Influence Factors On Microbial Identification By Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry

Objective Fifty eight strains,including forty reference strains and eighteen Cronobacter sakazakii isolated strains,were identified using VITEK MS,with the aim to research on the influence of sample preparation procedures and matrices on microbial discrimination,and its accuracy and reproducibility.The traceability of VITEK MS was also evaluated by cluster analysis.And two data acquisition software of VITEK MS and MALDI 7090 were used,for the purpose of comparing the differences of mass spectrograms between them.Methods All the strains were prepared respectively by three different preparation procedures,including direct transfer method,formic acid extraction method,and formic acid-acetonitrile extraction method,and CHCA matix were used to spot target;one Escherichia coli reference strain and one Staphylococcus aureus reference strain were prepared by formic acid method and four matrices(DHB,SA,FA,CHCA)were used to spot target;and then analyzed by VITEK MS,the results were compared with biochemical ones.A detailed analysis of mass spectrograms were obtained from the software of VITEK MS,then the influence of sample preparation procedures and matrices on the identification results was evaluated,and its accuracy and reproducibility were also investigated.And the traceability of Cronobacter sakazakii strains was researched by the cluster analysis of VITEK MS.The same strains were analyzed by MALDI 7090,and the mass spectrograms were compared with ones by VITEK MS.Results The results of the fifty five strains prepared by the three different preparation procedures were successfully identified to the species level(48/55,87.3%)and to the genus level(7/55,12.7%)by VITEK MS,in agreement with biochemical results,three strains had wrong identification.There was no significant difference in the results confidence among the three different preparation procedures(p>0.05),but the number of mass peaks and their relative intensities showed remarkable difference.The formic acid extraction method performed well on the number of mass peaks and their range.The results confidence of the four matrices were all above 99.9%,in agreement with biochemical results,and the matrix of CHCA had better performance on the the mass spectrograms.And the identification of VITEK MS had good accuracy and reproducibility.At similarity level of 80%,the Cronobacter sakazakii strains prepared by the formic acid extraction method were classified into 5 clusters by the cluster analysis of VITEK MS,and the contaminated pathway of the Cronobacter sakazakii were also speculated.There was no obvious difference in the peak positions and relative intensity between VITEK MS and MALDI 7090.Conclusion MALDI-TOF MS is considered as a reliable technique in the microbial identification.The formic acid extraction method and CHCA matrix are more suitable for the microbial identification by MALDI-TOF MS,which will be a potential technique for the Cronobacter sakazakii traceability.There is no obvious difference in the mass spectrograms between VITEK MS and MALDI 7090.