were identified. All effusions were collected by tympanocentesis in children with acute otitis media. ... strong argument for their involvement in acute otitis media.
Vol. 31, No. 6
JOURNAL OF CLINICAL MICROBIOLOGY, June 1993, p. 1667-1668
0095-1137/93/061667-02$02.00/0 Copyright © 1993, American Society for Microbiology
Coryneform Bacteria Isolated from Middle Ear Fluid MICHEL
SIMONET,`* DOMINIQUE DE BRIEL,2 ISABELLE BOUCOT,1 RAYMOND MINCK,2
MICHEL VERON' Laboratoire de Microbiologie, Faculte de Medecine Necker-Enfants Malades, 75730 Paris,1 and Institut de Bacteriologie, Faculte de Medecine, 67000 Strasbourg, France AND
Received 22 December 1992/Accepted 25 March 1993
Nineteen strains of facultatively anaerobic gram-positive rods isolated in pure culture from middle ear fluids identified. All effusions were collected by tympanocentesis in children with acute otitis media. Identification of microorganisms to the genus level was done by studying the cell wall composition. Sixteen strains contain meso-diaminopimelic acid and arabinogalactan polymer but lack mycolic acids; therefore, these strains do not belong to a previously described taxon. Because of similarities with Corynebacterium afermentans (Centers for Disease Control group ANF-1), we temporarily classified these mycolateless strains ANF-1 like. Isolation of these microorganisms in pure culture from middle ear fluids collected by tympanocentesis is a strong argument for their involvement in acute otitis media.
were
Acute otitis media (AOM) is a very common pediatric infection. This infection is caused by various bacterial species which spread into the middle ear from the nasopharyngeal region. In his 1992 report, Bluestone (1) indicated that the following bacterial species were isolated from otorrhea: Streptococcus pneumoniae (35% of specimens), Haemophilus influenzae (23%), Moraxella catarrhalis (14%), alphahemolytic streptococci and group A streptococci (3% each), and Staphylococcus aureus and Pseudomonas aeruginosa (1% each). Alpha-hemolytic streptococci and P. aeruginosa, which were considered contaminants a few years ago, are now recognized as true pathogens. Bluestone also reported that miscellaneous bacteria, without any details on their identity, were recovered from 28% of middle ear effusions. Though speculative, this group might include some microorganisms of uncertain pathogenicity. Nineteen outpatients, i.e., 12 boys and 7 girls aged 5 to 28 months, living in the Paris area, were referred to 12 different otolaryngologists for evaluation of AOM. None of them had been given antibiotics prior to bacteriological analysis of middle ear fluids. In all cases, middle ear fluids were taken by tympanocentesis. Disinfection of the external canal ear and the tympanic membrane by an antiseptic, which is unpleasant for children with painful ears and not completely efficient (8), was not performed prior to tympanocentesis. In addition, we had previously found by studying middle ear fluids collected by the procedure described here that (i) 25% of specimens were sterile (10), a rate in agreement with those reported in the literature (1), and (ii) a single pathogenic bacterial species was recovered in more than >80% of positive cultures (10). Fluids were aspirated into a Teflon catheter fitted with a tuberculin syringe, and specimens were inoculated into a transport agar medium (Portagerm; Biomerieux, Marcy l'Etoile, France) which prevented any direct examination. Samples were plated onto 5% horse blood and chocolate agar (Biomerieux) and incubated in 5% CO2 atmosphere for 18 to 48 h (10). Nineteen strains of facultatively anaerobic gram-positive rods were isolated from nineteen specimens in pure culture. After recognition as coryneform bacteria by Gram staining, these isolates were identified to the genus level by determination of their cell *
Corresponding author.
