EFFECT OF BIOCIDES ON BIOFILM BACTERIA FROM DENTAL PLAQUE

Abstract

The oral diseases (dental caries and periodontal diseases) are major public health problems and are found the most prevalent diseases of mankind. Accumulation and adherence of bacterial biofilm on tooth surfaces as dental plaque mainly cause for two human diseases like inflammatory periodontal disease and dental caries. The defensive immune systems and resident microbial flora are responsible for balance in oral health. Disruption of balance favours the colonisation of transient bacteria and commensal that may lead to localised infections e.g. periodontal, endodontic, and gingival infections. Oral cavity may provide suitable environment for the exchange and store of genetic material. Bacteria from the oral cavity have acquired resistance against antibiotics and/or biocides due to inadequate or extensive use of biocides and antibiotics in the field of dentistry and medicine. In this study five dental plaque samples were collected from five healthy individual for isolation, characterization and to check the efficacy of biocides on growth and biofilm formation of oral microbes. Susceptibility pattern of oral isolates were observed against different concentrations (0.1, 1, 10, 20, 30, 40, and 50 μg/ml) of four biocides/ commercially available mouth-washes by culture dependent method. These include Chlorhexidine (CHX) 0.2%, Benzidamine hydrochloride (BZD) 0.15%, Sodium fluoride 0.05% and potassium chloride 0.05%, and Benzidamine hydrochloride 0.15% and Chlorhexidine 0.2%). Thirty four (70%), 9 (18%), 2 (04%), 2 (04%) and 2 (04%) out of total 58 oral isolates were found with common biocides resistance pattern and showed reduce susceptibility against different concentrations 0.1, 1.0, 10, 20, 30 μgml-1of all four biocides respectively. All the 58 isolates were found sensitive at concentrations (40 and 50 μgml-1) of four biocides. Overall, 9 (15%) out of total fifty eight (58) plaque isolates were selected on different morphological basis and common resistance (1.0 μgml-1) against four biocides. Dental plaque bacteria were characterized morphologically, biochemically, physiologically and genetically; phylogenic analysis was also performed. The molecular characterization reveals that isolates N4A, B1E, N1D, N2C, N2D, N3C, N3D, N4E, and C2E belong to genera including Acinetobacter schindleri (JF837190), Morexiella acj (JF837191), Chryseobacterium culicis (KR002422), Chryseobacterium indologenes (KR002424), Acinetobacter johnsonii (KR002423), Enterobacter ludwigii (KR002425), Pseudomonas stutzeri (KC817808), Streptococcus salivarius (KC817808) and Bacillus cereus (JF837191) respectively. The isolates were variably resistant to multiple drugs including ampicillin, kanamycin, gentamicin and tetracycline, erythromycin, vancomycin and chloramphenicol. Seven out of nine isolates Summary 2 have shown resistance to ampicillin and tetracycline while erythromycin found the most effective drug with active susceptibility for six oral bacteria. Results indicated that all the plaque isolates were biofilm producers in the absence and presence of CHX within 24, 72 and 120 hours (hrs). High variation in biofilm forming ability of isolates was observed with exhibition of strong biofilm within 120 hrs followed by 72 hrs and 24 hrs. Study results directed that CHX+BZD with 1 μg/ml were found the most effective biocide for biofilm inhibition in 24 hrs. The effect of four biocides CHX, BZD, NaF+KCl and CHX+BZD (1.0 μg/ml stress) against biofilm bacteria in comparison with control (0 μg/ml stress) cells was analyzed statistically. It was found highly significant with P < 0.0001. Exposure of biofilms of these resistant species to undiluted commercial CHX mouthwash for intervals from 5 to 60 s indicated that the mouthwash was unlikely to eliminate them from dental plaque in vivo. Two species, Chryseobacterium indologenes and Chryseobacterium culicis were able to grow planktonically and form biofilms in the presence of 32 μg/ml CHX. Multidrug and CHX resistant C. indologenes demonstrated a 19-fold up-regulation of expression of gene CIN01S_RS05745 that encode the HlyD-like periplasmic adaptor protein of a tripartite efflux pump upon exposure to sub inhibitory concentration 16 μg/ ml of CHX suggesting that multidrug resistance may be mediated by this system. Bioinformatics analysis reveals the homology of this protein with RND family of transporters proteins (NCBI accession # TIGR01730) with e-value 2.08e-49. The signal of gene up regulation were also confirmed with the presence of 1250 bp amplification band of study gene flanking regions using reverse transcriptase PCR (RT-PCR). Northern hybridization reveals ̴6.3kb sized operon that encode for putative tripartite efflux pump. Furthermore, the ability of the selected actinomycetes to control biofilm of plaque bacteria was also accessed and observed that three out of ten Streptomyces strains named as S. erythrogriseus and S. labedae showed good antibacterial activity against Moraxella and Acinetobacter oral strains while S. macrosporeus can only inhibit biofilm formation by Bacillus cereus. Further study is required to show the transcriptomic picture of some CHX resistant genes in C. indologenes. This study has shown the resistance gene in emerging but uncommon pathogen; that will probably attract the researcher’s intension as new resistant bacterial species in dental setting. It has also pointed out the alarming situation with long term use of CHX (antiseptic/ oral care products) may cause resistance and/or may pose threat in the contribution of resistance gene to other dental microbiota. Future plan is to analyze the whole genome sequence data for genome comparison and for searching out CHX resistance efflux protein in newly Summary 3 sequenced C. culicis oral isolate. The whole genome sequencing data processing of C. culicis is going on for annotation using Rapid Annotation Subsystem Technology (RAST) tool.