玉米粉对水源性肠球菌抗生素耐药形成的影响
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摘要
目的:建立模型生态系统研究玉米粉引发水源性肠球菌对氯霉素(CHL)、红霉素(ERY)、氨苄西林(AMP)、土霉素(OTC)、万古霉素(VAN)和环丙沙星(CIP)耐药表型产生的影响,初步探讨玉米粉引发水源性肠球菌对红霉素和环丙沙星耐药的机理。
方法:从福州森林公园采集土样,检测土样中肠球菌对试验用6种抗生素的敏感性,结果表明这些土样中的肠球菌对这6种抗生素都敏感,可作为模型生态系统的底泥。模型生态系统建立后,将系统分为6个剂量组,分别添加8g/L、4g/L、1g/L、0.25g/L、0.05g/L和0g/L的灭菌玉米粉,每个剂量组两个重复。采集第0d、第1d、第3d、第7d、第14d、第30d、第40d、第61d和第130d系统中的泥样,检测泥样中肠球菌对6种抗生素的敏感性,了解玉米粉对水源性肠球菌抗生素耐药表型形成的影响;应用PCR方法检测ermB、mefA、gyrA和Esp等基因,序列测定和分析gyrA基因83位点和87位点是否发生突变;结晶紫染色法检测不同葡萄糖浓度对肠球菌生物膜形成的影响。
结果: (1)添加不同剂量玉米粉对肠球菌分离率的影响不同,影响程度和添加玉米粉的剂量呈正相关,添加8g/L玉米粉对肠球菌分离率影响最大,可使分离率从40%上升为100%,添加4g/L玉米粉时肠球菌的分离率最高只上升到80%,添加1g/L、0.25g/L、0.05g/L和0g/L玉米粉时肠球菌的分离率变化不大。
(2)添加不同剂量玉米粉没有引发肠球菌对氨苄西林和万古霉素的敏感性产生可检测到的影响。添加0.05g/L-8g/L玉米粉对环丙沙星的耐药性都产生了影响,且添加玉米粉的剂量越大产生的影响越大,环丙沙星耐药菌株的出现时间越早,耐药率也越大。添加8g/L玉米粉可在第1d就出现了耐环丙沙星的菌株,而在第7d时就使肠球菌对环丙沙星的耐药率达到了100%;添加4g/L玉米粉时对环丙沙星的影响虽然也在第1d就出现,但到了第14d时才出现了最高的耐药率85.7%;添加1g/L玉米粉对环丙沙星的影响较小,在第7d才出现25%的耐药率,影响最大时的耐药率也才达到50%。对氯霉素的影响也因添加玉米粉剂量不同而不同,添加8g/L和4g/L玉米粉时没有出现对氯霉素耐药的肠球菌,而添加0.05g/L-1g/L玉米粉时都出现了氯霉素耐药菌株,且耐药程度与添加玉米粉的剂量呈正相关,耐药率分别为20%、25%和55.5%。对红霉素和土霉素耐药性的影响情况较一致,耐药程度与添加玉米粉的剂量不完全呈正比,且耐药菌株出现的时间较晚。
(3)对红霉素耐药的28株肠球菌中,ermB基因的阳性率为78.6%,而对红霉素敏感的16株肠球菌均未检测到ermB耐药基因,另外,无论是对红霉素敏感的肠球菌还是对红霉素耐药的肠球菌均未检测到mefA耐药基因。模型生态系统中分离的102株肠球菌gyrA基因的阳性率为96.1%,但83位点和87位点均没有发生突变。
(4)表达肠球菌表面蛋白的Esp基因检出率为0%。不同浓度的葡萄糖对分离自模型生态系统中肠球菌的生物膜形成能力有不同的促进作用,1.0%葡萄糖比0.5%葡萄糖有更强的促进生物膜形成作用。
结论: (1)添加玉米粉可引发模型生态系统中的肠球菌对红霉素、土霉素、环丙沙星和氯霉素产生耐药,但对万古霉素和氨苄西林没有产生可检测到的影响。
(2)添加不同剂量的玉米粉引发水源性肠球菌对红霉素、土霉素、环丙沙星和氯霉素耐药的程度不同;对环丙沙星的影响程度与添加玉米粉的剂量呈正比,添加剂量越大对环丙沙星耐药性形成速度与程度的影响越大;对氯霉素的影响在添加0.05g/L-1g/L剂量区间,影响程度与添加玉米粉的剂量呈正相关;对土霉素和红霉素的影响较不规律,影响程度与添加玉米粉的剂量不完全呈正比,但整体趋势还是表现为添加剂量越大,影响程度越大。
(3)模型生态系统中的肠球菌对环丙沙星耐药不是由gyrA基因突变引起,但对红霉素的耐药绝大部分与ermB耐药基因有关。
(4)肠球菌生物膜的形成与Esp基因之间没有必然的联系,但肠球菌在葡萄糖诱导下具有形成生物膜的能力,且生物膜的形成与葡萄糖的浓度在一定范围内呈正相关。肠球菌耐药的产生与环境中葡萄糖的浓度以及生物膜的形成有一定的相关性。
Objective: Model ecosystems were established to study the effect of cornmeal on the resistant phenotype of water source enterococcus to chloramphenicol (CHL), erythromycin (ERY), ampicillin (AMP), oxytetracycline (OTC), vancomycin (VAN) and ciprofloxacin (CIP). The resistant mechanism of enterococcus to erythromycin and ciprofloxacin were studied as well.
