Ellen V. G. Frandsen

Biological significance of IgA1 proteases in bacterial colonization and pathogenesis: critical evaluation of experimental evidence*

IgA1 protease activity, which allows bacteria to cleave human IgA1 in the hinge region, represents a striking example of convergent evolution of a specific property in bacteria. Although it has been known since 1979 that IgA1 protease is produced by the three leading causes of bacterial meningitis in addition to important urogenital pathogens and some members of the oropharyngeal flora, the exact role of this enzyme in bacterial pathogenesis is still incompletely understood owing to lack of a satisfactory animal model. Cleavage of IgA1 by these post‐proline endopeptidases efficiently separates the monomeric antigen‐binding fragments from the secondary effector functions of the IgA1 antibody molecule. Several in vivo and in vitro observations indicate that the enzymes are important for the ability of bacteria to colonize mucosal membranes in the presence of S‐IgA antibodies. Furthermore, the extensive cleavage of IgA sometimes observed in vivo, suggests that IgA1 protease activity results in a local functional IgA deficiency that may facilitate colonization of other microorganisms and the penetration of potential allergens. It has been hypothesized that IgA1 protease activity of Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae, under special immunological circumstances, allows these bacteria to take advantage of specific IgA1 antibodies in a strategy to evade other immune factors of the human body. The decisive factor is the balance between IgA antibodies against surface antigens of the respective bacteria and their IgA1 protease. Recent studies have shown that serine‐type IgA1 proteases of H. influenzae, meningococci, and gonococci belong to a family of proteins used by a diverse group of Gramnegative bacteria for colonization and invasion.

Evidence of recombination in Porphyromonas gingivalis and random distribution of putative virulence markers.

ABSTRACT The association of Porphyromonas gingivalis to periodontal disease is not clearly understood. Similar proportions ofP. gingivalis may be cultivated from both inactive and actively degrading periodontal pockets. Differences in virulence among strains of P. gingivalis exist, but the molecular reason for this remains unknown. We examined the population structure ofP. gingivalis to obtain a framework in which to study pathogenicity in relation to evolution. Phylogenetic trees derived from the sequencing of fragments of four housekeeping genes, ahp, thy, rmlB, and infB, in 57 strains were completely different with no correlation between clustering of strains in the four dendrograms. Combining the various alleles of the four gene fragments sequenced resulted in 41 different sequence types. The index of association, IA, based on a single representative of each sequence type was 0.143 ± 0.202, indicating a population at linkage equilibrium. Inclusion of all isolates for the calculation of IA resulted in a value of 0.206 ± 0.171. This suggests an epidemic population structure supported by the finding of genetically identical strains in different parts of the world. We observed a random distribution of two virulence-associated mobile genetic elements, the ragB locus and the insertion sequence IS1598, among 132 strains tested. In conclusion, P. gingivalis has a nonclonal population structure characterized by frequent recombination. Our study suggests that particular genotypes, possibly with increased pathogenic potential, may spread successfully in the human population.

Confirmation of the Species Prevotella intermedia and Prevotella nigrescens

The elevation of the two genotypes of Prevotella intermedia to species rank as P. intermedia and Prevotella nigrescens has increased the need for reliable differentiation between the two taxa. In this study, 53 strains, including strains whose species affiliations were known as well as fresh dental plaque isolates, were subjected to a multilocus enzyme electrophoretic analysis, DNA analyses in which we used whole genomic DNA, rRNA sequences, and an oligonucleotide specific for the former P. intermedia genotype II (P. nigrescens) as probes, and a sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of soluble cellular proteins. All of these tests consistently separated the strains into the same two distinct groups corresponding to P. intermedia and P. nigrescens, confirming that the two species constitute two distinct populations of bacteria. Each of the tests used independently provided reliable identification to the species level. A previously reported heterogeneity in the pattern of human immunoglobulin A1 (IgA1) degradation was not confirmed. No differences between species were observed. All of the strains induced total degradation of IgA1 within 48 h, a property that may be a virulence factor in periodontal disease development. The enzymes responsible for IgA1 degradation were not inactivated by the physiological proteinase inhibitors alpha 2-macroglobulin and alpha 1-proteinase inhibitor.

Phylogenetic characterization and proposal of a new pigmented species to the genus Prevotella: Prevotella pallens sp.nov.

