AIMS To investigate the effects of the combined application of an N-acyl homoserine lactone (HSL) analog and antibiotics on biofilms of Porphyromonas gingivalis, a major pathogen of periodontal disease. METHODS AND RESULTS Antibiotics used were cefuroxime, ofloxacin and minocycline. A flow-cell model was used for biofilm formation. Samples were divided into four groups: control, analog-treated, antibiotic-treated and combined application groups. Biofilm cell survival was determined using adenosine triphosphate (ATP) bioluminescence and confocal laser microscopy (CLSM). In the combined application group, the ATP count in biofilm cells was significantly decreased compared with the antibiotic-treated group (Games-Howell test, P < 0·05). A combination of cefuroxime and the analog was most effective against the P. gingivalis biofilm. CLSM observations revealed that the proportion of dead cells was highest in the combined application group. CONCLUSIONS The combined application of the N-acyl HSL analog and antibiotics was effective at reducing the viability of P. gingivalis cells in biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY The combined application of the N-acyl HSL analog and antibiotics may be successful for eradicating infections involving bacterial biofilms, such as periodontitis.

Antibiotic resistance of biofilm-grown bacteria contributes to chronic infections such as marginal and periapical periodontitis, which are strongly associated with Porphyromonas gingivalis. Concurrent azithromycin (AZM) administration and mechanical debridement improve the clinical parameters of periodontal tissue in situ. We examined the in vitro efficacy of AZM against P. gingivalis biofilms. The susceptibility of adherent P. gingivalis strains 381, HW24D1, 6/26 and W83 to AZM, erythromycin (ERY), ampicillin (AMP), ofloxacin (OFX) and gentamicin (GEN) were investigated using a static model. The optical densities of adherent P. gingivalis cells were significantly decreased by using AZM and ERY at sub-MIC compared with controls in all the strains tested, except for the effect of ERY on strain W83. AMP and OFX inhibited P. gingivalis adherent cells at over their MICs, and GEN showed no inhibition in the static model. The effects of AZM and ERY against biofilm cells were investigated using a flow cell model. The ATP levels of P. gingivalis biofilms were significantly decreased by AZM at concentrations below the sub-MICshowever, ERY was not effective for inhibition of P. gingivalis biofilm cells at their sub-MICs. Furthermore, decreased density of P. gingivalis biofilms was observed three-dimensionally with sub-MIC AZM, using confocal laser scanning microscopy. These findings suggest that AZM is effective against P. gingivalis biofilms at sub-MIC levels, and could have future clinical application for oral biofilm infections such as chronic marginal and periapical periodontitis.

Chronological gene expression patterns of biofilm-forming cells are important to understand bioactivity and pathogenicity of biofilms. For Porphyromonas gingivalis ATCC 33277 biofilm formation, the number of genes differentially regulated by more than 1.5-fold was highest during the growth stage (312/2,090 genes), and some pathogen-associated genes were time-dependently controlled.

Asahi Y, Noiri Y, Igarashi J, Asai H, Suga H, Ebisu S. Effects of N-acyl homoserine lactone analogues on Porphyromonas gingivalis biofilm formation. J Periodont Res 2009; doi: 10.1111/j.1600-0765.2009.01228.x (c) 2009 John Wiley & Sons A/SBackground and Objective: The gram-negative anaerobic rod Porphyromonas gingivalis in oral biofilms is a primary etiological agent of periodontal disease. Biofilm formation of various gram-negative bacteria is regulated by a quorum-sensing circuit that relies on N-acyl homoserine lactones (HSLs). Some synthetic N-acyl HSL analogues act as quorum-sensing inhibitors and suppress biofilm formation in Pseudomonas aeruginosa. Development of chemical control agents against oral biofilms is necessary, because until now, biofilms have been removed only by mechanical debridement. The present study investigated the effect of N-acyl HSL analogues on P. gingivalis biofilm formation, with the aim of developing new drugs that inhibit oral biofilm formation. Material and Methods: A flow-cell model was used for P. gingivalis biofilm formation. Seventeen synthetic N-acyl HSL analogues were quantitatively assessed by spectrophotometry. The effects of three antagonistic compounds against P. gingivalis biofilm formation were further examined by confocal laser scanning microscopy, and investigated for primary attachment using spectrophotometry and phase contrast microscopy. Results: Ten out of 17 analogues affected P. gingivalis biofilm formation. Three out of 10 analogues significantly decreased biofilm-forming cells (p < 0.05), and these biofilm structures were less well formed three-dimensionally. There were no quantitative or qualitative differences in cell attachment between the control and the three analogue-treated groups. Conclusion: Three synthetic N-acyl HSL analogues inhibited biofilm formation in P. gingivalis. We suggest that these analogues influence the development stage of P. gingivalis biofilm formation.

