Effect of feed supplementation with humic substances on phenotypic resistance profiles of Escherichia coli isolates from the ceca of broiler chickens

Humic substances (HS), natural complex-forming feed additives, are believed to reduce the bioavailability of antimicrobials to the gut microbiome, thereby limiting the emergence and spread of antimicrobial resistance in the food chain. However, this hypothesis has not yet been adequately verified experimentally. Our study was focused on the isolation of Escherichia coli (E. coli) from the ceca of 80 broiler chickens bred for 37 d under controlled conditions. Chickens in the control and experimental groups (40 broilers each) were fed standard commercial feeds, but the diet for experimental chicken was permanently supplemented with HS (99.3% feed + 0.7% HS). After slaughter and evisceration at the end of fattening period, the cecal contents of all chickens were subjected to microbiological analysis aimed at the enumeration, isolation and genotypic identification of E. coli strains by the species-specific PCR method. In total, 58 E. coli strains from control chickens and 60 strains from HS-fed chickens were tested for resistance against 19 antimicrobials using the broth microdilution method based on minimum inhibitory concentration breakpoints. Resistance to at least one antimicrobial was detected in almost 90% of E. coli isolates from control chickens, but in only 68.33% of isolates from HS-fed broilers (P < 0.01). In the experimental group, a noticeable decrease in resistance rates for ampicillin (31.78%), trimethoprim (22.88%), sulfamethoxazol (20.86%), and tetracycline (19.71%) was observed. The effect for fluorochinolones was less pronounced (0.09 ≤ P < 0.90). Feed supplementation with HS also reduced the prevalence of multidrug resistance (20.0% vs. 43.1%; P < 0.01) and led to a complete elimination of co-resistance to six and more antimicrobial classes. Additionally, no extended-spectrum beta-lactamase production could be predicted for E. coli isolates from HS-fed broilers. This is probably the first experimental study demonstrating a direct beneficial effect of HS-supplemented diet on the phenotypic resistance profiles of E. coli isolates from the ceca of commercially raised broiler chickens, showing that HS as feed additives can effectively reduce the phenotypic expression of antimicrobial resistance in E. coli.