Neil Hunter

Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set

The design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by real-time PCR is reported. Broad specificity of the universal detection system was confirmed by testing DNA isolated from 34 bacterial species encompassing most of the groups of bacteria outlined in Bergeys Manual of Determinative Bacteriology. However, the nature of the chromosomal DNA used as a standard was critical. A DNA standard representing those bacteria most likely to predominate in a given habitat was important for a more accurate determination of total bacterial load due to variations in 16S rDNA copy number and the effect of generation time of the bacteria on this number, since rapid growth could result in multiple replication forks and hence, in effect, more than one copy of portions of the chromosome. The validity of applying these caveats to estimating bacterial load was confirmed by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical carious dentine samples. Taking these parameters into account, the number of anaerobic bacteria estimated by the universal probe and primers set in carious dentine was 40-fold greater than the total bacterial load detected by culture methods, demonstrating the utility of real-time PCR in the analysis of this environment.

Quantitative Analysis of Diverse Lactobacillus Species Present in Advanced Dental Caries

ABSTRACT Our previous analysis of 65 advanced dental caries lesions by traditional culture techniques indicated that lactobacilli were numerous in the advancing front of the progressive lesion. Production of organic acids by lactobacilli is considered to be important in causing decalcification of the dentinal matrix. The present study was undertaken to define more precisely the diversity of lactobacilli found in this environment and to quantify the major species and phylotypes relative to total load of lactobacilli by real-time PCR. Pooled DNA was amplified by PCR with Lactobacillus genus-specific primers for subsequent cloning, sequencing, and phylogenetic analysis. Based on 16S ribosomal DNA sequence comparisons, 18 different phylotypes of lactobacilli were detected, including strong representation of both novel and gastrointestinal phylotypes. Specific PCR primers were designed for nine prominent species, including Lactobacillus gasseri, L. ultunensis, L. salivarius, L. rhamnosus, L. casei, L. crispatus, L. delbrueckii, L. fermentum, and L. gallinarum. More than three different species were identified as being present in most of the dentine samples, confirming the widespread distribution and numerical importance of various Lactobacillus spp. in carious dentine. Quantification by real-time PCR revealed various proportions of the nine species colonizing carious dentine, with higher mean loads of L. gasseri and L. ultunensis than of the other prevalent species. The findings provide a basis for further characterization of the pathogenicity of Lactobacillus spp. in the context of extension of the carious lesion.

Quantitative Microbiological Study of Human Carious Dentine by Culture and Real-Time PCR: Association of Anaerobes with Histopathological Changes in Chronic Pulpitis

ABSTRACT The bacteria found in carious dentine were correlated with the tissue response of the dental pulps of 65 teeth extracted from patients with advanced caries and pulpitis. Standardized homogenates of carious dentine were plated onto selective and nonselective media under anaerobic and microaerophilic conditions. In addition, real-time PCR was used to quantify the recovery of anaerobic bacteria. Primers and fluorogenic probes were designed to detect the total anaerobic microbial load, the genera Prevotella and Fusobacterium, and the species Prevotella melaninogenica, Porphyromonas endodontalis, Porphyromonas gingivalis, and Micromonas (formerly Peptostreptococcus) micros. The pulpal pathology was categorized according to the cellular response and degenerative changes. Analysis of cultured bacteria showed a predominance of gram-positive microorganisms, particularly lactobacilli. Gram-negative bacteria were also present in significant numbers with Prevotella spp., the most numerous anaerobic group cultured. Real-time PCR analysis indicated a greater microbial load than that determined by colony counting. The total number of anaerobes detected was 41-fold greater by real-time PCR than by colony counting, while the numbers of Prevotella and Fusobacterium spp. detected were 82- and 2.4-fold greater by real-time PCR than by colony counting, respectively. Real-time PCR also identified M. micros, P. endodontalis, and P. gingivalis in 71, 60, and 52% of carious samples, respectively. Correlation matrices of the real-time PCR data revealed significant positive associations between M. micros and P. endodontalis detection and inflammatory degeneration of pulpal tissues. These anaerobes have been strongly implicated in endodontic infections that occur as sequelae to carious pulpitis. Accordingly, the data suggest that the presence of high levels of these bacteria in carious lesions may be indicative of irreversible pulpal pathology.

Molecular Analysis of Microbial Diversity in Advanced Caries

ABSTRACT Real-time PCR analysis of the total bacterial load in advanced carious lesions has shown that the total load exceeds the number of cultivable bacteria. This suggests that an unresolved complexity exists in bacteria associated with advanced caries. In this report, the profile of the microflora of carious dentine was explored by using DNA extracted from 10 lesions selected on the basis of comparable total microbial load and on the relative abundance of Prevotella spp. Using universal primers for the 16S rRNA gene, PCR amplicons were cloned, and approximately 100 transformants were processed for each lesion. Phylogenetic analysis of 942 edited sequences demonstrated the presence of 75 species or phylotypes in the 10 carious lesions. Up to 31 taxa were represented in each sample. A diverse array of lactobacilli were found to comprise 50% of the species, with prevotellae also abundant, comprising 15% of the species. Other taxa present in a number of lesions or occurring with high abundance included Selenomonas spp., Dialister spp., Fusobacterium nucleatum, Eubacterium spp., members of the Lachnospiraceae family, Olsenella spp., Bifidobacterium spp., Propionibacterium sp., and Pseudoramibacter alactolyticus. The mechanisms by which such diverse patterns of bacteria extend carious lesions, including the aspect of infection of the vital dental pulp, remain unclear.

Characteristics of a High Molecular Weight Extracellular Protein of Streptococcus mutans

A high molecular weight protein antigen, designated P1, has been isolated from the culture fluid of chemostat-grown Streptococcus mutans strain Ingbritt and shown to be free of other antigens including glucosyltransferase. Antiserum against the protein was used in rocket immunoelectrophoresis to confirm and extend the previous observation that there were major differences in the amount of the protein produced under different growth conditions. Physico-chemical and serological studies indicated that protein P1 was indistinguishable from antigens B, I/II and IF isolated in other laboratories. Mammalian tissue cross-reactivity of protein P1 was demonstrated by binding of antiserum to P1 to sections of normal rabbit tissues, particularly heart. There was also a statistically significant increase in the number of mononuclear leucocytes in heart tissue of rabbits which had been injected with protein P1, when compared with the levels in control uninjected rabbits; injection with whole cells of S. mutans Ingbritt did not produce this effect.

Measuring nasal bacterial load and its association with otitis media

BackgroundNasal colonisation with otitis media (OM) pathogens, particularly Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis, is a precursor to the onset of OM. Many children experience asymptomatic nasal carriage of these pathogens whereas others will progress to otitis media with effusion (OME) or suppurative OM. We observed a disparity in the prevalence of suppurative OM between Aboriginal children living in remote communities and non-Aboriginal children attending child-care centres; up to 60% and <1%, respectively. This could not be explained by the less dramatic difference in rates of carriage of respiratory bacterial pathogens (80% vs 50%, respectively). In this study, we measured nasal bacterial load to help explain the different propensity for suppurative OM in these two populations.MethodsQuantitative measures (colony counts and real-time quantitative PCR) of the respiratory pathogens S. pneumoniae, H. influenzae and M. catarrhalis, and total bacterial load were analysed in nasal swabs from Aboriginal children from remote communities, and non-Aboriginal children attending urban child-care centres.ResultsIn both populations nearly all swabs were positive for at least one of these respiratory pathogens. Using either quantification method, positive correlations between bacterial load and ear state (no OM, OME, or suppurative OM) were observed. This relationship held for single and combined bacterial respiratory pathogens, total bacterial load, and the proportion of respiratory pathogens to total bacterial load. Comparison of Aboriginal and non-Aboriginal children, all with a diagnosis of OME, demonstrated significantly higher loads of S. pneumoniae and M. catarrhalis in the Aboriginal group. The increased bacterial load despite similar clinical condition may predict persistence of middle ear effusions and progression to suppurative OM in the Aboriginal population. Our data also demonstrated the presence of PCR-detectable non-cultivable respiratory pathogens in 36% of nasal swabs. This may have implications for the pathogenesis of OM including persistence of infection despite aggressive therapies.ConclusionNasal bacterial load was significantly higher among Aboriginal children and may explain their increased risk of suppurative OM. It was also positively correlated with ear state. We believe that a reduction in bacterial load in high-risk populations may be required before dramatic reductions in OM can be achieved.

Hydrolysis of Interleukin-12 by Porphyromonas gingivalis Major Cysteine Proteinases May Affect Local Gamma Interferon Accumulation and the Th1 or Th2 T-Cell Phenotype in Periodontitis

ABSTRACT Porphyromonas gingivalis cysteine proteinases (gingipains) have been associated with virulence in destructive periodontitis, a disease process variously considered to represent an unregulated stimulation of either T helper type 1 (Th1)- or Th2-type cells. Critical in maintaining Th1 activity is the response of T lymphocytes to environmental interleukin 12 (IL-12) in the form of up-regulation of gamma interferon (IFN-γ) production. Here we demonstrate that in the presence or absence of serum, gingipains were able to hydrolyze IL-12 and reduce the IL-12-induced IFN-γ production from CD4+ T cells. However, the induction of IL-12 receptors on T cells by gingipains did not correlate with the enhancement of IFN-γ production. The gingipains cleaved IL-12 within the COOH-terminal region of the p40 and p35 subunit chains, which leads to IL-12 inactivity, whereas IL-2 in these assays was not affected. Inactivation of IL-12 by the gingipains could disrupt the cytokine balance or favor Th2 activities in the progression of periodontitis.

Failure of macrophage activation in destructive periodontal disease

Macrophage populations in 22 biopsies of untreated advanced periodontitis were compared with those in 26 biopsies of clinically healthy (minimally inflamed) gingival tissue. The immunohistochemical investigation used high specificity monoclonal antibodies, including a pan‐macrophage marker and probes for acute inflammatory, resident histiocytic, and reparative phenotypes. Macrophage expression of the functional activation markers MHC class II, basic fibroblast growth factor (bFGF), acid phosphatase (AP), and tartrate‐resistant acid phosphatase (TRAP) was also examined. The study showed that advanced periodontitis and minimally inflamed tissues displayed similar distribution patterns and numbers for the macrophage phenotypic markers: there were, however, regionally‐specific differences in the populations. In the advanced periodontitis lesion, there was little evidence of macrophage activation for the expression of HLA‐DR, bFGF, and TRAP, although strong expression of HLA‐DR and bFGF was observed in association with blood vessels. Macrophages expressing AP showed a distinct regional distribution; this, however, was not associated with foci of degenerate plasma cells. The apparent failure of recruitment and activation of macrophages may in part be both a cause and a consequence of the pathological features of this disease. Copyright

Expression patterns of E-cadherin, involucrin, and connexin gap junction proteins in the lining epithelia of inflamed gingiva

The structural integrity and functional differentiation of the lining epithelium were studied in relation to inflammatory changes associated with destructive periodontitis. In the different regions of lining epithelia from clinically healthy gingiva and periodontitis, comparisons were made of the expression patterns of E‐cadherin, which is critical in intercellular adhesion; of proteins associated with gap junction communication channels; and of involucrin, which is a key marker of differentiation in stratified epithelia. Filamentous actin (F‐actin), which is important in cell structural integrity, attachment, and migration, was also examined. Semiquantitative immunohistochemical analysis revealed that in both clinically healthy gingiva and lesions of advanced periodontitis, expression patterns of E‐cadherin, involucrin, and connexins 26 and 43 were similar, with a statistically significant reduction in staining intensity from the external oral epithelium, through the gingival sulcus, to the junctional epithelium or pocket epithelium, respectively. Furthermore, there was a striking reduction in staining for E‐cadherin, involucrin, and both connexins in the pathological lining epithelium of the periodontal pocket. These changes were associated with marked alterations of filamentous actin expression, collectively indicating profound perturbation of the epithelial structure. The data reported support the concept that the ability of the pathological lining epithelium to function as an effective barrier against the ingress of microbial products into the tissues is severely compromised. Copyright

Porphyrin-Mediated Cell Surface Heme Capture from Hemoglobin by Porphyromonas gingivalis

The porphyrin requirements for growth recovery of Porphyromonas gingivalis in heme-depleted cultures are investigated. In addition to physiologically relevant sources of heme, growth recovery is stimulated by a number of noniron porphyrins. These data demonstrate that, as for Haemophilus influenzae, reliance on captured iron and on exogenous porphyrin is manifest as an absolute growth requirement for heme. A number of outer membrane proteins including some gingipains contain the hemoglobin receptor (HA2) domain. In cell surface extracts, polypeptides derived from HA2-containing proteins predominated in hemoglobin binding. The in vitro porphyrin-binding properties of a recombinant HA2 domain were investigated and found to be iron independent. Porphyrins that differ from protoporphyrin IX in only the vinyl aspect of the tetrapyrrole ring show comparable effects in competing with hemoglobin for HA2 and facilitate growth recovery. For some porphyrins which differ from protoporphyrin IX at both propionic acid side chains, the modification is detrimental in both these assays. Correlations of porphyrin competition and growth recovery imply that the HA2 domain acts as a high-affinity hemophore at the cell surface to capture porphyrin from hemoglobin. While some proteins involved with heme capture bind directly to the iron center, the HA2 domain of P. gingivalis recognizes heme by a mechanism that is solely porphyrin mediated.