A novel method for measuring phenotypic colistin resistance in populations from chicken flocks.

Colistin is extensively used in animal production in many low- and middle-income countries. There is a need to develop methodologies to benchmark and monitor changes in resistance among mixed commensal bacterial populations in farms. We aimed to evaluate the performance of a broth microdilution method based on culturing a pooled suspension (30-50 organisms) obtained from each sample. To confirm the biological basis and sensitivity of the method, we cultured 16 combinations of one colistin-susceptible and one -1 encoded colistin-resistant in the presence of 2mg/L colistin. Optical density (OD) readings over time were used to generate a growth curve, and these values were adjusted to the values obtained in the absence of colistin (adjusted Area Under the Curve, AUC). The median limit of detection was 1 resistant in 10 susceptible colonies [1 - 3 quartile, 10:1 -10:1]. We applied this method to 108 pooled faecal samples from 36 chicken flocks from the Mekong Delta (Vietnam), and determined the correlation between this method and the prevalence of colistin resistance in individual colonies harvested from field samples, determined by the Minimum Inhibitory Concentration. The overall prevalence of colistin resistance at sample and isolate level (estimated from the AUC) was 38.9% [95%CI, 29.8-48.8%] and 19.4% (SD± 26.3%), respectively. Increased colistin resistance was associated with recent (2 weeks) use of colistin (OR=3.67) and other, non-colistin antimicrobials (OR=1.84). Our method is a sensitive and affordable approach to monitor changes in colistin resistance in populations from faecal samples over time. Colistin (polymyxin E) is an antimicrobial with poor solubility in agar-based media, and therefore broth microdilution is the only available method for phenotypic resistance. However, estimating colistin resistance in mixed populations is laborious since it requires individual colony isolation, identification and susceptibility testing. We developed a growth-based microdilution method suitable for pooled faecal samples. We validated the method by comparing it with individual MIC of 909 isolates; we then tested 108 pooled faecal samples from 36 healthy chicken flocks collected over their production cycle. A higher level of resistance was seen in flocks recently treated with colistin in water, although the observed generated resistance was short-lived. Our method is affordable, and may potentially be integrated into surveillance systems aiming at estimating the prevalence of resistance at colony level in flocks/herds. Furthermore, it may also be adapted to other complex biological systems, such as farms and abattoirs.