Biomedical subjects

P K Sreenivasan

Publications and source records attributed to P K Sreenivasan.

16 recordsLinked to original sources

PubMed2006

Individual microflora beget unique oral microcosms.

AIMS: To examine the efficacy of the multiple Sorbarod device (MSD) for the reproduction of inter-individual variations in oral microbiotas. The MSD supports sessile growth on parallel cellulose filters, perfused with artificial saliva. This enables biofilms (BF) to be grown and sampled, together with released cells in eluted medium (perfusates, PAs). METHODS AND RESULTS: Two sets of triplicate MSDs were established. One set was inoculated using fresh saliva from three separate volunteers; the second set was inoculated from one saliva donor. Both were incubated in an anaerobic cabinet. BF and PA were analysed at 24-h intervals by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. Hierarchical dendrograms were constructed in order to sort community fingerprints over time, based on community relatedness. The MSD supported complex oral communities, as evidenced by DGGE (>20 distinct DGGE bands) and confocal scanning laser microscopy. DGGE band sequencing revealed bacteriological diversity and a high incidence of anaerobic species, including Prevotella sp. Dendrograms demonstrated marked inter-individual variation in the relative species abundance within salivary inocula from different volunteers (DV) and each associated MSD (all >45%, majority c. 85% concordance). Less variation was shown between triplicate models established using saliva from a single volunteer (SV) (all >58%; majority c. 95% concordance). PAs clustered together with the associated biofilms and inocula in the majority of cases for the DV MSDs whilst SV MSD community profiles clustered between replicate MSDs. CONCLUSIONS: Data indicate that marked inter-individual variations in human salivary composition can be partially replicated in individualized MSD microcosms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the in vitro reproduction of individual oral microbiotas and suggests that taking inter-individual variability into account will increase the relevance of microcosm studies.

Bacterial Typing Techniques↗

PubMed2006

Media- and method-dependent variations in minimal inhibitory concentrations of antiplaque agents on oral bacteria.

AIMS: To determine minimal inhibitory concentrations (MICs) and the percentage of nonsusceptible bacteria-- those still cultivable above a threshold concentration--in human supragingival dental plaque and saliva for antiplaque/antimicrobial agents including triclosan (TCS) and trichlorocarbanilide (TCC), and a new potential antimicrobial, 2-t-butyl-5-(4-t-butylphenyl)-phenol (DTBBP). METHODS AND RESULTS: Broth and agar dilution-based MIC tests were performed using 28 oral and nonoral bacterial strains representing 17 species. MICs for TCS were lowest and more than 100-fold lower than DTBBP (P < 0.0005) by both methods. MICs for TCS were lower in broth-based tests compared with TCC. The additions of defibrinated blood to agar and horse serum to broth increased MICs--in the case of TCS, 10- to 15-fold. Significantly higher proportions of nonsusceptible plaque and salivary bacteria were recovered from agar media containing DTBBP or TCC compared with TCS (P < 0.05). CONCLUSIONS: TCS is a more effective antimicrobial agent than either TCC or DTBBP as determined by in vitro testing. SIGNIFICANCE AND IMPACT OF THE STUDY: The utility of in vitro testing for antiplaque agents as a predictor of in vivo efficacy is affected by the methods used.

Anti-Bacterial Agents↗

PubMed2005

Development and characterization of a simple perfused oral microcosm.

