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Biomedical subjects

P W Caufield

Publications and source records attributed to P W Caufield.

At least 19 recordsLinked to original sources

Diversity of lactobacilli in the oral cavities of young women with dental caries.

For nearly a century, lactobacilli (LB) in the oral cavity have been generally associated with dental caries. Here, we characterized the LB isolated from the saliva of 6 women with active caries using genetic-based taxonomical identification methods. From each subject, 30 isolates growing on Rogosa medium and presumed to be LB were analyzed. Of the 180 isolates, 176 were further characterized by biotyping, DNA melting points, DNA chromosomal fingerprinting, genotyping, and phylogenetic cluster assessment. We found a total of 30 unique genotypes of LB in the saliva of caries-active women, with each woman harboring between 2 and 8 distinct genotypes. Although Lactobacillus vaginalis, L. fermentum, and L. salivarius were found in 4 of 6 of the subjects, results from other studies using comparable methods show an entirely different array of LB associated with caries. These collective observations lead us to surmise that LB associated with dental caries are likely exogenous and opportunistic colonizers, arising from food or other reservoirs outside the oral cavity.

Adolescent↗

Genetic profiling of the oral microbiota associated with severe early-childhood caries.

The determination of the composition of the microbial community in the oral cavity is usually based on cultivation methods; however, nearly half of the bacteria in the saliva and the dental plaque are not cultivable. In this study, we evaluated the difference in oral microbial diversity between children with severe early-childhood caries (S-ECC) and caries-free (CF) controls by means of a cultivation-independent approach called denaturing gradient gel electrophoresis (DGGE). Pooled dental plaque samples were collected from 20 children aged 2 to 8 years. Total microbial genomic DNA was isolated from those subjects, and a portion of the 16S rRNA gene locus was PCR amplified by using universal primers. We observed that the mean species richness of the bacterial population was greater in the CF children (n = 12) (42 +/- 3.7) than in the S-ECC children (n = 8) (35 +/- 4.3); the difference was statistically significant (P = 0.005). The overall diversity of plaque samples as measured by the Shannon index was 3.5 for the S-ECC group and 3.7 for the CF group (P = 0.004). Differences in DGGE profiles were distinguished on the basis of a cluster analysis. Sequence analysis of excised DGGE bands consisted of 2.7 phylotypes, on average. After adjusting for the number of observed bands, we estimated that the S-ECC group exhibited 94.5 total phylotypes and that the CF group exhibited 113.4. These results suggest that the microbial diversity and complexity of the microbial biota in dental plaque are significantly less in S-ECC children than in CF children.

Bacterial Typing Techniques↗

Survey of oral microbial diversity using PCR-based denaturing gradient gel electrophoresis.

Polymicrobial biofilms in the human oral cavity exhibit marked diversity. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) surveys microbial diversity by displaying PCR-generated 16S rDNA fragments that migrate at different distances, reflecting the differences in the base-pair (i.e., % G+C) composition of the fragment. This study examined DGGE-generated diversity profiles of cultivable bacteria from individuals with different caries status. Initially, we developed a set of PCR-DGGE running conditions appropriate to oral bacteria. Next, we assessed migration standards from known oral bacterial reference strains. To test the methods, we profiled 20 bacterial saliva samples cultivated from young adults. The study produced a battery of species-specific 16S rDNA amplicons that could be used as a migration distance standard necessary for computer-assisted profile analysis. From the clinical samples, we found a significantly greater diversity of oral microbes in caries-free individuals compared with caries-active individuals (P = 0.01). These findings suggest thtat a portion of oral microbiota of caries-active individuals may be absent, suppressed, or replaced.

Actinomyces↗

Mode of delivery and other maternal factors influence the acquisition of Streptococcus mutans in infants.

