基质辅助激光解析电离飞行时间质谱检测耐甲氧西林金黄色葡萄球菌δ-毒素的应用

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the detection of MRSA δ-toxin

目的本研究采用基质辅助激光解析电离飞行时间质谱(MALDI-TOFMS)检测δ-毒素,评估其在耐甲氧西林金黄色葡萄球菌(MRSA)的分型和毒力表达中的作用。方法使用Bruker microflex MALDI-TOF仪器,采集2000~20000Da质量范围内,以正线性模式采集图谱,使用仪器配套的MALDI Biotyper 2.0软件分析MRSA菌株的原始图谱,产生的峰列表直接使用flexAnalysis、clinProTools 3.0软件分析。结果本研究中共检测83株MRSA,共有39(47.0%)株MRSA检出(3005±5)m/z信号峰,其中HA-MRSA19(22.9%)株,CA-MRSA20(26.5%)株,P=0.766,两者之间无显著性差异;33(39.8%)株MRSA检出(3035±5)m/z信号峰,其中HA-MRSA9(10.8%)株,CA-MRSA24(28.9%)株,P=0.003,两者之间有显著性差异;11(13.%)株MRSA既未检出(3005±5)m/z信号峰也未检出(3035±5)m/z信号峰,全部为HA-MRSA菌株。spa分型中检出(3005±5)m/z信号峰,15(18.1%)株为t062型,8株为t015(9.6%)型,4株为t030(4.8%)株,P=0,有显著性差异;检出(3035±5)m/z信号峰,31(37.3%)株为t437型,2(2.4%)株t8660型,P=0,有显著性差异;(3035±5)m/z作为spat437型特征信号ROC曲线下面积0.89,P=0。11株未检出δ-毒素,6株分离自骨关节标本,3株分离自呼吸道标本,1株分离自慢性溃疡的分泌物标本,1株分离自血液;在血平板的菌落特征,6株MRSA菌落形态发生改变,5株菌落形态正常。结论MALDI-TOF MS使用常规方法即可快速检测MRSA的δ-毒素,其质谱峰为(3005±5)m/z和(3035±5)m/z两种;(3035±5)m/z是δ-毒素的同基因变异体(HldG10S)的质谱峰,该峰可快速鉴别spat437型;不产生δ-毒素的菌株是agr调控系统失调的表现,与慢性感染、小菌落形成、无明显β-溶血有关。

Objective In this study, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect the δ-toxin, and to evaluate the role of δ-toxin production in methicillin-resistant Staphylococcus aureus (MRSA) typing and virulence expression. Methods The Bruker microflex MALDI-TOF was used to analyze collected proteins with the relative molecular weight ranging from 2,000 to 20,000 Da in a positive linear mode, and the software MALDI Biotyper 2 was used to analyze the original map of the MRSA strain. The generated list of peaks were analyzed directly by flexAnalysis and clinProTools3.0. Results A total of 83 MRSA were analyzed, and 39 (47.0%) MRSA showed peaks of (3005±5)m/z, including19 (22.9%) HA-MRSA and 20 ( 26.5%) CA-MRSA, showing no significant difference (P=0.766);33 (39.8%)MRSA were detected with peaks at (3035±5)m/z , including 9 (10.8%) HA-MRSA and 24 (28.9%) CA-MRSA, showing the statistically significant difference between the two (P=0.003);11 (13%) MRSA were not detected with any peak at (3005±5)m/z or (3035±5)m/z, and thus all strains were HA-MRSA. As to spa typing, peaks at (3005±5)m/z were detected, 15 (18.1%) belonged to t062, 8 (9.6%) were t015, and 4 (4.8%) were t030, showing statistically significant difference (P=0);peaks at (3035±5)m/z were also found, 31 (37.3%) were t437 , 2 ( 2.4%) were t8660, showing statistically significant difference (P=0);the peak at (3035±5)m/z was the characteristic for spa type t437, and its area under the ROC curve was 0.89 (P=0). The δ- toxin was not detected in 11 strains, among which 6 were isolated from bone and joint specimens, 3 were isolated from the respiratory tract specimens, one from the secretions of chronic ulcer specimens, one isolated from the blood. In the blood plate colonies, 6 strains of MRSA colony morphology changed, while 5 colonies were normal. Conclusions MALDI-TOF MS can be used to quickly detect the δ- toxin of MRSA, and their mass spectrum peaks were at (3005±5)m/z and (3035±5)m/z. The peak at (3035±5)m/z is the mass spectrum of the δ-toxin homologous variant (HldG10S), which can quickly identify spa t437. The absence of δ-toxin is a manifestation of the dysregulation of the agr regulatory system, which is associated with chronic infections, small colony formation, and no obvious beta hemolysis.