Microbiological insights and dermatological applications of live biotherapeutic products.

As our understanding of dermatological conditions advances, it becomes increasingly evident that traditional pharmaceutical interventions are not universally effective. The intricate balance of skin microbiota plays a pivotal role in the development of various skin conditions, prompting a growing interest in probiotics, or live biotherapeutic products (LBPs), as potential remedies. Specifically, the topical application of LBPs to modulate bacterial populations on the skin has emerged as a promising approach to alleviate symptoms associated with common skin conditions. This review considers LBPs and their application in addressing a wide spectrum of dermatological conditions with particular emphasis on three key areas: acne, atopic dermatitis, and wound healing. Within this context, the critical role of strain selection is presented as a pivotal factor in effectively managing these dermatological concerns. Additionally, the review considers formulation challenges associated with probiotic viability and proposes a personalised approach to facilitate compatibility with the skin's unique microenvironment. This analysis offers valuable insights into the potential of LBPs in dermatological applications, underlining their promise in reshaping the landscape of dermatological treatments while acknowledging the hurdles that must be overcome to unlock their full potential.

Variable effects of exposure to ionic silver in wound-associated bacterial pathogens.

Silver compounds are used in wound dressings to reduce bioburden. Where infection is not rapidly resolved, bacteria may be exposed to sub-therapeutic concentrations of antimicrobials over prolonged periods of time. In this study, a panel of chronic wound bacteria, Pseudomonas aeruginosa (two strains), Staphylococcus aureus, and Escherichia coli, were exposed to silver nitrate on agar. Phenotypic characterization was achieved using broth microdilution sensitivity testing, a crystal violet biofilm assay, and a wax moth pathogenesis model. Repeated exposure to ionic silver did not result in planktonic phenotypic silver resistance in any of the test panels, although S. aureus demonstrated reversible increases in minimum bactericidal concentration. An ulcer-derived P. aeruginosa exhibited marked reductions in biofilm eradication concentration as well as significantly increased biofilm formation and wax moth killing when compared to the same progenitor. These changes were reversible, trending towards baseline measurements following 10 passages on silver-free media. Changes in virulence and biofilm formation in the other test bacteria were generally limited. In summary, phenotypic adaptation following exposure to ionic silver was manifested other than through changes in planktonic susceptibility. Significant changes in pseudomonas biofilm formation and sensitivity could have implications for wound care regimes and therefore warrant further investigation.

An update on the prevalence of colistin and carbapenem-resistant Gram-negative bacteria in aquaculture: an emerging threat to public health.

Aquaculture has been recognized as a hotspot for the emergence and spread of antimicrobial resistance genes conferring resistance to clinically important antibiotics. This review gives insights into studies investigating the prevalence of colistin and carbapenem resistance (CCR) among Gram-negative bacilli in aquaculture. Overall, a high incidence of CCR has been reported in aquatic farms in several countries, with CCR being more prevalent among opportunistic human pathogens such as Acinetobacter nosocomialis, Shewanella algae, Photobacterium damselae, Vibrio spp., Aeromonas spp., as well as members of Enterobacteriaceae family. A high proportion of isolates in these studies exhibited wide-spectrum profiles of antimicrobial resistance, highlighting their multidrug-resistance properties (MDR). Several mobile colistin resistance genes (including, mcr-1, mcr-1.1, mcr-2, mcr-2.1, mcr-3, mcr-3.1, mcr-4.1, mcr-4.3, mcr-5.1, mcr-6.1, mcr-7.1, mcr-8.1, and mcr-10.1) and carbapenemase encoding genes (including, blaOXA-48, blaOXA-55, blaNDM, blaKPC, blaIMI, blaAIM, blaVIM, and blaIMP) have been detected in aquatic farms in different countries. The majority of these were carried on MDR Incompatibility (Inc) plasmids including IncA/C, and IncX4, which have been associated with a wide host range of different sources. Thus, there is a risk for the possible spread of resistance genes between fish, their environments, and humans. These findings highlight the need to monitor and regulate the usage of antimicrobials in aquaculture. A multisectoral and transdisciplinary (One Health) approach is urgently needed to reduce the spread of resistant bacteria and/or resistance genes originating in aquaculture and avoid their global reach.

Escherichia coli Nissle 1917 inhibits biofilm formation and mitigates virulence in Pseudomonas aeruginosa.