Complete 16S rRNA gene sequences of three representative strains of anaerobic, Gram-negative, pigmented, moderately saccharolytic, indole-positive bacteria isolated from the oral cavity of humans were determined. According to comparative analyses of the rRNA sequence data, this organism represents a previously unknown species within the genus Prevotella. In addition, 22 representative strains and 21 reference strains (including 11 Prevotella intermedia and 10 Prevotella nigrescens strains) were subjected to multilocus enzyme electrophoretic analysis. The strains were consistently separated into three clearly distinct groups, corresponding to their previous entities. On the basis of the present phylogenetic results that confirmed our biochemical and genetic data, we propose a new species, Prevotella pallens. The type strain is NCTC 13042 (= AHN 10371).

Endodontic photoactivated disinfection using a conventional light source: an in vitro and ex vivo study.

OBJECTIVE The antimicrobial effect of photoactivated disinfection (PAD) using toluidine blue and an LED lamp was tested on endodontic pathogens in planktonic suspension and after inoculation into extracted teeth. Irradiation time was limited to 30 seconds. STUDY DESIGN The effect of PAD on planktonic suspensions of Escherichia coli, Candida albicans, Enterococcus faecalis, Fusobacterium nucleatum, and Streptococcus intermedius was analyzed using Poisson regression. Moreover, cultures of S. intermedius were inoculated into prepared root canals of extracted molars. The effect of PAD performed immediately after inoculation or after overnight bacterial incubation was determined by a 2-sample t test. RESULTS Photoactivated disinfection yielded significant reductions (P < .001) in the viable counts of all organisms in planktonic suspension. The PAD treatment of S. intermedius in root canals yielded a mean log10 reduction of 2.60 (P < .001) immediately after inoculation and of 1.38 (P < .001) after overnight incubation. CONCLUSION Photoactivated disinfection using a conventional light source strongly reduces the number of viable endodontic pathogens in planktonic suspension and in root canals.

Diversity of Capnocytophaga species in children and description of Capnocytophaga leadbetteri sp. nov. and Capnocytophaga genospecies AHN8471

Bacteria of the genus Capnocytophaga form part of the resident oral flora in children and adults. They are recognized as opportunistic pathogens of various extra-oral infections. The significance of individual species in periodontal and extra-oral diseases is unclear, due to the inability of conventional phenotypic tests to identify clinical isolates to species level. Aiming at a clear distinction between species, we undertook a phylogenetic study of a collection of 102 Capnocytophaga strains including 62 oral isolates from children and 40 reference strains from oral and extra-oral infections representing the five known, human, oral Capnocytophaga species. The phylogeny was estimated on the basis of multilocus enzyme electrophoresis (MLEE) of 12 intracellular, housekeeping enzymes and by partial 16S rRNA gene sequencing, and was compared to phenotypic characteristics. The clustering profiles in the MLEE and sequence-based dendrograms were concordant and allowed identification of isolates to species level, based on co-clustering with reference strains. The study confirmed Capnocytophaga ochracea and Capnocytophaga sputigena as separate species, and underlined the problems of distinguishing between them by conventional phenotypic tests. The presence of two distinct clusters of oral isolates from children indicated the existence of novel species, supported by analysis of near-full-length 16S rRNA gene sequences and by DNA-DNA hybridization results. One cluster of weakly saccharolytic isolates without the ability to ferment sucrose is proposed as Capnocytophaga leadbetteri sp. nov. (type strain AHN8855(T)=CCUG 51857(T)=NCTC 13375(T)). Another cluster not phenotypically distinguishable from C. ochracea and C. sputigena is designated Capnocytophaga genospecies AHN8471 (represented by strain AHN8471=CCUG 51856=NCTC 13374).

Biochemical and genetic characterization of a Prevotella intermedia/nigrescens-like organism.

Thirty-three previously non-typable faintly pigmented Gram-negative anaerobic bacterial isolates, biochemically most closely related to Prevotella intermedia and Prevotella nigrescens, were analysed for enzymic reactions, cellular fatty acid (CFA) composition, electrophoretic mobility of malate and glutamate dehydrogenases, hybridization with P. intermedia and P. nigrescens species-specific oligonucleotide probes and, for genetic heterogeneity, by arbitrarily primed PCR (AP-PCR). P. intermedia ATCC 25611T and P. nigrescens ATCC 33563T were run in parallel for comparison. Twenty-nine isolates originated from the normal oral flora of 18 subjects (including five mother-child pairs), and four isolates from various infections. Except for a negative lipase reaction, enzymic profiles of the test isolates were similar to those of P. intermedia and P. nigrescens. Clustering of CFAs, electrophoretic mobility patterns, hybridization with DNA probes for P. intermedia and P. nigrescens, and AP-PCR band patterns of the test isolates differed from those of the type strains of P. intermedia and P. nigrescens, suggesting the existence, in humans, of a new anaerobic species of pigmented, moderately saccharolytic, indole-positive Gram-negative rods.