Characterization of ear fluid isolates of Alloiococcus otitidis from patients with recurrent otitis media

Abstract

Nineteen isolates of Alloiococcus otitidis from ear fluid samples collected by tympanostomy from patients at four geographic locations were identified by phenotypic characterization and genetic relatedness. Initial growth of A. otitidis isolates occurred after 3 days at 37؇C on brain heart infusion (BHI) agar with 5% rabbit blood. Heavy growth occurred in BHI broth supplemented with 0.07% lecithin and 0.5% Tween 80 after 4 days of incubation. The isolates were gram-positive cocci that divided on an irregular plane and produced metabolic lactic acid, pyrrolidonyl arylamidase, and leucine aminopeptidase. These cocci grew sparsely in 6.5% NaCl-BHI broth, were asaccharolytic on both fermentative and oxidative bases, and were cytochrome negative by the iron-porphyrin test. The cellular fatty acid profile of A. otitidis was distinguished from those of related genera and characterized by major amounts (>14%) of 16:0, 18:2, 18:1 9c, and 18:0 and smaller amounts of 14:0, 16:1 7c, 17:0, and 18:1 7c. Fifteen isolates demonstrated >69% relatedness by DNA-DNA hybridization. Four isolates plus the original 15 were confirmed as A. otitidis by dot blot hybridization with a digoxigenin-labeled nucleotide probe specific for this species. The intergenic space between the genes coding for the 16S and 23S rRNAs of alloiococci was amplified by PCR, analyzed by restriction fragment length polymorphism, and determined to consist of three different genetic types. Although ␤-lactamase negative, A. otitidis demonstrated intermediate levels of resistance to beta-lactams, including expanded-spectrum cephalosporins, and were resistant to trimethoprim-sulfamethoxazole and erythromycin. Alloiococcus otitidis was first isolated from the ear fluid samples of children in a clinical study of chronic otitis media in Buffalo, N.Y. (10). Fifteen A. otitidis isolates were collected from 320 tympanocentesis fluid samples collected during effusive episodes of 200 patients with otitis media. In a study in Atlanta, Ga., of patients undergoing tympanostomy and tube placement, A. otitidis was isolated from 5 of 108 ear fluid samples collected from young children with chronic otitis media during 1991 and 1992 (20). The children had otalgia and middle ear effusion, with reddened, thickened, and retractive tympanic membranes. Two of the 5 Atlanta isolates and 10 of the 15 New York isolates were the only organisms found in the chronically effusive ear fluid samples, indicating a potential association between this bacterium and chronic effusive otitis. In 1992, British researchers designated this organism a new genus and species, A. otitis, on the basis of sequence analysis of the 16S rRNA (1). This nomenclature was revised to A. otitidis in keeping with the rules of the Bacteriological Code (21). This study characterizes 19 of these isolates from four geographic locations to (i) determine an adequate growth medium for cultivation, (ii) identify physiological characteristics, (iii) confirm the identities of these isolates by using DNA-DNA homology and a dot blot ...

Isolation ofAlloiococcus otitidisfrom Indigenous and non-Indigenous Australian children with chronic otitis media with effusion

“…These plates were then incubated for a further 7 days before being discarding as negative. Previous authors reported sparse growth using this broth after 3 days of incubation (Bosley et al, 1995) and positive results but an unspecified incubation time for this broth medium (Faden & Dryja, 1989).…”

“…All media were incubated for a total of 7 days in 7.5% (v/v) carbon dioxide at 35 1C. The incubation period was longer than those previously described for isola-tion of A. otitidis: 4 days (Bosley et al, 1995), 5 days (Faden & Dryja, 1989), 5 days (Stroman et al, 2001).…”

“…Antibiotic susceptibility testing by disc diffusion and by E-Test s followed the Clinical and Laboratory Standards Institute (CLSI) guidelines, previously the National Committee of Clinical Laboratory Standards (NCCLS). This method has not been calibrated for A. otitidis; therefore, the guidelines for S. pneumoniae reported by previous authors were used (Bosley et al, 1995). Other authors have not stated which guidelines were used to designate sensitivity or resistance (Miguel et al, 2000;Stroman et al, 2001).…”

“…During the last decade, Alloiococcus otitidis has been implicated as a cause of OME (Faden & Dryja, 1989;Bosley et al, 1995;Hendolin et al, 1999). Sequence analysis of the 16S rRNA gene led to the proposition of a new genus and species, Alloiococcus otitis (Aguirre & Collins, 1992), revised to A. otitidis (Von Graevenitz, 1993).…”

Interleukin‐8 Secretion of Human Epithelial and Monocytic Cell Lines Induced by Middle Ear Pathogens

“…However, in a recent study using PCR for the detection of microbes, A. otitidis was found in over 40% of child OME cases (3,11) and was more frequently detected than other common pathogens of otitis media (11). There have been reports of the isolation of Alloiococcus otitidis strains which were resistant to both erythromycin and trimethoprimsulfamethoxazole and relatively resistant to beta-lac- tams (4). Therefore, this new bacterial species, A. otitidis, should be examined to determine whether or not it has a pathogenetic role in the occurrence of otitis media.…”