In the quest for mitigators of bacterial virulence, cell-free supernatants (CFS) from 25 human commensal and associated bacteria were tested for activity against Pseudomonas aeruginosa. Among these, Escherichia coli Nissle 1917 CFS significantly inhibited biofilm formation and dispersed extant pseudomonas biofilms without inhibiting planktonic bacterial growth. eDNA was reduced in biofilms following exposure to E. coli Nissle CFS, as visualized by confocal microscopy. E. coli Nissle CFS also showed a significant protective effect in a Galleria mellonella-based larval virulence assay when administrated 24 h before challenge with the P. aeruginosa. No inhibitory effects against P. aeruginosa were observed for other tested E. coli strains. According to proteomic analysis, E. coli Nissle CFS downregulated the expression of several P. aeruginosa proteins involved in motility (Flagellar secretion chaperone FliSB, B-type flagellin fliC, Type IV pilus assembly ATPase PilB), and quorum sensing (acyl-homoserine lactone synthase lasI and HTH-type quorum-sensing regulator rhlR), which are associated with biofilm formation. Physicochemical characterization of the putative antibiofilm compound(s) indicates the involvement of heat-labile proteinaceous factors of greater than 30 kDa molecular size.

Modulation of Biofilm Formation and Permeability in Streptococcus mutans during Exposure To Zinc Acetate.

The penetration of biofilms by antimicrobials is a potential limiting factor in biofilm control. This is relevant to oral health, as compounds that are used to control microbial growth and activities could also affect the permeability of dental plaque biofilm with secondary effects on biofilm tolerance. We investigated the effects of zinc salts on the permeability of Streptococcus mutans biofilms. Biofilms were grown with low concentrations of zinc acetate (ZA), and a transwell transportation assay was applied to test biofilm permeability in an apical-basolateral direction. Crystal violet assays and total viable counts were used to quantify the biofilm formation and viability, respectively, and short time frame diffusion rates within microcolonies were determined using spatial intensity distribution analysis (SpIDA). While the diffusion rates within biofilm microcolonies were not significantly altered, exposure to ZA significantly increased the overall permeability of S. mutans biofilms (P < 0.05) through decreased biofilm formation, particularly at concentrations above 0.3 mg/mL. Transport was significantly lower through biofilms grown in high sucrose conditions. IMPORTANCE Zinc salts are added to dentifrices to improve oral hygiene through the control of dental plaque. We describe a method for determining biofilm permeability and show a moderate inhibitory effect of zinc acetate on biofilm formation, and that this inhibitory effect is associated with increases in overall biofilm permeability.

Supramolecular architecture of a multi-component biomimetic lipid barrier formulation.

HYPOTHESIS: Biomimetic liquid crystalline systems are widely used in skin care cosmetics and topical pharmaceutical preparations. Our ability to rationally design such formulations, however, is hampered by our incomplete understanding of their structure on the nanoscale. EXPERIMENTS: Using polarized light microscopy and small-angle and wide-angle X-ray scattering, the molecular architecture and properties of a barrier formulation prepared from distearoylphosphatidylcholine mixed with long chain fatty acid and alcohols, with and without antimicrobial pentanediols are directly probed. The nature and composition of the phases identified are determined through small-angle neutron scattering studies using chain-deuterated components, and the detailed structure and dynamics of the gel network lamellae are determined through molecular dynamics simulations. FINDINGS: The formulations show molecular ordering with long and short periodicity lamellar phases and there is little change in these structures caused by changes in temperature, drying, or the application of shear stress. The diol-free formulation is demonstrated to be self-preserving, and the added pentanediols are shown to distribute within the interlamellar regions where they limit availability of water for microbial growth. In culmination of these studies, we develop a more complete picture of these complex biomimetic preparations, and thereby enable their structure-based design.

Exposure to a Manuka Honey Wound Gel Is Associated With Changes in Bacterial Virulence and Antimicrobial Susceptibility.

