Tentative Epidemiological Cut-off Values Research Articles

New generation fluoroquinolone sitafloxacin could potentially overcome the majority levofloxacin and moxifloxacin resistance in multidrug-resistant Mycobacterium tuberculosis.

Introduction. Pre-existing fluoroquinolones (FQs) resistance is a major threat in treating multidrug-resistant (MDR) tuberculosis. Sitafloxacin (Sfx) is a new broad-spectrum FQ.Hypothesis. Sfx is more active against drug-resistant Mycobacterium tuberculosis (Mtb) isolates.Aim. To determine whether there is cross-resistance between Sfx and ofloxacin (Ofx), levofloxacin (Lfx) and moxifloxacin (Mfx) in MDR Mtb.Methods. A total of 106 clinical Mtb isolates, including 23 pan-susceptible and 83 MDR strains, were analysed for Sfx, Lfx and Mfx resistance using MIC assay. The isolates were also subjected to whole-genome sequencing to analyse drug-resistant genes.Results. Sfx exhibited the most robust inhibition activity against Mtb clinical isolates, with a MIC50 of 0.0313 µg ml-1 and MIC90 of 0.125 µg ml-1, which was lower than that of Mfx (MIC50 = 0.0625 µg ml-1, MIC90 = 1 µg ml-1) and Lfx (MIC50 = 0.125 µg ml-1, MIC90 = 2 µg ml-1). We determined the tentative epidemiological cut-off values as 0.5 µg ml-1 for Sfx. Also, 8.43% (7/83), 43.37% (36/83), 42.17% (35/83) and 51.81% (43/83) MDR strains were resistant to Sfx, Mfx, Lfx and Ofx, respectively. Cross-resistance between Ofx, Lfx and Mfx was 80.43% (37/46). Only 15.22% (7/46) of the pre-existing FQs resistance isolates were resistant to Sfx. Among the 30 isolates with mutations in gyrA or gyrB, 5 (16.67%) were Sfx resistant. The combination of Sfx and rifampicin could exert partial synergistic effects, and no antagonism between Sfx and six clinically important anti-Mtb antibiotics was evident.Conclusion. Sfx exhibited superior activity against MDR isolates comparing to Lfx and Mfx, and could potentially overcome the majority pre-existing FQs resistance in Mtb strains.

Mapping the Global Spread of T. indotineae: An Update on Antifungal Resistance, Mutations, and Strategies for Effective Management.

The global spread of Trichophyton indotineae presents a pressing challenge in dermatophytosis management. This systematic review explores the current landscape of T. indotineae infections, emphasizing resistance patterns, susceptibility testing, mutational analysis, and management strategies. A literature search was conducted in November 2023 using Embase, PubMed, Scopus, and Web of Science databases. Inclusion criteria covered clinical trials, observational studies, case series, or case reports with T. indotineae diagnosis through molecular methods. Reports on resistance mechanisms, antifungal susceptibility testing, and management were used for data extraction. A total of 1148 articles were identified through the systematic search process, with 45 meeting the inclusion criteria. The global spread of T. indotineae is evident, with cases reported in numerous new countries in 2023. Tentative epidemiological cut-off values (ECOFFs) suggested by several groups provide insights into the likelihood of clinical resistance. The presence of specific mutations, particularly Phe397Leu, correlate with higher minimum inhibitory concentrations (MICs), indicating potential clinical resistance. Azole resistance has also been reported and investigated in T. indotineae, and is a growing concern. Nevertheless, itraconazole continues to be an alternative therapy. Recommendations for management include oral or combination therapies and individualized approaches based on mutational analysis and susceptibility testing. Trichophyton indotineae poses a complex clinical scenario, necessitating enhanced surveillance, improved diagnostics, and cautious antifungal use. The absence of established clinical breakpoints for dermatophytes underscores the need for further research in this challenging field.

