Study On Rapid Detection Of Pathogenic Microorganisms By Confocal Raman Microspectroscopy

Objective:The traditional detection methods of clinical microorganisms are at least 24~48 hours,which significantly reduce the effectiveness of clinical microbiology laboratory for rapid microbial detection and identification.Meanwhile,professional skills,operating skills and work experience are highly demanded for laboratory technicians.Moreover,drug-resistance of pathogenic microorganisms is increasing due to the misuse of broad-spectrum antibiotics.These would cause inadequacy of the initial therapy because the appropriate treatment may not be selected at early stage of the infection.Thus,rapid detection and identification of clinical pathogenic microorganisms will have a strong impact on the morbidity,mortality,and duration of hospitalization.The ability to rapidly and accurately identify the species of potent pathogens has received a high level of attention in clinical medicine,but this is still a challenge for clinical microbiology laboratory.In recent years,with the rapid development of instrument technology,Raman spectrometer gradually emerging in the application in the domain of biology and medicine,Raman spectroscopy technology appears new development constantly in the rapid identification and classification of microorganisms because of its rapid,efficient,sensitive,noninvasive,repeatability and other unique advantages.In our study,confocal Raman microspectroscopy is applied to rapidly detect and distinguish clinical common pathogenic microorganisms,and to explore the value of confocal Raman microspectroscopy in rapid detection of clinical pathogenic microorganisms.Methods:(1)Ten standard strains were detected by confocal Raman microspectroscopy,including gram-negative bacteria: Escherichia coli(E.coli)ATCC 25922,Klebsiella Pneumoniae(K.Pneumoniae)ATCC 700603,Acinetobacter baumannii(A.baumanii)ATCC 19606.Gram-positive bacteria: Staphylococcus aureus(S.aureus)ATCC 25923,Bacillus thuringiensis(B.thuringiensis)ATCC 10792,Bacillus cereus(B.cereus)ATCC 11778,Bacillus subtilis(B.subtilis)ATCC 6633.Fungi: Candida albicans(C.albicans)ATCC 10231,Candida tropical(C.tropical)ATCC 750 and Candida glabrata(C.glabrata)ATCC 64677.(2)The A.baumannii and E.coli(1:1)were mixed culture and detected by confocal Raman microspectroscopy.(3)The Raman spectra were analyzed by two multivariate analysis methods,principal component analysis(PCA)and hierarchical cluster analysis(HCA).Results:1.The Raman spectra of Gram-positive bacteria(E.coli,A.baumanii,and K.Pneumoniae),Gram-negative bacteria(S.aureus,B.subtilis,B.cereus and B.thuringiensis)and fungi(C.albicans,C.tropical and C.glabrata)were obtained by confocal Raman microscopy could be clearly and directly distinguished by naked eyes2.The good classification was achieved by multivariate chemical analysis methods(PCA and HCA)within the Raman spectra of same species.Meanwhile,the good clustering within the Raman spectra of Gram-negative bacteria,Gram-positive bacteria and fungi was achieved.3.Raman spectroscopy also showed obvious advantages in the detection of mixed bacteria,and a good identification was obtained for the mixed culture of E.coli and A.baumanii.Conclusion: Confocal Raman microspectroscopy has been applied to detection and identification of pathogenic microorganisms,which could be used to obtain Raman spectra for single bacterial cell detection without any enhancement substrate modification.And combined with multivariate analysis methods,10 pathogens were directly distinguished.Confocal Raman microspectroscopy possess a good applied prospect for the rapid detection of clinical pathogenic microorganisms.