[show abstract][hide abstract] ABSTRACT: In earlier studies we used molecular methods to identify the major bacterial consortia associated with advanced dentin caries. These consortia are dominated by bacteria from the families Lactobacillaceae, Streptococcaceae, Veillonellaceae (formerly Acidaminococcaceae), Eubacteriaceae, and Lachnospiraceae from the phylum Firmicutes; Coriobacteriaceae, Bifidobacteriaceae, and Propionibacteriaceae from the phylum Actinobacteria; and Prevotellaceae from the phylum Bacteroidetes, as well as fusobacteria. The phases of infection of vital pulp tissue by dentin microorganisms remain obscure. In the present study, fluorescence in situ hybridization was performed on sections of tissue embedded in resin. Probes for 16S rRNA corresponding to the major taxa of bacteria in carious dentin were used to provide information on the characteristics of pulp infection. Lactobacilli were prominent in 7 of 8 pulps determined to be at a limited stage of infection. Established infection (6 pulps) showed a more complex profile, with lactobacilli persisting in all of the lesions and with invasion of the necrotic regions of tissue by Bacteroidetes, fusobacteria, Lachnospiraceae, and Coriobacteriaceae in particular. Advanced infections (7 pulps) were characterized by mixed anaerobic species, with a strong representation by Coriobacteriaceae and Lachnospiraceae. Lactobacilli were not represented at this stage. Typically, groups of organisms were spatially isolated within the pulp tissue. Analysis indicated that lactobacilli could invade vital pulp tissue to achieve a very high biomass that was not associated with a detectable local inflammatory infiltrate. The findings establish that invasion of the dental pulp can be associated with a pronounced selection from the complex microbial populations within carious dentin, suggesting specific pathogenicity.
Journal of clinical microbiology 03/2010; 48(5):1732-40. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Marsupial mammals, born in an extremely atricial state with no functional immune system, offer a unique opportunity to investigate both the developing microbiome and its relationship to that of the mother and the potential influence of this microbiome upon the development of the immune system. In this study we used a well-established marsupial model animal, Macropus eugenii, the tammar wallaby, to document the microbiome of three related sites: the maternal pouch and saliva, and the gastrointestinal tract (GIT) of the young animal. We used molecular-based methods, targeting the 16S rDNA gene to determine the bacterial diversity at these study sites. In the maternal pouch, 41 unique phylotypes, predominantly belonging to the phylum Actinobacteria, were detected, while in the saliva, 48 unique phylotypes were found that predominantly belonged to the phylum Proteobacteria. The GIT of the pouch young had a complex microbiome of 53 unique phylotypes, even though the pouch young were still permanently attached to the teat and had only been exposed to the external environment for a few minutes immediately after birth while making their way from the birth canal to the maternal pouch. Of these 53 phylotypes, only nine were detected at maternal sites. Overall, the majority of bacteria isolated were novel species (<97 % identity to known 16S rDNA sequences), and each study site (i.e. maternal pouch and saliva, and the GIT of the pouch young) possessed its own unique microbiome.
[show abstract][hide abstract] ABSTRACT: A predominant kgp biovar colonized subgingival sites and buccal and tongue mucosa in 45 of 56 adults in an isolated community. The presence of biovars 381, W83, and W83v, but not HG66, correlated with the Porphyromonas gingivalis load at diseased sites. Biovars W83 and W83v poorly colonized tongue and buccal mucosa.
Journal of clinical microbiology 09/2009; 47(10):3350-2. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Methods for the optimal extraction of genomic DNA for real-time PCR enumeration of oral bacteria using the muramidase, mutanolysin, were developed using a simple in vitro oral flora model comprised of the facultative anaerobic gram-positive bacteria, Lactobacillus acidophilus and Streptococcus mutans, the gram-positive anaerobe, Parvimonas micra, and the gram-negative anaerobes, Porphyromonas gingivalis, Prevotella melaninogenica and Fusobacterium nucleatum. Traditional, as well as more elaborate, methods of quantifying bacterial numbers, including colony counting and estimation of DNA content using 4',6-diamino-2-phenylindole were compared in order to validate the real-time PCR approach. Evidence was obtained that P. gingivalis nuclease activity adversely affected the extraction of double-stranded DNA from this bacterium either alone or when it formed part of a consortium with the other bacteria. This nuclease activity could be overcome by treatment of the bacteria with either 20 mM diethyl pyrocarbonate or 70% ethanol at 4 degrees C overnight. A final purification of the DNA to remove any potential PCR inhibitors was added to the protocol in order to accurately quantify the amount of DNA by real-time PCR and hence the number of bacteria in a sample.
