synergy test.7 ESBLs were genotypically characterized by PCR for bla genes of the CTX-M, SHV and TEM families as previously described.8,9 Temocillin susceptibility testing and definition For each strain, temocillin MIC was determined by agar dilution (AD) as the reference method;10 analysis was performed according to CLSI guidelines with temocillin final concentrations of 2, 3, 4, 6, 8, 12, 16, 24, 32, 48, 64, 96 or 128 mg/L. These reference MIC values were then compared with: (i) susceptibility testing by disc diffusion using discs containing 30 μg of temocillin (Bio-Rad, Marnes-la-Coquette, France) according to CA-SFM 2016 guidelines; (ii) MIC determination by Etest (bioMérieux, Marcy-l’Étoile, France) according to the manufacturer’s recommendations; and (iii) MIC estimation by the AST-N236 card of the Vitek 2 automated system (bioMérieux) according to the manufacturer’s recommendations. Mueller–Hinton agar was used as the medium for AD, disc diffusion and Etest. Susceptibility categorization was determined according to the different clinical breakpoints in use in Europe: susceptible if MIC ≤8, 16 or ≤32 mg/L, corresponding to inhibition diameters for disc diffusion of ≥20, ≥15 or ≥12 mm, respectively.11,12 Discrepancies in susceptibility categorization were classified as follows: a major error when the reference method showed a susceptible result and the commercial method showed a resistant result; or a very major error when the reference method classified an isolate as resistant and the commercial method classified it as susceptible. As the AST-N236 Vitek 2 card provided an MIC range only from ≤4 to >16 mg/L, discrepancies of this method were evaluable only for the 8 and 16 mg/L clinical breakpoints. Statistics Continuous variables are expressed as mean ± SD. Rates of discrepancies in susceptibility categorization were compared between methods using Fisher’s exact test. A P value <0.05 was considered to be significant. Statistical analyses were performed using R software (www.R-project.org/). Results Bacterial strains A total of 762 Enterobacteriaceae (corresponding to 628 patients) were analysed comprising 658 (86.4%) E. coli, 42 (5.5%) Klebsiella spp., 25 (3.3%) Proteus mirabilis, 12 (1.6%) Citrobacter koseri, 12 (1.6%) Enterobacter spp. and 13 (1.7%) miscellaneous species. Thirty-seven (4.9%) isolates were ESBL producers including 30 (81.1%) CTX-M enzymes, 1 (2.7%) SHV enzyme and 3 (8.1%) TEM enzymes. Five isolates were AmpC-hyperproducing strains. Temocillin susceptibility Table 2 shows the susceptibility rate determined by AD and by the three routine tests according to the different clinical breakpoints. Susceptibility rates assessed by AD were 99.6%, 100% and 100% for the 8, 16 and 32 mg/L clinical breakpoints, respectively. Very similar rates were observed by disc diffusion and Etest, while Vitek 2 showed a significantly lower susceptibility rate of the E. coli isolates using a clinical breakpoint of 8 mg/L (94.7% versus 99.7%, P < 0.01). Table 2. Temocillin susceptibility rate of 762 Enterobacteriaceae isolates from community-acquired UTIs by four methods according to the three clinical breakpoints currently in use Bacterial species Susceptibility rate (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L AD disc Etest Vitek 2 AD disc Etest Vitek 2 AD disc Etest Vitek 2 All (n = 762) 99.6 98.9 98.9 95.3a 100 100 98.9 99.7 100 100 100 NE E. coli (n = 658) 99.7 99.1 99.1 94.7a 100 100 99.1 99.8 100 100 100 NE Other Enterobacteriaceae (n = 104) 99.0 98.1 98.1 99.0 100 100 98.1 99.0 100 100 100 NE Bacterial species Susceptibility rate (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L AD disc Etest Vitek 2 AD disc Etest Vitek 2 AD disc Etest Vitek 2 All (n = 762) 99.6 98.9 98.9 95.3a 100 100 98.9 99.7 100 100 100 NE E. coli (n = 658) 99.7 99.1 99.1 94.7a 100 100 99.1 99.8 100 100 100 NE Other Enterobacteriaceae (n = 104) 99.0 98.1 98.1 99.0 100 100 98.1 99.0 100 100 100 NE NE, not evaluable. a P < 0.01 versus AD, disc and Etest (two-by-two comparisons). Table 2. Temocillin susceptibility rate of 762 Enterobacteriaceae isolates from community-acquired UTIs by four methods according to the three clinical breakpoints currently in use Bacterial species Susceptibility rate (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L AD disc Etest Vitek 2 AD disc Etest Vitek 2 AD disc Etest Vitek 2 All (n = 762) 99.6 98.9 98.9 95.3a 100 100 98.9 99.7 100 100 100 NE E. coli (n = 658) 99.7 99.1 99.1 94.7a 100 100 99.1 99.8 100 100 100 NE Other Enterobacteriaceae (n = 104) 99.0 98.1 98.1 99.0 100 100 98.1 99.0 100 100 100 NE Bacterial species Susceptibility rate (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L AD disc Etest Vitek 2 AD disc Etest Vitek 2 AD disc Etest Vitek 2 All (n = 762) 99.6 98.9 98.9 95.3a 100 100 98.9 99.7 100 100 100 NE E. coli (n = 658) 99.7 99.1 99.1 94.7a 100 100 99.1 99.8 100 100 100 NE Other Enterobacteriaceae (n = 104) 99.0 98.1 98.1 99.0 100 100 98.1 99.0 100 100 100 NE NE, not evaluable. a P < 0.01 versus AD, disc and Etest (two-by-two comparisons). Table 3 shows the distribution of MIC values assessed by AD according to bacterial characteristics. A large proportion of E. coli isolates had very low MIC values (26.9% of strains with MIC ≤2 mg/L and 35.9% with MIC of 3 mg/L). This proportion was even more important for other Enterobacteriaceae (69.2% of strains with MIC ≤2 mg/L and 14.4% with MIC of 3 mg/L). Temocillin activity was significantly lower against the ESBL-producing strains. By AD, the susceptibility rate according to the 8 mg/L clinical breakpoint was 94.6% for the 37 ESBL-producing strains compared with 99.9% for the 725 non-ESBL-producing isolates (P < 0.01). Table 3. Distribution of temocillin MIC assessed by AD according