Methods: Soil samples were collected from forest park in Fu-zhou, enterococcus strains were isolated from these soil samples and the sensitivity of these strains to the six kinds of antibiotics were detected, the results showed that these enterococcus stains were sensitive to the six kinds of antibiotics, so they could be used as the sediment in model ecosystems. After the model ecosystems were established, they were divided into six groups, sterilization cornmeal were added at the doses of 8g/L, 4g/L, 1g/L, 0.25 g/L, 0.05 g/L and 0g/L respectively, each group had two replications. Enterococcus strains were isolated from the model ecosystems at the time of 0d, 1d, 3d, 7d, 14d, 30d, 40d, 61d and 130d, sensitivity test of these enterococcus strains to six kinds of antibiotics were detected; the ermB, mefA, gyrA and Esp gene of enterococcus strains were detected with the method of PCR, gyrA gene sequence was determined and the 83 site and 87 site were analyzed, in order to get the imformation of mutation; the impact of different concentration glucose on the enterococcus biofilm formation was tested by the method of crystallization purple stained.
Results: (1) The effect of cornmeal to the separation rate of enterococcus was different when different doses cornmeal were added, the more the cornmeal added the higher the separation rate was. when added 8g/L cornmeal, the rate of separation elevated from 40% to100%, while the rate of separation could only rose to 80% when 4g/L cornmeal were added; meanwhile, there were little changes on the separation rate of enterococcus when 1g/L, 0.25g/L, 0.05g/L and 0g/L cornmeal were added.
(2) There were no detactable resistant phenotype changes of enterococcus to vancomycin and ampicillin caused by different doses of cornmeal. The changes of resistant phenotype of enterococcus to ciprofloxacin were corelated to the dosages of cornmeal. when cornmeal was added at dose of 8g/L, the ciprofloxacin resistance strains appeared at one day, the resistance rate to ciprofloxacin reached 100% at 7d; when 4g/L cornmeal was added, ciprofloxacin resistance strains aslo appeared at one day, but the resistance rate to ciprofloxacin only reached to 87.5% at 7d; when 1g/L cornmeal was added, ciprofloxacin resistance strains appeared at 7d, the resistance rate to ciprofloxacin only reached to 50% at 14d and 30d. The changes of resistant phenotype of enterococcus to chloramphenicol were corelated to the dosages of cornmeal while it was in the ranges of 0.05 g/L to 1g/L, the resistanc rate was 20%, 25% and 55.5% respectively. when 8g/L and 4g/L cornmeal were added there were no cholramphenicol resistance strains detacted. The changes of resistant phenotype of enterococcus to erythromycin and oxytetracycline were similar, which were not corelated to the doses of cornmeal and the time of resistance strains could be detected was late.