Antimicrobial susceptibility testing and tentative epidemiological cut-off values for Lactobacillaceae family species intended for ingestion

IntroductionIn this work, 170 strains covering 13 species from the Lactobacillaceae family were analyzed to determine minimal inhibitory concentration (MIC) distributions to nine antimicrobial agents, and genes potentially conferring resistance. This allows a proposal of tentative Epidemiological Cut-Offs (ECOFFs) that follows the phylogeny for interpretation of resistance in the 13 species.MethodsThe 170 strains originated from different sources, geographical areas, and time periods. MICs for nine antibiotics were determined according to the ISO 10932 standard for lactobacillia and by a modified CLSI-method for Leuconostoc and Pediococcus which ensured sufficient growth. The strains were whole genome sequenced, subtyped by core genome analysis, and assessed for the presence of antibiotic resistance genes using the ResFinder and NCBI AMRFinder databases.Results and discussionThe data provide evidence that antimicrobial susceptibility follows phylogeny instead of fermentation pattern and accordingly, tentative ECOFFs were defined. For some species the tentative ECOFFs for specific antibiotics are above the cut-off values set by the European Food Safety Authority (EFSA) which are primarily defined according to fermentation pattern or at genus level. The increased tolerance for specific antibiotics observed for some species was evaluated to be innate, as only for one strain phenotypic resistance was found to be related to an acquired resistance gene. In general, more data are needed to define ECOFFs and since the number of isolates available for industrial relevant bacterial species are often limited compared to clinically relevant species, it is important; 1) that strains are unambiguously defined at species level and subtyped through core genome analysis, 2) MIC determination are performed by use of a standardized method to define species-specific MIC distributions and 3) that known antimicrobial resistance genes are determined in whole genome sequences to support the MIC determinations.

Tentative Epidemiological Cut-Off Values and Distribution of Resistance Genes in Aquatic Pseudomonas Species Isolated from Rainbow Trout

Epidemiological cut-off value (ECV) analysis for commonly used antimicrobials in aquaculture have not been established for many aquatic pathogens, including Pseudomonas. This study was the first to examine the categorization of 92 aquatic Pseudomonas isolates by calculating seven antimicrobials ECVs using two analytical methods: normalized resistance interpretation and ECOFFinder. Pseudomonas spp. isolates had decreased sensitivity to all antimicrobials examined except for doxycycline and ciprofloxacin. The PCR analysis of the 91 isolates of Pseudomonas spp. detected the tetracycline genes are predominant with the count of 41 genes, including tetA, tetC, tetD, tetM, tetS and tetH, following sulfonamide genes are in 21 isolates including sul1 and sul2, floR gene in 15 isolates and ermA gene in three isolates. Our findings provide an understanding of the antimicrobial categorization of Pseudomonas species, which are significant groups, subgroups, and species for aquaculture due to insufficiently defined breakpoints or cut-off values reported in CLSI and/or EUCAST.

Setting of the tentative epidemiological cut-off values of contezolid for Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae and Streptococcus agalactiae.

To set the tentative epidemiological cut-off values (TECOFFs) of contezolid for Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae and Streptococcus agalactiae based on the distributions of inhibition zone diameters and MICs. A total of 1358 non-duplicate clinical isolates of Gram-positive bacteria were collected from the patients across China from 2017 to 2020. The isolates were tested for susceptibility to contezolid and the comparator linezolid by broth microdilution and disc diffusion methods in three microbiology laboratories. The zone diameters and MICs of linezolid WT strains were used to set the WT TECOFFs of contezolid by normalized resistance interpretation calculations. Contezolid showed an aggregate MIC range from 0.03 to 8 mg/L and MIC90 value of 1-2 mg/L against all of the Gram-positive bacterial strains tested. The TECOFF of contezolid based on MIC distributions was 4 mg/L for both S. aureus and Enterococcus species, and 2 mg/L for S. pneumoniae and S. agalactiae. The TECOFF of contezolid based on zone diameter was 24 mm for S. aureus, 18 mm for E. faecalis, 20 mm for E. faecium and S. pneumoniae, and 17 mm for S. agalactiae. The epidemiological cut-off values of contezolid were set tentatively for selected Gram-positive bacteria using the MIC and zone diameter distributions. These data are helpful for clinical microbiologists and clinicians to interpret the antimicrobial susceptibility results of contezolid.

Antimicrobial Susceptibility Testing Using the MYCO Test System and MIC Distribution of 8 Drugs against Clinical Isolates of Nontuberculous Mycobacteria from Shanghai.

