Antibacterial Activity of Sodium Citrate against Oral Bacteria Isolated from Human Tongue Dorsum

J. Oral Biosci. 53 （1）：87−92, 2011

SHORT COMMUNICATION

Antibacterial Activity of Sodium Citrate against Oral Bacteria Isolated from Human Tongue Dorsum Reiko Watanabe1）, Kenichi Hojo1,2）,§, Seiji Nagaoka1）, Katsunori Kimura1）, Tomoko Ohshima2）and Nobuko Maeda2） 1）

Food Science Institute, Meiji Dairies Corporation

2）

Department of Oral Bacteriology, School of Dental Medicine, Tsurumi University 1）

540 Naruda, Odawara, Kanagawa 250−0862, Japan 2）

2−1−3 Tsurumi, Yokohama 230−8501, Japan 〔Received September 6, 2010；Accepted October 29, 2010〕 Key words：sodium citrate／tongue coating／hydrogen sulfide Abstract：Antibacterial activity of sodium citrate against tongue bacteria, especially hydrogen−sulfide（H2S） producers, was examined. Based on 16s rDNA partial sequences analysis, 691 isolates from 6 subjects were identified. In the present study, Prevotella histicola, P. melaninogenica, Veillonella atypica and V. dispar／V. parvula were detected as predominant H2S producing bacteria. Antibacterial assay showed that sodium citrate inhibited the growth of the representative strains of H2S producers at low concentrations although it similarly inhibited that of commensal bacteria, viz., Streptococcus parasanguinis and S. salivarius. When the effects of several pH conditions on antibacterial activity of sodium citrate against P. melaninogenica JCM6325T were examined, the activity was effective under neutral pH as well as under acidic conditions.

Introduction  Halitosis arises from a variety of sources including the sinuses, gastrointestinal tract, ingested food, lungs and oral cavity；however, the main cause is believed to originate above all in volatile sulfur compounds such as hydrogen sulfide（H2S）and methyl mercaptan, which are produced by oral bacteria1）. Several recent studies have suggested that tongue coating is closely associated with halitosis2―5）. Washio et al.5） recently reported that an increase in viable cells of Actinomyces, Prevotella, and Veillonella, which are ubiquitous oral bacteria, is responsible for oral §  

Correspondence author E−mail：kenichi_houjou@meiji−milk.com

malodor. Accordingly, it is likely that keeping a low level of bacterial cells on the tongue dorsum is of importance for fresh breath.  Sodium citrate is widely used as an acidulant, flavorant and preservative in foods and beverage as an economical and safe food additive. It has been reported that sodium citrate strongly inhibits the growth of opportunistic pathogenic bacteria, coagulase−negative staphylococci6）. Our previous study suggested that sodium citrate suppresses the growth of Streptococcus pneumoniae under neutral pH conditions7）. From these previous studies, we expected that sodium citrate is available for oral hygiene including the prevention of halitosis. In the present study, we isolated hydrogen−sulfide （H2S） producing−bacteria from human tongue dorsum and evaluated the antimicrobial activity of sodium citrate against 95 representa-

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R. Watanabe, et al：Inhibition of Oral Bacteria by Sodium Citrate

tive isolates. Materials and Methods   1 ．Bacterial isolation from tongue dorsum  Six healthy subjects（3 males and 3 females）aged 24 to 59（mean age±standard deviation：38.2±12.3 years） were recruited, and informed consent was obtained from each subject. The Ethical Committee of Meiji Dairies Corporation approved the study protocol. Bacterial isolation was carried out according to the procedure described by Washio et al.5） with minor modification. In order to collect tongue bacteria, an area of 1 cm2, predetermined by a window made of sterilized plain paper on the rear dorsal surface of the tongue, was firmly scraped with a sterilized cotton swab. Samples were suspended in 1 mL of trypticase soy broth without dextrose（BBL, USA）and were diluted in the same medium. One hundred microliters from each dilution was dispersed and spread onto Columbia blood agar base（Difco, USA） plates containing 4 mM L−cysteine HCl, 0.2 g／L lead acetate, 1 mg／L menadione, and 5 mg／L hemin. All plates were anaerobically（Anaero Pack System；Mitsubishi Gas Chemical Co., Japan）incubated at 37℃ for 5 days. Black or grey colonies were considered as H2S−producing bacteria.   2 ．Provisional identification of bacterial isolates  Bacterial isolates were identified by 16S rDNA partial sequence analysis. The primers for the amplifica−AGA GTT TGA TCC tion of 16S rDNA were 27F（5′ −ACC GCG GCT TGG CTC AG−3′）and 520R（5′ GCT GGC−3′）. PCR products were purified by using the GFX PCR DNA and Gel Band Purification kit （Amersham Biosciences, USA） and sequenced directly with the ABI PRISM Dye Terminator Cycle Sequencing Kit（AB Applied Biosystems, USA）and the 3100 Genetic Analyzer（ABI Applied Biosystems）. By using the BLAST algorithm, the results were compared with the sequences deposited in the DNA Data Bank of Japan. The isolates were identified based on more than 98％ sequence similarity.