The use of manuka honey for the topical treatment of wounds has increased worldwide owing to its broad spectrum of activity towards bacteria in both planktonic and biofilm growth modes. Despite this, the potential consequences of bacterial exposure to manuka honey, as may occur during the treatment of chronic wounds, are not fully understood. Here, we describe changes in antimicrobial susceptibility and virulence in a panel of bacteria, including wound isolates, following repeated exposure (ten passages) to sub-inhibitory concentrations of a manuka honey based wound gel. Changes in antibiotic sensitivity above 4-fold were predominantly related to increased vancomycin sensitivity in the staphylococci. Interestingly, Staphylococcus epidermidis displayed phenotypic resistance to erythromycin following passaging, with susceptibility profiles returning to baseline in the absence of further honey exposure. Changes in susceptibility to the tested wound gel were moderate (≤ 1-fold) when compared to the respective parent strain. In sessile communities, increased biofilm eradication concentrations over 4-fold occurred in a wound isolate of Pseudomonas aeruginosa (WIBG 2.2) as evidenced by a 7-fold reduction in gentamicin sensitivity following passaging. With regards to pathogenesis, 4/8 bacteria exhibited enhanced virulence following honey wound gel exposure. In the pseudomonads and S. epidermidis, this occurred in conjunction with increased haemolysis and biofilm formation, whilst P. aeruginosa also exhibited increased pyocyanin production. Where virulence attenuation was noted in a passaged wound isolate of S. aureus (WIBG 1.6), this was concomitant to delayed coagulation and reduced haemolytic potential. Overall, passaging in the presence of a manuka honey wound gel led to changes in antimicrobial sensitivity and virulence that varied between test bacteria.

Mitigation of the Toxic Effects of Periodontal Pathogens by Candidate Probiotics in Oral Keratinocytes, and in an Invertebrate Model.

The larvae of the wax moth Galleria mellonella and human oral keratinocytes were used to investigate the protective activity of the candidate oral probiotics Lactobacillus rhamnosus GG (LHR), Lactobacillus reuteri (LR), and Streptococcus salivarius K-12 (SS) against the periodontal pathogens Fusobacterium nucleatum (FN), Porphyromonas gingivalis (PG), and Aggregatibacter actinomycetemcomitans (AA). Probiotics were delivered to the larvae (i) concomitantly with the pathogen in the same larval pro-leg; (ii) concomitantly with the pathogen in different pro-legs, and (iii) before inoculation with the pathogen in different pro-legs. Probiotics were delivered as viable cells, cell lysates or cell supernatants to the oral keratinocytes concomitantly with the pathogen. The periodontal pathogens killed at least 50% of larvae within 24 h although PG and FN were significantly more virulent than AA in the order FN > PG > AA and were also significantly lethal to mammalian cells. The candidate probiotics, however, were not lethal to the larvae or human oral keratinocytes at doses up to 107 cells/larvae. Wax worm survival rates increased up to 60% for some probiotic/pathogen combinations compared with control larvae inoculated with pathogens only. SS was the most effective probiotic against FN challenge and LHR the least, in simultaneous administration and pre-treatment, SS and LR were generally the most protective against all pathogens (up to 60% survival). For P. gingivalis, LR > LHR > SS, and for A. actinomycetemcomitans SS > LHR and LR. Administering the candidate probiotics to human oral keratinocytes significantly decreased the toxic effects of the periodontal pathogens. In summary, the periodontal pathogens were variably lethal to G. mellonella and human oral keratinocytes and the candidate probiotics had measurable protective effects, which were greatest when administrated simultaneously with the periodontal pathogens, suggesting protective effects based on bacterial interaction, and providing a basis for mechanistic studies.

Visualization and Quantification of the Oral Hygiene Effects of Brushing, Dentifrice Use, and Brush Wear Using a Tooth Brushing Simulator.

Approaches that reproduce dental hygiene regimens under controlled conditions have applications in preclinical research. We have applied standardized, reproducible brushing regimes to typodonts coated in simulated or biological plaques to assess the effects on tooth cleaning of toothbrush/dentifrice regimens. Replicated typodonts were coated with OccludeTM or GlogermTM indicators to simulate plaque, and brushed reproducibly using a mechanical brushing simulator to compare the cleaning of occlusal surfaces before and after brushing with water or a dentifrice. An in vitro model using salivary inocula to cultivate oral biofilms on typodont surfaces was then developed to evaluate removal of disclosed plaque by new toothbrushes in comparison to toothbrushes with wear equivalent to 3 months of use. Analyses of typodonts brushed under controlled conditions significantly (p < 0.01) distinguished between brushed and unbrushed surfaces and between the use of water vs. dentifrice for the removal of simulated interproximal plaque (p < 0.05). New toothbrushes removed significantly (p < 0.05) more biological plaque from typodont surfaces than brushes that had been worn by repeated brushing. Through controlled and defined brushing of typodonts with simulated and biological plaques, the effectiveness of dental hygiene regimens was compared under reproducible conditions. Data indicate that the cleaning effectiveness of brushing was augmented by the addition of dentifrice and that new brushes were significantly more effective than brushes with substantial wear from previous use. Whilst we have focussed on the occlusal surfaces of molars and worn brushes, the method could be applied to a range of other tooth surfaces and oral hygiene regimens.

