European Committee On Antimicrobial Susceptibility Testing Breakpoints Research Articles

Impact of EUCAST ceftaroline breakpoint change on the susceptibility of methicillin-resistant Staphylococcus aureus isolates collected from patients with complicated skin and soft-tissue infections

ObjectivesIn 2018, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) introduced an intermediate breakpoint for ceftaroline against Staphylococcus aureus. The objective of this study was to compare data on resistance to ceftaroline among methicillin-resistant S. aureus (MRSA) isolates using versions 7.1 (March 2017) and 8.0 (January 2018) of the EUCAST breakpoints. MethodsParticipating centers were located in Africa, Asia, Europe, Oceania and South America. Isolates were collected from patients with complicated skin and soft-tissue infections and were cultured from integumentary sources. Methicillin resistance among S. aureus was confirmed locally using the oxacillin method. The CLSI broth microdilution method was used to measure ceftaroline MICs at the central laboratory. Versions 7.1 and 8.0 of the EUCAST breakpoints were used to interpret MIC data. ResultsBetween 2015 and 2016, 9559 isolates of S. aureus were collected, of which 5566 (58.2%) isolates were MRSA. Overall, the lowest rate of MRSA was in Asia (56.5%; 705/1247) and the highest rate was in Oceania (62.7%; 299/477). Using version 7.1 of the EUCAST breakpoints, 4.5% (250/5566) of all MRSA isolates were resistant to ceftaroline and when version 8.0 of the breakpoints was applied, 4.2% (235/5566) of MRSA were in the intermediate category and 0.3% (15/5566) of all isolates were considered resistant. ConclusionsBy applying version 8.0 of the EUCAST breakpoints, the majority of MRSA isolates that were resistant are now in the intermediate category for ceftaroline. Ceftaroline resistance among MRSA now appears rare.

The association between treatment appropriateness according to EUCAST and CLSI breakpoints and mortality among patients with candidemia: a retrospective observational study.

To evaluate the association between appropriate antifungal treatment and mortality among patients with candidemia using different breakpoint definitions. In a retrospective study, we included all adults with candidemia in a tertiary center between 2009 and 2015. We defined three versions of appropriate (covering) antifungal treatment, according to Clinical and Laboratory Standards Institute (CLSI) 2008, CLSI 2012, and European Committee on Antimicrobial Susceptibility Testing (EUCAST) (2017 update) breakpoints. For empiric treatment, we evaluated the association with 30-day mortality. For definitive treatment, we evaluated the association with 90-day mortality among patients surviving the first week after candidemia onset. Adjusted odds ratios (OR) from a bivariate logistic regression with 95% confidence intervals are reported. We identified 302 patients with 308 separate candidemia episodes. The crude 30-day mortality was 55% (168/308). Resistance to anidulafungin increased from 3.5 to 51.6% and to fluconazole from 15.2 to 44.1%, when applying CLSI 2008 and EUCAST definitions, respectively. Appropriate empirical treatment was significantly associated with lower 30-day mortality using the CLSI 2008 definitions, adjusted OR 0.56 (0.33-0.96). The associations were similar, though not statistically significant for EUCAST, 0.58 (0.33-1.00), and CLSI 2012, OR 0.62 (0.37-1.04). Appropriate definitive treatment according to CLSI 2012 and EUCAST was independently associated with lower 90-day mortality, ORs 0.31 (0.13-0.75) and 0.44 (0.23-0.8), respectively. With CLSI 2008, the association was similar but not statistically significant, OR 0.4 (0.11-1.41), with few isolates classified as resistant. Considering the major shift in resistance prevalence when applying CLSI 2008, CLSI 2012, and EUCAST breakpoint definitions, no major differences were observed in their association with mortality.

