ACOUSTIC TRAPPING FOR BACTEREMIA DIAGNOSIS WITH MALDI-MS

B. Hammarström1*, B. Nilson2, T. Laurell1,3,4, J. Nilsson1 and S. Ekström1,4 1Department of Measurement Technology and Industrial Electrical Engineering, Lund University, SWEDEN

2Clinical Microbiology, Labmedicin Skåne, SWEDEN 3Department of Biomedical Engineering, Dongguk University, KOREA

4CREATE Health, Lund University, SWEDEN ABSTRACT

A novel approach to sample preparation for MALDI-MS bacteria typing in blood-culture flasks, that uses acoustic trapping, is presented. Successful separation and typing of a E. coli in whole blood is demonstrated, without the use of any centrifugation steps. KEYWORDS: Acoustic Trapping, Bacteria Typing, Sample Preparation, Mass-spectrometry

INTRODUCTION

Identification of bacteria species with MALDI-MS has emerged as a new standard in clinical practice for microbial pathogen diagnosis [1]. This approach is used in more than 800 microbiology labs around the world, and more than 20 million analyses were performed last year. All of these analyses require time consuming manual sample preparation of hazardous samples before a diagnosis can be reached.

We present a novel, time-saving, and automatable method for bacteria purification from blood samples for bacteremia diagnosis where the traditional centrifugation and clean-up procedures are replaced by seed-particle aided acoustic trapping, figure 1. This technology addresses a number of difficulties encountered with the current standard sample preparation methods and provides a means to efficiently handle microbial pathogens in complex backgrounds (blood/urine).

Figure 1: (a) Seed-particle aided acoustic trapping retains a cluster of fluorescent GFP expressing E. coli against fluid flow. Note the black seed-particle beads. (b) Schematic of the ultrasonic standing wave used for non-contact particle retention in acoustic trapping.

The current standard protocol involves overnight culturing of patient samples in blood culture flasks to increase the bacteria concentration, followed by manual sample preparation in the form of selective-lysis and several centrifugation steps, figure 2. Our improved method for bacteria purification is based on a microfluidic platform for seed-particle aided acoustic trapping of the bacteria [2], enabling non-contact enrichment in low-cost disposable glass capillaries [3] with very efficient washing capability. This allows automation of the assay by removing the centrifugation steps currently used for enrichment and washing of bacteria from the infected blood.

978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 1108 17th International Conference on MiniaturizedSystems for Chemistry and Life Sciences27-31 October 2013, Freiburg, Germany

Figure 2: Top shows the manual labor intensive standard work flow for bacteria identification from blood culture. Below; the simplified workflow enabled by acoustic trapping for bacteria typing in blood samples circumvents all centrifugations, as aspiration into the capillary allows bacteria to be captured and separated from lysed plasma. The enriched bacteria are released as a10 µL water droplet into a bead-packed ISET well to reduce the fluid volume and form a crystal for MALDI-MS analysis.

EXPERIMENTAL AND RESULTS

We demonstrate enrichment and identification of Escherichia coli in whole blood cultivated in clinical blood culture flasks. At a concentration of approximately 107 bact. /mL the presence of bacteria was detected by the incubator and 20 µL of this blood was used for each analysis. The red and white cells in the blood were selectively lysed using a lysis buffer from the Bruker-Sepsityper kit. The lysate was directly aspirated in an acoustic trap arranged in a glass capillary (200x2000 µm2 cross-section). The trap was preloaded with a small number of 10 µm seed-particles allowing for acoustic trapping of the micron-sized bacteria and subsequent aspiration and dispensing of MilliQ water enabled efficient removal of plasma proteins. The trapped bacteria were released by dispensing a 10 µL water volume containing the bacteria cluster. The cluster was released into the previously presented ISET sample preparation platform [4] packed with 10 µm RP beads, in order to decrease the sample volume, lyse the bacteria and generate a small crystallized sample spot for the MALDI-detection. The bacteria were subsequently identified with MALDI-MS as E. coli using the proprietary Bruker-Biotyper software correlating the obtained spectra with the reference database, figure 3.

Figure 3: Characteristic spectra for E. coli identification obtained using the acoustic trapping protocol. Correlation with the Biotyper database results in a score of 2.00 corresponding to successful identification of the bacteria.

DISCUSSION The successful typing of bacteria from blood culture demonstrates an automated assay for bacteria identification

using MALDI-MS and microfluidics by removing three centrifugation steps. The value of an automatable microfluidic assay that can improve bacterial identification using only a very small sample is great, as clinical microbiology laboratories are in dire need of fast and reliable methods to analyze bacteria in order to provide the correct infection therapy for the patient.

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REFERENCES [1] S. Sauer,M. Kliem, "Mass spectrometry tools for the classification and identification of bacteria," Nat. Rev. Mi-

crobiol, vol. 8, pp. 74-82, 2009. [2] B. Hammarström, T. Laurell, J. Nilsson, “Seed particle-enabled acoustic trapping of bacteria and nanoparticles in

continuous flow systems,” Lab on a Chip, vol. 17, pp. 2251-7, 2012. [3] B. Hammarström, M. Evander, H. Barbeau, M. Bruzelius, J. Larsson, T. Laurell, J. Nilsson, “Non-contact

acoustic cell trapping in disposable glass capillaries,” Lab on a Chip, vol. 10, pp. 2251-2257, 2010. [4] S. Ekström, L. Wallman, D. Hök, G. Marko Varga and T. Laurell, "Miniaturized solid-phase extraction and

sample preparation for MALDI MS using using a microfabricated integrated selective enrichment target," Journal of Proteome Research, vol. 5, pp. 1071-1081, 2006.

CONTACT *B. Hammarström, tel: +46-46-2229371; Bjorn. Hammarstrom@elmat. lth. se

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