Antimicrobial susceptibility testing of Kingella kingae with broth microdilution and disk diffusion using EUCAST recommended media

ObjectivesIncreasing use of improved culture techniques and sensitive nucleic acid amplification assays have resulted in recognition of Kingella kingae as an important cause of invasive infections in young children, especially in septic arthritis, osteomyelitis, bacteraemia, and endocarditis. In 2016, EUCAST established clinical MIC breakpoints for K. kingae (published in EUCAST Clinical Breakpoint Tables v 7.0, 2017). The present study was carried out to produce MIC-zone diameter correlations for K. kingae on an international collection of isolates, with the aim of suggesting zone diameter breakpoints corresponding to the clinical MIC breakpoints.MethodsAntimicrobial susceptibility testing was performed for 18 clinically relevant agents on a collection of 159 clinical isolates of K. kingae. Broth microdilution MIC determination and disk diffusion were performed according to EUCAST recommendations for fastidious organisms.ResultsThe correlation between MICs and zone diameters was good for all agents with EUCAST breakpoints for K. kingae. β-lactamase was detected in 41 isolates (26%) and these isolates were resistant to aminopenicillins. These isolates were also resistant to trimethoprim-sulfamethoxazole. Resistance to tetracyclines was detected in 8% of all isolates. All resistant isolates were correctly categorized for these agents with the proposed zone diameter breakpoints. One isolate, resistant to erythromycin but susceptible to other macrolides, was categorized as susceptible with erythromycin disk diffusion. No resistance was detected for the cephalosporins, carbapenems, and fluoroquinolones tested.ConclusionBased on the results in this study, zone diameter breakpoints for K. kingae calibrated to EUCAST clinical MIC breakpoints were proposed and approved by EUCAST.     

A pharmacometric approach to define target site-specific breakpoints for bacterial killing and resistance suppression integrating microdialysis,time–kill curves and heteroresistance data: a case study with moxifloxacin

ObjectivesPharmacokinetic–pharmacodynamic (PK-PD) considerations are at the heart of defining susceptibility breakpoints for antibiotic therapy. However, current approaches follow a fragmented workflow. The aim of this study was to develop an integrative pharmacometric approach to define MIC-based breakpoints for killing and suppression of resistance development for plasma and tissue sites, integrating clinical microdialysis data as well as in vitro time–kill curves and heteroresistance information, exemplified by moxifloxacin against Staphylococcus aureus and Escherichia coli.MethodsPlasma and target site samples were collected from ten patients receiving 400 mg moxifloxacin/day. In vitro time–kill studies with three S. aureus and two E. coli strains were performed and resistant subpopulations were quantified. Using these data, a hybrid physiologically based (PB) PK model and a PK-PD model were developed, and utilized to predict site-specific breakpoints.ResultsFor both bacterial species, the predicted MIC breakpoint for stasis at 400 mg/day was 0.25 mg/L. Less reliable killing was predicted for E. coli in subcutaneous tissues where the breakpoint was 0.125 mg/L. The breakpoint for resistance suppression was 0.06 mg/L. Notably, amplification of resistant subpopulations was highest at the clinical breakpoint of 0.25 mg/L. High-dose moxifloxacin (800 mg/day) increased all breakpoints by one MIC tier.ConclusionsAn efficient pharmacometric approach to define susceptibility breakpoints was developed; this has the potential to streamline the process of breakpoint determination. Thereby, the approach provided additional insight into target site PK-PD and resistance development for moxifloxacin. Application of the approach to further drugs is warranted.     

Multicentre testing of the EUCAST broth microdilution reference method for MIC determination on Mycobacterium tuberculosis

