The Clinical and Laboratory Standards Institute has published epidemiological cut-off values for susceptibility data generated at 22°°C and read after 44-48 h for florfenicol, oxolinic acid and oxytetracycline against Aeromonas salmonicida. The cut-off values for the minimum inhibitory concentration (MIC) and disc diffusion were derived from data obtained by 1 laboratory and 2 laboratories respectively. The present work reports the generation of susceptibility data from additional laboratories and the calculation of provisional cut-off values from aggregations of these data with previously published data. With respect to MIC data, the provisional cut-off values, derived from aggregations of the data from 4 laboratories, were ≤4 µg ml-1 for florfenicol, ≤0.0625 µg ml-1 for oxolinic acid and ≤1 µg ml-1 for oxytetracycline. For disc diffusion data, the provisional cut-off values derived from aggregations of the data from 5 laboratories were ≥30 mm for florfenicol, ≥32 mm for oxolinic acid and ≥25 mm for oxytetracycline. In addition, a cut-off value of ≥29 mm for ampicillin was derived from the aggregation of data from 4 laboratories.

BACKGROUND: Aspergillus species cause a variety of serious clinical conditions with increasing trend in antifungal resistance. The present study aimed at evaluating hospital epidemiology and antifungal susceptibility of all isolates recorded in our clinical database since its implementation.
METHODS: Data on date of isolation, biological samples, patients\' age and sex, clinical settings, and antifungal susceptibility tests for all Aspergillus spp. isolated from 2015 to 2022 were extracted from the clinical database. Score test for trend of odds, non-parametric Mann Kendall trend test and logistic regression analysis were used to analyze prevalence, incidence, and seasonality of Aspergillus spp. isolates.
RESULTS: A total of 1126 Aspergillus spp. isolates were evaluated. A. fumigatus was the most prevalent (44.1%) followed by A. niger (22.3%), A. flavus (17.7%) and A. terreus (10.6%). A. niger prevalence increased over time in intensive care units (p-trend = 0.0051). Overall, 16 (1.5%) were not susceptible to one azole compound, and 108 (10.9%) to amphotericin B, with A. niger showing the highest percentage (21.9%). The risk of detecting A. fumigatus was higher in June, (OR = 2.14, 95% CI [1.16; 3.98] p = 0.016) and reduced during September (OR = 0.48, 95% CI [0.27; 0.87] p = 0.015) and October as compared to January (OR = 0.39, 95% CI [0.21; 0.70] p = 0.002. A. niger showed a reduced risk of isolation from all clinical samples in the month of June as compared to January (OR = 0.34, 95% CI [0.14; 0.79] p = 0.012). Seasonal trend for A. flavus showed a higher risk of detection in September (OR = 2.7, 95% CI [1.18; 6.18] p = 0.019), October (OR = 2.32, 95% CI [1.01; 5.35] p = 0.048) and November (OR = 2.42, 95% CI [1.01; 5.79] p = 0.047) as compared to January.
CONCLUSIONS: This is the first study to analyze, at once, data regarding prevalence, time trends, seasonality, species distribution and antifungal susceptibility profiles of all Aspergillus spp. isolates over a 8-year period in a tertiary care center. Surprisingly no increase in azole resistance was observed over time.

This work generates the data needed to set epidemiological cut-off values for disc-diffusion zone measurements of Vibrio cholerae. The susceptibility of 147 European isolates of non-O1/non-O139 V. cholerae to 19 antibiotics was established using a standardised disc diffusion method which specified incubation of Mueller Hinton agar plates at 35°C. Epidemiological cut-off values were calculated by analysis of the zone size data with the statistically based normalised resistance interpretation method. Cut-off values for 17 agents were calculated by analysis of the aggregated data from all 4 laboratories participating in this study. The cut-off values calculated were ≥18 mm for amoxicillin/clavulanate, ≥18 mm for amikacin, ≥19 mm for ampicillin, ≥27 mm for cefepime, ≥31 mm for cefotaxime, ≥24 mm for ceftazidime, ≥24 mm for chloramphenicol, ≥31 mm for ciprofloxacin, ≥16 mm for erythromycin, ≥ 27 mm for florfenicol, ≥16 mm for gentamicin, ≥23 mm for imipenem, ≥25 mm for meropenem, ≥29 mm for nalidixic acid, ≥28 mm for norfloxacin, ≥13 mm for streptomycin and ≥23 mm for tetracycline. For the other 2 agents the data from 1 laboratory was excluded from the censored aggregation because the data from that laboratory was considered excessively imprecise. The cut-off values for these 2 agents calculated for the aggregation of the data from 3 laboratories were ≥23 mm for trimethoprim and ≥24 mm for trimethoprim/sulfamethoxazole. These zone size data will be submitted to the Clinical Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST) for their consideration in setting international consensus epidemiological cut-off values for non O1/non-O139 V. cholerae.