Population pharmacokinetics and pharmacodynamics of fosfomycin in non–critically ill patients with bacteremic urinary infection caused by multidrug-resistant Escherichia coli

ObjectivesTo describe the population pharmacokinetics of fosfomycin for patients with bacteraemic urinary tract infection (BUTI). The analysis identified optimal regimens on the basis of pharmacodynamic targets and assessed the adequacy of Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) susceptibility breakpoints for Escherichia coli. MethodsData of 16 patients with BUTI caused by multidrug-resistant E. coli (FOREST clinical trial) received intravenous fosfomycin (4 g every 6 hours) were analysed. A population pharmacokinetic analysis was performed, and Monte Carlo simulations were undertaken using 4 g every 6 hours and 8 g every 8 hours. The probability of pharmacodynamic target attainment was assessed using pharmacodynamic targets for E. coli for static effect, 1-log drop in bacterial burden and resistance suppression. ResultsSixty-four plasma samples were collected over a single dosing interval (day 2 or 3 after starting fosfomycin treatment). Fosfomycin concentrations were highly variable. Pharmacodynamic target attainment analysis showed mild improvement by increasing fosfomycin dosing (4 g every 6 hours vs. every 8 hours). These dosages showed success for decreasing 1-log bacterial burden in 89% to 96% (EUCAST breakpoints) and 33% to 54% (CLSI breakpoints) of patients, but they were unable to reach bacterial resistance suppression targets. ConclusionsFosfomycin concentrations are highly variable—a fact partially explained by renal impairment. The present work supports the use of 4 g every 6 hours as an effective regimen for the treatment of non–critically ill patients with BUTI caused by multidrug-resistant E. coli, as higher dosages might increase toxicity but may not significantly increase efficacy. The current information may suggest that fosfomycin susceptibility breakpoints need to be reappraised.

From CLSI to EUCAST guidelines in the interpretation of antimicrobial susceptibility: What is the effect in our setting?

IntroductionImplementation of the breakpoints established in the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines in comparison with those of the Clinical and Laboratory Standards Institute (CLSI) means that the criteria for interpreting the susceptibility of some antimicrobials have been modified, resulting in changes in the reports of accumulated antibiotic susceptibility. MethodsThe effect of applying EUCAST breakpoints in 10,359 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus spp. was analysed. ResultsBy applying EUCAST breakpoints, most antimicrobial susceptibility percentages did not change or changed very slightly. However, a decrease in aminoglycoside susceptibility was observed in Gram-negative bacilli, mainly for amikacin and Pseudomonas aeruginosa (23.2%), although only 5.7% were completely resistant; a notably decrease in the percentage of isolates susceptible to aztreonam was also observed. There was also a marked increase in the number of Staphylococcus aureus strains resistant to clindamycin (51.5%) and aminoglycosides (gentamicin 43.1%). ConclusionsSwitching from CLSI to EUCAST criteria in some pathogens alters the percentages of resistance to several antimicrobials, and therefore the local epidemiology of the resistance. These changes should be implemented by a multidisciplinary group in order to analyse the influence of the new data on the empirical treatment protocols of each centre.

Aerococcus urinae and Aerococcus sanguinicola: Susceptibility Testing of 120 Isolates to Six Antimicrobial Agents Using Disk Diffusion (EUCAST), Etest, and Broth Microdilution Techniques.

Background: Aerococcus urinae and Aerococcus sanguinicola are relatively newcomers and emerging organisms in clinical and microbiological practice. Both species have worldwide been associated with urinary tract infections. More rarely cases of bacteremia/septicemia and infective endocarditis have been reported. Treatment options are therefore important. Just recently, European recommendations on susceptibility testing and interpretive criteria have been released.Objective:In this investigation 120 A. urinae and A. sanguinicola isolates were tested for susceptibility to six antimicrobial agents: Penicillin, cefotaxime, meropenem, vancomycin, linezolid, and rifampicin.Methods:Three susceptibility testing methods were used; disk diffusion according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST) standardized disk diffusion methodology and MIC determination with Etest and broth microdilution (BMD). All testing was performed with EUCAST media for fastidious organisms.Results:Data obtained in this study were part of the background data for establishing EUCAST breakpoints. MIC values obtained by Etest and BMD were well correlated with disk diffusion results.Conclusion:All isolates were found susceptible to all six antimicrobial agents: penicillin, cefotaxime, meropenem, vancomycin, linezolid, and rifampicin.

Cambio de CLSI a EUCAST en la interpretación de la sensibilidad a antimicrobianos: ¿cómo influye en nuestro medio?

