Anal. Chem. 1999, 71, 3416-3419
Direct Analysis and Identification of Helicobacter and Campylobacter Species by MALDI-TOF Mass Spectrometry Martin A. Winkler,*,† John Uher,† and Steven Cepa‡
Diagnostics Division and Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064
Campylobacter jejuni, Campylobacter fetus, and Campylobacter coli were compared with Helicobacter pylori and Helicobacter mustelae by direct analysis of individual cultured colonies in 50% methanol-water with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). H. pylori and Campylobacter species from blood agar culture produced unique, complex spectra with over 25 different ions in mass/charge (m/z) range from 2 000 to 62 000. A biomarker for H. pylori was centered around m/z 58 268, and H. mustelae was distinguished from H. pylori by its ions at m/z 49 608 and 57 231. Campylobacters could be distinguished from Helicobacters by their lack of ions around m/z 58 000 and 61 000 as well as distinguishing biomarkers of lower m/z: 10 074 and 25 478 for C. coli; m/z 10 285 and 12 901 for C. jejuni; m/z 10 726 and 11 289 for C. fetus. MALDI-TOF MS is a rapid and direct method for detection of these potentially pathogenic bacteria from culture. Recent studies1-3 have shown that bacteria such as Escherichia coli, Staphylococcus aureus, and Bacillus anthracis may be rapidly identified (less than 1 h) and distinguished from closely related species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of intact microorganisms. In this technique, positively charged ions are resolved after laser irradiation of intact microorganisms in the presence of a saturated solution of UV-absorbing matrix. The sample preparation method and the matrix composition, especially the organic solvents, have been found critical for the sensitivity and reproducibility of the method.4 Chemotaxonomy was demonstrated in a blind study,1 and distinguishing ions classified as biomarkers5 have been identified for some individual species.1,2 Haag et al.6 identified and * Corresponding author: (phone) (847) 938-7806; (fax) (847) 938-7550; (e-mail)
[email protected]. † Diagnostics Division. ‡ Pharmaceutical Products Division. (1) Holland, D.; Wilkes, J. G.; Rafii, F.; Sutherland, J. B.; Persons, C. C.; Voorhees, K. J.; Lay, J. O., Jr. Rapid Commun. Mass Spectrom. 1996, 10, 1227-1232. (2) Claydon, M. A.; Davey, S. N.; Edwards-Jones, V.; Gordon, D. B. Nature Biotechnol. 1996, 14, 1584-1586. (3) Krishnamurthy, T.; Ross, P. L. Rapid Commun. Mass Spectrom. 1996, 10, 1992-1996. (4) Wang, Z.; Russon, L.; Li, L.; Roser, D. C.; Long, S. R. Rapid Commun. Mass Spectrom. 1998, 12, 456-464.
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speciated Haemophilus by a rapid MALDI-TOF MS technique using a brief centrifugation step, and they could determine strain differences from different bacterial isolates. Welham et al.7 used a 20-min centrifugation step after bacterial harvest to detect ions in the 3-30 kDa range of Gram positive and negative bacteria. Partially purified extracts from lysed bacteria have also been analyzed.8,6,9 Campylobacter jejuni is a leading cause of gastrointestinal food poisoning in the United States, with up to 4 million suspected cases per year. Infections occur primarily from contaminated water, milk, or poultry.10 The related species Campylobacter fetus and Campylobacter coli are also causes of gastrointestinal illness. The Lior biotyping scheme11 uses hippurate hydrolysis, and nalidixic acid and cephalothin resistance, for discriminating Campylobacter species. Occasionally, C. jejuni can be difficult to distinguish from C. coli by the standard method of hippurate hydrolysis.12,13 PCR tests are available that recognize C. jejuni, C. coli14, and Campylobacter lari;15 species-specific PCR tests have also been developed.16,17 Pulsed field gel electrophoresis of restriction endonucleasecleaved DNA has been used to distinguish strains of C. jejuni isolates.18 Thermostable antigens for serotyping were first identified by Penner et al.19 Commercially available rapid diagnostic (5) Fenselau, C. In Mass Spectrometry for the Characterization of Microorganisms; Fenselau, C., Ed.; ACS Symposium Series Vol. 541; American Chemical Society: Washington, DC, 1994. (6) Haag, A. M.; Taylor, S. N.; Johnston, K. H.; Cole, R. B. J. Mass Spectrom. 