OBJECTIVE: Streptococcus parauberis is responsible for the development of streptococcosis in marine fish. The aim of the current study was to determine the antimicrobial susceptibility of aquatic Strep. parauberis strains, thus establishing laboratory-specific epidemiological cut-off (COWT) values to distinguish wild-type (WT) and nonwild-type (NWT) strains.
RESULTS: Using 220 Strep. parauberis isolates obtained from diseased Paralichthys olivaceus, Platichthys stellatus, and Sebastes schlegelii over 6 years from seven different locations in Korea, we established COWT values for eight common antimicrobial agents using the standard broth microdilution method. The COWT values calculated using MIC distribution with the NRI and ECOFFinder methods were the same or within one dilution step for the eight antimicrobials tested. Nine NWT isolates with decreased susceptibility to at least two antimicrobials and one of these isolates exhibited decreased susceptibility to six antimicrobial agents were identified using COWT values based on NRI.
CONCLUSIONS: Interpretive criteria for Strep. parauberis have not yet been established, and the findings of this study provide putative COWT values for eight antimicrobial agents frequently used in aquaculture in Korea.

OBJECTIVE: To determine the epidemiological cut-off values (ECVs) of ten antifungal agents in a wide range of yeasts and Aspergillus spp. using gradient concentration strips.
METHODS: The minimum inhibitory concentrations for amphotericin B, anidulafungin, caspofungin, micafungin, flucytosine, fluconazole, itraconazole, isavuconazole, posaconazole, and voriconazole, determined with gradient concentration strips at 35 French microbiology laboratories between 2002 and 2020, were retrospectively collected. Then, the ECVs were calculated using the iterative method and a cut-off value of 97.5%.
RESULTS: Minimum inhibitory concentrations were available for 17 653 clinical isolates. In total, 48 ECVs (including 32 new ECVs) were determined: 29 ECVs for frequent yeast species (e.g. Candida albicans and itraconazole/flucytosine, and Candida glabrata species complex [SC] and flucytosine) and rare yeast species (e.g. Candida dubliniensis, Candida inconspicua, Saccharomyces cerevisiae, and Cryptococcus neoformans) and 19 ECVs for Aspergillusflavus SC, Aspergillusfumigatus SC, Aspergillusnidulans SC, Aspergillusniger SC, and Aspergillusterreus SC.
CONCLUSIONS: These ECVs can be added to the already available gradient concentration strip-specific ECVs to facilitate minimum inhibitory concentration interpretation and streamline the identification of nonwild type isolates.

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.

BACKGROUND: Eumycetoma is a neglected tropical disease. It is a chronic inflammatory subcutaneous infection characterised by painless swellings which produce grains. It is currently treated with a combination of itraconazole and surgery. In an ongoing clinical study, the efficacy of fosravuconazole, the prodrug of ravuconazole, is being investigated. For both itraconazole and ravuconazole, no clinical breakpoints or epidemiological cut-off values (ECV) to guide treatment are currently available.
OBJECTIVE: To determine tentative ECVs for itraconazole and ravuconazole in Madurella mycetomatis, the main causative agent of eumycetoma.
METHODS: Minimal inhibitory concentrations (MICs) for itraconazole and ravuconazole were determined in 131 genetically diverse clinical M. mycetomatis isolates with the modified CLSI M38 broth microdilution method. The MIC distributions were established and used to determine ECVs with the ECOFFinder software. CYP51A sequences were sequenced to determine whether mutations occurred in this azole target gene, and comparisons were made between the different CYP51A variants and the MIC distributions.
RESULTS: The MICs ranged from 0.008 to 1 mg/L for itraconazole and from 0.002 to 0.125 mg/L for ravuconazole. The M. mycetomatis ECV for itraconazole was 1 mg/L and for ravuconazole 0.064 mg/L. In the wild-type population, two CYP51A variants were found for M. mycetomatis, which differed in one amino acid at position 499 (S499G). The MIC distributions for itraconazole and ravuconazole were similar between the two variants. No mutations linked to decreased susceptibility were found.
CONCLUSIONS: The proposed M. mycetomatis ECV for itraconazole is 1 mg/L and for ravuconazole 0.064 mg/L.

