ABSTRACT
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Asso ci a t i o n o f t h r e e p u t a t i v e p e r i o d o n t a l
pat hogens w it h chronic periodont it is in Brazilian
subj ect s
Cristiane GONÇALVES, Geisla Mary S. SOARES, Marcelo FAVERI, Paula Juliana PÉREZ-CHAPARRO, Eduardo LOBÃO, Luciene Cristina FIGUEIREDO, Gustavo Titonele BACCELLI, Magda FERES
Universidade de Guarulhos, Departamento de Periodontia, Guarulhos, SP, Brasil.
Corresponding address: Magda Feres - Centro de Pós-Graduação e Pesquisa - CEPPE - Universidade Guarulhos - Praça Tereza Cristina, 229 - Centro - Guarulhos - SP - Brazil - 07023-070 - e-mail: [email protected]
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bj ect ive: The aim of t his st udy was t o evaluat e t he associat ion of Porphyrom onas endodont alis, Filifact or alocis and Dialist er pneum osint es w it h t he occur r ence of periodont it is. Mat erial and Met hods: Thirt y subj ect s wit h chronic periodont it is ( ChP) and 10ZLWKSHULRGRQWDOKHDOWK3+ZHUHLQFOXGHGLQWKHVWXG\1LQHVXEJLQJLYDOELR¿OPVDPSOHV
w ere collect ed as follow s: i) PH group - from t he m esial/ buccal aspect of each t oot h in t w o random ly chosen cont ralat eral quadrant s; ii) ChP group - from t hree sit es in each of
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Result s: We found t he t hree species evaluat ed in a higher percent age of sit es and at higher levels in t he group w it h ChP t han in t he PH group ( p< 0.05, Mann-Whit ney t est ) . We also
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subj ect s wit h ChP were com pared wit h t hose from subj ect s wit h PH ( p< 0.05, Mann-Whit ney t est ) . I n addit ion, t he prevalence and levels of D. pneum osint es, and especially of F. alocis w ere very low in healt hy subj ect s ( 0.12x105 and 0.01x105, respect ively) . Conclusion: F.
alocis and D. pneum osint es m ight be associat ed w it h t he et iology of ChP, and t heir role in t he onset and progression of t his infect ion should be furt her invest igat ed. The role of P. endodont alis was less evident , since t his species was found in relat ively high levels and prevalence in t he PH group.
Keyw ords: Chronic periodont it is. Et iology. Pat hogens. Microbiology.
I N TRODUCTI ON
Th e o r a l cav i t y n a t u r a l l y h o st s h u n d r ed s o f sp eci es t h a t t o g et h er co n st i t u t e t h e o r a l m icr obiom e. Most of t hese m icr oor ganism s ar e com pat ible w it h a good oral healt h st at us, w hile som e of t hem are considered pat hogens t hat m ight t rigger infect ious processes, such as periodont it is. Kn o w l ed g e ab o u t t h e p er i o d o n t al p at h o g en s associat ed w it h t h e on set an d pr ogr ession of periodont al diseases has been largely concent rat ed o n t h e m i c r o o r g a n i s m s t h a t c o m p r i s e t h e “ su b g in g iv al m icr ob ial com p lex es” p r ev iou sly described by Socransky, et al.24,25 ( 2002, 1998) . Non et h eless, a r ecen t sy st em at ic r ev iew h as i n d i ca t e d t h e e x i st e n ce o f o t h e r ca n d i d a t e
per iodont al pat hogens20.
subj ect s1,2,5,7,10,14,15,18,21.However, t o our knowledge, no st udies t o dat e have com par ed t he pr evalence as w ell as t he levels of t hese t hr ee species in subj ect s w it h good per iodont al healt h and w it h disease.
Therefore, t he aim of t his st udy was t o evaluat e t h e associat ion of t h r ee p u t at iv e p er iod on t al p at h og en s, P. en d od on t alis, F. alocis an d D. pneum osint es w it h per iodont it is, by com par ing t heir levels and prevalence in subj ect s wit h chronic periodont it is ( ChP) and t hose in good per iodont al
healt h ( PH) . This knowledge m ight help t o est ablish m ore effect ive prevent ive and t reat m ent st rat egies for t hese infect ions.