to bacterial characteristics Number (%) isolates with temocillin MIC (mg/L) of: ≤2 3 4 6 8 12 16 24 ≥32 All isolates (n = 762) 249 (32.7) 251 (32.9) 133 (17.5) 98 (12.9) 28 (3.7) 1 (0.1) 2 (0.3) _ _ E. coli (n = 658) 177 (26.9) 236 (35.9) 125 (19.0) 93 (14.1) 25 (3.8) 1 (0.2) 1 (0.2) _ _ Other Enterobacteriaceae (n = 104) 72 (69.2) 15 (14.4) 8 (7.7) 5 (4.8) 3 (2.9) _ 1 (1.0) _ _ ESBL-producing isolates (n = 37) 5 (13.5) 8 (21.6) 11 (29.7) 9 (24.3) 2 (5.4) 1 (2.7) 1 (2.7) _ _ Number (%) isolates with temocillin MIC (mg/L) of: ≤2 3 4 6 8 12 16 24 ≥32 All isolates (n = 762) 249 (32.7) 251 (32.9) 133 (17.5) 98 (12.9) 28 (3.7) 1 (0.1) 2 (0.3) _ _ E. coli (n = 658) 177 (26.9) 236 (35.9) 125 (19.0) 93 (14.1) 25 (3.8) 1 (0.2) 1 (0.2) _ _ Other Enterobacteriaceae (n = 104) 72 (69.2) 15 (14.4) 8 (7.7) 5 (4.8) 3 (2.9) _ 1 (1.0) _ _ ESBL-producing isolates (n = 37) 5 (13.5) 8 (21.6) 11 (29.7) 9 (24.3) 2 (5.4) 1 (2.7) 1 (2.7) _ _ Light grey shading, MIC50; dark grey shading MIC90. Table 3. Distribution of temocillin MIC assessed by AD according to bacterial characteristics Number (%) isolates with temocillin MIC (mg/L) of: ≤2 3 4 6 8 12 16 24 ≥32 All isolates (n = 762) 249 (32.7) 251 (32.9) 133 (17.5) 98 (12.9) 28 (3.7) 1 (0.1) 2 (0.3) _ _ E. coli (n = 658) 177 (26.9) 236 (35.9) 125 (19.0) 93 (14.1) 25 (3.8) 1 (0.2) 1 (0.2) _ _ Other Enterobacteriaceae (n = 104) 72 (69.2) 15 (14.4) 8 (7.7) 5 (4.8) 3 (2.9) _ 1 (1.0) _ _ ESBL-producing isolates (n = 37) 5 (13.5) 8 (21.6) 11 (29.7) 9 (24.3) 2 (5.4) 1 (2.7) 1 (2.7) _ _ Number (%) isolates with temocillin MIC (mg/L) of: ≤2 3 4 6 8 12 16 24 ≥32 All isolates (n = 762) 249 (32.7) 251 (32.9) 133 (17.5) 98 (12.9) 28 (3.7) 1 (0.1) 2 (0.3) _ _ E. coli (n = 658) 177 (26.9) 236 (35.9) 125 (19.0) 93 (14.1) 25 (3.8) 1 (0.2) 1 (0.2) _ _ Other Enterobacteriaceae (n = 104) 72 (69.2) 15 (14.4) 8 (7.7) 5 (4.8) 3 (2.9) _ 1 (1.0) _ _ ESBL-producing isolates (n = 37) 5 (13.5) 8 (21.6) 11 (29.7) 9 (24.3) 2 (5.4) 1 (2.7) 1 (2.7) _ _ Light grey shading, MIC50; dark grey shading MIC90. Only 3 of 762 isolates were categorized as resistant according to AD using the 8 mg/L clinical breakpoint: one non-ESBL-producing C. koseri isolate with a temocillin MIC value of 16 mg/L, one ESBL-producing E. coli isolate with a temocillin MIC value of 12 mg/L and one ESBL-producing Enterobacter cloacae isolate with a temocillin MIC value of 16 mg/L. Accuracy of temocillin susceptibility testing Table 4 shows the rate of misclassification in the 762 isolates by the three routine tests compared with AD as the reference. No very major error was noticed for the total of the 6858 comparisons. According to the 8 mg/L clinical breakpoint, the rate of major error was significantly higher for Vitek 2 compared with disc diffusion and Etest (P < 0.01). All the 33 isolates misclassified by Vitek 2 according to the 8 mg/L clinical breakpoint (presented in Table S1, available as Supplementary data at JAC Online) were E. coli, among which only three strains were ESBL-producing isolates. While the temocillin MIC was estimated at 16 mg/L by Vitek 2 for all these 33 isolates, the MIC values varies from 2 to 8 mg/L by AD (mean: 6 mg/L). Only three of the strains misclassified by Vitek 2 were also misclassified by disc diffusion or Etest. Table 4. Rate of error in temocillin susceptibility categorization of 762 Enterobacteriaceae urinary isolates by three routine tests in comparison with the AD method as the reference method Routine test Major error (%) at clinical breakpoint of: Very major error (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L 8 mg/L 16 mg/L 32 mg/L Disc diffusion 0.7 0 0 0 0 0 Etest 0.7 0.1 0 0 0 0 Vitek 2 4.3a 0.3 NE 0 0 NE Routine test Major error (%) at clinical breakpoint of: Very major error (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L 8 mg/L 16 mg/L 32 mg/L Disc diffusion 0.7 0 0 0 0 0 Etest 0.7 0.1 0 0 0 0 Vitek 2 4.3a 0.3 NE 0 0 NE NE, not evaluable. a P < 0.01 versus disc diffusion and Etest. Table 4. Rate of error in temocillin susceptibility categorization of 762 Enterobacteriaceae urinary isolates by three routine tests in comparison with the AD method as the reference method Routine test Major error (%) at clinical breakpoint of: Very major error (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L 8 mg/L 16 mg/L 32 mg/L Disc diffusion 0.7 0 0 0 0 0 Etest 0.7 0.1 0 0 0 0 Vitek 2 4.3a 0.3 NE 0 0 NE Routine test Major error (%) at clinical breakpoint of: Very major error (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L 8 mg/L 16 mg/L 32 mg/L Disc diffusion 0.7 0 0 0 0 0 Etest 0.7 0.1 0 0 0 0 Vitek 2 4.3a 0.3 NE 0 0 NE NE, not evaluable. a P < 0.01 versus disc diffusion and Etest. Precise comparison of MIC values could be achieved only between AD and Etest. As shown in Figure 1 an overall very high correlation was observed with a Pearson’s r = 0.80 (P < 0.001), and very low differences between tests: absolute delta ≤2 mg/L for 95% of the isolates (mean = 0.86 ± 0.04 mg/L). Among the five isolates misclassified as resistant by Etest using the 8 mg/L clinical breakpoint, four were E. coli (of which one was an ESBL-producing isolate) and one was Serratia marcescens; Etest MICs were evaluated at 12 mg/L for these five strains, whereas AD MICs were measured at 8 mg/L for four of them and at 6 mg/L for the other one. Figure 1. View largeDownload slide Distribution of difference between temocillin MIC assessed by Etest and AD. For each strain, the difference (D) between temocillin MICs measured by Etest (MICEtest) and AD (MICAD) was calculated as follows: D = MICEtest − MICAD. A positive value indicates that the temocillin MICEtest was above the temocillin MICAD and a negative value indicates that the temocillin MICEtest was below the temocillin