硝酸镓联合抗菌药物抑制洋葱伯克霍尔德菌生物膜形成的研究

doi:10.3877/cma.j.issn.2095-5820.2019.01.009

目的

研究硝酸镓单用及联合头孢他啶或(和)美罗培南对洋葱伯克霍尔德菌生物膜形成的抑制作用。

方法

收集2017年1月至12月临床分离到的34株洋葱伯克霍尔德菌，微量肉汤稀释法检测其对硝酸镓及头孢他啶、复方磺胺甲恶唑、左氧氟沙星、哌拉西林/他唑巴坦、替加环素、美罗培南、氯霉素、米诺环素、头孢哌酮/舒巴坦9种抗菌药物的最低抑菌浓度(minimal inhibitory concentration，MIC)。结晶紫染色法定性、定量分析生物膜形成能力，比较不同生物膜形成能力菌株对硝酸镓及这9种抗菌药物的敏感性。将1.0×MIC的硝酸镓和头孢他啶、美罗培南单独应用及联合使用，检测其对洋葱伯克霍尔德生物膜的抑制率。

结果

培养48小时34株细菌均能形成稳定的生物膜。其中(弱)阳性10株，占29.4%；强阳性24株，占70.6%，生物膜(弱)阳性和强阳性菌株对头孢哌酮舒巴坦的耐药率差异具有统计学意义。加入硝酸镓前后菌株的生物膜形成光密度(optical density, OD)值、硝酸镓单用与联合头孢他啶后的OD值差异均具有统计学意义，但硝酸镓单用与联合美罗培南后的OD值差异无统计学意义。

结论

洋葱伯克霍尔德菌具有较强的生物膜形成能力，硝酸镓单用及联合头孢他啶对菌株生物膜形成具有明显抑制作用。

关键词: 洋葱伯克霍尔德菌, 生物膜, 硝酸镓, 抑制

Objective

To study the inhibitory effects of gallium nitrate alone and combined with ceftazidime or meropenem on biofilm formation of Burkholderia cepacia.

Methods

Thirty-four strains of Burkholderia cepacia were isolated from January to December 2017. The minimal inhibitory concentration (MIC) of Burkholderia cepacia to gallium nitrate, ceftazidime, compound sulfamethoxazole, levofloxacin, piperacillin/tazobactam, tegacycline, meropenem, chloramphenicol, minocycline and cefoperazone/sulbactam were detected by broth dilution method. Crystal violet staining was used to quantitatively and qualitatively analyze the ability of biofilm formation of, Burkholderia cepacia and to compare the sensitivity of those strains with different ability of biofilm formation to gallium nitrate and the nine antimicrobial agents. The effects of gallium nitrate and ceftazidime and meropenem on biofilm formation were evaluated separately or jointly at 1.0×MIC.

Results

All the 34 strains could form stable biofilms after 48 hours culture. Among them, 10 were weak postive (29.4%) and 24 were strong positive (70.6%). There was a significant difference in the resistant rate of cefoperazone and sulbactam between the weak and strong positive strains with biofilm. There were significant differences in optical density (OD) values of biofilm formed by the strains before and after treated with gallium nitrate alone or combined with ceftazidime, but there was no significant difference in OD values between treated with gallium nitrate alone and meropenem alone.

Conclusion

Burkholderia cepacia have strong biofilm formation ability. Gallium nitrate alone or in combined with ceftazidime have obvious inhibitory effect on the biofilm formation.

Key words: Burkholderia cepacia, Biofilm, Gallium nitrate, Inhibition

表1 34株临床分离的洋葱伯克霍尔德菌体外药敏分析结果(n=34)

抗菌药物	敏感率(%)	中介率(%)	耐药率(%)	MIC50(μg/ml)	MIC90(μg/ml)	MIC范围(μg/ml)
头孢他啶	64.7	23.5	11.8	8.0	32	0.25~32
复方磺胺甲恶唑	100.0	0.0	0.0	0.5	1	0.016~2
左氧氟沙星	38.2	5.9	55.9	8.0	32	0.25~>32
哌拉西林他唑巴坦	41.2	0.0	58.8	128.0	256	<0.125~>256
替加环素	85.3	11.8	2.9	1.0	4	0.125~32
美罗培南	61.8	23.5	14.7	4.0	16	0.125~32
氯霉素	91.2	2.9	5.9	8.0	8	<0.064~>256
米诺环素	100.0	0.0	0.0	1.0	4	0.064~4
头孢哌酮舒巴坦	47.0	11.8	41.2	32.0	256	<0.125~256
硝酸镓	…	…	…	512	1024	128~>1024

表1 34株临床分离的洋葱伯克霍尔德菌体外药敏分析结果(n=34)

