CHARACTERIZATION OF EAR FLUID ISOLATES OF ALLOIOCOCCUS-OTITIDIS FROM PATIENTS WITH RECURRENT OTITIS-MEDIA

Nineteen isolates of Alloiococcus otitidis from ear fluid samples coll ected by tympanostomy from patients at four geographic locations were identified by phenotypic characterization and genetic relatedness, Ini tial growth of A. otitidis isolates occurred after 3 days at 37 degree s C on brain heart infusion (BHI) agar with 5% rabbit blood, Heavy gro wth occurred in BHI broth supplemented with 0.07% lecithin and 9.5% Tw een 80 after 4 days of incubation, The isolates were gram-positive coc ci that divided on an irregular plane and produced metabolic lactic ac id, pyrrolidonyl arylamidase, and leucine aminopeptidase. These cocci grew sparsely in 6.5% NaCl-BHI broth, were asaccharolytic on both ferm entative and oxidative bases, and were cytochrome negative by the iron -porphyrin test, The cellular fatty acid profile of A. otitidis was di stinguished from those of related genera and characterized by major am ounts (greater than or equal to 14%) of 16:0, 18:2, 18:1 omega 9c, and 18:0 and smaller amounts of 14:0, 16:1 omega 7c, 17:0, and 18:1 omega 7c. Fifteen isolates demonstrated >69% relatedness by DNA-DNA hybridi zation., Four isolates plus the original 15 were confirmed as A. otiti dis by dot blot hybridization with a digoxigenin-labeled nucleotide pr obe 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 t o consist of three different genetic types, Although beta-lactamase ne gative, A, otitidis demonstrated intermediate levels of resistance to beta-lactams, including expanded-spectrum cephalosporins, and were res istant to trimethoprim-sulfamethoxazole and erythromycin.