Post-hydrogen Peroxide Effect in Peroxidogenic Oral Streptococci

Full Te ms & Condi ions o access and use can be ound a h ps://www. and online.com/ac ion/jou nalIn o ma ion?jou nalCode=zmeh20 Mic obial Ecology in Heal h and Disease ISSN: (P in ) 1651-2235 (Online) Jou nal homepage: www. and online.com/jou nals/zmeh20 Pos -hyd ogen Pe oxide E ec in Pe oxidogenic O al S ep ococci A. Ga cía-Mendoza, J. Liébana, A. Cas illo, A. De La Higue a & J. Gu ié ez To ci e his a icle: A. Ga cía-Mendoza, J. Liébana, A. Cas illo, A. De La Higue a & J. Gu ié ez (1993) Pos -hyd ogen Pe oxide E ec in Pe oxidogenic O al S ep ococci, Mic obial Ecology in Heal h and Disease, 6:1, 17-22, DOI: 10.3109/08910609309141557 To link o his a icle: h ps://doi.o g/10.3109/08910609309141557 © 1993 The Au ho (s). Published by Taylo & F ancis. Published online: 11 Jul 2009. Submi you a icle o his jou nal A icle iews: 106 View ela ed a icles MICROBIAL ECOLOGY IN HEALTH AND DISEASE VOL. 6: 1 7-22 (1 993) Pos -hyd ogen Pe oxide E ec in Pe oxidogenic O al S ep ococci A. GARCiA-MENDOZA*, J. LIEBANA, A. CASTILLO, A. DE LA HIGUERA and J. GUTIERREZ Dep nien 9 Mic obiology. School o ' Medicine and School q 'Odon ology , Uni e si y o G anada, E- 18071 G anada, spuin Recei ed 1 h Ap il 1992; e ised 14 Augus 1992 The e ec s o inhibi o y concen a ions o hyd ogen pe oxide on he g ow h o 11 s ains o ou pe oxidogenic species o o al s ep ococci (S ep ococcus o alis, S ep ococcus mi is, S ep ococcus sanguis and S ep ococcus sob inus) we e s udied. The e ec o H,O, was measu ed as he pos -hyd ogen pe oxide e ec (PHPE), de ined as he di e ence in he ime necessa y o he bac e ial popula ion in ba ch cul u e o inc ease by one decimal loga i hmic uni o he numbe o colony o ming uni s pe millili e, be ween cul u es cxposed o a concen a ion equal o he co esponding minimum inhibi o y concen a ion o H,O,, and non-exposed cul u es. No PHPE was shown by S. o alis NCTC 11427; o he s ains es ed ga e imes anging om 20 min (S. .sanguis JENA 2697) o 9 h 15 min (S. mi is OGS 232). The PHPE appea s o be s ain- and species-dependen . KEY wo n-Hyd ogen pe oxide; O al s ep ococci; Pos -hyd ogen pe oxide e ec INTRODUCTION Se e al species o o al s ep ococci p oduce hyd ogen pe oxide, 9* ' and he abili y o sali a o inhibi bac e ial g ow h has been aced o hyd ogen pe oxide p oduced by hese mic oo - ganism~.'~.'~.'~.~'.~~ The oxic ac ion o H202 on mammalian cells has been shown wi h cell cul u e echniques.'.'' and sali a y lac ope oxidase may play a ole in he de oxica ion o he o al O he s udies ha e in es iga ed he ela ion be ween he pe oxidogenic capaci y o o al s ep ococci and he a ailable subs a e, such as glucose.'4.19.22 In s ep ococci lacking cy och omes and ca a- la op o eins such as NADH-oxidase7 make oxygen consump ion possible, and gi e ise o supe oxide ions, hyd ogen pe oxide o wa e . O hese me aboli es, he i s wo a e highly oxic o he mic oo ganism, and a e apidly emo ed om he en i onmen by supe oxide dismu ases.8 and NADH-pe oxidase6 espec i ely. Howe e , a ce ain concen a ions, H202 inhibi s he g ow h o o al s ep ococci, and highe concen a ions can be bac e icidal. The e ec o inhibi o y concen a ions on he eco e y o hese mic oo ganisms has no *Au ho lo whom co espondence should be add essed: An onio Ga cia-Mendoza. Dep o. de Mic obioiogia. Facul ad de Medicina. A da. de Mad id no. I I, Uni e sidad de G anada, E-I807 I G anada. Spain. 