M ATERI AL AN D M ETH ODS
St udy populat ion
Ten p er iod on t ally h ealt h y su b j ect s an d 3 0 su b j e ct s w i t h Ch P w e r e se l e ct e d f r o m t h e populat ion r efer r ed t o t he Per iodont al Clinic of
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t r eat m ent . Det ailed m edical and dent al r ecor ds w er e obt ained, and one t rained and calibrat ed ex am in er p er f or m ed a f u ll- m ou t h p er iod on t al
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crit eria w ere invit ed t o part icipat e in t he st udy. All eligible subj ect s w er e infor m ed of t he nat ur e,
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in t h e st u dy an d sign ed an in f or m ed con sen t
GRFXPHQW 7KH *XDUXOKRV 8QLYHUVLW\¶VClin ical Resear ch Et hics Com m it t ee appr oved t he st udy prot ocol.
I nclusion and exclusion crit eria
I n clu sion cr it er ia f or p er iod on t ally h ealt h y
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wit h probing dept h ( PD) and/ or clinical at t achm ent
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sit es w it h gingival bleeding and/ or bleeding on probing ( BOP) . I nclusion crit eria for subj ect s w it h
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m m and BOP.
Exclusion cr it er ia w er e pr egnancy, lact at ion, cur r ent ly being a sm oker, ant im icr obial t herapies dur ing t he pr evious 6 m ont hs, m edical condit ions r e q u i r i n g p r o p h y l a ct i c a n t i b i o t i c co v e r a g e , co n t i n u o u s u se o f m o u t h - r i n se s co n t a i n i n g ant im icr obial agent s in t he pr eceding 3 m ont hs, system ic conditions that could affect the progression of p er iod on t it is an d lon g - t er m ad m in ist r at ion of an t i- in f lam m at or y an d im m u n osu p p r essiv e
m edicat ions.
Clinical evaluat ion
One exam iner perform ed t he clinical m onit oring and carried out all clinical m easurem ent s in a given
subj ect . Visible plaque ( 0/ 1) , gingival bleeding ( 0/ 1) , BOP ( 0/ 1) , suppurat ion ( 0/ 1) , PD and CAL were m easured at six sit es per t oot h ( m esiobuccal, b u ccal, d ist ob u ccal, d ist olin g u al, lin g u al an d m esiolingual) in all t eet h, excluding t hird m olars. PD an d CAL m easu r em en t s w er e r ecor d ed t o t h e n ear est m illim et er u sin g a Nor t h Car olin a per iodont al pr obe ( Hu- Fr iedy, Chicago, I L, USA) .
M icrobiological evaluat ion Sam ple collect ion
Aft er having r ecor ded t he clinical param et er s, su p r ag in g iv al p laq u e w as r em ov ed an d t h en nine individual subgingival plaque sam ples w ere collect ed per subj ect using st er ile 11/ 12 m
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subj ect s: sam ples were collect ed from t he m esial/ b u ccal asp ect of each t oot h in t w o r an d om ly chosen cont ralat eral quadrant s. Subj ect swit h ChP: sam ples w er e collect ed fr om t hr ee sit es in each of
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The sam ples w er e placed in separat e plast ic t ubes cont aining 0.15 m L of TE ( 10 m M Tr is- HCl, 1m M EDTA, pH 7.6) . One hundr ed m icr olit er s of 0.5 M NaOH w ere im m ediat ely added t o each t ube and t he sam ples w er e st or ed at - 80° C.
Check erboard DN A- DN A hybridizat ion
The sam ples w er e evaluat ed by checker boar d DNA– DNA h y b r i d i zat i on1 9 , 2 3 DW WKH *XDUXOKRV Universit y Laborat ory of Microbiology. The sam ples were boiled for 10 m in and 0.8 m l of 5 M am m onium acet at e w as added t o each sam ple. Th e DNA released was t hen placed int o t he ext ended slot s of a Minislot 30 apparat us ( I m m unet ics, Cam br idge, MA, USA) , concent rat ed on a 15/ 15 cm , posit ively charged, nylon m em brane ( Boehringer Mannheim ,
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of det ect ion for each of t he species.
