Antibiotic content of selective culture media for isolation of Capnocytophaga species from oral polymicrobial samples

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1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE Antibiotic content of selective culture media for isolation of Capnocytophaga species from oral polymicrobial samples E. Ehrmann 1,2,3, A. Jolivet-Gougeon 3,4, M. Bonnaure-Mallet 3,4 and T. Fosse 5 1 P^ole odontologie, CHU Nice, Nice, France 2 Faculte d odontologie, Universite de Nice Sophia-Antipolis, Nice, France 3 Equipe de Microbiologie, EA 1254, Universite de Rennes I, Rennes, France 4 P^ole microorganismes CHU Rennes, Rennes, France 5 Laboratoire de bacteriologie et epidemiologie moleculaire, CHU Nice, Nice, France Significance and Impact of the Study: Isolation of Capnocytophaga species is important for the proper diagnosis and treatment of the systemic infections they cause and for epidemiological studies of periodontal flora. We showed that in pure culture, a simple blood agar allowed the growth of all Capnocytophaga species. Nonetheless, in oral samples, because of the abundance of commensal competitive flora, selective media with antibiotics are necessary for the recovery of Capnocytophaga species. The demonstrated superiority of VCAT medium made its use essential for the optimal detection of this bacterial genus. This work showed that extreme caution should be exercised when reporting the isolation of Capnocytophaga species from oral polymicrobial samples, because the culture medium is a determining factor. Keywords antibiotic susceptibility, Capnocytophaga species, culture media, isolation, oral bacteria. Correspondence Martine Bonnaure-Mallet, Equipe de Microbiologie EA 1254, Universite de Rennes 1, 2 avenue du Professeur Leon Bernard, Rennes, France. martine.bonnaure@univ-rennes1.fr 2013/0113: received 18 January 2013, revised 21 May 2013 and accepted 28 May 2013 doi: /lam Abstract In oral microbiome, because of the abundance of commensal competitive flora, selective media with antibiotics are necessary for the recovery of fastidious Capnocytophaga species. The performances of six culture media (blood agar, chocolate blood agar, VCAT medium, CAP E medium, bacitracin chocolate blood agar and VK medium) were compared with literature data concerning five other media (FAA, LB, TSBV, Cap R and TBBP media). To understand variable growth on selective media, the MICs of each antimicrobial agent contained in this different media (colistin, kanamycin, trimethoprim, trimethoprim sulfamethoxazole, vancomycin, aztreonam and bacitracin) were determined for all Capnocytophaga species. Overall, VCAT medium (Columbia, 10% cooked horse blood, polyvitaminic supplement, 375 mg l 1 of colistin, 15 mgl 1 of trimethoprim, 1 mg l 1 of vancomycin and 05 mgl 1 of amphotericin B, Oxoid, France) was the more efficient selective medium, with regard to the detection of Capnocytophaga species from oral samples (P < 0001) and the elimination of commensal clinical species (P < 0001). The demonstrated superiority of VCAT medium, related to its antibiotic content, made its use indispensable for the optimal isolation of Capnocytophaga species from polymicrobial samples. Introduction The genus Capnocytophaga comprises a group of fastidious capnophilic, facultatively anaerobic Gram-negative bacilli that include eight species, namely C. ochracea, C. sputigena, C. gingivalis, C. granulosa, C. leadbetteri, C. haemolytica, C. canimorsus and C. cynodegmi (Ciantar et al. 2001b; Frandsen et al. 2008). The last two species, C. canimorsus and C. cynodegmi, are found in the oral cavity of dogs and cats and most commonly associated with dog-bite infections in humans (Brenner et al. 1989). The ecological niche for the first six species is the human Letters in Applied Microbiology 57, The Society for Applied Microbiology 303

