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 ¼ 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 log 10 colony-forming units (CFU)/filter] comprising considerable numbers of streptococci and Gramnegative species. PA contained ca 9-10 log 10 CFU ml )1 suggesting an apparent mean growth rate of 0AE1 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 Gramnegative 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 microecology of the oral cavity.

Quaternary ammonium compounds (QACs) are widely used as adjuncts to hygiene in domestic cleaning products. Current concern that the increased use of such biocides in consumer products might contribute to the emergence of antibiotic resistance has led us to examine the effects of a QAC-containing domestic cleaning fluid on the population dynamics and antimicrobial susceptibility of domestic sink drain biofilm communities. QAC susceptibilities of numerically dominant, culturable drain bacteria (15 genera, 17 species) were determined in vitro before and after repeated QAC exposure (14 passages). A fully characterized drain microcosm was then exposed to short-term (12 days) and long-term (3 months) dosing with a QAC-containing domestic detergent (QD). QAC exposure of isolated cultures caused both increases (three species) and circa twofold decreases (six species) in QAC susceptibility. The susceptibility of Ralstonia sp. was considerably decreased following 14 consecutive QAC passages. Control drain microcosm biofilms maintained dynamic stability, as evidenced by culture and denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial population densities were largely unaffected during short-term exposure to use levels of QD, although 50% QD caused circa 10-fold viability reductions. DGGE analysis supported these observations; identified the major microcosm genera as Pseudomonas, Pseudoalteromonas, Erwinia, and Enterobacter, and showed that aeromonads increased in abundance under 10 to 50% QD. Long-term exposure of the microcosms to QD did not significantly alter the pattern of antimicrobial susceptibility. These data demonstrate the recalcitrance of domestic drain biofilms toward QAC and that although repeated QAC exposure of drain isolates in pure culture results in susceptibility change in some test bacteria, such changes do not necessarily occur within complex communities.

This investigation provides molecular analyses of the periodontal microbiota in health and disease. Subgingival samples from 47 volunteers with healthy gingivae or clinically diagnosed chronic periodontitis were characterized by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the V2-V3 region of the eubacterial 16S rRNA gene. A hierarchical dendrogram was constructed from band patterns. All unique PCR amplicons (DGGE bands) were sequenced for identity. Samples were also analyzed for the presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis by multiplex PCR. Associations of patient age, gender, and smoking status together with the presence of each unique band and putative periodontal pathogens with disease were assessed by logistic regression. Periodontal pockets were colonized by complex eubacterial communities (10 to 40 distinct DGGE bands) with substantial individual variation in the community profile. Species diversity in health and disease was determined by the ShannonWeaver index of diversity and compared by the Mann-Whitney U test. Sequence analyses of DGGE amplicons indicated the occurrence of many nontypical oral species and eubacteria previously associated with this environment. With the exception of T. forsythensis, the putative pathogens were not detected by DGGE. Multiplex PCR, however, detected T. forsythensis, A. actinomycetemcomitans, and P. gingivalis in 9% 16%, and 29% of the patients with disease, respectively. The presence of A. actinomycetemcomitans was significantly associated with disease (P < 0.01). Statistical analyses indicated that the presence of Treponema socranskii and Pseudomonas sp. was a significant predictor of disease (P < 0.05) and that there was no significant difference (P > 0.05) in terms of eubacterial species diversity between health and disease.Periodontitis is a generic term relating to inflammation of the tissues supporting the teeth but is widely attributed to succession by polymicrobial communities (36,58,74). The etiology of the condition is further complicated by the presence of a complex resident subgingival microbiota that underlies both periodontal health and disease (22,45). Periodontitis is often self-limiting; invasion of bacteria beyond the gingival tissue is rare (32). No single etiologic agent has been identified; rather, specific groups and combinations of bacteria including Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythensis have been strongly associated with pathology (11, 32, 58). Emerging research now implicates both host genetic and immunological factors as being important in disease susceptibility (9,11,23,24), further demonstrating the complex nature of this condition.Plaque accumulates in the mouth at sites such as the gingival margin, where shear forces are low (36). Chronic bacterial colonization of this site, often in the absence of effective oral hygiene, leads to inflammation of the adjacent gingival tissue, termed gingivitis. Chronic gingivit...