MICAD. Figure 1. View largeDownload slide Distribution of difference between temocillin MIC assessed by Etest and AD. For each strain, the difference (D) between temocillin MICs measured by Etest (MICEtest) and AD (MICAD) was calculated as follows: D = MICEtest − MICAD. A positive value indicates that the temocillin MICEtest was above the temocillin MICAD and a negative value indicates that the temocillin MICEtest was below the temocillin MICAD. Discussion The two major results of this study performed on a large set of Enterobacteriaceae strains from community-acquired UTI were: (i) the high level of temocillin activity; and (ii) the good to excellent accuracy of the three methods routinely available for the susceptibility testing of this antibiotic. The temocillin MIC calculated by the reference method was very low for most Enterobacteriaceae, far below the most stringent critical concentration currently applied in some countries, e.g. France, i.e. 8 mg/L.13 It is noteworthy that the MIC50 and MIC90 observed here for the total set (3 and 6 mg/L) were in accordance with historical studies carried out more than 30 years ago14–17 before the abandonment of the development of this antibiotic. Since resistance of Enterobacteriaceae to most other β-lactam antibiotics has markedly increased during the three last decades, this confirms that temocillin resists hydrolysis by β-lactamases widely produced by community Enterobacteriaceae18–20 and suggests there is no co-selection by mechanisms such as cross-resistance, co-resistance or pleiotropic resistance.21 Nevertheless, two recent studies carried out in the Asia Pacific region and studying both community-acquired and healthcare-associated UTIs found lower temocillin susceptibility rates than our study (89%–93% according to the 8 mg/L clinical breakpoint)22,23 suggesting that our conclusion should be retained only for the community setting. ESBL-producing strains were significantly less susceptible than non-ESBL-producing isolates as found in other work.23,24 In the literature, temocillin susceptibility rates (according to the 8 mg/L clinical breakpoint) of ESBL-producing Enterobacteriaceae vary from 61% to 93%.2–4,24–27 Such discrepancies suggest that other resistance mechanisms (such as altered outer membrane permeability) might be responsible for temocillin resistance.28,29 The second major result is the very good to excellent accuracy of routine susceptibility testing methods for temocillin. Indeed, the concordance scores with the reference method were exceptionally high (i.e. >98%) for disc diffusion and Etest, and still good (i.e. >94%) for Vitek 2. As Vitek 2 is widely used in many laboratories such discrepancies may have clinical impact. Moreover, 30 out of the 33 (91%) isolates misclassified as resistant by Vitek 2 were correctly reported as susceptible by the two other routine methods, as previously reported for another automated system (Phoenix®).6 This finding suggests that disc diffusion or Etest should be run to analyse temocillin susceptibility in resistant isolates assessed by the Vitek 2 method. The overall very good accuracy of routine susceptibility testing methods for temocillin observed in this work might be related to the low proportion (4.9%) of ESBL-producing isolates. This differs from two former studies in which the proportion of ESBL-producing isolates was much higher.5,6 Indeed, Rodriguez-Villalobos et al.5 evaluated the reliability of four commercial methods for a collection of 153 E. coli strains, 58% of which were ESBL-producing strains. According to the 8 mg/L clinical breakpoint, total discrepancy rates for Etest and Vitek 2 AST-N045/N046 cards were 15% and 14%, respectively.5 Similarly, Patel et al.,6 who assessed the accuracy of Etest and the Phoenix automated system for 281 Enterobacteriaceae, 10% of which were ESBL-producing strains, found that the total discrepancy rate was 4% for Etest and 34% for the Phoenix® card (according to the 8 mg/L clinical breakpoint).6 Interestingly, in our work, a vast majority (83%) of community-acquired UTI-causing strains were highly susceptible to temocillin (temocillin MIC ≤4 mg/L) with an MIC far below the lowest clinical breakpoint (i.e. 8 mg/L). This finding indicates that despite issues concerning clinical breakpoints and therapeutic regimen (i.e. 4 or 6 g per day),30 temocillin may be safely used to treat community-acquired UTIs with 4 g per day as 2 g every 12 h would provide a sufficient T0–24>MIC of the free drug concentration in 96% of our isolates.31 This is consistent with a retrospective study involving patients infected by Enterobacteriaceae that found a clinical cure rate of 97% with 2 g of temocillin every 12 h.32 A potential limitation of this study is the relatively low rate of ESBL-producing Enterobacteriaceae and the rarity of temocillin-resistant strains, which may underestimate categorization errors, especially very major errors. Nonetheless, the rate of ESBL-producing strains was in accordance with those reported in other recent works studying strains isolated from community-acquired UTIs in northern Europe and North America.33,34 Therefore, the current work reflects temocillin susceptibility testing performances in daily practice in areas sharing similar epidemiology in terms of community-acquired ESBL-producing Enterobacteriaceae. In conclusion, temocillin showed a high level of activity against Enterobacteriaceae from community-acquired UTIs and good to excellent reliability of routine methods for susceptibility testing in such a setting. Taken together, our results suggest that temocillin could be proposed as a first-line treatment of febrile community-acquired UTIs and as a carbapenem-sparing agent whenever ESBL-producing strains are suspected or confirmed. Funding The epidemiological study on community-acquired UTIs (ClinicalTrials.gov identifier NCT02292160) was funded by a grant from the French Ministry of Health.  