AIMS: To validate perfused, inline, filter-based fermentation systems (multiple Sorbarod devices, MSD) for their ability to maintain stable oral bacterial communities. MSD enable replicate (n=5) microcosm biofilms (BF) to be established and sampled, together with their perfusates (PA, cells in eluted medium). METHODS AND RESULTS: Fresh saliva from human volunteers was used to inoculate MSD, incubated in an anaerobic cabinet and perfused with artificial saliva at 7 ml h(-1). BF within Sorbarod filters and cells eluted in the PA were analysed at 24-h intervals by differential bacteriological culture and checkerboard DNA-DNA hybridization (CKB, 40 oral species). Dynamic stability was apparent after 2-3 days within both BF and PA as evidenced by culture, CKB data and pH measurements. BF harboured large numbers of anaerobic species and facultative anaerobes [ca 10-11 log10 colony-forming units (CFU)/filter] comprising considerable numbers of streptococci and Gram-negative species. PA contained ca 9-10 log(10) CFU ml(-1) suggesting an apparent mean growth rate of 0.1 h(-1) for the BF, as a whole corresponding to a mean generation time of 10 h. CKB analysis revealed considerable bacterial diversity within the respective MSD. Inter-individual variations in the relative species abundance of inocula was broadly reproduced in the MSD (BF and PA), although considerable variation was apparent between triplicate models established using saliva from one saliva donor or from three individual donors. The dominance of Gram-negative species, indicated by culture was supported by CKB analysis (major species, Prevotella melaninogenica and Fusobacterium nucleatum). CONCLUSIONS: Data obtained from the various analytical approaches showed a high degree of congruence. The MSD enables the maintenance of complex, stable salivary microcosms and represents a simple, reproducible tool for modelling individual oral bacterial ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the utility of the MSD for studying the micro-ecology of the oral cavity.

Bacteria↗

PubMed2005

An ex-vivo multiplexed antibacterial test on oral microflora.

BACKGROUND/AIMS: Characterized clinical strains of oral bacteria are utilized to examine the antimicrobial efficacy of oral care formulations. A demonstration of antimicrobial effects of formulations on microbial samples obtained from the human mouth offers advantages, i.e. incorporates the considerable microbial diversity of this environment with bacteria harvested from naturally occurring biofilms to form the focus of this investigation. MATERIALS AND METHODS: Samples of oral flora from each adult subject were briefly treated (2 min) with test and control formulations and plated on appropriate agar for multiplexed antimicrobial effects on functional groups of oral bacteria associated with specific conditions. RESULTS: Ex-vivo treatments of oral samples with the triclosan/copolymer dentifrice demonstrated significant dose-dependent antimicrobial effects compared with a control formulation on anaerobic and facultative oral bacteria from a group of 16 volunteers (P < 0.05) with reproducible effects observed in three separate trials (P < 0.05). Similarly, significant dose-dependent effects were noted with chlorhexidine mouthrinses (P < 0.05). A simultaneous assessment of the effects of these formulations on several functional classes of oral bacteria associated with specific oral conditions such as dental caries and oral malodor demonstrated multiplexed antimicrobial activity (P < 0.05). CONCLUSIONS: A rapid procedure using small volumes of human oral samples and well-known formulations demonstrates ex-vivo multiplexed antimicrobial effects on functional groups of bacteria with both dentifrices and mouthrinses. Modifications of this approach may include antibacterial effects over time with formulations at clinically relevant concentrations, effects of novel agents or samples from subjects stratified on the basis of their clinical status.

Adolescent↗

PubMed2004

Antimicrobial effects of a new therapeutic liquid dentifrice formulation on oral bacteria including odorigenic species.

The control of oral malodor is well-recognized in efforts to improve oral health. Antimicrobial formulations can mitigate oral malodor, however, procedures to assess effects on oral bacteria including those implicated in halitosis are unavailable. This investigation examined the antimicrobial effects of a new liquid triclosan/copolymer dentifrice (test) formulation that demonstrated significant inhibition of oral malodor in previous organoleptic clinical studies. Procedures compared antimicrobial effects of the test and control formulations on a range of oral micro-organisms including members implicated in halitosis, substantive antimicrobial effects of formulations with hydroxyapatite as a surrogate for human teeth and ex vivo effects on oral bacteria from human volunteers. With Actinomyces viscosus, as a model system, the test formulation demonstrated a dose-dependent effect. At these concentrations the test formulation provided significant antimicrobial effects on 13 strains of oral bacteria including those implicated in bad breath at selected posttreatment time points. Treatment of hydroxyapatite by the test dentifrice resulted in a significant and substantive antimicrobial effect vs. controls. Oral bacteria from subjects treated ex vivo with the test dentifrice resulted in significant reductions in cultivable oral bacteria and odorigenic bacteria producing hydrogen sulfide. In summary, microbiological methods adapted to study odorigenic bacteria demonstrate the significant antimicrobial effects of the test (triclosan/copolymer) dentifrice with laboratory and clinical strains of oral bacteria implicated in bad breath.