2 Isolation of Capnocytophaga species E. Ehrmann et al. oral cavity. They are associated with varying degrees of periodontal health, and translocation of Capnocytophaga sp. from oral origin may be responsible for bacteraemia and systemic infections, which can be severe, especially in immunocompromised patients (Martino et al. 2001; Wang et al. 2007). Currently, for paediatric patients, in the context of haematopoietic transplant, an initial microbiological examination, which includes blood culture, urine culture and a throat swab, is required. Capnocytophaga spp. is part of the bacterial flora found in throat swabs, and their antibiotic resistance has been widely described by many authors (trimethoprim, Hawkey et al. 1987; vancomycin, Forlenza et al. 1981; amoxicillin, Jolivet-Gougeon et al. 2000). Although vancomycin is an antibiotic active against Gram-positive bacteria, it is partially active against Gram-negative strict anaerobes. In case of febrile neutropenia (with a neutrophil count <500 mm 3 ), antibiotics will be started no later than 2 h after the beginning of the fever. The choice of antibiotic treatments will consider bacterial species isolated in the initial microbiological examination. However, the associated polymorphic flora makes their isolation difficult. Indeed, this bacterial genus is a member of the difficult to cultivate group (Vergnaud 2009). Data regarding the isolation frequency of Capnocytophaga spp. from oral clinical samples are conflicting due to the different microbial procedures employed, the fastidious growth of this bacterial genus and the rapid growth of the competitive flora of the oral microbiome (Rummens et al. 1985; 2001a; Ehrmann et al. 2011). Specific culture media are required to isolate these organisms from oral samples. Some selective media have been used, but no study has demonstrated the superiority of any one method. Isolation of Capnocytophaga species is important for the proper diagnosis and treatment of the systemic infections they cause and for epidemiological studies of periodontal flora (Rummens et al. 1986; Martino et al. 2001; Sixou et al. 2006). The first objective of this investigation was to perform a qualitative comparison of the growth and isolation of the different Capnocytophaga species on six commercial culture media and to compare these results to media already described in the literature. The second objective was to demonstrate a relationship between the susceptibility of different Capnocytophaga species to antimicrobial agents contained in the different media as related to the medium content (i.e. antimicrobial concentration in each medium). Results and discussion Table 1 presents a compilation of literature data concerning prior investigations about all culture media used for the growth and isolation of Capnocytophaga spp., but no study has demonstrated the superiority of any one method (Table 1). Only the nonselective culture media Fastidious Anaerobe Agar (FAA, lab M) ( 2001a) allowed the growth of each species in culture alone, but the problem of isolating each different Capnocytophaga species from a polymicrobial sample using selective medium has not been solved (Leadbetter et al. 1979; Slots 1982; Mashimo et al. 1983; Rummens et al. 1985; 2001a). This difficulty is due, in part, to insufficient data on the profile of the natural antibiotic susceptibility of different Capnocytophaga species ( 2001a). Tables 2 and 3 present the results of our own investigations about culture media used for the growth and isolation of Capnocytophaga spp. The nonselective media, blood agar (BA) and chocolate blood agar (CBA), allowed the growth of the five reference strains and ten clinical isolates in pure culture (Table 2). With equivalent results obtained by FAA medium (Table 1), we showed that simple blood agar can be used for the growth of all Capnocytophaga species. However, these nonselective media presented the major inconvenience of allowing the growth of numerous commensal bacteria from the oral cavity, which disrupted the optimal detection of Capnocytophaga spp. from polymicrobial samples (Rummens et al. 1985; 2001a). Therefore, selective media, containing antimicrobial agents, must necessarily be tested and used. On selective media, the growth, in pure culture, of the different reference and clinical Capnocytophaga spp. was variable. In the literature, the best selective medium for the growth of Capnocytophaga spp. was TBBP (11/14), followed by Cap R (8/14) and TSBV (5/14) (Table 1). The media VCAT (11/15), VK (12/15) and CAP E (13/15) were significantly more efficient than TSBV medium (P < 005) and seemed more successful or comparable than Cap R medium and TBBP (P > 005). Consequently, VCAT, VK, CAP E, Cap R and TBBP media were further evaluated and Capnocytophaga species isolation from plaque samples compared. The last part of Tables 1 and 3 shows the performance of all media to detect Capnocytophaga spp. from subgingival polymicrobial samples and their ability to inhibit contaminating species. In our study (Table 3), we