Distribution Characteristics Of The Bacterial Community And ARGs In The Chicken Cloaca,aerosol,and Workers’ Nasopharynx In A Chicken Farm

ObjectiveBy investigating the microbial community structure and ARGs/MGEs abundance in the chicken cloaca,aerosol and nasopharynx of workers in a poultry farm in Henan Province,to expolre the transmission rules of bacterial taxon and ARGs in breeding animals and human,so as to provide clear clues for controlling the transmission of antibiotic resistance and preventing the occupational hazards related to breeding.Methods1.A large chicken farm in Jiaozuo City,Henan Province was selected as the research site.Cloacal swab samples of two day-old chicken（CA and CB）,aerosol samples inside and outside the chicken coop（AI and AO）,nasopharynx swabs of the first-line and non-first-line workers（FL and NFL）were collected.2.For aerosol samples,the formula was used to calculate the concentration of microorganisms in the air;then the total DNA of aerosol,cloacal swab and worker’s nasopharynx swab samples were extracted.3.The V3-V4 region of 16S r RNA gene was amplified and sequenced by Illumina novaseq technology to characterize the composition of microbial community structure in different sample types.4.High-throughput quantitative PCR（HT-q PCR）was used to quantify 116 ARGs/MGEs in farm environment,16S r RNA gene was used as internal reference gene,and relative abundance was calculated by 2^-△△Ct method.5.The original sequencing data was processed by QIIME software.The data ofαdiversity,dominant taxa,and the relative abundance of ARGs/MGEs among different groups were analyzed by SPSS 21.0.NMDS analysis and network analysis were performed by Gephi 0.9.Results1.A total of 42 samples of different types were collected,including 20 cloacal swab samples of two day-old chicken（10 samples at age of 190 and 520 days,respectively）;3 aerosol samples in chicken house,1 aerosol sample in workers’living area outside chicken house;and 18 nasopharyngeal swab samples of farm workers,including 9 first-line workers and 9 non first-line workers.2.The total bacterial concentration of aerosols inside the chicken house was3.117×10⁴ CFU/m³,while the concentration of the living area（outside the chicken house）was 1.505×10³ CFU/m³.The peak value of aerosols both inside and outside the chicken house appeared at level 1,and were mainly concentrated in the particle size with a diameter>3.3μm.3.The results of Illumina high-throughput sequencing showed that there was no significant difference in the four indices ofαdiversity among the three sample types（C,A and N）（P>0.05）.β-diversity analysis showed that the cloaca swab samples（C）and the workers’nasopharyngeal swab samples（N）overlapped each other,indicating that the microbial communities of the two were closer,while the aerosol samples inside and outside the chicken house（AI and AO）were far from these,and the aggregation degree of worker’s nasopharynx swab samples were the highest（PERMANOVA,stress=0.0563）.There were significant differences in nasopharyngeal microorganisms between first-line workers and non first-line workers.MND1,RS＿045,Burkholderia,I025,Planctomycetia and Chloroflexi were significantly higher in the first-line workers group,while 19 taxa such as Gammaproteobacteria,Pseudomonadales,Moraxelaceae and Haemophilus were significantly higher in the non first-line workers group（P<0.05）.There were 603 OTUs in chicken cloaca,aerosols,and workers’nasopharynx,mainly including Rothia,Corynebacterium,Streptococcus,Lactobacillus,and Prevotella.4.All 116 ARGs/MGEs included in this study were detected,and the order of the detected quantity in each group was:CB>AI>CA>FL>AO>NFL.The abundance distribution of ARGs/MGEs in cloacal swab（C）and aerosol（AI）in chicken house was similar,but it was significantly different from that of aerosol（AO）in workers’living area and ARGs/MGEs in workers’nasopharynx（N）（PERMANOVA,stress=0.073）.In this study,12 ARGs were found in all 42 samples,tet M,erm B,bla TEM and erm X were mainly found in cloacal swab（C）,chicken house aerosol（AI）and workers’nasopharynx（N）;tet32 and erm K-01 were mainly found in chicken cloacal swab（C）and worker throat swab（N）;tet W,aad A1 and str B were mainly found in chicken cloacal swabs（C）and aerosols inside the coop（AI）.Conclusions1.The concentration of aerosol bacteria inside the chicken house of the farm exceeds the limit,so corresponding measures should be taken to reduce the health risk of workers.2.There are consistency between the bacterial community structure of workers’nasopharynx and chicken cloaca,as well as,between the abundance of ARGs in aerosols and chicken cloaca.Bacterial communities and ARGs could be transmitted from animals to workers through aerosols,and other ways may also affect the microbial community in workers’nasopharynx.