ObjectivesThe first objective of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee for antimycobacterial susceptibility testing (AMST), launched in 2016, was to set a reference method for determining the MICs of antituberculous agents, since many protocols are used worldwide and a consensus one is needed for the determination of microbiological breakpoints.MethodsDuring 2017 and 2018, MIC determination protocols were evaluated prospectively in a multicentre study within the four AMST laboratories. MIC results were obtained for isoniazid, levofloxacin and amikacin on the reference strain Mycobacterium tuberculosis H37Rv ATCC 27294. Broth microdilution (BMD) in Middlebrook 7H9 and solid medium dilution (SMD) in Middlebrook 7H10 were performed using two inoculum concentrations. MICs were interpreted with regard to visual and 99% inhibition after 7, 14 or 21 days of incubation for BMD and 21 days for SMD.ResultsFollowing the EUCAST reference protocol, intra- and inter-assay agreements were within ±1 MIC dilution for >95% of the observations for the three drugs in both methods. MIC values, presented as MIC mode (range) for BMD and SMD respectively, were: 0.03 (0.015–0.06) mg/L and 0.12 (0.06–0.25) mg/L for isoniazid, 0.25 mg/L (0.25–0.5) and 0.5 mg/L (0.12–0.5) for levofloxacin, and 0.5 mg/L (0.5–1.0) and 0.5 mg/L (0.5–1.0) for amikacin.ConclusionsBoth SMD and BMD were reproducible and eligible as a reference method for MIC determination of the Mycobacterium tuberculosis complex (MTBC). BMD was finally selected as the EUCAST reference method. From now on it will be used to set epidemiological cut-off values and clinical breakpoints of new and old antituberculous agents.     

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.     

Carbapenemase-producing Klebsiella pneumoniae: (when) might we still consider treating with carbapenems?

Infections caused by carbapenemase-producing Klebsiella pneumoniae (CPKP) are increasing in frequency worldwide. CPKP isolates exhibit extensive drug resistance phenotypes, complicate therapy, and limit treatment options. Although CPKP isolates are often highly resistant to carbapenems, a proportion of these have relatively low MICs for carbapenems, raising the question of whether this class of agents has any therapeutic potential against CPKP infections. Results from animal studies and patient outcome data indicate that carbapenems retain meaningful in vitro activity against CPKP isolates with carbapenem MICs of ≤4 mg/L. Accumulating clinical experience also suggests that the therapeutic efficacy of carbapenems against CPKP isolates with MICs of ≤4 mg/L is enhanced when these agents are administered in combination with another active antibiotic. The results of human pharmacokinetic/pharmacodynamic studies are in line with the above observations; it is highly probable that a high-dose/prolonged-infusion regimen of a carbapenem would attain a time above the MIC value of 50% for CPKP isolates with MICs up to 4 mg/L, ensuring acceptable drug exposure and favourable treatment outcome. The analyses summarized in this review support the notion that carbapenems have their place in the treatment of CPKP infections and that the currently proposed EUCAST clinical breakpoints could direct physicians in making treatment decisions.     

Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates – the EUCAST broth microdilution reference method for MIC determination

ScopeSeveral methods are used worldwide for antibiotic susceptibility testing (AST) for the Mycobacterium tuberculosis complex (MTBC). The variability in the results obtained with these methods hampers setting epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines. Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MICs) need to be standardized for MTBC isolates for old and new agents. Our objective was to establish a standardized reference method for MIC determination for MTBC.MethodsThe EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared protocols of MIC determination with regard to medium, inoculum preparation, antituberculous agent preparation, incubation, reading of the results and interpretation.RecommendationsThe EUCAST reference method of MIC determination for MTBC is the broth microdilution method in Middlebrook 7H9-10% OADC medium. The final inoculum is a 10⁵ CFU/mL suspension, obtained from a 10⁻² dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonies with glass beads before suspending them in sterile water. The culture is maintained in a U-shaped 96-well polystyrene microtitre sterile plate with a lid incubated at 36° ± 1°C. Reading is done using an inverted mirror as soon as the 1:100 diluted control (i.e. 10³ CFU/mL suspension) shows visual growth. The MIC, expressed in mg/L, is the lowest concentration that inhibits visual growth. Mycobacterium tuberculosis H37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within the range 0.03–0.12 for isoniazid, 0.12–0.5 for levofloxacin and 0.25–1 mg/L for amikacin.ConclusionsThe EUCAST reference method for MTBC was endorsed by EUCAST after public consultation and will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference method is not primarily intended to be used under routine conditions and the AST methods will need to be calibrated against this reference method to be used with EUCAST breakpoints.     