  3 ．Minimum inhibitory concentration  Minimum inhibitory concentration（MIC）of sodium citrate against 95 representative tongue isolates was determined by a broth dilution method8）. The isolates were randomly selected from the subjects. In this study, cation−adjusted Mueller−Hinton broth （BD） with 50 mg／L calcium and 25 mg／L magnesium was used. When streptococci were assessed, 5％ horse hemolysate was added into the broth. The MIC for Veillonella isolates was assessed in cation−adjusted Brain Heart Infusion（BHI, BBL）instead of Muller− Hinton broth. For the isolates belonging to the genus Prevotella, BHI supplemented with 5 mg／L hemin and 1 mg／L menadion（BHI−HK）was used. Assay media with twofold serial dilutions of sodium citrate（0.4― 102.4 mg／mL）were prepared. Bacterial suspensions, prepared in saline by harvesting from fresh overnight cultures on sheep blood agar plates, were adjusted to MacFarland 0.5 turbidity, and a tenfold dilutions of these bacterial suspensions were then prepared. Five microliters of each dilution was added to 100 μL of the assay media in a microtiter plate. The cultures were incubated anaerobically for 18―24 h at 37℃. MIC was defined as the lowest concentration for inhibiting visible growth.   4 ．Effect of pH on the antibacterial activity against P. melaninogenica  The antibacterial activity was assessed under several pH conditions. BHI−HK with 25 mM sodium citrate or 100 mM sodium lactate was prepared, and medium pH was adjusted to 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 using 1−N HCl or 4−N NaOH. All media were filtrated by 0.45 μm membrane filter before use. P. melaninogenica JCM6325T was tested in this study. The cultures were incubated anaerobically for 72 h at 35℃. After incubation, the growth of P. melaninogenica was measured by optical density at 600 nm. Results  In this study, we used Columbia blood agar base plates containing cysteine and lead acetate. Therefore, the colony colour provided information about H2S production. Almost all isolates belonging to the

89 Table 1 Composition of commensal bacteria on the tongue dorsum Putative bacterial species Actinobacteria  Actinomyces graevenitzii  Actinomyces lingnae  Actinomyces naeslundii  Actinomyces odontolyticus  Actinomyces sp.  Rothia mucilaginosa Bacteroidetes  Porphyromonas sp.  Prevotella aurantiaca  Prevotella histicola  Prevotella intermedia  Prevotella melaninogenica  Prevotella nanceiensis  Prevotella pallens  Prevotella salivae  Prevotella shahii  Prevotella sp.  Prevotella sp.  Prevotella sp. Firmicutes  Abiotrophia defectiva  Catonella morbi  Eubacterium saburreum  Eubacterium sulci  Gemella sanguinis  Granulicatella adiacens  Megasphaera sp.  Mogibacterium vescum  Oribacterium sinus  Peptostreptococcus sp.  Streptococcus mitis related−species  Streptococcus parasanguinis  Streptococcus salivarius  Streptococcus sp.  Streptococcus sp.  Streptococcus sp.  Veillonella atypica  Veillonella dispar／parvula  Veillonella sp. Fusobacteria  Fusobacterium periodonticum  Leptotrichia sp. Proteobacteria  Campylobacter showae  Haemophilus parahaemolyticus  Haemophilus parainfluenzae No. of isolates Total bacterial counts（cfu／cm2）

