Journal Search Engine
Search Advanced Search Adode Reader(link)
Download PDF Export Citaion korean bibliography PMC previewer
ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.43 No.4 pp.201-207

Antibody-based Screening of Porphyromonas gingivalis Proteins Specifically Produced in Patients with Chronic Periodontitis

Hye-Jung Kim1, Seok-Woo Lee1,2*
1Department of Dental Education, School of Dentistry, Dental Research Institute, Chonnam National University, Gwangju 61186, Korea
2Department of Periodontology, School of Dentistry, Dental Research Institute, Chonnam National University, Gwangju 61186, Korea
Correspondence to: Seok-Woo Lee, D.D.S., M.S., Ph.D., Departments of Dental Education and Periodontology School of Dentistry Chonnam National University, 77 Yongbong-ro, Buk-gu Gwangju 61186, Korea Tel: 82-62-530-5820, Fax: 82-62-530-5810 E-mail:
September 27, 2018 December 10, 2018 December 14, 2018


Porphyromonas gingivalis is among the major etiological pathogens of chronic periodontitis. The virulence mechanisms of P. gingivalis is yet to be identified as its activity is largely unknown in actual disease process. The purpose of this study is to identify antigens of P. gingivalis expressed only in patients with chronic periodontitis using a unique immunoscreening technique. Change Mediated Antigen Technology (CMAT), an antibody-based screening technique, was used to identify virulence-associated proteins of P. gingivalis that are expressed only during infection stage in patients having chronic periodontitis. Out of 13,000 recombinant clones screened, 22 tested positive for reproducible reactivity with rabbit hyperimmune anti-sera prepared against dental plaque samples acquired from periodontitis patients. The DNA sequences of these 18 genes were determined. CMAT-identified protein antigens of P. gingivalis included proteins involved in energy metabolism and biosynthesis, heme and iron binding, drug resistance, specific enzyme activities, and unknown functions. Further analysis of these genes could result in a novel insight into the virulence mechanisms of P. gingivalis.


    Chonnam National University
    CRI 17025-1
    © The Korean Academy of Oral Biology. All rights reserved.

    This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.


    Dental plaque is a biofilm containing more than 500 different bacterial species [1-3], and behaves as a dynamic community affecting the host, which can lead to periodontal disease resulting from dysbiosis state [4]. It has been found that a population change toward increased composition of gram-negative bacteria within the dental plaque biofilm can lead to the development of periodontal diseases [2,5]. It is therefore crucial to understand the complicated interactions between the host and anaerobic gram-negative bacteria to better understand the microbial pathogenesis of periodontal diseases.

    Although numerous data have been accumulated for the molecular pathogenicity of P. gingivalis [6-9], the exact mechanisms by which P. gingivalis behave in actual infectious processes are not fully understood. In order to assess the in vivo activity of P. gingivalis in periodontal disease, we applied a novel antibody-screening technique termed change mediated antigen technology (CMAT). CMAT is an immune-screening technique used to identify bacterial antigens produced only in the host when cells undergo change such as in infection [10]. This technology utilizes hyperimmune antisera raised against bacterial cells obtained from disease sites, which are later adsorbed with bacterial cells grown in vitro. This procedure produce an antibody probe that is reactive only with in vivo-expressed proteins. Using this approach, we were able to identify in vivo-induced antigens of Enterococcus faecalis, a major endodontic pathogen in our previous study [11]. The purpose of the present study was to detect in vivo-expressed proteins of P. gingivalis using CMAT.

    Materials and Methods

    Bacterial strains and growth conditions

    P. gingivalisATCC 33277 was purchased from American Type Culture Collection (Manassas, VA, USA) and cultivated on tryptic soy agar (Becton Dickson, Sparks, MD, USA) supplemented with 0.5% (w/v) yeast extract (Becton Dickson), 5% (v/v) fetal bovine serum (Hyclone, Logan, UT, USA), and 0.001% N-acetylmuramic acid (Sigma, St. Louis, MO, USA) at 37ºC under anaerobic conditions (80% N2, 10% CO2, 10% H2) for 5-7 days using a Forma 1025 Anaerobic Chamber (Thermo, Marietta, OH, USA). P. gingivalis cells were collected from the agar plates and washed, and kept at –80ºC until used. E. coli BL21(DE3)/pLysS Competent Cells (Stratagene, La Jolla, CA, USA) were grown on LB agar plates or broth containing kanamycin (30 μg/ml) at 37 ºC overnight.

