Emenike R.K. Eribe

Assessment of periradicular microbiota by DNA‐DNA hybridization

Abstract – In the present study the “checkerboard” DNA‐DNA hybridization technique was used to identify bacteria in periapical endodontic lesions of asymptomatic teeth. Thirty‐four patients with root‐filled teeth and apical periodontitis were divided into two groups, each containing 17 patients. In Group 1, a marginal incision was performed during surgery to expose the lesion, and in Group 2, a submarginal incision was applied. The gingiva and mucosa were swabbed with an 0.2% chlorhexidine gluconate solution prior to surgery. Bacterial DNA was identified in all samples from the two groups using 40 different whole genomic probes. The mean number (±SD) of species detected was 33.7±3.3 in Group 1 and 21.3±6.3 in Group 2 (P<0.001). The majority of the probe‐detected bacteria were present in more lesions from Group1 than from Group 2. The differences were most notable for Campylobacter gracilis, Porphyromonas endodontalis, Propionibacterium acnes, Capnocytophaga gingivalis, Fusobacterium nucleatum ssp. nucleatum, Fusobacterium nucleatum ssp. polymorphum, Prevotella intermedia, Treponema denticola, Streptococcus constellatus and Actinomyces naeslundii I. Bacterial species such as Actinobacillus actinomycetemcomitans and Bacteroides forsythus were detected in more than 60% of the lesions from both groups. Also, P. endodontalis was abundant in periapical tissue. The data supported the idea that following a marginal incision, bacteria from the periodontal pocket might reach the underlying tissues by surgeon‐released bacteremia. The study provided solid evidence that bacteria invade the periapical tissue of asymptomatic teeth with apical periodontitis. The detection of much more bacteria with the “checkerboard” DNA‐DNA hybridization method than has previously been recovered by anaerobic culture indicated that the endodontic (and periodontal) microfloras should be redefined using molecular methods.

Dysgonomonas gen. nov. to accommodate Dysgonomonas gadei sp. nov., an organism isolated from a human gall bladder, and Dysgonomonas capnocytophagoides (formerly CDC group DF-3)

Results of a polyphasic taxonomic study on an unknown Gram-negative, facultatively anaerobic, coccobacillus-shaped organism isolated from an infected human gall bladder are presented. Phenotypic and molecular taxonomic studies revealed the organism to be close to, but distinct from, organisms designated CDC (Centers for Disease Control and Prevention) group DF-3. The unknown bacterium was readily distinguished from reference strains of Bacteroides, Prevotella, Porphyromonas and related taxa by 16S rRNA gene sequencing, biochemical tests, analysis of cellular long-chain fatty acids and electrophoretic analysis of whole-cell proteins. Based on the results of the present study, it is proposed that the unknown bacterium be classified in a new genus, Dysgonomonas, as Dysgonomonas gadei sp. nov. (type strain CCUG 42882T = CIP 106420T). In addition, a new species, Dysgonomonas capnocytophagoides sp. nov., is proposed to accommodate strains previously belonging to CDC group DF-3. The type species of the genus Dysgonomonas is Dysgonomonas gadei.

Strain differentiation in Bacteroides fragilis by RAPD and Dendron computer-assisted gel analysis.

Bacteroides fragilis is the anaerobe most frequently isolated from human infections. Strains of this species are not easily distinguished by phenotypic tests. It is important to make this distinction because virulence may vary between strains and because B. fragilis seems to be a heterogeneous species. The aim of this study was to assess the utility of randomly amplified polymorphic DNA (RAPD) analysis for differentiation of 46 strains of B. fragilis. Twenty‐seven of the strains belonged to Johnsons DNA homology group I and 8 to group II, while 11 strains had not been assigned to any of these groups (NI group). The primers OPA16 and 18 were chosen among 30 primers tested for optimal RAPD analysis. OPA18 gave best discrimination, revealing a total of 15 genotypes while OPA16 gave 13. The gels obtained after RAPD analysis were evaluated with the Dendron computer‐assisted program. Most strains showed similarity levels (SAB) within 70%. Strain clusters thus established were not always in agreement with DNA homology since strains from both homology groups fell in the same cluster. Similarly, strains of the NI group fell among the group I and II homology strains. RAPD was useful for differentiation of B. fragilis strains and thus probably suitable for epidemiological studies. On the other hand, DNA‐DNA homology, comparing the entire genome of strains rather than a few random priming sites, would be more reliable for taxonomy. Computer‐assisted gel analysis made it possible to objectively evaluate multiple banding patterns, thereby increasing the reliability of the RAPD analysis.

