Prevalence and antimicrobial resistance of Enterococcus spp. isolated from animal feed in Japan.

The rising prevalence of antimicrobial resistance (AMR) of bacteria is a global health problem at the human, animal, and environmental interfaces, which necessitates the "One Health" approach. AMR of bacteria in animal feed are a potential cause of the prevalence in livestock; however, the role remains unclear. To date, there is limited research on AMR of bacteria in animal feed in Japan. In this study, a total of 57 complete feed samples and 275 feed ingredient samples were collected between 2018 and 2020. Enterococcus spp. were present in 82.5% of complete feed (47/57 samples), 76.5% of soybean meal (62/81), 49.6% of fish meal (55/111), 33.3% of poultry meal (22/66), and 47.1% of meat and bone meal (8/17) samples. Of 295 isolates, E. faecium (33.2% of total isolates) was the dominant Enterococcus spp., followed by E. faecalis (14.2%), E. hirae (6.4%), E. durans (2.7%), E. casseliflavus (2.4%), and E. gallinarum (1.0%). Of 134 isolates which were tested for antimicrobial susceptibility, resistance to kanamycin was the highest (26.1%), followed by erythromycin (24.6%), tetracycline (6.0%), lincomycin (2.2%), tylosin (1.5%), gentamicin (0.8%), and ciprofloxacin (0.8%). All Enterococcus spp. exhibited susceptibility to ampicillin, vancomycin, and chloramphenicol. Of 33 erythromycin-resistant isolates, only two showed a high minimum inhibitory concentration value (>128 µg/mL) and possessed ermB. These results revealed that overall resistance to antimicrobials is relatively low; however, animal feed is a source of Enterococcus spp. It is essential to elucidate the causative factors related to the prevalence of AMR in animal feed.

[Validation Study on a Method of Determination of Nosiheptide in Formula Feeds by HPLC-FL].

A determination method of nosiheptide in formula feeds by HPLC-FL was developed and validated, including an inter-laboratory study. Formula feeds were extracted with acetone after adding acetic acid. Liquid chromatographic separation was performed using a ZORBAX Eclipse XDB-C18 column, with acetonitrile and water containing acetic acid as the mobile phase. Detection of NH was carried out with a fluorescence detector. Recovery tests and an inter-laboratory study were conducted using chicken and swine formula feeds fortified with nosiheptide at 0.5-27 mg-potency/kg. Mean recoveries in recovery tests ranged from 91.4 to 103%, and the repeatability in terms of relative standard deviation was within 7.8%. Mean recoveries of the inter-laboratory study ranged from 98.4 to 108%, the repeatability and reproducibility in terms of the relative standard deviations were within 8.1% and 13% respectively, and the HorRat values ranged from 0.21 to 0.75.

Investigation of whether CLSI broth microdilution method is applicable for MICs Determination of Enterococcus species.

The broth microdilution (BMD) method is an antimicrobial susceptibility testing method defined as a guideline by the Clinical and Laboratory Standards Institute (CLSI). To date, the Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM), has adopted the agar dilution (AD) method for monitoring antimicrobial resistances targeting isolates of Enterococcus spp., found in the fecal flora of food-producing animals, as indicator bacteria. However, the AD method is tedious, and time-consuming. In order to examine whether it could be replaced with the BMD method, the two methods were compared in terms of the correlation of MICs. In this study, the BMD results agreed with the AD results within ±1 log(2) dilutions in 72.3% of cases, except for the antimicrobial feed additive, Nosiheptide (NHT). Similarly, except for NHT, the MIC(50) and MIC(90) values obtained by the two methods were well correlated. In conclusion, our results indicate that the BMD method might be suitable for antimicrobial susceptibility testing targeting Enterococcus spp..