IntroductionImplementation of the breakpoints established in the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines in comparison with those of the Clinical and Laboratory Standards Institute (CLSI) means that the criteria for interpreting the susceptibility of some antimicrobials have been modified, resulting in changes in the reports of accumulated antibiotic susceptibility. MethodsThe effect of applying EUCAST breakpoints in 10,359 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus spp. was analysed. ResultsBy applying EUCAST breakpoints, most antimicrobial susceptibility percentages did not change or changed very slightly. However, a decrease in aminoglycoside susceptibility was observed in Gram-negative bacilli, mainly for amikacin and Pseudomonas aeruginosa (23.2%), although only 5.7% were completely resistant; a notably decrease in the percentage of isolates susceptible to aztreonam was also observed. There was also a marked increase in the number of Staphylococcus aureus strains resistant to clindamycin (51.5%) and aminoglycosides (gentamicin 43.1%). ConclusionsSwitching from CLSI to EUCAST criteria in some pathogens alters the percentages of resistance to several antimicrobials, and therefore the local epidemiology of the resistance. These changes should be implemented by a multidisciplinary group in order to analyse the influence of the new data on the empirical treatment protocols of each centre.

In vitro antimicrobial susceptibility patterns of Propionibacterium acnes isolated from patients with acne vulgaris.

Propionibacterium acnes has been implicated in the development of acne vulgaris. Rampant use of topical and systemic antibiotics for acne vulgaris has led to resistance due to selective pressure. This study aimed to determine antibiotic resistance of P. acnes. A total of 102 samples were collected from acne lesions and cultured onto sheep's blood agar and brain-heart infusion agar supplemented with 5 g/L glucose and 2 mg/L furazolidone) (BHIg) under aerobic and anaerobic conditions. Species identification was done by conventional methods and the VITEK2 Compact system. The isolates were tested for penicillin, erythromycin, clindamycin, ciprofloxacin, nadifloxacin, and tetracycline by E-test, and minimum inhibitory concentration (MIC) of minocycline was determined by agar dilution on BHIg. MIC results were interpreted as per EUCAST (European Committee on Antimicrobial Susceptibility Testing) and CLSI (Clinical Laboratory Standards Institute) guidelines. P. acnes was the most common anaerobe (66%) isolated. Resistance rates using EUCAST and CLSI breakpoints were 10.6% and 6.1%, 7.6% and 0%, 7.8% and 0% for erythromycin, clindamycin, and minocycline, respectively. Tetracycline resistance was observed in 9.2% isolates irrespective of the interpretative criteria used. MIC50 and MIC90 values for nadifloxacin (0.25 and 1 µg/mL) were found to be twofold lower than those for ciprofloxacin (0.5 and 1 µg/mL). Similarly, MIC50 and MIC90 values for minocycline (0.125 and 0.5 µg/mL) were also two- to threefold lower than those for tetracycline (0.38 and 1 µg/mL). To the best of our knowledge, this is the first study focusing on P. acnes resistance from India.

Development of a national EUCAST challenge panel for antimicrobial susceptibility testing

A challenge panel of bacterial strains useful for clinical laboratories to validate their European Committee on Antimicrobial Susceptibility Testing (EUCAST) antimicrobial susceptibility test (AST) system was established. A total of 117 strains, obtained from Belgian Reference Centres (n = 57) and from routine clinical samples (n = 60) was selected based on resistance pattern. These strains were analysed in seven different laboratories by three different automated AST systems (Vitek (n = 2), Phoenix (n = 2) and Microscan (n = 2)) and by disc diffusion from five different manufacturers (Rosco (n = 2), Becton-Dickinson (n = 2), Biomérieux (n = 1), Bio-rad (n = 1) and i2a (n = 1)). To select the challenge panel, selection criteria were set for categorical agreement between the different systems and the number of very major errors, major errors and minor errors. Very major and major errors for at least two antibiotics were observed in 43% of all strains, leading to the exclusion of these strains from the selected panel. In only 10% of all tested strains was there 100% categorical agreement for all antibiotics. Finally, 28 strains (14 Gram-positive and 14 Gram-negative) covering a wide spectrum of resistance mechanisms were selected. Pilot-testing of this challenge panel in 20 laboratories mainly confirmed the results of the validation study. Only six strains withheld for the pilot study could not be used as challenge strain due to an overall (very) major error rate of >5% for a particular antibiotic (n = 5) or for two antibiotics (n = 1). To conclude, this challenge panel should facilitate the implementation and use of EUCAST breakpoints in laboratories.