S. mutans plays a key role in dental caries. The extent to which perinatal events influence the acquisition of S. mutans is unclear. We hypothesized that several maternal factors, including the mode of delivery, influence the initial acquisition of S. mutans in infants. A prospective cohort study was conducted in 156 mother-infant pairs. The study found that maternal gestational age (p = 0.04), S. mutans level (p = 0.02), caries score (p = 0.02), sexually transmitted disease (STD) infection experience (p = 0.01), and family income (p = 0.03) had significant effects on the acquisition of S. mutans. Among infants who became infected, those delivered by Caesarean section acquired S. mutans 11.7 mos earlier than did vaginally delivered infants (p = 0.038). C-section infants harbored a single genotype of S. mutans that was identical to that of their mothers (100% fidelity). Analysis of the data demonstrated the possible perinatal influences on infants' acquisition of a member of the cariogenic microbiota, and its potential effect on caries outcome.

Analysis of Variance↗

Genetic diversity of Actinomyces naeslundii genospecies 2 in mother-child pairs.

Actinomyces naeslundii genospecies 2 (gsp-2) are members of the autochthonous oral flora. Chromosomal DNA fingerprinting (CDF) with SmaI revealed extensive genetic diversity among A. naeslundii gsp-2 strains within individual mothers and children. There was a low prevalence of genotype match among A. naeslundii gsp-2 strains between all mother and child pairs.

Actinomyces↗

Genetic characterization of the oral Actinomyces.

Actinomyces are difficult to identify using serological and biochemical methods but genotyping is an efficient and reliable means of bacterial characterization and can be used to determine clonal identity. The purpose here was to genotype 13 American type culture collection (ATCC) reference strains representing six different oral Actinomyces spp. by using chromosomal DNA fingerprinting (CDF), arbitrarily primed-polymerase chain reaction (AP-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In CDF analysis, BamHI, BstEII and SmaI yielded digestion patterns revealing characteristic differences among the known Actinomyces spp., with SmaI demonstrating optimal resolution. Amplicons generated by AP-PCR with primer OPB-07 displayed banding patterns that permitted discrimination of all Actinomyces strains tested. PCR-RFLP with MnlI digests generated fragment patterns that also characterized the reference strains. Collectively, genotypic profiles generated by CDF, AP-PCR and PCR-RFLP permitted differentiation of all 13 ATCC Actinomyces strains. SmaI CDF analysis of 18 clinical isolates of catalase-positive A. naeslundii genospecies 2 revealed extensive genetic diversity among these strains. These molecular approaches may be useful in determining genetic diversity within oral Actinomyces populations and fidelity of Actinomyces transmission between mother and child.

Actinomyces↗

Effect of amino acid substitutions in conserved residues in the leader peptide on biosynthesis of the lantibiotic mutacin II.

The lantibiotic mutacin II, produced by Streptococcus mutans T8, is a ribosomally synthesized peptide antibiotic that contains thioether amino acids such as lanthionine and methyllanthionine as a result of post-translational modifications. The mutacin II leader peptide sequence shares a number of identical amino acid residues with class AII lantibiotic leader peptides. To study the role of these conservative residues in the production of active antimicrobial mutacin, 15 mutations were generated by site-directed mutagenesis. The effects of these substitutions vary from no effect to complete block-out. Mutations G-1A, G-2A, I-4D, and L-7K completely blocked the production of mature mutacin. Other mutations (I-4V, L-7M, E-8D, S-11T/A, V-12I/A, and E-13D) had no detectable effect on mutacin production. The changes of Glu-8 to Lys, Val-12 to Leu, Glu-13 to Lys reduced the mutacin production level to about 75%, 50%, and 10% of the wild-type, respectively. Thus, our data indicated that some of these conserved residues are essential for the mutacin biosynthesis, whereas others are important for optimal biosynthesis rates.

Amino Acid Sequence↗

Complete nucleotide sequence and characterization of pUA140, a cryptic plasmid from Streptococcus mutans.

