Biomedical subjects
Publications and source records attributed to J Pratten.
Twelve out of 96 Veillonella spp. isolated from oral samples harbored tetracycline resistance genes. The most common resistance gene was tet(M). A tet(M)-positive Veillonella dispar strain was shown to transfer a Tn916-like element to four Streptococcus spp. by conjugation at a frequency of 5.2 x 10(-6) to 4.5 x 10(-5) per recipient.
Stable microbial communities associated with health can be disrupted by altered environmental conditions. Periodontal diseases are associated with changes in the resident oral microflora. For example, as gingivitis develops, a key change in the microbial composition of dental plaque is the ascendancy of Actinomyces spp. and gram-negative rods at the expense of Streptococcus spp. We describe the use of an in vitro model to replicate this population shift, first with a dual-species model (Actinomyces naeslundii and Streptococcus sobrinus) and then using a microcosm model of dental plaque. The population shift was induced by environmental changes associated with gingivitis, first by the addition of artificial gingival crevicular fluid and then by a switch to a microaerophilic atmosphere. In addition to the observed population shifts, confocal laser scanning microscopy also revealed structural changes and differences in the distribution of viable and nonviable bacteria associated with the change in environmental conditions. This model provides an appropriate system for the further understanding of microbial population shifts associated with gingivitis and for the testing of, for example, antimicrobial agents.
Phosphate-based glass fibres (PGF) of the general formula Na(2)O-CaO-P(2)O(5) are degradable in an aqueous environment, and therefore can function as antibacterial delivery systems through the inclusion of ions such as copper. In this study, PGF with varying amounts of copper oxide (CuO) were developed for potential uses in wound healing applications. PGF with 0, 1, 5 and 10 mol% CuO were produced with different diameters and characterised in terms of structural and antibacterial properties. The effect of CuO and fibre pulling speed on the glass properties were investigated using rapid differential scanning calorimetry, differential thermal analysis and X-ray diffraction. The effect of two fibre diameters on short-term (3 h) attachment and killing against Staphylococcus epidermidis were investigated and were related to their rate of degradation in deionised water, as well as copper ion release measured using ion chromatography. Thermal analysis showed that there was a significant increase in the PGF glass transition temperature as the CuO content increased. There was a significant decrease in the rate of degradation with increasing CuO content and an increase in fibre diameter. Over 6 h, both the amount and rate of copper ions released increased with CuO content, as well as a reduction in fibre diameter thus increasing the surface area to volume ratio. There was a decrease in the number of viable staphylococci both attached to the CuO-containing fibres and in the surrounding environment.
AIMS: To isolate bacteriophages lytic for oral pathogens from human saliva, dental plaque and mature biofilms constituted from saliva-derived bacteria. METHODS AND RESULTS: Saliva and dental plaque samples from healthy volunteers and from patients with gingivitis and periodontitis were examined for the presence of lytic bacteriophage using a panel of oral pathogens and bacteria isolated from the samples. Samples were also enriched for bacteriophage using static culture techniques and mature biofilms. A limited number of samples contained bacteriophage particles that were visualized using electron microscopy. Cultures yielded phage infecting non-oral bacteria (Proteus mirabilis) but no bacteriophage specific for recognized oral pathogens were found. Some micro-organisms from the oral microflora elaborated antibacterial substances that inhibited growth of other residents of the oral cavity. CONCLUSIONS: Unlike other ecosystems, the composition of the oral cavity does not appear to be heavily influenced by interactions between bacteriophages and their hosts. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophage for control of oral infections may need to be obtained from other sources. Antibacterial substances derived from some members of the oral microflora warrant investigation as potential antibiotics.
The aim of this study was to develop an in vitro model of the tongue microflora in order to assess anti-malodour compounds before clinical evaluation. Biofilms, derived from the tongue microflora, were grown in a constant depth film fermentor (CDFF) with nutrients supplied in the form of mucin- and serum-containing artificial saliva. Differential agars and a halimeter were used to determine the bacterial microflora and production of volatile sulphur compounds (VSCs), respectively. The resulting biofilms had a bacterial population which contained, on average, 29% streptococci, 48% Gram-negative anaerobes and 2.5% with an H2S-producing phenotype. When the biofilms were pulsed with either chlorhexidine or zinc acetate there was a reduction in the number of H2S-producing bacteria, however these counts subsequently recovered as pulsing continued. The generation of VSCs was correlated to the viable counts of the H2S-producing bacteria. By pulsing with anti-malodour compounds over time we observed a reduction in the quantity of VSCs produced and a change in the composition of the plaque to one which contained fewer H2S-producing bacteria.