    Construction of genomic expression libraries of P. gingivalisATCC 33277

    P. gingivalis genomic DNA was extracted using a G-spinTM Genomic DNA Extraction Kit for Bacteria (iNtRON Biotechnology, Inc., Gyeonggi-do, Korea) according to the manufacturer’s instruction. The genomic DNA was then cut by sonication using a SONOPULS Ultrasonic homogenizer (Bandelin, Berlin, Germany) for 6 seconds at the 100% power setting in order to produce DNA fragments sized 1–5 kb. After they were fractionated by agarose gel electrophoresis to eliminate very small and too large fragments, the DNA fragments were treated with the End-ItTM DNA End-Repair Kit (Epicentre, Madison, WI, USA) and subjected to phenolchloroform extraction procedure to remove T4 polynucleokinase activity. A recombinant plasmid vector, pET-30c(+) (Novagen, Madison, WI, USA), was treated with EcoRV, and subsequently purified with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). pET-30c(+)/EcoRV and blunt-ended P. gingivalis DNA fragments (1-5 kb) were ligated using T4 DNA ligase (New England Biolab, Ipswich, MA, USA). Then, the mixture was used to transform E.coli DH5α (Takara, Daejon, Korea), and transformants were chosen on LB plates containing kanamycin antibiotics. Ten randomly selected colonies were screened using colony PCR with T7 promoter primers and checked to ensure self-ligation frequency was lower than 10%. Then, purified recombinant DNA mixture was used to transform E. coli BL21(DE3)/pLysS Competent Cells (Stratagene, La Jolla, CA, USA). Transformed cells were grown on LB agar plates containing kanamycin (30μg/ml) at 37 ºC overnight.

    Collecting dental plaque samples containing P. gingivalis and preparing hyperimmune antisera

    The protocol for collecting the dental plaque samples was approved by the Institutional Review Board (Approval number: 1-2008-05-22, Chonnam National University Hospital, Gwangju, Korea). Dental plaque samples were obtained from 10 patients who had moderate or severe chronic periodontitis, collected from the subgingival pockets of maxillary first molars. After supragingival plaque and calculus were removed, sterile #14 Gracey curette was inserted into the subgingival pockets, and plaque samples were obtained with a single stroke. The plaque samples were transferred aseptically to 1.5 ml microcentrifuge tubes containing 1 ml TE buffer, vortexed for 10 s, and centrifuged at 100 rpm for 10s to collect samples. Then, the presence of P. gingivalis in the samples was confirmed by PCR analysis as described before [12]. P. gingivalis-containing samples were immediately formalin fixed to preserve the integrity of protein antigens present at the time of harvest. Finally, tissue samples were pooled together and immediately stored at -70ºC until use. The samples were then sent to a commercial vendor (Young In Frontier, Seoul, Korea) for preparing rabbit polyclonal antibodies. The presence of high-titer rabbit polyclonal antibodies produced against P. gingivalis was confirmed by ELISA and western blot analysis.

    Serum adsorption

    In order to obtain an antibody probe that reacted only with in vivo-expressed P. gingivalis antigens, hyper-immune rabbit anti-sera were adsorbed with P. gingivalis whole cells cultivated in vitro. First, the rabbit polyclonal antiserum was mixed with 1 x 109P. gingivalis cells with slow agitation (30 rpm) at 4˚C for 1 h; the cells were removed by centrifugation at 5,000 × g for 10 min, and the antisera were obtained. This step was repeated 4 times. Cell lysates were bound to NC membrane and added to the serum after whole cells were removed and then were agitated overnight at 4˚C; the next day, the serum was collected. To remove antibodies reacted with E. coli proteins, the serum was agitated again by adding nitrocellulose membrane-bound E. coli BL21 harboring only pET-30c(+).