0891L060X,93/010017-06 $08.00 @ 1993 by John Wiley & Sons. L d. '. ' '.' been clea ly documen ed, bu is a ac o ha needs o be aken in o accoun in a emp s o explain he selec i e unc ion o H,O,. As a s ong bu uns able oxidan ,'s,'8 he 'in i o' ac ion o H,02 is likely o be ansien . I is he e o e o in e es o s udy he abili y o o al s ep ococci exposed b ie ly o H202 o eco e hei op imal g ow h a e. The p esen s udy in es iga ed he pos -hyd ogen pe oxide e ec (PHPE) on he de elopmen o pe oxidogenic species equipped wi h NADH- pe oxidase and NADH-~xidase,'~ as a ac o ha may in luence he in e ela ions in he o al ca i y. MATERIALS AND METHODS Mic oo ganisms es ed The e e ence s ains we e S ep ococcus o alis NCTC 1 1427, S. mi is NCTC 3 16 1, S. sanguisNCTC 7863 and S. sanguis JENA 2697. Au och honous s ains om he collec ion o he Mic obiology Labo a o y, Uni e si y o G anada Hospi al, we e S. mi is OGS 218, S. mi is OGS 232, S. mi is OGS 628, S. sob inus OGS 4 15, S. sob inus OGS 324, S. .soh inus OGS 529 and S. mi is OGS 420 (OGS= Odm ologia G anada S ep ococcus). These s ains we e iden i ied acco ding o he c i e ia desc ibed by Ha die' and Loesche." 18 A. GARC~A-MENDOZA ETAL. Minimum inhibi o y concen a ion and minimum bac e icidal Concen a ion T yp icase soy b o h (TSB) (Sco 4900-5207) was used in all assays. Comme cial hyd ogen pe oxide (Pan eac 141076), 30 pe cen w / ol., was i a ed acco ding o he echnique o Be n and Be gmeye ,' and used o p epa e se ial double dilu ions in ubes con aining 5ml TSB. The inal dilu ions anged om 900 o 1.46 pg/ml; some in e media e dilu ions we e also used wi h S. mi is OGS 420. The ubes we e inocula ed wi h 0.1 ml o bac e ial suspension ( u bidi y 0.5 on he Mac a land scale), iable coun s anged om lo6 o lo7 c. .u./ml de e mined by coun ing in pla e, p epa ed om loga i hmic g ow h phase cul u es in TSB. Colony o ming uni s om con ol (TSB wi hou H,O,) and es (TSB wi h H,O,) ubes we e coun ed on Mi is Sali a ius Aga (MSA, Di co 0298-01 -0) pla es; a e 24 h anae obic incuba ion (He aeus 433 incuba o , PA 262) a 36 1 "C. E aluu ion o pos -hyd ogen pe oxide e lec Au oma ic me hod. To ubes con aining 5 ml TSB and H,O, a he MIC o each s ain es ed was added 0.5 ml o a bac e ial suspension (Tu bidi y 0.5 on he Mac a land scale), wi h iable coun s anging om lo6 o lo7 c. .u./ml. Con ols wi hou H,O, we e also p epa ed o each s ain. All ubes we e incuba ed o 1 h a 36 1 "C, a e which 2 ml o he con en s we e ans e ed o au oma ic analy- sis bo les (O ganon Teknika 52269) and placed in a BacT/Ale incuba o - eade (O ganon Tecknika Mic obial De ec ion Sys em 03 1 BT5024). This au oma ed sys em con inuously de ec s posi i e samples as hey appea , based on