Antimicrobial susceptibility and typing of Neisseria gonorrhoeae strains from Southern Spain, 2012–2014

BackgroundGonorrhoea remains an important health problem worldwide. The latest European guidelines have recommended the introduction of dual antimicrobial therapy due to the increase in its resistance to antimicrobial agents. MethodsIn the present study, the susceptibility to some antibiotics was evaluated, and the typing of Neisseria gonorrhoeae strains was also performed. All Neisseria gonorrhoeae (NG) strains isolated from January 2012 to October 2014 were included in this work. Gonococcal isolates were tested for susceptibility according to the recommendations of both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). A total of 65 isolates were typed by the NG multi-antigen sequence types (NG-MAST) technique. ResultsThe most frequent types found were ST 1407, ST 5405, ST 2992, and ST 5120. If CLSI and EUCAST criteria were applied, an ST 9807 type was found non-susceptible to ceftriaxone and cefixime (MIC 0.5μg/mL). When only EUCAST breakpoints were taken into account, three strains were also resistant to cefixime (MIC 0.25μg/mL) and two isolates were resistant to ceftriaxone (MIC 0.19 and 0.25μg/mL, respectively). The majority of strains were resistant to ciprofloxacin, and all Neisseria gonorrhoeae strains were susceptible to spectinomycin; twenty-five percent of isolates were resistant to azithromycin. ConclusionsThe implementation of antimicrobial surveillance programs at regional level should be part of an overall gonococcal infection control strategy. Efforts should be made to perform antimicrobial susceptibility, a “cured test” in all gonorrhoea cases, and identify treatment failures to verify emerging resistance. Some types have been associated with decreased susceptibility to cephalosporins, making molecular typing a useful tool to predict antimicrobial resistance.

Widespread implementation of EUCAST breakpoints for antibacterial susceptibility testing in Europe.

The European Committee on Antimicrobial Susceptibility Testing (EUCAST) was established to harmonise clinical antimicrobial breakpoints and to define breakpoints for new agents in Europe. Data from the European Antimicrobial Resistance Surveillance Network (EARS-Net) external quality assessment (EQA) exercises from 2009 to 2012, from the United Kingdom External Quality Assessment Scheme (UK NEQAS) from November 2009 to March 2013 and data collected by EUCAST through a questionnaire in the first quarter of 2013 were analysed to investigate implementation of EUCAST guidelines in Europe. A rapid change to use of EUCAST breakpoints was observed over time. Figures for implementation of EUCAST breakpoints at the end of the studied period were 61.2% from EARSNet data and 73.2% from UK NEQAS data. Responses to the EUCAST questionnaire indicated that EUCAST breakpoints were used by over 50% of laboratories in 18 countries, by 10 to 50% of laboratories in eight countries and by less than 10% in seven countries. The EUCAST disk diffusion method was used by more than 50% of laboratories in 12 countries, by 10 to 50% of laboratories in ten countries and by less than 10% in eleven countries. EUCAST guidelines implementation is essential to ensure consistent clinical reporting of antimicrobial susceptibility results and antimicrobial resistance surveillance.

Antimicrobial resistance among anaerobes isolated from clinical specimens in Kuwait hospitals: Comparative analysis of 11-year data

Our objective was to compare the antimicrobial resistance trends among clinically relevant anaerobes against 9 different antibiotics over two periods, 2008–2012 and 2002–2007. Antimicrobial susceptibility testing was performed by determining the MICs using E test method. The interpretation of results was according to the breakpoints recommended by the Clinical Laboratory and Standard Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST). A total of 2240 clinically significant isolates were collected between 2008 and 2012 in four teaching hospitals in Kuwait. The commonest isolates were Bacteroides fragilis (40.4%), Prevotella bivia (18.6%), Peptostreptococcus spp. (13.8%) and Bacteroides ovatus (11.1%). According to CLSI and EUCAST breakpoints used for the 2008–2012 and 2002–2007 isolates, high resistance rates to amoxicillin–clavulanic acid, clindamycin, penicillin and piperacillin were noted among the Gram-negative isolates. They ranged between 0 and 0–62.1 and 62.1%, and 0 and 0–59.1 and 62.1%, respectively against clindamycin, 0 and 0–34.5 and 45.3%, and 0 and 0–45 and 57.5%, respectively against piperacillin and 0 and 0–24.2 and 24.2%, and 0 and 0–23.1 and 30.6%, respectively against amoxicillin–clavulanic acid. The mean interpretative results by both CLSI and EUCAST during the 2008–2012 and 2002–2007 periods showed that the B. fragilis isolates were highly resistant to penicillin (100 vs 100%), clindamycin (43.7 vs 44.2%), piperacillin (35.8 vs 42.7%) and amoxicillin–clavulanic acid (13.2 vs 14%), respectively. When compared with 2002–2007, the CLSI, but not EUCAST, demonstrated statistically significant decreased resistance to clindamycin (P < 0.03). However, both interpretative criteria showed demonstrable statistically significant decrease in resistance rates to imipenem (P < 0.00097 vs P < 0.00074), meropenem (P < 0.000006 vs P < 0.0407) and piperacillin (P < 0.000017 vs P < 0.0461). Our data shows that there is a need for periodic monitoring of the susceptibility testing for anaerobic bacteria in the face of increasing resistance rates as well as to guide in the empirical therapy of anaerobic infections.