[show abstract][hide abstract] ABSTRACT: Cathelicidins are important components of the innate immune system and have been identified in skin and epithelia of a range of mammals. In this study molecular techniques, including RACE-PCR, were used to identify the full cDNA sequence of a cathelicidin gene, MaeuCath8, from the Australian marsupial, the tammar wallaby, Macropus eugenii. This cathelicidin was not homologous to other such genes previously isolated from a tammar wallaby mammary gland EST library, however, it did contain 4 conserved cysteine residues which characterise the pre-propeptide and had 80% identity with a previously isolated bandicoot cathelicidin. Reverse transcriptase-PCR established the expression profile of MaeuCath8 in a range of tissues, including spleen, thymus, gastrointestinal tract, skin and liver, of the tammar wallaby from birth to adulthood. Expression of MaeuCath8 was observed in spleen and gastrointestinal tract of newborn animals and was observed in most tissues by 7 days post-partum. The results indicate that pouch young could synthesize their own antimicrobial peptides from an early age suggesting that this ability most likely plays a role in protecting the pouch young from infection prior to the development of immunocompetence.
Veterinary Immunology and Immunopathology 12/2008; 127(3-4):269-76. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Antibodies to the human cathelicidin, hCAP18 have been used to examine epithelial tissues of adult and pouch young marsupials. Immunoreactivity was observed in skin, gastrointestinal tract, lung and mammary node of adults as well as skin, gastrointestinal tract, lung and bone marrow of pouch young. The locations of expression were similar to that reported in human tissues. Although the antibody to hCAP18 is primarily directed at the C-terminal antimicrobial peptide LL37, our observations suggest recognition of a common conserved element of this cathelicidin and lead us to conclude that the epithelial tissues of marsupials are sites of production of cathelicidin. This is consistent with observations in other mammals but is the first report of expression of these compounds in marsupials.
Tissue and Cell 08/2008; 40(6):459-66. · 1.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: The bacterial diversity of the openings of the urogenital and anal tracts of the adult female tammar wallaby, Macropus eugenii, was determined in order to ascertain whether the physical proximity of the openings of these tracts within the cloaca affected the two populations of bacteria. Terminal restriction fragment length polymorphism (T-RFLP) analyses of 42 wallabies identified 81 different terminal fragments, indicative of diverse and complex microbiomes at these anatomical locations. Subsequent amplified rDNA restriction analysis (ARDRA) identified 72 phylotypes from the urogenital tract and 50 from the anal tract. Twenty-two of these phylotypes were common to both tracts. Phylogenetic analysis of sequenced 16S rDNA showed that 83 % of the phylotypes were unidentified species based on the premise that any sequence possessing <97 % homology to a known bacterial species or phylotype was novel. Thus, despite the close proximity of the openings of the urogenital and anal tracts within the cloaca, the two sites retained a diverse range of distinct bacteria, with only a small percentage of overlapping species.
[show abstract][hide abstract] ABSTRACT: The crystal structure of GcnA, an N-acetyl-beta-D-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal alpha-helical domain has not been observed previously and forms a large dimerisation interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical (beta/alpha)(8) TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a family 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-beta-D-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.
Journal of Molecular Biology 04/2008; 377(1):104-16. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: For Indigenous Australian children living in remote communities, onset of otitis media commences within weeks of birth and is associated with early nasopharyngeal colonisation with multiple respiratory bacterial pathogens: Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The high prevalence of eardrum perforation and the failure of standard therapies to cure or prevent OM in this population require urgent attention. The objective of this study was to measure the changes in nasopharyngeal bacterial flora between birth and first episode of otitis media.
For 10 randomly selected Indigenous children with early onset otitis media, S. pneumoniae, H. influenzae, M. catarrhalis, S. aureus, and total bacterial load were enumerated in serial nasopharyngeal swabs using real-time quantitative PCR.
Between 0 and 3 weeks of age, all 10 infants had bilaterally normal ears. At 3-6 weeks of age, seven of eight infants examined had otitis media. By 6-13 weeks of age, all 10 infants had otitis media. The relative density of respiratory pathogens among total nasopharyngeal flora increased significantly with onset of otitis media, and the majority of children became colonised with the three respiratory pathogens. There was no association between OM onset and S. aureus load.
Onset of otitis media between 3 and 6 weeks of life was associated with a significant increase in all major bacterial OM pathogens (S. pneumoniae, H. influenzae, M. catarrhalis), as well as total bacterial load in the nasopharynx. Interventions to prevent acquisition of multiple OM pathogens in the first weeks of life are needed.
