Epidemiological Investigation Of Peudomonas Aeruginosa In Breeding Poultry Field And Rapid Detection Of PA By Cross Priming Amplification

Pseudomonas aeruginosa(PA)is a common opportunistic pathogen of zoonotic infection.This bacterium is one of the most common pathogens in hospitals,which often causes high mortality of patients.At the same time,this bacterium has pathogenicity to many kinds of animals,such as livestock and poultry,and has important public health significance.Pathogenic PA has been isolated from almost all animals,with the highest separation rate of avian PA.At present,with the continuous expansion of the scale of the breeding industry,PA-caused epidemics are on the rise,causing harm to the breeding industry.However,currently,little is known about the epidemic situation and drug resistance data of animal-derived pseudomonas aeruginosa,so it is of great significance to carry out epidemiological research on animal-derived pseudomonas aeruginosa.In order to learn about the prevalence of PA in Guangdong province from breeding poultry field,in this research,we collected dead embryos and their environmental samples total up to 1224 samples from breeding poultry field in different regions of Guangdong province from March 2018 to January 2019,including 655 chickens,318 pheasants,40geese,18 quails and 193 environmental samples.A total of 233 strains of PA including 37strains(19.17%)from environmental samples and 196 strains(19.01%)from dead embryos,including 75 strains of chickens(11.45%),109 strains of pheasants(34.28%)and 12 strains of geese(30%)were isolated(the isolation rate was 19.04%).22 kinds of commonly used antibiotic drug sensitivity test showed that 233 strains of PA were strongly resistant to sulfamethoxazole(99.1%),tetracycline(77.3%)and chloramphenicol(76.0%),followed by cefotaxime(16.3%),and the proportion of multiple drug resistant PA was up to 67.4%,and appeared a certain proportion of PA strains which resistanted to imipenem,piperacillin,cefepime and amikacin.27 kinds of resistance genes of all the isolates were detected by PCR,a total of 22 kinds of drug-resistant genetic testing positive,the detection rate of opr D₂ of carbapenems was the highest(29.18%);And qnr D(11.59%)was the main resistance gene of fluoroquinolones,which was consistent with the drug resistance rate of phenotypes.The highest detection rates of aminoglycosides were aac(3)-II and ant(3")-I,both of which were 10.73%.The main chloramphenicol group was cml A(16.31%),the tetracycline group had the highest detection rate of tet A(10.30%),and the sulfonamides qac E?1-sul1 detection rate was 13.73%.Five resistance genes of metallo-?-lactamases(VIM,IMP,SPM,GIM,NDM)and aac(3)-I of aminoglycoside and mcr-1 were negative.We picked up part of the isolates from the various regions(wide resistance spectrum was preferred),a total of 118 strains were analysed by multilocus sequence typing(MLST),the results showed that 118 strains of PA can be divided into 28 ST types,presented a highly diverse,indicated that PA was given priority to with sporadic propagated;ST-260 types accounted for the most,followed by ST-2100 and ST-3202.Among them,9 ST types were newly discovered in this study which indicated quick evolution speed;The results of ST Typing and drug resistance spectrum analysis showed that the drug resistance spectrum of the same ST type or similar clonal group was not necessarily the same.The 28 ST types contained only 3 clonal complexes,and the Bio Numerics soft analysis showed that the PA in environment were closely related to that in dead embryos.In view of the fact that PA is an important human and animal pathogen,the current PA detection methods have their own shortcomings,therefore,a simple,accurate,rapid and economical molecular detection method was established to provide technical support for the diagnosis of PA diseases.This research adopts the technology of cross priming amplification(CPA),targered at opr I specific genes of PA,designed a set of cross amplification primer labeled by FAM and Biotin,with a nucleic acid test strip was established to detect PA.The reaction was amplified at 62°C for 45 minutes,the sensitivity of PA-CPA was 100 times more than PCR with high specificity.This established constant-temperature CPA of PA detection method did not require a PCR instrument,DNA amplification can be done in just one water bath or metal bath,and the closed nucleic acid detection test strip makes the result judgment intuitive and objective.It can effectively avoid aerosol pollution and can be applied to the rapid detection of PA.In summary,the 11 poultry farms in Guangdong province not only have different degrees of PA infection in their dead embryos,but also a considerable proportion of PA contamination in their surrounding environment in this study.The strains also showed strong drug resistance and could not be ignored.Therefore,it is necessary to strengthen the management of feeding,timely clean and disinfect the incubator and its surrounding environment,create good breeding conditions,and prevent cross-infection of drug-resistant bacteria;In addition,when using antibacterial drugs,it should be properly use according to the results of drug sensitivity test to reduce the emergence of multi-drug resistant strains.Finally,the PA-CPA detection method established in this study can be applied to the rapid detection of PA,providing technical support for the diagnosis and prevention of diseases caused by PA infection.