Study On The Contamination Of Florfenicol Resistance Genes In Soils Adjacent To Swine Feedlots

As a phenicols antibiotic, florfenicol (FFC) is widely used in the world and is the largest used veterinary medicine in our country. It mainly used for prevention and treatment of respiratory tract diseases and enteric infections in veterinary clinics. With the extensive use of FFC in livestock, the great concerns has been arisen for FFC resistance, but it is still unknown whether the administration of FFC has resulted in the emergence and dissemination of florfenicol resistance genes (FRGs), especially the multi-resistance gene cfr and optrA which confer the resistance to the promising agent -linezolid-against infections caused by gram-positive bacteria. The aim of this study is to investigate the relationship between the usage of the FFC and contamination of the FRGs in the soil environment.In this study, Firstly, the prevalence of FRGs was investigated in soils adjacent to swine feedlots. Six pig farms keeping use of FFC and irrigation with wastewater/manure were studied. Among these six pig farms, three of them (HN-S-1, HN-S-2 and HN-S-3) were using florfenicol extensively and long-termly, while the other three pig farms (HN-S-4, HN-S-5 and HN-S-6). were with less and short-term usage. We applied a culture-independent method with quantitative polymerase chain reaction (qPCR) to investigate the abundance of FRGs (fexA、fexB、cfr、 optrA、floR and pexA) in soil samples from six swine feedlots. The result showed that all of these six pig farms were contaminated with FRGs. The gene optrA was detected in all of these pig farms, and cfr was also detected in five of these soil samples, but no FRGs was detected in control samples (over 5km away from the pig farm). The qPCR result showed the relative abundance of FRGs from the farms HN-S-4, HN-S-5, and HN-S-6 was much lower than that from the farms HN-S-1, HN-S-2, and HN-S-3. Of the six farms, and the abundance of cfr, optrA, and fexA was significantly higher in soils from HN-S-2 and HN-S-3 comparing to the other four farms (p<0.001) and fexB was only detected in soils from HN-S-2 and HN-S-3. This result indicated that the prevalence and abundance of FRGs might be affected by the administration of FFC in the farms, especially the highlighted resistance genes cfr and optrA in clinical medicine.Secondly, the absolute abundance of the FRGs in soil samples was further quantified using Illumina HiSeq 2500 combined with high-throughput sequencing-based metagenomic analysis. Transposase including IS256, IS6100,IS26, IS1216,ISEnfa4, and ISEnfa5, which played an important role in the horizontal transfer of FRGs, were also identified. The result showed the overall prevalence of FRGs was significantly higher in the long-standing farms (HN-S-1, HN-S-2 and HN-S-3) than the newly established farms (HN-S-4, HN-S-5 and HN-S-6), which was consistent with the qPCR assays. The total number of FRGs in each sample and the abundance of these genes were highly correlated with the levels of transposases in the soil samples (r2=0.9833,p<0.0001). Furthermore, according to metagenomic analysis, other kinds of antibiotic resistance genes have also been found in these soils, including aminoglycosides, tetracyclines, macrolides, and so on. In addition, the analyses of bacterial community compositions and resultant observations showed that the relative abundance of phyla and the possible FRGs-carrying genera in the soils adjacent to the farms was much higher than that in the control samples. All of these results suggest that horizontal gene transfer may have aided in the enrichment of resistance genes through the transfer of mobile genetic elements among soil resistance bacteria.Thirdly, a soil metagenomics library was constructed using pZE21-MCS1 as vector. We screened several resistance clones resistance to FFC by using the FFC selective plates from this library, and identified resistance genes successfully including floR, soxS and marA/R. Besides, a fosmid metagenomics library with 204,000 clones was successfully constructed and screened with FFC for FFC resistance strains, and 11 positive clones were acquired. Two active clones exhibited 16-fold increases in the MICs for FFC compared to the host strain were selected to sequence and screen by subclone library. However, the FFC positive clones was tested as negative for the known FRGs or variant, and the insert fragment may be present a novel FFC resistance gene. The result was still under further verification, but both of these ways provided new methods for determining new resistance genes from antibiotic resistance bacteria.In conclusion, the occurrence of FRGs was investigated in soil samples collected from swine farms. Our findings indicated that the administration of florfenicol in swine husbandry could promote the prevalence of FRGs in soils adjacent to swine feedlots, especially the linezolid resistance genes cfr and optrA. These resistance genes might spread into human from environment via food chain and cause potential risk to food safety and human health, which need further investigation.