have identified all Capnocytophaga isolates at the species level to perform a qualitative comparison of all media proposed. The identification of species from this bacterial genus was achieved by sequencing the 16S rrna gene (Frandsen et al. 2008), which makes any protocol concerning this micro-organism more intensive. Overall, VCAT was the more efficient selective medium, with regard to the recovery of Capnocytophaga spp. from plaque samples (80%; P < 0001) and the elimination of contaminants (75%; P < 0001) (Tables 1 and 3). After VCAT, the best selective medium was VK (20%; Table 3), which was quantitatively comparable (P > 005) 304 Letters in Applied Microbiology 57, The Society for Applied Microbiology

3 E. Ehrmann et al. Isolation of Capnocytophaga species Table 1 Meta-analysis of the growth and isolation of Capnocytophaga spp. on solid media Media FAA LB BA TSBV Cap R TBBP Studies Leadbetter et al. (1979); Rummens et al. (1985) Slots (1982); Rummens et al. (1985); Mashimo et al. (1983); ; Rummens et al. (1985) Antibiotic content (mg l 1 ) No antibiotic No antibiotic No antibiotic Vancomycin (5) Bacitracin (75) Trimethoprim (25) Vancomycin (5) Polymyxin B (1,8) Amphotericin B (25) Bacitracin (50) Polymyxin B (100) Pure culture No. species able to grow (No. strains tested) C. ochracea 6 (6) 5 (6) NT 4 (6) 5 (6) 6 (6) C. sputigena 1 (1) 1 (1) NT 0 (1) 1 (1) 1 (1) C. gingivalis 2 (2) 2 (2) NT 0 (2) 0 (2) 1 (2) C. granulosa 2 (2) 2 (2) NT 0 (2) 0 (2) 2 (2) C. haemolytica 1 (1) 1 (1) NT 1 (1) 1 (1) 1 (1) C. leadbetteri NT NT NT NT NT NT C. canimorsus 1 (1) 1 (1) NT 0 (1) 1 (1) 0 (1) C. cynodegmi 1 (1) 0 (1) NT 0 (1) 0 (1) 0 (1) Total 14 (14) 12 (14) NT 5 (14) 8 (14) 11 (14) Plaque samples No. plates with Capnocytophaga spp. (No. samples) 4 (5) 4 (5) 20 (725); 727% 0 (5) 315 (730); 4315% 178 (735); 2421% No. plates without contaminant (No. samples) NT NT 8 (725); 11% NT 183 (725); 2524% 70 (730); 96% NT, not tested. Table 2 Growth of the ATCC Capnocytophaga type strains and clinical isolates on five culture media commonly found in bacteriology laboratories, tested in this study Media BA/CBA VCAT CAP E BCBA VK Antibiotic content (mg l 1 ) No antibiotic Vancomycin (1) Colistin (375) Amphotericin B (05) Trimethoprim (15) Colistin (10) Aztreonam (2) Bacitracin (50) Vancomycin (75) Kanamycin (100) Pure culture No. species able to grow (No. strains tested) C. ochracea 3 (3) 3 (3) 3 (3) 2 (3) 3 (3) C. sputigena 3 (3) 3 (3) 2 (3) 2 (3) 2 (3) C. gingivalis 3 (3) 2 (3) 3 (3) 2 (3) 3 (3) C. granulosa 3 (3) 1 (3) 3 (3) 1 (3) 1 (3) C. haemolytica 1 (1) 1 (1) 1 (1) 1 (1) 1 (1) C. leadbetteri 1 (1) 1 (1) 0 (1) 0 (1) 1 (1) C. canimorsus 1 (1) 0 (1) 1 (1) 0 (1) 1 (1) C. cynodegmi NT NT NT NT NT Total 15 (15) 11 (15) 13 (15) 8 (15) 12 (15) NT, not tested. with Cap R (4315%) and TBBP (2421%) for the isolation of Capnocytophaga spp. (Mashimo et al. 1983; Rummens et al. 1985; 2001a; Table 1). Qualitatively, it is noteworthy that, in one sample (no. 9), VK was the only selective medium that allowed the culture of one C. granulosa strain (Table 3). The CAP E medium was quantitatively less selective and successful than Cap R medium (P < 005). However, CAP E Letters in Applied Microbiology 57, The Society for Applied Microbiology 305

4 Isolation of Capnocytophaga species E. Ehrmann et al. Table 3 Evaluation of the five media for the detection of Capnocytophaga species and the inhibition of the contaminating flora, from oral plaque samples BA VCAT CAP E BCBA VK C. spp GC C. spp GC C. spp GC C. spp GC C. spp GC Plaque samples C. sputigena C. spp C. sputigena C. gingivalis C. ochracea C. leadbetteri, C. spp 10 4 C. sputigena 5 1 C. granulosa C. ochracea C. spp C. sputigena C. spp C. ochracea C. granulosa C. sputigena C. ochracea C. spp C. sputigena C. sputigena C. ochracea C. granulosa C. leadbetteri C. ochracea C. leadbetteri C. spp C. ochracea Plates with Capnocytophaga spp. (No. samples) 3 (20), 15% 16 (20), 80% 2 (20), 10% 0 (20), 0% 4 (20), 20% Plates without contaminant (No. samples) 0 (20), 0% 15 (20), 75% 0 (20) 0 (20), 0% 4 (20), 20% C. spp, Capnocytophaga spp.; GC, growth of contaminants;, no growth; +, 1 to 10 2 colonies; ++,10 3 to 10 4 colonies; +++, >10 5 colonies. allowed the identification of one C. gingivalis and one C. granulosa strain in two different samples, whereas other media did not (Table 3). The last medium, BCBA, was uninteresting due to an overgrowth of contaminants (Table 3). In our study, BA