Jpn. J. Infect. Dis., 64, 327-329, 2011

Short Communication Rapid Identification of hominis by MALDI-TOF Mass Spectrometry during Infective Endocarditis

Fráedáeric Wallet1,2*, Caroline Loäƒez1,2, Christophe Decoene1,2, and Renáe Courcol1,2 1University of Lille Nord de France, Lille; and 2CHU Lille, Lille, France (Received April 8, 2011. Accepted May 11, 2011)

SUMMARY:WereportanewcaseofCardiobacterium hominis endocarditis identified during an acute coronary syndrome. The positivity of the blood cultures was confirmed rapidly (50 h) as a result of im- provements to the automated detection system, whereby it is no longer necessary to incubate the vials for long periods of time when Aggregatibacter-Cardiobacterium-Eikenella-Kingella infections is sus- pected. The phenotype-based VITEK 2 NH identification system is not able to distinguish between the two species of Cardiobacterium, as it does not contain C. valvarum in its library. The method for 16S rRNA gene sequence analysis is able to separate the two species but is not available in all laboratories. We used MALDI-TOF mass spectrometry, as an alternative, to rapidly distinguish between C. hominis and C. valvarum, because both species are contained in the system library.

A 60-year-old man, without coronary history, was this Gram-negative rod was pleiomorphic, and pairs, hospitalized in the cardiologic ward for an atypical chest short chains, and filaments could be seen. Some of these constrictive pain. Initial examination showed that his organisms retained a variable amount of Gram-positive pulse was regular at 80/min and that his blood pressure stain in the end or in central portions. The subcultures was 140/70 mm Hg. He had a diastolic murmur at the only grew on 5z blood Columbia agar and on ``choco- left sternal border with hyperpulsability of the peripher- late'' agar at 379Cin10z CO2, producing in 24 h, al arteries. His body temperature was 37.39C. There small (1 mm in diameter), grey, moist, round, raised, were no symptoms of heart failure or peripheral cutane- and non-hemolytic colonies. Identification of the Gram- ous endocarditis symptoms, nor was the spleen palpa- negative bacillus isolated in the blood culture was rap- ble. Transesophageal echocardiography confirmed sig- idly performed by depositing a thin smear of this strain nificant aortic insufficiency and showed a large valvular on a MALDI-steel plate. A saturated solution of a- vegetation on a right coronary aortic cusp with prolapse cyano-4-hydroxycinnamic acid in 50z acetonitrile and into the left ventricular outflow tract. There was no 2.5z trifluoroacetic acid (1.1 mL)wasappliedtothe periannular abscess. The C-reactive protein concentra- bacterial smear and dried. Measurements were per- tion was 24 mg/L (normal range º6 mg/L). The leuko- formed with a Microflex mass spectrometer (Bruker cyte count was 9.1 × 109/L, 75z of which was poly- Daltonik, Wissembourg, France) using the FlexControl morphonuclear, the hemoglobin level was 11.8 g/dL, software (version 3.0). Mass spectra were acquired in a and the platelet count was 170 × 109/L. Four blood cul- linear positive extraction mode ranging from 2,000 to ture sets were collected using BacT/Alert (bioMáerieux, 20,000 Da. The spectrum was imported into the Marcy l'Etoile, France) before starting antibiotic ther- BioTyper software (version 2.0; Bruker, Karlsruhe, apy with amoxicillin (12 g/d), gentamycin (240 mg/d), Germany). The BioTyper database contains the spectra and levofloxacin (750 mg/d). The four aerobic blood of approximately 3,739 species and is regularly updated cultures were found to be positive after 50 h of incuba- by the Bruker Company. The result of the pattern- tion. The diagnosis of endocariditis was confirmed on matching process was expressed with a score of 1.9, giv- the basis of a positive direct examination of blood cul- ing DSM 8339T as first choice ture using a pleiomorphic Gram-negative rod. Before (a score of 1.9 was considered to be identification at spe- the antibiogram was available, the therapy was modi- cies level [1]) (Fig. 1). Phenotypical identification using fied: amoxicillin was replaced with cefotaxime (7 g/d). the VITEK 2 NH system (bioMáerieux) performed on The infectious process was favorable, and the aortic in- this oxidase-positive, -negative, indole-produc- sufficiency was treated using a mechanical prosthesis. ing strain indicated C. hominis (very good identifica- The culture obtained from the native valve was sterile. tion, with a probability of 93z). An in vitro susceptibil- The history of this patient, after re-examination, re- ity test, using a disk diffusion technique on Mueller- vealed a dermatological lesion on the right arm treated Hinton agar supplemented with 5z horse blood, with corticoids (prednisolone) per os 15 days before the showed that the organism was susceptible to , beginning of the illness. amoxicillin plus clavulanic acid, piperacillin, cefota- Direct examination showed that the morphology of xime, aminoglycosides, co-trimoxazole, vancomycin, and fluorinated-quinolones. The isolate was resistant to *Corresponding author: Mailing address: Centre de erythromycin. No b-lactamase was detected by the Biologie-Pathologie, Institut de Microbiologie, Bld du Pr nitrocefin test. Leclercq, 59037 Lille Cedex, France. E-mail: frederic. To determine the involvement of C. hominis in the wallet＠chru-lille.fr endocarditic process and to confirm its conventional