A Current Perspective on Daptomycin for the Clinical Microbiologist

SUMMARY

Daptomycin is a lipopeptide antimicrobial with in vitro bactericidal activity against Gram-positive bacteria that was first approved for clinical use in 2004 in the United States. Since this time, significant data have emerged regarding the use of daptomycin for the treatment of serious infections, such as bacteremia and endocarditis, caused by Gram-positive pathogens. However, there are also increasing reports of daptomycin nonsusceptibility, in Staphylococcus aureus and, in particular, Enterococcus faecium and Enterococcus faecalis. Such nonsusceptibility is largely in the context of prolonged treatment courses and infections with high bacterial burdens, but it may occur in the absence of prior daptomycin exposure. Nonsusceptibility in both S. aureus and Enterococcus is mediated by adaptations to cell wall homeostasis and membrane phospholipid metabolism. This review summarizes the data on daptomycin, including daptomycin''s unique mode of action and spectrum of activity and mechanisms for nonsusceptibility in key pathogens, including S. aureus, E. faecium, and E. faecalis. The challenges faced by the clinical laboratory in obtaining accurate susceptibility results and reporting daptomycin MICs are also discussed.     

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.     

In vitro activity of the novel siderophore cephalosporin,cefiderocol, in Gram-negative pathogens in Europe by site of infection

ObjectivesWe assessed the activity of the novel siderophore cephalosporin, cefiderocol and selected other antibacterial agents against Gram-negative bacterial isolates in Europe.MethodsIsolates were obtained between 2013 and 2018 from European countries participating in the SIDERO-WT and SIDERO-Proteeae multinational surveillance studies. Isolates were categorised by infection site, focusing on bloodstream infections, hospital-acquired/ventilator-associated bacterial pneumonia (HABP/VABP), complicated intra-abdominal infections and complicated urinary tract infections. Cefiderocol activity was compared with ceftazidime–avibactam, ceftolozane–tazobactam, colistin and meropenem using standard susceptibility testing methods. European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were used to interpret susceptibility data.ResultsIsolates (n = 20 911) were collected from 145 sites in 24 countries in Europe, the highest proportion (34%) being from patients with HABP/VABP. Enterobacterales (66.6% of isolates) were more frequent than glucose non-fermenting species (33.4%) overall, with some differences between infection sites. Across all infection sites, the MIC₅₀/MIC₉₀ for cefiderocol was ≤0.5/≤2 mg/L for Enterobacter spp., ≤0.25/<2 mg/L for Klebsiella spp., 0.12/2 mg/L for Acinetobacter spp., ≤0.25/1 mg/L for Pseudomonas aeruginosa and ≤0.12/≤0.5 mg/L for Stenotrophomonas maltophilia. Across all infection sites, cefiderocol MICs were ≤2 mg/L for ≥96% of Enterobacter spp., ≥95% of Klebsiella spp., ≥90% of Acinetobacter spp. and ≥99% of Pseudomonas aeruginosa and Stenotrophomonas maltophilia isolates. Cefiderocol maintained high activity in carbapenem-resistant isolates, and the difference in activity between carbapenem-resistant (percentage susceptibility at EUCAST breakpoint: E. coli 77.8%, Klebsiella spp. 69.2%, Pseudomonas aeruginosa 97.5%, Acinetobacter spp. 90.7%, Stenotrophomonas maltophilia 99.6%) and carbapenem-susceptible (percentage susceptibility at EUCAST breakpoint: E. coli 99.4%, Klebsiella spp. 98.0%, Pseudomonas aeruginosa 99.7%, Acinetobacter spp. 94.9%) isolates was lower for cefiderocol than other agents.ConclusionsCefiderocol had excellent activity against all Gram-negative species, independent of key infection site and carbapenem MIC. Cefiderocol is a useful addition to the therapeutic options available for these difficult-to-treat infections.     

Dalbavancin exposure in vitro selects for dalbavancin-non-susceptible and vancomycin-intermediate strains of methicillin-resistant Staphylococcus aureus

