Nguyen Nhung Thi, Phuong Yen Nguyen Thi, Ky Thien Nguyen Van, Van Cuong Nguyen, Kiet Bach Tuan, Campbell James, Thwaites Guy, Baker Stephen, Geskus Ronald B, Carrique-Mas Juan
Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam
Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam.
Appl Environ Microbiol. 2021 Mar 1;87(5). doi: 10.1128/AEM.02597-20. Epub 2020 Dec 18.
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.
在许多低收入和中等收入国家，黏菌素广泛应用于动物生产中。有必要开发方法来对养殖场中混合共生细菌群体的耐药性变化进行基准测试和监测。我们旨在评估一种基于培养从每个样本中获得的混合悬浮液（30 - 50个生物体）的肉汤微量稀释法的性能。为了确认该方法的生物学基础和敏感性，我们在2mg/L黏菌素存在的情况下培养了16种组合，其中一种是对黏菌素敏感的，另一种是编码对黏菌素耐药的。随着时间的推移读取光密度（OD）值以生成生长曲线，并将这些值调整为在无黏菌素情况下获得的值（调整后的曲线下面积，AUC）。检测限的中位数为10个敏感菌落中有1个耐药菌[第1 - 3四分位数，10:1 - 10:1]。我们将此方法应用于来自越南湄公河三角洲36个鸡群的108份混合粪便样本，并确定了该方法与通过最低抑菌浓度测定的从田间样本收获的单个菌落中黏菌素耐药性患病率之间的相关性。在样本和分离株水平上黏菌素耐药性的总体患病率（根据AUC估计）分别为38.9%[95%置信区间，29.8 - 48.8%]和19.4%（标准差±26.3%）。黏菌素耐药性增加与近期（2周）使用黏菌素（比值比 = 3.67）和其他非黏菌素类抗菌药物（比值比 = 1.84）有关。我们的方法是一种敏感且经济实惠的方法，可用于监测粪便样本中群体黏菌素耐药性随时间的变化。黏菌素（多粘菌素E）是一种在基于琼脂的培养基中溶解度较差的抗菌药物，因此肉汤微量稀释是唯一可用于表型耐药性检测的方法。然而，估计混合群体中的黏菌素耐药性很费力，因为它需要单个菌落的分离、鉴定和药敏试验。我们开发了一种适用于混合粪便样本的基于生长的微量稀释法。我们通过将其与909株分离株的个体最低抑菌浓度进行比较来验证该方法；然后我们测试了在其生产周期内收集的来自36个健康鸡群的108份混合粪便样本。在最近在饮水中使用黏菌素的鸡群中观察到更高水平的耐药性，尽管观察到产生的耐药性是短暂的。我们的方法经济实惠，并且有可能整合到旨在估计鸡群/畜群中菌落水平耐药性患病率的监测系统中。此外，它也可能适用于其他复杂的生物系统，如养殖场和屠宰场。