Given the increased incidence and prevalence of nontuberculous mycobacterial (NTM) diseases and the natural resistance of NTM to multiple antibiotics, in vitro susceptibility testing of different NTM species against drugs from the MYCO test system and new applied drugs is required. A total of 241 NTM clinical isolates were analyzed, including 181 slowly growing mycobacteria (SGM) and 60 rapidly growing mycobacteria (RGM). The Sensititre SLOMYCO and RAPMYCO panels were used for testing susceptibility to commonly used anti-NTM antibiotics. Furthermore, MIC distributions were determined against 8 potential anti-NTM drugs, including vancomycin (VAN), bedaquiline (BDQ), delamanid (DLM), faropenem (FAR), meropenem (MEM), clofazimine (CLO), cefoperazone-avibactam (CFP-AVI), and cefoxitin (FOX), and epidemiological cutoff values (ECOFFs) were analyzed using ECOFFinder. The results showed that most of the SGM strains were susceptible to amikacin (AMK), clarithromycin (CLA), and rifabutin (RFB) from the SLOMYCO panels and BDQ and CLO from the 8 applied drugs, while RGM strains were susceptible to tigecycline (TGC) from the RAPMYCO panels and also BDQ and CLO. The ECOFFs of CLO were 0.25, 0.25, 0.5, and 1 μg/mL for the mycobacteria M. kansasii, M. avium, M. intracellulare, and M. abscessus, respectively, and the ECOFF of BDQ was 0.5 μg/mL for the same four prevalent NTM species. Due to the weak activity of the other 6 drugs, no ECOFF was determined. This study on the susceptibility of NTM includes 8 potential anti-NTM drugs and a large sample size of Shanghai clinical isolates and demonstrates that BDQ and CLO had efficient activities against different NTM species in vitro, which can be applied to the treatment of NTM diseases. IMPORTANCE We designed customized panel that contains 8 repurposed drugs, including vancomycin (VAN), bedaquiline (BDQ), delamanid (DLM), faropenem (FAR), meropenem (MEM), clofazimine (CLO), cefoperazone-avibactam (CFP-AVI), and cefoxitin (FOX) from the MYCO test system. To better understand the efficacy of these 8 drugs against different NTM species, we determined the MICs of 241 NTM isolates collected in Shanghai, China. We attempted to define the tentative epidemiological cutoff values (ECOFFs) for the most prevalent NTM species, which is an important factor in setting up the breakpoint for a drug susceptibility testing. We used the MYCO test system as an automatic quantitative drug sensitivity test of NTM and extended the method to BDQ and CLO in this study. The MYCO test system complements commercial microdilution systems that currently lack BDQ and CLO detection.

Antimicrobial susceptibility profiles and tentative epidemiological cutoff values of Legionella pneumophila from environmental water and soil sources in China.

Legionnaires’ disease (LD), caused by Legionella, including the most prevalent Legionella pneumophila, has been treated primarily with antibiotics. Environmental water and soil are the reservoirs for L. pneumophila. Studying antimicrobial susceptibility using a large number of isolates from various environmental sources and regions could provide an unbiased result. In the present study, antimicrobial susceptibility of 1464 environmental L. pneumophila isolates that were derived from various environmental water and soil sources of 12 cities in China to rifampin (RIF), erythromycin (ERY), clarithromycin (CLA), azithromycin (AZI), ciprofloxacin (CIP), moxifloxacin (MOX), levofloxacin (LEV), and doxycycline (DOX) was investigated, and minimum inhibitory concentration (MIC) data were obtained. We show that regarding macrolides, ERY was least active (MIC90 = 0.5 mg/L), while CLA was most active (MIC90 = 0.063 mg/L). A total of three fluoroquinolones have similar MICs on L. pneumophila. Among these antimicrobials, RIF was the most active agent, while DOX was the most inactive one. We observed different susceptibility profiles between serogroup 1 (sg1) and sg2-15 or between water and soil isolates from different regions. The ECOFFs were ERY and AZI (0.5 mg/L), RIF (0.002 mg/L), CIP, CLA and MOX (0.125 mg/L), LEV (0.063 mg/), and DOX (32 mg/L). Overall, two fluoroquinolone-resistant environmental isolates (0.14%) were first documented based on the wild-type MIC distribution. Not all azithromycin-resistant isolates (44/46, 95.65%) harbored the lpeAB efflux pump. The MICs of the ERY and CLA on the lpeAB + isolates were not elevated. These results suggested that the lpeAB efflux pump might be only responsible for AZI resistance, and undiscovered AZI-specific resistant mechanisms exist in L. pneumophila. Based on the big MIC data obtained in the present study, the same defense strategies, particularly against both CLA and RIF, may exist in L. pneumophila. The results determined in our study will guide further research on antimicrobial resistance mechanisms of L. pneumophila and could be used as a reference for setting clinical breakpoints and discovering antimicrobial-resistant isolates in the clinic, contributing to the antibiotic choice in the treatment of LD.