1998, 33, 750-756. (7) Welham, K. J.; Domin, M. A.; Scannell, D. E.; Cohen, E.; Ashton, D. S. Rapid Commun. Mass Spectom. 1998, 12, 176-180. (8) Cain, T. C.; Lubman, D. M.; Weber, W. J., Jr. Rapid Commun. Mass Spectrom. 1994, 8, 1026-1030. (9) Krishnamurthy, T.; Ross, P. L.; Rajamani, U. Rapid Commun. Mass Spectrom. 1996, 10, 883-888. (10) Altekruse, S. F.; Cohen, M. L.; Swerdlow, D. L. Emerging Infect. Dis. 1997, 3, 285-293. (11) Lior, H. J. Clin. Microbiol. 1984, 20, 636-640. (12) Penner, J. L. Clin. Microbiol. Rev. 1988, 1, 157-172. (13) Tenover, F. C.; Carlson, L.; Barbagallo, S.; Nachamkin, I. J. Clin. Microbiol. 1990, 28, 1284-1287. (14) Oyofo, B. A.; Thornton, S. A.; Burr, D.; Trust, T. J.; Pavlovskis, O. R.; Guerry, P. J. Clin. Microbiol. 1992, 30, 2613-2619. (15) Giesendorf, B. A. J.; Van Belkum, A.; Koeken, A.; Stegeman, H.; Niesters, H. G. M.; Quint, W. G. V. J. Clin. Microbiol. 1993, 31, 1541-1546 (16) Eyers, M.; Chapelle, S.; Camp, G. V.; Goossens, H.; Wachter, R. D. J. Clin. Microbiol. 1993, 31, 3340-3343. (17) Giesendorf, B. A. J.; Quint, W. G. V.; Henkens, M. H. C.; Stegeman, H.; Huf, F. A.; Niesters, H. G. M. Appl. Environ. Microbiol. 1992, 58, 38043808. (18) Hanninen, M.-L.; Pajarre, S.; Klossner, M.-L.; Rautelin, H. J. Clin. Microbiol. 1998, 36, 1787-1789. 10.1021/ac990135r CCC: $18.00
© 1999 American Chemical Society Published on Web 07/02/1999
tests for Campylobacters include immunoassays20,21 and DNA probe-based diagnostics.22,23 Helicobacter pylori is a curved, microaerophilic, Gram negative organism which colonizes the stomachs of many mammalian species and causes gastritis in primates. Chronic gastritis in man has been associated with a number of diseases, including peptic ulcer, gastric lymphoma, gastric atrophy, and gastric carcinoma.25 H. pylori can be detected in biopsies by urease stains or light microscopy after giemsa stain, in individuals by breath testing with isotopically labeled urea, serologically by immunoassays,26 and by direct culture. In addition, restriction endonuclease gel methods have been used to type strains,27 and more recently, PCR has been used to demonstrate considerable diversity among strains of H. Pylori.28 Intact Helicobacter species have also been analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to differentiate species and establish taxonomy.29 In this study, we investigate MALDI-TOF MS as a rapid method to identify and distinguish C. fetus, C. coli, and C. jejuni, as well as H. pylori and Helicobacter mustelae, using biomarkers. The nutrient-rich medium we needed to grow Helicobacter species, sheep blood agar medium, contributes background ions, so we developed a direct method for identifying bacterial biomakers in the range of 10 000-70 000 Da excluding background ions and without bacterial lysis or centrifugation. The blood agar medium has the advantage of supporting the growth of most microorganisms under appropriate aerobic, anaerobic, or microaerophilic conditions,30 so it would make possible their growth and identification by MALDI-TOF MS, provided a sufficiently large database of biomarkers for bacteria exists. This study will show the feasibility of such an approach and will add to the database of MALDI-TOF MS biomarkers for pathogenic bacteria, especially in the higher mass range. Databases already exist for bacteria analyzed by mass spectrometry using thermal methods.31 MATERIALS AND METHODS Bacterial Strains and Growth Conditions. C. fetus strains 19438, 27374 and 25936, C. jejuni strains 33292 and 43464, C. coli strains 43474, 33559, and 43482, H. pylori strains 43526, 43579, and 43629, and H. mustelae 43774 were all obtained from the American Type Culture Collection (ATCC). H. pylori strains were grown in a 12% CO2 incubator under humidified conditions. (19) Penner, J. L.; Hennessy, J. N.; Congi, R. V. Eur. J. Clin. Microbiol. 