J. Med. Microbiol. Ð Vol. 50 2001), 42±48 # 2001 The Pathological Society of Great Britain and Ireland ISSN 0022-2615 ORAL MICROBIOLOGY The sensitivity of Porphyromonas gingivalis and Fusobacterium nucleatum to different pseudo)halide-peroxidase combinations compared with mutans streptococci RIIKKA IHALIN, VUOKKO LOIMARANTA, MARIANNE LENANDER-LUMIKARI and JORMA TENOVUO Institute of Dentistry, Turku Immunology Centre and TuBS, University of Turku, Turku, Finland Previous studies have shown that the peroxidase system with iodide is particularly effective against Actinobacillus actinomycetemcomitans. In the present study, the effects of iodide, chloride and thiocyanate in combinations with lactoperoxidase LP) and myeloperoxidase MP) on the viability of Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and S. rattus were analysed. Bacteria were incubated in buffer solution containing peroxidase, substrates) and H2O2 all in oral physiological concentrations), and plated after 0, 0.5 and 1 h. The oxidation product of iodide was the most bactericidal against all the bacteria tested. The effect was signi®cantly weaker on mutans streptococci. Physiological concentrations of thiocyanate abolished the effects of LP-H2O2-iodide and MP-H2O2-iodide/chloride combinations. Thiocyanate-peroxidase systems have already been used in oral hygiene products. The incorporation of iodide into these products could make them much more potent against periodontal pathogens, and also help to prevent transmission of these pathogens from person to person via saliva. Introduction MP. In gingival crevicular ¯uid, CIÀ is most abundant c.90mM) and the amount of IÀ 4.3, SD 1.9 ìM)is Human oral ¯uids, saliva and gingival crevicular ¯uid, only about one-tenth of the SCNÀ concentration contain peroxidase enzymes which are part of the 37 SD 24 ìM) [3, 4]. In stimulated whole saliva, the innate host defence system. Salivary peroxidase SP) is ClÀ concentration is in the range 10±56 mM and the IÀ secreted by major salivary glands and is found only in concentration is c.10SD7ìM [5]. The salivary saliva, whereas myeloperoxidase MP) originates from concentrations of SCNÀ vary and depend, for example, polymorphonuclear leucocytes PMNLs) and ®lters into on diet and smoking habits. The normal range of saliva from gingival crevicular ¯uid. The amount of salivary SCNÀ for non-smokers is 0.5±2 mM, but MP in gingival crevicular ¯uid, and thereafter also in concentrations as high as 6 mM can be found in saliva saliva, depends on the periodontal status [1]. In from smokers [6, 7]. The peroxidase systems have been periodontally healthy persons, the amount of MP in shown to inhibit bacterial [8±14], fungal [15, 16] and whole saliva is c.3:6 ìg=ml, which is twice the viral [17±19] viability. amount of SP 1:9 ìg=ml) [2]. The complete perox- idase system consists of three components: a perox- Actinobacillus actinomycetemcomitans and Porphyro- idase enzyme, hydrogen peroxide H2O2) and an monas gingivalis are among the main pathogens in oxidisable substrate such as a halide or a pseudohalide. periodontitis. Fusobacterium nucleatum has been Both SP and MP can oxidise thiocyanate SCNÀ) and proposed as the major cause of initial peridontal iodide IÀ), whereas chloride CIÀ) is oxidised only by irritation and it usually maintains its proportion in the peridontal ¯ora when gingivitis progresses to perio- dontitis [20]. The complete lactoperoxidase LP) Received 6 Dec. 1999; revised version accepted 19 May system has been used in some oral hygiene products, 2000. mainly to prevent caries. Although the use of such Corresponding author: Dr. R. Ihalin e-mail: riikka.ihalin@ products elevates the amount of hypothiocyanous acid/ utu.®). hypothiocyanite ion HOSCN/OSCNÀ) in saliva [21], P. GINGIVALIS, F. NUCLEATUM AND PEROXIDASE SYSTEMS 43 their effects on the levels of Streptococcus mutans, LP and MP activities were analysed by Nbs-SCN assay lactobacilli or the total oral ¯ora in vivo have not been [26]. In the assay, peroxidases in the sample oxidise À signi®cant [21±23]. However, there is some evidence 4.0 mM SCN in the presence of 100 ìM H2O2 to of their favourable in¯uences on the condition of the produce hypothiocyanite ions OSCNÀ). OSCNÀ ions gingiva in patients who suffer from oral dryness [22]. oxidise 5-thio-2-nitrobenzoic acid TNB), which is produced from 5,59-dinitrobis-2-nitrobenzoic acid) The effect of peroxidase systems on A. actinomyce- DTNB) by reduction with 2-mercaptoethanol. The temcomitans has been studied extensively [10, TNB concentration was adjusted to c.50ìM with 2- 13, 24, 25]. An earlier study demonstrated a superior mercaptoethanol. The absorbance of the reaction mix- effect of the oxidation product of IÀ compared with