Actinomyces viscosus↗

PubMed2004

The effects of a chlorhexidine mouthrinse on culturable microorganisms of the tongue and saliva.

A randomized and cross-over design clinical study with normal adult volunteers examined the effects of a 0.12% chlorhexidine (CHX) mouthrinse and a control rinse on a range of microflora including those implicated in halitosis on the tongue and saliva. Saliva and tongue scrapings were collected from 13 subjects prior to treatments with additional samples collected at 3 h post-treatment cultured on media to enumerate anaerobic, Gram-positive and Gram-negative bacteria, odorigenic bacteria producing hydrogen sulphide (H2S) and oral bacteria with proteolytic activity. In comparison to the control, rinsing with CHX demonstrated statistically significant reductions that ranged from 81-90% for tongue microflora with a 89-95% decrease noted on salivary flora (p<0.05). The effects of CHX on anaerobic, Gram-positive and Gram-negative bacteria are in accord with those noted on odorigenic bacteria producing H2S or proteolytic activity. These results help to explain previous clinical results on the malodor efficacy of CHX and highlight microbiological approaches for bacteria implicated in malodor.

Adult↗

PubMed2004

A simple approach to examine early oral microbial biofilm formation and the effects of treatments.

BACKGROUND/AIMS: A simple in vivo approach to examine early dental plaque formation in the human mouth and to determine the effects of common dietary and oral hygiene procedures on biofilm formation is reported. METHODS: A custom designed device that fits securely behind the teeth of the mandibular arch provides a surface for microbial colonization. This device is prepared with denture acrylic and can be repeatedly used by the subject, exposing a large and constant surface area for microbial accumulation. RESULTS: Large numbers of oral bacteria colonized the device by 2 h; these increased significantly by 4 h (P < 0.05). Bacterial colonization increased significantly after rinsing with a sucrose solution (P < 0.05) but remained unaffected after rinsing with water, a commercially available fluoride mouthrinse without antimicrobial agents, or brushing with a fluoride dentifrice (P > 0.05). Rinsing with mouthrinses formulated with chlorhexidine, cetylpyridinium chloride or triclosan/copolymer significantly inhibited colonization (P < 0.05). A dose-dependent inhibition was noted with chlorhexidine rinses (P < 0.05). Brushing with a triclosan/copolymer dentifrice significantly inhibited microbial colonization compared with a control (P < 0.05). CONCLUSION: This simple approach was useful for examining the effects of common dietary and oral hygiene procedures. Significant biofilm inhibitory effects were noted with formulations that demonstrated efficacy in previous clinical studies.

Actinomyces viscosus↗

PubMed2004

Effects of low dose chlorhexidine mouthrinses on oral bacteria and salivary microflora including those producing hydrogen sulfide.

BACKGROUND/AIMS: Clinical studies have demonstrated the considerable effects of chlorhexidine on dental plaque and oral microbiota as well as improvements in indices of oral health. This investigation examined the efficacy of lower concentrations of chlorhexidine. METHODS: Mouthrinses with 0.03%, 0.06%, 0.12% chlorhexidine and a control rinse without chlorhexidine were examined. Alamar blue, an oxidation-reduction dye with fluorescent end-points proportional to bacterial viability, was used to determine bacterial viability. Further clinical studies examined the effects of these rinses on salivary bacteria and on bacteria producing hydrogen sulfide (H(2)S) and implicated in halitosis. RESULTS: In laboratory tests, a significant dose-dependent effect was observed with Actinomyces viscosus as a model system using the Alamar blue procedure (P < 0.05). Clinical studies examined the effects 1.5 h and 3 h post-treatment on salivary bacteria and bacteria producing H(2)S. The first study compared the control rinse with the 0.03% and 0.06% chlorhexidine rinses; a second study compared the effects of the control rinse and the 0.06% and 0.12% chlorhexidine mouthrinses. In both studies, chlorhexidine rinses demonstrated significant dose-dependent effects post-treatment on salivary bacteria vs. the control rinse (P < 0.05). Significant decreases in H(2)S-producing bacteria were noted with these chlorhexidine rinses vs. the control rinse (P < 0.05). CONCLUSION: The results highlight the dose-dependent relationships noted in laboratory and clinical tests which have potential implications for the use of lower doses of chlorhexidine to inhibit oral bacteria, including those implicated in halitosis.