Candida species distribution and antifungal susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing and new vs. old Clinical and Laboratory Standards Institute clinical breakpoints: a 6-year prospective candidaemia survey from the fungal infection network of Switzerland

We analyzed the species distribution of Candida blood isolates (CBIs), prospectively collected between 2004 and 2009 within FUNGINOS, and compared their antifungal susceptibility according to clinical breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in 2013, and the Clinical and Laboratory Standards Institute (CLSI) in 2008 (old CLSI breakpoints) and 2012 (new CLSI breakpoints). CBIs were tested for susceptiblity to fluconazole, voriconazole and caspofungin by microtitre broth dilution (Sensititre® YeastOne™ test panel). Of 1090 CBIs, 675 (61.9%) were C. albicans, 191 (17.5%) C. glabrata, 64 (5.9%) C. tropicalis, 59 (5.4%) C. parapsilosis, 33 (3%) C. dubliniensis, 22 (2%) C. krusei and 46 (4.2%) rare Candida species. Independently of the breakpoints applied, C. albicans was almost uniformly (>98%) susceptible to all three antifungal agents. In contrast, the proportions of fluconazole- and voriconazole-susceptible C. tropicalis and F-susceptible C. parapsilosis were lower according to EUCAST/new CLSI breakpoints than to the old CLSI breakpoints. For caspofungin, non-susceptibility occurred mainly in C. krusei (63.3%) and C. glabrata (9.4%). Nine isolates (five C. tropicalis, three C. albicans and one C. parapsilosis) were cross-resistant to azoles according to EUCAST breakpoints, compared with three isolates (two C. albicans and one C. tropicalis) according to new and two (2 C. albicans) according to old CLSI breakpoints. Four species (C. albicans, C. glabrata, C. tropicalis and C. parapsilosis) represented >90% of all CBIs. In vitro resistance to fluconazole, voriconazole and caspofungin was rare among C. albicans, but an increase of non-susceptibile isolates was observed among C. tropicalis/C. parapsilosis for the azoles and C. glabrata/C. krusei for caspofungin according to EUCAST and new CLSI breakpoints compared with old CLSI breakpoints.

Evaluation of the Clinical and Laboratory Standards Institute phenotypic confirmatory test to detect the presence of extended-spectrum β-lactamases from 4005 Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae and Proteus mirabilis isolates

A subset of Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae and Proteus mirabilis isolates collected for the Study for Monitoring Antimicrobial Resistance Trends that were positive for the Clinical and Laboratory Standards Institute (CLSI) extended-spectrum β-lactamase (ESBL) phenotypic confirmatory test (n = 3245) or had an ertapenem MIC of ≥0.5 µg ml(-1) (n = 293), or both (n = 467), were analysed for ESBL genes. Most ESBL phenotype E. coli or K. pneumoniae possessed an ESBL gene (95.8 and 88.4 %, respectively), and this was 93.1 % if carbapenem-non-susceptible K. pneumoniae were removed. This rate was lower for P. mirabilis (73.4 %) and K. oxytoca (62.5 %). Virtually all ESBL-positive isolates (99.5 %) were cefotaxime non-susceptible [CLSI or European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints)]. Fewer isolates (82 %) were ceftazidime non-susceptible (CLSI breakpoints). In addition, 21.1 % of E. coli, 25 % of K. oxytoca and 78.7 % of P. mirabilis isolates were ceftazidime susceptible but ESBL positive. This suggests that CLSI breakpoints for ceftazidime are too high to detect ESBLs. The lower EUCAST breakpoints detected ESBLs in E. coli and K. oxytoca better, but 59.6 % of ESBL-positive isolates of P. mirabilis were ceftazidime susceptible. For isolates with ertapenem MICs ≥0.5 µg ml(-1), more accurate ESBL phenotype analysis was observed for E. coli and K. pneumoniae (sensitivity >95 % for both, specificity 94.4 and 54.1 %, respectively). If carbapenemase-positive K. pneumoniae were excluded, the specificity increased to 78 %. The positive predictive values for the ESBL phenotypic test with E. coli and K. pneumoniae were 97.6 and 81.8 %, respectively, and negative predictive values were 75.9 and 95.2 %, respectively. We therefore suggest that it would be prudent to confirm phenotypic ESBL-positive P. mirabilis, K. pneumoniae and K. oxytoca with molecular analysis.