Etests were provided without charge by Eumedica and AST-N236 cards were provided without charge by bioMérieux. Transparency declarations K. A. and M. E. each received a travel grant from Eumedica. All other authors: none to declare. Supplementary data Table S1 is available as Supplementary data at JAC Online. References 1 Alexandre K , Fantin B. Pharmacokinetics and pharmacodynamics of temocillin . Clin Pharmacokinet 2018 ; 57 : 287 – 96 . Google Scholar CrossRef Search ADS PubMed 2 Zykov IN , Sundsfjord A , Småbrekke L et al. The antimicrobial activity of mecillinam, nitrofurantoin, temocillin and fosfomycin and comparative analysis of resistance patterns in a nationwide collection of ESBL-producing Escherichia coli in Norway 2010-2011 . Infect Dis (Lond) 2016 ; 48 : 99 – 107 . 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In vitro activity of temocillin against prevalent extended-spectrum β-lactamases producing Enterobacteriaceae from Belgian intensive care units . Eur J Clin Microbiol Infect Dis 2007 ; 26 : 777 – 83 . Google Scholar CrossRef Search ADS PubMed 25 Mischnik A , Baumert P , Hamprecht A et al. Susceptibility to penicillin derivatives among third-generation cephalosporin-resistant Enterobacteriaceae recovered on hospital admission . Diagn Microbiol Infect Dis 2017 ; 87 : 71 – 3 . Google Scholar CrossRef Search ADS PubMed 26 Fournier D , Chirouze C , Leroy J et al. Alternatives to carbapenems in ESBL-producing Escherichia coli infections . Med Mal Infect 2013 ; 43 : 62 – 6 . Google Scholar CrossRef Search ADS PubMed 27 Rodriguez-Villalobos H , Malaviolle V , Frankard J et al. In vitro activity of temocillin against extended spectrum β-lactamase-producing Escherichia coli . J Antimicrob Chemother 2006 ; 57 : 771 – 4 . Google Scholar CrossRef Search ADS PubMed 28 Mutters NT , Zimmermann S , Kaase M et al. Activity of temocillin, mecillinam, ceftazidime, and ceftazidime/avibactam against carbapenem-non-susceptible Enterobacteriaceae without carbapenemase production . Eur J Clin Microbiol Infect Dis 2015 ; 34 : 2429 – 37 . Google Scholar CrossRef Search ADS PubMed 29 Livermore DM , Warner M , Mushtaq S et al. What remains against carbapenem-resistant Enterobacteriaceae? Evaluation of chloramphenicol, ciprofloxacin, colistin, fosfomycin, minocycline, nitrofurantoin, temocillin and tigecycline . Int J Antimicrob Agents 2011 ; 37 : 415 – 9 . Google Scholar CrossRef Search ADS PubMed 30 Laterre P-F , Wittebole X , Van de Velde S et al. Temocillin (6 g daily) in critically ill patients: continuous infusion versus three times daily administration . J Antimicrob Chemother 2015 ; 70 : 891 – 8 . Google Scholar CrossRef Search ADS PubMed 31 De Jongh R , Hens R , Basma V et al. Continuous versus intermittent infusion of temocillin, a directed spectrum penicillin for intensive care patients with nosocomial pneumonia: stability, compatibility, population pharmacokinetic studies and breakpoint selection . J Antimicrob Chemother 2008 ; 61 : 382 – 8 . Google Scholar CrossRef Search ADS PubMed 32 Balakrishnan I , Awad-El-Kariem FM , Aali A et al. Temocillin use in England: clinical and microbiological efficacies in infections caused by extended-spectrum and/or derepressed AmpC β-lactamase-producing Enterobacteriaceae . J Antimicrob Chemother 2011 ; 66 : 2628 – 31 . Google Scholar CrossRef Search ADS PubMed 33 Morrill HJ , Morton JB , Caffrey AR et al. Antimicrobial resistance of Escherichia coli urinary isolates in the Veterans Affairs health care system . Antimicrob Agents Chemother 2017 ; 61 : e02236-16 . Google Scholar CrossRef Search ADS PubMed 34 Rossignol L , Vaux S , Maugat S et al. Incidence of urinary tract infections and antibiotic resistance in the outpatient setting: a cross-sectional study . Infection 2017 ; 45 : 33 – 40 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Temocillin against Enterobacteriaceae isolates from community-acquired urinary tract infections: low rate of resistance and good accuracy of routine susceptibility testing methods

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Abstract

Abstract Background Temocillin is an old ‘revived’ antibiotic that may play an important role in the treatment of febrile urinary tract infection (UTI). Data regarding its activity against current Enterobacteriaceae isolates as well as the performance of routine susceptibility testing methods are, however, scarce. Objectives To determine the MICs of temocillin for Enterobacteriaceae strains reflecting the current epidemiology and to analyse the accuracy of three commercial methods. Methods Enterobacteriaceae isolates causing community-acquired UTI were prospectively collected from September 2015 to January 2017 in two French centres. Temocillin MIC was determined by agar dilution (AD) as the reference method and then compared with: (i) susceptibility testing by disc diffusion; (ii) MIC determination by Etest; and (iii) MIC estimation by the Vitek 2 automated system. Results A total of 762 Enterobacteriaceae were analysed comprising 658 (86.4%) Escherichia coli and 37 (4.9%) ESBL-producing isolates. Susceptibility rate assessed by AD was 99.6% according to the 8 mg/L clinical breakpoint and was significantly lower against the ESBL-producing isolates than the non-ESBL-producing isolates (94.6% versus 99.9%, P < 0.01). The MIC50 and MIC90 for the total set were 3 and 6 mg/L, respectively. According to the 8 mg/L clinical breakpoint, the major error rate was <1% for disc diffusion and Etest, and significantly higher for Vitek 2 (4.3%, P < 0.01), but still low. No very major