EUCAST Methodology Research Articles

Efficacy of Pivmecillinam for Treatment of Lower Urinary Tract Infection Caused by Extended-Spectrum β-Lactamase–Producing Escherichia coli and Klebsiella pneumoniae

To evaluate the clinical and bacteriological efficacy of pivmecillinam against lower urinary tract infection (UTI) caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae, patients treated for lower UTI with pivmecillinam (n=8) were studied. Patients treated with nitrofurantoin (n=3) and trimethoprim (n=3) or a combination of these agents with pivmecillinam (n=3) were included as a control group. Antimicrobial susceptibility was determined with EUCAST methodology. Bacteriologic cure was defined as <10(3) CFU/ml at follow-up (30 days), and clinical cure as resolved UTI symptoms after completed treatment. All patients receiving pivmecillinam had good clinical response (8/8), but bacteriological cure rates were low (2/8). However, none of the patients with persisting bacteriuria had a relapse of UTI symptoms within 6 months. All isolates were susceptible to the given antimicrobial. Most isolates belonged to the CTX-M-1 group (n=11, 65%) or CTX-M-9-group (n=4, 24%). Four E. coli isolates belonged to the international clone O25b-ST131 (25%). In conclusion, pivmecillinam had good clinical activity against lower UTI caused by ESBL-producing Enterobacteriaceae, but bacteriological cure rates were low. The persistent bacteriuria appears to be of little clinical importance, but larger clinical studies are needed to determine the usefulness of pivmecillinam in infections caused by ESBL-producing bacteria.

Wild-type MIC distribution and epidemiological cut-off values in clinical Legionella pneumophila serogroup 1 isolates

ObjectivesThe purpose of this study was to establish wild-type (WT) distributions and determine the epidemiological cut-off values (ECOFF) in clinical L. pneumophila serogroup 1 isolates for 10 antimicrobials commonly used for the treatment of Legionella infections using a method feasible in a routine clinical laboratory. MethodsMICs of 183 clinical L. pneumophila serogroup 1 isolates, collected as part of an outbreak detection program, were tested using E-test methodology on buffered charcoal yeast extract agar supplemented with α-ketoglutarate (BCYE-α). The MICs were read after 2 days of incubation at 35 °C with increased humidity and without CO2. ECOFFs were determined according to EUCAST methodology and expressed as WT ≤ X mg/L. ResultsAll antimicrobials showed a WT distribution, although the width varied from 2 two-fold dilutions to 8 dilutions, depending on antibiotic class. The ECOFFs determined were 1.0 mg/L for ciprofloxacin, 0.50 mg/L for levofloxacin, 1.0 mg/L for moxifloxacin, 1.0 mg/L for erythromycin, 1.0 mg/L for azithromycin, 0.50 mg/L for clarithromycin, 1.0 mg/L for cefotaxime, 0.032 mg/L for rifampicin, 16 mg/L for tigecycline, and 8 mg/L for doxycycline. ConclusionAll isolates were inhibited by low concentrations of the fluoroquinolones and macrolides tested, with somewhat higher MICs for the fluoroquinolones. Rifampicin was found to be the most active against L. pneumophila isolates in vitro. These data can be used as a reference for the detection of resistance in clinical L. pneumophila isolates and as a setting of clinical breakpoints.

In vitro activity of CXA-101 plus tazobactam (CXA-201) against CTX-M-14– and CTX-M-15–producing Escherichia coli and Klebsiella pneumoniae

CXA-101, a novel cephalosporin with good antipseudomonal activity, was evaluated against a consecutive and polyclonal collection of extended-spectrum-β-lactamase (ESBL)–producing Escherichia coli (n = 149) and Klebsiella pneumoniae (n = 20), mainly CTX-M-15- (69%) or CTX-M-14 producing (22%). A total of 41% of the E. coli isolates belonged to the international clone O25b-ST131. Broth microdilution versus CXA-101, CXA-tazobactam 4 and 8 mg/L (CXA-201), ceftazidime–tazobactam (CAT), ceftazidime–clavulanate (CAC), piperacillin–tazobactam (TZP), amoxicillin–clavulanate (ACL), ampicillin–sulbactam (ASU), and other comparators was performed, using EUCAST methodology and breakpoints. Susceptibility to CXA-201 was 96% (tazobactam 8 mg/L, tentative breakpoint S ≤ 1 mg/L), CAT 93%, CAC 95%, ACL 24%, ASU 2%, TZP 58%, ciprofloxacin 25%, levofloxacin 30%, gentamicin 54%, tobramycin 34%, amikacin 90%, and tigecycline 98%. Ninety-four percent of the TZP-resistant and all ACL-resistant isolates were CXA-201 susceptible. CXA-201 has good in vitro activity against ESBL-producing Enterobacteriaceae and might be a future therapeutic option for infections caused by TZP- and ACL-resistant isolates.

