TL;DR: Testing of the CSF pathogen and contaminant probes against DNA from over 60 different strains indicated that, with the exception of the coagulase-negative Staphylococcus probes, these probes provided the correct identification of bacterial species known to be found in CSF.
Abstract: A set of broad-range PCR primers for the 16S rRNA gene in bacteria were tested, along with three series of oligonucleotide probes to detect the PCR product. The first series of probes is broad in range and consists of a universal bacterial probe, a gram-positive probe, a Bacteroides-Flavobacterium probe, and two probes for other gram-negative species. The second series was designed to detect PCR products from seven major bacterial species or groups frequently causing meningitis: Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, Escherichia coli and other enteric bacteria, Listeria monocytogenes, and Staphylococcus aureus. The third series was designed for the detection of DNA from species or genera commonly considered potential contaminants of clinical samples, including cerebrospinal fluid (CSF): Bacillus, Corynebacterium, Propionibacterium, and coagulase-negative Staphylococcus spp. The primers amplified DNA from all 124 different species of bacteria tested. Southern hybridization testing of the broad-range probes with washes containing 3 M tetramethylammonium chloride indicated that this set of probes correctly identified all but two of the 102 bacterial species tested, the exceptions being Deinococcus radiopugnans and Gardnerella vaginalis. The gram-negative and gram-positive probes hybridized to isolates of two newly characterized bacteria, Alloiococcus otitis and Rochalimaea henselii, as predicted by Gram stain characteristics. The CSF pathogen and contaminant probe sequences were compared with available sequence information and with sequencing data for 32 different species. Testing of the CSF pathogen and contaminant probes against DNA from over 60 different strains indicated that, with the exception of the coagulase-negative Staphylococcus probes, these probes provided the correct identification of bacterial species known to be found in CSF.
TL;DR: It is suggested that the outer ear canal may serve as a reservoir for normally commensal microbes that can contribute to pathogenesis upon introduction into the middle ear.
Abstract: Molecular-phylogenetic sequence analyses have provided a new perspective on microbial communities by allowing the detection and identification of constituent microorganisms in the absence of cultivation. In this study we used broad-specificity amplification of ribosomal DNA (rDNA) genes to survey organisms present in the human outer ear canal. Samples were obtained from 24 individuals, including members of three extended families, in order to survey the resident microbiota and to examine microbial population structures in individuals related by familial or household associations. To examine the stability of the microbial populations, one individual was sampled four times and another twice over a 14-month period. We found that a distinct set of microbial types was present in the majority of the subjects sampled. The two most prevalent rDNA sequence types that were identified in multiple individuals corresponded closely to those of Alloiococcus otitis and Corynebacterium otitidis, commonly thought to be associated exclusively with infections of the middle ear. Our results suggest, therefore, that the outer ear canal may serve as a reservoir for normally commensal microbes that can contribute to pathogenesis upon introduction into the middle ear. Alternatively, culture analyses of diseases of the middle ear may have been confounded by these contaminating commensal organisms.
TL;DR: The partial 16S rRNA sequence of an unknown bacterium that was originally isolated from middle ear fluids of children with persistent otitis media was determined by reverse transcription and revealed that this bacterium represents a new line of descent.
Abstract: The partial 16S rRNA sequence of an unknown bacterium that was originally isolated from middle ear fluids of children with persistent otitis media was determined by reverse transcription. A comparison of this sequence with sequences from other gram-positive species having low guanine-plus-cytosine contents revealed that this bacterium represents a new line of descent, for which the name Alloiococcus otitis gen. nov., sp. nov., is proposed. The type strain is strain NCFB 2890.
TL;DR: The findings suggest a bacterial etiology for OME, and association of A. otitis with the three other species implies that this organism might have the capability of augmenting bacterial colonization in the middle ear.
Abstract: Background. The etiology of otitis media with effusion (OME) is unclear. Although the majority of effusions show inflammation, culture methods yield positive results for bacteria in only 20 to 30% of cases. Methods. The polymerase chain reaction was used for detection of three upper respiratory tract pathogens, Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae, and a fairly recently described bacterium, Alloiococcus otitis (A. otitidis), that is solely found in OME. The study included 67 middle ear effusions that were collected from 48 pediatric OME patients during ventilation tube placement. Results. PCR tested positive for 57 (85.1%) of the middle ear effusions. Thirty-one (46.3%) A. otitis-, 12 (17.9%) H. influenzae-, 25 (37.3%) M. catarrhalis- and 14 (20.9%) S. pneumoniae-positive effusions were obtained. All four study organisms showed similar distribution in effusions of various duration (P = 0.72) and in different effusion types (P = 0.59). Only the proportion of M. catarrhalis-positive effusions was lowered by recent antimicrobial therapy (P < 0.05). Although the study organisms had equal distributions among singly and multiply positive specimens (P = 0.90), A. otitis was detected significantly more often with one of the three other species (15 of 19, 78.9%) than the other species with each other (4 of 19, 21.1%, P < 0.001). Conclusions. The findings suggest a bacterial etiology for OME. Association of A. otitis with the three other species implies that this organism might have the capability of augmenting bacterial colonization in the middle ear.
TL;DR: Many bacterial species were detected by PCR, whereas with culture-based approaches, no bacterial growth was detected for ten of the 12 patients, so the method may have general usefulness in characterising bacterial populations at the site of infection.