wall composition according to "Section 15. Irregular, Nonsporing Gram-Positive Rods" of Bergey's Manual of Systematic Bacteriology (7). The presence of meso-diaminopimelic acid (meso-DAP) plus arabinogalactan polymer was detected by thin-layer chromatography (11), and mycolic acids were analyzed by high-performance liquid chromatography (3). The classical procedure of Hollis and Weaver (6), recommended by the Centers for Disease Control (CDC), was used for identification to the species level. Among the 19 isolates, we identified 2 as Corynebacterium pseudodiphtheriticum and 1 as Brevibacterium epidermidis. The remaining 16 isolates could not be classified; however, as reported in Table 1, these bacteria shared many phenotypic characteristics with C. afennentans subsp. afermentans (CDC group ANF-1). The reference strain LCDC 88-199, kindly provided by P. E. Ewan (Laboratory Centre for Disease Control, Ottawa, Ontario, Canada), and 11 strains of C. afermentans previously described (9) were used as controls. Like C. afermentans, these bacteria grew well under aerobic or microaerophilic conditions and weakly under anaerobic conditions. However, the following two major differences with this latter species were noted. (i) The unclassified coryneforms gave convex, whitish or creamish colonies of 1-mm diameter on blood agar after a 72-h incubation period at 37°C, whereas under the same culture conditions, C. afernentans is characterized by an overall pleomorphic appearance with flat, whitish or creamish centered opaque colonies of 1.5-mm diameter. (ii) The sixteen strains contain no mycolic acids, unlike C. afermentans. Hence, these bacteria cannot belong to the genus Corynebacterium, even though both arabinogalactan and mesoDAP are present in the cell wall. Thus, we propose to call these mycolateless nonfermentative coryneforms ANF-1 like. These coryneforms can be differentiated from C. afermentans by colony morphology on blood agar, the presence of a DNase (15 of 16 strains), and trypsin (16 of 16 strains) activity. Taxonomic aspects of coryneform CDC groups and their relationships with the genus Corynebacterium were recently reported (4). Group ANF-1 exhibits all phenotypic features consistent with the genus Corynebacterium and is characterized by a guanine plus cytosine (G+C) content ranging from 66 to 68%. This coryneform taxon group, defined temporarily by the CDC, is a new nonfermentative Corynebacte1667
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J. CLIN. MICROBIOL.
NOTES
TABLE 1. Phenotypic characteristics of C. afennentans and ANF-1-like strains
af ntrains) Test Test or characteristic stan) (11 stains)
.1
Colony morphology on blood agar Hemolysis Whitish pigment Motility at 25°C Catalase Oxidase Nitrate reduction Nutrient broth with 6.5% NaCl Acid produced from: Glucose Lactose Mannitol Trehalose Sucrose Galactose Maltose Mannose Xylose Voges-Proskauer Methyl red Urease Indole production DNase Gelatin hydrolysis Esculin hydrolysis Simmons citrate TSI slant/butt' H2S (TSI)
Flat
sANF-1-like strains (16 strains) Convex
We gratefully acknowledge H. Malnick from the Public Health Laboratory Service (London, United Kingdom), who has confirmed the phenotypic characteristics of ANF-1-like bacteria (strain FLO).
-I-
-I-
Enzyme production (API-ZYM): Alkaline phosphatase Esterase Esterase lipase Lipase Leucine arylamidase Valine arylamidase
Cystine arylamidase Trypsin a-Chymotrypsin Acid phosphatase
Phosphohydrolase ot-Galactosidase 1-Galactosidase 13-Glucuronidase ot-Glucosidase 1-Glucosidase
N-acetyl-3-glucosaminidase a-Mannosidase a-Fucosidase a
TSI, triple
sugar
hosts (2). Arcanobacterium (Cotynebacterium) haemolyticum is now recognized as a causative agent of pharyngitis in young immunocompetent adults (2). To the best of our knowledge, coryneforms have not been mentioned in the literature as microorganisms involved in AOM, probably because they are considered contaminants. In a 4-year bacteriological study of more than 1,100 patients with AOM, we isolated these microorganisms from middle ear effusions in approximately 5% of the patients (5). Because coryneforms were recovered in pure culture from fluids collected by tympanocentesis, we postulated that these bacteria are responsible for AOM. In all but three cases, bacteria belonging to an unknown genus were recovered. This homogeneity is a strong argument for the involvement of these microorganisms in AOM. Interestingly, of 32 ANF-1 strains referred to the CDC for identification, 8 have been isolated from the ear (6), but since the differential characteristics mentioned above have not been considered, it is not possible to assess the role of the typical or atypical ANF-1 bacteria at this site. Additional reports from other microbiologists are necessary to confirm our hypothesis. All isolates were susceptible by the disk method to amoxicillin plus clavulanate, which is one of the therapeutic options for AOM (1).
