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

1	Baylan O. An opportunistic pathogen frequently isolated from immunocompromised patients: Burkholderia cepacia complex[J]. Mikrobiyol Bul, 2012,46(2):304-318.
2	Ocak F, Gözalan A, Ozçelik U, et al. Isolation frequency of Burkholderia cepacia from cystic fibrosis patients[J]. Mikrobiyol Bul, 2002,36(1):1-10.
3	Kuzumoto K, Kubota N, Ishii K, et al. Successful cessation of transmitting healthcare- associated infections due to Burkholderia cepacia complex in a neonatal intensive care unit in a Japanese children′s hospital[J]. Eur J Med Res, 2011,16(2):537-542.
4	Liao CH, Chang HT, Lai CC, et al. Clinical characteristics and outcomes of patients with Burkholderia cepacia bacteremia in an intensive care unit[J]. Diagn Microbiol Infect Dis, 2011,70(2):260-266.
5	胡付品，郭燕，朱德妹，等. 2016年中国CHINET细菌耐药性监测[J]. 中国感染与化疗杂志, 2017,17(5):481-491.
6	葛新，邢杰. 硝酸镓联合氧氟沙星抑制尿路致病性大肠杆菌生物膜形成[J]. 中国现代应用药学, 2014,31(11):1330-1333.
7	Peeters E, Nelis HJ, Coenye T. Resistance of planktonic and biofilm-grown Burkholderia cepacia complex isolates to the transition metal gallium[J]. J Antimicrob Chemother, 2008,61(26):1062-1065.
8	吴浩昕，李蓉，葛新. 硝酸镓对临床分离金黄色葡萄球菌生物膜的体外清除作用[J]. 中国感染控制杂志, 2015,14(4):223-226.
9	Marti S, Rodrlguez-Bafio J, Catel-Ferreira M, et a1. Bioflm formation at the solid-liquid and air-liquid interfaces by Acinetobacterspecies[J]. BMC Res Notes, 2011,12(4):5.
10	韩欣欣，李庆淑，申丽婷，等. 鲍曼不动杆菌生物膜形成能力与生物膜相关基因及耐药性之间的关系[J]. 中华危重病急救医学, 2014,26(9):639-643.
11	Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the natural environment to infectious diseases[J]. Nat Rev Microbiol, 2004,2(2):95-108.
12	Lenz Ailyn P, Williamson Kerry S, Pitts B, et al. Localized gene expression in Pseudomonas aeruginosa biofilms[J]. Appl Environ Microbiol, 2008,74(14):4463-4471.
13	Landini P, Antoniani D, Burgess JG, et a1. Molecular mechanisms of compounds afecting bacterial bioflm formation and dispersal[J]. Appl Microbiol Biotechnol, 2010,86(3):813-823.
14	Xu KD, McFeters GA, Stewart PS. Biofilm resistance to antimicrobial agents[J]. Microbiol, 2000,146(3):547-549.
15	胡晓丰，史云，戚丽华，等. 金黄色葡萄球菌生物膜形成机制研究进展[J]. 生物技术通讯, 2014,25(5):714-718.
16	茅国峰，何秋丽，沈少卿. 浙江绍兴地区洋葱伯克霍尔德菌的基因分型及其耐药性[J]. 国际流行病学传染病学杂志, 2015,42(6):376-379.
17	Rao RS, Karthika RU, Singh SP, et a1. Correlation between biofilm production and multiple drug resistance in imipenem resistant clinical isolates of Acinetobacter baumannii[J]. Indian J Med Microbiol, 2008,26(4):333-337.
18	Soriano F, Huelves L, Naves P, et a1. In vitro activity of ciprofloxacin, moxifloxacin, vancomycin and erythromycin against planktonic and biofilm forms of Corynebacterium urealyticum[J]. J Antimicrob Chemother, 2009,63(2):353-356.
19	Espinal P, Marti S, Vila J. Effect of biofilm formation on the survival of Acinetobaeter baumannii on dry surfaces[J]. J Hosp Infect, 2012,80(1):56-60.
20	Wood P, Caldwell DE, Evans E, et al. Surface-catalysed disinfection of thick Pseudomonas aeruginosa biofilms[J]. J Appl Microbiol, 1998,84(6):1092-1098.
21	Jones MN. Use of liposomes to deliver bactericides to bacterial biofilms[J]. Methods Enzymol, 2005,391(9):211-228.
22	Yanagihara K, Tomono K, Imamura Y, et al. Effect of clarithromycin on chronic respiratory infection caused by Pseudomonas aeruginosa with biofilm formation in an experimental murine model[J]. J Antimicrob Chemother, 2002,49(5):867-870.
23	Zaitseva J, Granik V, Belik A, et al. Effect of nitrofurans and NO generators on biofilm formation by Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia 370[J]. Res Microbiol, 2009,160(5):353-357.
24	Dales L, Ferris W, Vandemheen K, et al. Combination antibiotic susceptibility of biofilm- grown Burkholderia cepacia and Pseudomonas aeruginosa isolatedfrom patients with pulmonary exacerbations of cystic fibrosis[J]. Eur J Clin Microbiol Infect Dis, 2009,28(10):1275-1279.
25	Yang L, Barken KB, Skindersoe ME, et al. Effects of iron on DNA release and biofilm development by Pseudomonas aeruginosa[J]. Microbiology, 2007,153(5):1318-1328.
26	Kaneko Y, Thoendel M, Olakanmi O, et al. The transition metal gallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobial and antibiofilm activity[J]. J Clin Invest, 2007,117(4):877-888.

