Early detection of drug resistance contributes to combating drug-resistant bacteria and improving patient outcomes. Microbial testing in the laboratory is essential for treating infectious diseases because it can provide critical information related to identifying pathogenic bacteria and their resistance profiles. Despite these clinical requirements, conventional phenotypic testing is time-consuming. Additionally, recent rapid drug resistance tests are not compatible with fastidious bacteria such as Streptococcus and Haemophilus species. In this study, we validated the feasibility of direct bacteria counting using highly sensitive quantitative flow cytometry. Furthermore, by combining flow cytometry and a nucleic acid intercalator, we constructed a highly sensitive method for counting viable fastidious bacteria. These are inherently difficult to measure due to interfering substances from nutrients contained in the medium. Based on the conventional broth microdilution method, our method acquired a few microliter samples in a time series from the same microplate well to exclude the growth curve inconsistency between the samples. Fluorescent staining and flow cytometry measurements were completed within 10 min. Therefore, this approach enabled us to determine antimicrobial resistance for these bacteria within a few hours. Highly sensitive quantitative flow cytometry presents a novel avenue for conducting rapid antimicrobial susceptibility tests.

Early detection of drug resistance contributes to combating drug-resistant bacteria and improving patient outcomes. Microbial testing in the laboratory is essential for treating infectious diseases because it can provide critical information related to identifying pathogenic bacteria and their resistance profiles. Despite these clinical requirements, conventional phenotypic testing is time-consuming. In addition, recent rapid drug resistance tests are not compatible with fastidious bacteria such as Streptococcus and Haemophilus species.In this study, we validated the feasibility of direct bacteria counting using highly sensitive quantitative flow cytometry. Furthermore, by combining flow cytometry and a nucleic acid intercalator, we constructed a highly sensitive method for counting viable fastidious bacteria. These are inherently difficult to measure due to interfering substances from nutrients contained in the medium. Based on the conventional broth microdilution method, our method acquired a few microliter samples in a time series from the same microplate well to exclude the growth curve inconsistency between the samples. Fluorescent staining and FCM measurements were completed within 10 minutes. Therefore, this approach enabled us to determine antimicrobial resistance for these bacteria within a few hours. Highly sensitive quantitative flow cytometry presents a novel avenue for conducting rapid antimicrobial susceptibility tests.