Breakpoints for antifungal agents: An update from EUCAST focussing on echinocandins against Candida spp. and triazoles against Aspergillus spp.

Candida and Aspergillus infections have emerged as significant pathogens in recent decades. During this same time, broad spectrum triazole and echinocandin antifungal agents have been developed and increasingly used. One consequence of widespread use is leading to the emergence of mutants with acquired resistance mutations. Therefore, accurate susceptibility testing and appropriate clinical breakpoints for the interpretation of susceptibility results have become increasingly important. Here we review the underlying methodology by which breakpoints have been selected by EUCAST (European Committee on Antimicrobial Susceptibility Testing). Five parameters are evaluated: dosing regimens used; EUCAST MIC distributions from multiple laboratories, species and compound specific epidemiological cut off values (upper MIC limits of wild type isolates or ECOFFs), pharmacokinetic/pharmacodynamic relationships and targets associated with outcome and finally clinical data by species and MIC when available. The general principles are reviewed followed by a detailed review of the individual aspects for Candida species and the three echinocandins and for Aspergillus and the three mould-active azoles. This review provides an update of the subcommittee on antifungal susceptibility testing (AFST) of the EUCAST methodology and summarises the current EUCAST breakpoints for Candida and Aspergillus. Recommendations about applicability of antifungal susceptibility testing in the routine setting are also included.

Contemporary potencies of minocycline and tetracycline HCL tested against Gram-positive pathogens: SENTRY Program results using CLSI and EUCAST breakpoint criteria

Tetracycline class agents vary widely in their activity against emerging important antimicrobial-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter spp. Also, published susceptibility breakpoints are discordant between the Clinical and Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and regulatory-approved documents. We have assessed the impact of these differences for tetracycline HCL and minocycline when tested against contemporary Gram-positive pathogens. The SENTRY Antimicrobial Surveillance Program (2011) compared minocycline and tetracycline HCL activity via reference methods (M07-A9) using a worldwide collection of S. aureus (SA; 4917 strains with 1955 MRSA), Streptococcus pneumoniae (SPN; 1899), S. pyogenes (GRA; 246), and S. agalactiae (GRB; 217). Regardless of applied categorical breakpoints, minocycline exhibited wider coverage (% susceptible) than tetracycline HCL of 4.5–11.8/0.5–2.6/1.4–2.3/0.4–0.4% for MRSA/SPN/GRB/GRA, respectively. Lower EUCAST susceptible breakpoints produced reduced susceptibility rates for minocycline ranging from no difference (≤0.5 μg/mL) for GRA to −8.9% (≤1 μg/mL) for MRSA (97.2% susceptible by CLSI; 88.3% by EUCAST). Use of tetracycline HCL-susceptible results to predict minocycline susceptibility was very accurate (99.0–100.0%), with absolute categorical agreement rates ranging from 92.1% to 98.4% (CLSI) to 98.4% to 99.6% (EUCAST) for streptococci; greatest predictive error was noted using the CLSI breakpoints (14.7%) compared to EUCAST criteria (only 5.0%; acceptable), both for MRSA testing dominated by false-resistant results for minocycline. In conclusion, minocycline demonstrates continued superior in vitro activity compared to tetracycline HCL when testing SA (especially MRSA) and pathogenic streptococci. When testing tetracyclines, laboratories must recognize the expanded spectrum of minocycline against certain pathogens and utilize methods minimizing interpretive error. We conclude that EUCAST breakpoint criteria (≤0.5 or ≤1 μg/mL) represent the most conservative (better recognize strains with tet resistance mechanisms) and accurate tetracycline breakpoint guidelines for testing contemporary isolates of Gram-positive cocci.