The aim of this study was to investigate the accumulation and composition of microcosm dental plaque on different orthodontic bonding materials using an in vitro model. Microcosm plaques were grown on discs of a range of bonding materials in a constant depth film fermentor. The biofilms were derived from human saliva and supplied with artificial saliva as a source of nutrients. The number of viable bacteria in the biofilms was determined and the streptococci present were identified to species level. The results showed that there was no significant difference in bacterial accumulation between different bonding materials, however, biofilms grown on materials which were fluoride releasing, did not contain Streptococcus mutans. This in vitro study has shown that the use of fluoride-releasing bonding materials may support the growth of supragingival plaque, which does not contain S. mutans.
The aim of this study was to compare culture-based bacterial isolation methods with direct amplification and cloning of 16S rRNA genes from oral biofilms grown in an in vitro model. The model used was a constant depth film fermentor which was inoculated with pooled human saliva. The use of culture techniques and cloning resulted in the identification of 36 different bacterial species from the saliva inoculum and from the biofilms. Of these, only five were detected solely by molecular methods. Three taxa were detected which, according to the databases, were unidentified. Using the molecular methods of detection, differences in the number of species observed were found using different 16S rRNA gene primers and numbers of PCR cycles. We have shown that microcosm supragingival plaque biofilms grown in a fermentor consisted of a community most of the members of which could be cultivated on laboratory media.
The aim of this study was to investigate the effects of tetracycline administration on the viability and antibiotic resistance profiles of microcosm dental plaques. A constant depth film fermenter was used to generate multi-species biofilms, which were grown for 216 h before tetracycline was added. The composition of the microcosm plaques was determined by viable counting on selective and non-selective media. The prevalence of antibiotic-resistant organisms was determined on antibiotic-containing media. Before administration of tetracycline, the biofilms had a total viable anaerobic count of 7 x 10(7) cfu per biofilm. They contained 7% lactobacilli, 19% streptococci and 2% Actinomyces spp. Immediately after pulsing with tetracycline, the composition of the biofilms changed and they consisted of 30% lactobacilli, 1.5% streptococci and 3% Actinomyces spp., with a total anaerobic count of 1 x 10(7) cfu per biofilm. The pre-valence and composition of the antibiotic-resistant microflora changed dramatically after the addition of tetracycline, with the proportion of the microflora displaying resistance to tetracycline increasing from 6% to 45%. Corresponding changes in the proportions of the microflora displaying resistance to other antibiotics were as follows: 5-28% for erythromycin, 1-5% for vancomycin and 0.4-3% for ampicillin. The results of this study have shown that the addition of tetracycline to microcosm dental plaques alters their composition and enriches for bacteria resistant to tetracycline and other unrelated agents.
Many of the genes encoding the virulence factors for Staphylococcus aureus are controlled by the accessory gene regulator (agr) and staphylococcal accessory regulator (sar). This regulation may be affected by the environment in which the organisms are grown. In the majority of ecosystems, bacteria grow attached to surfaces and form biofilms. We used S. aureus strains containing mutations inactivating agr and sar to determine whether the presence of these genes influences the attachment of the bacterium to a surface. We also used strains harbouring reporter constructs of the agr and sar operons to determine their expression in biofilms. The attachment study results showed that the sarA mutant strain adhered better to glass than did the agrA mutant or the wild type. There was an increased adherence to fibronectin-coated glass for all three strains compared to glass. Thus, these adhesion studies demonstrate that agr and sar have pleiotrophic effects on the surface expression of molecules responsible for binding to different substrata. In the biofilms higher numbers of bacteria and the greatest expression were observed at the base, but there were no observable differences between the reporter constructs. Expression of the agr and sar reporter fusions was significantly higher in the deepest layers of the biofilms where the greatest numbers of bacteria were also observed, perhaps as one might expect for genes that are regulated in a cell density dependent fashion.