    To confirm adsorption efficiency, ELISA was performed as described before [13]. Briefly, a 96-well plate was coated with P. gingivalis protein extracts (0.1mg/100μl) in a coating buffer (0.1M sodium carbonate, pH9.6) overnight at 4℃. After washing, 2% skim milk in PBS was added and incubated for 1 hr at 37℃, followed by re-washing with PBS. Then, adsorbed rabbit antisera were added to each well using serial dilution starting at the ratio of 1:1000 and incubated at 37℃ for 2 hr. After the incubation, the plated was washed 3 times with 1× TBS-Tween. Then, 50 μl of 1:10000 diluted goat anti-rabbit IgG conjugated with horse radish peroxidase (Abcam, Hanam, Korea) was added to each well and incubated for 1 hr at 37℃. After washing with TBS-Tween (3X), TMB solution (GenDEPOT, Barker, TX, USA) was mixed and incubated for 2 min. The reaction was interrupted by adding 1N H2SO4 (100 μl). The reactivity was measured with absorbance at 450 nm in a spectrometer.

    Screening for recombinant proteins uniquely expressed in vivo

    Recombinant E. coli clones were detected using the adsorbed rabbit antisera as described previously [10]. Briefly, E. coli clones showing kanamycin resistance, about 200 per plate, were transferred to a nitrocellulose membrane. P. gingivalis and E. coli containing pET-30(+) were spotted on the membrane as positive and negative controls for screening, respectively. The membrane was then transferred to a LB agar plate containing 1 mM IPTG to induce protein expression. After the incubation at 37˚C for 3 hrs, the adhered cells were lysed by chloroform vapor. Then, the membrane was treated with PBS (5% skim milk) for 1 h, washed with PBS-Tween 20, and subjected to overnight incubation with the adsorbed rabbit antisera (1:500) at 4˚C. After the incubation, the membrane was screened with goat anti-rabbit IgG conjugated with horse radish peroxidase (1:1,000) for 1 hr at room temperature. 1-Step Chloronaphthol (4CN) (Pierce Biotechnology, Rockford, IL, USA) was used to identify reactive clones. The reactive clones were removed from the master plate and subsequently cultivated on a LB-kanamycin plate, and their relativities were confirmed.

    Identifying genes isolated by CMAT

    Plasmid DNA from positive clones was isolated with a QIAprep Spin Miniprep Kit (Qiagen), and DNA sequences of inserted fragments were determined using a T7 promoter primer with an ABI Prism 377 automatic DNA sequencer at GenoTech (Daejeon, Korea). Identified gene sequences were compared with the Oral Pathogen Sequence Databases (http://www.o, where the entire genome sequence of P. gingivalisATCC 33277 is available. Additionally, these gene sequences were compared with the DNA and protein databases using BLAST ( and analyzed with Invitrogen Vector NTI software (Thermo, CA).

    Predicting functions of antigens identified by CMAT

    Functional classification of identified antigens was based, when available, on published studies of identified proteins of P. gingivalis. When functional studies for P. gingivalis were not available in the literature, protein functional classification was predicted on the description posted on the Oral Pathogen Sequence Databases.


    Adsorption of rabbit hyper-immune antisera

    Hyper-immune rabbit antisera produced against dental plaque samples containing P. gingivalis were successively adsorbed against P. gingivalis cells grown in vitro. Adsorption efficiency was determined by analyzing the reactivity of antisera using ELISA after each adsorption step with in vitro-grown P. gingivalis. Fully adsorbed serum exhibited significantly lower reactivity with in vitro-grown P. gingivalis (as shown in Figure 1). This result suggested that the adsorption process efficiently removed antibodies against most P. gingivalis proteins produced during in vitro cultivation.

    Genomic library construction and screening

    Ten colonies were randomly selected, and their plasmids were subjected to DNA sequencing to ensure the presence of the DNA inserts present in the recombinant plasmids. It was found that 8 out of 10 colonies had DNA inserts in the pET-30c(+) vector. A total of approximately 13,000 recombinant clones were screened with hyper-immune rabbit antisera. Initially, 28 immunoreactive clones were detected. Twenty-two of these reproducibly exhibited reactivity with the adsorbed serum, and these were chosen for further study in gene sequencing.

    Identification and functional classification of P. gingivalis genes identified by CMAT

    Sequencing of the DNA inserts from the 22 positive clones revealed that 18 ORFs had been identified; the gene sequences from four clones could not be obtained. These gene sequences were analyzed using a BLAST program against the total genome sequence of P. gingivalis in the database at NCBI (http://www. CMAT-identified IVI genes and their predicted functions are listed in Table 1.

