MALDI-TOF Mass Spectrometry Analysis of gram-positive, catalase-negative Cocci not Belonging to the Streptococcus or Enterococcus Genera and Benefits of Database Extension Jens Jørgen Christensen1,3, Rimtas Dargis1,3, Monja Hammer3, Ulrik Stenz Justesen2, Xiaohui C. Nielsen1 Michael Kemp2,3 and the Danish MALDI-TOF study group* Departments of Clinical Microbiology, 1Slagelse Hospital, Slagelse and 2Odense University Hospital, Odense and 3Department of Microbiological Surveillance and Research, Statens Serum Institut, Copenhagen, Denmark Correspondance: Jens Jørgen Christensen , Dept. of Clinical Microbiology, Slagelse Hospital, 4200 Slagelse, Denmark email:[email protected] INTRODUCTION Within the last decade and especially within the last five years MALDI-TOF MS has been a major contribution to clinical microbiology. Catalase-negative, gram-positive cocci, not belonging to the streptococci or the enterococci (of which many taxons may cause serious invasive infection), has become increasingly well characterized and the number of taxonomic entities steadily growing thereby complicating their identification. MALDI-TOF MS was applied on 51 collection strains within this group of bacteria in order to look for benefits of supplementing the existing database with new entries and the identification setups were challenged with 90 well-characterized strains. METHODS TABLE 2. Allocation of 51 collection and 90 challenge strains to interpretation groups based on obtained score values. The meaning of score values obtained were: MALDI-TOF MS was done with a Bruker Daltronics microflex LT instrument. 2.300 ... 3.000: highly probable species identification (marked (+++) and green in reports); 2.000 ... 2.299: secure genus identification, probable species identification (marked (++) and green in reports); 1.700 ... 1.999: probable genus identification (marked (+) and yellow in reports); 0.000 ... 1.699: not reliable identification The database included in the BioTyper v. 2.0.43.1 software was used singly or (marked (-) and red in reports). in combination with a database extension with consensus mass spectra Identification database 2.300 - 3.000 2.000 - 2.299 1.700 - 1.999 0.000 - 1.699 generated on 51 collection strains (representing 16 genera and 51 species, ( +++ ) ( ++ ) ( + ) ( - ) green green yellow red Table 1). Moreover, 90 well-characterized challenge strains from the Danish ______________________________________________________________________________________________________________________________________ Biotyper (v. 2.0) national reference laboratory were examined. All collection strains (n=51) 6 18 7 20 Preparation of strains for MALDI-TOF MS and identification (using Maldi Challenge strains (n=90): 15 35 18 22 Aerococcus species (n=35) 7 22 5 1 Biotyper Automation Control 2.0.43.1 software), was performed as Gemella species (n=23) 0 5 5 13 recommended by the manufacturer. Briefly, a small amount of an overnight Abiotrophia/Granulicatella species (n=10) 1 1 7 1 culture was added to the spot, followed by 70% formic acid to obtain extraction, Mixed group of collection strains (n=22)a 7 8 0 7b before adding the matrix solution (saturated solution of HCCA). For each Biotyper + database extensionc collection strain, a mass spectrum profile (MSP), based on 24 separate All collection strains (n=51) 38 13 0 0 determinations (MALDI Biotyper 2.0SR1 (build 223.8)) was created and stored Challenge strains (n=90): 39 39 12 0 Aerococcus species (n=35) 15 18 2 0 in a separate library that could be combined with the standard database. Gemella species (n=23) 6 13 4 0 Abiotrophia/Granulicatella species (n=10) 7 2 1 0 Mixed group of collection strains (n=22)a 11 6 5d 0 ______________________________________________________________________________________________________________________________________________ RESULTS a: Genera: Globicatella (n=5), Lactococcus (n=5), Leuconostoc (n=5), Rothia (n=3), Facklamia (n=1), Vagococcus (n=1), Helcococcus (n=1), Alloioicoccus (n=1). N=22. Collection strains: When using the combined database all collection strains b: Genera: Globicatella (n=5), Alloioicoccus (n=1), Rothia mucilaginosa (n=1) were categorized as obtaining either highly probable species identification c: self-created mass spectrum profiles (MSPs) (n=39) or secure genus identification and probable species identification d: Genera: Globicatella (n=2), Lactococcus (n=1), Leuconostoc (n=1), R. mucilaginosa (n=1) (n=13). Using the BioTyper (v. 2.0.43.1 ) database singly, allocated seven and 21 strains to either obtaining probable genus identification or no reliable