RESUMEN

Background: There is growing interest in understanding gut microbiome dynamics, to increase the sustainability of livestock production systems and to better understand the dynamics that regulate antibiotic resistance genes (i.e., the resistome). High-throughput sequencing of RNA transcripts (RNA-seq) from microbial communities (metatranscriptome) allows an unprecedented opportunity to analyze the functional and taxonomical dynamics of the expressed microbiome and emerges as a highly informative approach. However, the isolation and preservation of high-quality RNA from livestock fecal samples remains highly challenging. This study aimed to determine the impact of the various sample storage and RNA extraction strategies on the recovery and integrity of microbial RNA extracted from selected livestock (chicken and pig) fecal samples. Methods: Fecal samples from pigs and chicken were collected from conventional slaughterhouses. Two different storage buffers were used at two different storage temperatures. The extraction of total RNA was done using four different commercially available kits and RNA integrity/quality and concentration were measured using a Bioanalyzer 2100 system with RNA 6000 Nano kit (Agilent, Santa Clara, CA, USA). In addition, RT-qPCR was used to assess bacterial RNA quality and the level of host RNA contamination. Results: The quantity and quality of RNA differed by sample type (i.e., either pig or chicken) and most significantly by the extraction kit, with differences in the extraction method resulting in the least variability in pig feces samples and the most variability in chicken feces. Considering a tradeoff between the RNA yield and the RNA integrity and at the same time minimizing the amount of host RNA in the sample, a combination of storing the fecal samples in RNALater at either 4 °C (for 24 h) or -80 °C (up to 2 weeks) with extraction with PM kit (RNEasy Power Microbiome Kit) had the best performance for both chicken and pig samples. Conclusion: Our findings provided a further emphasis on using a consistent methodology for sample storage, duration as well as a compatible RNA extraction approach. This is crucial as the impact of these technical steps can be potentially large compared with the real biological variability to be explained in microbiome and resistome studies.

Asunto(s)

Microbioma Gastrointestinal , Microbiota , Porcinos , Animales , Ganado/genética , ARN/genética , Microbiota/genética , Microbioma Gastrointestinal/genética , Heces/microbiología

RESUMEN

The emergence of resistance against common antibiotics in the gut microbiota is a major issue for both human and livestock health. This highlights the need for understanding the impact of such application on the reservoir of antibiotic resistance genes in poultry gut and devising means to circumvent the potential resistome expansion. Phytogenic feed additives (PFAs) are potential natural alternative to antibiotic to improve animal health and performance, supposedly via positively affecting the gut microbial ecosystem, but there is little systematic information available. In this time-course study, we applied a shotgun meta-transcriptomics approach to investigate the impact of a PFA product as well as the commonly used antibiotic, zinc bacitracin either at AGP concentration or therapeutic concentration on the gut microbiome and resistome of broiler chickens raised for 35 days. Over the course of the trial, PFA treatments increased the abundance of Firmicutes such as Lactobacillus and resulted in a lower abundance of Escherichia, while the latter group increased significantly in the feces of chickens that received either AGP or AB doses of bacitracin. Tetracycline resistance and aminoglycoside resistance were the predominant antibiotic resistance gene (ARG) classes found, regardless of the treatment. PFA application resulted in a decrease in abundance of ARGs compared to those in the control group and other antibiotic treatment groups. In summary, the findings from this study demonstrate the potential of phytogenic feed additives could be an alternative to antibiotics in poultry farming, with the added benefit of counteracting antimicrobial resistance development.