ABSTRACT
Objective To establish Brucella peptide mass fingerprint database by MicroflexTMLT MALDI-TOF mass spectrometer ,and evaluate its application value in the identification of clinical isolates of Brucella. Methods In October 2013 ,a suspected Brucella strain was isolated in the microbiology laboratory of the hos-pital.It was confirmed as Brucella meltensis by VITEK ? 2 Compact automatic microbial analysis system and 16S rDNA gene sequencing.Brucella peptide mass fingerprint database was established with this strain ac-cording to the manufacturer's recommended method.Two suspected Brucella strains isolated in the laboratory in April 2015 and June 2016 were tested for Gram stain ,Kozlowski stain and biochemical reactions such as oxi-dase ,catalase and rapid urease activity.MicroflexTM LT mass spectrometer was used to collect the mass spec-trums of these two strains and three suspected Brucella strains isolated from other hospitals.When these spectrums were comparatively analyzed ,self-built database was added on the basis of the MALDI Biotyper da-tabase.The 16S rDNA gene sequencing was also used to identify the above-mentioned bacteria.Results Ac-cording to the recommended self-built database construction process ,Brucella peptide mass fingerprint data-base based on BRUxh1 was obtained successfully.The suspected Brucella strains isolated in April 2015 and June 2016 showed small Gram-negative coccobacillus under microscopic examination of stained smear ,and were positive for oxidase ,catalase ,urease activity (within 5 min).The identification results of mass spectrom- etry for the 5 suspected strains were all Brucella (mass spectrometry score :2.407 ,2.475 ,2.436 ,2.466 and 2.397).The 5 strains were confirmed as Brucella by 16S rDNA gene sequencing.Two strains among bacteria from other hospitals were falsely identified as Roseomonas gilardii by the VITEK?2 Compact system.Conclu-sion The combination of MALDI Biotyper database and self-built Brucella database can be used to compara-tively analyze the mass spectrometry of clinical isolates.Accurate identification of Brucella can be achieved by this means.The method which is fast and sensitive has high application value for rapid clinical diagnosis of Brucellosis.
ABSTRACT
Objective·To directly detect the bacteria in positive blood culture specimens by the separation gel tube combined with MicroflexTM matrixassisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS),perform direct antimicrobial susceptibility test based on the results of mass spectrometry,and preliminary explore the redesign of conventional positive blood culture specimen processing flow.Methods·449 positive-alarm blood culture specimens of monobacterial infections in clinical microbiology laboratory of Xirhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine from September 1,2015 to August 31,2016 were collected and prepared according to the new positive blood culture specimens processing flow.The new redesigned processing flow included smear staining and microscopy,separation gel/mass spectrometry direct identification,bacteria film/mass spectrometry identification,pure colony/mass spectrometry identification,direct antimicrobial susceptibility test,and conventional antimicrobial susceptibility test,etc.According to the results of microscopic examination,identification test,and antimicrobial susceptibility test,level Ⅰ direct microscopic examination report,positive blood culture level Ⅱ (direct identification/bacteria film identification or direct antimicrobial susceptibility) report,and level Ⅲ final report were provided sequentially.Results·With the new redesigned processing flow,bacterial specieslevel identification reports for 82.2％,95.8％,and 100％ of positive blood samples could be obtain at 10 am and 17 pm on the current day and 10 am in the next morning,respectively,and be sent to clinical departments.Initial antimicrobial susceptibility reports for the bacteria that were considered as clinical significant pathogens by preliminary assessment could be provided in the next day of blood culture positive alarm with a higher coincidence rate as compared to the results of traditional antimicrobial susceptibility tests.Conclusion·The time from blood culture positive alarm to the provision of initial identification and antimicrobial susceptibility reports is shorter by 1-2 d for the redesigned processing flow than for the traditional one,which can provide fast and accurate etiologic diagnosis evidence for bloodstream infections for clinicians and is important for improving early diagnosis and treatment of clinical bloodstream infections.