[1]	王青青, 耿翠芝, 苏晓雨, 彭玉晓, 秦明祎, 刘风侠. 乳腺癌芳香化酶抑制剂相关肌肉骨骼不良反应的研究进展[J]. 中华乳腺病杂志(电子版), 2023, 17(04): 243-249.
[2]	马敏榕, 李聪, 周勤. 宫颈癌治疗研究现状[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(05): 497-504.
[3]	林昌盛, 战军, 肖雪. 上皮性卵巢癌患者诊疗中基因检测及分子靶向药物治疗[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(05): 505-510.
[4]	刘星辰, 刘娟, 魏宝宝, 刘洁, 刘辉. XIAP与XAF1异常表达与卵巢癌的相关性分析[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(04): 419-427.
[5]	张利, 张阳, 马菁菁, 喻哲昊, 葛亮, 孙林春. 细胞壁锚定蛋白SasX调控RNAⅢ参与金黄色葡萄球菌ST239克隆生物膜形成及致病性相关研究[J]. 中华实验和临床感染病杂志(电子版), 2023, 17(04): 252-259.
[6]	张龙飞, 高涌, 张懿刚, 余朝文, 刘德朗, 刘亚, 陈世远. 载脂蛋白B、性激素结合球蛋白及金属蛋白酶组织抑制剂-1在皖北地区原发性下腔静脉隔膜型布加综合征的临床意义[J]. 中华普通外科学文献(电子版), 2023, 17(04): 252-256.
[7]	常剑, 邱峰, 毛郁琪. 摄食抑制因子-1与腹腔镜结直肠癌根治术后肝转移的关系分析[J]. 中华普外科手术学杂志(电子版), 2023, 17(05): 502-505.
[8]	巨春蓉, 何建行, 钟南山. 咪唑立宾在器官移植领域的应用及展望[J]. 中华移植杂志(电子版), 2023, 17(04): 227-231.
[9]	中国器官移植发展基金会器官移植受者健康管理专家委员会, 中国医师协会器官移植医师分会, 中华医学会器官移植学分会, 国家肝脏移植质控中心. 肝移植受者雷帕霉素靶蛋白抑制剂临床应用中国专家共识(2023版)[J]. 中华移植杂志(电子版), 2023, 17(04): 193-204.
[10]	刘恒, 侯宇川. 膀胱癌新型灌注药物的研究进展[J]. 中华腔镜泌尿外科杂志(电子版), 2023, 17(05): 445-451.
[11]	杨秀君, 崔梦莹, 张丹, 曲仙智, 苗云皓, 盛基尧, 郑戈, 刘水, 张学文. 信迪利单抗联合仑伐替尼成功转化治疗不可切除肝癌一例[J]. 中华肝脏外科手术学电子杂志, 2023, 12(05): 581-584.
[12]	梁文龙, 曹杰, 黄庆, 林泳, 黄红丽, 杨平, 李冠炜, 胡鹤. 信迪利单抗联合瑞戈非尼治疗晚期结直肠癌的疗效与安全性分析[J]. 中华消化病与影像杂志(电子版), 2023, 13(06): 409-413.
[13]	郑秀丽, 倪敏. 功能性便秘患者的直肠肛门抑制反射特征分析100例[J]. 中华临床医师杂志(电子版), 2023, 17(08): 870-875.
[14]	董青, 丁飞, 郭浩, 李峰. Nesfatin-1/NUCB2在幽门螺杆菌感染相关早期胃癌患者中的表达及临床意义[J]. 中华临床医师杂志(电子版), 2023, 17(07): 783-789.
[15]	熊亚琼, 田文泽, 冷雪春, 尤振兵, 韦欣琪. 集束化干预在肺癌术后顽固性咳嗽中的应用[J]. 中华胸部外科电子杂志, 2023, 10(04): 207-212.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

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

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

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