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Year : 2014  |  Volume : 32  |  Issue : 4  |  Page : 419--422

Use of MALDI-TOF MS technique for rapid identification of bacteria from positive blood cultures

Sung Kuk Hong, Beung Ki Chang, Sang Hoon Song, Eui-Chong Kim 
 Department of Laboratory Medicine, Seoul National University Hospital, Seoul, Korea

Correspondence Address:
Eui-Chong Kim
Department of Laboratory Medicine, Seoul National University Hospital, Seoul
Korea

Abstract

We evaluated the feasibility of same-day routine aerobic bacterial identification using the following procedures: Picking colonies from 4 and 6 h incubated subculture from positive blood culture bottle and analyzing them by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The matched identification rate of this procedure at the species level was 80.6% (141/175) for the 4-h cultures compared with overnight cultures and 90.9% (159/175) for the 6-h cultures. Thus, our technique provides an easy and rapid method for identification of aerobic bacteria in routine clinical microbiology laboratories.

How to cite this article:
Hong SK, Chang BK, Song SH, Kim EC. Use of MALDI-TOF MS technique for rapid identification of bacteria from positive blood cultures .Indian J Med Microbiol 2014;32:419-422

How to cite this URL:
Hong SK, Chang BK, Song SH, Kim EC. Use of MALDI-TOF MS technique for rapid identification of bacteria from positive blood cultures . Indian J Med Microbiol [serial online] 2014 [cited 2019 Nov 17 ];32:419-422
Available from: http://www.ijmm.org/text.asp?2014/32/4/419/142261

Full Text

 Introduction



Rapid identification of pathogens that cause potentially life-threatening sepsis is important in clinical settings. [1],[2] Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) permits faster identification of pathogens than conventional phenotypic methods. [3] Additionally, the time required for MALDI-TOF MS could be further decreased by direct extraction of bacteria from positive blood culture bottles, which does not require additional overnight incubation. However, previous studies, especially those using charcoal-containing BacT/Alert® (bioMeriéux, Marcy l'Etoile, France) blood culture bottles, showed unfavourable results, including species identification rates of 57-95% for Bactec™ (Becton Dickinson, NJ, USA) systems [4],[5],[6],[7],[8],[9] and 32-80.7% for the BacT/Alert® (bioMeriéux) system. [10],[11],[12]

The aim of this study was thus to evaluate the feasibility of same-day routine aerobic bacterial identification by picking colonies from 4 and 6 h incubated subculture from positive blood culture bottle and analyzing them MALDI-TOF MS.

 Materials and Methods



In total, 197 blood culture-positive aerobic bottles referred for blood culture testing from April 2012 to May 2012 were included. Two types of blood culture systems were used for the blood cultures: The BD BACTEC™ FX Blood Culture System (Becton Dickinson) and the BacT/ALERT® 3D System (bioMeriéux). Broth was taken from the positive blood culture bottles and inoculated to two blood agar plates (BAP) and one MacConkey agar plate and incubated in a CO 2 (5%) environment at 35°C. One BAP was used for serial testing by MALDI-TOF MS, and the others were used for conventional biochemical identification tests. Isolates were identified using two biochemical bacterial identification systems: The MicroScan WalkAway-96 plus system (Siemens, CA, USA) or the VITEK 2 system (bioMérieux). Mass spectra were generated using a VITEK® MS Axima® Assurance mass spectrometer (bioMérieux) with single-use FlexiMass MALDI target plates. Two spots of the isolates incubated for 4 and 6 h were inoculated to disposable plates, and one spot of the overnight cultures was used for testing by VITEK® MS. Although there were no well-defined bacterial colonies at 4 and 6 h, two spots were inoculated to the plate using a 1-μL sterile plastic loop. The incubation times were selected using experimental cultures, in which many routine clinical bacteria were seen on the BAP with incubation from 4 to 6 h. In the two-spot analysis, matched or single-identified organisms with a confidence value over 90 were considered to be correctly identified, but even when the confidence value was over 90, unmatched organisms between two spots were considered to be incorrectly identified. Sequencing of the 16S rRNA gene was used to resolve discordant results at the genus level between biochemical methods and MALDI-TOF MS using the MicroSeq 500 system (Applied Biosystems, Foster City, CA, USA).