Addressing the Antimicrobial Resistance of Ruminant Mycoplasmas Using a Clinical Surveillance Network.

Antimicrobial resistance (AMR) surveillance of mycoplasmas of veterinary importance has been held back for years due to lack of harmonized methods for antimicrobial susceptibility testing (AST) and interpretative criteria, resulting in a crucial shortage of data. To address AMR in ruminant mycoplasmas, we mobilized a long-established clinical surveillance network called “Vigimyc.” Here we describe our surveillance strategy and detail the results obtained during a 2-year monitoring period. We also assess how far our system complies with current guidelines on AMR surveillance and how it could serve to build epidemiological cut-off values (ECOFFs), as a first attainable criterion to help harmonize monitoring efforts and move forward to clinical breakpoints. Clinical surveillance through Vigimyc enables continuous collection, identification and preservation of Mycoplasma spp. isolates along with metadata. The most frequent pathogens, i.e., M. bovis and species belonging to M. mycoides group, show stable clinicoepidemiological trends and were included for annual AST. In the absence of interpretative criteria for ruminant mycoplasmas, we compared yearly minimum inhibitory concentration (MIC) results against reference datasets. We also ran a SWOT (Strengths, Weaknesses, Opportunities, Threats) analysis on the overall service provided by our AMR surveillance strategy. Results of the 2018–2019 surveillance campaign were consistent with the reference datasets, with M. bovis isolates showing high MIC values for all antimicrobial classes except fluoroquinolones, and species of the Mycoides group showing predominantly low MIC values. A few new AMR patterns were detected, such as M. bovis with lower spectinomycin MICs. Our reference dataset partially complied with European Committee on Antimicrobial Susceptibility Testing (EUCAST) requirements, and we were able to propose tentative epidemiological cut-off values (TECOFFs) for M. bovis with tilmicosin and spectinomycin and for M. mycoides group with tilmicosin and lincomycin. These TECOFFs were consistent with other published data and the clinical breakpoints of Pasteurellaceae, which are often used as surrogates for mycoplasmas. SWOT analysis highlighted the benefit of pairing clinical and antimicrobial resistance surveillance despite the AST method-related gaps that remain. The international community should now direct efforts toward AST method harmonization and clinical interpretation.

Employing MIC Data for Mink Pathogens to Propose Tentative Epidemiological Cut-Off Values: A Step Toward Rationalizing Antimicrobial Use in Mink.

Optimizing antimicrobial dosage regimens and development of breakpoints for antimicrobial susceptibility testing are important prerequisites for rational antimicrobial use. The objectives of the study were (1) to produce MIC data for four mink pathogens and (2) to employ these MIC data to support the development of tentative epidemiological cut-off values (TECOFFs), which may be used for future development of mink-specific antimicrobial dosages and breakpoints. Broth microdilution was used to establish MIC distributions for 322 mink bacterial isolates of clinical origin from six European mink-producing countries. The included species were E. coli (n = 162), S. delphini (n = 63), S. canis (n = 42), and P. aeruginosa (n = 55). Sixty-four E. coli isolates and 34 S. delphini isolates were whole-genome sequenced and analyzed for antimicrobial resistance genes. No EUCAST MIC data are available on S. delphini and S. canis, hence tentative ECOFFs were suggested for the majority of the tested antimicrobials. For E. coli and P. aeruginosa, the wildtype distributions were in accordance with EUCAST data. Overall, the genotypes of the sequenced isolates were in concordance with the phenotypes. These data constitute an important piece in the puzzle of developing antimicrobial dosages and clinical breakpoints for mink. Until pharmacokinetic and clinical data become available, the (tentative) ECOFFs can be used for monitoring resistance development and as surrogates for clinical breakpoints.

Antimicrobial susceptibility of Corynebacterium diphtheriae and Corynebacterium ulcerans in Germany 2011-17.