Oral Microbiota in Severe Early Childhood Caries in Thai Children and Their Families: A Pilot Study.

Thailand has a comparatively high prevalence of severe early childhood caries (S-ECC). S-ECC adversely affects the quality of life for children and their caregivers and represents a considerable economic burden. We have assessed the bacteriological composition of unstimulated saliva, dental plaque, and degraded dentine in a Thai cohort, including children with S-ECC and children without cavities; their siblings, and their primary caregivers. Samples were collected during a dental examination and patients were scored for plaque accumulation and their decayed, missing, and filled teeth (dmft) index. Samples were analyzed using differential bacteriological counting and gel-based eubacterial DNA profiling. Plaque Lactobacillus abundance correlated significantly with S-ECC. Whilst Lactobacillus counts were significantly higher in children with S-ECC than in their siblings and primary caregivers (five families), the opposite trend was apparent for cavity-free children. Counts of Gram-negative anaerobes were significantly lower in children with S-ECC than orally healthy children. S-ECC correlated significantly with plaque index scores, dmft, and with Lactobacillus abundance in a highly predictive manner. DNA profiles showed significant homology between families but not within non-cavity and S-ECC groups. In conclusion, salivary and plaque Lactobacillus counts were significantly associated with S-ECC in the Thai subjects. Lactobacillus counts in the children were not correlated with those of their siblings and primary caregivers. Individuals could be significantly differentiated based on family but not on caries status.

Synthesis and antibacterial activities of enamine derivatives of dehydroacetic acid.

Dehydroacetic acid is a common pyrone derivative used commercially as an antibacterial and antifungal agent. Based on the synthesis of dehydroacetic acid (1) from N-hydroxysuccinimdyl acetoacetate, a novel series of enamine-based derivatives were synthesised in order to improve the antibacterial activity of dehydroacetic acid. The antibacterial activities of the synthesised analogues were evaluated against Escherichia coli and Staphylococcus aureus. Derivative 4d (N-Ph) was identified as the most potent inhibitor of S. aureus growth. Overall, derivative 4b (N-Me) showed the best broad-spectrum activity with five-fold greater minimum inhibitory concentration and 11-fold greater minimum biocidal concentration against E. coli when compared to dehydroacetic acid, in addition to improved antibacterial activity against S. aureus.

Fatty Acid Supplementation Reverses the Small Colony Variant Phenotype in Triclosan-Adapted Staphylococcus aureus: Genetic, Proteomic and Phenotypic Analyses.

Staphylococcus aureus can develop a small colony variant (SCV) phenotype in response to sub-lethal exposure to the biocide triclosan. In the current study, whole genome sequencing was performed and changes in virulence were investigated in five Staphylococcus aureus strains following repeated exposure to triclosan. Following exposure, 4/5 formed SCV and exhibited point mutations in the triclosan target gene fabI with 2/4 SCVs showing mutations in both fabI and fabD. The SCV phenotype was in all cases immediately reversed by nutritional supplementation with fatty acids or by repeated growth in the absence of triclosan, although fabI mutations persisted in 3/4 reverted SCVs. Virulence, determined using keratinocyte invasion and Galleria mellonella pathogenicity assays was significantly (p < 0.05) attenuated in 3/4 SCVs and in the non-SCV triclosan-adapted bacterium. Proteomic analysis revealed elevated FabI in 2/3 SCV and down-regulation in a protein associated with virulence in 1/3 SCV. In summary, attenuated keratinocyte invasion and larval virulence in triclosan-induced SCVs was associated with decreases in growth rate and virulence factor expression. Mutation occurred in fabI, which encodes the main triclosan target in all SCVs and the phenotype was reversed by fatty acid supplementation, demonstrating an association between fatty acid metabolism and triclosan-induced SCV.

Arginine Exposure Decreases Acidogenesis in Long-Term Oral Biofilm Microcosms.