Actinomyces viscosus↗

PubMed2003

A rapid procedure to ascertain the antimicrobial efficacy of oral care formulations.

A rapid method examining the antimicrobial efficacy of oral care formulations with alamar blue, an oxidation-reduction dye with fluorescent end-points, is described. Significant correlations between increasing viable plate counts of the oral bacteria Actinomyces viscosus, Streptococcus sanguis, Streptococcus mutans and Actinobacillus actinomycetemcomitans and increased alamar fluorescence were noted. Metabolically active bacteria reduced alamar with the reduced dye found in the cell-free filtrate. Insignificant alamar reductions were noted in the absence of bacteria or by spent culture supernatants. The efficacy of mouthrinses with clinically proven antiplaque agents such as chlorhexidine or cetylpyridinium chloride were determined by alamar blue. In a model system with A. viscosus, triclosan dentifrices demonstrate a dose-dependent effect on bacteria. Human salivary bacteria demonstrate increasing alamar fluorescence with increasing plate counts. A clinical study examined the effects of rinsing with chlorhexidine or cetylpyridinium chloride mouthrinses in comparison with a placebo mouthrinse and water on salivary bacteria. Rinsing with chlorhexidine resulted in the least number of bacteria by alamar and plate count methods. In summary, the current study demonstrates the utility of alamar blue to examine the antimicrobial effects of oral care formulations in laboratory and clinical studies.

Actinomyces viscosus↗

PubMed1994

Isolation and characterization of deletion derivatives of pDL282, an Actinobacillus actinomycetemcomitans/Escherichia coli shuttle plasmid.

Recent reports have described the construction of several shuttle plasmids (D.J. LeBlanc, L. L. Lee, A. Al-Jaibat, P. K. Sreenivasan, and P. M. Fives-Taylor, Oral Micro. Immunol. (1993) 8, 94-99) and the development of an efficient transformation system for Actinobacillus actinomycetemcomitans (P. K. Sreenivasan, D. J. LeBlanc, L. L. Lee, and P. M. Fives-Taylor, Infect. Immun. (1991) 59, 4621-4627), a gram-negative bacterium implicated in human periodontal disease. This report presents results from further studies on pDL282, an A. actinomycetemcomitans-Escherichia coli shuttle plasmid. A. actinomycetemcomitans containing pDL282 lost the plasmid at a rapid rate when cultured in antibiotic free medium. Intact pDL282 was maintained for 20 generations or more when the host cells were grown in the presence of ampicillin, or ampicillin plus spectinomycin. However, prolonged incubation in the presence of spectinomycin only resulted in the emergence of one or the other of two unique deletion derivatives of pDL282, designated pPK1 and pPK2. Whereas A. actinomycetemcomitans was efficiently transformed with pDL282 (5.7 kb), pPK1 (3.6 kb), and pPK2 (2.5 kb), E. coli was transformed only by the two largest species. Like the parent molecule, pPK1 and pPK2 were rapidly lost from A. actinomycetemcomitans hosts in the absence of antibiotic selection. Neither pPK1 nor pPK2 suffered any further deletions following prolonged cultivation in the presence of spectinomycin. The minimal replicon of pVT736-1, the A. actinomycetemcomitans-derived plasmid component of pDL282, was located within a 1400 bp DNA fragment of pPK1 and pPK2.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinobacillus↗

PubMed1994

Phase variation in pneumococcal opacity: relationship between colonial morphology and nasopharyngeal colonization.