International Journal of Pediatric Otorhinolaryngology 02/2008; 72(1):57-61. · 1.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Selective culture of human carious dentine for Veillonella strains resulted in the isolation of two strains of a Gram-negative, coccus-shaped bacterium that has not been described previously. Comparative 16S rRNA and dnaK gene sequence analysis indicated that the two strains were homogeneous and comprised a distinct lineage within the genus Veillonella, phylogenetically most closely related to Veillonella rodentium. This was supported by DNA-DNA hybridization, which showed clearly that the two strains were similar and distinct from other Veillonella species, and the production of major cellular fatty acids (C(13 : 0) and C(17 : 1)omega8), which is consistent with other members of the genus Veillonella. Based on these observations, strains RBV81 and RBV106(T) represent a novel species, for which the name Veillonella denticariosi sp. nov. is proposed, with the type strain RBV106(T) (=CIP 109448(T) =CCUG 54362(T) =DSM 19009(T)).
International journal of systematic and evolutionary microbiology 01/2008; 57(Pt 12):2844-8. · 2.11 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nasal 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.
Quantitative 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.
In 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.
Nasal 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.
[show abstract][hide abstract] ABSTRACT: Mature biofilm and planktonic cells of Streptococcus mutans cultured in a neutral pH environment were subjected to comparative proteome analysis. Of the 242 protein spots identified, 48 were significantly altered in their level of expression (P<0.050) or were unique to planktonic or biofilm-grown cells. Among these were four hypothetical proteins as well as proteins known to be associated with the maintenance of competence or found to possess a cin-box-like element upstream of their coding gene. Most notable among the non-responsive genes were those encoding the molecular chaperones DnaK, GroEL and GroES, which are considered to be up-regulated by sessile growth. Analysis of the rest of the proteome indicated that a number of cellular functions associated with carbon uptake and cell division were down-regulated. The data obtained were consistent with the hypothesis that a reduction in the general growth rate of mature biofilms of S. mutans in a neutral pH environment is associated with the maintenance of transformation without the concomitant stress response observed during the transient state of competence in bacterial batch cultures.
[show abstract][hide abstract] 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.
Journal of Clinical Microbiology 03/2005; 43(2):843-9. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Compared with traditional two-dimensional (2D) proteome analysis of Streptococcus mutans grown as a biofilm from a planktonic culture at steady state (Rathsam et al., Microbiol. 2005, 151, 1823-1837), the use of 2D fluorescence difference gel electrophoresis (DIGE) led to a 3-fold increase in the number of identified protein spots that were significantly altered in their level of expression (P < 0.050). Of the 73 identified proteins, only nine were up-regulated in biofilm grown cells. The results supported the previously surmised hypothesis that general metabolic functions were down-regulated in response to a reduction in growth rate in mature S. mutans biofilms. Up-regulation of competence proteins without any concomitant increase in stress-responsive proteins was confirmed, while the levels of glucosyltransferase C (GtfC), involved in glucan formation, O-acetylserine sulfhyrylase (cysteine synthetase A; CsyK), implicated in the formation of [Fe-S] clusters, and a hypothetical protein encoded by the open reading frame, SMu0188, were also up-regulated.
Journal of Proteome Research 01/2005; 4(6):2161-73. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Previous analysis of the microbiology of advanced caries by culture and real-time PCR emphasized the high incidence and abundance of gram-negative anaerobic species, particularly Prevotella-like bacteria. The diversity of Prevotella-like bacteria was further explored by analyzing pooled bacterial DNA from lesions of carious dentine. This was achieved by amplification of a region of the 16S ribosomal DNA with a Prevotella genus-specific forward primer and a universal bacterial reverse primer, followed by cloning and sequencing. Cultured Prevotella species commonly associated with oral tissues constituted only 12% of the Prevotella clones isolated from advanced carious lesions. The remaining 88% consisted of a diverse range of phylotypes. These included five clusters of previously recognized but uncultured oral Prevotella spp. and a major cluster containing Prevotella-like bacteria most closely related to uncharacterized rumen bacteria. Cluster-specific primers were designed, and the numbers of bacteria within clusters were quantified by real-time PCR, confirming the abundance of these organisms. The data indicated that advanced dental caries provides a unique environment for a complex array of novel and uncultured Prevotella and Prevotella-like bacteria which, in some cases, may dominate the diverse polymicrobial community associated with the disease.
Journal of Clinical Microbiology 12/2004; 42(11):5238-44. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Paired subgingival plaque samples representing the most-diseased and least-diseased sites were collected from 34 adult patients with diagnosed chronic periodontitis. The percentage of Porphyromonas gingivalis relative to the total anaerobic and gram-negative bacterial load at each site was determined by real-time PCR. Based on variations in the noncatalytic C terminus of the Lys-gingipain (Kgp), it was reasoned that DNA sequence variation in the 3'-coding region of the kgp gene might determine functional biotypes. Perusal of the available sequence information in GenBank indicated three such forms of the kgp gene corresponding to P. gingivalis strains HG66, 381, and W83. Analysis of patient samples revealed the presence of a fourth genotype (W83v) that showed duplication of a sequence recognized by the W83 reverse primer. The four biotypes, HG66, 381, W83, and W83v, were present in the study group in the ratio 8:11:6:5, respectively. Each subject was colonized by one predominant biotype, and only three patients were colonized by a trace amount of a second biotype.
