两株禽绿脓杆菌的分离鉴定与致病性研究
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摘要
[目的]从临汾、平遥等地养殖场采集发病鸡病料,对禽绿脓杆菌进行分离鉴定,及耐药分析和致病性研究。[方法]通过分离培养,形态特征观察,生化试验,16S rDNA基因PCR扩增与测序分析,药敏试验及动物致病性试验,以鉴定禽绿脓杆菌。[结果]经鉴定共分离2株禽绿脓杆菌,分离菌株G1株和G2株接种雏鸡在24 h内死亡率可达到100%,将分离株的16S rDNA基因序列与NCBI中绿脓杆菌序列进行Blast比对,分离菌株与绿脓杆菌不同菌株的同源性高达99%以上,分离菌株对环丙沙星、恩诺沙星、亚胺培南、哌拉西林和替卡西林等抗生素敏感。[结论]本研究通过对山西地区禽绿脓杆菌分离株的鉴定,旨在为进一步研究其致病机制、临床用药和治疗提供理论依据。
[Objective] The diseased chicken materials were collected from poultry farms in Pingyao and Jiexiu city, and the Pseudomonas aeruginosa strains were isolated and identified. The drug resistance analysis and pathogenicity study were conducted. [Methods] Pseudomonas aeruginosa strains were isolated and cultured from the disease carried material, and identification was conducted coupled with morphological observation and biochemical tests. 16 S rDNA gene PCR amplification and sequencing analysis was performed. Drug sensitivity and animal pathogenicity test were carried out to further verify the new strains.[Results] Two strains of Pseudomonas aeruginosa were isolated and identified, and the mortality of chicken inoculated with the isolated strains G1 and G2 was reached up to 100% within 24 hours.Blast sequence alignment analysis with 16 S rDNA gene sequence indicated a more than 99% homologous relationship between the new isolated and other identified strains of Pseudomonas aeruginosa registered in NCBI, and new strains were highly sensitive to ciprofloxacin, enrofloxacin, imipenem, piperacillin, and ticarcillin. [Conclusion] The identification of new strains of Pseudomonas aeruginosa in Shanxi province provided a theoretical basis for further study of the pathogenic mechanism, and pathogenicity.
[Objective] The diseased chicken materials were collected from poultry farms in Pingyao and Jiexiu city, and the Pseudomonas aeruginosa strains were isolated and identified. The drug resistance analysis and pathogenicity study were conducted. [Methods] Pseudomonas aeruginosa strains were isolated and cultured from the disease carried material, and identification was conducted coupled with morphological observation and biochemical tests. 16 S rDNA gene PCR amplification and sequencing analysis was performed. Drug sensitivity and animal pathogenicity test were carried out to further verify the new strains.[Results] Two strains of Pseudomonas aeruginosa were isolated and identified, and the mortality of chicken inoculated with the isolated strains G1 and G2 was reached up to 100% within 24 hours.Blast sequence alignment analysis with 16 S rDNA gene sequence indicated a more than 99% homologous relationship between the new isolated and other identified strains of Pseudomonas aeruginosa registered in NCBI, and new strains were highly sensitive to ciprofloxacin, enrofloxacin, imipenem, piperacillin, and ticarcillin. [Conclusion] The identification of new strains of Pseudomonas aeruginosa in Shanxi province provided a theoretical basis for further study of the pathogenic mechanism, and pathogenicity.
引文
[1]Goldberg J B. Why is Pseudomonas aeruginosa a pathogen[J]. F1000 Biology Report, 2010, 2(1):29.
[2]Walkty A, Decorby M, Nichol K, et al. Antimicrobial susceptibility of Pseudomonas aeruginosa isolates obtained from patients in Canadian intensive care units as part of the Canadian National Intensive Care Unit study[J]. Diagnostic Microbiology and Infectious Disease, 2008, 61(2):0-221.
[3]牛钟相, 李雅林. 动物绿脓杆菌病研究进展[J].动物医学进展, 2003, 24(1):16-18.
