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

Colistin (polymyxin E) is an antimicrobial with poor solubility in agar-based media, and therefore, broth microdilution is the only available method for determining phenotypic resistance. However, estimating colistin resistance in mixed Escherichia coli populations is laborious, since it requires individual colony isolation, identification, and susceptibility testing. ABSTRACT Colistin is extensively used in animal production in many low- and middle-income countries. There is a need to develop methods 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 Escherichia coli suspension (30 to 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 mcr-1-carrying colistin-resistant E. coli isolate in the presence of 2 mg/liter colistin. Readings of optical density at 600 nm (OD600) 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 [AUCadj]). The median limit of detection was 1 resistant in 104 susceptible colonies (1st to 3rd quartile, 102:1 to 105:1). We applied this method to 108 pooled fecal 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 MIC. The overall prevalences of colistin resistance at the sample and isolate levels (estimated from the AUCadj) were 38.9% (95% confidence intervals [CI], 29.8 to 48.8%) and 19.4% ± 26.3% (± values are standard deviations [SD]), respectively. Increased colistin resistance was associated with recent (2 weeks) use of colistin (odds ratio [OR] = 3.67) and other, noncolistin antimicrobials (OR = 1.84). Our method is a sensitive and affordable approach to monitor changes in colistin resistance in E. coli populations from fecal samples over time. IMPORTANCE Colistin (polymyxin E) is an antimicrobial with poor solubility in agar-based media, and therefore, broth microdilution is the only available method for determining phenotypic resistance. However, estimating colistin resistance in mixed Escherichia coli populations is laborious, since it requires individual colony isolation, identification, and susceptibility testing. We developed a growth-based microdilution method suitable for pooled fecal samples. We validated the method by comparing it with individual MIC determinations for 909 E. coli isolates; we then tested 108 pooled fecal 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.