he change in he pH o he medium esul ing om bac e ial acid p oduc ion. The PHPE was de ined as he di e ence be ween ime o de ec ion o g ow h in cul u es exposed o H,O, and in non-exposed cul u es. Viable coun s me hod. Tubes con aining TSB, H,O, and bac e ial suspension we e p epa ed as desc ibed abo e o au oma ic de ec ion, and incu- ba ed o 1 h a 36k 1°C. The con en s o each ube we e added o 100 ml E lenmeye lasks con aining 75 ml TSB, and incuba ed a 36 1 "C. Pe iodically, 2 ml aliquo s we e aken o p epa e se ial dilu ions anging om lo-' o lo-", depending on he du a ion o incuba ion. The dilu ions we e used o inocula e Mi is Sali a ius Aga pla es, which we e hen incuba ed o 48 h a 36+ 1°C anae obically. Table 1. Minimum inhibi o y concen a ion (MIC) and minimum bac e icidal concen a ion (MBC) o H,02 in cul u es o pe oxidogenic o al s ep ococci MIC H,02 MBC H202 Mic oo ganism (Pgiml) (clglml) S. o alis NCTC 11427 S. mi is NCTC 3 16 1 S. sanguis JENA 2697 S. sanguis NCTC 7863 S. mi is OGS 21 8 S. mi is OGS 232 S. mi is OGS 628 S.sob inus OGS 324 S. sob inus OGS 41 5 S. sob inus OGS 529 S. mi is OGS 420 7.0 7.0 7.0 7.0 14.1 14.1 14.1 14.1 14.1 7.0 2.3 14.1 14.1 14.1 14.1 28.2 28.2 28.2 28.2 28.2 14.1 3.5 All expe imen s we e pe o med in iplica e. A e coun ing he numbe s o colonies, he concen- a ion o c. .u. pe millili e o o iginal cul u e was calcula ed con en ionally. The PHPE was de ined as he di e ence in ime needed o he numbe o c. .u. pe millili e o inc ease by one decimal loga i hmic uni be ween unexposed cul u es and cul u es incuba ed in he p esence o H,O, a he le el o he MIC o he pa icula s ain. RESULTS Table 1 shows he MIC and MBC o H,O, o he di e en s ains o o al s ep ococci assayed. Th ee alues o MIC we e ob ained: 2.3 pg/ml (S. mi is OGS 420), 7.0 pg/ml (S. o alis NCTC 1 1427, S. mi is NCTC 3161, S. sanguis JENA 2697, S. sanguis NCTC 7863, S. sob inus OGS 529), and 14.1 pg/ml o he emaining i e s ains. The imes un il g ow h in unexposed cul u es and cul u es exposed o H,O,, oge he wi h he PHPE measu ed wi h he au oma ic sys em (BacT/Ale ), a e shown in Table 2. The igu es gi en a e he means ( s anda d de ia ions) o iplica e de e mi- na ions. The PHPE anged om 52 min in S. mi is OSG 420 o 6 h 31 min in S. mi is s ains OSG 232 and OSG 628. Li le o no conco dance be ween his me hod and he esul s ob ained a e iable coun s was obse ed (Table 3). The only s ain ha showed no delay in PHPE a e exposu e was S. o alis NCTC 11427; all o he s ains showed PHPE POST-H,OI EFFECT IN ORAL STREPTOCOCCI 19 Table 2. Reco e y imes o cul u es o pe oxidogenic o al s ep ococci a e ans e al o medium wi h (T,) o wi hou H,O, (TJ. de e mined au oma ically (BacT/Ale ). The pos -hyd ogen pe oxide e ec (PHPE) was calcula ed by sub ac ing T, om Ti Mic oo ganism PHPE (h) S. o alis NCTC 1 1427 S. mi is NCTC 3 16 1 S. ~onguis JENA 2697 S. sanguis NCTC 7863 S. mi is OGS 21 8 S. mi is OGS 232 S. mi is OGS 628 S. soh inus OGS 324 S. soh inus OGS 41 5 S. soh inus OGS 529 S. mi is OGS 420 9:41 *0:18* 7:41 $0:33 8:Ol $0:23 1 I :22 0:35 14: 52 