Recent Exposure to Caspofungin or Fluconazole Influences the Epidemiology of Candidemia: a Prospective Multicenter Study Involving 2,441 Patients

A prospective multicenter surveillance program on yeast bloodstream infections was implemented in the Paris, France, area without restrictions on ward of hospitalization (intensive care unit, hematology, and surgery) or age (adults and children). The present analysis concerns 2,618 isolates collected over 7 years from 2,441 patients. Centralized species identification and antifungal susceptibility testing using the EUCAST methodology were performed. Almost 10% (232/2,441) of the patients had recently (≤30 days) been treated with antifungal drugs. We analyzed the effect of recent exposure to fluconazole (n = 159) or caspofungin (n = 61) on the proportions of the five major Candida species. For both drugs, preexposure was associated with a decreased prevalence of Candida albicans in favor of less drug-susceptible species (C. glabrata and C. krusei for the former and C. parapsilosis and, to a lesser extent, C. glabrata and C. krusei for the latter; P = 0.001). In the multivariate analysis, the risk of being infected with an isolate with decreased susceptibility to fluconazole was independently associated with an age of ≥15 years (odds ratio [OR] = 2.45; 95% confidence interval [CI] = 1.39 to 4.31; P = 0.002) and with recent exposure to fluconazole (OR = 2.17; 95% CI = 1.51 to 3.13; P < 0.001), while the risk of being infected with an isolate with decreased susceptibility to caspofungin was independently associated with an age <15 years (OR = 2.53; 95% CI = 1.43 to 4.48; P = 0.001) and with recent exposure to caspofungin (OR = 4.79; 95% CI = 2.47 to 9.28; P < 0.001). These findings could influence future recommendations for the management of candidemia.

Antifungal Susceptibility Profile of Human-Pathogenic Species of Lichtheimia

Forty-four isolates belonging to human pathogenic species of Lichtheimia were tested against nine antifungal agents by using the EUCAST methodology. No remarkable differences were found between the clinical species, although L. ramosa showed slightly higher MICs for all drugs. Amphotericin B was the most active drug. Among azole drugs, posaconazole had the best activity in vitro and voriconazole was inactive. Echinocandins showed activity for some isolates, suggesting a potential role in combination therapy.

Estudo comparativo entre as metodologias preconizadas pelo CLSI e pelo EUCAST para avaliação da atividade antifúngica

The international organizations CLSI and EUCAST developed reference methodologies for activity evaluation antifungal. The aim of this work was to compare the recommended methodologies by the CLSI and EUCAST in the antifungal activity evaluation of crude extracts of Azadirachta indica and green propolis. The results showed that the MIC values determined by the EUCAST methodology were smaller than that determined by the CLSI. Nevertheless, both methodologies were satisfactory to detect and evaluate antifungal activity of the crude extracts and isolated compounds. The EUCAST methodology showed advantage by making possible to obtain results in less time.

Breakthrough Aspergillus fumigatus and Candida albicans Double Infection during Caspofungin Treatment: Laboratory Characteristics and Implication for Susceptibility Testing