The abuse of antibiotics in aquaculture has led to the increasing rate of antibiotic resistance of aquatic bacteria including Aeromonas, which is an increasing threat to environmental and human health. To date, no epidemiological cut-off values (COWT) for Aeromonas spp. have been established by the Clinical and Laboratory Standards Institute nor the European Commission on Antimicrobial Susceptibility Testing. In this study, commercially prepared minimum inhibitory concentration (MIC) test 96-well plates (dry-form plates) were used to determine the MIC of eight antimicrobial agents against 556 Aeromonas strains. The obtained MIC distributions were simulated and analyzed by NRI and ECOFFinder to obtain tentative COWT values for Aeromonas spp. The COWT values of eight kinds of representative antimicrobial agents including trimethoprim-sulfamethoxazole, erythromycin, doxycycline, neomycin, colistin, florfenicol, enrofloxacin, and ceftazidime for Aeromonas spp. were established and were 0.25, 64/32, 4/2, 8, 4, 1, 0.062/0.125, and 0.5 μg/mL, respectively. Results showed that Aeromonas spp. had a very high proportion of non-wild-type strains to enrofloxacin, florfenicol, and doxycycline, which are the most widely used antimicrobials in aquaculture. The COWT values for Aeromonas spp. obtained in this study can contribute to the final establishment of COWT for Aeromonas spp. internationally.

Increasing antimicrobial resistance (AMR) colonizing domesticated animals is a global concern threatening food safety. This study aimed at determining the prevalence of multidrug resistance (MDR) and epidemiological cut-off values (ECVs) of E. coli isolated from poultry and pigs in Mwanza, Tanzania. This cross-sectional study was conducted between June and August 2021, involving 297 pigs, 191 broilers, and 203 layers. Rectal and cloacal swabs were collected and processed following standard guidelines. ECVs were determined using normalized resistance interpretation (NRI), a computer software, and descriptive analysis was performed using STATA version 13.0. The overall prevalence of MDR E.coli was 63.2%, whereas poultry (87.5% layers and 86.3% broilers) were more colonized than pigs (31.8%) (p < 0.001). Based on ECVs of antibiotics tested, E. coli from broilers, layers, and pigs exhibited different resistance patterns hence different populations. Exotic breed (p < 0.001) and recent antimicrobial use (p < 0.001) significantly predicted colonization with MDR E. coli. Veterinary officers should implement regulations that prohibit the inappropriate use of antimicrobial agents in livestock keeping.

The emergence of antimicrobial-resistant bacteria is an enormous challenge to public health. Aeromonas hydrophila and Aeromonas veronii are opportunistic pathogens in fish. They exert tremendous adverse effects on aquaculture production, owing to their acquired antibiotic resistance. A few Clinical and Laboratory Standards Institute (CLSI) epidemiological cut-off values (ECVs) against Aeromonas spp. are available. We evaluated antimicrobial susceptibility by establishing 8 ECVs using two analytical methods, normalized resistance interpretation and ECOFFinder. We detected antimicrobial resistance genes in two motile Aeromonas spp. isolated from aquatic animals. Results showed that 89.2% of A. hydrophila and 75.8% of A. veronii isolates were non-wild types according to the oxytetracycline ECVCLSI and ECVNRI, respectively. The antimicrobial resistance genes included tetA, tetB, tetD, tetE, cat, floR, qnrA, qnrB, qnrS, strA-strB, and aac(6\')-1b. The most common tet gene in Aeromonas spp. isolates was tetE, followed by tetA. Some strains carried more than one tet gene, with tetA-tetD and tetA-tetE found in A. hydrophila; however, tetB was not detected in any of the strains. Furthermore, 18.6% of A. hydrophila and 24.2% of A. veronii isolates showed presumptive multidrug-resistant phenotypes. The emergence of multidrug resistance among aquatic aeromonads suggests the spread of drug resistance and difficult to treat bacterial infections.