Development of a rapid diagnostic method for antimicrobial susceptibility testing of Enterobacterales using a drug...

The global spread of drug-resistant bacteria represents a critical public health challenge, underscoring the need for rapid and reliable antibiotic susceptibility testing. Recently developed drug susceptibility testing microfluidic (DSTM) devices enable rapid assessment by analyzing antibiotic-induced bacterial morphological changes. However, interpretation of these changes is complex and subjective, necessitating automation. In this study, we developed an image analysis-based interpretation system for DSTM devices and evaluated its performance and practical utility.

Using reference strains and 975 Enterobacterales isolates collected in Japan, we developed an algorithm that assigns susceptibility categories based on DSTM results. Eight antibiotics were evaluated: ampicillin (ABPC), cefazolin (CEZ), cefmetazole (CMZ), cefotaxime (CTX), cefepime (CFPM), meropenem (MEPM), levofloxacin (LVFX), and amikacin (AMK). Performance was assessed using 59 clinical isolates and 10 standard strains, with results compared with those obtained using the Clinical and Laboratory Standards Institute M07 broth microdilution method.

Category agreement was highest for CEZ and LVFX (98.6%) and lowest for CMZ (82.6%). Major error rates were 0% for CFPM, LVFX, MEPM, and AMK, whereas ABPC and CTX showed the highest major error rate (7.2%). No very major errors were observed for CEZ; however, MEPM showed a relatively high very major error rate (13.0%). Although discrepancies occurred between algorithm-based and morphology-based interpretations for some antibiotics, morphological transitions generally corresponded to susceptibility profiles in most strains.

Image analysis-based automation reduced subjectivity associated with conventional visual evaluation and yielded highly reproducible results, enabling quickly determination of Enterobacterales antimicrobial susceptibility within 3 h.