AIM: The bactericidal effect of four antimicrobial agents was investigated against single-species biofilms derived from a range of root canal isolates. METHODOLOGY: Single-species biofilms of Prevotella intermedia, Peptostreptococcus micros, Streptococcus intermedius, Fusobacterium nucleatum and Enterococcus faecalis were generated on membrane filter discs and subjected to 15 min or 1 h incubation with 5 p.p.m. colloidal silver, 2.25% sodium hypochlorite (NaOCl), 0.2% chlorhexidine, 10% iodine or phosphate buffered saline (PBS) as a control. The antimicrobial activity of the agents was neutralized and the bacterial cells were harvested from the discs by vortexing, serially diluted in reduced transport fluid, plated on fastidious anaerobe agar containing 5% horse blood, incubated anaerobically and colony-forming units calculated. RESULTS: Iodine and NaOCl were more effective than chlorhexidine except against P. micros and P. intermedia where they were all 100% effective. Iodine and NaOCl elicited a 100% kill after 1 h incubation for all strains used. However, after 15 min, they showed differing bactericidal effects depending on the strain. None of the agents were effective against F. nucleatum after 15 min but NaOCl, iodine and chlorhexidine were all effective after 1 h. Colloidal silver was generally ineffective. CONCLUSIONS: The effectiveness of a particular agent was dependent on the nature of the organism in the biofilm and on the contact time. NaOCl was generally the most effective agent tested, followed by iodine. However the clinical efficacy of these agents must be considered in light of the complex root canal anatomy and polymicrobial nature of root canal infections.
AIM: The aim of this study was to develop an in vitro model to replicate microbial microleakage at a tooth/ restoration interface using a constant depth film fermentor (CDFF). METHODOLOGY: Amalgam restorations were placed in machined bovine dentine cylinders and sealed externally with varnish, leaving a 1-mm perimeter exposed around the tooth/restoration interface. The dentine cylinders were housed in a CDFF and 300-microm thick microcosm dental plaques were grown over their exposed surfaces. The biofilms were maintained with a mucin-containing artificial saliva for up to 8 weeks. Cylinders were aseptically removed from the CDFF (at 1, 2, 4, & 8 weeks) and surface-decontaminated with validated protocols prior to splitting and sampling of apposing amalgam and dentine surfaces. Scanning electron microscopy (SEM) was used to ascertain the position and structure of the bacterial aggregates. Bacterial viability was determined by vital staining of the bacteria in situ. RESULTS: At all sampling times, SEM showed cocci, rods and filaments on both amalgam and dentine surfaces; some originated as cascades from the surface biofilm and extended into the tooth/restoration microspace. Vital staining showed the majority of bacteria from both dentine and amalgam surfaces to be viable. CONCLUSION: This preliminary investigation showed that the CDFF may be a valuable tool for the in vitro study of the dynamics of microbial microleakage around dental restorations.
Microcosm dental plaques were grown from an inoculum of human saliva in a constant-depth film fermentor. The inoculum contained four tetracycline-resistant streptococcal species, each of which contained a Tn916-like element. This element was shown to transfer to other streptococci both in filter-mating experiments and within the biofilms in the fermentor.
The aim of this study was to investigate the structure of intact oral biofilms using confocal laser scanning microscopy (CLSM). Mixed-species biofilms were grown on enamel discs in a constant depth film fermentor. The biofilms were fed with a mucin-containing artificial saliva with or without sucrose supplementation. Biofilms were examined using a Wild-Leitz CLSM, operating in reflected light mode. The microstructure of non-supplemented biofilms was revealed to be complex, with stacks of bacteria developing over time, separated by clear channels. Sucrose-supplemented biofilms appeared to colonise the substratum more rapidly. The results of this study have shown that using CLSM it is possible to examine the structure of oral biofilms grown under conditions similar to those which would exist in vivo.
Microcosmic dental plaques were grown in artificial saliva and supplemented with either milk or fluoridated milk. The presence of fluoride in the milk increased the pH of the biofilms and reduced the proportions of streptococci, demonstrating that in this model, fluoridation of milk produces biofilms with reduced cariogenic potential.