0:3S 14:51 $0:40 18: 12 0:38 10:22 0:42 13:52 0:39 10:42 $ 0:45 952 0:30 6:38 0:22 6:OO k 0:25 650 - 0: 18 9: 10 0: 15 9:51 0:28 8:20 0:3 1 11:41 0:25 750 0:21 9:30 022 8:21 k 0:35 9:OO 0:3S 3:03+0:11 1:41+0:16 1:ll 0:20 2: 12 0: I0 S:OI O:18 6:3 I +0:25 6:3 1 0:22 2:32 0:15 4:22 *0:26 221 0:26 0:52 O:I3 All expe imen s we e pe o med in iplica e *S anda d de ia ions. Table 3. Reco e y imes o cul u es o pe oxidogenic o al s ep ococci a e ans e al o medium wi h (Ti) o wi hou H,O, (T2), de e mined by coun ing iable cells me hod. The pos -hyd ogen pe oxide e ec (PHPE) was calcula ed by sub ac ing T, om T, S. o alis NCTC 1 1427 S. mi is NCTC 3161 S. sanguis JENA 2697 S. sanguis NCTC 7863 S. mi is OGS 21 8 S. mi is OGS 232 S. mi is OGS 628 S. soh inus OGS 324 S. soh inus OGS 4 I 5 S. soh inus OGS 529 S. mi is OGS 420 430 0:21* 5:40 0:35 5:50 0:38 9:20 0:42 6: 10 +0:26 15:30 0:50 10:30+0:45 830 + 0:43 6: 10 0:32 720 0:35 4:OO 0: 18 4:35 0:20 - 450 0:25 5:30$0:31 4:50+0:22 2:40 k 0: 1 1 6:15 +0:34 635 +0:35 3:45 0: 12 1 :50 +0:09 4:30 0: 16 300 0: 10 0:os k 0: 10 0:so 0: 1s 0:20 0: 10 430 k 0:20 3:30 0: 16 9: 15 0:29 3:SS&0:22 4:45 5 0:22 4:20 0:25 250 0: 13 1:00+0:08 All expe imen s we e pe o med in iplica e. *S anda d de ia ions. anging om 20 min (S. sanguis JENA 2697) o 9 h 15 min (S. mi is OGS 232). The g ow h cu es o he ou di e en s ains, based on coun s o iable cells in con ol cul u es and cul u es exposed o H,O,, a e shown in Figu e la-d. DISCUSSION The colonisa ion o di e en habi a s in he o al ca i y by s ep ococci is in luenced by many ac- o ~,~ including he e ec s o g ow h-inhibi ing sub- s ances such as hyd ogen pe o~ide.'~,'~,~~.~~ Th is 20 A. GARC~A-MENDOZA ET AL. 14' a h . l4 1 S. o alis NCTC 11427 S. mi is NCTC 3161 -*- Nonexposed -w- Exposed 10 / 4- 4l 2 - l4 3 12- n 10- 8- S. sanguis NCTC 7863 -*- Nonexpo ed -- Exposed 0 12 3 4 5 6 7 8 91011 Oi 0 12 3 4 5 6 7 8 91011 Time (h) Time (h) - 14- l2 2 S. sanguis JENA 2697 -*- Nonexposed -w- Exposed 11 2 0 0 1 2 3 4 5 6 7 8 9 101112131415 0 < 01234567891011 Time (h) Time (h) Figu e lad. Pos -hyd ogen pe oxide e ec in ou s ains exposed and non-exposed o he minimum inhibi o y concen a ion o HA me aboli e modi ies he de elopmen o some pe - oxidogenic s ains hemsel es, and o s ains equipped wi h NADH-pe oxidase. The e ec o H20, is in luenced by he amoun exc e ed, and by he mic oo ganism's esis ance. The ange o con- cen a ions o H202 able o inhibi g ow h o pe oxidogenic s ep ococci 'in i o' is na ow, excep in he case o S. mi is OGS 420. Bac e icidal concen a ions o H,O, we e usually double he inhibi o y concen a ions. I is di icul o ela e hese indings wi h he si ua ion in he open ecosys- em o he o al ca i y in li ing o ganisms, whe e he e a e coun less in e ela ions be ween mic obes, sali a and cells.' Mo eo e , in quan i a i e e ms a leas , he pe oxidogenic capaci y o o al s ep o- cocci appea s o be s ain o species dependen . Fo example, S. sanguis NCTC 7863 p oduces ela i ely li le H20, (0.84 pg/ml) in compa ison wi h S. o alis NCTC 11427 (6.7 pg/ml) (unpublished