Arginine is an important pH-elevating agent in the oral cavity. It has been incorporated in oral hygiene formulations to mitigate sensitivity and to prevent caries. In this investigation, the effects of sustained arginine dosing of dental plaque microcosms on bacteriological composition and pH were evaluated under controlled conditions. Plaque microcosms were established in constant-depth film fermentors (CDFFs) using salivary inocula and fed continuously with artificial saliva. To simulate resting and cariogenic states, the CDFFs were supplemented with sterile water or 5% sucrose, respectively. Plaques were then dosed twice daily with a dentifrice with 1.5% arginine arginine added (DA) or without arginine (DN). This regimen continued for over 3 weeks, after which fermentors were maintained without dosing. Microcosms were analyzed by differential viable counting, with a pH microelectrode, and by eubacterial DNA profiling. Sucrose dosing was associated with significantly (P < 0.001) decreased pH, significantly (P < 0.05) increased counts of total aerobes, Gram-negative anaerobes, aciduric species, acidogenic species, arginine utilizing species, bifidobacteria, lactobacilli and streptococci, and significant (P < 0.05) changes in DNA profiles. Plaques dosed with DA had a significantly (P < 0.001) higher pH than those dosed with DN, with or without sucrose supplementation. Dosing with DA but not DN significantly (P < 0.05) decreased counts of all functional bacterial groups apart from the total anaerobes in cariogenic plaques, and in resting plaques, dosing with DA significantly (P < 0.05) decreased counts of streptococci, lactobacilli, bifidobacteria, and acidogenic bacteria. In summary, sustained exposure of oral microcosms to arginine in formulation significantly increased plaque pH and significantly reduced the viability of cariogenic bacterial species. IMPORTANCE Arginine is used in dental health formulations to help prevent dental cavities. This study assessed the effects of the long-term dosing of laboratory dental plaques with an arginine dentifrice. Data indicate that the addition of arginine dentifrice during sucrose challenge significantly increased plaque pH, thus potentially mitigating cariogenesis. Counts of several functional groups of bacteria associated with tooth decay were significantly decreased in the laboratory plaques during exposure to the arginine dentifrice.

Faucicola mancuniensis gen. nov., sp. nov., isolated from the human oropharynx.

An aerobic, Gram-stain-negative, non-motile coccus, designated strain GVCNT2(T), was isolated from the tonsils of a healthy adult female. Cells were oxidase- and catalase-positive, positive for the production of esterase (C4), esterase lipase (C8) and leucine arylamidase, and weakly positive for naphthol-AS-BI-phosphohydrolase and alkaline phosphatase. Cells were also capable of hydrolysing DNA. Growth was observed at 20-37 °C and in the presence of up to 1.5% NaCl. Phylogenetic analysis of near full-length 16S rRNA gene sequences indicated that the strain exhibited closest sequence similarity to Moraxella boevrei ATCC 700022(T) (94.68%) and an uncultured, unspeciated bacterial clone (strain S12-08; 99%). The major fatty acids were C18:1ω9c, C18 : 0, C16:0 and C16:1ω6c/C16:1ω7c. The DNA G+C content of strain GVCNT2(T) was 40.7 mol%. The major respiratory quinone identified was Q-8. Strain GVCNT2(T) exhibited a comparable phenotypic profile to other members of the genus Moraxella but could be distinguished based on its ability to produce acid (weakly) from d-glucose, melibiose, l-arabinose and rhamnose and on its ability to hydrolyse DNA. On the basis of phenotypic and phylogenetic differences from other members of the family Moraxellaceae, strain GVCNT2(T) is considered to represent a novel species of a new genus, for which the name Faucicola mancuniensis gen. nov., sp. nov. is proposed. The type strain of Faucicola mancuniensis is GVCNT2(T) ( =DSM 28411(T) =NCIMB 14946(T)).

Bacteriological effects of dentifrices with and without active ingredients of natural origin.

