J. Gen. Appl. Microbiol., 64, 55–61 (2018) doi 10.2323/jgam.2017.06.003 „2018 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation Full Paper Inhibitory effect of Lactococcus lactis on the bioactivity of periodontopathogens (Received February 27, 2017; Accepted June 28, 2017; J-STAGE Advance publication date: January 25, 2018) Hyun-Seung Shin,1 Dong-Heon Baek,2 and Sung-Hoon Lee2,* 1 Department of Periodontology, College of Dentistry, Dankook University, Cheonan, Republic of Korea 2 Department of Oral Microbiology and Immunology, College of Dentistry, Dankook University, Cheonan, Republic of Korea Lactococcus lactis is a probiotic bacterium that pro- Key Words: antibacterial activity; L. lactis; duces various bacteriocins. Periodontopathogens probiotics; neutralizing activity; induce inflammation and halitosis through the ac- periodontopathogens tions of lipopolysaccharide (LPS) and trypsin-like enzymes. The purpose of this study was to investi- gate the inhibitory effects of L. lactis on the Introduction bioactivity of periodontopathogens. To investigate the antimicrobial peptide of L. lactis, the spent cul- Lactococcus lactis is a Gram-positive and spherical- ture medium (SCM) of L. lactis was treated with or shaped facultative anaerobic bacterium (Brooijmans et al., without proteinase K after collection by centrifu- 2007). This bacterium is widely used in the production of gation, and the antibacterial activity of SCM fermented dairy foods such as cheese, yogurt, and sour against periodontopathogens was assessed. To cream as a probiotic bacterium (Karpinski and evaluate the neutralizing effect of L. lactis on hali- Szkaradkiewicz, 2013; Kimoto-Nira et al., 2014; van tosis, SCM of periodontopathogens was mixed with Hylckama Vlieg et al., 2006). Also, L. lactis produces an L. lactis suspension, and the levels of volatile various bacteriocins including diacetin, lactococcin and sulfur compounds (VSCs) were measured by gas nisin (Ali et al., 1995; Dussault et al., 2016; Holo et al., chromatography. LPS from the 1991), and these bacteriocins exhibit bactericidal or bac- periodontopathogens was extracted by an LPS ex- teriostatic effects on Gram-positive and Gram-negative traction kit with little modification, and THP-1 cells pathogens (Ali et al., 1995; Arques et al., 2015). Further- as a monocytic cell line were treated with the ex- more, L. lactis antagonizes pathogenic bacteria in the host tracted LPS in the presence or absence of UV-killed gut through its antimicrobial metabolites, such as hydro- L. lactis. The production of inflammatory cytokines gen peroxide, acetaldehyde, and ethanol organic acids was analyzed by ELISA. The SCM of L. lactis ex- (Enan et al., 2013). This bacterium improves human health hibited antimicrobial activity against the by providing nutritional benefits and helping to balance periodontopathogens, whereas the proteinase K- cholesterol and bile salts (Enan et al., 2013; Tanaka et al., treated SCM showed little antimicrobial activity. 1999) and is therefore considered a beneficial bacterium. In addition, the L. lactis suspension had a neutral- Periodontitis is a chronic inflammatory condition of the izing effect on the VSCs produced by gingiva with a polymicrobial etiology. Porphyromonas periodontopathogens, and UV-killed L. lactis inhib- gingivalis, Tannerella forsythia and Treponema denticola ited the production of IL-6 and TNF-a induced by are directly associated with this condition and are there- the LPS. These results suggest that L. lactis may be fore referred to as periodontopathogens or red complex a useful probiotic to prevent and treat periodonti- bacteria (Socransky and Haffajee, 2002; Socransky et al., tis and halitosis. 1998). These Gram-negative obligate anaerobic bacteria exist within a biofilm that forms in subgingival pockets, with Fusobacterium nucleatum serving as a bridge bacte- rium to the supragingival biofilm, which consists mostly *Corresponding author: Sung-Hoon Lee, Department of Oral Microbiology and Immunology, College of Dentistry, Dankook University, San 29 Anseo-dong, Dongnam-gu, Cheonan 330-714, Republic of Korea. Tel: +82-41-550-1867 Fax: +82-41-550-1859 E-mail: [email protected] None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. 56 SHIN, BAEK, and LEE of streptococci (Socransky and Haffajee, 2005). The li- L. lactis was treated with proteinase K (100 mg/ml) at 55∞C popolysaccharide (LPS) of these bacteria acts as an im- for 30 min and heated to inactivate proteinase K at 100∞C mune stimulator by inducing gingival inflammation and for 10 min. Proteinase K-treated SCM was assayed as de- activating osteoclasts via Toll-like receptors (TLR2 or scribed above. TLR4) that trigger the expression of various cytokines, All four periodontopathogens were counted with a bac- which in turn causes alveolar bone resorption (Kim and terial counting chamber (Marienfeld, Lauda-Konigshofen, 6 Lee, 2014; Lee, 2015; Lee and Baek, 2013; Socransky et Germany) and then diluted to 3 ¥ 10 cells/ml in the re- al., 1998). Another characteristic