中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2015年
4期
285-289
,共5页
欧阳净%熊丽蓉%冯伟%孙凤军%陈勇川
歐暘淨%熊麗蓉%馮偉%孫鳳軍%陳勇川
구양정%웅려용%풍위%손봉군%진용천
葡萄球菌,表皮%生物膜%抑制肽
葡萄毬菌,錶皮%生物膜%抑製肽
포도구균,표피%생물막%억제태
Staphylococcus epidermidis%Biofilms%Inhibitory peptide
目的 研究表皮葡萄球菌生物膜抑制肽(简称抑制肽)对表皮葡萄球菌早期黏附及生物膜形成的影响. 方法 采用多肽合成仪合成抑制肽,其纯度为96.8％、相对分子质量为874.4.(1)采用终浓度为1～256 μg/mL的抑制肽培养表皮葡萄球菌ATCC 35984(下同)菌液,不含菌液的M-H肉汤为空白对照,观察抑制肽对该菌的MIC(样本数为3).(2)采用含终浓度为16、32、64、128、256 μg/mL抑制肽的胰蛋白胨大豆肉汤(TSB)培养液培养表皮葡萄球菌菌液(设为相应浓度抑制肽组),以TSB培养基培养前述菌液作为阴性对照组,从培养即刻起每小时观察该菌的生长情况(结果以吸光度值表示),绘制该菌培养24 h内的生长曲线(每组各时相点样本数为3).(3)采用含终浓度为16、32、64、128、256 μg/mL抑制肽的TSB培养液培养表皮葡萄球菌菌液(设为相应浓度抑制肽组),以TSB培养基培养前述菌液作为阴性对照组,培养4h检测该菌黏附情况,培养20 h检测该菌生物膜的形成情况(结果均以吸光度值表示,样本数均为10).(4)采用含128 μg/mL抑制肽的TSB培养液培养表皮葡萄球菌菌液(设为128μg/mL抑制肽组),以TSB培养基培养前述菌液作为阴性对照组,培养24 h后采用激光扫描共聚焦显微镜观察该菌的黏附与生物膜形成情况(样本数为3).对数据行单因素方差分析、LSD检验、Dunnett T3检验. 结果 (1)抑制肽对表皮葡萄球菌的MIC大于256 μg/mL. (2)与阴性对照组比较,各浓度抑制肽组表皮葡萄球菌培养24 h内的生长曲线无明显变化.(3)256、128、64、32 μg/mL抑制肽组培养4h表皮葡萄球菌的黏附情况分别为0.20 ±0.04、0.27 ±0.03、0.35±0.04、0.40 ±0.04,明显少于阴性对照组(0.53±0.10,P＜0.05或P＜0.01);16 μg/mL抑制肽组培养4 h该菌的黏附情况(0.47±0.09)与阴性对照组相近(P＞0.05).256、128、64 μg/mL抑制肽组培养20 h的表皮葡萄球菌生物膜形成情况分别为0.49 ±0.10、0.68 ±0.06、0.93 ±0.13,明显少于阴性对照组(1.21 ±0.18,P＜0.05或P＜0.01);32、16 μg/mL抑制肽组培养20 h该菌生物膜形成情况分别为1.18 ±0.22、1.15 ±0.26,与阴性对照组相近(P值均大于0.05).(4)激光扫描共聚焦显微镜显示,培养24 h后阴性对照组黏附的表皮葡萄球菌较多,形成的生物膜结构致密;128 μg/mL抑制肽组黏附的表皮葡萄球菌较少,形成的生物膜结构较为疏松. 结论 抑制肽能显著抑制表皮葡萄球菌早期黏附及生物膜形成,但其结构需要进一步修饰.