Contemporary tetracycline susceptibility testing: doxycycline MIC methods and interpretive criteria (CLSI and EUCAST) performance when testing Gram-positive pathogens

International susceptibility testing breakpoint organizations and regulatory agencies have markedly differing interpretive criteria for the tetracycline class. Here we examined the magnitude of these differences for doxycycline and tetracycline hydrochloride (HCL) when tested against a collection of 13,176 Gram-positive cocci from a worldwide surveillance network (SENTRY Antimicrobial Surveillance Program, 2010). Clinical and Laboratory Standards Institute (CLSI) breakpoints are routinely higher, usually 4-fold, compared to those of the European Committee on Antimicrobial Susceptibility Testing (EUCAST); however, CLSI recently (2013) modified Streptococcus pneumoniae breakpoints (≤2 μg/mL in 2012) to ≤0.25 and ≤1 μg/mL for doxycycline and tetracycline HCL, respectively. We report that these changes are a promising step toward international breakpoint harmonization, but lack a comprehensive approach needed for testing tetracyclines against all Gram-positive cocci. Generally, EUCAST breakpoint criteria showed i) lower spectrums (reduced susceptibility rates) for the tetracyclines, but highest for doxycycline versus all species examined; ii) greater test accuracy (lower predictive categorical errors), especially for tetracycline to predict doxycycline susceptibility (99.91%); and iii) zone diameter correlate breakpoints which are generally available online. Molecular tests for tet resistance genes demonstrate that tet (K) and tet (M) containing strains can occur in the susceptible population of MIC results by both CLSI and EUCAST breakpoint criteria. In summary, doxycycline continues to show greater comparative potency versus tetracycline HCL against all monitored Gram-positive species and the international harmonization of tetracycline breakpoints should be a priority, as the most recent CLSI update only addressed 1 streptococcal species and 2 tetracycline agents.

Clinical breakpoint changes and their impact on surveillance of antimicrobial resistance in Escherichia coli causing bacteraemia

Dutch laboratories are currently changing their breakpoint criteria from mostly Clinical Laboratory and Standards Institute (CLSI) breakpoints to European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. To evaluate the impact of these changes, we studied antimicrobial resistance trends of Escherichia coli in blood specimens from January 2008 to January 2012 using CLSI and EUCAST breakpoints and compared them with the antimicrobial susceptibility test (AST) interpretations reported by Dutch laboratories participating in the Infectious Disease Surveillance Information System for Antibiotic Resistance (ISIS-AR). ISIS-AR collects AST interpretations, including underlying minimal inhibitory concentrations (MICs) of routinely cultured bacterial species on a monthly basis from Dutch laboratories. MICs of Etests or automated systems were reinterpreted according to the CLSI 2009 and EUCAST 2010 guidelines. Trends in non-susceptibility (i.e. intermediate resistant and resistant) over time were analysed by the Cochran–Armitage test for trend. The effects of the change from CLSI to EUCAST breakpoints on non-susceptibility were small. There were no differences in non-susceptibility to amoxicillin, amoxicillin/clavulanic acid, cefuroxim, gentamicin and co-trimoxazol and only small differences (1–1.5%) for ciprofloxacin between AST interpretations by CLSI or EUCAST. However, for ceftazidime, and cefotaxime/ceftriaxone the proportion of non-susceptibility was substantially higher when EUCAST breakpoints were used (2–3%). The effects on time trends of the change in guidelines were limited, with only substantial differences for the oxymino-cephalosporins. Our study shows that the implementation of EUCAST breakpoints has a limited effect on the proportion of non-susceptible isolates and time trends in E. coli for most, but not all, antimicrobial agents.