(3) In the 28 strains of erythromycin resistant enterococcus, the rate of ermB gene was 78.6% while the rate was 0% in the 16 strains of erythromycin sensitive enterococcus; meanwhile there were no mefA gene detected in both erythromycin sensitive or resistant enterococcus. In the 102 strains of enterococcus which were isolated from model ecosystems, the rate of gyrA gene was 96.1%; but there were no mutation detected at the site of 83 and 87 in the gyrA gene.
(4) The were no Esp gene detected in the 102 enterococcus strains, which function was expressing enterococcus surface protein. Both concentration of glucose could induce the formation of biofilm in the enterococcus isolated from model ecosystems. 1% glucose has the better effect on the induction of biofilm formation than 0.5% glucose.
Conclusion: (1) There were no detactable resistant phenotype changes of enterococcus to vancomycin and ampicillin caused by different doses of cornmeal. Meanwhile, resistant phenotype of enterococcus to ciprofloxacin, chloramphenicol, erythromycin and oxytetracycline changed while different doses of cornmeal were added into the model ecosystems.
(2) Different doses of cornmeal have different effects on the formation of resistance and the level of resistance of enterococcus to erythromycin, oxytetracycline, ciprofloxacin and chloramphenicol. In the case of ciprofloxacin, the higher the dose was the earlier the resistant enterococcus appeared and the higher the resistant level was. In the case of chloramphenicol, while the doses of cornmeal were at the ranges of 0.05 g/L to 1g/L, it has similar effect on the form of resistance to ciprofloxacin. In the cases of erythromycin and oxytetracycline there were no obvious relationship between the doeses of cornmeal and the form and the level of resistance.
(3) In the model ecosystem, the appearance of ciprofloxacin resistant enterococcus was not caused by the mutation of gyrA gene; while, the appearance of erythromycin resistant enterococcus has strong correlation to the appearance of ermB gene.
(4) Esp gene did not necessary correlate to the formation of Enterococcus biofilm. In certain concentration range, the ablity of enterococcus form biofilm was correlated to the concentration of glucose. There was certain relationship between antibiotics resistance of enterococcus and the concentration of glucose in the emvironment and the formation of biofilm.
方法:从福州森林公园采集土样,检测土样中肠球菌对试验用6种抗生素的敏感性,结果表明这些土样中的肠球菌对这6种抗生素都敏感,可作为模型生态系统的底泥。模型生态系统建立后,将系统分为6个剂量组,分别添加8g/L、4g/L、1g/L、0.25g/L、0.05g/L和0g/L的灭菌玉米粉,每个剂量组两个重复。采集第0d、第1d、第3d、第7d、第14d、第30d、第40d、第61d和第130d系统中的泥样,检测泥样中肠球菌对6种抗生素的敏感性,了解玉米粉对水源性肠球菌抗生素耐药表型形成的影响;应用PCR方法检测ermB、mefA、gyrA和Esp等基因,序列测定和分析gyrA基因83位点和87位点是否发生突变;结晶紫染色法检测不同葡萄糖浓度对肠球菌生物膜形成的影响。