allowed the isolation of Capnocytophaga spp. from only three samples (3/20; 15%), which is comparable with previous findings (727%; Rummens et al. 1985) (Tables 1 and 3). However, this medium allowed, in one sample, the culture of a C. granulosa strain that was not found with other media (Table 3). Despite several dilutions of the inoculum, massive overgrowth with other commensal micro-organisms was responsible for the low recovery rate of Capnocytophaga species on BA. To understand variable growth on selective media, we analysed the minimum inhibitory concentration (MIC) values of antibiotics contained in these media according to the different Capnocytophaga species (Table 4), which is the first time for this bacterial genus. Capnocytophaga granulosa, C. gingivalis, C. leadbetteri and C. canimorsus presented relatively low MIC values (025 to 12 mg l 1 ) (Table 4) for vancomycin in the same order of magnitude of media containing vancomycin (1, 5or75 lg ml 1 for VCAT, Cap R and VK, respectively). Minimum range MICs and MIC50s were variable according to studies (02 8 and mg l 1, respectively), probably due to specific strain differences (Table 4) (Sutter et al. 1981; Rummens et al. 1986; Roscoe et al. 1992; Forlenza et al. 1981). The lowest vancomycin concentration (1 mg l 1 ) in VCAT medium can explain the better growth on this medium (P < 0001). The other change between VCAT and Cap R, which were the best selective media described in the literature (for Capnocytophaga spp. detection from oral sample), is the replacement of polymyxin B by colistin in the same antibiotic polypeptide group. All Capnocytophaga species were resistant to colistin (Sutter et al. 1981) or polymyxin (Mashimo et al. 1983; Rummens et al. 1986) with high MICs (Table 4). We also observed an important variation in susceptibility to bacitracin (minimum range MICs mg l 1 and MIC50s 8 to >200 mg l 1 ) in our study and in the literature (Table 4) (Sutter et al. 1981; Mashimo et al. 1983). This relative susceptibility could explain why not all strains of Capnocytophaga species were capable of 306 Letters in Applied Microbiology 57, The Society for Applied Microbiology

5 E. Ehrmann et al. Isolation of Capnocytophaga species Table 4 MICs of antibiotics contained in selective media for the isolation of Capnocytophaga spp. Antibiotics Total No. tested strains MIC (mg l 1 ) (No. tested strains) Range values 50% 90% C. gin (3) C. och (3) C. spu (3) C. gra (3) C. lea (1) C. can (1) C. hae (1) References Colistin 15 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 This study >128 >128 >128 Sutter et al. (1981) Kanamycin 15 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 This study >128 >128 >128 Sutter et al. (1981) Trimethoprim 15 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 This study >128 >128 >128 Rummens et al. (1986) 33 <003 >64 >64 >64 Hawkey et al. (1987) Trimethoprim- Sulfamethoxazole 15 1 >32 >32 > >32 > >32 NT 1 This study Vancomycin > This study 41 8 > Rummens et al. (1986) 27 4 > Sutter et al. (1981) Forlenza et al. (1981) Roscoe et al. (1992) Aztreonam > NT 64 This study > Rummens et al. (1986) 33 < >64 Hawkey et al. (1987) NT Bremmelgaard et al. (1989) 120 NT 025 NT Foweraker et al. (1990) 34* Jolivet-Gougeon et al. (2000) NT Jolivet-Gougeon et al. (2000) Bacitracin > NT 32 This study >200 >200 NT Mashimo et al. (1983) Sutter et al. (1981) Amphotericin B 41 > 128 >128 >128 Rummens et al. (1986) C. gin, C. gingivalis; C.och, C. ochracea; C. spu, C. sputigena; C. gra, C. granulosa; C. lea, C. leadbetteri; C. can, C. canimorsus; C. hae, C. haemolytica; NT, no tested. *Beta-lactamase positive isolates. Beta-lactamase negative isolates. Letters in Applied Microbiology 57, The Society for Applied Microbiology 307

6 Isolation of Capnocytophaga species E. Ehrmann et al. growing on TBBP and BCBA, which contained 50 mg l 1 of bacitracin. In our study, the MICs were determined by E-test (Biomerieux), which has been proved to be similar to performing the agar dilution reference method (Bremmelgaard et al. 1989; Kawakami et al. 1998; Jorgensen 2004). Contradictory data on susceptibility are probably due to differences in methodology, but also to the proportion of different species tested in our study. All species were polymyxin B, trimethoprim, amphotericin B, colistin and kanamycin resistant according to the literature (Table 4). Growth inhibition of some Capnocytophaga spp. on selective media was essentially linked to