Using MALDI-TOF MS analysis for rapid discrimination of MRSA MLST types Jang-Jih Lu1,2,Tsui-Ping Liu1, Shih-Cheng Chang1,2 1Department of Laboratory Medicine, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan 2Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan Email: [email protected]

73 0:F8 MS Ra w 71 0:F9 MS Ra w

5000 6422.794

6000

Intens.[a.u.] Intens.[a.u.] Introduction 4000 Results ST5 ST5 4000 3000 2978.985 2000 6551.531 Staphylococcus aureus is one of the common causes of 3027.802 3054.785 2964.900 3175.943 2000 Table 1: MRSA and MSSA associated peaks by MALDI TOF 1000 community-associated infections and health care related 234 0:F1 MS Ra w 234 0:F1 MS Ra w 3005.919 MRSA(129 isolates) MSSA(77 isolates) 5000

6000

Intens.[a.u.] Intens.[a.u.] pathogen. It can usually cause wound infection, ST45 4000 ST45 6549.206 4000 3000 MS peaks (m/z) no. of isolate % of isolate no. of isolate % of isolate 6420.525 osteomyelitis, and even serious invasive infections, such as 2000 6610.628 2000 2978.373 3053.663 3174.805 2415 67 51.9 2 2.6 1000 sepsis and pneumonia. Epidemiology studies have shown 86 0:E4 MS Ra w 70 0:E5 MS Ra w 3058.565 5000 2431 65 50.4 0 0 3074.390

6000

Intens.[a.u.] Intens.[a.u.] that there are close relationship between the results of ST59 4000 6550.723 ST59

4000 3000 2879 51 39.5 2 2.6 6422.151 genotyping from multilocus sequence typing (MLST) and 2000 2000 characteristics of MRSA strains, including hospital- 6593 77 59.7 7 9.1 1000 200 0:E8 MS Ra w 38 0:E11 MS Ra w Appear of any above 103 79.8 11 14.3 2978.890 5000

6000 Intens.[a.u.] Intens.[a.u.] 6421.658 acquired, community-associated strain and even the 2965.568 ST239 4000 ST239 peaks 4000 3000 none of above peaks 6590.270 26 20.1 66 85.7 2937.113 3177.333 2916.384 antibiogram of the strain. However, pulsed-field gel 2000 6548.995 6611.749 2000 electrophoresis (PFGE) and MLST for epidemiological The associated peaks in MRSA and MSSA: (Table 1) 1000 2900 2950 3000 3050 3100 3150 6400 6450 6500 6550 6600 6650 m /z m /z studies are is not suitable for clinical microbiology The 129 MRSA strains and 77 MSSA strains were directly prepared, Fig. 1：The related peaks of MRSA major clonal laboratories due to costly and labor-intensive.. introduced into mass spectrometry and performed mass spectrum analysis. We The flow process of identification of MLST type of MRSA clonal lineages: Recently, matrix-assisted laser desorption-time of flight could discriminate MRSA and MSSA by whether 2415, 2431, 2879 and 6593 We generalized the flow diagram for MLST identification by mass spectrum mass spectrometry (MALDI-TOF MS) has been utilized in m/z peaks are present or not. from these particular peaks (Fig. 2) and the predictive results. (Table 3) many clinical microbiology laboratories. It could not only Table 2: MRSA STs 5,45,59,239 clonal lineages associated peaks by MALDI TOF 3892 m/z identify bacterial species effectively and rapidly, but is an ST5 ( 28 isolates) ST45 ( 8 isolates) ST59 ( 27 isolates) ST239 ( 62 isolates) Yes No alternative solution for molecular methods. MS peaks (m/z) no. of isolate% of isolate no. of isolate% of isolate no. of isolate% of isolate no. of isolate% of isolate ST45, ST239 or ST59 Our purposes of this study were to discriminate MRSA and 3877 1 3.6 6 75 16 59.3 24 38.7 MSSA strain and identify the MRSA MLST type by protein 3892 15 53.6 0 0 0 0 0 0 3070 m/z mass fingerprints of MALDI-TOF MS analysis. 6552 28 100 8 100 26 96.3 19 30.6 No 6593 6 21.4 3 37.5 8 29.6 56 90.3 Yes Materials and Methods 3070 0 0 0 0 25 92.6 1 1.6 ST45 or ST239 Strain resource 3077 13 46.4 4 50 0 0 21 33.9 6593m/z The 206 strains of S. aureus, including 129 MRSA and 77 3084 1 3.6 0 0 17 63 2 3.2 MSSA, have been isolated from clinical blood samples since 4515 0 0 4 50 0 0 0 0 No Yes 4808 0 0 4 50 0 0 0 0 2009 to 2014 in Chung Gung Memorial hospital. ST45 or others Bacterial identification by MALDI-TOF MS 4515 or 4808 0 0 6 75 0 0 0 0 4515 or 4808 m/z The fresh bacterial colonies that grew on blood agar plate The related peaks of MRSA major clonal lineage ST5, ST45, ST59, and ST239 for 24 hours were picked and smeared onto a MALDI steel (Table 2, Fig. 1) No Yes target plate to thin films. 1 ul 70% formic acid were added We used MALDI-TOF MS to identify 125 known MRSA MLST sequence type for on films, dried at room temperature and following 1 ul comparison. As Table 2 and Fig. 1 shown, different MRSA STs have its unique matrix solution (50 % acetonitrite contain 1% α-cyano-4- peaks. To sum up, peaks at 3892 (ST5), 4515, 4808 (ST45), 6593(ST239), and 3070, hydroxycinnamic acid and 2.5% trifluoroacetic acid) were 3084 (ST59) m/z were each type’s specific signals. Fig. 2: Peak rules for MRSA major clonal lineages identification