Osteoradionecrosis contains a wide variety of cultivable and non-cultivable bacteria

Abstract Background: Direct microscopy, 1 1The chief editor, Ingar Olsen, has had no part in the review and decision process of this paper.anaerobic culture and DNA–DNA hybridization have previously demonstrated an association between microorganisms and osteoradionecrosis (ORN). The purpose of our study was to use culture independent molecular techniques to detect bacteria in necrotic bone lesions of the mandible after radiation therapy. Design: Bacterial DNA was extracted from eight deep medullar specimens from resected mandibles (six cases), including one patient with relapse. 16S rRNA genes were PCR amplified, cloned, transformed into Escherichia coli and sequenced to determine species identity and closest relatives. Results: From the analysis of 438 clones, 59 predominant species were detected, 27% of which have not been cultivated. The predominant species detected from radionecrotic mandibles were Campylobacter gracilis, Streptococcus intermedius, Peptostreptococcus sp. oral clone FG014, uncultured bacterium clone RL178, Fusobacterium nucleatum, and Prevotella spp. The study demonstrated intersubject variability of the bacteria present in ORN. In contrast to the diverse bacterial profile detected in primary infection, only a few members of the oral indigenous flora were identified from the relapse case. Conclusions: Diverse bacterial profiles in specimens of ORN in marrow spaces of the mandible were detected by culture independent molecular techniques. To better understand the pathogenesis and to improve the therapy of the infection, detection of all members of the complex bacterial flora associated with ORN is necessary.

Strain differentiation in Bacteroides fragilis by ribotyping and computer-assisted gel analysis

It is important to distinguish between Bacteroides fragilis strains as their virulence may vary and as B. fragilis seems to be a heterogeneous species. The aim of our study was to evaluate ribotyping for differentiation of 46 strains of B. fragilis and for assessment of strain heterogeneity within and between the two DNA‐DNA homology strain groups established in this species. Twenty‐seven strains belonged to Johnsons DNA homology group I and eight to group II. Eleven strains had not been assigned to any group (NI group). DNA from all strains was cut with BglI, EcoRI and HindIII. Restriction fragment length polymorphisms were investigated using a non‐radioactive digoxigenin‐labelled cDNA probe transcribed from Escherichia coli 16S+23S rRNA. Ribotyping with BglI was most discriminatory, revealing a total of 26 different patterns by visual inspection of gels. EcoRI followed with 20 patterns and HindIII with 13 patterns. The gels from ribotyping were processed using the Dendron computer‐assisted program. Strain clusters established using Dendron were not always in agreement with homology‐based strain groups. Strains of the NI group fell into both homology groups. Ribotyping, as it is based on a relatively small portion of the genome, is useful for strain distinction in epidemiological studies with B. fragilis, whereas DNA‐DNA homology, using the entire genome, is more reliable for taxonomy. The Dendron computer‐assisted program, which enabled objective assessment of multiple banding patterns, increased the reliability of ribotyping.

Leptotrichia species in human infections II

ABSTRACT Leptotrichia species are non-motile facultative anaerobic/anaerobic bacteria that are found mostly in the oral cavity and some other parts of the human body, in animals, and even in ocean sediments. Valid species include L. buccalis, L. goodfellowii, L. hofstadii, L. honkongensis, L. shahii, L. trevisanii, and L. wadei. Some species require serum or blood for growth. All species ferment carbohydrates and produce lactic acid that may be involved with tooth decay. Acting as opportunistic pathogens, they are involved in a variety of diseases, and have been isolated from immunocompromised but also immunocompetent individuals. Mucositis, oral lesions, wounds, and abscesses may predispose to Leptotrichia septicemia. Because identification of Leptotrichia species by phenotypic features occasionally lead to misidentification, genetic techniques such as 16S rRNA gene sequencing is recommended. Early diagnosis and treatment of leptotrichia infections is important for positive outcomes. Over the last years, Leptotrichia species have been associated with several changes in taxonomy and new associations with clinical diseases. Such changes are reported in this updated review.