We have used heterotrophic plate counts, together with live-dead direct staining and denaturing gradient gel electrophoresis (DGGE), to characterize the eubacterial communities that had formed as biofilms within domestic sink drain outlets. Laboratory microcosms of these environments were established using excised biofilms from two separate drain biofilm samples to inoculate constant-depth film fermentors (CDFFs). Drain biofilms harbored 9.8 to 11.3 log 10 cells of viable enteric species and pseudomonads/g, while CDFF-grown biofilms harbored 10.6 to 11.4 log 10 cells/g. Since live-dead direct staining revealed various efficiencies of recovery by culture, samples were analyzed by DGGE, utilizing primers specific for the V2-V3 region of eubacterial 16S rDNA. These analyses showed that the major PCR amplicons from in situ material were represented in the microcosms and maintained there over extended periods. Sequencing of amplicons resolved by DGGE revealed that the biofilms were dominated by a small number of genera, which were also isolated by culture. One drain sample harbored the protozoan Colpoda maupasi, together with rhabtidid nematodes and bdelloid rotifers. The microcosm enables the maintenance of stable drain-type bacterial communities and represents a useful tool for the modeling of this ecosystem.Clinical epidemiologists have long recognized the potential of sink drains in hospital wards to harbor pathogens. Several studies have identified sink drains within medical-surgical intensive-care wards (19,22,30,35) and cystic fibrosis units (24) as possible sources of infection. Despite the increased information relating to the occurrence of bacterial biofilms and their reported involvement in the biofouling of domestic drains (7), there are few reports in the literature concerning the ecology and microbiology of this environment. The persistence (1) and significance (10) of biofilms in virtually all environments is widely acknowledged. Studies in the home (14, 34) have identified the potential health risks of microbial contamination. Scott et al. (34) identified possible pathogens in the kitchen, toilet, and bathrooms in Ͼ200 homes in the United Kingdom. More recent studies (3,8,9) have demonstrated that homes represent an environment into which bacterial, viral, and fungal pathogens are continuously introduced in association with food, people, and pets. Studies by Cogan et al. (8,9) showed that detergent-based cleaning was relatively ineffective in controlling the spread of salmonella and campylobacter to kitchen surfaces during the preparation of contaminated poultry. Despite such concerns, there have been few investigations into the bacterial composition of biofilms within domestic sink drains. As with hospital drains, the pipe work presents a variety of solid surfaces that are suitable substrates for biofilm formation (7, 26). Biofilm has been implicated in a high proportion of slow-running drains in the United States (7). Domestic drains are subject to intermittent wetting, periodic feeding with a plet...

Recent concern that the increased use of triclosan (TCS) in consumer products may contribute to the emergence of antibiotic resistance has led us to examine the effects of TCS dosing on domestic-drain biofilm microcosms. TCS-containing domestic detergent (TCSD) markedly lowered biofouling at 50% (wt/vol) but was poorly effective at use levels. Long-term microcosms were established and stabilized for 6 months before one was subjected to successive 3-month exposures to TCSD at sublethal concentrations (0.2 and 0.4% [wt/vol]). Culturable bacteria were identified by 16S rDNA sequence analysis, and their susceptibilities to four biocides and six antibiotics were determined. Microcosms harbored ca. 10 log 10 CFU/g of biofilm, representing at least 27 species, mainly gamma proteobacteria, and maintained dynamic stability. Viable cell counts were largely unaffected by TCSD exposure, but species diversity was decreased, as corroborated by denaturing gradient gel electrophoresis analysis. TCS susceptibilities ranged widely within bacterial groups, and TCS-tolerant strains (including aeromonads, pseudomonads, stenotrophomonads, and Alcaligenes spp.) were isolated before and after TCSD exposure. Several TCS-tolerant bacteria related to Achromobacter xylosoxidans became clonally expanded during dosing. TCSD addition did not significantly affect the community profiles of susceptibility to the test biocides or antibiotics. Several microcosm isolates, as well as reference bacteria, caused clearing of particulate TCS in solid media. Incubations of consortia and isolates with particulate TCS in liquid led to putative TCS degradation by the consortia and TCS solubilization by the reference strains. Our results support the view that low-level exposure of environmental microcosms to TCS does not affect antimicrobial susceptibility and that TCS is degradable by common domestic biofilms.