Compounds of natural origin are increasingly used as adjuncts to oral hygiene. We have adopted four distinct approaches to assess the antibacterial activity of dentifrices containing natural active ingredients against oral bacteria in several test systems. Corsodyl Daily (CD), Kingfisher Mint (KM), and Parodontax fluoride (PF) were compared to a dentifrice containing fluoride (Colgate Cavity Protection [CCP]) and one containing triclosan (Colgate Total [CT]). The growth inhibitory and bactericidal potency of the formulations were determined for 10 isolated oral bacteria. Effects of single exposures of simulated supragingival plaques were then determined by epifluorescence and confocal microscopy, while the effects of repeated exposures were quantified by viable counting. Additionally, dense plaques, maintained in continuous culture, were repeatedly dosed, and the outcome was assessed by viable counting and eubacterial DNA profiling. The test dentifrices exhibited variable specificity and potency against oral bacteria in axenic culture. Of the herbal formulations, KM caused the largest viability reductions in simulated supragingival plaques, with CT causing the greatest reductions overall. Following single exposures, CD caused moderate reductions, while PF had no effect. After multiple dosing, all formulations significantly reduced numbers of total, facultative, and Gram-negative anaerobes, but only KM and CT caused greater reductions than the fluoride control. KM also reduced counts of streptococci (rank order of effectiveness: CT > KM > CCP > PF > CD). Marked changes in eubacterial DNA profiles were not detected for any herbal formulation in dense plaques, although KM markedly reduced viable counts of streptococci, in agreement with supragingival data. While both nonherbal comparators displayed antibacterial activity, the triclosan-containing formulation caused greater viability reductions than the herbal and nonherbal formulations.

Oral bacterial communities in individuals with type 2 diabetes who live in southern Thailand.

Type 2 diabetes mellitus is increasingly common in Thailand and elsewhere. In the present investigation, the bacteriological composition of saliva and supragingival plaque in Thai diabetics with and without active dental caries and in nondiabetics was determined by differential culture and eubacterial DNA profiling. Potential associations between fasting blood sugar and glycosylated hemoglobin (biomarkers of current and historical glucose control, respectively) with decayed, missing, and filled teeth and with salivary Streptococcus and Lactobacillus counts were also investigated. The incidence of active dental caries was greater in the Thai diabetics than in nondiabetics, and the numbers of total streptococci and lactobacilli were significantly higher in supragingival plaque from diabetics than in nondiabetics. Lactobacillus counts in the saliva and supragingival plaque of diabetics with active caries were significantly higher than those in diabetics without active caries. Oral eubacterial DNA profiles of diabetic versus nondiabetic individuals and of diabetics with active caries versus those without active caries could not be readily differentiated through cluster analysis or multidimensional scaling. The elevated caries incidence in the Thai diabetics was positively associated with numbers of bacteria of the acidogenic/acid-tolerant genera Streptococcus and Lactobacillus. Lactobacillus bacterial numbers were further elevated in diabetics with active caries, although salivary eubacterial DNA profiles were not significantly altered.

An in vitro comparison of dentifrice formulations in three distinct oral microbiotas.

OBJECTIVES: In vitro biofilm models, representative of some aspects of nascent, supra-gingival plaques (Hydroxyapatite Disc Biofilm Models), developed supra-gingival plaques (Modified Drip-flow Biofilm Reactors) and sub-gingival plaques (Multiple Sorbarod Devices) were used to compare the antimicrobial effects of a triclosan-containing dentifrice with a stannous fluoride and zinc lactate combination. DESIGN: Triplicate salivary biofilm microcosms were maintained for 2d (hydroxyapatite discs), 5d (Sorbarods) or up to 6d (drip flow reactors). Dentifrice slurries (10%, w/v) were added once to the discs and repeatedly to the Drip Flow Reactors and Sorbarods. Plaques were analysed by differential culture and gravimetrically. RESULTS: Whilst both dentifrices were comparably effective at reducing viability and plaque accumulation in mature supragingival plaques, the triclosan dentifrice produced comparatively larger reductions in total streptococci and anaerobes in nascent plaques (p<0.05) and greater reductions in Gram-negative anaerobes and streptococci in subgingival plaques. CONCLUSIONS: We have used a multi-model approach to determine the effectiveness and specificity of dentifrices against compositionally distinct plaques. Whilst both formations reduced bacterial viability and plaque accumulation, their effects could be differentiated in nascent and deep plaques where the triclosan dentifrice caused larger viability reductions.

Evaluation of the specificity and effectiveness of selected oral hygiene actives in salivary biofilm microcosms.