    It was determined from sequence analysis that many of these genes were enzymes implicated in housekeeping functions with intermediary energy metabolism and biosynthesis. Also, a small number of enzymes that possessed peptidase activity, and heme and iron binding proteins were also identified. In addition, proteins involved in drug resistance and proteins with unknown function were identified.


    It is not known how P. gingivalis actually behaves within the host and ultimately mediates periodontal infection that can lead to tissue destruction. Logically, it can be assumed that genes (proteins) expressed in vivo are essential in survival in the host and serve as potential virulence factors. It was our major objective to identify proteins from P. gingivalis that were exclusively produced in vivo during the actual disease process. For this purpose, we used an immunoscreening technique called CMAT in the present study; CMAT was designed to detect immunoreactive proteins produced only in vivo. The technique is a modification of the in vivo-induced antigen technology (IVIAT) that has allowed many researches to identify IVI proteins directly from human hosts rather than from potentially misleading animal models [10,14]. In many pathogenesis studies, IVIAT has been used to identify potential virulence-associated antigens from Candida albicans [15], Vibrio cholera [16], Mycobacterium tuberculosis [17], Vibrio vulnificus [18], Escherichia coli [19], Salmonella enterica [20], Group A Streptococcus [21], and periodontal pathogens including Aggregatibacter actinomycetemcomitans [22], P. gingivalis [23], and Tannerella forsythia [13].

    Many IVI genes identified by CMAT in this study were enzymes associated with housekeeping functions including energy metabolism, nucleotide biosynthesis, and other cellular processes, as shown in Table 1. These genes were similar to those identified in previous IVIAT studies [13,24], and they are thought to play roles that are not required during in vitro growth [25]. It has been observed that housekeeping genes are not constitutively expressed at the transcriptional level in bacteria as was previously assumed [26-28], indicating that housekeeping metabolism is a dynamic process that is highly capable of adapting to different growth conditions. Therefore, it is plausible that these genes are up-regulated, and thus expressed only in vivo

    Two important proteins related to the physiology and virulence of P. gingivalis were identified: HmuR and ferritin. HmuR, a TonB-dependent hemoglobin receptor, is a component of the hmu operon system that mediates heme and iron acquisition by P. ginigvalis [29,30]. Heme binding activity can directly affect virulence of P. gingivalis, as heme was demonstrated to have a virulence‐enhancing effect [31]. It was also shown that HmuR is required for P. gingivalis growth [32]. Separately, ferritin is one of the intracellular iron-storage proteins, and this protein may be important for P. gingivalis survival under iron-depleted conditions [33]. Because heme and iron are required by most bacterial pathogens for infection development and progression [30,34], in vivo expression of HmuR and ferritin may be responsible for enhancing the virulence potential for P. gingivalis inside the host. We found it of interest that we identified a putative transport multidrug efflux protein that could be recognized as the major player in antimicrobial resistance. It is plausible for P. gingivalis to express a novel antimicrobial resistance using this mechanism inside the host, in addition to other conventional antimicrobial resistance mechanisms [35-37].

    We also identified dipeptidyl peptidase 7 (DPP7) that possessed aminopeptidase activity; this enzyme is involved in detaching dipeptides from the N-terminal end of oligopeptides and is therefore crucial for the growth of P. gingivalis utilizing amino acids as energy sources. It was suggested that this protein is involved in facilitating dental plaque development and exerts cytotoxicity, leading to increased pathogenicity of P. gingivalis [38]. We also identified another enzyme, PorU, with the cysteine-type peptidase activity that is involved in the hydrolysis of peptide bonds. It is very likely that both DPP7 and PorU utilize peptides in vivo given that P. gingivalis is asaccharolytic, and therefore can only metabolize amino acids.

    In summary, we identified 18 IVI antigenic bacterial proteins of P. gingivalis that are expressed uniquely in vivo and are reactive with rabbit hyperimmune sera produced against P. gingivalis contained in the dental plaque samples. The results of this pilot study suggest that CMAT is a useful approach to identifying and characterizing potential virulence factors of P. gingivalis. The antigens identified in this study can be further analyzed by conventional techniques in order to refine the exact functions. Subsequent studies can test the functional roles of these identified genes by producing isogenic mutants for the purpose of studies using animal or in vitro models. These efforts will ultimately allow us to achieve better insights into the virulence mechanisms of P. gingivalis. This effort can lead to the advancement of new diagnostic, therapeutic, and preventive measures for periodontal and other infections associated with P. gingivalis.