St at ist ical analysis
Each individual clinical param et er as w ell as m ean cou n t s ( x 1 05) of each bact er ial species evaluat ed and t he per cent age of sit es colonized by t hese species w ere com put ed per subj ect and t hen acr oss subj ect s in bot h gr oups. The Mann–
:KLWQH\ WHVW ZDV XVHG WR VHHN VLJQL¿FDQFH RI differences bet ween t he t wo groups for age, clinical an d m icr obiological par am et er s, an d apply in g Bonfer r oni’s cor r ect ion for m ult iple com par isons. Thus, fr om t his com put at ion, a p value of < 0.016
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p< 0.05. Sim ilarly, individual values of p< 0.003 and
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at p< 0. 01 and p< 0. 001, r espect ively26. A Chi-square t est was used t o com pare differences in t he fr equency of gender. Levels of t he t hr ee bact er ial sp ecies w er e av er ag ed sep ar at ely w it h in t w o
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subj ect , and t hen acr oss subj ect s w it h ChP. The
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of differ ences bet w een t hese t w o cat egor ies. The
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RESULTS
Table 1 pr esent s t he dem ographic and clinical charact er ist ics of t he subj ect s w it h ChP and PH.
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differences bet w een t he t w o groups for all clinical param et er s evaluat ed ( p< 0.05) . The m ean age and per cent age of fem ales did not differ bet w een gr oups ( p> 0.05) .
Figur es 1 and 2 pr esent t he m ean per cent age of sit es colonized by F. alocis, D. pneum osint es an d P. en d od on t alis an d t h e m ean cou n t s of t hese species, r espect iv ely, in t he t w o clinical g r o u p s. Al l t h r ee sp eci es w er e p r esen t i n a st at ist ically sig n if ican t , h ig h er p er cen t ag e of sit es and m ean count s in subj ect s w it h ChP t han in t hose in PH ( p< 0.05) . The m ean levels of F. alocis, D. pneum osint es and P. endodont alis in t he healt hy subj ect s w er e 0.01x105,0.12x105 and 1.89x 105, r espect ively ; and 1.77x 105,7.61x 105 and 4.01x 105, r espect ively, in t he per iodont it is
subj ect s. Figure 3 shows t he m ean count s ( x105) of t he t hree m icrobial species in subj ect s wit h PH and
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ChP. F. alocis and D. pneum osint es w er e pr esent
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shallow and deep sit es of periodont it is subj ect s in com par ison w it h t he levels obser ved in healt hy subj ect s ( p< 0.05) . The levels of P. endodont alis
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(n=10) Q
Mann-Whitney
Mean±SD Mean±SD (p)
Number of females
9 22 0.73889
Age 38.3±6.8 42.2±6.4 0.26
PD 1.8±0.2 3.7±0.7 0.0
CAL 0.9±0.2 4.5±1.3 0.0
% sites with:
Plaque 25.5±10.1 80.0±15.6 0.0
Gingival bleeding
2.0±0.8 27.7±15.3 0.0
Bleeding on probing
3.1±1.2 78.9±14.1 0.0
Suppuration 0.0±0.0 1.1±0.8 0.00001
PD: probing depth; CAL: clinical attachment level; SD: standard deviation
Table 1- Clinical parameters and demographic
characteristics of the two groups
Figure 1- Mean percentage of sites colonized by the three bacterial species evaluated. The Mann-Whitney test was used
of subj ect s w it h ChP and PH.
DI SCUSSI ON
We f ou n d t h e t h r ee can d id at e p er iod on t al pat hogens evaluat ed in t his st udy in st at ist ically
VLJQL¿FDQWKLJKHUSHUFHQWDJH of sit es and in higher levels in subj ect s w it h ChP t han in periodont ally healt hy indiv iduals. This dat a is in agr eem ent w it h pr ev iou s inv est igat ion s t h at also sh ow ed an associ at i on of P. en d od on t al i s1 , 6 , 1 0 , 1 5 - 1 7, F. alocis1 - 3 , 6 , 8 , 1 0 , 1 4 , 1 5 , 2 1 an d D. p n eu m osin t es7 , 8 , 1 4 , 1 8 w it h periodont al diseases. I n addit ion, a recent Syst em at ic Review, w hich evaluat ed t he w eight of evidence for t he exist ence of novel per iodont al
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t ax a m igh t be associat ed w it h t h e et iology of per iodont it is. The aut hor s pr oposed t hat t her e is m oderat e evidence in t he lit erat ur e t o suppor t
t h e r o l e o f P. en d o d o n t al i s an d F. al o ci s as per iodont al pat hogens, and som e evidence for D. pneum osint es20.
