血培养阳性标本分离胶促凝管联合SDS 前处理与固体培养基短时培养菌膜应用MALDI-TOF MS 快速鉴定最佳时间探讨

目的　探讨血培养阳性标本分离胶促凝管联合十二烷基硫酸钠（sodium dodecyl sulfate，SDS）前处理与固体培养基短时培养菌膜应用基质辅助激光解析电离飞行时间质谱（matrix-assisted laser desorption ionization time of flightmass spectrometry，MALDI-TOF MS）快速鉴定的最佳时间。方法　收集2020 年8 月～ 2021 年1 月来源于咸阳市第一人民医院微生物室的243 瓶血培养阳性（单数菌）样本，采用MALDI-TOF MS 技术对分离胶促凝管联合SDS 前处理标本直接鉴定，同时对固体培养基短时培养（6h 内）菌膜鉴定，以传统培养（24h）方法鉴定结果为金标准，分析MALDI-TOF MS 快速鉴定血培养阳性菌株的最佳方法和时间。结果　243 瓶血培养阳性标本共鉴定出病原菌44 种，分离率前五位的细菌为大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌、金黄色葡萄球菌和表皮葡萄球菌。SDS 前处理法直接鉴定总符合率为75.72%（184/243），其中革兰阴性菌和革兰阳性菌符合率分别为84.97%（130/153）和60.00%（54/90）；固体培养基生长2h，4h 和6h 的菌膜鉴定总符合率依次为44.44%（108/243），72.02%（175/243）和90.53%（220/243），其中革兰阴性菌依次为59.48%（91/153），83.01%（127/153）和92.81%（142/153），革兰阳性菌依次为18.89%（17/90）,53.33%（48/90）和86.67%（78/90）。金黄色葡萄球菌SDS 前处理法直接鉴定和2h，4h 生长菌膜鉴定符合率均高于凝固酶阴性葡萄球菌（χ2=4.795，12.083, 9.035，均P ＜ 0.01）。革兰阴性菌SDS 前处理法直接鉴定和2h，4h 生长菌膜鉴定符合率均高于革兰阳性菌（χ2=17.880，37.808，24.758, 均P ＜ 0.01)。结论　MALDI-TOF MS 可对血流感染（bloodstreaminfection，BSI）主要病原菌进行快速直接鉴定，根据革兰染色结果联合固体培养基短时培养菌膜鉴定，可进一步缩短细菌鉴定时间，为临床抗感染治疗提供早期依据。

Discussion on the Best Time of Blood Culture Positive Samples for Rapid Identification by MALDI-TOF MS Using Separation Gel Accelerating Tube Combined with SDS Pretreatment and Short-term Culture of Biofilm on Solid Medium

Objective　To explore the best time for rapid identification of blood culture positive samples by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) using separation gel coagulation promoting tube combined with sodium dodecyl sulfate（SDS）pretreatment and short-term culture of bacterial membrane in solid medium. Methods　243 bottles of blood culture positive (singular bacteria) samples from Department of Clinical Laboratory, Xianyang First People’s Hospital from August 2020 to January 2021 were collected. The samples pretreated by gel promoting tube combined with SDS were directly identified by MALDI-TOF MS technology. At the same time, the bacterial membrane was identified by short-term culture in solid medium (within 6h). The identification results of traditional culture (24h) method were taken as the gold standard to analyze the best method and time for rapid identification of blood culture positive strains by MALDI-TOF MS. Results　A total of 44 kinds of pathogenic bacteria were identified in 243 blood culture positive samples. The top five bacteria were Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis. The total coincidence rate of direct identification by SDS pretreatment method was 75.72% (184 / 243), of which the coincidence rates of Gram-negative bacteria and Gram-positive bacteria were 84.97% (130 / 153) and 60.00% (54 / 90), respectively. The total coincidence rates of bacterial membrane identification in solid medium grown for 2h, 4h and 6h were 44.44% (108 / 243), 72.02% (175 / 243) and 90.53% (220 / 243), respectively. Among them Gram negative bacteria were successively 59.48% (91 / 153), 83.01% (127 / 153) and 92.81% (142 / 153), respectively, and Gram positive bacteria were successively 18.89% (17 / 90), 53.33% (48 / 90) and 86.67% (78 / 90), respectively. The coincidence rates between SDS pretreatment identification and identification of biofilm after growing for 2h and 4h for Staphylococcus aureus were higher than those of coagulase negative Staphylococcus (χ2=4.795, 12.083, 9.035，all P ＜ 0.01）.For Gram-negative bacteria were higher than those of Gram-positive bacteria, the differences were statistically significant (χ2=17.880，37.808，24.758, all P<0.01). Conclusion　MALDI-TOF MS can quickly and directly identify the main pathogens of blood flow infection. According to the results of Gram staining combined with short-term culture of bacterial membrane in solid medium, the identification time can be further shortened, the identification accuracy can be improved, and the needs of early clinical etiological diagnosis and timely intervention of blood flow infection can be basically met.