of periodontopathogens spective media. Twenty microliters of the is their ability to induce halitosis, an oral malodor. periodontopathogen suspensions was inoculated into the Periodontopathogens secrete trypsin-like enzymes that prepared wells. The plates were incubated at 37∞C in an produce volatile sulfur compounds (VSCs) such as hydro- anaerobic chamber for 36 h, and optical densities were gen sulfide(H2S), methyl mercaptan (CH3SH), and dime- measured at 600 nm using a microplate reader (BioTek, thyl sulfide ([CH3]2S) in the presence of methionine and Winooski, VT, USA). cysteine in human serum protein (Lee and Baek, 2014). Co-cultivation of L. lactis and periodontopathogens. To VSCs are responsible for halitosis. investigate the effect of L. lactis on periodontitis when This study investigated the effects of L. lactis on the they co-existed, co-cultivation of L. lactis and F. periodontopathogens, F. nucleatum, P. gingivalis, T. for- nucleatum, P. gingivalis, T. forsythia, or T. denticola was sythia, and T. denticola by focusing on their bioactivity; performed. L. lactis and periodontopathogens were co- namely, their growth, induction of inflammation, and pro- cultivated by Millicell® cell culture insert (Millipore, duction of VSCs. Billerica, MA, USA). In the cases of F. nucleatum and P. gingivalis, BHI broth supplemented with hemin and vita- Materials and Methods min K was used, and in the cases of T. forsythia and T. denticola, a mixture of BHI and an equal volume of a spe- Bacterial strain and culture conditions. L. lactis HY449 cific medium was used. The prepared media were dis- was gratefully received from Yakult (Korea Yakult Com, pensed into two new tubes, and L. lactis and Gyeonggi, Korea) and was cultivated in brain heart infu- periodontopathogens were inoculated into each tube. Cul- sion (BHI) broth (BD Bioscience, San Jose, CA, USA) at ture inserts were placed into the wells of a 12-well plate. 37 C under anaerobic conditions (5% H , 10% CO , and ∞ 2 2 The suspension of periodontopathogens and L. lactis were 85% N ). F. nucleatum ATCC 25586 and P. gingivalis 2 dispensed into the inside and basolateral side, respectively, ATCC 33277 were cultured anaerobically with BHI broth of the insert, with the recommended volumes. The plates supplemented with hemin (1 g/ml) and vitamin K (0.2 m were incubated at 37 C in an anaerobic chamber for 36 h, g/ml) at 37 C, anaerobically. T. forsythia ATCC 43037 ∞ m ∞ and the periodontopathogens were counted by a bacterial and T. denticola ATCC 35405 were cultured in modified counting chamber (Marienfeld). new oral spirochete (mNOS) broth (Lee et al., 2010) and tryptone-yeast extract-gelatin-volatile fatty acid-serum Measurement of VSCs. VSC levels were measured in gas (TYGVS) broth respectively, at 37∞C (Ohta et al., 1986) from the SCM from the periodontopathogen cultures mixed in an anaerobic atmosphere. or unmixed with various volumes of L. lactis suspension. After cultivation for 36 h, the SCM (1 ml) of Antibacterial activity of L. lactis against periodontopathogens were transferred to 50-ml conical periodontopathogens. The antimicrobial susceptibility of tubes to which 1, 2, or 3 ml of L. lactis suspension was periodontopathogens to L. lactis was determined by a mini- added. The mixtures were filled to 5 ml with fresh BHI mum inhibitory concentration assay in a microplate, ac- media, and the control group was filled with 4 ml of fresh cording to methods recommended by Clinical and Labo- BHI medium. The preparations were vortexed for 30 s. ratory Standards Institute (CLSI) (Hecht et al., 2007). Five VSC gas was collected above the mixed solution using a milliliters of L. lactis (1 107 cells/ml) was inoculated ¥ 10 ml syringe, and one milliliter of VSC gas was injected into 50 ml fresh BHI broth, and the bacteria were culti- into Oral ChromaTM gas chromatograph (FIS Inc., Itami, vated for 24 h under aerobic conditions. The bacterial sus- Hyogo, Japan), and the level of VSCs was measured. pension was centrifuged at 7,000 ¥ g, and the supernatant (spent culture medium) was transferred into a new tube Lipopolysaccharide extraction. LPS was extracted from and then filtrated through a polyvinylidene fluoride filter F. nucleatum, P. gingivalis, T. forsythia, and T. denticola (pore size 0.22 mm). To investigate susceptibility, 20–180 by an LPS extraction kit with little modification as de- ml of BHI broth containing hemin (1 mg/ml) and vitamin scribed by Lee (2015). After cultivation in the respective K (0.2 mg/ml) was dispensed into each well (three rows) media, the periodontopathogens were harvested by cen- from the 10th column to the 1st column in a 96-well poly- trifugation at 6,500 ¥ g for 10 min at 4∞C and then washed styrene plate (SPL Life Sciences, Gyeonggi, Korea) using with cold phosphate buffered saline (PBS; pH 7.0). Then, a multi-channel pipette. After adding hemin and menadi- the periodontopathogens were mixed with lysis buffer and one to the spent culture medium (SCM) of the probiotics, vortexed until the bacteria pellet disappeared.