结果: (1)添加不同剂量玉米粉对肠球菌分离率的影响不同,影响程度和添加玉米粉的剂量呈正相关,添加8g/L玉米粉对肠球菌分离率影响最大,可使分离率从40%上升为100%,添加4g/L玉米粉时肠球菌的分离率最高只上升到80%,添加1g/L、0.25g/L、0.05g/L和0g/L玉米粉时肠球菌的分离率变化不大。
(2)添加不同剂量玉米粉没有引发肠球菌对氨苄西林和万古霉素的敏感性产生可检测到的影响。添加0.05g/L-8g/L玉米粉对环丙沙星的耐药性都产生了影响,且添加玉米粉的剂量越大产生的影响越大,环丙沙星耐药菌株的出现时间越早,耐药率也越大。添加8g/L玉米粉可在第1d就出现了耐环丙沙星的菌株,而在第7d时就使肠球菌对环丙沙星的耐药率达到了100%;添加4g/L玉米粉时对环丙沙星的影响虽然也在第1d就出现,但到了第14d时才出现了最高的耐药率85.7%;添加1g/L玉米粉对环丙沙星的影响较小,在第7d才出现25%的耐药率,影响最大时的耐药率也才达到50%。对氯霉素的影响也因添加玉米粉剂量不同而不同,添加8g/L和4g/L玉米粉时没有出现对氯霉素耐药的肠球菌,而添加0.05g/L-1g/L玉米粉时都出现了氯霉素耐药菌株,且耐药程度与添加玉米粉的剂量呈正相关,耐药率分别为20%、25%和55.5%。对红霉素和土霉素耐药性的影响情况较一致,耐药程度与添加玉米粉的剂量不完全呈正比,且耐药菌株出现的时间较晚。
(3)对红霉素耐药的28株肠球菌中,ermB基因的阳性率为78.6%,而对红霉素敏感的16株肠球菌均未检测到ermB耐药基因,另外,无论是对红霉素敏感的肠球菌还是对红霉素耐药的肠球菌均未检测到mefA耐药基因。模型生态系统中分离的102株肠球菌gyrA基因的阳性率为96.1%,但83位点和87位点均没有发生突变。
(4)表达肠球菌表面蛋白的Esp基因检出率为0%。不同浓度的葡萄糖对分离自模型生态系统中肠球菌的生物膜形成能力有不同的促进作用,1.0%葡萄糖比0.5%葡萄糖有更强的促进生物膜形成作用。
结论: (1)添加玉米粉可引发模型生态系统中的肠球菌对红霉素、土霉素、环丙沙星和氯霉素产生耐药,但对万古霉素和氨苄西林没有产生可检测到的影响。
(2)添加不同剂量的玉米粉引发水源性肠球菌对红霉素、土霉素、环丙沙星和氯霉素耐药的程度不同;对环丙沙星的影响程度与添加玉米粉的剂量呈正比,添加剂量越大对环丙沙星耐药性形成速度与程度的影响越大;对氯霉素的影响在添加0.05g/L-1g/L剂量区间,影响程度与添加玉米粉的剂量呈正相关;对土霉素和红霉素的影响较不规律,影响程度与添加玉米粉的剂量不完全呈正比,但整体趋势还是表现为添加剂量越大,影响程度越大。
(3)模型生态系统中的肠球菌对环丙沙星耐药不是由gyrA基因突变引起,但对红霉素的耐药绝大部分与ermB耐药基因有关。
(4)肠球菌生物膜的形成与Esp基因之间没有必然的联系,但肠球菌在葡萄糖诱导下具有形成生物膜的能力,且生物膜的形成与葡萄糖的浓度在一定范围内呈正相关。肠球菌耐药的产生与环境中葡萄糖的浓度以及生物膜的形成有一定的相关性。
Objective: Model ecosystems were established to study the effect of cornmeal on the resistant phenotype of water source enterococcus to chloramphenicol (CHL), erythromycin (ERY), ampicillin (AMP), oxytetracycline (OTC), vancomycin (VAN) and ciprofloxacin (CIP). The resistant mechanism of enterococcus to erythromycin and ciprofloxacin were studied as well.
Methods: Soil samples were collected from forest park in Fu-zhou, enterococcus strains were isolated from these soil samples and the sensitivity of these strains to the six kinds of antibiotics were detected, the results showed that these enterococcus stains were sensitive to the six kinds of antibiotics, so they could be used as the sediment in model ecosystems. After the model ecosystems were established, they were divided into six groups, sterilization cornmeal were added at the doses of 8g/L, 4g/L, 1g/L, 0.25 g/L, 0.05 g/L and 0g/L respectively, each group had two replications. Enterococcus strains were isolated from the model ecosystems at the time of 0d, 1d, 3d, 7d, 14d, 30d, 40d, 61d and 130d, sensitivity test of these enterococcus strains to six kinds of antibiotics were detected; the ermB, mefA, gyrA and Esp gene of enterococcus strains were detected with the method of PCR, gyrA gene sequence was determined and the 83 site and 87 site were analyzed, in order to get the imformation of mutation; the impact of different concentration glucose on the enterococcus biofilm formation was tested by the method of crystallization purple stained.
