Influences of orthodontic appliances on oral populations of mutans streptococci.

A study was conducted to assess the influence of fixed appliances on the mutans streptococcal in a group of patients receiving orthodontic treatment. Mutans streptococcal counts in stimulated saliva of 27 patients were obtained at baseline, and at 1-month intervals for 4 months. The difference in mutans streptococcal counts at baseline and at the 4-month sampling was less than an order of magnitude in 18 of the patients, between 1 and 2 orders of magnitude in eight of the patients, and 3 orders of magnitude in one patient. Restriction endonuclease digests of genomic DNA from representative mutans streptococci isolates taken from baseline and 4-month saliva samples, as well as from 4-month tooth and appliance surfaces, were examined by pulsed field gel electrophoresis, after restriction endonuclease digestion. Results of the DNA banding patterns associated with isolates from 19 patients showed that, for 12 patients, all isolates examined represented the same clone of Streptococcus mutans, whereas for six patients two different S. mutans clones were detected. One patient yielded three different clones of S. mutans. A much larger number of baseline, as well as post-appliance, isolates will have to be examined from each patient in future studies, in order to determine if the number of different S. mutans clones harbored by individual patients is related to orthodontic treatment.

Subgingival microbiota of indigenous Indians of Central America.

OBJECTIVE: To define the subgingival microbial profiles of adult subjects from a previously identified rural community of indigenous Indians in Guatemala, Central America. MATERIALS AND METHODS: A full-mouth periodontal examination was performed in 114 adult subjects from 45 families. Plaque samples were collected from both deep and shallow periodontal pockets and checkerboard DNA-DNA hybridization was employed to identify 17 species previously associated with periodontitis or health. RESULTS: Plaque deposits and gingivitis were universal and widespread, and periodontal pocketing > or =5 mm was highly prevalent (84% of subjects). Streptococcus sanguis, Actinomyces naeslundii genospecies 2 and Fusobacterium nucleatum were significantly more prevalent in shallow sites. At the subject level, Actinomyces naeslundii and Peptostreptococcus micros were significantly more prevalent in periodontally-healthy subjects. Actinobacillus actinomycetemcomitans was not detected in any sample. CONCLUSION: There was no association between periodontal disease status and presence of suspected periodontal pathogens. These latter results conflict somewhat with those from treated populations. However, in this population where extensive plaque deposits and gingivitis are universal, the presence of putative pathogens may be more reflective of the local environment.

Genome of the bacterium Streptococcus pneumoniae strain R6.

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

Nucleotide sequence and analysis of conjugative plasmid pVT745.

The complete nucleotide sequence and genetic map of pVT745 are presented. The 25-kb plasmid was isolated from Actinobacillus actinomycetemcomitans, a periodontal pathogen. Two-thirds of the plasmid encode functions related to conjugation, replication, and replicon stability. Among potential gene products with a high degree of similarity to known proteins are those associated with plasmid conjugation. It was shown that pVT745 derivatives not only mobilized a coresident nontransmissible plasmid, pMMB67, but also mediated their own conjugative transfer to different A. actinomycetemcomitans strains. However, transfer of pVT745 derivatives from A. actinomycetemcomitans to Escherichia coli JM109 by conjugation was successful only when an E. coli origin of replication was present on the pVT745 construct. Surprisingly, 16 open reading frames encode products of unknown function. The plasmid contains a conserved replication region which belongs to the HAP (Haemophilus-Actinobacillus-Pasteurella) theta replicon family. However, its host range appears to be rather narrow compared to other members of this family. Sequences homologous to pVT745 have previously been detected in the chromosomes of numerous A. actinomycetemcomitans strains. The nature and origin of these homologs are discussed based on information derived from the nucleotide sequence.

An effective strategy, applicable to Streptococcus salivarius and related bacteria, to enhance or confer electroporation competence.

Despite the large number of techniques available for transformation of bacteria, certain species and strains are still resistant to introduction of foreign DNA. Some oral streptococci are among the organisms that can be particularly difficult to transform. We performed a series of experiments that involved manipulation of growth and recovery media and cell wall weakening, in which the electroporation conditions, cell concentration, and type and concentration of the transforming plasmid were varied. The variables were optimized such that a previously untransformable Streptococcus salivarius strain, ATCC 25975, could be transformed reproducibly at a level of 10(5) transformants per microg of DNA. The technique was used to introduce a plasmid into other strains of S. salivarius, including a fresh isolate. Moreover, the same technique was applied successfully to a wide range of oral streptococci and other gram-positive bacteria.

Detection of Tn917-like sequences within a Tn916-like conjugative transposon (Tn3872) in erythromycin-resistant isolates of Streptococcus pneumoniae.