Diphtheria is mainly caused by diphtheria-toxin-producing strains of Corynebacterium diphtheriae and Corynebacterium ulcerans. The recommended first-line antibiotic is penicillin or erythromycin, but reliable susceptibility data are scarce. To define WT MIC distributions of 12 antimicrobial agents and provide data for the determination of tentative epidemiological cut-off values (TECOFFs) for potentially toxigenic corynebacteria and to evaluate the potential usefulness of a gradient test (Etest) for susceptibility testing of penicillin, erythromycin and clindamycin. For the 421 human or veterinary isolates from the period 2011-17, MICs of 12 antimicrobial agents were determined. Etest performance was evaluated for penicillin, erythromycin and clindamycin. MIC distributions were characterized and TECOFFs could be set for 11 out of 24 antibiotic/species combinations. The current EUCAST clinical breakpoints, predominantly determined for Corynebacterium species other than C. diphtheriae and C. ulcerans, divide the WT MIC distributions of penicillin and clindamycin, thereby making reproducible susceptibility testing of C. diphtheriae and C. ulcerans difficult. For erythromycin, 4% of C. diphtheriae and 2% of C. ulcerans had MICs higher than those for WT isolates. Phenotypically detectable resistance to other antibiotics was rare. Etest underestimated MICs of penicillin and lower concentrations needed to be included for erythromycin, while for clindamycin the Etest was not a good surrogate method. MIC distributions based on reference broth microdilution for potentially toxigenic Corynebacterium spp. were developed. For five and six agents, TECOFFs were suggested for C. diphtheriae and C. ulcerans, respectively, but for Corynebacterium pseudotuberculosis the number of isolates was too low.

In vitro antifungal activity of a novel topical triazole PC945 against emerging yeast Candida auris.

ObjectivesManagement of Candida auris infection is difficult as this yeast exhibits resistance to different classes of antifungals, necessitating the development of new antifungals. The aim of this study was to investigate the susceptibility of C. auris to a novel antifungal triazole, PC945, optimized for topical delivery.MethodsA collection of 50 clinical isolates was obtained from a tertiary care hospital in North India. Nine isolates from the UK, 10 from a CDC panel (USA) and 3 from the CBS-KNAW culture collection (Japanese and South Korean isolates) were also obtained. MICs (azole endpoint) of PC945 and other triazoles were determined in accordance with CLSI M27 (third edition). Quality control strains were included [Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258)].ResultsSeventy-four percent of isolates tested showed reduced susceptibility to fluconazole (≥64 mg/L). PC945 (geometric mean MIC = 0.058 mg/L) was 7.4-fold and 1.5-fold more potent than voriconazole and posaconazole, respectively (both P < 0.01). PC945 MIC values correlated with those of voriconazole or posaconazole, and only three isolates were found to be cross-resistant between PC945 and other azoles. ERG11 sequence analysis revealed several mutations, but no correlation could be established with the MIC of PC945. Tentative epidemiological cut-off values (ECOFFs) evaluated by CLSI’s ECOFF Finder (at 99%) with 24 h reading of MICs were 1, 4 and 1 mg/L for PC945, voriconazole and posaconazole, respectively. MIC values for quality control strains of all triazoles were in the normal ranges.ConclusionsPC945 was found to be a more potent inhibitor than posaconazole, voriconazole and fluconazole of C. auris isolates collected globally, warranting further laboratory and clinical evaluations.

Antifungal susceptibility profiles of rare ascomycetous yeasts.

To generate antifungal susceptibility patterns for Trichomonascus ciferrii (Candida ciferrii), Candida inconspicua (Torulopsis inconspicua) and Diutina rugosa species complex (Candida rugosa species complex), and to provide key parameters such as MIC50, MIC90 and tentative epidemiological cut-off values (TECOFFs). Our strain set included isolates of clinical origin: C. inconspicua (n = 168), D. rugosa species complex (n = 90) [Candida pararugosa (n = 60), D. rugosa (n = 26) and Candida mesorugosa (n = 4)], Pichia norvegensis (Candida norvegensis) (n = 15) and T. ciferrii (n = 8). Identification was performed by MALDI-TOF MS or internal transcribed spacer sequencing. Antifungal susceptibility patterns were generated for azoles, echinocandins and amphotericin B using commercial Etest and the EUCAST broth microdilution method v7.3.1. Essential agreement (EA) was calculated for Etest and EUCAST. C. inconspicua, C. pararugosa and P. norvegensis showed elevated azole MICs (MIC50 ≥0.06 mg/L), and D. rugosa and C. pararugosa elevated echinocandin MICs (MIC50 ≥0.06 mg/L). EA between methods was generally low (<90%); EA averaged 77.45%. TECOFFs were suggested for C. inconspicua and D. rugosa species complex. Rare yeast species tested shared high fluconazole MICs. D. rugosa species complex displayed high echinocandin MICs, while C. inconspicua and P. norvegensis were found to have high azole MICs. Overall, the agreement between EUCAST and Etest was poor and therefore MIC values generated with Etest cannot be directly compared with EUCAST results.

Antimicrobial Susceptibility Testing and Tentative Epidemiological Cutoff Values for Five Bacillus Species Relevant for Use as Animal Feed Additives or for Plant Protection.

Bacillus megaterium (n = 29), Bacillus velezensis (n = 26), Bacillus amyloliquefaciens (n = 6), Bacillus paralicheniformis (n = 28), and Bacillus licheniformis (n = 35) strains from different sources, origins, and time periods were tested for the MICs for nine antimicrobial agents by the CLSI-recommended method (Mueller-Hinton broth, 35°C, for 18 to 20 h), as well as with a modified CLSI method (Iso-Sensitest [IST] broth, 37°C [35°C for B. megaterium], 24 h). This allows a proposal of species-specific epidemiological cutoff values (ECOFFs) for the interpretation of antimicrobial resistance in these species. MICs determined by the modified CLSI method were 2- to 16-fold higher than with the CLSI-recommended method for several antimicrobials. The MIC distributions differed between species for five of the nine antimicrobials. Consequently, use of the modified CLSI method and interpretation of resistance by use of species-specific ECOFFs is recommended. The genome sequences of all strains were determined and used for screening for resistance genes against the ResFinder database and for multilocus sequence typing. A putative chloramphenicol acetyltransferase (cat) gene was found in one B. megaterium strain with an elevated chloramphenicol MIC compared to the other B. megaterium strains. In B. velezensis and B. amyloliquefaciens, a putative tetracycline efflux gene, tet(L), was found in all strains (n = 27) with reduced tetracycline susceptibility but was absent in susceptible strains. All B. paralicheniformis and 23% of B. licheniformis strains had elevated MICs for erythromycin and harbored ermD The presence of these resistance genes follows taxonomy suggesting they may be intrinsic rather than horizontally acquired. Reduced susceptibility to chloramphenicol, streptomycin, and clindamycin could not be explained in all species.IMPORTANCE When commercializing bacterial strains, like Bacillus spp., for feed applications or plant bioprotection, it is required that the strains are free of acquired antimicrobial resistance genes that could potentially spread to pathogenic bacteria, thereby adding to the pool of resistance genes that may cause treatment failures in humans or animals. Conversely, if antimicrobial resistance is intrinsic to a bacterial species, the risk of spreading horizontally to other bacteria is considered very low. Reliable susceptibility test methods and interpretation criteria at the species level are needed to accurately assess antimicrobial resistance levels. In the present study, tentative ECOFFs for five Bacillus species were determined, and the results showed that the variation in MICs followed the respective species. Moreover, putative resistance genes, which were detected by whole-genome sequencing and suggested to be intrinsic rather that acquired, could explain the resistance phenotypes in most cases.

Comparison of EUCAST and CLSI Reference Microdilution MICs of Eight Antifungal Compounds for Candida auris and Associated Tentative Epidemiological Cutoff Values.