1983, 2, 378-383. (20) Hodinka, R. L.; Gilligan, P. H. J. Clin. Microbiol. 1988, 26, 47-49. (21) Nachamkin, I.; Barbagallo, S. J. Clin. Microbiol. 1990, 28, 817-818. (22) Popovic-Uroic, T.; Patton, C. M.; Wachsmuth, I. K.; Roeder, P. Lab. Med. 1991, 22, 533-539. (23) Romaniuk, P. J.; Trust, T. J. Microbiol. Lett. 1987, 43, 331-335. (24) Cover, T. L.; Blaser, M. J. Adv. Intern. Med. 1996, 41, 85-117. (25) Graham, D. Y.; Evans, D. J.; Peacock, J.; Baker, J. T.; Schrier, W. H. Am. J. Gastroenterol. 1996, 91, 942-948. (26) Langenberg, W.; Rauws, E. A. J.; Widjojokusumo, A.; Tytgat, G. N. J.; Zanen, H. C. J. Clin. Microbiol. 1986, 24, 414-417. (27) Majewski, S. I. H.; Goodwin, C. S. J. Infect. Dis. 1988, 157, 465-471. (28) Akopyanz, N.; Bukanov, N. O.; Westblom, T. U.; Kresovich, S.; Berg, D. E. Nucleic Acids Res. 1992, 20, 5137-5142. (29) Ferguson, D. A.; Dwight W. Lambe, J. J. Clin. Microbiol. 1984, 20, 453460. (30) Forbes, B. A.; Granato, P. A. In Manual of Clinical Microbiology; Murray, D. R., Ed.; ASM Press: Washington, DC, 1995. (31) Basile, F.; Beverly, M. B.; Abbas-Hawks, C.; Mowry, C. D.; Voorhees, K. J.; Hadfield, T. L. Anal. Chem. 1998, 70, 1555-1562.
Figure 1. MALDI-TOF mass spectrum of blood agar medium used to grow all bacterial strains.
Campylobacter species were grown using the BBL CampyPak Plus system (Becton Dickinson Microbiology Systems, Sparks, MD) in anaerobic jars, which produces conditions conducive to the primary isolation and cultivation of microaerophilic organisms. All organisms were grown at 37 °C on sheep blood agar medium (Gibco-BRL, Life Technologies, Gaithersburg, MD). MALDI-TOF Mass Spectrometry. A Perseptive Biosystems Voyager DE mass spectrometer incorporating delayed extraction with MALDI was used in positive ion mode. The instrument was calibrated with a mixture of horse cardiac apomyoglobin and bovine serum albumin (each from Sigma Chemicals, St. Louis, MO) and checked prior to analysis to be within 0.1% mass accuracy for each standard. In a biosafety cabinet, an individual colony approximately 1 mm in diameter was picked after 3 days growth using a Texwipe microabsorbent applicator stick (Texwipe Industries,Upper Saddle River, NJ) and immersed in 40 µL of a 50% methanol-water solution. Controls were also taken by touching the applicator to blood agar medium and immersing it in 50% methanol-water. Samples and controls were also suspended in 0.1% trifluoroacetic acid (TFA) for comparison. The mixtures were briefly vortexed and 0.5 µL was spotted on the sample plate and overlaid with 0.5 µL of sinapinic acid matrix, which was a 100 mM solution of sinapinic acid in a 1:1:1 mixture of acetonitrile, methanol, and water (Hewlett-Packard, Palo Alto, CA). A total of 150-250 laser shots were accumulated from each sample. A low-mass gate was set to 500 Da, and delayed extraction was set to 200 ns at 25 kV. All spectra were smoothed with the Savitzky-Golay 19-point algorithm.32 Helicobacter strains were also analyzed in a blind study where an attempt was made to correctly identify the three coded strains based only on mass spectra. Biomarker data were acquired by analyzing samples immediately after harvest. Samples were also analyzed at 1 and 2 h after bacterial harvest to determine stabilities of the bacteria-solvent preparations. RESULTS Reproducible bacterial MALDI-TOF MS spectra required bacterial colonies of sufficient size. The relatively slowly growing Helicobacters and Campylobacters required 3 days growth on the nutrient-rich blood agar medium to produce colonies of sufficient size for analysis. Figure 1 shows a particularly intense example of the background spectrum of the blood agar media, chosen to (32) Savitzky, A.; Golay, M. J. E. Anal. Chem. 1964, 36, 1627-1638.
