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

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 degrees 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 ( > or = 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 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 beta-lactamase negative, A. otitidis demonstrated intermediate levels of resistance to beta-lactams, including expanded-spectrum cephalosporins, and were resistant to trimethoprim-sulfamethoxazole and erythromycin.

Heterogeneity of rRNA gene restriction patterns of multiresistant serotype 6B Streptococcus pneumoniae strains.

Three multiresistant serotype 6B Streptococcus pneumoniae strains were isolated from the middle ear fluids of children undergoing tympanostomy in Atlanta. Because multiresistant 6B pneumococci have been reported to spread from a single clone, the three isolates were compared with 13 other multiresistant 6B pneumococci by hybridization of endonuclease-restricted DNA fragments with a digoxigenin-labeled cDNA probe complementary to 16 and 23S rRNAs (ribotyping). The ear isolates were heterogeneous, whereas six of the other pneumococcal isolates were alike, indicating a need for additional studies to determine the possibility of clonal spread.

Phenotypic characterization, cellular fatty acid composition, and DNA relatedness of aerococci and comparison to related genera.

Aerococci can be misidentified as streptococci, enterococci, pediococci, lactococci, or leuconostocs. To distinguish the genus and determine if another species is needed in the present taxon, we analyzed 37 aerococci for cellular fatty acids and compared them with 377 strains of gram-positive cocci, including the species type strains from each of the related genera. The cellular fatty acid profile of aerococci was distinguishable from other genera. Two relatively novel fatty acids found in the aerococci were identified as C16:1 omega 9c and C16:1 omega 9t. Eleven strains of aerococci (including a strain originally identified as "Gaffkya" species) were chosen for DNA-DNA reassociation studies with the type strain Aerococcus viridans ATCC 11563; DNAs from eight of these strains were more than 75% related to the type strain and had 1 to 4% divergence in related sequences. The remaining three strains were 60 to 70% related to the type strain, had 7 to 11.5% divergence, and may represent a second species, Aerococcus genospecies 2. beta-Glucuronidase, alpha-galactosidase, and beta-galactosidase were useful in characterizing the aerococci.

Identification of monounsaturated fatty acids of Aerococcus viridans with dimethyl disulfide derivatives and combined gas chromatography-mass spectrometry.

Location of the double-bond position of monounsaturated fatty acids of Aerococcus viridans was accomplished by combined gas chromatography (GC)-mass spectrometry analysis of dimethyl disulfide (DMDS) derivatives. The monoenoic fatty acids from whole bacterial cells were converted to methyl esters and then to DMDS adducts and analyzed by capillary GC-mass spectrometry. The mass spectra of DMDS adducts gave an easily recognizable molecular ion (M+) and two major diagnostic ions attributable to fragmentation between the two CH3S groups located at the original site of unsaturation. Two relatively novel acids that distinguish aerococci from bacteria of closely related genera were identified as C16:1 omega 9c and C16:1 omega 9t from their mass spectrometry fragmentation patterns and retention characteristics on nonpolar capillary GC columns.

Separation of Haemophilus influenzae type b subtypes by numerical analysis.

Outer membrane proteins (OMPs) were prepared from 20 previously established subtypes of Haemophilus influenzae type b. Proteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (11% acrylamide). Each lane in the gels contained two internal molecular weight standards. One central lane contained a range of molecular weight standards, which were used to calculate a molecular weight curve for each gel. Migration distances of the OMPs were determined with a soft laser-scanning densitometer, and the distances were normalized by using the mean migration distances of the internal standards. The protein patterns of all subtypes were compared by a recently described method (B.D. Plikaytis, G.M. Carlone, and B.B. Plikaytis, J. Gen. Microbiol. 132:2653-2660, 1986). All subtypes could be differentiated by this method. The ability to store and compare numerous OMP patterns from different isolates of H. influenzae type b, separated with a single homogeneous polyacrylamide gel, will facilitate the continued development of a subtyping system based on these proteins.

Susceptibility of relatively penicillin-resistant Streptococcus pneumoniae to newer cephalosporin antibiotics.

Antimicrobial susceptibilities were performed at the Centers for Disease Control on 3400 Streptococcus pneumoniae isolates that were collected during a national survey of serotype-distribution of pneumococci found in normally sterile body fluids. The results showed 126 isolates (3.7%) to be relatively resistant to penicillin (RPR). The RPR strains were tested for susceptibility to cefuroxime, ceftriaxone, cefotaxime, cefamandole, cefaclor, ceftazidime, and moxalactam. These newer generation cephalosporin drugs were tested either because of their ability to penetrate into the cerebrospinal fluid (CSF) or for their activity against pneumococci. Three hundred ninety-one pneumococci were tested with 179 resistant to at least one antimicrobial. The RPR strains were not categorically resistant to the cephalosporins but were fourfold more resistant to them than were the penicillin-susceptible strains. The three most effective antimicrobials in the study for RPR were cefuroxime, cefotaxime, and ceftriaxone [corrected]. Each gave MICs that were attainable in CSF for RPR. Fifty percent of the RPR were inhibited by 0.06 mg/ml and 90% by 0.25 micrograms/ml of these antimicrobials. The least effective were cefaclor, moxalactam, and ceftazidime.

Comparative evaluation of the API 20S and AutoMicrobic gram-positive identification systems for non-beta-hemolytic streptococci and aerococci.

The API 20S system (Analytab Products, Plainview, N.Y.) and the AutoMicrobic Gram-Positive Identification system (GPI; Vitek Systems, Hazelwood, Mo.) were evaluated for their capacity to identify the non-beta-hemolytic streptococci and aerococci to the species level. The 20S system identified 86% (six of seven strains) of nonhemolytic group B streptococci, whereas 100% of the same group B streptococcal strains were correctly identified by the GPI system. With both systems 99% (134 of 135 strains) of four species of group D enterococcus strains and 92% (24 of 26 strains) of the Aerococcus spp. strains were identified. The 20S system identified 84% (41 of 49 strains) of three species of group D non-enterococcus strains. The GPI system identified 96% of the same group D non-enterococcus strains. The 20S system identified 84% (190 of 226 strains) of 10 species of viridans streptococci; however, supplemental conventional tests were required to identify 49% (110 of the 226 strains) of the viridans strains to the species level. The GPI system identified 79% of the same viridans streptococci without the need for supplemental tests. Both systems identified 84% (161 of 192 strains) of the seven most commonly occurring viridans Streptococcus spp. The 20S system identified 82% (75 of 92 strains) and the GPI system identified 84% (54 of 64 strains) of Streptococcus pneumoniae.

Rapid identification of enterococci.

A 4-h method was devised to differentiate the non-beta-hemolytic streptococci into three categories: enterococci, group D nonenterococci, and viridans streptococci. All of the Streptococcus faecalis, 90% of the Streptococcus faecium (enterococci), and 96% of the Streptococcus bovis biotype I (group D nonenterococci) cultures were correctly identified by the 4-h method. The less commonly isolated group D cultures had lower rates of correct identification by this method. None of the viridans streptococci was identified incorrectly.