MALDI-TOF MS鉴定一株小菌落变异型金黄色葡萄球菌

doi:10.3877/cma.j.issn.2095-5820.2018.01.009

目的

探讨运用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ ionization time of flight mass spectrometry，MALDI-TOF MS)技术对金黄色葡萄球菌小菌落变异型的特征性蛋白指纹图谱进行深度研究的价值。

方法

分别采用传统生化鉴定、16S rRNA测序技术和MALDI-TOF MS技术对1株小菌落变异型金黄色葡萄球菌进行鉴定，通过质谱鉴定系统VITEK MS的质谱科研模式获取金黄色葡萄球菌小菌落突变株(small colony variants, SCV)的质谱图，分析代表金黄色葡萄球菌agr和sigB系统的m/z表达强度，并用SARAMIS软件构建基于质谱峰谱特征的系统发育树。

结果

使用MALDI-TOF MS技术鉴定该菌株为金黄色葡萄球菌，与传统生化鉴定、16S rRNA测序技术的鉴定结果一致。其菌落形态符合典型SCV菌株的特征，预示生物膜形成的PSMα3 (m/z 2634.4)、PSMα1(m/z 2286.8)和PSMα4(m/z 2198.6)的表达强度分别为100%、76.1%和32.3%，反映急性早期感染状态的agr调控系统的delta毒素强度只有10%；反映慢性、复发性感染的sigB调控系统的峰谱相对强度均<25%，该菌株和科研菌种库SCV的系统发育树近似度>70%。

结论

MALDI-TOF MS技术不仅能够快速准确鉴定不典型金黄色葡萄球菌，且生成的质谱图能够初步提示菌株在宿主体内的感染状态，表明该技术具有极大的应用前景。

关键词: 基质辅助激光解吸电离飞行时间质谱, 辅助基因调节子(agr)，delta毒素，sigB基因, 金黄色葡萄球菌, 小菌落变异型

Objective

To explore the feasibility of using matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) to study the variation of Staphylococcus aureus colony, and to study the characteristic protein fingerprint of bacteria in depth.

Methods

Traditional biochemical identification, 16S rRNA gene sequencing and MALDI-TOF MS were used to identify the isolate. The MS for the Staphylococcus aureus SCV strain was obtained by vitek MS, and the m/z expression intensity of Staphylococcus aureus agr and sigB systems was analyzed. Phylogenetic tree based on peak spectrum of mass spectrum was constructed by SARAMIS software.

Results

The isolate was identified as Staphylococcus aureus by MALDI-TOF MS, the results obtained from the other two methods mentioned above including traditional biochemical identification and 16S rRNA gene seqencing were consistant with the MALDI-TOF MS. The colony morphology was in line with the characteristic of typical SCV strain, and the expression intensity related with biofilm formation was 100% for PSMα3 (m/z 2634.4); 76.1% for PSMα1 (m/z 2286.8) and 32.3% for PSMα4 (m/z 2198.6). The delta toxin intensity of agr regulatory system reflecting acute early infection was only 10%. The relative intensity of peak spectrum of sigB regulatory system reflecting chronic and recurrent infection was less than 25%. The phylogenetic tree approximation of the strain and SCV was more than 70%.

Conclusions

MALDI-TOF MS technology not only can identify the atypical Staphylococcus aureus, quickly and accurately, but also can preliminarily indicate the infection status of the strain in the host, and thus this technology has great application prospect.

Key words: Matrix-assisted laser desorption/ionization time of flight mass spectrometry, Accessory gene regulator(agr), delta toxin, sigB gene, Staphylococcus aureus, Small colony variants

图1 SCV菌落在血平板和MH平板的生长情况

表1 金黄色葡萄球菌在VITEK2 Compart的药敏结果

抗生素	MIC(μg/ml)	药敏结果
青霉素	≥0.5	耐药
苯唑西林	≥4	耐药
庆大霉素	≤0.5	敏感
环丙沙星	≤0.5	敏感
左氧氟沙星	≤0.12	敏感
莫西沙星	≤0.25	敏感
诱导型克林霉素耐药	—	阴性
红霉素	≤0.25	敏感
克林霉素	≤0.25	敏感
奎奴普汀/达福普汀	≤0.25	敏感
利奈唑胺	1	敏感
万古霉素	≤0.5	敏感
四环素	≤1	敏感
替加环素	≤0.12	敏感
呋喃妥因	≤16	敏感
复方新诺明	≤10	敏感
头孢西丁筛选	—	阳性