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Table 1. Significant Biomarkers for Bacteria from MALDI-TOF Mass Spectrometry mass/charge (m/z)
bacteria Helicobacter mustalae 43774 Helicobacter pylori 43629 Campylobacter fetus 27374 Campylobacter jejuni 43464 Campylobacter coli 43474 blood agar
12 833 13 239 10 726 10 285 10 074 7 549
25 066 24 336
49 608
57 231 58 268
11 289 12 901 25 478 Medium: Background Ions 8 598 11 415
Figure 2. MALDI-TOF mass spectrum of Helicobacter pylori ATCC strain 43629.
clearly delineate any ions due to the growth medium. The ions are primarily due to the ovine R-hemoglobin chain at mass/charge (m/z) of 15 113; its double charged ion can also be seen at m/z 7549. The ions attributed to background are summarized along with other biomarker ions in Table 1. Biomarkers ions were unique to a bacterial species and were not confounded by background ions. Biomarkers could not be within (0.1% mass of any of the background ions identified in Table 1, nor could they be within (0.1% of ions observed with other bacterial species. Our initial experiments with dilution of the bacterial colonies into 0.1% TFA showed variability in the spectra depending on the length of time between colony dilution and analysis. Dilution into 50% methanol preserved bacteria for up to 1 h prior to analysis. Campylobacter ions above m/z 30 000 lost intensity after 1 h of dilution into 50% methanol; the H. pylori ions at m/z 58 268 were significantly less intense at 2 h. The spectrum for H. pylori ATCC strain 43629 is shown in Figure 2. Ions common to all three H. pylori strains appear at 61.4-61.5, 58.3, and 44.1-44.3 kDa (for masses for strain 43629 see Figure 2). Ions of m/z less than 10 000 were not suitable as biomarkers due to their complexity in this range in both Helicobacters and Campylobacters and overlap with background ions. While the three strains of Helicobacter showed some variation in spectra, these differences were within the error of the method (0.1%) and the strains could not be distinguished in the blind study we performed. However, H. mustelae ions of m/z 57 231 varied significantly from those of H. pylori (Table 1), and H. mustelae also showed biomarker ions at m/z 49 608. Spectra of C. jejuni ATCC strain 43464 (Figure 3), C. coli ATCC strain 43474 (Figure 4), and C. fetus ATCC strain 27374 showed numerous ions in the range 2-45 kDa. C. fetus lacked ions with m/z greater than 36 000 (Figure 5), and distinguishing biomarkers were only in the m/z range 10 000-12 000 (Figure 6). Identifying 3418 Analytical Chemistry, Vol. 71, No. 16, August 15, 1999
15 113
16 167
Figure 3. MALDI-TOF mass spectrum of Campylobacter jejuni ATCC strain 43464.
Figure 4. MALDI-TOF mass spectrum of Campylobacter coli ATCC strain 43474.
biomarker ions for Campylobacters were primarily at m/z 10 074 and 25 478 (C. coli), m/z 10 285 and 12 901 (C. jejuni), and m/z 10 726 and 11 289 (C. fetus). Both C. jejuni and C. coli showed ions in the m/z 43 861-43 874 range. DISCUSSION Improved sample stability was achieved by dilution of cultures in 50% methanol, as opposed to 0.1% trifluoroacetic acid used in previous direct methods.1-3 The methanol-water mixture may fix the bacteria and preserve its structure until suspension in the organic solvents of the matrix and may reduce degradation due to proteases. However, a few ions, such as those above m/z 30 000 in Campylobacters and around m/z 58 000 in Helicobacters, were still less stable at times after 1 h and were best seen in analyses performed in less than 1 h.
Figure 5. MALDI-TOF mass spectrum of Campylobacter fetus strain 27374.
Figure 6. MALDI-TOF mass spectrum of Campylobacter fetus strain 27374 in the 10 000-12 000 m/z range to resolve biomarkers.