ObjectivesDalbavancin is a lipoglycopeptide active against methicillin-resistant Staphylococcus aureus (MRSA). Its long half-life (8.5–16 days) allows for once-weekly or single-dose treatments but could prolong the mutant selection window, promoting resistance and cross-resistance to related antimicrobials such as vancomycin. The objective of this study was to evaluate the capacity of post-distributional pharmacokinetic exposures of dalbavancin to select for resistance and cross-resistance in MRSA.MethodsWe simulated average, post-distributional exposures of single-dose (1500 mg) dalbavancin (fCmax 9.9 μg/mL, β-elimination t_1/2 204 h) in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model for 28 days (672 h) against five MRSA strains and one methicillin-susceptible strain (MSSA). Samples were collected at least daily, and surviving colonies were enumerated and screened for resistance on drug-free and dalbavancin-supplemented medium respectively. Isolates from resistance screening plates were subjected to whole-genome sequencing (WGS) and susceptibly testing against dalbavancin, vancomycin, daptomycin, and six β-lactams with varying penicillin-binding protein (PBP) affinities.ResultsDalbavancin was bactericidal against most strains for days 1–4 before regrowth of less susceptible subpopulations occurred. Isolates with eight-fold increases in dalbavancin MIC were detected as early as day 4 but increased 64–128-fold in all models by day 28. Vancomycin and daptomycin MICs increased 4–16-fold, exceeding the susceptibly breakpoints for both antibiotics; β-lactam MICs generally decreased by two-to eight-fold, suggesting a dalbavancin–β-lactam seesaw effect, but increased by eight-fold or more in certain isolates. Resistant isolates carried mutations in a variety of genes, most commonly walKR, apt, stp1, and atl.ConclusionsIn our in vitro system, post-distributional dalbavancin exposures selected for stable mutants with reduced susceptibility to dalbavancin, vancomycin, and daptomycin, and generally increased susceptibility to β-lactams in all strains of MRSA tested. The clinical significance of these findings remains unclear, but created an opportunity to genotype a unique collection of dalbavancin-resistant strains for the first time. Mutations involved genes previously associated with vancomycin intermediate susceptibility and daptomycin non-susceptibility, most commonly walKR-associated genes.     

Area of technical uncertainty for susceptibility testing of amoxicillin/clavulanate against Escherichia coli: analysis of automated system,Etest and disk diffusion methods compared to the broth microdilution reference

ObjectivesThe European Committee on Antimicrobial Susceptibility Testing (EUCAST) recently warned about an area of technical uncertainty (ATU) of amoxicillin/clavulanate (AMX/C) disk susceptibility testing against members of the Enterobacterales. Thus, we aimed to compare the reliability of three routine methods and to evaluate the impact of the ATU.Methods286 Escherichia coli strains (including 159 AMX-resistant strains) were categorized for the two EUCAST AMX/C breakpoints by disk diffusion (Bio-Rad), the Phoenix automated system (Becton Dickinson) and the Etest (AES) compared to the broth microdilution reference method.ResultsBy microdilution, 84.2% of strains were AMX/C-susceptible using the urinary breakpoint (MIC ≤32 mg/L) and 62.2% using the systemic breakpoint (MIC ≤8 mg/L), with 63.6% of MICs between 4 and 16 mg/L. For the systemic breakpoint, category agreement (CA) and very major error (VME) were unacceptable for the Etest (71.7% and 27.3%), disk (73.1% and 23.4% at 19-mm cut-off) and to a lesser extent for the Phoenix system (83.6% and 10.5%). For disks, an unacceptable VME rate was observed for diameters up to 22 mm, probably due to overcharged disks. For the Etest, VMEs were high at 6 mg/L (46/63) and 8 mg/L (22/29). For the urinary breakpoint, CA was more acceptable for disk (88.9%) and Etest (84.3%) but was unevaluable for Phoenix.ConclusionAMX/C susceptibility testing of E. coli for systemic breakpoint was unreliable with the three routine methods, explained mainly by the high prevalence (~60%) of strains with microdilution MICs around the breakpoint (8 mg/L). Our data confirmed the EUCAST 19–20-mm ATU for disk and suggest introducing ATU for Etest MIC values of 6 and 8 mg/L.     

Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories

With the support of ESCMID and European countries, EUCAST has developed a disk diffusion test with zone diameter breakpoints correlated with the EUCAST clinical MIC breakpoints. The development of the EUCAST disk diffusion method and quality control criteria are described, together with guidance on quality control and implementation of the method in clinical microbiology laboratories. The method includes the use of Mueller–Hinton agar without supplements for non-fastidious organisms and with 5% mechanically defibrinated horse blood and 20 mg/L β-NAD for fastidious organisms, a standardized inoculum resulting in confluent growth, an incubation time of 16–20 h, a reading guide on how to read zone diameters on individual species-agent combinations and zone diameter breakpoints calibrated to the EUCAST clinical MIC breakpoints. EUCAST recommendations are described in detail and updated regularly on the EUCAST website (http://www.eucast.org).     