identification (Table 2). Challenge strains: Strains belonging to the genera Aerococcus, Gemella and Abiotrophia/Granulicatella dominated, but 1-5 strains each belonged to eight other genera. By MALDI-TOF MS analysis all strains were allocated to the presumed genera. Strains from the Aerococcus genus (n = 35, Table 2) and the following list of genera with 1-5 strains each, were convincingly identified to the species level using both database set ups: Abiotrophia, Lactococcus, Leuconostoc, Rothia, Facklamia, Helcococcus and Vagococcus. Strains belonging to the genera Gemella, Granulicatella, Globicatella and Alloiococcus, with few exceptions, only obtained convincing identifications when using the combined database. The MSP Dendrogram for Aerococcus strains (Fig. 1) and the genera Gemella, Granulicatella and Abiotrophia (Fig. 2) convincingly delineated the included species. Fig.1: Dendrogram for collection and challenge strains of Fig. 2: Dendrogram for collection and challenge strains of TABLE 1. Collection strains used for database extension and species included in the BioTyper Aerococcus. v. 2.0.43.1 software. Gemella, Granulicatella and Abiotrophia. CCUG no. in the BioTyper v.2.0.43.1 Abiotrophia defectiva CCUG 27639 + Aerococcus christensenii CCUG 28831 + CONCLUSION Aerococcus sanguinicola CCUG 4100 + Aerococcus suis CCUG 52530 - • MALDI-TOF MS seems very promising for identification of strains being catalase-negative, Aerococcus urinae CCUG 36881 + Aerococcus urinaeequi CCUG 28094 - Aerococcus urinaehominis CCUG 42038 B + gram-positive cocci, not belonging to the streptococci or the enterococci. Aerococcus viridans CCUG 4311 + Alloiococcus otitis CCUG 32997 + Dolosicoccus paucivorans CCUG 39307 - Dolosigranulum pigrum CCUG 33392 - Facklamia hominis CCUG 36813 + • The possibility of creating your own consensus mass spectra, opens up for its use in specialized Facklamia ignava CCUG 37419 - Facklamia languida CCUG 37842 + settings. Facklamia miroungae CCUG 42728 - Facklamia sourekii CCUG 28783ª - Facklamia tabacinasalis CCUG 30090 - Gemella asaccharolytica CCUG 57045 - Gemella bergeri CCUG 37817 + • Dendrograms are useful for demonstrating the ability of MALDI-TOF MS to differentiate between Gemella cuniculi CCUG 42726 - Gemella haemolysans CCUG 37985 + Gemella morbillorum CCUG 18164 - the included species and to visualize intra-species similarity or variation when more strains are Gemella palaticanis CCUG 39489 - Gemella sanguinis CCUG 37820 + examined. Globicatella sanguinis CCUG 32999 - Globicatella sulfidifaciens CCUG 44365 - Granulicatella adiacens CCUG 27809 + Granulicatella balaenopterae CCUG 37380 + Granulicatella elegans CCUG 38949 + Helcococcus kunzii CCUG 32213 + REFERENCES Bizzini A, G. Greub. 2010. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolution in clinical microbial identification. Clin. Helcococcus ovis CCUG 37441 - Helcococcus sueciensis CCUG 47334 - Microbiol. Infect. 16(11):1614-1619. Ignavigranum ruoffiae CCUG 37658 - Lactococcus garvieae CCUG 32208 + Emonet S., H.N. Shah, A. Cherkaoui, J. Schrenzel. 2010. Application and use of various mass spectrometry methods in clinical microbiology. Clin. Microbiol. Lactococcus lactis subsp. cremoris CCUG 21953 + Infect. 16(11):1604-1613. Lactococcus lactis subsp. hordniae CCUG 32210 - Lactococcus lactis subsp. lactis CCUG 7980 + Ruoff K.L. Aerococcus, Abiotrophia, and other aerobic, catalase-negative, gram-positive cocci. 2007. p. 443-454. In P.R. Murray (ed. in chief). Manual of Leuconostoc lactis CCUG 30064 + th Leuconostoc mesenteroides (subsp. mesenteroides) CCUG 30066 + clinical microbiology. 9 edition. American Society for Microbiology, Washington DC. Leuconostoc mesenteroides subsp. cremoris CCUG 21965 + Leuconostoc mesenteroides subsp. dextranicum CCUG 30065 - Pediococcus acidilactici CCUG 32235 + Pediococcus parvulus CCUG 28439 - *)The Danish MALDI-TOF MS study group. Participants from Departments of Clinical Microbiology in Denmark: C.Ø. Andersen (Hvidovre), H. Friis (Slagelse), Pediococcus pentosaceus CCUG 32205 + Rothia aeria CCUG 51932 + T.G. Jensen (Odense), M. Tvede (RH), P. Kjældgaard (Sønderborg), S.E. Eriksen (Skejby), I.P. Jensen (Hillerød), S. Lomborg (Herning), J. Prag (Viborg), J. Rothia amarae CCUG 47294 + Rothia dentocariosa CCUG 35437 + Møller (Vejle), D. Fuglsang-Damgaard (Aalborg), S. Hartzen (Esbjerg), J.O. Jarløv (Herlev). Participants from Statens Serum Institut, Department of Rothia mucilaginosa CCUG 20962 + Microbiological Diagnostics: Annemarie Hesselbjerg, Elsa Vilhelmsen. CCUG: Received from Culture Collection, University of of Göteborg .