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Dugoni School of Dentistry Faculty Articles Arthur A. Dugoni School of Dentistry

1-1-2005 Association between coinfection of , Actinobacillus actinomycetemcomitans and denticola and periodontal tissue destruction in L. L. Chen

Y. M. Wu

J. Yan

W. L. Sun

Y. Z. Sun

See next page for additional authors

Follow this and additional works at: https://scholarlycommons.pacific.edu/dugoni-facarticles Part of the Biochemistry Commons, Immunity Commons, Immunology of Infectious Disease Commons, and the Medical Immunology Commons

Recommended Citation Chen, L. L., Wu, Y. M., Yan, J., