br Conclusion In summary during

Conclusion In summary, during H1N1 virus infection, the abundance of upper respiratory tract flora was reduced compared with that in healthy individuals; however Pseudomonas was a dominant genus in the upper respiratory tract. The changes in the upper respiratory tract flora might closely relate to the occurrence and progression of secondary bacterial infection.
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Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging as a rapid and reliable tool for the identification of microorganisms. This technique compares protein profiles from the sample generated with MALDI-TOF MS against proprietary profiles of reference isolates in the system database. Briefly, a single colon or a centrifuged aliquot of liquid culture can be either directly or following a chemical extraction step spotted on a MALDI target plate and then mixed with matrix solution. Laser dependent ionization of bacterial peptides and proteins generates species-specific profiles of spectra allowing bacterial identification by comparison with database profiles of well characterized reference strains. Thus, MALDI-TOF MS has been proven reliable for rapid and accurate identification of various microorganisms, including Gram-positive bacteria Enterobacteriaceae, non-fermenting bacteria, mycobacteria, and fungi. Bacterial identification performed by MALDI-TOF MS was also showed to exceed conventional phenotypical methods and may perform as well as bacterial gene amplification, which remains the gold-standard for microorganism identification (4.13). Additionally, in the case of fastidious bacteria, MALDI-TOF MS has also been shown to decrease workload and the need for more expensive and time-consuming instruments. Recently, the several studies have demonstrated that the identification of Sulfo-NHS-LC-Biotin Supplier bacteria by MALDI-TOF MS are as accurately as 16S rDNA sequence analysis but more rapidly in hours versus days. However, these studies used chemical extraction for bacterial preparation before MALDI-TOF MS identification which can be time-consuming and labor-intensive. The timesaving alternative is to directly smear the cultured colony onto the MALDI target plate without any additional preparation with extraction. In this communication, we compared colony direct smear method with chemical extraction method for MALDI-TOF MS (Bruker Daltonics) identification of clinically relevant anaerobic bacteria. A collection of 216 clinical isolates of anaerobic bacteria from our laboratory was used for this study. The strain identification was determined by conventional phenotypic methods. The strains were analyzed by MALDI-TOF MS as described previously in a blind manner and the phenotypical identification was also performed. Briefly, for direct smear method, a couple of colonies were smeared on the target plate, then 2µl of 70% formic acid was added to colony smear, after air drying for about 5min to allow the formic acid to evaporate, each spot was overlaid with 2µl of Biotyper standard matrix solution and then dried under fume hood for about 10min. Once dry, the samples were subjected to MALDI-TOF analysis. For chemical extraction, the standard protocol was performed according to the Bruker Biotyper’s recommendation. Chemical extraction and direct smear were performed on colony at the same time point after 48h of incubation. A total of 216 anaerobic bacteria were analyzed by Biotyper 3.0 software. According to the criteria recommended by the manufacturer, scores higher than 2.00 provide secure species identification; scores between 1.70 and 2.00 allow identification to the genus leave; scores below 1.70 indicate that the identification was not reliable. Bruker Biotyper version 3.0 database was used at the time of this study for identification. Following chemical extraction, 14/216 strains did not lead to reliable results (score<1.70) whereas 176/216 identifications were secured to both the genus and species (score>2.00) and 26/216 identifications were secured to the genus only (score between 1.70 and 2.00) (). Conversely in the group of bacteria identified directly on smear, 22/216 did not lead to reliable identification. Our data also indicated that the cutoff of 1.70 might be sufficient to accurately identify anaerobic bacteria. Therefore, using the recommended minimal reliability score threshold for secured-to-genus identification (1.70), 202/216 anaerobic bacteria were identified to the level of species as well following chemical extraction versus 194/216 by direct smear whereas 14/216 remained unidentified after chemical extraction versus 22/216 by direct smear. However, this difference was not statistically significant (Chi square test, 0.05<<0.1), leading to the suggestion that direct smear may be non-inferior to chemical reliable results whereas 155/216 anaerobic bacteria were secured to both the genus and species and 39 strains were secured to the genus only (). Chemical extraction has been proposed to “clean” the spectra profile of background artifacts that affect the identification of typical peaks of bacteria species. Indeed, direct smear was associated with an overall decrease in the score values, i.e., Thirty-nine species from direct smear had score between 1.70 and 2.00 while only 26 species from chemical extraction had score between 1.70 and 2.00 (). Direct smear also failed to identify 9 isolates which were from one of , , , , , , , , and species.