抗菌药物	敏感率(%)	中介率(%)	耐药率(%)	MIC50(μg/ml)	MIC90(μg/ml)	MIC范围(μg/ml)
头孢他啶	64.7	23.5	11.8	8.0	32	0.25~32
复方磺胺甲恶唑	100.0	0.0	0.0	0.5	1	0.016~2
左氧氟沙星	38.2	5.9	55.9	8.0	32	0.25~>32
哌拉西林他唑巴坦	41.2	0.0	58.8	128.0	256	<0.125~>256
替加环素	85.3	11.8	2.9	1.0	4	0.125~32
美罗培南	61.8	23.5	14.7	4.0	16	0.125~32
氯霉素	91.2	2.9	5.9	8.0	8	<0.064~>256
米诺环素	100.0	0.0	0.0	1.0	4	0.064~4
头孢哌酮舒巴坦	47.0	11.8	41.2	32.0	256	<0.125~256
硝酸镓	…	…	…	512	1024	128~>1024

图1 高倍镜下观察洋葱伯克霍尔德菌生物膜形成(10×40倍)

表2 不同生物膜形成能力的菌株对抗菌药物的耐药率比较

抗菌药物	耐药率%		χ²	P
抗菌药物	弱阳性(n=10)	强阳性(n=24)	χ²	P
头孢他啶	0.0	16.7	1.889	0.169
复方磺胺异恶唑	0.0	0.0	...	...
左氧氟沙星	50.0	58.3	0.199	0.656
哌拉西林他唑巴坦	50.0	62.5	0.455	0.500
替加环素	10.0	0.0	2.473	0.116
美罗培南	10.0	16.7	0.250	0.617
氯霉素	10.0	4.2	0.434	0.510
米诺环素	0.0	0.0	...	...
头孢哌酮舒巴坦	30.0	45.8	3.969	0.046

表2 不同生物膜形成能力的菌株对抗菌药物的耐药率比较

抗菌药物	耐药率%		χ²	P
抗菌药物	弱阳性(n=10)	强阳性(n=24)	χ²	P
头孢他啶	0.0	16.7	1.889	0.169
复方磺胺异恶唑	0.0	0.0	...	...
左氧氟沙星	50.0	58.3	0.199	0.656
哌拉西林他唑巴坦	50.0	62.5	0.455	0.500
替加环素	10.0	0.0	2.473	0.116
美罗培南	10.0	16.7	0.250	0.617
氯霉素	10.0	4.2	0.434	0.510
米诺环素	0.0	0.0	...	...
头孢哌酮舒巴坦	30.0	45.8	3.969	0.046

表3 硝酸镓单用及分别联合头孢他啶和美罗培南光密度与抑制率比较

组别	光密度M(P₂₅, P₇₅)	抑制率% M(P₂₅, P₇₅)
①生理盐水组	1.052(0.310,2.232)	…
②硝酸镓组	0.098(0.092,0.131)	89.8(67.7,95.1)
③CAZ组	0.102(0.096,0.118)	89.9(68.3,95.2)
④MEM组	0.095(0.089,0.105)	90.7(69.7,95.4)
⑤硝酸镓＋CAZ组	0.152(0.128,0.197)	85.8(48.5,92.6)
⑥硝酸镓＋MEM组	0.119(0.097,0.163)	88.2(64.2,94.3)
⑦CAZ＋MEM组	0.088(0.082,0.098)	91.0(70.7,95.9)
⑧硝酸镓＋CAZ＋MEM组	0.111(0.101,0.151)	88.0(57.0,93.9)
检验统计量H值	142.410	8.546
P值	<0.01	0.201

表3 硝酸镓单用及分别联合头孢他啶和美罗培南光密度与抑制率比较

组别	光密度M(P₂₅, P₇₅)	抑制率% M(P₂₅, P₇₅)
①生理盐水组	1.052(0.310,2.232)	…
②硝酸镓组	0.098(0.092,0.131)	89.8(67.7,95.1)
③CAZ组	0.102(0.096,0.118)	89.9(68.3,95.2)
④MEM组	0.095(0.089,0.105)	90.7(69.7,95.4)
⑤硝酸镓＋CAZ组	0.152(0.128,0.197)	85.8(48.5,92.6)
⑥硝酸镓＋MEM组	0.119(0.097,0.163)	88.2(64.2,94.3)
⑦CAZ＋MEM组	0.088(0.082,0.098)	91.0(70.7,95.9)
⑧硝酸镓＋CAZ＋MEM组	0.111(0.101,0.151)	88.0(57.0,93.9)
检验统计量H值	142.410	8.546
P值	<0.01	0.201

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Study of the inhibitory effect of gallium nitrate combined with antimicrobial agents on biofilm formation of Burkholderia cepacia

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Study of the inhibitory effect of gallium nitrate combined with antimicrobial agents on biofilm formation of Burkholderia cepacia