Alt hough levels of t he t hr ee bact er ial species evaluat ed in t his st udy w er e elevat ed in subj ect s w it h ChP in com parison w it h periodont ally healt hy indiv iduals ( Figur es 1 and 2) , t heir lev els and p r ev alen ce in h ealt h y su b j ect s also p r ov id ed essent ial inform at ion as regards t heir possible role in t he et iology of t he disease. F. alocis was present in only 8% of t he sit es of healt hy individuals and
DWLQVLJQL¿FDQWOHYHOVD. pneum osint es was also present at very low levels in healt hy individuals, but was det ect ed in 42% of t he sit es evaluat ed. We found P. endodont alis in alm ost 50% of t he sit es
of healt hy subj ect s and at relat ively high levels. This dat a m ight indicat e t hat P. endodont alis is an oppor t unist ic bact er ial species or an accessor y p a t h o g e n t h a t m a y n o t t r i g g e r t h e d i se a se
Figure 2- Mean counts (x105) of the three bacterial species evaluated. The Mann-Whitney test was used to assess
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)LJXUH Mean counts (x105) of the three bacterial species evaluated in periodontally health and in sites with probing
pr ocess, but w hose levels m ay incr ease w hen t he
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t o t h e disease pr ocess1 2. Appar en t ly, F. alocis r et ains t he charact er ist ics of a so called “ keyst one pat hogen”, m icroorganism s t hat have t he pot ent ial t o in it iat e p er iod on t al d est r u ct ion b y cau sin g a d y sb iosis in t h e su b g in g iv al ecosy st em3 , 1 3. Alt h ou gh it s (F. alocis) pr ev alen ce an d lev els in healt hy subj ect s was ver y low in t his st udy, Ar uni, et al.3,4 ( 2014,2011) show ed t hat F. alocis has som e unique vir ulence fact or s t hat favor it s
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m ay m ediat e P. gingivalis proliferat ion, suggest ing t hat t his species can play a pivot al role in m icrobial com m unit y dynam ics.
The st r engt h of t his st udy was t he num ber of subgingival plaque sam ples analyzed for t he levels and pr evalence of t he t hr ee bact er ial species. Alt hough t he num ber of subj ect s included in t his st udy was r elat ively sm all, 360 plaque sam ples w er e in d iv id u ally an aly zed . To d at e, m ost of t h e associat ion st u dies t h at h av e inv est igat ed F. alocis, D. pn eu m osin t es or P. en dodon t alis evaluat ed fewer sam ples1,6,9,10,15,16,18,21. I n addit ion, t h ese st u dies h av e gen er ally on ly det er m in ed t h e p r ev alen ce or ab u n d an ce ( p r op or t ion ) of
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I ndeed, st udies have r ecognized t hat evaluat ing a large num ber of plaque sam ples and quant ifying t he m icr oor ganism s ar e cr it ical r equisit es w hen t r ying t o est ablish an associat ion bet w een cer t ain m icr oor ganism s and t he onset and pr ogr ession of periodont it is11.
I t is im por t an t t o m en t ion t h at associat ion
st u d i e s o n l y p r o v i d e t h e i n i t i a l i n f o r m a t i o n n ecessar y t o su ggest a possible lin k bet w een a m i cr o o r g a n i sm a n d a n i n f e ct i o n . Fu r t h e r
HYDOXDWLRQVDUHQHFHVVDU\WRFRQ¿UPt his t ype of associat ion, such as clinical ( i.e. risk assessm ent a n d i n t e r v e n t i o n a l st u d i e s) ; h o st - r e sp o n se st udies, and invest igat ions int o t he m echanism s of pat hogenicit y of t he suspect ed pat hogens.
CON CLUSI ON
I n conclusion, t he result s of t his invest igat ion suggest ed an associat ion of D. pneum osint es, and especially of F. alocis w it h t he et iology of periodont it is. The role of P. endodont alis was less evident , since w e found t his species in relat ively high levels and prevalence in periodont ally healt hy subj ect s. This dat a m ight guide fut ure st udies on t he act ual r ole of t hese t hr ee bact er ial species in t he et iology of per iodont it is and help t o est ablish a m or e effect ive t r eat m ent for t hese infect ions.
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int er est w it h r espect t o t he aut hor ship and/ or publicat ion of t his art icle.
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