[4]Tam V H, Schilling A N, Neshat S, et al. Optimization of Meropenem Minimum Concentration/MIC Ratio To Suppress In Vitro Resistance of Pseudomonas aeruginosa[J]. Antimicrobial Agents and Chemotherapy, 2005, 49(12):4920-4927.
[5]Salimi H, Owlia P, Yakhchali B, et al. Characterization of Pseudomonas aeruginosa in burn patients using PCR-restriction fragment length polymorphism and random amplified polymorphic DNA analysis.[J]. Iranian Journal of Medical Sciences, 2015, 35(3):236-241.
[6]Patel J B, Leonard D G B, Pan X, et al. Sequence-Based Identification of Mycobacterium Species Using the MicroSeq 500 16S rDNA Bacterial Identification System[J]. Journal of Clinical Microbiology, 2000, 38(1):246-251.
[7]Lau S, Ng K, Woo P, et al. Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures[J]. Journal of Clinical Pathology, 2006, 59(2):219-222.
[8]Hall L, Doerr K A, Wohlfiel S L, et al. Evaluation of the MicroSeq System for Identification of Mycobacteria by 16S Ribosomal DNA Sequencing and Its Integration into a Routine Clinical Mycobacteriology Laboratory[J]. Journal of Clinical Microbiology, 2003, 41(4):1447.
[9]Vandercam B, Jeumont S, Cornu O, et al. Amplification-Based DNA Analysis in the Diagnosis of Prosthetic Joint Infection[J]. The Journal of Molecular Diagnostics, 2008, 10(6):537-543.
[10]陈晨. 貂源绿脓杆菌的分离鉴定及其致病性研究[D]. 济南:山东农业大学, 2015.
[11]Lau G W, Hassett D J, Ran H, et al. The role of pyocyanin in Pseudomonas aeruginosa infection.[J]. Trends in Molecular Medicine, 2004, 10(12):599-606.
[12]Knepp J H, Geahr M A, Forman M S, et al. Comparison of automated and manual nucleic acid extraction methods for detection of enterovirus RNA.[J]. Journal of Clinical Microbiology, 2003, 41(8):3532.
[13]Pirnay J P, Matthijs S, Colak H, et al. Global Pseudomonas aeruginosa biodiversity as reflected in a Belgian river[J]. Environmental Microbiology, 2005, 7(7):969-980.
[14]Bosshard P P, Abels S, Zbinden R, et al. Ribosomal DNA sequencing for identification of aerobic gram-positive rods in the clinical laboratory (an 18-month evaluation).[J]. Journal of Clinical Microbiology, 2003, 41(9):4134.
[15]Hafiane A, Ravaoarinoro M. Characterization of Pseudomonas aeruginosa strains isolated from cystic fibrosis patients by different typing methods.[J]. Pathologie Biologie, 2011, 59(5):e109-e114.
[16]张香斋, 张艳英, 李蕴玉,等. 鸡源绿脓杆菌的分离鉴定及药敏试验[J].中国畜牧兽医, 2015, 42(6):1602-1607.
[17]周学章, 田东林, 邓旭明. 雏鸡绿脓杆菌病病原菌的分离和鉴定[J]. 中兽医医药杂志, 2004, 23(3):19-21.
[18]Gootz T D. Global dissemination of beta-lactamases mediating resistance to cephalosporins and carbapenems[J]. Expert Review of Anti-Infective Therapy, 2004, 2(2):317.
[19]Sader H S, Rhomberg P R, Jones R N. In vitro activity of beta-lactam antimicrobial agents in combination with aztreonam tested against metallo-beta-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii[J]. Journal of Chemotherapy, 2005, 17(6):622-627.
[20]Livermore D M. Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: our worst nightmare?[J]. Clinical Infectious Diseases, 2002, 34(5):634-640.
[21]Stover C K, Pham X Q, Erwin A L, et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen[J]. Nature, 2000, 406(6799): 959.
[2]Walkty A, Decorby M, Nichol K, et al. Antimicrobial susceptibility of Pseudomonas aeruginosa isolates obtained from patients in Canadian intensive care units as part of the Canadian National Intensive Care Unit study[J]. Diagnostic Microbiology and Infectious Disease, 2008, 61(2):0-221.