A series of macrolide-lincosamide-streptogramin B (MLS)-resistant pneumococcal isolates of a variety of serotypes was examined and was found to contain Tn917-like elements by DNA-DNA hybridization. Like Tn1545, Tn917 also encodes an ermAM gene but does not mediate resistance to other antimicrobial agents. Furthermore, nucleotide sequence analyses of the DNAs flanking three of the Tn917-like elements revealed that they were inserted into orf9 of a Tn916-like element in a composite transposon-like structure (Tn3872). Other MLS-resistant strains appeared to contain Tn1545-like elements that had suffered a deletion of sequences including the aphA-3 sequences responsible for kanamycin resistance. Thus, the MLS resistance phenotype in pneumococci appears to be mediated by the ermAM present on a much wider variety of genetic elements than was previously appreciated.

Functional analysis of pVT745, a plasmid from Actinobacillus actinomycetemcomitans.

Plasmid pVT745 is a 25.1-kb replicon isolated from Actinobacillus actinomycetemcomitans strain VT745. A previous report described the hybridization of pVT745 in 5 strain-specific patterns to chromosomal DNA from 15 other A. actinomycetemcomitans strains. However, pVT745 does not share homology with the chromosome of the strain from which it was isolated, VT745. It was hypothesized that the shared areas of homology might represent insertion sequence elements and/or transposons possibly encoding resistance to one or more antibiotics. An antibiogram of strain VT745 demonstrated that this strain was uniformly susceptible to all antibiotics examined. Because insertion sequence elements and transposons are mobile genetic elements, a series of cell passaging experiments, followed by Southern hybridization was conducted in a attempt to detect transposition of pVT745 homologous DNA within the chromosomes of several A. actinomycetemcomitans strains. The results of these experiments suggested stability of the homologous DNA both within the chromosome and on the plasmid. It was also possible that pVT745 represented a lysogenic bacteriophage. Phage induction experiments were conducted under conditions that induced a previously described A. actinomycetemcomitans lysogenic phage, but no phage could be induced from strain VT745. Attempts to obtain isolates of VT745 cured of pVT745 were also unsuccessful.

Identification of a maintenance system on rolling circle replicating plasmid pVT736-1. dgalli@iusd.iupui.edu.

Distribution of plasmid molecules to the two daughter cells at cell division is of major importance for their stable inheritance. Several mechanisms that control equipartitioning of low-copy-number plasmids have been described in molecular terms. However, no homologous or analogous systems have been identified for intermediate or high-copy-number plasmids, including rolling circle replicating (RCR) plasmids. It has been suggested that distribution of such plasmids at cell division relies solely on random segregation. Plasmid pVT736-1 is a 2 kb RCR plasmid that was isolated from the Gram-negative capnophilic coccobacillus Actinobacillus actinomycetemcomitans. The plasmid contains a DNA region of approximately 0.8 kb that is associated with its segregational stability. An operon that consists of two genes (orf3 and orf2) is followed by a putative cis-acting site that contains an integration host factor (IHF) binding site, flanked by several repeats. Mutations in orf2 resulted in plasmid instability. In addition, this DNA region was able to stabilize partially a heterologous replicon, p15A. Homologues or analogues of the pVT736-1 stabilization system have been detected on numerous plasmid and bacterial genomes.

Antibiotic resistance among enterococci isolated from clinical specimens between 1953 and 1954.

Two hundred twenty group D streptococci isolated from 1953 to 1954 from patients in the Washington, D.C., area were characterized. All were susceptible to ampicillin, vancomycin, and gentamicin; none produced beta-lactamase activity. High-level resistance to streptomycin was expressed by 117 strains, and 2 strains were resistant to >8 microg of chloramphenicol per ml. Three isolates were resistant to both erythromycin and lincomycin, and DNA from these hybridized to an ermAM probe. Of 118 strains resistant to tetracycline and minocycline, genomic DNA from 63 was examined for homology to tet(M), tet(O), and tet(S). DNA from 20 strains hybridized to tet(M), DNA from 37 strains hybridized to tet(S), and DNA from none of the strains hybridized to tet(O).

Scope of practice for APNs (advanced practice nurses). Discussion.

A bill for Advanced Practice Nurse (APN) Prescriptive Authority was introduced into the Michigan state legislature in 1996, and again in 1997. The legislation would add registered professional nurses with specialty certification to the list of health practitioners granted independent prescribing rights. Currently, licensed dentists, medical doctors, osteopathic doctors, podiatrists, certified optometrists and veterinarians comprise the list of prescribers.

Structural and segregational stability of various replicons in Actinobacillus actinomycetemcomitans.

