Study On The Suspected Hemorrhagic Pneumonia Pathogenic Bacteria And Related Genes Of Drug Resistance In Malay Pangolins Based On High Throughput Sequencing

Many literatures have pointed out that the influence of antimicrobial resistance(AMR)and Virulence factors on public health safety in protective animals is becoming more and more important.At present,the monitoring and attention on bacterial resistance in clinic is not deep enough and comprehensive,and the research on bacterial resistance has not formed a complete the oretical basis.Malay pangolin is one of the most important species of wild animal protection at present,because of its unique medicinal characteristics,they have long been subjected to poaching and illegal traffic,or even cross-border smuggling.However,at present,the breeding of pangolins is still in the exploratory stage all over the world.Pangolins often suffer from a variety of diseases,and the therapeutic effect on pangolins is not obvious.In this study,some cases of suspected hemorrhagic pneumonia of Malay pangolins that receiving and curing by Terrestrial Wildlife Rescue and Epidemic Diseases Surveillance Center of Guangxi were detected and analyzed to investigated the pathogenic bacteria and drug resistance carried by them,and the results are summarized as follows.Part Ⅰ Screening of pathogenic bacteria in suspected hemorrhagic pneumonia of Malay pangolins based on high throughput sequencingObjective It provides a good genomic data reference for analyzing the screening of pathogenic bacteria in Malay pangolin pneumonia.This study wants to further clarify the pathogenic microorganisms that cause the suspected hemorrhagic pneumonia of Malay pangolins,and lay a solid foundation for the prevention and treatment of pangolins’ disease.Methods In this study,we collected lung tissues from Malay pangolins suspected of hemorrhagic pneumonia and non-pneumonia dead Malay pangolins.We extracted DNA from all the tissues and sequenced the whole genome of Malay pangolins’ lung tissues suspected of pneumonia.Finally,the sequencing results were analyzed,and PCR was used to verify lung tissues of Malay pangolins suspected of hemorrhagic pneumonia and non-pneumonia death.Results 37 bacteria were found in the lung tissues of 16 cases of Malay pangolins suspected of hemorrhagic pneumonia with macrogenome sequencing,mainly including Pseudomonas,Staphylococcus,Streptococcus,Bacillus,Clostridium,Mycoplasma,Spiroplasma,Ureaplasma,Corynebacterium,Lactobacillus,Lactococcus,Enterococcus,Proteus and Morganella.PCR results showed that Pseudomonas and Bacillus were detected in the lung tissues of the suspected hemorrhagic pneumonia dead Malay pangolins and non-pneumonia dead Malay pangolins,while Staphylococcus,Streptococcus and Clostridium and Morgenella were found only in the lung tissues of the suspected hemorrhagic pneumonia dead Malay pangolins.Conclusion Through sequencing analysis and PCR verification of the whole genome,we found a variety of bacteria in the samples of the suspected hemorrhagic pneumonia dead Malay pangolins,and we inferred that there may be a synergistic effect of many bacteria on the cause of death of Malay pangolins.In order to further clarify the etiology of Malay pangolins,we need to carry out more in-depth research and exploration.Part Ⅱ Drug resistance genes of dominant strains associated with suspected hemorrhagic pneumonia of Malay pangolinsObjective In order to further verify the above results and implement targeted detection,it is expected to obtain more detailed experimental data to support clinical diagnosis.Methods In this study,we re-collected the dead individuals of Malay pangolins with suspected hemorrhagic pneumonia during the rescue period,collected the lung samples from the dead Malay pangolins for culture and purification of bacteria,and determined the bacteria by 16 s r DNA universal primer PCR,and so on.We sequenced the whole genome of positive bacteria.The results of bacterial PCR sequencing were mapped to the data of NCBI database to determine the species of bacteria.In addition,the cultured and purified bacteria were sequenced by DNA small fragment library(PE150),and the drug resistance genes were analyzed after splicing and assembling,and the drug resistance of pathogenic bacteria was discussed.Results Bacterial culture and sequencing showed that all of bacterial species belonged to staphylococcus,and staphylococcus aureus can be found in almost samples.Combined with clinical data,we selected two dominant bacteria(staphylococcus haemolyticus and staphylococcus aureus)to sequence the whole genome,and analyzed the drug resistance genes.These two strains carry many types of antibiotic resistance genes(including vancomycin,teicoplanin,bacterin,bacillus subtilis antibiotics,sodium antibiotics,macrolides,lincosamide,streptogramin_b,etc).At the same time,clinical treatment showed poor treatment on bacteria carrying these antibiotic resistant genes.Conclusion This study showed that staphylococcus was the dominant flora,in the lung tissues of the suspected hemorrhagic pneumonia dead Malay pangolins,especially staphylococcus aureus.These staphylococci carry many types of antibiotic resistance genes,which may pose a threat animal health.