Investigation On Bacterial Resistance Of Migratory Birds And The Relationship With Their Habitat Environment

In order to investigate the bacterial resistance of migratory birds and the correlation with habitat ecological factors,samples of migratory bird feces and ecological factors（cattle feces,sheep feces,soil and water samples）were collected at Qinghai Lake in 2019.Combined with the advantages of metagenomic,High-Throughput real-time PCR（HT-qPCR）and traditional bacterial isolating method,the distribution of antibiotic resistance genes（ARGs）and the microbiome of various samples were analyzed.By means of spearman correlation analysis,network analysis and principal component analysis,the correlation between ARGs and microbiome of all samples,as well as the correlation between ARGs of different samples were analyzed to provide theoretical guidance for the tracing and transmission of ARGs in migratory birds.The results of metagenomic sequencing revealed the microbial communities and resistome of migratory birds and ecological factors.The microbiome of migratory birds was significantly different from that of cattle,sheep,soil and water,and the diversity of microbiome in migratory birds was lower than that of other samples（P<0.01）.Among them,the relative abundance of Enterobacteriaceae in migratory birds was significantly higher than that of other samples.The results of source tracker analysis showed that 40.05%of the microbiome of migratory birds may come from cattle,8.37%from sheep,and 2.15%from soil environment.The diversity of resistance genes of migratory birds was higher than that of ecological factors,including 14 kinds of resistance genes which were common in clinic.It is worth noting that multidrug resistance genes are relatively abundant in all kinds of samples,and the main mechanism of multidrug resistance is the expression of efflux pump genes.In order to further explore the distribution and abundance of ARGs in each sample,vital resistance genes were selected to be detected by HT-qPCR.The results showed that the abundance of vital resistance genes in migratory birds was higher than that in ecological factors.The total abundance of vital resistance genes in migratory birds was significantly lower than that in soil and cattle manure（P<0.01）,among whichβ-lactam,tetracycline,quinolones and vancomycin resistant genes were the most significant.It is worth noting that bla _NDM,a carbapenem resistant gene,has a high detection rate in migratory birds and ecological factor samples.The vital resistance genes in migratory bird samples were significantly correlated with cattle,sheep and natural environment and the correlation with environmental samples was stronger（Spearman’s r>0.6,P<0.01）.There was a significant correlation between MEGs and vital resistance genes in all samples,including the elements Intl1,Tp614,IS613,ISApl1 and transposase genes（Spearman’s r>0.3,P<0.01）.According to the correlation analysis of vital resistance genes and microbiome,it was found that add A5,arm A,bcr B,bla _CMY,bla _IMP,bla _OXA-1/30,bla _VIM,drf A12,tet C,tet M,tetx3 and van A resistance genes were common in all samples（Spearman’s r>0.7,P<0.05）,indicating that these resistance genes may be transmitted through related bacteria in each sample.Three strains were found to carry bla _NDM-5,identified as carbapenem resistant Enterobacter hormaechei.These Enterobacter hormaechei were clones from different bird samples,and their MLST type were ST63.The results of second-generation sequencing of these strains showed that bla _CTX-M-55,bla _TEM-1 and bla _NDM-5 were carried,making them multidrug-resistant strains,among which bla _CTX-M-55 was located in IncHI2 plasmid and bla _NDM-5 in IncX3 plasmid,with the risk of horizontal transmission.Combined with metagenomic,HT-qPCR and bacterial isolating method,the drug resistance of migratory birds and the relationship between migratory birds and ecological factors were described comprehensively.As a member of wild animals,migratory birds carry a variety of ARGs.Although the abundance of antibiotic genes in migratory bird is low,they still have the risk of transmission.Moreover,the ecological factors in the habitat of migratory birds have a greater impact on the microbiome and ARGs of migratory birds.