da a). Because H202 is labile and eadily inac i a ed in he POST-HIOI EFFECT IN ORAL STREPTOCOCCI en i onmen , 'in i o' an ibac e ial ac i i y is likely o be ansien ,possiblycausingalagin heg ow ho bac e ia loca ed nea he H,O, p oduce . Hyd ogen pe oxide may hus be an impo an ecological ac o in he o al ca i y: s ains ha a e mo e esis an o he e ec s o H,O, may well ha e an ad an age in he compe i ion o a speci ic habi a . The indings ob ained wi h he au oma ed sys em o g ow h de ec ion di e ed ma kedly om hose ob ained by pe iodic coun ing o iable cells nie hod. The au oma ed me hod de ec s he dec ease in pH caused by bac e ial acid p oduc ion, whe eas he iable coun s depends on he appea ance o c. .11. on cul u e pla es. Al hough he la e me hod is con- side ably mo e labou in ensi e, he da a gene a ed a e mo e in o ma i e, allowing he e alua ion o g ow h cu es, a ea u e no a ailable wi h he au oma ed me hod. When de e mined on he basis o iable cell coun s, he e ec o H,O, on g ow h was appa en in all s ains excep he highly pe oxidogenic (unpublished da a) S. omlis NCTC 11427. I seems easonable o assume ha his s ain is able o wi h- s and concen a ions o H,O, app oaching ha o he HzOz i exc e es. Thee ec so exposu e o HzO, a ied widely in he o he s ains. sugges ing ha pe oxidogenic o al s ep ococci espond di e en ly o H,02 depending on he species and s ain. In conclusion, g ow h o 10 o 11 pe oxidogenic o al s ep ococci s udied was a ec ed by exposu e o he MIC ( o a gi en s ain) o H,O,. This e ec may well in luence mic obial colonisa ion o o al habi a s, and ha e some bea ing on he in e ela ionships be ween bac e ia in he mou h. ACKNOWLEDGEMENTS This s udy was pa ially suppo ed by he Andalusian Regional Go e nmen h ough he esea ch p ojec 'Mic obiology, Immunology and Epidemiology o O al Diseases'. We hank D M. Hayes o he Depa mcn o Biochemis y. Medical School. Uni e si y o R is ol. o supplying he e e ence s ains and o p o iding help ul commen s on [his s udy, and Ms Ka en Shashak o ansla ing he o iginal manusc ip in o English. REFERENCES Age A, Go don JL. (1984). Di e en ial e cc(s o hyd ogen pe oxide on indices o endo helial cell unc ion. Jou n l o ' E.ype ini i d Medicine 159, 592-603. Be n E. Be gmeye HU. (1965). Ino ganic pe oxides. in: Be gmeye HU (ed) Me ho s o Enzjwiu ic Ani lysis, I s cdn. Academic. Basel. p. 633. 3. 4. 5. 6. 7. 8. 9. 10. 1 I. 12. 13. 14. 15. 16. 17. 18. 21 Ca lsson J. (1989). Mic obiology o plaque- associa ed pe iodon al disease. In: Lindhe J (ed) Tex book o ' Clinical Pe iodon o ogy, 2nd edn. Munksgaa d, Copenhagen, pp. 129-130. Ca lsson J. Iwami Y, Yamada T. (1983). Hyd ogen pe oxide exc e ion by o al s ep ococci and e ec o lac ope oxidasc- hiocyana e-hyd ogen pe oxidc. In ec ion and Immuni y 40,70-80. DiGuiseppi J, F ido ich I. (1982). Oxygen oxici y in S ep ococcus .mnguis. The ela i e impo ancc o supe oxide and hyd oxyl adicals. Jou nal o ' Biological C'he ~iis i~j~ 257,4046 405 I . Dolin MI. (1955). The DPNH-oxidizing enzymes o S p ococciis, a~cal~.~. 