error was noticed. Conclusions Temocillin showed a high level of activity against Enterobacteriaceae from community-acquired UTI and good to excellent reliability of routine methods for susceptibility testing in such a setting. Introduction Temocillin (6-α-methoxy derivative of ticarcillin) is an old ‘revived’ antibiotic from the penicillin family currently available in several European countries (Belgium, France, Luxembourg and the UK) that has very attractive characteristics: narrow spectrum mainly restricted to Enterobacteriaceae, high resistance to hydrolysis by numerous β-lactamases including ESBL and hyperproduced AmpC, and minimal risk of Clostridium difficile infection. Thus, it may act as an alternative to carbapenems for the treatment of serious Enterobacteriaceae infections such as systemic urinary tract infections (UTIs).1 As for other forgotten antibiotics, some microbiological data are, however, scarce. Studies are still in progress to better define the clinical breakpoints of temocillin, while Enterobacteriaceae are currently categorized susceptible for MIC values ≤8, ≤16 or ≤32 mg/L depending on the country (Table 1).1 If the in vitro activity of temocillin against ESBL-producing isolates has largely been described,2–4 data are paradoxically lacking for non-ESBL-producing isolates still commonly encountered in community-acquired UTIs. Table 1. Clinical breakpoints of temocillin currently in use in different countries where this antibiotic is available Country MIC (mg/L) susceptible resistant Belgiuma ≤16 ≥32 Franceb ≤8 >8 UK, systemic infectionc ≤8 >8 UK, uncomplicated UTIc ≤32 >32 Country MIC (mg/L) susceptible resistant Belgiuma ≤16 ≥32 Franceb ≤8 >8 UK, systemic infectionc ≤8 >8 UK, uncomplicated UTIc ≤32 >32 a See Fuchs et al.11 b See Comité de l’Antibiogramme de la Société Française de Microbiologie.13 c See BSAC.12 Table 1. Clinical breakpoints of temocillin currently in use in different countries where this antibiotic is available Country MIC (mg/L) susceptible resistant Belgiuma ≤16 ≥32 Franceb ≤8 >8 UK, systemic infectionc ≤8 >8 UK, uncomplicated UTIc ≤32 >32 Country MIC (mg/L) susceptible resistant Belgiuma ≤16 ≥32 Franceb ≤8 >8 UK, systemic infectionc ≤8 >8 UK, uncomplicated UTIc ≤32 >32 a See Fuchs et al.11 b See Comité de l’Antibiogramme de la Société Française de Microbiologie.13 c See BSAC.12 Moreover, the performance of the three commercial methods commonly used to determine susceptibility (disc diffusion, Etest and the Vitek 2 automated instrument system) are not well described for the temocillin/Enterobacteriaceae pairs. Two previous studies concerning temocillin have reported marked discrepancies (up to 36%) between routine tests and a reference method,5,6 but these studies were biased by an overrepresentation of strains from healthcare-associated infections. In this context, we aimed to precisely determine the MIC of temocillin for a large set of Enterobacteriaceae strains reflecting the current epidemiology of community-acquired UTIs and to assess the accuracy of disc diffusion, Etest and the Vitek 2 automated instrument system. Materials and methods Bacterial set Enterobacteriaceae isolates causing community-acquired UTIs were prospectively collected from September 2015 to January 2017 in two French centres—a private practice laboratory (analysing ∼4000 urine samples per year) and a teaching hospital—as part of an ongoing epidemiological study on community-acquired UTIs. Identification to the species level was performed routinely using either Vitek 2 or MALDI-TOF technology. Reliable clinical data were prospectively collected for patients by a dedicated staff, to ensure that no strain was hospital acquired or healthcare related. Duplicate specimens defined as the same species from the same patient within 3 months from the first positive culture were excluded. Escherichia coli ATCC 25922 was used as the reference strain for quality control in each series. Ethics Participating patients received an information letter and provided written informed consent. This study was approved by the Medical Research Ethics Committee of the Rouen University Hospital (ClinicalTrials.gov identifier: NCT02292160). Susceptibility testing Each strain was first tested against first-line antibiotics by disc diffusion or the automated system, according to French national recommendations (CA-SFM 2016 guidelines, www.sfm-microbiologie.org/). ESBL production was screened for by using the double-disc synergy test.7 ESBLs were genotypically characterized by PCR for bla genes of the CTX-M, SHV and TEM families as previously described.8,9 Temocillin susceptibility testing and definition For each strain, temocillin MIC was determined by agar dilution (AD) as the reference method;10 analysis was performed according to CLSI guidelines with temocillin final concentrations of 2, 3, 4, 6, 8, 12, 16, 24, 32, 48, 64, 96 or 128 mg/L. These reference MIC values were then compared with: (i) susceptibility testing by disc diffusion using discs containing 30 μg of temocillin (Bio-Rad, Marnes-la-Coquette, France) according to CA-SFM 2016 guidelines; (ii) MIC determination by Etest (bioMérieux, Marcy-l’Étoile, France) according to the manufacturer’s recommendations; and (iii) MIC estimation by the AST-N236 card of the Vitek 2 automated system (bioMérieux) according to the manufacturer’s recommendations. Mueller–Hinton agar was used as the medium for AD, disc diffusion and Etest. Susceptibility categorization was determined according to the different clinical breakpoints in use in Europe: susceptible if MIC ≤8, 16 or ≤32 mg/L, corresponding to inhibition diameters for disc diffusion of ≥20, ≥15 or ≥12 mm, respectively.11,12 Discrepancies in susceptibility categorization were classified