％ isolatates from each subject A

B

C

D

E

F

― ― ― 25.5 ― 1.0

― ― ― 31.1 ― ―

― 0.7 ― 0.7 ― ―

― ― 1.7 10.8 0.8 3.3

― 4.3 1.4 27.1 ― 1.4

1.2 ― ― 4.7 ― ―

― ― 12.7 ― 2.0 ― 4.9 1.0 ― 1.0 ― ―

― ― 6.7 2.2 8.9 ― 6.7 2.2 ― 2.2 2.2 4.4

― ― 6.8 ― 9.5 ― ― ― ― ― ― 4.7

1.2 ― 0.8 ― 6.6 0.4 1.2 ― ― 0.8 ― 3.7

1.4 1.4 ― ― 8.6 1.4 ― ― ― 1.4 ― ―

― ― 3.5 ― 1.2 1.2 4.7 7.1 1.2 ― 1.2 15.3

― ― 2.0 ― 1.0 ― ― 1.0 1.0 ― ― 13.7 10.8 ― 2.0 ― 6.9 12.7 ―

― ― ― 2.2 ― ― ― 4.4 2.2 ― 11.1 ― 6.7 ― ― ― 2.2 ― ―

― ― 0.7 ― ― 5.4

3.7 ―

0.7 6.1 ― 18.9 2.7 17.6 3.4 ― ― 2.0 19.6 ―

0.4 ― 3.7 0.4 0.8 0.4 ― 19.1 7.1 15.8 3.3 ― 0.4 3.7 8.7 ―

― ― 7.1 1.4 ― ― 4.3 ― ― ― 17.1 ― ― ― ― ― ― 12.9 ―

― 1.2 ― ― ― 1.2 4.7 1.2 5.9 4.7 ― 4.7 14.1 2.4 ― ― 14.1 3.5 1.2

― 1.0

2.2 ―

― ―

0.8 ―

5.7 ―

― ―

― ― ― 102 1.0×108

― 2.2 ― 45 4.5×107

― ― 0.7 148 1.5×108

― ― ― 241 2.4×107

1.4 ― 1.4 70 7.0×106

― ― ― 85 8.5×106

R. Watanabe, et al：Inhibition of Oral Bacteria by Sodium Citrate

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Table 2 Minimum inhibitory concentration of sodium citrate on representative oral isolates Organism Actinomyces odontlyticus Prevotella histicola Prevotella melaninogenica Streptococcus parasanguinis Streptococcus salivarius Veillonella atypica Veillonella disper／parvula

No. of isolates tested

0.4

0.8

1.6

3.2

6.4

12.8

25.6

51.2

102.4

27 13 7 14 13 9 12

― ― ― ― ― 1 ―

― ― ― ― ― 1 ―

― ― ― ― ― 1 ―

― ― ― ― ― ― ―

3 4 4 2 ― 1 ―

2 6 ― 11 13 ― ―

7 2 3 1 ― 5 12

12 1 ― ― ― ― ―

3 ― ― ― ― ― ―

No. of isolates with MIC at sodium citrate（mg／mL）

genera Prevotella and Veillonella formed entirely black colonies or bull’s−eye colonies with black in the centres, suggesting that they greatly or moderately produce H2S. On the other hand, the isolates belonging to the genera Actinomyces and Streptococcus formed white or light grey colonies, or bull’s−eye colonies with grey or black in the centres. Therefore, these groups seemed to be non− or low−H2S producers. The other bacterial species apparently had high or middling ability of H2S production because they formed entirely or at least partly black colonies.  Based on 16s rDNA partial sequences analysis, 691 isolates from 6 subjects were identified. The sequences of almost all isolates corresponded to those of known species, while a few strains did not. In addition, partial sequences analysis could not clearly discriminate between Veillonella dispar and V. parvula, and between Streptococcus mitis and its closely related−species, e.g., S. infantis, S. cristatus and S. sanguinis. As shown in Table 1, the genera of Actinomyces, Prevotella, Streptococcus and Veillonella were dominantly detected. A. odontolyticus and P. melaninogenica were isolated from all subjects.  Table 2 shows the antibacterial activity of sodium citrate. In this study, P. histicola, P. melaninogenica, V. atypica and V. dispar／V. parvula were tested as representative H2S−producers because these species were dominant and prevalent in the subjects. The MICs of sodium citrate for these isolates were between 0.4 and 25.6 mg／mL, except one strain of P. histicola. A. odontolyticus seemed to be slightly more tolerant as compared with other species. Non− or low−H2S producers, namely S. parasanguinis and S. salivarius,

Fig. 1 Effect of medium pH on the bacteriostatic activity of sodium citrate and sodium lactate against Prevotella melaninogenica JCM6325T. Symbols：circles, 25 mM sodium citrate；squares, 100 mM sodium lactate. Experimens were carried out three times independently and average values are plotted.

were sensitive to sodium citrate as well as the representative H2S−producers.  The characteristics of the inhibitory activity of sodium citrate were examined in comparison with sodium lactate. Since the bactericidal activity of sodium lactate was modest compared with that of sodium citrate（data not shown）, an assay of sodium lactate was carried out using higher concentrations . As shown in （100 mM）than sodium citrate（25 mM） Fig.  1, the bacteriostatic activity of sodium lactate against P. melaninogenica JCM6325T was dependent on pH. The activity was decreased under neutral pH conditions. In contrast, the antimicrobial activity of sodium citrate was not influenced by the medium pH.