11. The enzymes u ilizing oxy- gen, cy och omec. pe oxide and 2.6-dichlo ophenol- indophenol o e icyanide as oxidan s. A chi es o Biochemis y and Biopliysics 5541 5435. Doh MI. (1961 ). Cy och ome-independen elec- on anspo enzymes o bac e ia. In: Gunsalus IC, S anie RY (eds) The B Jc e ia, Vol 2. Academic, New Yo k, pp. 425-460. F ido ich I. (1 975). Supe oxide dis nu ases. Annual Re ieicx o Biochemis y 44, 147-1 59. Hamada S, Slade HD. (1980). Biology immu- nology, and ca iogenici y o S ep ococcus mu ans. Mic obiological Re iews 44, 33 1-384. Hamon CB. Klebano SJ. (1973). A pe oxidase- media ed, S ep ococcus mi s-dependen an imic o- bial sys em in sali a. Jou zul y ' E.ype imen 1 Medicine 137,438450. Ha die JM. (1986). O al s ep ococci. In: Snea h PHA, Mai NS. Sha pe ME (eds) Be geys Manual q ' Sys ema ic Bac e iology, Vol 2. Williams & Wilkins. Bal imo e. pp. 10541063. Ho niann ME. Meneghini R. (1979). Ac ion o hyd ogen pe oxide on human ib oblas in cul u e. Pho ocheniis j, cmd Pho obiology 30, 15 1-1 55. Holmbe g K. Hallande HO. (1973). P oduc ion o bac e icidal concen a ions o hyd ogen pe oxide by S ep ococcus sanguis. A chi s o O al Biology 18, 423434. LeBien TW. B omel MC. (1975). An ibac e ial p op- e ies o a pe oxidogenic s ain o S epplococcus nii io (mi is). Canadiu~iJou nalcl 'Mic~ obio~o~~y21,101-103. Lehninge AL. (I 978). Enzimas de oxido- educcibn y anspo e elec bnico. In: Lehninge AL (ed) Bioqui nicu, 2nd edn. Ediciones Omega, S.A., Ba celona. p. 518. Loesche WJ. (1986). Roleo S ep ococc.u. niu a isin human den al decay. Mic obiological Re iews 50, McLeod JW, Go don J. (1922). P oduc ion o hyd ogen pe oxide by bac e ia. Biochemical Jou nal Mo cillo J. (1 977). Oxidacion- educcion. In: Mo cillo J. (ed) Temas Busicos d~ Qi imica. Alhamb a, Mad id, p. 294. 353-380. 16,499-504. 22 A. GARC~A-MENDOZA ETAL. 23. Whiley RA, F ase H, Ha die JM, Beigh on D. (1990). Pheno ypic di e en ia ion o S ep ococcus in e medius, S ep ococcus cons ella us, and S ep o- coccus anginosus wi hin he ‘S ep ococcus mille i g oup’. Jou nal o Clinical Mic obiology 28, Willcox MDP, D ucke DB. (1988). Pa ial cha ac- e isa ion o he inhibi o y subs ances p oduced by S ep ococcus o alis and ela ed species. Mic obios 55,135-145. 25. Zi zelsbe ge W, Go z F, Schlei e KM. (1984). Dis ibu ion o supe oxide dismu ases, oxidases and NADH-pe oxidase in a ious s ep ococci. FEMS Mic obiologjj Le e s 21,243-246. 1497-1 50 1. 24. 19. Thomas EL, Pe a KA. (1983). Oxygen me abolism o S ep ococcus mu ans: up ake o oxygen and elease o supe oxide and hyd ogen pe oxide. Jou nal o Bac e iology 154, 1236-1244. Thomas EL, Pe a KA, Smi h KW, Chwang AK. (1983). Inhibi ion o S ep ococcus mu ans by he lac ope oxidase an imic obial sys em. In ec ion and Immuni y 39,761-718. Thompson R, Johnson A. (1951). The inhibi o y ac ion o sali a on he diph he ia bacillus: hyd ogen pe oxide, he inhibi o y agen p oduced by sali a y s ep ococci. Jou nal o In ec ious Diseases 88, 22. Ve nazza TR, Mel ille TH. (1979). Inhibi o y ac i i y o S ep ococcus mi is agains o al bac e ia. Mic obios 26,95-101. 20. 21. 81-85.