| The giant panda(Ailuropoda melanoleuca)is a rare and endangered species of wild animal unique to China,which is susceptible to bacterial diseases that threaten its population’s health.There have been reports of giant pandas dying from bacterial diseases such as Proteus mirabilis,Klebsiella pneumoniae,and Escherichia coli both domestically and abroad.However,the traditional bacterial isolation and culture methods currently used are cumbersome with long detection cycles,making it difficult to meet the clinical need for early diagnosis and treatment of diseases.Based on this,this study explores a multiplex PCR method that can simultaneously detect Proteus mirabilis,Klebsiella pneumoniae,and Escherichia coli from giant panda sources and applies it to the clinical fecal samples of giant pandas.At the same time,the virulence factors and drug resistance of clinically isolated Proteus mirabilis were studied,with the aim of making timely and reliable clinical diagnoses of infections caused by these bacteria in giant pandas,and providing theoretical guidance for the epidemiological prevention and clinical medication of Proteus mirabilis from giant panda sources,and enriching the biological data of giant pandas.(1)Establishment and application of a multiplex PCR method for detecting Proteus mirabilis,Klebsiella pneumoniae,and Escherichia coli from giant panda sourcesThis study successfully developed a triple-PCR detection method targeting the ure R gene of Proteus mirabilis,the pho E gene of Klebsiella pneumoniae,and the Pho A gene of Escherichia coli,respectively,for the rapid and accurate identification of these three pathogenic bacteria originating from giant pandas.After optimizing the annealing temperature and primer concentration,this PCR method could amplify the expected target fragments for each pathogenic bacteria without false positives.The specificity test showed that only the target pathogenic bacteria were amplified,indicating a good specificity of the method.The detection limits for Proteus mirabilis,Klebsiella pneumoniae,and Escherichia coli were 0.87 ng,0.48 ng,and 0.2 ng,respectively.The method also demonstrated good reproducibility for eight consecutive weeks.When applied to 100 clinical samples from the Chengdu Research Base of Giant Panda Breeding,the detection rates of Proteus mirabilis,Klebsiella pneumoniae,and Escherichia coli were 30%(30/100),90%(90/100),and 100%(100/100),respectively.The consistency of the results between the triple-PCR method and single-PCR and pathogen isolation methods was 100%.In summary,this study established a reliable and rapid detection method for identifying the three pathogenic bacteria originating from giant pandas,providing a valuable tool for the diagnosis of bacterial diseases in giant pandas and guiding clinical treatment decisions.(2)Research on virulence factors and antibiotic resistance of Proteus mirabilis from giant pandasThis study isolated and identified Proteus mirabilis from 150 fecal samples of117 giant pandas in three different age groups,namely subadults,adults,and seniors,collected from the Chengdu Research Base of Giant Panda Breeding.The isolation and identification were performed using PCR,K-B technique,and high-throughput fluorescence quantitative PCR technology.The drug-resistant phenotype(28antibiotics),resistant genotype(53 resistant genes),and eight typical virulence factors of Proteus mirabilis originating from giant pandas were detected and analyzed,and the isolation rates and drug resistance of Proteus mirabilis from giant pandas of different ages and sexes were compared.The results showed that a total of 45 Proteus mirabilis strains were isolated and identified from the 150 giant panda fecal samples.The isolation rates of Proteus mirabilis strains from subadults,adults,and seniors were 28.13%(9/32),37.35%(31/83),and 14.29%(5/35),respectively.The difference in strain isolation rates among different age groups was extremely significant(P <0.01),while the difference in strain isolation rates between different genders was significant(P < 0.05).Moreover,all 45 Proteus mirabilis strains carried seven virulence factors,including mrp A(100%),atf C(97.78%),zap A(97.78%),atf A(95.56%),pmf A(95.56%),ure C(91.11%),and ucc A(84.44%).None of the strains carried the rsb A virulence factor.The isolates showed different degrees of resistance to 28 kinds of antibiotics,with the highest resistance rate to tetracycline antibiotics(tetracycline and doxycycline were 77.78% and 62.22%,respectively).The difference in antibiotic resistance of pandas with different sexes and ages showed that the resistance rates of male pandas to kanamycin and ciprofloxacin were 50.00% and37.50%,respectively,which were significantly higher than that of female pandas(P <0.05).The drug resistance rate to tetracycline and doxycycline in the elderly group was significantly lower than that in the subadult group and the adult group(P < 0.05).Among the 45 isolates,24 showed multi-drug resistance,accounting for 53.33%(24/45)of total samples.A total of 44 drug resistance genes were detected,and the top ten drug-resistance genes in carrying rate were tet J(100%),flo R(93.3%),sul2(93.33%),aad A2-01(91.11%),aac(6’)-Ib(aka aac A4)-01(86.67%),aac(6’)-Ib(aka aac A4)-02(86.67%),dfr A1(86.67%),aph A1(aka kan R)(82.22%),bla CTX-M-01(75.56%),amp C-09(66.67%).In conclusion,this study has successfully established a multiplex PCR method for detecting Proteus mirabilis,Klebsiella pneumoniae,and Escherichia coli in giant pandas.This method exhibits good specificity and sensitivity and can provide significant value for clinical applications and promotion.Moreover,the study found that the virulence factor carrying rate of Proteus mirabilis isolated from captive giant pandas was above 80%.Results showed varying degrees of drug resistance to commonly used clinical antibiotics.The phenomenon of multiple drug resistance was also prevalent,with the highest detection rates for the tetracycline tet J and Amide alcohols resistance gene flo R. |
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