When colonies of encapsulated isolates of Streptococcus pneumoniae are viewed with oblique, transmitted light on a transparent surface, they are heterogeneous in appearance because of variation in opacity. There is spontaneous phase variation among at least three discernible phenotypes at frequencies from 10(-3) to 10(-6). The ability to detect differences in opacity varies according to serotype, but variation is independent of capsule expression. Electron microscopy shows no difference in chain length but suggests that autolysis occurs earlier in the growth of the transparent variant. There was no identifiable difference in membrane protein profiles of opaque and transparent variants of the same strain. In an infant rat model of nasopharyngeal carriage, there was no significant colonization by opaque variants. Efficient and stable colonization by the transparent variants was observed, suggesting a selective advantage for this phenotype in the nasopharynx. In contrast, there was no difference in the incidence of bacteremia or in the 50% lethal dose among the variants following their intraperitoneal inoculation. These results suggest that phase variation which is marked by differences in colonial morphology may provide insight into the interaction of the pneumococcus with its host.

Animals↗

PubMed1993

Identification of plasmids in Actinobacillus actinomycetemcomitans and construction of intergeneric shuttle plasmids.

A collection of 39 isolates of Actinobacillus actinomycetemcomitans, obtained from laboratories located in 5 different geographical regions of the United States, was examined for the presence of plasmid DNA. Only 2 of the strains examined, designated VT736 and VT745, harbored detectable plasmids. Strain VT736 contained a 1.9 kb plasmid species (pVT736-1) and a larger ( > 30 kb) species (pVT736-2). Both plasmids were detected in the covalently closed circular DNA fraction of dye buoyant density gradients. However, only the smaller plasmid was observed in agarose gels containing plasmid-enriched cell lysates prepared by a rapid screening procedure. Strain VT745 contained a single, 24 kb, plasmid (pVT745) that was observed consistently in plasmid-enriched lysates, as well as in the plasmid band of dye buoyant density gradients. A restriction endonuclease map of pVT736-1 was constructed. The plasmid contained one site each for the enzymes HincII, KpnI and XhoI, located 600 to 700 bp from each other on the pVT736-1 map. HincII-digested pVT736-1 DNA could not be cloned in Escherichia coli. However, intact pVT736-1 digested with KpnI or XhoI could be cloned in E. coli on pUC19 or pGEM7Zf(-), respectively. KpnI-digested pVT736-1 was cloned in both orientations on pUC19, but XhoI-digested pVT736-1 was clonable in only one orientation on pGEM7Zf(-). Each of the 3 types of chimeric plasmid constructs provided a potential A. actinomycetemcomitans/E. coli shuttle plasmid for the development of a genetic transfer system in A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗

PubMed1993

Factors influencing the growth and viability of Actinobacillus actinomycetemcomitans.

The metabolic requirements for the routine growth of Actinobacillus actinomycetemcomitans were investigated by the addition of nutrients to conventional bacteriological and tissue culture media. Commonly used tissue culture media required fetal bovine serum as an additive to sustain bacterial growth rates comparable to those obtained with bacteriological media. The addition of increasing concentrations of yeast extract to bacteriological medium increased the growth rate of several A. actinomycetemcomitans strains. In an attempt to identify the components of yeast extract that enhanced the growth of A. actinomycetemcomitans, a number of vitamins, essential and non-essential amino acids were tested for their role in promoting growth. The addition of L-cystine resulted in bacterial growth rates comparable to those with yeast extract. Thiamine increased the growth of several A. actinomycetemcomitans strains but did not result in growth rates comparable to those with yeast extract. The addition of physiological concentrations of steroid hormones to bacteriological medium enhanced the growth of A. actinomycetemcomitans. Additional iron compounds and fat-soluble vitamins had no influence on A. actinomycetemcomitans growth. However, the requirement of iron for bacterial growth remains unclear. The optimal pH range for growth of A. actinomycetemcomitans was between pH 7.0-8.0 in a medium containing 0.5-1% NaCl. Several interesting observations on the viability of A. actinomycetemcomitans were made. A rapid reduction of A. actinomycetemcomitans viability occurred following suspension in distilled water. The presence of the detergent Triton X-100 at concentrations above 2% (v/v) also decreased the viability of A. actinomycetemcomitans within 10 min.