BACKGROUND: Malasseziaglobosa constitutes a part of the normal flora of human skin, but may induce IgE production in atopic dermatitis (AD). However, information on M.globosa allergens is scant. OBJECTIVE: To identify the major M. globosa allergens by using proteomic analysis. METHODS: Immunoglobulin E (IgE) immunoblotting and cross-inhibition tests for M. globosa allergens were performed using sera from AD patients and control subjects. These allergens were identified and characterized using the proteomics approach involving a combination of two-dimensional (2D) electrophoresis, mass spectrometry, and bioinformatics tools. We cloned the cDNA of this allergen using sequences obtained by 5'- and 3'-rapid amplification of cDNA ends polymerase chain reaction. RESULTS: The sera of the AD patients had IgE-reactive 40-45-kDa protein components. By 2D immunoblotting, we detected a 42-kDa protein spot with an isoelectric point (pI) of 4.8; the protein was highly reactive to IgE and was designated MGp42. Full-length MGp42 cDNA contained a 1908-bp open reading frame encoding 635 amino acid residues (calculated molecular mass, 69.7kDa; pI, 6.02). The N-terminal MGp42 sequence started from the 250th residue (Asp-250) of the deduced amino acid sequence and consisted of 386 amino acid residues; these results are consistent with those of 2D immunoblotting. MGp42 showed sequence similarity to members of the heat shock protein 70 (hsp70) family. Immunoblot inhibition tests revealed no IgE cross-reactivity between MGp42 and human HSP70. CONCLUSIONS: MGp42 may be a cleavage product of intact HSP70. This novel M. globosa allergen could be useful for the diagnosis of AD.

We had clinical grounds to suspect that patients with autism had greater propofol requirements during dental procedures than patients with intellectual impairment without autism. This hypothesis was tested by an audit of a standard anaesthetic technique. The audit was approved by our Hospital Ethics Committee. We compared the propofol requirements and effect using a standardised protocol during dental treatment in 56 autistic patients (age range three to 35 years) and 56 intellectually impaired patients (age range four to 42 years). Patients in each disability group were divided into three subgroups by age: six years or younger, seven to 19 years and 20 years or older. Combative patients received oral midazolam premedication, other patients received a single intravenous bolus of midazolam at induction. Otherwise, standardised propofol boluses and infusion were the only anaesthetic agents used. The propofol infusion rates of the intellectually impaired group showed significant decline with age (propofol rate of requirement mg x kg(-1) x h(-1), mean [SD]):< six years 13.6 (3.6), seven to 19 years 9.5 (3.0)(P = 0.008 cf < six years group),> 19 years group 8.5 (2.4)(P = 0.001 cf < six years group). The propofol requirement was greater in the autism group than in the intellectual disability group, and the proportion of the cases where bolus propofol administration was needed after induction was significantly higher in the autistic patient group than in the intellectually impaired patients (P < 0.002). This suggests that autistic patients have greater propofol requirements for anaesthesia during ordinary dental treatment compared with intellectually impaired patients.

We previously mapped a locus for porcine intramuscular fat content (IMF) by linkage analysis to a 17.1-cM chromosome interval on Sus scrofa chromosome 7 (SSC7) flanked by microsatellite markers SW1083 and SW581. In this study, we identified 34 microsatellite markers and 14 STSs from the 17.1-cM IMF quantitative trait loci (QTL) region corresponding to HSA14q and aligned those loci using the INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel. We then constructed a 5.2-Mb porcine bacterial artificial chromosome (BAC) contig of this region that was aligned using the RH panel. Finally, the IMF QTL was fine-mapped to 12.6 cM between SJ169 and MM70 at the 0.1% chromosome-wise significance level by genotyping the previously studied F(2) resource family with 17 additional microsatellites. We also demonstrated that the SJ169-MM70 interval spans approximately 3.0 Mb and contains at least 12 genes: GALC, GPR65, KCNK10, SPATA7, PTPN21, FLJ11806, EML5, TTC8, CHES1, CAP2P1, CHORDC2P and C14orf143.