目的 研究錶皮葡萄毬菌生物膜抑製肽(簡稱抑製肽)對錶皮葡萄毬菌早期黏附及生物膜形成的影響. 方法 採用多肽閤成儀閤成抑製肽,其純度為96.8％、相對分子質量為874.4.(1)採用終濃度為1～256 μg/mL的抑製肽培養錶皮葡萄毬菌ATCC 35984(下同)菌液,不含菌液的M-H肉湯為空白對照,觀察抑製肽對該菌的MIC(樣本數為3).(2)採用含終濃度為16、32、64、128、256 μg/mL抑製肽的胰蛋白胨大豆肉湯(TSB)培養液培養錶皮葡萄毬菌菌液(設為相應濃度抑製肽組),以TSB培養基培養前述菌液作為陰性對照組,從培養即刻起每小時觀察該菌的生長情況(結果以吸光度值錶示),繪製該菌培養24 h內的生長麯線(每組各時相點樣本數為3).(3)採用含終濃度為16、32、64、128、256 μg/mL抑製肽的TSB培養液培養錶皮葡萄毬菌菌液(設為相應濃度抑製肽組),以TSB培養基培養前述菌液作為陰性對照組,培養4h檢測該菌黏附情況,培養20 h檢測該菌生物膜的形成情況(結果均以吸光度值錶示,樣本數均為10).(4)採用含128 μg/mL抑製肽的TSB培養液培養錶皮葡萄毬菌菌液(設為128μg/mL抑製肽組),以TSB培養基培養前述菌液作為陰性對照組,培養24 h後採用激光掃描共聚焦顯微鏡觀察該菌的黏附與生物膜形成情況(樣本數為3).對數據行單因素方差分析、LSD檢驗、Dunnett T3檢驗. 結果 (1)抑製肽對錶皮葡萄毬菌的MIC大于256 μg/mL. (2)與陰性對照組比較,各濃度抑製肽組錶皮葡萄毬菌培養24 h內的生長麯線無明顯變化.(3)256、128、64、32 μg/mL抑製肽組培養4h錶皮葡萄毬菌的黏附情況分彆為0.20 ±0.04、0.27 ±0.03、0.35±0.04、0.40 ±0.04,明顯少于陰性對照組(0.53±0.10,P＜0.05或P＜0.01);16 μg/mL抑製肽組培養4 h該菌的黏附情況(0.47±0.09)與陰性對照組相近(P＞0.05).256、128、64 μg/mL抑製肽組培養20 h的錶皮葡萄毬菌生物膜形成情況分彆為0.49 ±0.10、0.68 ±0.06、0.93 ±0.13,明顯少于陰性對照組(1.21 ±0.18,P＜0.05或P＜0.01);32、16 μg/mL抑製肽組培養20 h該菌生物膜形成情況分彆為1.18 ±0.22、1.15 ±0.26,與陰性對照組相近(P值均大于0.05).(4)激光掃描共聚焦顯微鏡顯示,培養24 h後陰性對照組黏附的錶皮葡萄毬菌較多,形成的生物膜結構緻密;128 μg/mL抑製肽組黏附的錶皮葡萄毬菌較少,形成的生物膜結構較為疏鬆. 結論 抑製肽能顯著抑製錶皮葡萄毬菌早期黏附及生物膜形成,但其結構需要進一步脩飾.
목적 연구표피포도구균생물막억제태(간칭억제태)대표피포도구균조기점부급생물막형성적영향. 방법 채용다태합성의합성억제태,기순도위96.8％、상대분자질량위874.4.(1)채용종농도위1～256 μg/mL적억제태배양표피포도구균ATCC 35984(하동)균액,불함균액적M-H육탕위공백대조,관찰억제태대해균적MIC(양본수위3).(2)채용함종농도위16、32、64、128、256 μg/mL억제태적이단백동대두육탕(TSB)배양액배양표피포도구균균액(설위상응농도억제태조),이TSB배양기배양전술균액작위음성대조조,종배양즉각기매소시관찰해균적생장정황(결과이흡광도치표시),회제해균배양24 h내적생장곡선(매조각시상점양본수위3).(3)채용함종농도위16、32、64、128、256 μg/mL억제태적TSB배양액배양표피포도구균균액(설위상응농도억제태조),이TSB배양기배양전술균액작위음성대조조,배양4h검측해균점부정황,배양20 h검측해균생물막적형성정황(결과균이흡광도치표시,양본수균위10).(4)채용함128 μg/mL억제태적TSB배양액배양표피포도구균균액(설위128μg/mL억제태조),이TSB배양기배양전술균액작위음성대조조,배양24 h후채용격광소묘공취초현미경관찰해균적점부여생물막형성정황(양본수위3).대수거행단인소방차분석、LSD검험、Dunnett T3검험. 결과 (1)억제태대표피포도구균적MIC대우256 μg/mL. (2)여음성대조조비교,각농도억제태조표피포도구균배양24 h내적생장곡선무명현변화.(3)256、128、64、32 μg/mL억제태조배양4h표피포도구균적점부정황분별위0.20 ±0.04、0.27 ±0.03、0.35±0.04、0.40 ±0.04,명현소우음성대조조(0.53±0.10,P＜0.05혹P＜0.01);16 μg/mL억제태조배양4 h해균적점부정황(0.47±0.09)여음성대조조상근(P＞0.05).256、128、64 μg/mL억제태조배양20 h적표피포도구균생물막형성정황분별위0.49 ±0.10、0.68 ±0.06、0.93 ±0.13,명현소우음성대조조(1.21 ±0.18,P＜0.05혹P＜0.01);32、16 μg/mL억제태조배양20 h해균생물막형성정황분별위1.18 ±0.22、1.15 ±0.26,여음성대조조상근(P치균대우0.05).(4)격광소묘공취초현미경현시,배양24 h후음성대조조점부적표피포도구균교다,형성적생물막결구치밀;128 μg/mL억제태조점부적표피포도구균교소,형성적생물막결구교위소송. 결론 억제태능현저억제표피포도구균조기점부급생물막형성,단기결구수요진일보수식.
