奶牛源性双歧杆菌和乳杆菌的分离鉴定及耐药性研究
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摘要
为了筛选性能优良且对机体有益的耐药性双歧杆菌和乳杆菌,扩大双歧杆菌、乳杆菌的应用范围,本研究采用乳酸菌鉴别培养基和微生物厌氧培养技术从呼和浩特地区两个牧场25头乳犊牛粪便中分离到49株双歧杆菌属和乳杆菌属的细菌,并采用纸片法对分离菌进行了药物敏感性试验。结果显示:49株菌对所选12种抗菌药均有不同程度的耐性,其中,49株菌全部对甲硝唑耐药;45株菌对氨基糖苷类耐药;37株菌对喹喏酮类耐药;对四环素类、氯霉素类、红霉素类的耐药菌株分别为:10株、10株和19株。
对其中7株耐药性较强的菌株进行形态、生理生化特性鉴定及16SrRNA序列分析,证明7株菌为:3株植物乳杆菌、2株链双歧杆菌、2株豚双歧杆菌。
采用试管二倍稀释法及杯碟法对这7株耐药菌进一步进行试验后发现,7株菌对12种抗菌药同时具有不同程度的耐药性。其中,3株植物乳杆菌除1株菌对四环素、左氧氟沙星中度敏感外,对其它抗菌药均有耐药性;2株豚双歧杆菌中除1株菌对氯霉素中度敏感外,对其它抗菌药均有耐药性;2株链双歧杆菌除对四环素中度敏感、其中1株对氨苄西林敏感外,对其它抗菌药均有耐药性。试验菌株均对甲硝唑和氨基糖苷类抗菌药表现出较为一致的耐性。
在对7株菌进行喹喏酮类耐药机制的研究时发现,采用琼脂凝胶电泳技术检测其质粒,7株菌内均未提取出质粒;采用荧光分光光度法,用2,4-二硝基酚作为抑制剂证明6株菌内存在药物主动外排系统;采用PCR技术和gyrA的QRDR序列分析表明,试验菌株有DNA旋转酶gyrA基因的突变。
结论:从未使用过抗菌药的乳犊牛粪便中分离到49株双歧杆菌和乳杆菌属的菌株,这些菌株对12种抗菌药均有不同程度的耐药性,其中对甲硝唑的耐药率达到100%;对氨基糖苷类的耐药率达87.8%以上;对四环素类、氯霉素类、红霉素类的耐药率在20.4%-38.8%之间;对喹喏酮类的耐药率在70%以上。对喹喏酮类药物产生耐药与质粒介导无关,而与其存在主动外排系统以及DNA旋转酶gyrA基因突变有关。
To screen the beneficial Bifidobacterium and Lactobacillus with high performance and drug resistance, widen the range and mode of its utilization, the isolation, identification, bionomics and resisitance of Bifidobacterium and Lactobacillus strains isolated from feces of cows in Huhhot were studied .
The 49 strains of Bifitobacteria and Lactobacilli from the fresh feces of calf never used antimicrobials were isolated and its drug resistance research were carried out. The morphological, physiological, biochemical characteristics and identification of multiple PCR and 16SrRNA sequence analysis of 7 strains with higher drug resistance of 49 strains were studied, the results showed that 3 strains belong to Lactobacillus plantarum, 2 strains belong to Bifidobacterium choleyium and 2 strains belonged to Bifidobacterium catemulatum.
The K-B method was adopted to determine the drug resistance of these 49 strains, the results showed that these strains had a certain drug resistance to 12 kinds of antimicrobials. Among the total, the strains of drug resistance of 49 strains were 49 to metronidazole, 45 to aminoglycosides, 10 to tetracycline, 10 to chloramphenicol, 19 to Chloramphenicol, and about 37 to fluoroquinolones.
The cylinder plate method was adopted to study the resistance of 7 strains; the results showed that these strains had a resistance to 12 kinds of antimicrobials. Among the total, 3 trains of Lactobacillus plantarum were resistant to all antimicrobial except 1 strain was sensitive to tetracycline and Levofloxacin, 2 strains of Bifidobacterium choleyium were resistant to all antimicrobial except 1 strain was sensitive to chlormycetin, 2 strains of Bifidobacterium catemulatum were sensitive to tetracycline, and resistant to else antimicrobial except 1 strain was sensitive to ampicillin. These test organism were higher resistant to aminoglycosides and arilin. The resistance degree of these strains were different for several antibacterials because of the kinds of strains.
The R-plasmid of 7 strains were detected by agar gel electrophoresis, their active efflux system were determined by fluorospectrophotometry, and their QRDR sequence of gyrA gene were detected by PCR technique and gene sequencing, the results showed that there is active efflux system in these strains but no R-plasmid in these strains, and the gyrA gene had happened mutation
Conculsions: 49 strains of Bifidobacterium and Lactobacillus were resistant to 12 kinds of antimicrobials at different degree, among the total, the resistance to arilin reached 100%, the resistance to aminoglycosides reached 87.8%, the resistance to tetracyclines, chlormycetin and erythromycin were low (20.4%-38.8%), and the resistance to fluoroquinolones were more than 70%. 7 strains of Bifidobacterium and Lactobacillus hade higher multidrug resistance. Among the total, had higher resistance to aminoglycosides and arilin. The drug resistant mechanism of test organism to fluoroquinolones had no relationship to mediation of plasmid, but had relationship to their active efflux system and the mutation of gyrA gene.