Approximately 5% of strains of Streptococcus mutans contain plasmid DNA. Strain UA140 harbors a 5.6-kb cryptic plasmid, pUA140, with an overall G+C content of 32.7%. Five open reading frames (ORF), encoding peptides of larger than 100 amino acid residues, were initially designated as ORF1 to ORF5. These five ORFs were located on the same strand of pUA140. ORF1 (258 amino acids) resembled a replication protein, Rep. Upstream of the putative Rep gene, a double-stranded origin for plasmid replication that showed strong similarity to those of a number of plasmids in the pT181 family was identified. Further upstream was a region constituting the single-stranded origin of replication. A single-stranded DNA intermediate was detected during plasmid replication. Taken together, these results suggest that pUA140 replicated by the rolling circle replication mechanism but exhibited several characteristics that differ from those of other members of the pT181 plasmid family.

Amino Acid Motifs↗

Differentiation of Streptococcus mutans and Streptococcus sobrinus via genotypic and phenotypic profiles from three different populations.

Routine identification of Streptococcus mutans and Streptococcus sobrinus is generally based upon growth on various selective media, colony morphology and biochemical characteristics. We examined various approaches of differentiating these two species through a combination of the conventional phenotypic methodology with chromosomal DNA fingerprint (CDF) and arbitrarily primed polymerase chain reaction (AP-PCR) methods. Initially, ten ATCC type strains and 20 randomly selected clinical isolates of mutans streptococci (MS) were characterized and grouped into two major types based on patterns generated by the CDF using HaeIII digestion. The CDF's patterns with restriction fragments equal to or greater than 6.6 kb were defined as the CDF-1 group. The CDF's patterns with restriction fragments less than 6.6 kb were defined as the CDF-2 group. Both groups were then examined for biotype, serotype, and composition of DNA via thermal denaturation. AP-PCR was applied and evaluated for the capability of delineating S. mutans from S. sobrinus strains. Results of this study showed that all CDF-1 strains fit within a G+C range of 36.2% to 42.2%, whereas the CDF-2 strains had a G+C range of 45.8% to 47.0%. The serotyping assay exhibited 100% sensitivity, 90% specificity and 86.7% agreement with the CDF. The biotyping assay presented the poorest specificity (38.5%), indicating the highest variability. The capability of AP-PCR in differentiation of S. mutans from S. sobrinus was comparable to the CDF method, suggesting that either of these two approaches can and may serve as a viable alternative method to serotyping or biotyping of MS.

Analysis of Variance↗

Phenotypic and genotypic diversity of Streptococcus sanguis in infants.

Streptococcus sanguis comprises a heterogeneous group of oral streptococci indigenous to the oral cavity of humans. A total of 289 isolates from an infant population (n=37) were tentatively identified as S. sanguis on the basis of the distinctive colony morphology as shown on MM10-sucrose non-selective medium. These isolates were divided into four biovars of S. sanguis as determined by an extended panel of biochemical attributes. Chromosomal DNA was extracted from each isolate, and an AP-PCR fingerprint profile was obtained to allow study of the diversity within and among the infants. In this study, all four biovars of S. sanguis were detected in the infants. A wide genotypic diversity of S. sanguis was observed among these isolates; on average, each infant harbored 2.7 unique amplitypes as shown by the AP-PCR fingerprints. To explore the phylogenic relationship among these S. sanguis isolates, 20 strains representing the four biovars were selected at random for sequencing of their 16S rDNA and 16S-23S rDNA intergenic spacer chromosomal loci. Two major sequence patterns were identified within the 16S rDNA sequences. A phylogenic analysis showed that members from each of the four biovars of S. sanguis bore close relationship with the type-strain ATCC 10556 sequence, and that all of the isolates representing the four biovars could be clustered into two main phylotypes. The biovars were distributed throughout the phylotypes, indicating no correlation between the genetic and phenotypic groupings.

Bacterial Typing Techniques↗

The group I strain of Streptococcus mutans, UA140, produces both the lantibiotic mutacin I and a nonlantibiotic bacteriocin, mutacin IV.