Few vectors suitable for cloning in Actinobacillus actinomycetemcomitans, a periodontal pathogen, have been described. These plasmids were based either on the native A. actinomycetemcomitans replicon, pVT736-1, or derivatives of the IncP and IncQ family of plasmids. Their usefulness as cloning vectors is limited because of instability or size. Therefore, the ability of additional replicons to function in A. actinomycetemcomitans was evaluated. Derivatives of p15A, ColE1/f1, and pWV01 transformed A. actinomycetemcomitans efficiently and exhibited no structural instability in the new host. The stable maintenance of A. actinomycetemcomitans-specific DNA fragments inserted into the p15A derivative, pDMG4, demonstrated the ability of this plasmid to function as a cloning vector. In addition, pVT736-1 was used to clone selectable markers directly into A. actinomycetemcomitans. These constructs were structurally and segregationally stable.

Use of a novel mobilizable vector to inactivate the scrA gene of Streptococcus sobrinus by allelic replacement.

The virulence factors of the cariogenic bacterium Streptococcus sobrinus have been difficult to assess because of a lack of tools for the genetic manipulation of this organism. The construction of an Escherichia coli-Streptococcus shuttle vector, pDL289, that can be mobilized into S. sobrinus by the conjugative plasmid pAM beta 1 was described in a previous report. The vector contains pVA380-1 for replication and mobilization in streptococci, the pSC101 replicon for maintenance in E. coli, a kanamycin resistance marker that functions in both hosts, and the multiple cloning site and lacZ from pGEM7Zf(-). pDL289 is stable with or without selection in several species of Streptococcus. In this study, a derivative with a deletion in the minus origin of the pVA380-1 component of pDL289 was constructed. This derivative, pDL289 delta 202, was less stable than pDL289 in Streptococcus gordonii Challis, Streptococcus mutans, and S. sobrinus. Both pDL289 and pDL289 delta 202 were mobilizable by pAM beta 1 into S. sobrinus, with frequencies of 3 x 10(-6) and 1 x 10(-7) transconjugants per recipient CFU, respectively. The cloned scrA gene of S. sobrinus 6715-10 coding for the EIISuc of the sucrose-specific phosphoenolpyruvate phosphotransferase system was interrupted by the insertion of a streptococcal spectinomycin resistance gene active in E. coli and streptococci. The interrupted scrA gene was subcloned into both pDL289 and pDL289 delta 202. Each recombinant plasmid was introduced into the DL1 strain of S. gordonii Challis, which was then used as a recipient for the conjugative transfer of pAM beta 1. The latter plasmid was used to mobilize each recombinant plasmid from S. gordonii Challis DL1 to S. sobrinus 6715-10RF. Subsequently, recombinants derived from a double-crossover event were isolated on the basis of resistance to spectinomycin and susceptibility to kanamycin. Recombinational events were confirmed by Southern hybridization, and the inactivation of the EII Suc in double crossovers was confirmed by phosphotransferase system assays. This is the first report of allelic replacement in S. sobrinus.

Transcriptional analysis of rolling circle replicating plasmid pVT736-1: evidence for replication control by antisense RNA.

Several plasmids have been described in Actinobacillus actinomycetemcomitans, a gram-negative coccobacillus. Recently, the nucleotide sequence of pVT736-1, a cryptic plasmid of A. actinomycetemcomitans VT736, was determined. This plasmid possesses all the features necessary for rolling circle replication. The present study involved a transcriptional analysis of pVT736-1. Results of Northern (RNA) blot analyses and primer extension studies indicated that the two open reading frames identified in pVT736-1 are each preceded by at least one promoter. Expression of these promoters varied with growth phase. In addition, an antisense RNA (Cop RNA) appeared to control the synthesis of the putative replication protein. To our knowledge, this is the first rolling circle replicating plasmid isolated from a gram-negative organism that has been subjected to such detailed analysis.

Transformation of Bifidobacterium longum with pRM2, a constructed Escherichia coli-B. longum shuttle vector.

An Escherichia coli-Bifidobacterium longum shuttle vector, designated pRM2, was constructed by cloning a B. longum plasmid and an enterococcal spectinomycin resistance gene into a commercial E. coli vector. The plasmid was successfully introduced into B. longum cells by electroporation and into E. coli cells by both electroporation and chemical transformation.

Characterization of pVT736-1, a rolling-circle plasmid from the gram-negative bacterium Actinobacillus actinomycetemcomitans.

The presence of plasmid DNA in Actinobacillus actinomycetemcomitans has been reported. Recently, the construction of an Escherichia coli/A. actinomycetemcomitans shuttle vector, based on the A. actinomycetemcomitans-derived 2.0-kb plasmid pVT736-1, was described (D. J. LeBlanc, A. R. Abu-Al-Jaibat, P. K. Sreenivasan, and P. M. Fives-Taylor, Oral Microbiol. Immunol. (1993) 8, 94-99). The current study was initiated with the determination of the nucleotide sequence of pVT736-1, which revealed the presence of two open reading frames (ORFs) encoding proteins of 293 (ORF1) and 95 (ORF2) amino acids. Evidence that pVT736-1 replicates via a single-stranded (ss) intermediate included: (i) the presence of ssDNA in cells and in cell-free supernatant, (ii) the presence of conserved sequence motifs in the predicted ORF1 protein that are typical of initiator (Rep) proteins associated with replication by a rolling-circle mode, and (iii) 39% amino acid identity between the putative Rep proteins of pVT736-1 and Pf3, a filamentous ssDNA bacteriophage of Pseudomonas aeruginosa. A putative plus origin of replication in pVT736-1 was located upstream of ORF1, in a 200-bp region with potential for secondary hairpin structures. The identification in gram-negative bacteria of extrachromosomal DNA (other than bacteriophage) that replicates by a rolling-circle mode and shows no extensive homology to plasmids from gram-positive organisms is rather unique.