Journal of Clinical Microbiology 09/2004; 42(8):3873-6. · 4.07 Impact Factor
[show abstract][hide abstract] 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.
Journal of Clinical Microbiology 08/2004; 42(7):3128-36. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: An N-acetyl-beta-D-glucosaminidase (GcnA) from Streptococcus gordonii FSS2 was cloned and sequenced. GcnA had a deduced molecular mass of 72,120 Da. The molecular weight after gel-filtration chromatography was 140,000 Da and by SDS-PAGE was 70,000 Da, indicating that the native protein was a homodimer. The deduced amino acid sequence had significant homology to a glycosyl hydrolase from Streptococcus pneumoniae and the conserved catalytic domain of the Family 20 glycosyl hydrolases. GcnA catalysed the hydrolysis of the synthetic substrates, 4-methylumbelliferyl (4MU)-N-acetyl-beta-D-glucosaminide, 4MU-N-acetyl-beta-D-galactosaminide, 4-MU-beta-D-N,N'-diacetylchitobioside, and 4-MU-beta-D-N,N',N''-chitotrioside as well as the respective chito-oligosaccharides. GcnA was optimally active at pH 6.6 and 42 degrees C. The Km for 4-MU-beta-D-N,N',N''-chitotrioside, 45 microM, was the lowest for all the substrates tested. Hg2+, Cu2+, Fe2+, and Zn2+ completely inhibited while Co2+, Mn2+, and Ni2+ partially inhibited activity. S. gordonii FSS2 and a GcnA negative mutant grew equally well on chito-oligosaccharides as substrates. The S. gordonii sequencing projects indicate two further N-acetyl-beta-D-glucosaminidase activities.
Biochemical and Biophysical Research Communications 07/2004; 319(2):439-47. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Streptococcus mutans is an important pathogen in the initiation of dental caries as the bacterium remains metabolically active when the environment becomes acidic. The mechanisms underlying this ability to survive and proliferate at low pH remain an area of intense investigation. Differential two-dimensional electrophoretic proteome analysis of S. mutans grown at steady state in continuous culture at pH 7.0 or pH 5.0 enabled the resolution of 199 cellular and extracellular protein spots with altered levels of expression. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified 167 of these protein spots. Sixty-one were associated with stress-responsive pathways involved in DNA replication, transcription, translation, protein folding and proteolysis. The 61 protein spots represented isoforms or cleavage products of 30 different proteins, of which 25 were either upregulated or uniquely expressed during acid-tolerant growth at pH 5.0. Among the unique and upregulated proteins were five that have not been previously identified as being associated with acid tolerance in S. mutans and/or which have not been studied in any detail in oral streptococci. These were the single-stranded DNA-binding protein, Ssb, the transcription elongation factor, GreA, the RNA exonuclease, polyribonucleotide nucleotidyltransferase (PnpA), and two proteinases, the ATP-binding subunit, ClpL, of the Clp family of proteinases and a proteinase encoded by the pep gene family with properties similar to the dipeptidase, PepD, of Lactobacillus helveticus. The identification of these and other differentially expressed proteins associated with an acid-tolerant-growth phenotype provides new information on targets for mutagenic studies that will allow the future assessment of their physiological significance in the survival and proliferation of S. mutans in low pH environments.
[show abstract][hide abstract] ABSTRACT: Two-dimensional gel electrophoretic analysis of the proteome of Streptococcus mutans grown at a steady state in a glucose-limited anaerobic continuous culture revealed a number of proteins that were differentially expressed when the growth pH was lowered from pH 7.0 to pH 5.0. Changes in the expression of metabolic proteins were generally limited to three biochemical pathways: glycolysis, alternative acid production and branched-chain amino acid biosynthesis. The relative level of expression of protein spots representing all of the enzymes associated with the Embden-Meyerhof-Parnas pathway, and all but one of the enzymes involved in the major alternative acid fermentation pathways of S. mutans, was identified and measured. Proteome data, in conjunction with end-product and cell-yield analyses, were consistent with a phenotypic change that allowed S. mutans to proliferate at low pH by expending energy to extrude excess H(+) from the cell, while minimizing the detrimental effects that result from the uncoupling of carbon flux from catabolism and the consequent imbalance in NADH and pyruvate production. The changes in enzyme levels were consistent with a reduction in the formation of the strongest acid, formic acid, which was a consequence of the diversion of pyruvate to both lactate and branched-chain amino acid production when S. mutans was cultivated in an acidic environment.