Bactericidal activity of daptomycin and vancomycin alone or in combination with tobramycin, netilmicin or ampicillin against enterococcus

The in vitro bactericidal activity of daptomycin and vancomycin alone or in combination was studied against enterococci in a microtiter checkerboard assay and by kill-kinetic experiments. Daptomycin was more active than vancomycin and was bactericidal. Better Fractional Bactericidal Concentration indices were observed with combinations of vancomycin with aminoglycosides, but in kill-kinetic studies, the combinations of 4 MICs of daptomycin with 4 mg/l of tobramycin or netilmicin produced the more lethal effect; these combinations were even more lethal than ampicillin and aminoglycosides in the same conditions. While vancomycin and ampicillin were antagonistic, synergistic FBC indices were observed with daptomycin and ampicillin in combination, but at 4 MICs of each antibiotic, the combination was less bactericidal than ampicillin alone. The bactericidal effect obtained with daptomycin in combination with aminoglycosides suggest that further evaluation of these combinations in enterococcal endocarditis could have a clinical interest if this bactericidal effect was confirmed by in vivo studies.     

Catheter-related bloodstream infection caused by Enterococcus spp.

The role of Enterococcus spp. as a cause of catheter-related bloodstream infections (CR-BSI) is almost unexplored. We assessed the incidence and clinical characteristics of enterococcal CR-BSI (ECR-BSI) over an 8-year period in our hospital. We performed a retrospective study (January 2003 to December 2010) in a large teaching institution. We recorded the incidence, and the microbiological and clinical data from patients with ECR-BSI. The incidence per 10 000 admissions for enterococcal BSI and ECR-BSI was 25 and 1.7, respectively. ECR-BSI was the fourth leading cause of CR-BSI in our institution (6%). A total of 75 episodes of ECR-BSI were detected in 73 patients (6% of all enterococcal BSI). The incidence of ECR-BSI increased by 17% annually (95% CI 19.0–21.0%) during the study period. Nineteen percent of ECR-BSI episodes were polymicrobial. Overall mortality was 33%. ECR-BSI is an emerging and increasingly common entity with a high mortality. This finding should be taken into account when selecting empirical treatment for presumptive CR-BSI.     

Changing Italian nosocomial-community trends and heteroresistance in Staphylococcus aureus from bacteremia and endocarditis

Bloodstream infections due to Staphylococcus aureus (BSI) are serious infections both in hospitals and in the community, possibly leading to infective endocarditis (IE). The use of glycopeptides has been recently challenged by various forms of low-level resistance. This study evaluated the distribution of MSSA and MRSA isolates from BSI and IE in 4 Italian hospitals, their antibiotic susceptibility—focusing on the emergence of hVISA—and genotypic relationships. Our results demonstrate that the epidemiology of MRSA is changing versus different STs possessing features between community-acquired (CA)- and hospital-acquired (HA)-MRSA groups; furthermore, different MSSA isolated from BSI and IE were found, with the same backgrounds of the Italian CA-MRSA. The hVISA phenotype was very frequent (19.5%) and occurred more frequently in isolates from IE and in both the MSSA and MRSA strains. As expected, hVISA were detected in MRSA with vancomycin minimum inhibitory concentrations (MICs) of 1–2?mg/l, frequently associated with the major SCCmec I and II nosocomial clones; this phenotype was also detected in some MSSA strains. The few cases of MR-hVISA infections evaluated in our study demonstrated that 5 out of 9 patients (55%) receiving a glycopeptide, died. Future studies are required to validate these findings in terms of clinical impact.     