    This research was supported in part by a grant (CRI 17025-1) from Chonnam National University Hospital Biomedical Research Institute.


    Effectiveness of antibody adsorption.

    Rabbit antisera were adsorbed with P. gingivalis whole cells cultivated in vitro, and the results were confirmed with ELISA.


    P. gingivalis protein antigens identified by CMAT


    1. Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, et al. Bacterial diversity in human subgingival plaque. J Bacteriol 2001;183:3770-83.
    2. Kroes I, Lepp PW, Relman DA. Bacterial diversity within the human subgingival crevice. Proc Natl Acad Sci U S A 1999;96:14547-52.
    3. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 2005;43:5721-32.
    4. Marsh PD. Dental plaque as a microbial biofilm. Caries Res 2004;38:204-11.
    5. Socransky SS, Haffajee AD. Evidence of bacterial etiology: a historical perspective. Periodontol 2000 1994;5:7-25.
    6. Holt SC, Ebersole JL. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia: the “red complex”, a prototype polybacterial pathogenic consortium in periodontitis. Periodontol 2000 2005;38:72-122.
    7. Forng RY, Champagne C, Simpson W, Genco CA. Environmental cues and gene expression in Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans. Oral Dis 2000;6:351-65.
    8. O'Brien-Simpson NM, Veith PD, Dashper SG, Reynolds EC. Antigens of bacteria associated with periodontitis. Periodontol 2000 2004;35:101-34.
    9. Nakayama M, Ohara N. Molecular mechanisms of Porphyromonas gingivalis-host cell interaction on periodontal diseases. Jpn Dent Sci Rev 2017;53:134-40.
    10. Handfield M, Brady LJ, Progulske-Fox A, Hillman JD. IVIAT: a novel method to identify microbial genes expressedspecifically during human infections. Trends Microbiol 2000;8:336-9.
    11. Lee SW, Shet UK, Park SW, Lim HP, Yun KD, Kang SS, et al. Identification of Enterococcus faecalis antigens specifically expressed in vivo. Restor Dent Endod 2015;40:306-11.
    12. Fouad AF, Barry J, Caimano M, Clawson M, Zhu Q, Carver R, et al. PCR-based identification of bacteria associated with endodontic infections. J Clin Microbiol 2002;40:3223-31.
    13. Yoo JY, Kim HC, Zhu W, Kim SM, Sabet M, Handfield M, et al. Identification of Tannerella forsythia antigens specifically expressed in patients with periodontal disease. FEMS Microbiol Lett 2007;275:344-52.
    14. Rollins SM, Peppercorn A, Hang L, Hillman JD, Calderwood SB, Handfield M, et al. in vivo induced antigen technology (IVIAT). Cell Microbiol 2005;7:1-9.
    15. Cheng S, Clancy CJ, Checkley MA, Handfield M, Hillman JD, Progulske-Fox A, et al. Identification of Candida albicans genes induced during thrush offers insight into pathogenesis. Mol Microbiol 2003;48:1275-88.
    16. Hang L, John M, Asaduzzaman M, Bridges EA, Vanderspurt C, Kirn TJ, et al. Use of in vivo-induced antigen technology(IVIAT) to identify genes uniquely expressed during human infection with Vibrio cholerae. Proc Natl Acad Sci U S A2003;100:8508-13.
    17. Deb DK, Dahiya P, Srivastava KK, Srivastava R, Srivastava BS. Selective identification of new therapeutic targets of Mycobacterium tuberculosis by IVIAT approach. Tuberculosis (Edinb) 2002;82:175-82.
    18. Kim YR, Lee SE, Kim CM, Kim SY, Shin EK, Shin DH, et al. Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients. Infect Immun 2003;71:5461-71.
    19. John M, Kudva IT, Griffin RW, Dodson AW, McManus B, Krastins B, et al. Use of in vivo-induced antigen technology for identification of Escherichia coli O157:H7 proteins expressed during human infection. Infect Immun 2005; 73:2665-79.