表1 金黄色葡萄球菌在VITEK2 Compart的药敏结果

抗生素	MIC(μg/ml)	药敏结果
青霉素	≥0.5	耐药
苯唑西林	≥4	耐药
庆大霉素	≤0.5	敏感
环丙沙星	≤0.5	敏感
左氧氟沙星	≤0.12	敏感
莫西沙星	≤0.25	敏感
诱导型克林霉素耐药	—	阴性
红霉素	≤0.25	敏感
克林霉素	≤0.25	敏感
奎奴普汀/达福普汀	≤0.25	敏感
利奈唑胺	1	敏感
万古霉素	≤0.5	敏感
四环素	≤1	敏感
替加环素	≤0.12	敏感
呋喃妥因	≤16	敏感
复方新诺明	≤10	敏感
头孢西丁筛选	—	阳性

图2 16S rRNA的PCR扩增产物琼脂糖凝胶电泳图

表2 VITEK MS检测SW菌株的质谱图峰谱表达情况m/z值(%)

菌株编号	生物膜相关PSMs				agr系统	sigB系统
菌株编号	PSMα4 2198	PSMα1 2289	PSMα2 2307	PSMα3 2636/2685	delta毒素3007/3037	graF (SAS030) 5032	SAS049 5525	SA0772 6889
SCV1	33.4	18.1	47.9	17.5/100.0	44.7	1.6	<1	13.7
SCV2	3.0	<1	6.0	100.0	37.3	14.9	13.1	81.9
SCV3	2.9	<1	6.2	100.0	37.2	14.9	13.1	81.9
SCV4	2.3	<1	6.3	92.3	36.2	20.9	16.9	100.0
SCV5	29.2	27.5	79.1	100.0	44.5	4.2	17.7	100.0
SCV6	24.7	26.9	80.8	100.0	43.4	8.6	29.1	22.2
SCV7	9.0	7.6	51.6	100.0	51.3	5.7	10.6	7.7
SCV8	4.0	4.5	42.1	100.0	28.0	5.8	5.2	6.1
53	32.2	76.1	91.4	100.0	10.5	10.7	27.3	16.3

表2 VITEK MS检测SW菌株的质谱图峰谱表达情况m/z值(%)

菌株编号	生物膜相关PSMs				agr系统	sigB系统
菌株编号	PSMα4 2198	PSMα1 2289	PSMα2 2307	PSMα3 2636/2685	delta毒素3007/3037	graF (SAS030) 5032	SAS049 5525	SA0772 6889
SCV1	33.4	18.1	47.9	17.5/100.0	44.7	1.6	<1	13.7
SCV2	3.0	<1	6.0	100.0	37.3	14.9	13.1	81.9
SCV3	2.9	<1	6.2	100.0	37.2	14.9	13.1	81.9
SCV4	2.3	<1	6.3	92.3	36.2	20.9	16.9	100.0
SCV5	29.2	27.5	79.1	100.0	44.5	4.2	17.7	100.0
SCV6	24.7	26.9	80.8	100.0	43.4	8.6	29.1	22.2
SCV7	9.0	7.6	51.6	100.0	51.3	5.7	10.6	7.7
SCV8	4.0	4.5	42.1	100.0	28.0	5.8	5.2	6.1
53	32.2	76.1	91.4	100.0	10.5	10.7	27.3	16.3

图3 53号菌株的质谱图

图4 质控菌株ATCC25923的质谱图

图5 质控菌株ATCC29213的质谱图

图6 基于SARAMIS^?软件构建的金黄色葡萄球菌SARAMIS构建的系统发育树

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Identification of Staphylococcus aureus small colony variant by MALDI-TOF MS

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Identification of Staphylococcus aureus small colony variant by MALDI-TOF MS