Oral bacterial microcosms, established using saliva inocula from three individuals, were maintained under a feast-famine regime within constant-depth film fermenters. Steady-state communities were exposed four times daily, postfeeding, to a chlorhexidine (CHX) gluconate-containing mouthwash (CHXM) diluted to 0.06% (wt/vol) antimicrobial content. The microcosms were characterized by heterotrophic plate counts and PCRdenaturing gradient gel electrophoresis (DGGE). CHXM caused significant decreases in both total anaerobe and total aerobe/facultative anaerobe counts (P < 0.05), together with lesser decreases in gram-negative anaerobes. The degree of streptococcal and actinomycete inhibition varied considerably among individuals. DGGE showed that CHXM exposure caused considerable decreases in microbial diversity, including marked reductions in Prevotella sp. and Selenomonas infelix. Pure-culture studies of 10 oral bacteria (eight genera) showed that Actinomyces naeslundii, Veillonella dispar, Prevotella nigrescens, and the streptococci were highly susceptible to CHX, while Lactobacillus rhamnosus, Fusobacterium nucleatum, and Neisseria subflava were the least susceptible. Determination of the MICs of triclosan, CHX, erythromycin, penicillin V, vancomycin, and metronidazole for microcosm isolates, before and after 5 days of CHXM exposure, showed that CHXM exposure altered the distribution of isolates toward those that were less susceptible to CHX (P < 0.05). Changes in susceptibility distributions for the other test agents were not statistically significant. In conclusion, population changes in plaque microcosms following repeated exposure to CHXM represented an inhibition of the most susceptible flora with a clonal expansion of less susceptible species.Chlorhexidine (CHX), a cationic bis-biguanide biocide with low mammalian toxicity and broad-spectrum antibacterial (6) activity, was first described in 1954 (5). The primary mechanism of action of this biocide is membrane disruption, causing concentration-dependent growth inhibition and cell death (18). Secondary interactions causing inhibition of proteolytic and glycosidic enzymes may also be significant (15). With respect to dental hygiene applications, the cationic nature of CHX enables it to bind to tooth surfaces and oral mucosa, reducing pellicle formation and increasing substantivity through controlled release of the agent (2). The efficacy of CHX in reducing oral bacterial viability (14,36,42), strongly inhibiting plaque regrowth, and preventing gingivitis (25) has been demonstrated in many studies (7). Relatively few investigations have considered longer-term effects of CHX use. An early study, however, demonstrated that oral treatment of human volunteers with CHX resulted in a 30 to 50% reduction in total bacterial counts with an associated reduction in counts of Streptococcus mutans (38).Recent reports have demonstrated that the chlorinated diphenylether antibacterial triclosan (TCS) can select for mutants in the FabI gene of Escherichia coli at sublethal co...

This study demonstrates the utility of DGGE for the analysis of dental plaque, especially with respect to unculturable bacteria. Results question the assumptions of reproducibility of plaque microcosms established in non-replicated CDFFs made on the basis of selective media. Feeding regimes, particularly those involving complex nutrients, will dramatically affect population dynamics.

The coaggregation ability of bacteria isolated from a freshwater biofilm was compared to those derived from the coexisting planktonic population. Twenty-nine morphologically distinct bacterial strains were isolated from a 6-month-old biofilm, established in a glass tank under high-shear conditions, and 15 distinct strains were isolated from the associated re-circulating water. All 44 strains were identified to genus or species level by 16S rDNA sequencing. The 29 biofilm strains belonged to 14 genera and 23.4% of all the possible pair-wise combinations coaggregated. The 15 planktonic strains belonged to seven genera and only 5.8% of all the possible pair-wise combinations coaggregated. Therefore, compared to the planktonic population, a greater proportion of the biofilm strains coaggregated. It is proposed that coaggregation influences biofilm formation and species diversity in freshwater under high shear.