[3]牛钟相, 李雅林. 动物绿脓杆菌病研究进展[J].动物医学进展, 2003, 24(1):16-18.
[4]Tam V H, Schilling A N, Neshat S, et al. Optimization of Meropenem Minimum Concentration/MIC Ratio To Suppress In Vitro Resistance of Pseudomonas aeruginosa[J]. Antimicrobial Agents and Chemotherapy, 2005, 49(12):4920-4927.
[5]Salimi H, Owlia P, Yakhchali B, et al. Characterization of Pseudomonas aeruginosa in burn patients using PCR-restriction fragment length polymorphism and random amplified polymorphic DNA analysis.[J]. Iranian Journal of Medical Sciences, 2015, 35(3):236-241.
[6]Patel J B, Leonard D G B, Pan X, et al. Sequence-Based Identification of Mycobacterium Species Using the MicroSeq 500 16S rDNA Bacterial Identification System[J]. Journal of Clinical Microbiology, 2000, 38(1):246-251.
[7]Lau S, Ng K, Woo P, et al. Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures[J]. Journal of Clinical Pathology, 2006, 59(2):219-222.
[8]Hall L, Doerr K A, Wohlfiel S L, et al. Evaluation of the MicroSeq System for Identification of Mycobacteria by 16S Ribosomal DNA Sequencing and Its Integration into a Routine Clinical Mycobacteriology Laboratory[J]. Journal of Clinical Microbiology, 2003, 41(4):1447.
[9]Vandercam B, Jeumont S, Cornu O, et al. Amplification-Based DNA Analysis in the Diagnosis of Prosthetic Joint Infection[J]. The Journal of Molecular Diagnostics, 2008, 10(6):537-543.
[10]陈晨. 貂源绿脓杆菌的分离鉴定及其致病性研究[D]. 济南:山东农业大学, 2015.
[11]Lau G W, Hassett D J, Ran H, et al. The role of pyocyanin in Pseudomonas aeruginosa infection.[J]. Trends in Molecular Medicine, 2004, 10(12):599-606.
[12]Knepp J H, Geahr M A, Forman M S, et al. Comparison of automated and manual nucleic acid extraction methods for detection of enterovirus RNA.[J]. Journal of Clinical Microbiology, 2003, 41(8):3532.
[13]Pirnay J P, Matthijs S, Colak H, et al. Global Pseudomonas aeruginosa biodiversity as reflected in a Belgian river[J]. Environmental Microbiology, 2005, 7(7):969-980.
[14]Bosshard P P, Abels S, Zbinden R, et al. Ribosomal DNA sequencing for identification of aerobic gram-positive rods in the clinical laboratory (an 18-month evaluation).[J]. Journal of Clinical Microbiology, 2003, 41(9):4134.
[15]Hafiane A, Ravaoarinoro M. Characterization of Pseudomonas aeruginosa strains isolated from cystic fibrosis patients by different typing methods.[J]. Pathologie Biologie, 2011, 59(5):e109-e114.
[16]张香斋, 张艳英, 李蕴玉,等. 鸡源绿脓杆菌的分离鉴定及药敏试验[J].中国畜牧兽医, 2015, 42(6):1602-1607.
[17]周学章, 田东林, 邓旭明. 雏鸡绿脓杆菌病病原菌的分离和鉴定[J]. 中兽医医药杂志, 2004, 23(3):19-21.
[18]Gootz T D. Global dissemination of beta-lactamases mediating resistance to cephalosporins and carbapenems[J]. Expert Review of Anti-Infective Therapy, 2004, 2(2):317.
[19]Sader H S, Rhomberg P R, Jones R N. In vitro activity of beta-lactam antimicrobial agents in combination with aztreonam tested against metallo-beta-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii[J]. Journal of Chemotherapy, 2005, 17(6):622-627.
[20]Livermore D M. Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: our worst nightmare?[J]. Clinical Infectious Diseases, 2002, 34(5):634-640.
[21]Stover C K, Pham X Q, Erwin A L, et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen[J]. Nature, 2000, 406(6799): 959.