as follows: a major error when the reference method showed a susceptible result and the commercial method showed a resistant result; or a very major error when the reference method classified an isolate as resistant and the commercial method classified it as susceptible. As the AST-N236 Vitek 2 card provided an MIC range only from ≤4 to >16 mg/L, discrepancies of this method were evaluable only for the 8 and 16 mg/L clinical breakpoints. Statistics Continuous variables are expressed as mean ± SD. Rates of discrepancies in susceptibility categorization were compared between methods using Fisher’s exact test. A P value <0.05 was considered to be significant. Statistical analyses were performed using R software (www.R-project.org/). Results Bacterial strains A total of 762 Enterobacteriaceae (corresponding to 628 patients) were analysed comprising 658 (86.4%) E. coli, 42 (5.5%) Klebsiella spp., 25 (3.3%) Proteus mirabilis, 12 (1.6%) Citrobacter koseri, 12 (1.6%) Enterobacter spp. and 13 (1.7%) miscellaneous species. Thirty-seven (4.9%) isolates were ESBL producers including 30 (81.1%) CTX-M enzymes, 1 (2.7%) SHV enzyme and 3 (8.1%) TEM enzymes. Five isolates were AmpC-hyperproducing strains. Temocillin susceptibility Table 2 shows the susceptibility rate determined by AD and by the three routine tests according to the different clinical breakpoints. Susceptibility rates assessed by AD were 99.6%, 100% and 100% for the 8, 16 and 32 mg/L clinical breakpoints, respectively. Very similar rates were observed by disc diffusion and Etest, while Vitek 2 showed a significantly lower susceptibility rate of the E. coli isolates using a clinical breakpoint of 8 mg/L (94.7% versus 99.7%, P < 0.01). Table 2. Temocillin susceptibility rate of 762 Enterobacteriaceae isolates from community-acquired UTIs by four methods according to the three clinical breakpoints currently in use Bacterial species Susceptibility rate (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L AD disc Etest Vitek 2 AD disc Etest Vitek 2 AD disc Etest Vitek 2 All (n = 762) 99.6 98.9 98.9 95.3a 100 100 98.9 99.7 100 100 100 NE E. coli (n = 658) 99.7 99.1 99.1 94.7a 100 100 99.1 99.8 100 100 100 NE Other Enterobacteriaceae (n = 104) 99.0 98.1 98.1 99.0 100 100 98.1 99.0 100 100 100 NE Bacterial species Susceptibility rate (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L AD disc Etest Vitek 2 AD disc Etest Vitek 2 AD disc Etest Vitek 2 All (n = 762) 99.6 98.9 98.9 95.3a 100 100 98.9 99.7 100 100 100 NE E. coli (n = 658) 99.7 99.1 99.1 94.7a 100 100 99.1 99.8 100 100 100 NE Other Enterobacteriaceae (n = 104) 99.0 98.1 98.1 99.0 100 100 98.1 99.0 100 100 100 NE NE, not evaluable. a P < 0.01 versus AD, disc and Etest (two-by-two comparisons). Table 2. Temocillin susceptibility rate of 762 Enterobacteriaceae isolates from community-acquired UTIs by four methods according to the three clinical breakpoints currently in use Bacterial species Susceptibility rate (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L AD disc Etest Vitek 2 AD disc Etest Vitek 2 AD disc Etest Vitek 2 All (n = 762) 99.6 98.9 98.9 95.3a 100 100 98.9 99.7 100 100 100 NE E. coli (n = 658) 99.7 99.1 99.1 94.7a 100 100 99.1 99.8 100 100 100 NE Other Enterobacteriaceae (n = 104) 99.0 98.1 98.1 99.0 100 100 98.1 99.0 100 100 100 NE Bacterial species Susceptibility rate (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L AD disc Etest Vitek 2 AD disc Etest Vitek 2 AD disc Etest Vitek 2 All (n = 762) 99.6 98.9 98.9 95.3a 100 100 98.9 99.7 100 100 100 NE E. coli (n = 658) 99.7 99.1 99.1 94.7a 100 100 99.1 99.8 100 100 100 NE Other Enterobacteriaceae (n = 104) 99.0 98.1 98.1 99.0 100 100 98.1 99.0 100 100 100 NE NE, not evaluable. a P < 0.01 versus AD, disc and Etest (two-by-two comparisons). Table 3 shows the distribution of MIC values assessed by AD according to bacterial characteristics. A large proportion of E. coli isolates had very low MIC values (26.9% of strains with MIC ≤2 mg/L and 35.9% with MIC of 3 mg/L). This proportion was even more important for other Enterobacteriaceae (69.2% of strains with MIC ≤2 mg/L and 14.4% with MIC of 3 mg/L). Temocillin activity was significantly lower against the ESBL-producing strains. By AD, the susceptibility rate according to the 8 mg/L clinical breakpoint was 94.6% for the 37 ESBL-producing strains compared with 99.9% for the 725 non-ESBL-producing isolates (P < 0.01). Table 3. Distribution of temocillin MIC assessed by AD according to bacterial characteristics Number (%) isolates with temocillin MIC (mg/L) of: ≤2 3 4 6 8 12 16 24 ≥32 All isolates (n = 762) 249 (32.7) 251 (32.9) 133 (17.5) 98 (12.9) 28 (3.7) 1 (0.1) 2 (0.3) _ _ E. coli (n = 658) 177 (26.9) 236 (35.9) 125 (19.0) 93 (14.1) 25 (3.8) 1 (0.2) 1 (0.2) _ _ Other Enterobacteriaceae (n = 104) 72 (69.2) 15 (14.4) 8 (7.7) 5 (4.8) 3 (2.9) _ 1 (1.0) _ _ ESBL-producing isolates (n = 37) 5 (13.5) 8 (21.6) 11 (29.7) 9 (24.3) 2 (5.4) 1 (2.7) 1 (2.7) _ _ Number (%) isolates with temocillin MIC (mg/L) of: ≤2 3 4 6 8 12 16 24 ≥32 All isolates (n = 762) 249 (32.7) 251 (32.9) 133 (17.5) 98 (12.9) 28 (3.7) 1 (0.1) 2 (0.3) _ _ E. coli (n = 658) 177 (26.9) 236 (35.9) 125 (19.0) 93 (14.1) 25 (3.8) 1 (0.2) 1 (0.2) _ _ Other Enterobacteriaceae (n = 104) 72 (69.2) 15 (14.4) 8 (7.7) 5 (4.8) 3 (2.9) _ 1 (1.0) _ _ ESBL-producing isolates (n = 37) 5 (13.5) 8 (21.6) 11 (29.7) 9 (24.3) 2 (5.4) 1 (2.7) 1 (2.7) _ _ Light grey shading, MIC50; dark grey shading MIC90. Table 3. Distribution of temocillin MIC assessed by AD according to bacterial characteristics Number (%) isolates with temocillin MIC (mg/L) of: ≤2 3 4 6 8 12 16 24 ≥32 All isolates (n = 762) 249 (32.7) 251 (32.9) 133 (17.5) 98 (12.9) 28 (3.7) 1 (0.1) 2 (0.3) _ _ E. coli (n = 658) 177 (26.9) 236 (35.9) 125 (19.0) 93 (14.1) 25 (3.8) 1 (0.2) 1 (0.2) _ _ Other Enterobacteriaceae (n = 104) 72 (69.2) 15 (14.4) 8 (7.7) 5 (4.8) 3 (2.9) _ 1 (1.0) _ _ ESBL-producing isolates (n = 37) 5 (13.5) 8 (21.6) 11 (29.7) 9 (24.3) 2 (5.4) 1 (2.7) 1 (2.7) _ _ Number (%) isolates with temocillin MIC (mg/L) of: ≤2 3 4 6 8 12 16 24 ≥32 All isolates (n = 762) 249 (32.7) 251 (32.9) 133 (17.5) 98 (12.9) 28 (3.7) 1 (0.1) 2 (0.3) _ _ E. coli (n = 658) 177 (26.9) 236 (35.9) 125 (19.0) 93 (14.1) 25 (3.8) 1 (0.2) 1 (0.2) _ _ Other Enterobacteriaceae (n = 104) 72 (69.2) 15 (14.4) 8 (7.7) 5 (4.8) 3 (2.9) _ 1 (1.0) _ _ ESBL-producing isolates (n = 37) 5 (13.5) 8 (21.6) 11 (29.7) 9 (24.3) 2 (5.4) 1 (2.7) 1 (2.7) _ _ Light grey shading, MIC50; dark grey shading MIC90. Only 3 of 762 isolates were categorized as resistant according to AD using the 8 mg/L clinical breakpoint: one non-ESBL-producing C. koseri isolate with a temocillin MIC value of 16 mg/L, one ESBL-producing E. coli isolate with a temocillin MIC value of 12 mg/L and one ESBL-producing Enterobacter cloacae isolate with a temocillin MIC value of 16 mg/L. Accuracy of temocillin susceptibility testing Table 4 shows the rate of misclassification in the 762 isolates by the three routine tests compared with AD as the reference. No very major error was noticed for the total of the 6858 comparisons. According to the 8 mg/L clinical breakpoint, the rate of major error was significantly higher for Vitek 2 compared with disc diffusion and Etest (P < 0.01). All the 33 isolates misclassified by Vitek 2 according to the 8 mg/L clinical breakpoint (presented in Table S1, available as Supplementary data at JAC Online) were E. coli, among which only three strains were ESBL-producing isolates. While the temocillin MIC was estimated at 16 mg/L by Vitek 2 for all these 33 isolates, the MIC values varies from 2 to 8 mg/L by AD (mean: 6 mg/L). Only three of the strains misclassified by Vitek 2 were also misclassified by disc diffusion or Etest. Table 4. Rate of error in temocillin susceptibility categorization of 762 Enterobacteriaceae urinary isolates by three routine tests in comparison with the AD method as the reference method Routine test Major error (%) at clinical breakpoint of: Very major error (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L 8 mg/L 16 mg/L 32 mg/L Disc diffusion 0.7 0 0 0 0 0 Etest 0.7 0.1 0 0 0 0 Vitek 2 4.3a 0.3 NE 0 0 NE Routine test Major error (%) at clinical breakpoint of: Very major error (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L 8 mg/L 16 mg/L 32 mg/L Disc diffusion 0.7 0 0 0 0 0 Etest 0.7 0.1 0 0 0 0 Vitek 2 4.3a 0.3 NE 0 0 NE NE, not evaluable. a P < 0.01 versus disc diffusion and Etest. Table 4. Rate of error in temocillin susceptibility categorization of 762 Enterobacteriaceae urinary isolates by three routine tests in comparison with the AD method as the reference method Routine test Major error (%) at clinical breakpoint of: Very major error (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L 8 mg/L 16 mg/L 32 mg/L Disc diffusion 0.7 0 0 0 0 0 Etest 0.7 0.1 0 0 0 0 Vitek 2 4.3a 0.3 NE 0 0 NE Routine test Major error (%) at clinical breakpoint of: Very major error (%) at clinical breakpoint of: 8 mg/L 16 mg/L 32 mg/L 8 mg/L 16 mg/L 32 mg/L Disc diffusion 0.7 0 0 0 0 0 Etest 0.7 0.1 0 0 0 0 Vitek 2 4.3a 0.3 NE 0 0 NE NE, not evaluable. a P < 0.01 versus disc diffusion and Etest. Precise comparison of MIC values could be achieved only between AD and Etest. As shown in Figure 1 an overall very high correlation was observed with a Pearson’s r = 0.80 (P < 0.001), and very low differences between tests: absolute delta ≤2 mg/L for 95% of the isolates (mean = 0.86 ± 0.04 mg/L). Among the five isolates misclassified as resistant by Etest using the 8 mg/L clinical breakpoint, four were E. coli (of which one was an ESBL-producing isolate) and one was Serratia marcescens; Etest MICs were evaluated at 12 mg/L for these five strains, whereas AD MICs were measured at 8 mg/L for four of them and at 6 mg/L for the other one. Figure 1. View largeDownload slide Distribution of difference between temocillin MIC assessed by Etest and AD. For each strain, the difference (D) between temocillin MICs measured by Etest (MICEtest) and AD (MICAD) was calculated as follows: D = MICEtest − MICAD. A positive value indicates that the temocillin MICEtest was above the temocillin MICAD and a negative value indicates that the temocillin MICEtest was below the temocillin MICAD. Figure 1. View largeDownload slide Distribution of difference between temocillin MIC assessed by Etest and AD. For each strain, the difference (D) between temocillin MICs measured by Etest (MICEtest) and AD (MICAD) was calculated as follows: D = MICEtest − MICAD. A positive value indicates that the temocillin MICEtest was above the temocillin MICAD and a negative value indicates that the temocillin MICEtest was below the temocillin MICAD. Discussion The two major results of this study performed on a large set of Enterobacteriaceae strains from community-acquired UTI were: (i) the high level of temocillin activity; and (ii) the good to excellent accuracy of the three methods routinely available for the susceptibility testing of this antibiotic. The temocillin MIC calculated by the reference method was very low for most Enterobacteriaceae, far below the most stringent critical concentration currently applied in some countries, e.g. France, i.e. 8 mg/L.13 It is noteworthy that the MIC50 and MIC90 observed here for the total set (3 and 6 mg/L) were in accordance with historical studies carried out more than 30 years ago14–17 before the abandonment of the development of this antibiotic. Since resistance of Enterobacteriaceae to most other β-lactam antibiotics has markedly