Candida auris is an emerging multidrug-resistant yeast. So far, all but two susceptibility testing studies have examined ≤50 isolates, mostly with the CLSI method. We investigated CLSI and EUCAST MICs for 123 C. auris isolates and eight antifungals and evaluated various methods for epidemiological cutoff (ECOFF) determinations. MICs (in milligrams per liter) were determined using CLSI method M27-A3, and the EUCAST E.Def 7.3. ANOVA analysis of variance with Bonferroni's multiple-comparison test and Pearson analysis were used on log2 MICs (significance at P values of <0.05). The percent agreement (within ±0 to ±2 2-fold dilutions) between the methods was calculated. ECOFFs were determined visually, statistically (using the ECOFF Finder program and MicDat1.23 software with 95% to 99% endpoints), and via the derivatization method (dECOFFs). The CLSI and EUCAST MIC distributions were wide, with several peaks for all compounds except amphotericin B, suggesting possible acquired resistance. Modal MIC, geometric MIC, MIC50, and MIC90 values were ≤1 2-fold dilutions apart, and no significant differences were found. The quantitative agreement was best for amphotericin B (80%/97% within ±1/±2 dilutions) and lowest for isavuconazole and anidulafungin (58%/76% to 75% within ±1/±2 dilutions). We found that 90.2%/100% of the isolates were amphotericin B susceptible based on CLSI/EUCAST methods, respectively (i.e., with MICs of ≤1 mg/liter), and 100%/97.6% were fluconazole nonsusceptible by CLSI/EUCAST (MICs > 2). The ECOFFs (in milligrams per liter) were similar across the three different methods for itraconazole (ranges for CLSI/EUCAST, 0.25 to 0.5/0.5 to 1), posaconazole (0.125/0.125 to 0.25), amphotericin B (0.25 to 0.5/1 to 2), micafungin (0.25 to 0.5), and anidulafungin (0.25 to 0.5/0.25 to 1). In contrast, the estimated ECOFFs were dependent on the method applied for voriconazole (1 to 32) and isavuconazole (0.125 to 4). CLSI and EUCAST MICs were remarkably similar and confirmed uniform fluconazole resistance and variable acquired resistance to the other agents.

Aeromonas Diversity and Antimicrobial Susceptibility in Freshwater-An Attempt to Set Generic Epidemiological Cut-Off Values.

The importance of the role of environment in the dissemination of antimicrobial resistant bacteria is now well recognized. Thus, bacterial indicators to monitor the phenomena are required. The Aeromonas genus is autochthonous in the aquatic environment and easy to detect in any water type, such as freshwater, or wastewater. These microorganisms are also causing infections in humans and animals (including fish). Furthermore, as Aeromonas spp. is able to acquire antimicrobial resistance mechanisms, it is candidate for indicator bacteria to follow antimicrobial resistance dissemination in aquatic environments. Unfortunately, to date, interpretation criteria for Aeromonas spp. for antimicrobial susceptibility tests are scarce in the literature. No epidemiological cut-off values for Aeromonas are currently available at EUCAST to interpret Minimum Inhibitory Concentrations (MIC). The only interpretation criteria available are clinical breakpoints from CLSI that are adapted from Enterobacteriaceae. Based on the results of MIC distributions obtained for a collection of environmental isolates of Aeromonas, this study aimed at proposing tentative epidemiological cut-off values (COWT) for Aeromonas spp. assessing whether the genus is an acceptable level of definition. Thus, 233 isolates collected from 16 rivers were identified at species level using Maldi-Tof (Bruker). Eleven different species were identified, the most abundant were A. bestiarum (n = 54), A. salmonicida (n = 45), A. sobria (n = 41), and A. eucrenophila (n = 37). 96-well micro-plates containing different concentrations of 15 antimicrobials, namely cefotaxime, ceftazidime, chloramphenicol, colistin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, nalidixic acid, oxolinic acid, streptomycin, temocillin, tetracycline, and trimethoprim-sulfamethoxazole, were prepared. The broth micro-dilution method was used to determine the antimicrobial susceptibility of each isolate. The estimation of COWT values was satisfactory obtained at genus level for all antimicrobials except cefotaxime and erythromycin. This first step is an invitation for other research teams to increase the amount of antimicrobial resistance data collected. Then, robustness of our proposed provisional generic epidemiological cut-off values could be assessed by testing antimicrobial susceptibility of various Aeromonas collections.