    20. Harris JB, Baresch-Bernal A, Rollins SM, Alam A, LaRocque RC, Bikowski M, et al. Identification of in vivo-induced bacterial protein antigens during human infection with Salmonella enterica serovar Typhi. Infect Immun 2006; 74:5161-8.
    21. Salim KY, Cvitkovitch DG, Chang P, Bast DJ, Handfield M, Hillman JD, et al. Identification of group A Streptococcus antigenic determinants upregulated in vivo. Infect Immun 2005;73:6026-38.
    22. Cao SL, Progulske-Fox A, Hillman JD, Handfield M. in vivo induced antigenic determinants of Actinobacillus actinomycetemcomitans. FEMS Microbiol Lett 2004;237:97-103.
    23. Song YH, Kozarov EV, Walters SM, Cao SL, Handfield M, Hillman JD, et al. Genes of periodontopathogens expressed during human disease. Ann Periodontol 2002;7:38-42.
    24. Gu H, Zhu H, Lu C. Use of in vivo-induced antigen technology (IVIAT) for the identification of Streptococcus suis serotype 2 in vivo-induced bacterial protein antigens. BMC Microbiol 2009;9:201.
    25. Lee HR, Rhyu IC, Kim HD, Jun HK, Min BM, Lee SH, et al. In-vivo-induced antigenic determinants of Fusobacterium nucleatum subsp. nucleatum. Mol Oral Microbiol 2011; 26:164-72.
    26. Savli H, Karadenizli A, Kolayli F, Gundes S, Ozbek U, Vahaboglu H. Expression stability of six housekeeping genes: A proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR.J Med Microbiol 2003;52:403-8.
    27. Vandecasteele SJ, Peetermans WE, Merckx R, Van Eldere J. Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under different conditions. J Bacteriol 2001;183:7094-101.
    28. Widada J, Nojiri H, Kasuga K, Yoshida T, Habe H, Omori T. Quantification of the carbazole 1,9a-dioxygenase gene by real-time competitive PCR combined with co-extraction of internal standards. FEMS Microbiol Lett 2001;202:51-7.
    29. Smalley JW, Olczak T. Heme acquisition mechanisms of Porphyromonas gingivalis - strategies used in a polymicrobial community in a heme-limited host environment. Mol Oral Microbiol 2017;32:1-23.
    30. Olczak T, Sroka A, Potempa J, Olczak M. Porphyromonas gingivalis HmuY and HmuR: further characterization of a novel mechanism of heme utilization. Arch Microbiol2008;189:197-210.
    31. McKee AS, McDermid AS, Baskerville A, Dowsett AB, Ellwood DC, Marsh PD. Effect of hemin on the physiology and virulence of Bacteroides gingivalis W50. Infect Immun 1986;52:349-55.
    32. Wu J, Lin X, Xie H. Regulation of hemin binding proteins by a novel transcriptional activator in Porphyromonas gingivalis.J Bacteriol 2009;191:115-22.
    33. Ratnayake DB, Wai SN, Shi Y, Amako K, Nakayama H, Nakayama K. Ferritin from the obligate anaerobePorphyromonas gingivalis: purification, gene cloning and mutant studies. Microbiology 2000;146 ( Pt 5):1119-27.
    34. Olczak T, Simpson W, Liu X, Genco CA. Iron and heme utilization in Porphyromonas gingivalis. FEMS Microbiol Rev 2005;29:119-44.
    35. Delmar JA, Su CC, Yu EW. Bacterial multidrug efflux transporters. Annu Rev Biophys 2014;43:93-117.
    36. Saleem HG, Seers CA, Sabri AN, Reynolds EC. Dental plaque bacteria with reduced susceptibility to chlorhexidine aremultidrug resistant. BMC Microbiol 2016;16:214.
    37. Kulik EM, Lenkeit K, Chenaux S, Meyer J. Antimicrobial susceptibility of periodontopathogenic bacteria. J AntimicrobChemother 2008;61:1087-91.
    38. Kurita-Ochiai T, Seto S, Suzuki N, Yamamoto M, Otsuka K, Abe K, et al. Butyric acid induces apoptosis in inflamedfibroblasts. J Dent Res 2008;87:51-5.