Results: (1) The effect of cornmeal to the separation rate of enterococcus was different when different doses cornmeal were added, the more the cornmeal added the higher the separation rate was. when added 8g/L cornmeal, the rate of separation elevated from 40% to100%, while the rate of separation could only rose to 80% when 4g/L cornmeal were added; meanwhile, there were little changes on the separation rate of enterococcus when 1g/L, 0.25g/L, 0.05g/L and 0g/L cornmeal were added.
(2) There were no detactable resistant phenotype changes of enterococcus to vancomycin and ampicillin caused by different doses of cornmeal. The changes of resistant phenotype of enterococcus to ciprofloxacin were corelated to the dosages of cornmeal. when cornmeal was added at dose of 8g/L, the ciprofloxacin resistance strains appeared at one day, the resistance rate to ciprofloxacin reached 100% at 7d; when 4g/L cornmeal was added, ciprofloxacin resistance strains aslo appeared at one day, but the resistance rate to ciprofloxacin only reached to 87.5% at 7d; when 1g/L cornmeal was added, ciprofloxacin resistance strains appeared at 7d, the resistance rate to ciprofloxacin only reached to 50% at 14d and 30d. The changes of resistant phenotype of enterococcus to chloramphenicol were corelated to the dosages of cornmeal while it was in the ranges of 0.05 g/L to 1g/L, the resistanc rate was 20%, 25% and 55.5% respectively. when 8g/L and 4g/L cornmeal were added there were no cholramphenicol resistance strains detacted. The changes of resistant phenotype of enterococcus to erythromycin and oxytetracycline were similar, which were not corelated to the doses of cornmeal and the time of resistance strains could be detected was late.
(3) In the 28 strains of erythromycin resistant enterococcus, the rate of ermB gene was 78.6% while the rate was 0% in the 16 strains of erythromycin sensitive enterococcus; meanwhile there were no mefA gene detected in both erythromycin sensitive or resistant enterococcus. In the 102 strains of enterococcus which were isolated from model ecosystems, the rate of gyrA gene was 96.1%; but there were no mutation detected at the site of 83 and 87 in the gyrA gene.
(4) The were no Esp gene detected in the 102 enterococcus strains, which function was expressing enterococcus surface protein. Both concentration of glucose could induce the formation of biofilm in the enterococcus isolated from model ecosystems. 1% glucose has the better effect on the induction of biofilm formation than 0.5% glucose.
Conclusion: (1) There were no detactable resistant phenotype changes of enterococcus to vancomycin and ampicillin caused by different doses of cornmeal. Meanwhile, resistant phenotype of enterococcus to ciprofloxacin, chloramphenicol, erythromycin and oxytetracycline changed while different doses of cornmeal were added into the model ecosystems.
(2) Different doses of cornmeal have different effects on the formation of resistance and the level of resistance of enterococcus to erythromycin, oxytetracycline, ciprofloxacin and chloramphenicol. In the case of ciprofloxacin, the higher the dose was the earlier the resistant enterococcus appeared and the higher the resistant level was. In the case of chloramphenicol, while the doses of cornmeal were at the ranges of 0.05 g/L to 1g/L, it has similar effect on the form of resistance to ciprofloxacin. In the cases of erythromycin and oxytetracycline there were no obvious relationship between the doeses of cornmeal and the form and the level of resistance.
(3) In the model ecosystem, the appearance of ciprofloxacin resistant enterococcus was not caused by the mutation of gyrA gene; while, the appearance of erythromycin resistant enterococcus has strong correlation to the appearance of ermB gene.
(4) Esp gene did not necessary correlate to the formation of Enterococcus biofilm. In certain concentration range, the ablity of enterococcus form biofilm was correlated to the concentration of glucose. There was certain relationship between antibiotics resistance of enterococcus and the concentration of glucose in the emvironment and the formation of biofilm.
引文
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