A tetracycline resistance profile was established from a microcosm dental plaque in a constant depth film fermenter. The fermenter was inoculated with a Bacillus subtilis strain which contained the conjugative transposon, Tn5397, which confers tetracycline resistance upon its host. After 6 hour and 24 hour the tetracycline resistance profile of the biofilm was redetermined and a tetracycline resistant Streptococcus species was isolated. A molecular analysis of this strain confirmed that Tn5397 was present in the genomic DNA of the isolate. These data represent the first report, to our knowledge, of intergeneric transfer of a conjugative transposon in a mixed species biofilm and demonstrates the ability of conjugative transposons to disseminate antibiotic resistance genes in a mixed species environment.
The aims of this study were to evaluate the effects of repeated chlorhexidine gluconate (CHG) pulsing on the viability and bacterial composition of microcosm dental plaques derived from human saliva. The biofilms were grown on bovine enamel discs in a constant-depth film fermentor fed with an artificial saliva which was supplemented thrice daily with sucrose. The microcosm plaques had total viable anaerobic counts of 5 x 10(8) CFU per mm2 and consisted of 12% Actinomyces spp., 85% streptococci, and 0.2% Veillonella spp. When pulsed twice daily with 0.2% CHG, there was an immediate 1.3-log10 reduction in the total viable (anaerobic) count. However, as pulsing continued, the viable counts recovered, and after 4 days, the anaerobic count reached its pre-CHG-pulsing level, although the bacterial composition of the biofilms had changed. The results of this study show that twice-daily pulsing with 0.2% CHG over a 4-day period was ineffective at reducing the total anaerobic viable count of the biofilms but did alter their bacterial composition.
The aims of this study were to evaluate the growth of Streptococcus sanguis on hydroxyapatite, bovine enamel and polytetrafluoroethylene substrata in a constant depth film fermentor, and to determine the effects of three antimicrobial-containing mouthwashes on biofilm formation and bacterial viability on hydroxyapatite and enamel. There was little difference in the final cell density (5 x 10(4) cfu mm-2) of the Strep. sanguis biofilm on the three substrata. When hydroxyapatite-grown biofilms were exposed to the mouthwashes for 1 min, the one containing triclosan (T) proved the most effective. The chlorhexidine-containing mouthwash (CX) also achieved significant kills. The T-containing mouthwash was the most effective at killing biofilms grown on enamel. Pre-treatment of hydroxyapatite with CX, cetylpyridium chloride (CPC) or T for 1 min resulted in undetectable biofilm formation after 8 h. After 8 h of growth, only biofilms grown on enamel discs pre-treated with CX showed a reduction in the number of viable organisms. In conclusion, the results of this study have shown that while growth of Strep. sanguis on hydroxyapatite and enamel were similar, the ability of antimicrobial agents to prevent the accumulation of viable bacteria depended on the nature of the substratum.
The aim of this study was to determine the effect of pulsing chlorhexidine gluconate, at concentrations commonly used in mouthwashes, on Streptococcus sanguis biofilms and microcosm dental plaques in vitro. Biofilms were grown on bovine enamel and nutrients were supplied in the form of artificial saliva. Pulsing experiments were carried out on steady-state biofilms using 0.05 or 0.2% chlorhexidine solutions delivered twice daily for 1 min. In a separate study, the enamel discs on which the biofilms were formed were pre-treated with chlorhexidine and pulsed directly after inoculation and then at regular intervals. With both concentrations of chlorhexidine used, a c.2 log10 reduction in the viable counts of S. sanguis was achieved with the initial pulse, but as pulsing continued, the bacterial population recovered, albeit not to the previous level. A c.1 log10 reduction in the total viable counts of the microcosm plaques was seen after the first pulse with 0.2% chlorhexidine. The total count then recovered rapidly and, after the fifth pulse, the total viable counts were not significantly different from those before pulsing. The total counts then remained at a similar level throughout the course of the experimental runs. Pre-treatment of the enamel discs with 0.2% chlorhexidine before inoculation produced viable counts of c.10(5) cfu/mm2, a 1 log10 reduction compared with untreated discs. After pulsing with 0.2% chlorhexidine at 8 h, a 3 log10 reduction was seen in the total aerobic and anaerobic counts, but again the viable counts subsequently increased despite twice-daily chlorhexidine pulsing. Regardless of the nature of the biofilm, pulsing initially achieved substantial kills, but the viability of the biofilms subsequently increased despite continued pulsing. Chlorhexidine was effective at reducing the viability of microcosm plaques when it was applied to the substratum before exposure to bacteria and subsequently pulsed on to the biofilms.