Characterization of plasmid pVT745 isolated from Actinobacillus actinomycetemcomitans.

A 25.1-kb plasmid, pVT745, was isolated from Actinobacillus actinomycetemcomitans strain VT745. This plasmid hybridized under stringent conditions to chromosomal DNA from 15 A. actinomycetemcomitans isolates obtained from geographically diverse regions of the United States. Southern blot analyses of HincII-digested A. actinomycetemcomitans genomic DNA revealed five strain-specific patterns of hybridization, which clearly indicated that intact pVT745 was not inserted into any of the genomes. Plasmid pVT745 was digested with BamHI and PstI into three, non-overlapping fragments of approximately 7.0, 8.0, and 10.0 kb. The fragments were cloned in Escherichia coli on the low copy number vector pGB2. Although these three fragments exhibited no cross-hybridization, genomic DNA from isolates representing each of the strain-specific patterns hybridized with two or three of the cloned fragments. The results suggest that two or more unique sequences within pVT745 also may be present in genomic DNA obtained from various strains of A. actinomycetemcomitans.

Identification and characterization of a mobilization gene in the streptococcal plasmid, pVA380-1.

The 4.2-kb cryptic streptococcal plasmid, pVA380-1, has a broad host range and replicates via a rolling circle mode. The nucleotide base sequence of the 2.5-kb basic replicon of this plasmid, containing a plus origin, a minus origin, and a rep gene, was reported previously. The completed nucleotide base sequence of pVA380-1 revealed the presence of two additional open reading frames, ORF2 and ORF3. The protein predicted by ORF2 shared 74% amino acid identity with the Mob protein of the Group B streptococcal plasmid, pMV158. The 170 bases immediately 5' to ORF2 and to mob of pMV158 shared 87% homology and included a putative -10 region and a RSa site. A derivative of pVA380-1 into which a kanamycin resistance gene was inserted outside of the above 170 base region and ORF2 was mobilized by the conjugative streptococcal plasmid, pAM beta 1, into Enterococcus faecalis and Streptococcus sobrinus. Interruption of ORF2 by the same kanamycin resistance gene abolished the mobilizability of pVA380-1.

Sequence analysis of scrA and scrB from Streptococcus sobrinus 6715.

The complete nucleotide sequences of Streptococcus sobrinus 6715 scrA and scrB, which encode sucrose-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system and sucrose-6-phosphate hydrolase, respectively, have been determined. These two genes were transcribed divergently, and the initiation codons of the two open reading frames were 192 bp apart. The transcriptional initiation sites were determined by primer extension analysis, and the putative promoter regions of these two genes overlapped partially. The gene encoding enzyme IIScr, scrA, contained 1,896 nucleotides, and the molecular mass of the predicted protein was 66,529 Da. The hydropathy plot of the predicted amino acid sequence indicated that enzyme IIScr was a relatively hydrophobic protein. The gene encoding sucrose-6-phosphate hydrolase, scrB, contained 1,437 nucleotides. The molecular mass of the predicted protein was 54,501 Da, and the encoded enzyme was hydrophilic. The predicted amino acid sequences of the two open reading frames exhibited approximately 45 and 70% identity with those encoded by scrA and scrB, respectively, from Streptococcus mutans GS5. Homology also was observed between the N-terminal region of the S. sobrinus 6715 enzyme IIScr and other enzyme IIs specific for the glucopyranoside molecule, all of which generate glucopyranoside-6-phosphate during translocation and phosphorylation of the respective substrates. The sequence of the C-terminal domain of the S. sobrinus 6715 enzyme IIScr shared significant homology with enzyme IIIGlc from Escherichia coli and Salmonella typhimurium and with the C-terminal domain of enzyme IIBgl from E. coli, indicating that the two functional domains, enzyme IIScr and enzyme IIIScr, were covalently linked as a single polypeptide in S. sobrinus 6715. The deduced amino acid sequence of the gene product of S. sobrinus scrB shared strong homology with sucrase from Bacillus subtilis, Klebsiella pneumoniae, and Vibrio alginolyticus, suggesting conservation based on the physiological roles of these proteins.