Method for Measuring Phenotypic Colistin Resistance in Escherichia coli Populations from Chicken Flocks

Abstract

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 Escherichia coli 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 mcr-1 encoded colistin-resistant E. coli in the presence of 2mg/L colistin. Optical density (OD600nm) 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, AUCadj). The median limit of detection was 1 resistant in 104 susceptible colonies [1st - 3rd quartile, 102:1 –105:1]. 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 AUCadj) 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 E. coli populations from faecal 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 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 faecal samples. We validated the method by comparing it with individual MIC of 909 E. coli 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.

Modelling the impact of antimicrobial use and external introductions on commensal E. coli colistin resistance in small‐scale chicken farms of the Mekong delta of Vietnam

“…Here, the collection of detailed and reliable AMU data on a weekly basis allows us to account for the dynamics of the effect of AMU on AMR. We find that our measure of colistin resistance is associated with the use of antimicrobials during the 1.73 weeks preceding the sample collection, similarly to (Nhung et al., 2021 ). The reason may be that AMU selects for colistin resistant E. coli strains that were already present at low level or that were introduced to the farm.…”

“…For each sample, this measure was repeated twice and averaged. The method, validated by Nhung et al (2021), aims to obtain an aggregated estimate of colistin resistance among a representative sample of the whole E. coli population circulating in the guts of all the chickens of the flock of each farm and at each time point.…”

Impact of antimicrobial use on abundance of antimicrobial resistance genes in chicken flocks in Vietnam

“…13–16 Studies in the Mekong Delta region have documented an increase in colistin and gentamicin resistance in Escherichia coli from two-month-old chickens compared with day-old chicks. 14 , 15 In contrast, decreases in prevalence of resistance to tetracyclines and sulphonamides have been reported in E. coli isolated from day-old chicks to maturity in laying hens in Europe. 16 Using longitudinal data from chicken flocks in the Mekong Delta (Vietnam), a modelling study on colistin resistance indicated that AMU in the early stage of production or introduction from hatcheries is far less determinant than importation from the environment and use in latter stages.…”