The microbiological effects of biocidal products used for the enhancement of oral hygiene relate to the active compound(s) as well as other formulation components. Here, we test the specificities of selected actives in the absence of multiple excipients. Salivary ecosystems were maintained in tissue culture plate-based hydroxyapatite disc models (HDMs) and modified drip-flow biofilm reactors (MDFRs). Test compounds stannous fluoride (SF), SDS, triclosan (TCS), zinc lactate (ZL) and ZL with SF in combination (ZLSF) were delivered to the HDMs once and four times daily for 6 days to MDFRs. Plaques were characterized by differential viable counting and PCR-denaturing gradient gel electrophoresis (DGGE). TCS and SDS were the most effective compounds against HDM plaques, significantly reducing total viable counts (P<0.05), whilst SF, ZL and ZLSF were comparatively ineffective. TCS exhibited specificity for streptococci (P<0.01) and Gram-negative anaerobes (P<0.01) following a single dosing and also on repeated dosing in MDFRs. In contrast to single exposures, multiple dosing with ZLSF also significantly reduced all bacterial groups, whilst SF and ZL caused significant but transient reductions. According to PCR-DGGE analyses, significant (P<0.05) reductions in eubacterial diversity occurred following 6 day dosing with both TCS and ZLSF. Concordance of MDFR eubacterial profiles with salivary inocula ranged between 58 and 97%. TCS and ZL(SF) exhibited similar specificities to those reported for formulations. TCS was the most potent antibacterial, after single and multiple dosage regimens.

An in vitro evaluation of hydrolytic enzymes as dental plaque control agents.

The plaque-control potential of commercially available amylase, lipase and protease was evaluated by observing their effects on coaggregation and on bacterial viability within various plaque microcosms. A quantitative coaggregation assay indicated that protease significantly inhibited the extent of coaggregation of Actinomyces naeslundii and Streptococcus oralis (P <0.05) and of Porphyromonas gingivalis and S. oralis. Amylase significantly (P <0.05) increased the coaggregation of A. naeslundii versus Fusobacterium nucleatum and A. naeslundii versus P. gingivalis. Concomitant challenge of constant-depth film fermenter-grown plaques with the enzymes did not result in detectable ecological perturbations (assessed by differential culture and denaturing gradient gel electrophoresis). Similar dosing and analysis of multiple Sorbarod devices did not reveal increases in bacterial dispersion which could result from disaggregation of extant plaques. A short-term hydroxyapatite colonization model was therefore used to investigate possible enzyme effects on early-stage plaque development. Whilst culture did not indicate significant reductions in adhesion or plaque accumulation, a vital visual assay revealed significantly increased aggregation frequency following enzyme exposure. In summary, although hydrolytic enzymes negatively influenced binary coaggregation, they did not cause statistically significant changes in bacterial viability within plaque microcosms. In contrast, enzyme exposure increased aggregation within extant plaques.

In vitro study of the effect of cationic biocides on bacterial population dynamics and susceptibility.

Cationic biocides (CBs) are widely used in domestic and public hygiene and to control biofouling and microbial contamination in industry. The increased use of biocides has led to concern regarding possible reductions in biocide effectiveness. Domestic drain microcosms were stabilized for 5 months and then exposed to polyhexamethylene biguanide (PHMB) at 0.1, 0.2, and 0.4g liter(-1) over 6 months and characterized throughout by differential culture, together with eubacterial-specific PCR-denaturing gradient gel electrophoresis. Additionally, MICs and minimal bactericidal concentrations (MBCs) for bacteria previously isolated from a domestic drain (n = 18) and the human skin (n = 13) were determined before, during, and after escalating, sublethal exposure (14 passages) to two quaternary ammonium compounds (QAC1 and QAC2), the bisbiguanide chlorhexidine (CHX), and PHMB. Exposure of the drain microcosm to PHMB did not decrease the total viable count although significant (P < 0.01) decreases in recovery were observed for the gram-positive cocci with associated clonal expansion of pseudomonads (from ca. 0.1% of the population to ca. 10%). This clonal expansion was also manifested as elevations in bacteria that could grow in the presence of PHMB, CHX, and QAC1. Decreases in susceptibility (greater than twofold) occurred for 10/31 of the test bacteria for QAC1, 14/31 for QAC2, 10/31 for CHX, and 7/31 for PHMB. Exposure of microcosms to PHMB targeted gram-positive species and caused the clonal expansion of pseudomonads. In terms of prolonged-sublethal passage on CBs, exposure to all the biocides tested resulted in susceptibility decreases for a proportion of test bacteria, but refractory clones were not generated.