Objective To study the effects of inhibitory peptide of Staphylococcus epidermidis (SE) biofilm (briefly referred to as inhibitory peptide) on adhesion and biofilm formation of SE at early stage.Methods By using peptide synthesizer,the inhibitory peptide was synthesized with purity of 96.8％ and relative molecular mass of 874.4.(1) Solution of SE ATCC 35984 (the same below) was cultivated with inhibitory peptide in the final concentrations of 1-256 μg/mL,and the M-H broth without bacteria solution was used as blank control.The MIC of the inhibitory peptide against SE was determined (n =3).(2) Solution of SE was cultivated with trypticase soy broth (TSB) culture solution containing inhibitory peptide in the final concentrations of 16,32,64,128,and 256 μg/mL (set as inhibitory peptide groups in corresponding concentration),and solution of SE being cultivated with TSB culture medium was used as negative control group.Growth of SE was observed every one hour from immediately after cultivation (denoted as absorbance value),and the growth curve of SE during the 24 hours of cultivation was drawn,with 3 samples in each group at each time point.(3) Solution of SE was cultivated with TSB culture solution containing inhibitory peptide in the final concentrations of 16,32,64,128,and 256 μg/mL (set as inhibitory peptide groups in corresponding concentration),and solution of SE being cultivated with TSB culture medium was used as negative control group.Adhesive property of SE was observed after cultivation for 4 hours (denoted as absorbance value,n =10);biofilm formation of SE was observed after cultivation for 20 hours (denoted as absorbance value,n =10).(4) Solution of SE was cultivated with TSB culture solution containing inhibitory peptide in the final concentration of 128 μg/mL (set as 128 μg/mL inhibitory peptide group),and solution of SE being cultivated with TSB culture medium was used as negative control group.Adhesive property of SE and its biofilm formation were observed with confocal laser scanning microscope (CLSM),and the sample numbers were both 3.Data were processed with one-way analysis of variance,LSD test,and Dunnett T3 test.Results (1) The MIC of inhibitory peptide against SE exceeded 256 μg/mL.(2) There was no significant difference in the growth curve of SE between inhibitory peptide groups in different concentrations and negative control group.(3) After 4 hours of cultivation,the absorbance values of adhesive property of SE in 256,128,64,and 32 μg/mL inhibitory peptide groups were respectively 0.20 ± 0.04,0.27 ± 0.03,0.35 ± 0.04,and 0.40 ± 0.04,which were significantly lower than the absorbance value in negative control group (0.53 ±0.10,P ＜ 0.05 or P ＜ 0.01);the absorbance value of adhesive property of SE in 16 μg/mL inhibitory peptide group was 0.47 ± 0.09,which was close to the absorbance value in negative control group (P ＞ 0.05).After 20 hours of cultivation,the absorbance values of biofilm formation of SE in 256,128,and 64 μg/mL inhibitory peptide groups were respectively 0.49 ± 0.10,0.68 ± 0.06,and 0.93 ± 0.13,which were significantly less than the absorbance value in negative control group (1.21 ± 0.18,P ＜ 0.05 or P ＜ 0.01);the absorbance values of biofilm formation in 32 and 16 μg/mL inhibitory peptide groups were respectively 1.18 ± 0.22 and 1.15 ± 0.26,which were close to the absorbanee value in negative control group (with P values above 0.05).(4) CLSM showed that more adhering bacteria and compact structure of biofilm were observed in negative control group,but less adhering bacteria and loose structure of biofilm were observed in 128 μg/mL inhibitory peptide group.Conclusions The inhibitory peptide can inhibit adhesion and biofilm formation of SE at early stage,but its structure still needs to be further modified.