4 then added on films again. Mass spectrum analysis was 110 0:F10 MS, Smoothed x10 4812.600 28 0:A10 MS Ra w 1400 1.5 Table 3: correct rate of MRSA clonal lineages identification

1200 Intens.[a.u.]

Intens.[a.u.] 3890.988

MALDI BioTyper (Microflex; Bruker Daltonics) with linear 1000 ST5 ST5 1.0 no. of MRSA 800 4444.934 4589.021 0.5 positive model, and the analytic region was 2000-20000 Da. 600 4860.899 total no. ST5 ST59 ST239 ST45 other ST type 400 0.0 4 12 0:F6 MS, Smoothed x10 98 0:E2 MS Multilocus Sequence Typing (MLST) 1400 1.5 4807.630 129 28 27 62 8 4

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Intens.[a.u.] Intens.[a.u.] ST45 ST45 129 MRSA strains were sequenced for 7 housekeeping 1000 1.0 correct isolates no. 89 15 20 50 3 1 800

0.5 600 genes, including carbamate , shikimate 4447.647 4592.054 4512.985 4861.445 400 correct rate (%) 69 53.5 74 80.6 37.5 25 0.0 x104 1400 16 0:E2 MS, Smoothed 16 0:E10 MS Ra w dehydrogenase, , , 1.5

1200

Intens.[a.u.] Intens.[a.u.] 1000 Conclusion ST59 1.0 ST59 4828.196 phosphate acetyltransferase , triosephosphate , 800 600 0.5 We could detect a part of MRSA early by analyzing the peaks of MALDI- 4446.313 and acetyl coenzyme A acetyltransferase genes. Sequencing 400 4845.032 200 0.0 4 22 0:E10 MS, Smoothed x10 2 0:A5 MS Ra w TOF MS and comprehend the MLST ST type whenever identifying bacterial 1400 3875.186 1.5

1200 Intens.[a.u.] results of these genes were compared to S. aureus MLST Intens.[a.u.] 4813.115 1000 strain in clinical microbiology laboratory. It is helpful to provide physicians ST239 1.0 ST239 database (http://asureus.mlst.net/) to acquire allelic number 800 600 4844.680 0.5 4445.690 for treatment information to patients and further investigation of 4589.707 400 and sequence type. 200 0.0 3800 3820 3840 3860 3880 3900 3920 3940 3960 3980 4400 4450 4500 4550 4600 4650 4700 4750 4800 4850 epidemiological outbreak. m /z m /z

MRSA(129 isolates) MSSA(77 isolates) MS peaks (m/z) no. of isolate % of isolate no. of isolate % of isolate 2415 67 51.9 2 2.6 2431 65 50.4 0 0 2879 51 39.5 2 2.6 6593 77 59.7 7 9.1 Appear of any above peaks 103 79.8 11 14.3 none of above peaks 26 20.1 66 85.7