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Discussion  It has been reported that the members of the genera Eubacterium, Fusobacterium, Porphyromonas, Prevotella, Propionibacterim and Veillonella have a high capacity to produce H2S from serum or L−cysteine9）. In contrast, Actinomyces and Streptococcus generated H2S less than the former genera9）. In the present study, Actinomyces, Prevotella, Streptococcus and Veilonella were dominantly isolated from the tongue dorsum. Judging from the colony colour, almost all isolates of Prevotella and Veilonella were likely to produce a great deal of H2S, whereas Actinomyces and Streptococcus seemed to produce less H2S. Our results thus correspond with the in vitro data reported by Persson and co−workers9）.  Many previous studies have reported that many different kinds of bacterial species inhabit the tongue dorsum2―5）. In the present study, at least 43 bacterial species were detected on the tongue dorsum of 6 subjects. Almost all of these species corresponded with the bacterial species reported by the previous studies2―5）.  The results presented in this study showed that sodium citrate inhibited the growth of P. histicola, P. melaninogenica, V. atypica and V. dispar／V. parvula at concentrations between 0.4 and 51.2 mg／mL although it similarly inhibited that of commensal bacteria, viz., Streptococcus parasanguinis and S. salivarius. Importantly, our study shows that the antimicrobial activity of sodium citrate against P. melaninogenica JCM6325T was effective under neutral pH conditions. In general, organic acids and their salts are effective at low pH because they exert the antimicrobial activity by intracellular transition in the undissociated form and by subsequent dissociation within the bacterial cell10）. In fact, the antimicrobial activity of sodium lactate was dependent on pH. On the other hand, it is reported that disodium ethylenediaminetetraacetic acid （EDTA） , a chelating agent, is effective at high pH11）. EDTA is thought to lead to increased permeability of bacterial outer membrane and to subsequently cause lysis and loss of viability12）. Although the exact mechanism of the antibacterial activity of sodium cit-

rate has not been identified yet, we speculate that the antimicrobial activity of sodium citrate may be attributed to chelating activity because it is also a strong chelating agent.  In conclusion, sodium citrate inhibited the growth of tongue bacteria including H2S−producing bacteria. Further studies are needed to clarify the exact antibacterial mechanism and the efficacy for the prevention of oral malodor. References R. and Green1）Scully, C., el−Maaytah, M., Porter, S.  man, J.：Breath odor：etiopathogenesis, assessment and management. Eur. J. Oral Sci. 105：287―293, 1997. 2）Haraszthy, V.  I., Zambon, J.  J., Sreenivasan, P.  K., Zambon, M.  M., Gerber, D., Rego, R. and Parker, C.  J.： Identification of oral bacterial species associated with halitosis. J. Am. Dent. Assoc. 138：1113―1120, 2007. 3）Kakuta, E., Ohshima, T., Nakagawa, Y. and Maeda, N.：Relationship between two Bacterial species and oral malodor. Tsurumi Univ. Dent. J. 35：1―8 2009. 4）Kazor, C.  E., Mitchell, P.  M., Lee, A.  M., Stokes, L.  N., Loesche, W.  J., Dewhirst, F.  E. and Paster, B.  J.： Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients. J. Clin. Microbiol. 41：558―563, 2003. 5）Washio, J., Sato, T., Koseki, T. and Takahashi, N.： Hydrogen sulfide−producing bacteria in tongue biofilm and their relationship with oral malodour. J. Med. Microbiol. 54：889―895, 2005. 6）Lee, Y.  L., Thrupp, L., Owens, J., Cesario, T. and Shanbrom, E.：Bactericidal activity of citrate against Gram−positive cocci. Lett. Appl. Microbiol. 33：349 ―351, 2001. 7）Nagaoka, S., Murata, S., Kimura, K., Mori, T. and Hojo, K.：Antimicrobial activity of sodium citrate against Streptococcus pneumoniae and several oral bacteria Lett. Appl. Microbiol. 51：546―551, 2010. 8）Japan Society of Chemotherapy：Method of MIC determination by broth microdilution method. Chemother 38：102―105, 1990.（in Japanese） 9）Persson, S., Edlund, M.  B., Claesson, R. and Carlsson, J.：The formation of hydrogen sulfide and methyl mercaptan by oral bacteria. Oral Microbiol. Immunol. 5：195―201, 1990.

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Taguchi, F.：Effect of pH on preferential antibacterial−activity of ethylenediaminetetraacetic acid（EDTA） Jpn. J. Bacteriol. 47：625―629, 1992.（in Japanese） 12）Vaara, M.：Agents that increase the permeability of the outer membrane. Microbiol. Rev. 56：395―411, 1992.