vancomycin, bacitracin or aztreonam. However, the use of these antibiotics was justified by their ability to eliminate the overgrowth of the most frequently contaminated clinical specimens observed on nonselective media. In conclusion, this work showed that extreme caution should be exercised when reporting the isolation of Capnocytophaga species from oral polymicrobial samples, because the culture medium is a determining factor in isolating the full range of Capnocytophaga species. For the isolation of all Capnocytophaga species from polymicrobial samples, we showed that no selective or nonselective medium was tested in the literature or this study was perfectly ideal because of differences in antimicrobial agent susceptibility between species and/or isolates. However, the demonstrated superiority of VCAT medium made its use indispensable for the optimal isolation of Capnocytophaga species from polymicrobial samples. For initial microbiological examination of throat swap in haematology oncology patient in the context of haematopoietic transplant, VCAT is the best medium to use for the detection of resistant Capnocytophaga spp. Materials and methods Two nonselective culture media, blood agar medium (BA; Columbia, 5% sterile sheep blood, Bio-Rad Laboratories, Marnes-la-Coquette, France) and chocolate blood agar medium (CBA; Columbia, 10% cooked horse blood, Bio- Rad Laboratories), were used, as well as four selective culture media: CAP E medium (Columbia, 5% sterile sheep blood, 10 mg l 1 of colistin and mg l 1 of aztreonam, Oxoid, Dardilly, France), VCAT medium (Columbia, 10% cooked horse blood, polyvitaminic supplement, 375 mg l 1 of colistin, 15 mgl 1 of trimethoprim, 1mgl 1 of vancomycin and 05 mgl 1 of amphotericin B, Oxoid), bacitracin chocolate blood agar medium (BCBA; Columbia, 10% cooked sheep blood, polyvitaminic supplement, 50 mg l 1 of bacitracin, Bio-Rad Laboratories) and VK medium (Sheadler, 5% sheep blood, 100 mg l 1 of kanamycin and 75 of vancomycin, Becton Dickinson, Le Pont de Claix, France). Five Capnocytophaga reference strains, C. sputigena ATCC 33612, C. gingivalis ATCC 33624, C. ochracea ATCC 33596, C. granulosa ATCC and C. haemolytica ATCC 51501, and ten clinical Capnocytophaga isolates (CS) from human supragingival or dog-bite infection samples (Laboratoire de Bacteriologie, H^opital Archet, Centre Hospitalier Universitaire de Nice, France) were used. Species identification was performed by 16S rrna sequencing (Frandsen et al. 2008). For growth evaluation, all strains were suspended and inoculated as described previously ( 2001a). All plates were incubated for 3 days at 37 C. BA and VK media were incubated in an anaerobic atmosphere, while CBA, BCBA, VCAT and CAPE were incubated in 5% CO 2. Twenty samples of subgingival plaque from patients in the Department of Haematology (H^opital Archet, Centre Hospitalier Universitaire Nice, France) were collected as described previously (Sixou et al. 2006), serially diluted and inoculated on the culture media. The growth of Capnocytophaga spp. and other micro-organisms from the oral flora (contaminating species) (CFU ml 1 ) was measured. Capnocytophaga identification was performed by 16S rrna sequencing (Frandsen et al. 2008). R software ( for Windows (Microsoft, Issy-les-Moulineaux, France) was used for the statistical analysis of Capnocytophaga spp. and contaminant growth compared with literature data (chisquare or Fisher s exact test according to size groups). A P-value of less than 005 was considered as significant. The MICs of the 15 strains were determined by the E-test method (Biomerieux, Craponne, France) (Vergnaud 2009) for each antimicrobial agent contained in the different media tested: colistin, kanamycin, trimethoprim, trimethoprim sulfamethoxazole, vancomycin, aztreonam and bacitracin. Acknowledgements This project was partially supported by a grant from the Ministere de la Sante, Projet Hospitalier de Recherche Clinique 2004, CHU de Nice promoteur (T.F.) and by a grant from IFRO and the Conseil Regional de Bretagne. Conflict of interest The authors declare that they have no conflict of interest. References Bremmelgaard, A., Pers, C., Kristiansen, J.E., Korner, B., Heltberg, O. and Frederiksen, W. (1989) Susceptibility testing of Danish isolates of Capnocytophaga and CDC group DF-2 bacteria. APMIS 97, Letters in Applied Microbiology 57, The Society for Applied Microbiology

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