increased during the three last decades, this confirms that temocillin resists hydrolysis by β-lactamases widely produced by community Enterobacteriaceae18–20 and suggests there is no co-selection by mechanisms such as cross-resistance, co-resistance or pleiotropic resistance.21 Nevertheless, two recent studies carried out in the Asia Pacific region and studying both community-acquired and healthcare-associated UTIs found lower temocillin susceptibility rates than our study (89%–93% according to the 8 mg/L clinical breakpoint)22,23 suggesting that our conclusion should be retained only for the community setting. ESBL-producing strains were significantly less susceptible than non-ESBL-producing isolates as found in other work.23,24 In the literature, temocillin susceptibility rates (according to the 8 mg/L clinical breakpoint) of ESBL-producing Enterobacteriaceae vary from 61% to 93%.2–4,24–27 Such discrepancies suggest that other resistance mechanisms (such as altered outer membrane permeability) might be responsible for temocillin resistance.28,29 The second major result is the very good to excellent accuracy of routine susceptibility testing methods for temocillin. Indeed, the concordance scores with the reference method were exceptionally high (i.e. >98%) for disc diffusion and Etest, and still good (i.e. >94%) for Vitek 2. As Vitek 2 is widely used in many laboratories such discrepancies may have clinical impact. Moreover, 30 out of the 33 (91%) isolates misclassified as resistant by Vitek 2 were correctly reported as susceptible by the two other routine methods, as previously reported for another automated system (Phoenix®).6 This finding suggests that disc diffusion or Etest should be run to analyse temocillin susceptibility in resistant isolates assessed by the Vitek 2 method. The overall very good accuracy of routine susceptibility testing methods for temocillin observed in this work might be related to the low proportion (4.9%) of ESBL-producing isolates. This differs from two former studies in which the proportion of ESBL-producing isolates was much higher.5,6 Indeed, Rodriguez-Villalobos et al.5 evaluated the reliability of four commercial methods for a collection of 153 E. coli strains, 58% of which were ESBL-producing strains. According to the 8 mg/L clinical breakpoint, total discrepancy rates for Etest and Vitek 2 AST-N045/N046 cards were 15% and 14%, respectively.5 Similarly, Patel et al.,6 who assessed the accuracy of Etest and the Phoenix automated system for 281 Enterobacteriaceae, 10% of which were ESBL-producing strains, found that the total discrepancy rate was 4% for Etest and 34% for the Phoenix® card (according to the 8 mg/L clinical breakpoint).6 Interestingly, in our work, a vast majority (83%) of community-acquired UTI-causing strains were highly susceptible to temocillin (temocillin MIC ≤4 mg/L) with an MIC far below the lowest clinical breakpoint (i.e. 8 mg/L). This finding indicates that despite issues concerning clinical breakpoints and therapeutic regimen (i.e. 4 or 6 g per day),30 temocillin may be safely used to treat community-acquired UTIs with 4 g per day as 2 g every 12 h would provide a sufficient T0–24>MIC of the free drug concentration in 96% of our isolates.31 This is consistent with a retrospective study involving patients infected by Enterobacteriaceae that found a clinical cure rate of 97% with 2 g of temocillin every 12 h.32 A potential limitation of this study is the relatively low rate of ESBL-producing Enterobacteriaceae and the rarity of temocillin-resistant strains, which may underestimate categorization errors, especially very major errors. Nonetheless, the rate of ESBL-producing strains was in accordance with those reported in other recent works studying strains isolated from community-acquired UTIs in northern Europe and North America.33,34 Therefore, the current work reflects temocillin susceptibility testing performances in daily practice in areas sharing similar epidemiology in terms of community-acquired ESBL-producing Enterobacteriaceae. In conclusion, temocillin showed a high level of activity against Enterobacteriaceae from community-acquired UTIs and good to excellent reliability of routine methods for susceptibility testing in such a setting. Taken together, our results suggest that temocillin could be proposed as a first-line treatment of febrile community-acquired UTIs and as a carbapenem-sparing agent whenever ESBL-producing strains are suspected or confirmed. Funding The epidemiological study on community-acquired UTIs (ClinicalTrials.gov identifier NCT02292160) was funded by a grant from the French Ministry of Health.  Etests were provided without charge by Eumedica and AST-N236 cards were provided without charge by bioMérieux. Transparency declarations K. A. and M. E. each received a travel grant from Eumedica. All other authors: none to declare. Supplementary data Table S1 is available as Supplementary data at JAC Online. References 1 Alexandre K , Fantin B. Pharmacokinetics and pharmacodynamics of temocillin . Clin Pharmacokinet 2018 ; 57 : 287 – 96 . Google Scholar CrossRef Search ADS PubMed 2 Zykov IN , Sundsfjord A , Småbrekke L et al. The antimicrobial activity of mecillinam, nitrofurantoin, temocillin and fosfomycin and comparative analysis of resistance patterns in a nationwide collection of ESBL-producing Escherichia coli in Norway 2010-2011 . Infect Dis (Lond) 2016 ; 48 : 99 – 107 . 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Incidence of urinary tract infections and antibiotic resistance in the outpatient setting: a cross-sectional study . Infection 2017 ; 45 : 33 – 40 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Apr 6, 2018

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