Strains of Streptococcus mutans produce at least three mutacins, I, II, and III. Mutacin II is a member of subgroup AII in the lantibiotic family of bacteriocins, and mutacins I and III belong to subgroup AI in the lantibiotic family. In this report, we characterize two mutacins produced by UA140, a group I strain of S. mutans. One is identical to the lantibiotic mutacin I produced by strain CH43 (F. Qi et al., Appl. Environ. Microbiol. 66:3221-3229, 2000); the other is a nonlantibiotic bacteriocin, which we named mutacin IV. Mutacin IV belongs to the two-peptide, nonlantibiotic family of bacteriocins produced by gram-positive bacteria. Peptide A, encoded by gene nlmA, is 44 amino acids (aa) in size and has a molecular mass of 4,169 Da; peptide B, encoded by nlmB, is 49 aa in size and has a molecular mass of 4,826 Da. Both peptides derive from prepeptides with glycines at positions -2 and -1 relative to the processing site. Production of mutacins I and IV by UA140 appears to be regulated by different mechanisms under different physiological conditions. The significance of producing two mutacins by one strain under different conditions and the implication of this property in terms of the ecology of S. mutans in the oral cavity are discussed.

Actinomycetales↗

Genotypic diversity of mutans streptococci in Brazilian nursery children suggests horizontal transmission.

Streptococcus mutans strains were isolated from cohorts of Brazilian nursery school children and genotyped by arbitrarily primed PCR and restriction fragment length polymorphism analysis. Of 24 children with two to five S. mutans isolates, 29% carried two or more genotypes. The presence of matching genotypes of S. mutans among children attending one nursery suggests horizontal transmission.

Brazil↗

The antimicrobial approach to caries management.

Dental caries is an infectious disease of bacterial origin. The use of antimicrobial agents to reduce or eliminate the bacteria associated with caries follows the approach used to combat other infectious diseases of humans. Unfortunately, only a few dozen studies have sufficient resolving power to make inferences as to the anticaries efficacy of the antimicrobial approach to caries management. Here, we comment on the findings of the RTI/UNC review concerning antimicrobials, discuss additional findings not covered in that review, and make recommendations based upon both the available literature and from our own experience. Even though the studies published thus far are inconclusive or lack sufficient demonstration of efficacy to recommend a specific approach involving antimicrobial agents in routine clinical practice, several pieces of information from these studies suggest future avenues of investigation.

Adolescent↗

Biochemical structural analysis of the lantibiotic mutacin II.

Mutacin II is a post-translationally modified lantibiotic peptide secreted by Streptococcus mutans T8, which inhibits the energy metabolism of sensitive cells. The deduced amino acid sequence of promutacin II is NRWWQGVVPTVSYECRMNSWQHVFTCC, which is capable of forming three thioether bridges. It was not obvious, however, how the three thioether bridges are organized. To examine the bridging, the cyanogen bromide cleavage products of mutacin II and its variants generated by protein engineering, C15A, C26A, and C15A/C26A, were analyzed by mass spectrometry. Analysis of the wild type molecule and the C15A variant excluded several possibilities and also indicated a high fidelity of formation of the thioether bridges. This allowed us to further resolve the structure by analysis (mass spectrometry and tandem mass spectrometry) of the cyanogen bromide cleavage fragments of the C26A and C15A/C26A mutants. Nuclear magnetic resonance analysis established the presence of one and two dehydrobutyrine residues in mutacin II and the C15A variant, respectively, thus yielding the final structure. The results of this investigation showed that the C-terminal part contains three thioether bridges connecting Cys residues 15, 26, and 27 to Ser/Thr residues 10, 12 and 19, respectively, with Thr(25) being modified to dehydrobutyrine.

Amino Acid Sequence↗

Dental caries. An infectious and transmissible disease.

Of the infectious diseases that affect humans, dental caries may be the most prevalent. Pediatric primary medical care providers are usually the first health care providers to examine the oral cavity of a child and so need to be able to recognize suspicious dental lesions. This article provides information on recognizing caries and reviews the epidemiology, pathogenesis, and treatment of caries and odontogenic infections.

Anti-Bacterial Agents↗

Purification and biochemical characterization of mutacin I from the group I strain of Streptococcus mutans, CH43, and genetic analysis of mutacin I biosynthesis genes.