327 Fig. 1. (A) Spectrum of C. hominis (up) compared with the result of the database C. hominis DSM 8339T (down) with a score of 1.980. (B) Spectrum of C. hominis (up) compared with the result of the database C. valvarum DSM17211T (down) with a score of 0.042. identification, the 16S rRNA gene was immediately se- tractII kit; Macherey Nagel, Hoerdt, France), amplifi- quenced from the colonies, and a further sample was cation was performed using the same primers. After obtained from a fragment of the valve preserved at purification of the product using a SpinX kit (Costar, －809C. Using the primers described by Gauduchon et Cambridge, Mass., USA), the 470-bp fragment ob- al. (2), a 16S rDNA bacterial fragment of 478 bp was tained was sequenced and compared with NCBI Gen- amplified from the and sequenced on an auto- Bank entries using the BLAST algorithm, giving 99z mated sequencer (377 ABI Prism; PE Applied (468/470 bp) identity with C. hominis strain 6573 (Gen- Biosystems, Foster City, Calif., USA). It was then com- Bank accession no. M35014). From the two amplifica- pared with NCBI GenBank entries giving 98z (474/478 tions, the second species of Cardiobacterium (C. valva- bp) identity with C. hominis strain 6573 (GenBank ac- rum) showed 97z identity with C. valvarum strain cession no. M35014). After DNA was extracted from MDA3079 (GenBank accession no. AF506987). This the fragment of the aortic valve tissue (Nucleospin Ex- second species of Cardiobacterium was never mentioned

328 by the MALDI-TOF mass spectrometry knowing that tification of HACEK clinical isolates by matrix-assisted laser this species belongs to the bank of the MALDI-TOF. desorption ionization—time of flight mass spectrometry. J. Clin. Microbiol., 49, 1104–1106. Aggregatibacter-Cardiobacterium-Eikenella-Kingella 2. Gauduchon, V., Chalabreysse, L., Etienne, J., et al. (2003): (ACEK) microorganisms are reported to cause 3z of all Molecular diagnosis of infective endocarditis by PCR amplifica- endocarditis cases. ACEK microorganisms frequently tion and direct sequencing of DNA from valve tissue. J. Clin. colonize the oropharynx, are slow growing, and their Microbiol., 41, 763–766. growth is enhanced by the presence of CO . C. hominis 3. Slotnick, I.J. and Dougherty, M. (1964): Further characterization 2 of an unclassified group of bacteria causing endocarditis in man: was isolated for the first time in 1962 from a patient Cardiobacterium hominis gen. et sp. n. Antonie van Leeuwen- with endocarditis and classified as a Pasteurella-like or- hoek, 30, 261–272. ganism. In 1964, Slotnick and Dougherty described the 4. Dewhirst, F.E., Paster, B.J., La Fontaine, S., et al. (1990): organism and gave it the present name (3). C. hominis Transfer of Kingella indologenes (Snell and Lapage 1976) to the was recently reclassified with Suttonella indologenes in genus Suttonella gen. nov. as Suttonella indologenes comb. nov.; transfer of Bacteroides nodosus (Beveridge 1941) to the genus the family on the basis of 16S rRNA Dichelobacter gen. nov. as comb. nov.; sequence studies (4). Since the original publication, and assignement of the genera Cardiobacterium, Dichelobacter, more than 90 cases of endocarditis have been document- and Suttonella to Cardiobacteriaceae fam. nov. in the gamma di- ed (5–11). Because of the low virulence of the bacteri- vision of on the basis of 16S rRNA sequence com- parisons. Int. J. Syst. 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(2006): Utility illustrates the ease and rapidity of MALDI-TOF mass of extended blood culture incubation for isolation of Haemophi- spectrometry, which has good discrimination power to lus, , Cardiobacterium, Eikenella,andKingella differentiate rapidly between the two species of Cardio- organisms: a retrospective multicenter evaluation. J. Clin. bacterium, eliminating the need to perform molecular Microbiol., 44, 257–259. 14. Habib, G., Hoen, B., Tornos, P., et al. (2009): Guidelines on the PCR (15–18). Nevertheless, this new technique does not prevention, diagnosis, and treatment of infective endocarditis propose additional biochemical or immunological tests (new version 2009) The task force on the prevention, diagnosis, if the MALDI-TOF result is not suitable (score º1.9). and treatment of infective endocarditis of the European Society Improvements to conventional techniques (automated of Cardiology (ESC). Eur. Heart. J., 30, 2369–2413. 15. 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