对其中7株耐药性较强的菌株进行形态、生理生化特性鉴定及16SrRNA序列分析,证明7株菌为:3株植物乳杆菌、2株链双歧杆菌、2株豚双歧杆菌。
采用试管二倍稀释法及杯碟法对这7株耐药菌进一步进行试验后发现,7株菌对12种抗菌药同时具有不同程度的耐药性。其中,3株植物乳杆菌除1株菌对四环素、左氧氟沙星中度敏感外,对其它抗菌药均有耐药性;2株豚双歧杆菌中除1株菌对氯霉素中度敏感外,对其它抗菌药均有耐药性;2株链双歧杆菌除对四环素中度敏感、其中1株对氨苄西林敏感外,对其它抗菌药均有耐药性。试验菌株均对甲硝唑和氨基糖苷类抗菌药表现出较为一致的耐性。
在对7株菌进行喹喏酮类耐药机制的研究时发现,采用琼脂凝胶电泳技术检测其质粒,7株菌内均未提取出质粒;采用荧光分光光度法,用2,4-二硝基酚作为抑制剂证明6株菌内存在药物主动外排系统;采用PCR技术和gyrA的QRDR序列分析表明,试验菌株有DNA旋转酶gyrA基因的突变。
结论:从未使用过抗菌药的乳犊牛粪便中分离到49株双歧杆菌和乳杆菌属的菌株,这些菌株对12种抗菌药均有不同程度的耐药性,其中对甲硝唑的耐药率达到100%;对氨基糖苷类的耐药率达87.8%以上;对四环素类、氯霉素类、红霉素类的耐药率在20.4%-38.8%之间;对喹喏酮类的耐药率在70%以上。对喹喏酮类药物产生耐药与质粒介导无关,而与其存在主动外排系统以及DNA旋转酶gyrA基因突变有关。
To screen the beneficial Bifidobacterium and Lactobacillus with high performance and drug resistance, widen the range and mode of its utilization, the isolation, identification, bionomics and resisitance of Bifidobacterium and Lactobacillus strains isolated from feces of cows in Huhhot were studied .
The 49 strains of Bifitobacteria and Lactobacilli from the fresh feces of calf never used antimicrobials were isolated and its drug resistance research were carried out. The morphological, physiological, biochemical characteristics and identification of multiple PCR and 16SrRNA sequence analysis of 7 strains with higher drug resistance of 49 strains were studied, the results showed that 3 strains belong to Lactobacillus plantarum, 2 strains belong to Bifidobacterium choleyium and 2 strains belonged to Bifidobacterium catemulatum.
The K-B method was adopted to determine the drug resistance of these 49 strains, the results showed that these strains had a certain drug resistance to 12 kinds of antimicrobials. Among the total, the strains of drug resistance of 49 strains were 49 to metronidazole, 45 to aminoglycosides, 10 to tetracycline, 10 to chloramphenicol, 19 to Chloramphenicol, and about 37 to fluoroquinolones.
The cylinder plate method was adopted to study the resistance of 7 strains; the results showed that these strains had a resistance to 12 kinds of antimicrobials. Among the total, 3 trains of Lactobacillus plantarum were resistant to all antimicrobial except 1 strain was sensitive to tetracycline and Levofloxacin, 2 strains of Bifidobacterium choleyium were resistant to all antimicrobial except 1 strain was sensitive to chlormycetin, 2 strains of Bifidobacterium catemulatum were sensitive to tetracycline, and resistant to else antimicrobial except 1 strain was sensitive to ampicillin. These test organism were higher resistant to aminoglycosides and arilin. The resistance degree of these strains were different for several antibacterials because of the kinds of strains.
The R-plasmid of 7 strains were detected by agar gel electrophoresis, their active efflux system were determined by fluorospectrophotometry, and their QRDR sequence of gyrA gene were detected by PCR technique and gene sequencing, the results showed that there is active efflux system in these strains but no R-plasmid in these strains, and the gyrA gene had happened mutation
Conculsions: 49 strains of Bifidobacterium and Lactobacillus were resistant to 12 kinds of antimicrobials at different degree, among the total, the resistance to arilin reached 100%, the resistance to aminoglycosides reached 87.8%, the resistance to tetracyclines, chlormycetin and erythromycin were low (20.4%-38.8%), and the resistance to fluoroquinolones were more than 70%. 7 strains of Bifidobacterium and Lactobacillus hade higher multidrug resistance. Among the total, had higher resistance to aminoglycosides and arilin. The drug resistant mechanism of test organism to fluoroquinolones had no relationship to mediation of plasmid, but had relationship to their active efflux system and the mutation of gyrA gene.
引文
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