Comparison of Three Statistical Methods for Establishing Tentative Wild-Type Population and Epidemiological Cutoff Values for Echinocandins, Amphotericin B, Flucytosine, and Six Candida Species as Determined by the Colorimetric Sensititre YeastOne Method

The Sensititre YeastOne (SYO) method is a widely used method to determine the susceptibility of Candida spp. to antifungal agents. CLSI clinical breakpoints (CBP) have been reported for antifungals, but not using this method. In the absence of CBP, epidemiological cutoff values (ECVs) are useful to separate wild-type (WT) isolates (those without mechanisms of resistance) from non-WT isolates (those that can harbor some resistance mechanisms), which is the goal of any susceptibility test. The ECVs for five agents, obtained using the MIC distributions determined by the SYO test, were calculated and contrasted with those for three statistical methods and the MIC(50) and modal MIC, both plus 2-fold dilutions. The median ECVs (in mg/liter) (% of isolates inhibited by MICs equal to or less than the ECV; number of isolates tested) of the five methods for anidulafungin, micafungin, caspofungin, amphotericin B, and flucytosine, respectively, were as follows: 0.25 (98.5%; 656), 0.06 (95.1%; 659), 0.25 (98.7%; 747), 2 (100%; 923), and 1 (98.5%; 915) for Candida albicans; 8 (100%; 352), 4 (99.2%; 392), 2 (99.2%; 480), 1 (99.8%; 603), and 0.5 (97.9%; 635) for C. parapsilosis; 1 (99.2%; 123), 0.12 (99.2%; 121), 0.25 (99.2%; 138), 2 (100%; 171), and 0.5 (97.2%; 175) for C. tropicalis; 0.12 (96.6%; 174), 0.06 (96%; 176), 0.25 (98.4%; 188), 2 (100%; 209), and 0.25 (97.6%; 208) for C. glabrata; 0.25 (97%; 33), 0.5 (93.9%; 33), 1 (91.9%; 37), 4 (100%; 51), and 32 (100%; 53) for C. krusei; and 4 (100%; 33), 2 (100%; 33), 2 (100%; 54), 1 (100%; 90), and 0.25 (93.4%; 91) for C. orthopsilosis. The three statistical methods gave similar ECVs (within one dilution) and included ≥ 95% of isolates. These tentative ECVs would be useful for monitoring the emergence of isolates with reduced susceptibility by use of the SYO method.

Antimicrobial susceptibility testing of lactic acid bacteria and bifidobacteria by broth microdilution method and Etest

We applied two methods of broth microdilution and Etest for measuring minimal inhibition concentration (MIC) of lactic acid bacteria and bifidobacteria for 15 antimicrobial agents to compare the feasibility, reproducibility, and equivalence of the two methods. Both methods were originally described by the European projects PROSAFE and ACE-ART. In 84% combinations of strains and antimicrobial agents MIC differences between the two methods were within one Log 2 dilution. In the case of rifampicin the difference between the two methods was more than ten fold. We further determined MICs of 70 strains (14 strains of Lactobacillus delbrueckii ssp. bulgaricus, 16 strains of Lactococcus lactis, 30 strains of Streptococcus thermophilus, and 10 strains of Bifidobacterium longum) by the broth microdilution method. In most cases, MIC distributions were uni-modal and within 5 Log 2 dilutions except for the MIC distribution of L. lactis to the aminoglycoside group which was broader. These data are a good basis for improving knowledge of antimicrobial susceptibility of lactic acid bacteria and bifidobacteria, and can be used to revise tentative epidemiological cut-off values.

Biosafety assessment of probiotics used for human consumption: recommendations from the EU-PROSAFE project

On June 26–27, 2006, 60 academic and industry scientists gathered during the PROSAFE workshop to discuss recommendations on taxonomy, antibiotic resistance, in vitro assessment of virulence and in vivo assessment of safety of probiotics used for human consumption. For identification of lactic acid bacteria (LAB) intended for probiotic use, it was recommended that conventional biochemical methods should be complemented with molecular methods and that these should be performed by an expert lab. Using the newly developed LAB Susceptibility test Medium (LSM), tentative epidemiological cut-off values were proposed. It was recommended that potentially probiotic strains not belonging to the wild-type distributions of relevant antimicrobials should not be developed as future products for human or animal consumption. Furthermore, it was recommended that the use of strains harbouring known and confirmed virulence genes should be avoided. Finally, for in vivo assessment of safety by investigating strain pathogenicity in animal models, the rat endocarditis model appeared to be the most reliable model tested in the PROSAFE project. Moreover, consensus was reached for approving the necessity of a human colonisation study in a randomised placebo-controlled double-blind design; however, further discussions are needed on the details of such as study.