the ture was determined at 412 nm before and 15 s after other pseudo)halides on A. actinomycetemcomitans the addition of H2O2, and the amount of oxidation [10]. However, A. actinomycetemcomitans is only one product produced was counted from the decrease in of the pathogens in periodontitis. Therefore, this study absorbance: 1 mU corresponds to 1 ìM oxidising focused on P. gingivalis and F. nucleatum. These product of TNB produced in 1 min. Stock solutions results were compared with previous and current of LP and MP were prepared and the activity was ®ndings on the susceptibility of A. actinomycetemco- determined. The peroxidase stock solutions were mitans, S. mutans and S. rattus to the peroxidase divided into tubes and kept frozen À208C) until used. system. These results will help further development of Both LP and MP sustained their activities when stored antimicrobial oral hygiene products. at À208C in phosphate buffer. Determination of the effective H O concentration Materials and methods 2 2 Bacteria and growth conditions P. gingivalis was chosen as a test bacterium because of its greater sensitivity to the peroxidase systems than F. A clinical isolate of P. gingivalis TUPg007 kindly nucleatum, as observed in preliminary studies. The provided by Dr K. Kari, University of Helsinki, H2O2 concentrations were varied 0±40 ìM and Finland) and F. nucleatum ATCC 10953 were grown 0±10 ìM in tests with LP-SCNÀ 1 mM) and LP-IÀ on Brucella Blood Agar Becton Dickinson, Cockeys- 5 ìM) combinations, respectively. The LP concentra- ville, MD, USA) with frozen sheep blood 5% and non- tion was 5 ìg=ml. The bacteria were incubated for selective CVE-plates containing trypticase Becton 30 min with LP and either SCNÀ or IÀ and the Dickinson) 1%, yeast extract Difco, Detroit, Michigan, appropriate concentration of H2O2, then plated on USA) 0.5%, NaCl 0.5%, glucose 0.2%, L-tryptophan Brucella blood agar and numbers of cfu were 0.02%, Bacto-agar Difco) 1.5%, frozen sheep blood determined as described previously [10]. 5%, respectively. Before each experiment, S. mutans ATCC 25175 and S. rattas ATCC 19645 were grown Effects of the peroxidase systems on the viability on Blood Agar Gibco BRL, Paisley) with frozen sheep of bacteria blood 5%. After incubation, S. mutans and S. rattus were plated on MS-plates containing Mitis-salivarius The antibacterial effects of different pseudo)halide agar Difco) 9% and potassium tellurite 15 mg=L. An combinations of IÀ,ClÀ and SCNÀ as substrates of anaerobic atmosphere CO2 10%, N2 80%, H2 10%) both LP and MP systems were examined as described and a temperature of 378C were used for growth of the in detail by Ihalin et al. [10]. Bovine LP, RZ 0.74 anaerobic bacteria P. gingivalis and F. nucleatum) and Sigma) and human MP, RZ 0.76 a generous gift aCO2-rich atmosphere CO2 7%, O2 19%, N2 74%) at from the late Professor B. MaÊnsson-Rahemtulla, 378C for S. mutans and S. rattus. Before each Birmingham, AL, USA) were used at a ®nal concen- experiment, F. nucleatum, S. mutans and S. rattus were tration of 5 ìg 22 mU=ml and 4 ìg 22 mU=ml, grown for 4 days and P. gingivalis for 6 days. The respectively. The IÀ concentration was 5 ìM, the SCNÀ bacteria were suspended in the test media to OD600: concentration was either 50 ìM or 1 mM and the H2O2 0.90 SD 0.01 for P. gingivalis, S. mutans and S. rattus, concentration was 1.25 ìM or 5 ìM. After incubation and 0.97 SD 0.02 for F. nucleatum, corresponding to c. of bacteria, and the appropriate components of the 6.3 3 108, 1.7 3 109, 1.7 3 109 and 1.1 3 108 cfu=ml, peroxidase system, on a shaking water bath at 378C for respectively. 1h, 5ìl of a reducing reagent, dithiotreitol DTT), were added to a ®nal concentration of 1 mM to abolish the oxidation. Samples were taken immediately before Media and chemical assays the addition of H2O2 0 min) and after incubation for Solution I [10] was used in the experiments with IÀ 30 and 60 min. Ten-fold dilutions of the samples were À and SCN . It contained 9 mM Na2HPO4,24mM made in peptone water containing DTT ± tryptone KH2PO4, 1.5 mM MgSO4 and 67 mM Na2SO4. When Difco) 2.5%, thiotone E peptone Becton Dickinson) ClÀ was used as a substrate, Solution II 9 mM 2.5%, NaCl 5%, DTT 0.02% ± in all experiments with Na2HPO4,24mM KH2PO4, 1.5 mM MgSO4) was used. anaerobic bacteria. In experiments with S. mutans and The pH of the media was adjusted to 6.5. S. rattus, DTT was excluded from the dilution medium. 44 R. IHALIN ET AL. After each experiment, dilutions were plated to growth of P. gingivalis; after incubation for 30 min the enumerate the number of cfu and the growth conditions log10 cfu=ml was still 5.4 SD 0.1. were as described previously. The incubation time for P. gingivalis was 7 days, for F.