Isavuconazole MIC distribution of 29 yeast species responsible for invasive infections (2015–2017)

ObjectivesIsavuconazole is a recent extended-spectrum triazole with activity against yeasts. However, few data are available about the in vitro activity of rare yeast species. We report the MIC distribution of isavuconazole compared with fluconazole for a large collection of common or rare yeasts.MethodsIsavuconazole and fluconazole MICs were determined using the EUCAST method for 1457 clinical isolates, mainly recovered from invasive infections, belonging to 29 species. They were sent to the National Reference Centre for Invasive Mycoses & Antifungals between January 2015 and October 2017 and species identification was performed using a polyphasic approach (matrix-assisted laser desorption/ionization time of flight analysis and a molecular method).ResultsIsavuconazole had effective in vitro activity against Cryptococcus neoformans (MIC90 < 0.25 mg/L), the five most common Candida spp. (MIC90 ≤ 0.5 mg/L for Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei) and also against the majority of rare species, including Candida kefyr and Candida lusitaniae. A few isolates of C. albicans (0.7%, 3/404), C. glabrata (2.7%, 5/184), C. tropicalis (1.0%, 1/96) and C. parapsilosis (0.8%, 1/127) exhibited MIC ≥4 mg/L. All were also resistant to fluconazole according to the EUCAST breakpoints. Some isolates with isavuconazole MIC ≥4 mg/L were also observed among rarer species: Meyerozyma guilliermondii (8.7%, 2/23), Wickerhamomyces anomalus (10.0%, 1/10). Other rare species Saprochaete clavata, Magnusiomyces capitatus, and Rhodotorula mucilaginosa had high MIC50 (≥1 mg/L) and MIC90 (≥4 mg/L) and could be considered as resistant to isavuconazole.ConclusionsWe confirmed the good in vitro activity of isavuconazole against common Candida, Cryptococcus species and the majority of the rare yeast species studied.     

Burkholderia pseudomallei multi-centre study to establish EUCAST MIC and zone diameter distributions and epidemiological cut-off values

ObjectivesMelioidosis, caused by Burkholderia pseudomallei, requires intensive antimicrobial treatment. However, standardized antimicrobial susceptibility testing (AST) methodology based on modern principles for determining breakpoints and ascertaining performance of methods are lacking for B. pseudomallei. This study aimed to establish MIC and zone diameter distributions on which to set epidemiological cut-off (ECOFF) values for B. pseudomallei using standard EUCAST methodology for non-fastidious organisms.MethodsNon-consecutive, non-duplicate clinical B. pseudomallei isolates (9–70 per centre) were tested at eight study centres against eight antimicrobials by broth microdilution (BMD) and the EUCAST disc diffusion method. Isolates without and with suspected resistance mechanisms were deliberately selected. The EUCAST Development Laboratory ensured the quality of study materials, and provided guidance on performance of the tests and interpretation of results. Aggregated results were analysed according to EUCAST recommendations to determine ECOFFs.ResultsMIC and zone diameter distributions were generated using BMD and disc diffusion results obtained for 361 B. pseudomallei isolates. MIC and zone diameter ECOFFs (mg/L; mm) were determined for amoxicillin-clavulanic acid (8; 22), ceftazidime (8; 22), imipenem (2; 29), meropenem (2; 26), doxycycline (2; none), tetracycline (8; 23), chloramphenicol (8; 22) and trimethoprim-sulfamethoxazole (4; 28).ConclusionsWe have validated the use of standard BMD and disc diffusion methodology for AST of B. pseudomallei. The MIC and zone diameter distributions generated in this study allowed us to establish MIC and zone diameter ECOFFs for the antimicrobials studied. These ECOFFs served as background data for EUCAST to set clinical MIC and zone diameter breakpoints for B. pseudomallei.     

How to: perform antifungal susceptibility testing of microconidia-forming dermatophytes following the new reference EUCAST method E.Def 11.0, exemplified by Trichophyton

BackgroundAntifungal drug resistance in dermatophytes was first reported shortly after the turn of the millennium and has today been reported in Trichophyton and occasionally in Microsporum, but not in Epidermophyton species. Although drug resistance in dermatophytes is not routinely investigated, resistance in Trichophyton spp. is increasingly reported worldwide. The highest rates are observed in India (36% and 68% for terbinafine (MIC ≥4 mg/L) and fluconazole (MICs ≥16 mg/L), respectively), and apparently involve the spread of a unique clade related to the Trichophyton mentagrophytes/Trichophyton interdigitale complex.ObjectivesThe European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has released a new method (E.Def 11.0) for antifungal susceptibility testing against microconidia-forming dermatophytes including tentative MIC ranges for quality control strains and tentative breakpoints against Trichophyton rubrum and T. interdigitale. Here, the details of the new procedure E.Def 11.0 are described.SourcesThis technical note is based on the multicentre validation of the EUCAST dermatophyte antifungal susceptibility testing method, the mould testing method (E.Def 9.3.2) and the updated quality control tables for antifungal susceptibility testing document, v 5.0 (available on the EUCAST website).ContentsThe method is based on the EUCAST microdilution method for moulds but significant differences include: (a) an altered test medium selective for dermatophytes; (b) an altered incubation time and temperature; and (c) a different end-point criterion (spectrophotometric determination) of fungal growth. It can easily be implemented in laboratories already performing EUCAST microdilution methods and has been validated for terbinafine, voriconazole, itraconazole and amorolfine against T. rubrum and T. interdigitale.ImplicationsThis standardized procedure with automated end-point reading will allow broader implementation of susceptibility testing of dermatophytes and so facilitate earlier appropriate therapy. This is important, as resistance is rapidly emerging and largely underdiagnosed.     