 Results



Among the 197 blood-culture-positive aerobic bottles included in the study, 22 blood-culture-positive specimens were excluded from further analysis: Polymicrobial (12), Candida spp. (5) and fastidious bacteria (5). The residual 175 monomicrobial specimens, of which 89 (50.9%) contained Gram-positive bacteria and 86 (49.1%) contained Gram-negative bacteria, were included for further analysis.

The concordance rate of MALDI-TOF MS and biochemical methods was 98.9% (173/175) at the genus level and 81.7% (143/175) at the species level. There were two discordant cases between MALDI-TOF MS and biochemical methods at the genus level: Gemella morbillorum and Paenibacillu spabuli by MALDI-TOF MS and Streptococcuss anguinis and Bacillus sp. by biochemical methods, respectively. These species were confirmed as Gemella sp. and Paenibacillus sp. by 16S rRNA sequencing.

The matched identification rate of the plating procedure at the species or genus level was 80.6% (141/175) in the 4-h cultures and 90.9% (159/175) in the 6-h cultures [Table 1]. There was a statistically significant difference in the matched identification rate of this plating procedure at the species level between 4- and 6-h cultures among Gram-positive bacteria (P < 0.05) but no difference in Gram-negative bacteria. The matched identification rates of Gram-positive bacteria were 66.3% (59/89) and 84.3% (75/89) for the 4-hour cultures and 6-hour cultures, respectively, and the matched identification rates for Gram-negative bacteria were 95.3 % (82/86) and 97.7 % (84/86), respectively.{Table 1}

Regardless of the incubation times, there were no statistically significant differences in the matched identification rate for this plating procedure depending on the blood culture systems, but this result might be due to uneven distribution of the blood culture system in the tested strains. Therefore, further analysis was performed separately using Escherichia coli, Klebsiella pneumoniae and total staphylococcus spp. but no statistically differences were observed.

 Discussion



MALDI-TOF MS is a rapid and accurate method for microbial identification that generates and detects characteristic mass spectra composed of mass-to-charge ratio (m/z) peaks with varying intensities. The spectra are then compared with a database for identification at the genus and species level. [13] Although MALDI-TOF MS can be a useful tool for detection of antibiotic resistance in some cases, it cannot replace standard susceptibility testing because resistance to antibiotics is a complex process. [14]

For rapid microbial identification using MALDI-TOF MS, the plating procedure used in this study has an advantage in that it is not affected by the blood culture system. Direct extraction procedures have limitations such as the influence of the blood culture system and complicated procedures for extraction of bacterial proteins. [11] It is known that current MALDI-TOF MS data software analysis is not able to reliably identify all microorganisms present in polymicrobial blood cultures. [7],[8],[9] Without this common limitation of MALDI-TOF MS system, the plating procedure is difficult to adapt for routine anaerobic bacteria identification and need more time than direct extraction method. However, the plating procedure of the present study has advantages; it is not influenced by the blood culture system and is inexpensive and easy to adapt in microbiology laboratories. Therefore, the BacT/ALERT® 3D System (bioMeriéux) and MALDI-TOF MS without direct extraction are good alternatives for microbiology laboratories.

Based on our results, there are two possible options for adapting this plating procedure when conducting Gram-staining from blood culture-positive bottles simultaneously for rapid identification of aerobic bacteria: 6-h culture for Gram-positive and -negative bacteria or 4-h culture for Gram-negative bacteria and 6-h culture for Gram-positive bacteria. By adapting these plating procedures in microbiology laboratories, same-day reporting of positive blood culture specimens obtained in the morning will be possible.

This study has limitations in that relatively few species were analysed because clinical samples were used and clinical pathogens causing septicemia such as Candida spp. and fastidious bacteria were not evaluated. However, our study includes MALDI-TOF MS results for major clinical aerobic bacteria cultured for 4 and 6 h, which is useful data for rapid bacterial identification in microbiology laboratories.

In conclusion, MALDI-TOF MS shows good performance for routine aerobic bacterial identification and the procedure of picking colonies from cultures incubated for 4 and 6 h followed by analysis using MALDI-TOF MS is an easy and rapid method for identification of clinical aerobic bacteria in typical microbiology laboratories.

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