iron.
rium species with the proposed name C. afermnentans and two proposed subspecies (9). On the other hand, since ANF-1-like bacteria lack mycolic acids, they probably belong to an unidentified genus. After decades of confusion about their clinical significance, coryneforms have been recognized as opportunistic pathogens in humans. Several species, i.e., Corynebacterium jeikeium (CDC group JK), Corynebacterium urealyticum (CDC group D-2), C. pseudodiphtheriticum (C. hofinannii), Rhodococcus (Corynebacterium) equi, and Cotynebacterium ulcerans, are now known to cause diseases such as endocarditis, pneumonitis, and cutaneous and urinary tract infections, especially in immunocompromised
REFERENCES 1. Bluestone, C. D. 1992. Current therapy for otitis media and criteria for evaluation of new antimicrobial agents. Clin. Infect. Dis. 14(Suppl. 2):S197-S203. 2. Coyle, M. B., and B. J. Lipski. 1990. Coryneform bacteria in infectious diseases: clinical and laboratory aspects. Clin. Microbiol. Rev. 3:227-246. 3. De Briel, D., F. Couderc, P. Riegel, F. Jehl, and R. Minck. 1992. High-performance liquid chromatography of corynomycolic acids as a tool in identification of Corynebacterium species and related organisms. J. Clin. Microbiol. 30:1407-1417. 4. De Briel, D., P. Riegel, F. Jehl, and R. Minck. 1992. Taxonomics of coryneform CDC taxon groups, abstr. R-17, p. 291. Abstr. 92nd Gen. Meet. Am. Soc. Microbiol. American Society for Microbiology, Washington, D.C. 5. Gehanno, P., B. Cohen, P. Berche, and I. Boucot. 1991. Epidemiology of acute otitis media in children in France between 1987-1991. Recent advances in otitis media, abstr. 149. Proceedings of the 5th International Symposium of the Ohio State University and the Deafness Research Foundation, Fort Lauderdale, Fla. 6. Hollis, D. G., and R. E. Weaver. 1981. Gram-positive organisms: a guide to identification. Special Bacteriology Section, Centers for Disease Control, Atlanta. 7. Jones, D., and M. D. Collins. 1986. Irregular, nonsporing grampositive rods, p. 1261-1266. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams and Wilkins Co., Baltimore. 8. Odio, C. M., H. Kusmiesz, S. Sharon, and J. D. Nelson. 1985. Comparative treatment trial of augmentin versus cefaclor for acute otitis media with effusion. Pediatrics 75:819-826. 9. Riegel, P., D. De Briel, G. Prevost, F. Jehl, H. Monteil, and R. Minck. 1993. Taxonomic study of Corynebacterium group ANF-1 strains: proposal of Corynebacterium afermentans sp. nov. containing the subspecies C afermentans subsp. afermentans subsp. nov. and C. afermentans subsp. lipophilum subsp. nov. Int. J. Syst. Bacteriol. 43:287-292. 10. Simonet, M., M. Veron, and P. Gehanno. 1988. Etude bacteriologique de l'otite moyenne aigue chez l'enfant non hospitalise. Med. Mal. Infect. 10:445-451. 11. Staneck, J. L., and G. D. Roberts. 1974. Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography. Appl. Microbiol. 28:226-231.