In previous direct analysis of whole bacteria by MALDITOF,1-3,6 ions above m/z 17 000 were not observed. H. pylori may (33) Mobley, H. L. T.; Island, M. D.; Hausinger, R. P. Microbiol. Rev. 1995, 59, 451-480. (34) Dunn, B. E.; Campbell, G. P.; Perez-Perez, G. I.; Blaser, M. J. Biol. Chem. 1990, 265, 9464-9469. (35) Maeda, M.; Hidaka, M.; Nakamura, A.; Masaki, H.; Uozumi, T. J. Bacteriol. 1994, 176, 432-442. (36) Doig, P.; Trust, T. J. Infect. Immun. 1994, 62, 4526-4533. (37) Tomb, J. F.; White, O.; Kerlavage, A.; Clayton, R. A.; Sutton, G. G.; Fleischmann, R. D.; Ketchum, K. A.; Klenk, H. P.; Gill, S.; Dougherty, B. A.; Nelson, K.; Quackenbush, J.; Zhou, L.; Kirkness, E. F.; Peterson, S.; Loftus, B.; Richardson, D.; Dodson, R.; Khalak, H. G.; Glodek, A.; McKenney, K.; Fitzegerald, L. M.; Lee, N.; Adams, M. D.; Hickey, E. K.; Berg, D. E.; Gocayne, J. D.; Utterback, T. R.; Peterson, J. D.; Kelley, J. M.; Cotton, M. D.; Weidman, J. M.; Fujii, C.; Bowman, C.; Watthey, L.; Wallin, E.; Hayes, W. S.; Borodovsky, M.; Karp, P. D.; Smith, H. O.; Fraser, C. M.; Venter, J. C. Nature 1997, 388, 539-547. (38) Vandamme, P.; Pot, B.; Gillis, M.; Vos, P. D.; Kersters, K.; Swings, J. Microbiol. Rev. 1996, 60, 407-438. (39) Vandamme, P.; Dewettinck, D.; Kersters, K. Syst. Appl. Microbiol. 1992, 15, 402-408.
be unusual in having ions of mass over 50 kDa in such abundance; H. pylori and H. mustelae ions in the broad peak at m/z 61 423 may be the urease B gene products, which have predicted masses of 60.3, 60.5, and 61.6 kDa,33 and purified as 61 kDa.34 The urease from Bacillus pasteurii has a predicted mass of 61 398 Da.35 Masses at 80, 60, 51, 50, 48, and 31 kDa were observed in a study that used SDS-PAGE to resolve surface antigens of H. pylori;36 ions of some of these masses were found in our study as well. The complete genome of H. pylori has now been sequenced,37 and it may be possible to assign the predicted masses of some of these gene products to some of the ions observed here. Although Haag et al.6 were able to distinguish strains from isolates of Haemophilus ducreyi by a rapid MALDI-TOF MS method, we could not distinguish between the three H. pylori strains in our blind study, though it differentiated H. pylori from H. mustelae as well as differentiating the three species of Campylobacters. To differentiate the three Campylobacter species studied, biomarkers in the 10-12 kDa range were required. While the majority of ions in all species studied were below 10 kDa in size, these were not useful as biomarkers because of their complexity and the possibility that some ions could be confused with background ions from the culture medium. Since Helicobacter is considered to be closely related to Campylobacter,38 it is not unexpected that many of the ions observed for Campylobacter seem to be a subset of those observed for H. pylori and H. mustelae. Computer-aided numerical analysis of SDS-PAGE bands from intact bacteria have been used to differentiate C. jejuni subspecies, C. lari, C. coli, and Campylobacter upsaliensis, as well as many strains of these species.39 Acid-phenol extracts and water extracts of Campylobacters have also been analyzed and in some cases stained with enzymatic stains, as reviewed in Vandamme et al.38 Since it requires minimal sample preparation and is more rapid and accurate compared with PAGE techniques, MALDI-TOF MS could be useful for confirmation of Campylobacter speciation after culture or as a rapid means of direct detection of Campylobacter or Helicobacter in foods, beverages, or manufactured products without use of a dedicated probe, PCR, or immunodiagnostic test. The MALDI-TOF mass spectrometric method also demonstrates the feasibility of a general identification method for bacteria following growth on blood agar media under appropriate environmental conditions. ACKNOWLEDGMENT We thank Dr. Vincent Varitek (retired from Abbott Laboratories) for supplying Helicobacter cultures during initial studies, and Dr. Catherine Fenselau for her critical reading of the manuscript.
Received for review February 10, 1999. Accepted May 17, 1999. AC990135R
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