Caspofungin is used for the treatment of acute invasive candidiasis and as salvage treatment for invasive aspergillosis. We report characteristics of isolates of Candida albicans and Aspergillus fumigatus detected in a patient with breakthrough infection complicating severe gastrointestinal surgery and evaluate the capability of susceptibility methods to identify candin resistance. The susceptibility of C. albicans to caspofungin and anidulafungin was investigated by Etest, microdilution (European Committee on Antibiotic Susceptibility Testing [EUCAST] and CLSI), disk diffusion, agar dilution, and FKS1 sequencing and in a mouse model. Tissue was examined by immunohistochemistry, PCR, and sequencing for the presence of A. fumigatus and resistance mutations. The MICs for the C. albicans isolate were as follows: >32 microg/ml caspofungin and 0.5 microg/ml anidulafungin by Etest, 2 microg/ml caspofungin and 0.125 microg/ml anidulafungin by EUCAST methods, and 1 microg/ml caspofungin and 0.5 microg/ml anidulafungin by CLSI methods. Sequencing of the FKS1 gene revealed a mutation leading to an S645P substitution. Caspofungin and anidulafungin failed to reduce kidney CFU counts in animals inoculated with this isolate (P > 0.05 compared to untreated control animals), while both candins completely sterilized the kidneys in animals infected with a control isolate. Disk diffusion and agar dilution methods clearly separated the two isolates. Immunohistochemistry and sequencing confirmed the presence of A. fumigatus without FSK1 resistance mutations in liver and lung tissues. Breakthrough disseminated aspergillosis and candidiasis developed despite an absence of characteristic FKS1 resistance mutations in the Aspergillus isolates. EUCAST and CLSI methodology did not separate the candin-resistant clinical isolate from the sensitive control isolate as well as did the Etest and agar methods.

Multicentre determination of quality control strains and quality control ranges for antifungal susceptibility testing of yeasts and filamentous fungi using the methods of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST)

A multicentre study involving seven laboratories was performed using techniques recommended by the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST) to evaluate and propose quality control ranges and strains for susceptibility testing of fermentative yeasts and filamentous fungi. Participating laboratories tested the susceptibilities of a panel of 12 encoded isolates to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and posaconazole. In total, 15 lots of assay medium were tested, with one lot being common to all laboratories, and 18 144 MIC values were determined. Intra- and inter-laboratory agreements and intra-class correlation coefficients (ICCs) of the results for each drug/strain/lot combination were calculated. An average value of 85% agreement was selected for validation purposes. The average percentage of intra-laboratory agreement was 90-95%, with ICC values of 0.90-0.95 (p <0.01). Inter-laboratory reproducibility was also high, with 92% agreement and an ICC of 0.97 (p <0.01). The reproducibility was somewhat better with the common lot of assay medium (96% agreement) than with the different lots (91% agreement), but this difference was not significant. Two isolates that showed trailing growth had agreement percentages below the 85% limit selected for validation purposes and were therefore excluded from the panel of quality control strains. The recommended EUCAST methodologies were found to be highly reproducible and reliable for susceptibility testing of yeasts and filamentous fungi. Ten isolates are proposed for use as quality control strains with these EUCAST procedures.

P1812 In vitro activity of tigecycline against pathogens from Turkey –T.E.S.T. Program 2006

CXA-101, a novel cephalosporin with good antipseudomonal activity, was evaluated against a consecutive and polyclonal collection of extended-spectrum-β-lactamase (ESBL)–producing Escherichia coli (n = 149) and Klebsiella pneumoniae (n = 20), mainly CTX-M-15- (69%) or CTX-M-14 producing (22%). A total of 41% of the E. coli isolates belonged to the international clone O25b-ST131. Broth microdilution versus CXA-101, CXA-tazobactam 4 and 8 mg/L (CXA-201), ceftazidime–tazobactam (CAT), ceftazidime–clavulanate (CAC), piperacillin–tazobactam (TZP), amoxicillin–clavulanate (ACL), ampicillin–sulbactam (ASU), and other comparators was performed, using EUCAST methodology and breakpoints. Susceptibility to CXA-201 was 96% (tazobactam 8 mg/L, tentative breakpoint S ≤ 1 mg/L), CAT 93%, CAC 95%, ACL 24%, ASU 2%, TZP 58%, ciprofloxacin 25%, levofloxacin 30%, gentamicin 54%, tobramycin 34%, amikacin 90%, and tigecycline 98%. Ninety-four percent of the TZP-resistant and all ACL-resistant isolates were CXA-201 susceptible. CXA-201 has good in vitro activity against ESBL-producing Enterobacteriaceae and might be a future therapeutic option for infections caused by TZP- and ACL-resistant isolates.