The impact of inoculation methods on bacterial aggregation and antimicrobial susceptibility testing

Antimicrobial susceptibility testing (AST) has been a gold standard for guiding physician treatment practices for accurate antibiotic prescription, of which the broth microdilution method is recommended and commonly used. Different inoculation methods and preparative steps of bacterial suspension for AST are applied in various settings, which are based on the assumption that planktonic broth cultures contain homogeneous single cells. However, this has been challenged by the common observation of non-surface-attached aggregates in shaking cultures. Whether different inoculation methods producing different aggregation patterns contribute to inconsistent AST results has not been systematically investigated. To address this, we evaluated how routinely used inoculation methods for Pseudomonas aeruginosa influence aggregation levels in liquid cultures, using 2D micrographs and novel mathematical indexes designated as the Entropic Coefficient (EC) and Dispersion Coefficient (DC). The aggregation levels of bacterial populations generated by different inoculation methods prior to AST were highly variable and were successfully differentiated by EC and DC using fluorescent or bright-field micrographs. The susceptibility of P. aeruginosa towards antibiotics was then measured using inocula prepared with these different methods. In line with the variations in aggregation patterns reflected by micrographs, different inoculation methods also resulted in inconsistent AST results, where, in general, directly resuspending agar colonies in broth yielded more consistent MIC results compared with other preparations. Our work indicates that different inoculation methods influence aggregation behaviours and AST results and provides rapid and inexpensive methods to quantify aggregation levels using 2D micrographs.