Comparison of antimicrobial susceptibility interpretation among Enterobacteriaceae using CLSI and EUCAST breakpoints

PurposeTo determine the difference in antimicrobial susceptibility of various antibiotics using the CLSI & EUCAST breakpoints.MethodsIn this non interventional, retrospective observational study, we reviewed minimum inhibitory concentrations (MIC) of various antibiotics routinely reported for Enterobacteriaceae clinical isolates, from an automated microbiology identification system (VITEK-2). These MICs were then analysed using both CLSI 2019 and EUCAST 2019 guidelines and classified as per the breakpoints into various categories.ResultsThe concordance rates of the antimicrobial susceptibility for various drugs ranged from 78.2% to 100% among two breakpoints. Perfect agreement with κ = 1 (p < 0.001) was observed for only three antimicrobials ceftriaxone, levofloxacin and trimethoprim-sulfamethoxazole. The changes in antimicrobial susceptibility interpretation for cefepime, ciprofloxacin, amoxicillin clavulanic acid was majorly in Intermediate category.ConclusionThe change in interpretation of the susceptibility will lead to change in the usage of antibiotics especially due to recent change in definition of I by EUCAST. There is need of more studies in this aspect to ascertain clinical implication of change in antimicrobial susceptibility.     

The contribution of pharmacokinetic–pharmacodynamic modelling with Monte Carlo simulation to the development of susceptibility breakpoints for Neisseria meningitidis

This study used pharmacokinetic-pharmacodynamic (PK-PD) modelling and MICs of 15 antimicrobial agents, derived from testing a large international culture collection, to assist in the development of interpretative criteria, i.e., breakpoints, for Neisseria meningitidis. PK parameters, protein binding, percentage penetration into cerebrospinal fluid (CSF), and the variability of these values, were extracted from the published literature for the 15 agents. PK-PD parameters have not been developed specifically for N. meningitidis in animal or human studies. Thus, it was necessary to invoke PK-PD targets from other organisms that cause infections at similar sites. The PK-PD targets utilised were: time above the MIC for at least 50% of the dosing interval for all beta-lactams, chloramphenicol, sulphafurazole and trimethoprim-sulphamethoxazole; an AUC/MIC ratio of >or=25 for the tetracyclines and macrolides; and an AUC/MIC ratio of >or=125 for the fluoroquinolones. A 10 000-subject Monte Carlo simulation was designed with the usual dosing regimens of each antimicrobial agent at MIC values of 0.03-64 mg/L in both serum and CSF. The PK-PD breakpoint was defined as the MIC at which the calculated target attainment was >or=95%. Using these assumptions, the proposed PK-PD breakpoints were: azithromycin, 0.125 mg/L; doxycycline, 0.25 mg/L; cefotaxime, ciprofloxacin and levofloxacin, 0.5 mg/L; penicillin G, meropenem, rifampicin, tetracycline and minocycline, 1 mg/L; chloramphenicol and sulphafurazole, 2 mg/L; and ampicillin, ceftriaxone and trimethoprim-sulphamethoxazole, 4 mg/L. Proposed PK-PD breakpoints applicable to CSF were: penicillin and cefotaxime, 0.06 mg/L; rifampicin, 0.125 mg/L; ceftriaxone, meropenem and trimethoprim-sulphamethoxazole, 0.25 mg/L; ampicillin, 0.5 mg/L; and chloramphenicol, 1 mg/L.