Impact of changes in CLSI and EUCAST breakpoints for susceptibility in bloodstream infections due to extended-spectrum β-lactamase-producing Escherichia coli

The impact of recent changes in and discrepancies between the breakpoints for cephalosporins and other antimicrobials, as determined by CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST), was analysed in patients with bloodstream infections caused by extended-spectrum β-lactamase (ESBL) producing Escherichia coli in Spain, was analysed. We studied a cohort of 191 episodes of bloodstream infection caused by ESBL-producing E. coli in 13 Spanish hospitals; the susceptibility of isolates to different antimicrobials was investigated by microdilution and interpreted according to recommendations established in 2009 and 2010 by CLSI, and in 2011 by EUCAST. Overall, 58.6% and 14.7% of isolates were susceptible to ceftazidime, and 35.1% and 14.7% to cefepime using the CLSI-2010 and EUCAST-2009/2011 recommendations, respectively (all isolates would have been considered resistant using the previous guidelines). Discrepancies between the CLSI-2010 and the EUCAST-2011 recommendations were statistically significant for other antimicrobials only in the case of amikacin (98.4% versus 75.9% of susceptible isolates; p <0.01). The results varied depending on the ESBL produced. No significant differences were found in the percentage of patients classified as receiving appropriate therapy, following the different recommendations. Four out of 11 patients treated with active cephalosporins according to CLSI-2010 guidelines died (all had severe sepsis or shock); these cases would have been considered resistant according to EUCAST-2011. In conclusion, by using current breakpoints, extended-spectrum cephalosporins would be regarded as active agents for treating a significant proportion of patients with bloodstream infections caused by ESBL-producing E. coli.

Comparison of antimicrobial pharmacokinetic/pharmacodynamic breakpoints with EUCAST and CLSI clinical breakpoints for Gram-positive bacteria

This study compared the susceptibility breakpoints based on pharmacokinetic/pharmacodynamic (PK/PD) models and Monte Carlo simulation with those defined by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for antibiotics used for the treatment of infections caused by Gram-positive bacteria. A secondary objective was to evaluate the probability of achieving the PK/PD target associated with the success of antimicrobial therapy. A 10000-subject Monte Carlo simulation was executed to evaluate 13 antimicrobials (47 intravenous dosing regimens). Susceptibility data were extracted from the British Society for Antimicrobial Chemotherapy database for bacteraemia isolates. The probability of target attainment and the cumulative fraction of response (CFR) were calculated. No antibiotic was predicted to be effective (CFR≥90%) against all microorganisms. The PK/PD susceptibility breakpoints were also estimated and were compared with CLSI and EUCAST breakpoints. The percentages of strains affected by breakpoint discrepancies were calculated. In the case of β-lactams, breakpoint discrepancies affected <15% of strains. However, higher differences were detected for low doses of vancomycin, daptomycin and linezolid, with PK/PD breakpoints being lower than those defined by the CLSI and EUCAST. If this occurs, an isolate will be considered susceptible based on CLSI and EUCAST breakpoints although the PK/PD analysis predicts failure, which may explain treatment failures reported in the literature. This study reinforces the idea of considering not only the antimicrobial activity but also the dosing regimen to increase the probability of clinical success of an antimicrobial treatment.

Cefixime and ceftriaxone susceptibility of Neisseria gonorrhoeae in Italy from 2006 to 2010

Neisseria gonorrhoeae resistance to cephalosporins, the currently recommended treatment, and treatment failures with cefixime have been reported worldwide. The purposes of the present study were (i) to examine the susceptibility of N. gonorrhoeae isolates isolated in Italy from 2006 through 2010 to cefixime (n = 293) taking into account both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical And Laboratory Standards Institute (CLSI) criteria for categorization; (ii) to determine the contribution to decreased/resistant susceptibility of mutations in the penA, mtrR, ponA and porB1b genes in a subsample of isolates; and (iii) to genotype the isolates showing decreased susceptibility or resistance to cefixime, by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and by pulsed-field gel electrophoresis (PFGE) to identify the predominant genotypes. Minimum inhibitory concentrations (MICs) were determined by the E-test and agar dilution method on 293 isolates and results were interpreted according to both EUCAST 2010 (MIC R >0.12 mg/L) and CLSI 2008 (MIC R >0.25 mg/L) criteria. All isolates showed full susceptibility to ceftriaxone, whereas those with a MIC for cefixime ≥0.125 mg/L were on the increase from 2008 through 2010. The same penA gene alterations were found among isolates with MICs close to the EUCAST breakpoint as the resistant ones, and they belong to ST1407. Seven isolates, belonging to various sequence types, showed a different por allele, though similar to the por 908 allele present in ST1407. PFGE divided strains ST1407 into two main groups confirming their genetic relationship.