Previously, we reported isolation and characterization of mutacin III and genetic analysis of mutacin III biosynthesis genes from the group III strain of Streptococcus mutans, UA787 (F. Qi, P. Chen, and P. W. Caufield, Appl. Environ. Microbiol. 65:3880-3887, 1999). During the same process of isolating the mutacin III structural gene, we also cloned the structural gene for mutacin I. In this report, we present purification and biochemical characterization of mutacin I from the group I strain CH43 and compare mutacin I and mutacin III biosynthesis genes. The mutacin I biosynthesis gene locus consists of 14 genes in the order mutR, -A, -A', -B, -C, -D, -P, -T, -F, -E, -G, orfX, orfY, orfZ. mutA is the structural gene for mutacin I, while mutA' is not required for mutacin I activity. DNA and protein sequence analysis revealed that mutacins I and III are homologous to each other, possibly arising from a common ancestor. The mature mutacin I is 24 amino acids in size and has a molecular mass of 2, 364 Da. Ethanethiol modification and peptide sequencing of mutacin I revealed that it contains six dehydrated serines, four of which are probably involved with thioether bridge formation. Comparison of the primary sequence of mutacin I with that of mutacin III and epidermin suggests that mutacin I likely has the same bridging pattern as epidermin.

Amino Acid Sequence↗

Natural history of Streptococcus sanguinis in the oral cavity of infants: evidence for a discrete window of infectivity.

The heterogeneous group of oral bacteria within the sanguinis (sanguis) streptococci comprise members of the indigenous biota of the human oral cavity. While the association of Streptococcus sanguinis with bacterial endocarditis is well described in the literature, S. sanguinis is thought to play a benign, if not a beneficial, role in the oral cavity. Little is known, however, about the natural history of S. sanguinis and its specific relationship with other oral bacteria. As part of a longitudinal study concerning the transmission and acquisition of oral bacteria within mother-infant pairs, we examined the initial acquisition of S. sanguinis and described its colonization relative to tooth emergence and its proportions in plaque and saliva as a function of other biological events, including subsequent colonization with mutans streptococci. A second cohort of infants was recruited to define the taxonomic affiliation of S. sanguinis. We found that the colonization of the S. sanguinis occurs during a discrete "window of infectivity" at a median age of 9 months in the infants. Its colonization is tooth dependent and correlated to the time of tooth emergence; its proportions in saliva increase as new teeth emerge. In addition, early colonization of S. sanguinis and its elevated levels in the oral cavity were correlated to a significant delay in the colonization of mutans streptococci. Underpinning this apparent antagonism between S. sanguinis and mutans streptococci is the observation that after mutans streptococci colonize the infant, the levels of S. sanguinis decrease. Children who do not harbor detectable levels of mutans streptococci have significantly higher levels of S. sanguinis in their saliva than do children colonized with mutans streptococci. Collectively, these findings suggest that the colonization of S. sanguinis may influence the subsequent colonization of mutans streptococci, and this in turn may suggest several ecological approaches toward controlling dental caries.

Age Factors↗

Associations of MHC genes with levels of caries-inducing organisms and caries severity in African-American women.

The aim of this investigation was to evaluate the relationship between MHC alleles at the HLA-DRB1, DQB1 and TNFa microsatellite loci and levels of oral bacteria that play a role in the etiology of dental caries, and the DMFS index in 186 AA primparous women. The average age of the cohort was 20.8+/-3.7 years. The median DMFS index was 9 (range 0-68). High levels of S. mutans were positively associated with DRB1*3 and DRB1*4 presence (p < or = 0.005). DRB1*8 was positively associated with higher levels of S. mutans as a percentage of total Streptococci (p = 0.04). DRB1*1 was positively associated with high levels L. casei (p = 0.04). DQB1 alleles were not observed associated with oral bacterial levels. TNFa allele 103 was negatively associated (p = 0.04), and TNFa 117 was positively associated (p = 0.007), with high levels of L. acidophilus. No significant associations were observed between any DRB1, DQB1 or TNFa allele and the DMFS index. These results support an hypothesis of an association between host HLA class II and TNFa genetic profile and colonization of S. mutans, L. casei, and L. acidophilus thought to be pathogens involved in the etiology of dental caries.

Adolescent↗