Aggregatibacter actinomycetemcomitans↗

PubMed1993

Requirements for invasion of epithelial cells by Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, an oral bacterium implicated in human periodontal disease, was recently demonstrated to invade cultured epithelial cells (D. H. Meyer, P. K. Sreenivasan, and P. M. Fives-Taylor, Infect. Immun. 59:2719-2726, 1991). This report characterizes the requirements for invasion of KB cells by A. actinomycetemcomitans. The roles of bacterial and host factors were investigated by using selective agents that influence specific bacterial or host cell functions. Inhibition of bacterial protein synthesis decreased invasion, suggesting the absence of a preformed pool of proteins involved in A. actinomycetemcomitans invasion. Inhibition of bacterial and eukaryotic energy synthesis also decreased invasion, confirming that A. actinomycetemcomitans invasion is an active process. Bacterial adherence to KB cells was indicated by scanning electron microscopy of infected KB cells. Further, the addition of A. actinomycetemcomitans-specific serum to the bacterial inoculum reduced invasion substantially, suggesting a role for bacterial attachment in invasion. Many of the adherent bacteria invaded the epithelial cells under optimal conditions. Inhibitors of receptor-mediated endocytosis inhibited invasion by A. actinomycetemcomitans. Like that of many facultatively intracellular bacteria, A. actinomycetemcomitans invasion was not affected by eukaryotic endosomal acidification. These are the first published observations describing the requirements for epithelial cell invasion by a periodontopathogen. They demonstrate that A. actinomycetemcomitans utilizes a mechanism similar to those used by many but not all invasive bacteria to gain entry into eukaryotic cells.

Aggregatibacter actinomycetemcomitans↗

PubMed1991

Transformation of Actinobacillus actinomycetemcomitans by electroporation, utilizing constructed shuttle plasmids.

Actinobacillus actinomycetemcomitans, a periodontal pathogen, has been strongly implicated in human periodontal disease. Advances in the molecular analysis of A. actinomycetemcomitans virulence factors have been limited due to the unavailability of systems for genetic transfer, transposon mutagenesis, and gene complementation. Slow progress can be traced almost exclusively to the lack of gene vector systems and methods for the introduction of DNA into A. actinomycetemcomitans. An electrotransformation system that allowed at least five strains of A. actinomycetemcomitans to be transformed with stable shuttle plasmids which efficiently replicated in both Escherichia coli and A. actinomycetemcomitans was developed. One plasmid, a potential shuttle vector designated pDL282, is 5.7 kb in size, has several unique restriction enzyme sites, and codes for resistance to spectinomycin and ampicillin. E. coli and A. actinomycetemcomitans were transformed with equal efficiencies of approximately 10(5) transformants per micrograms of DNA. Similar transformation efficiencies were obtained whether the plasmid DNA was isolated from A. actinomycetemcomitans or E. coli. In addition, frozen competent cells of A. actinomycetemcomitans yielded comparable efficiencies of transformation. Restriction enzyme analysis of pDL282 isolated after transformation confirmed the presence of intact donor plasmids. A plasmid isolated from A. pleuropneumoniae was also capable of transforming some isolates of A. actinomycetemcomitans, although generally at a lower frequency. The availability of these shuttle plasmids and an efficient transformation procedure should significantly facilitate the molecular analysis of virulence factors of A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗

PubMed1991

Evidence for invasion of a human oral cell line by Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, an oral bacterial species associated with periodontal disease, was found to invade human cell lines. Invasion was demonstrated by recovery of viable organisms from gentamicin-treated KB cell monolayers and by light and electron microscopy. Internalization occurred through a cytochalasin D-sensitive process. Invasion efficiencies of some A. actinomycetemcomitans strains were comparable to those of invasive members of the family Enterobacteriaceae. Differences in invasiveness were correlated with bacterial colonial morphology. Smooth variants invaded more proficiently than rough variants. A. actinomycetemcomitans can undergo a smooth-to-rough colonial morphology shift which results in the loss of invasiveness. Coordinated regulation of genes involved in the rough-to-smooth phenotypic transitions may play a role in the episodic nature of periodontal disease.

Actinobacillus↗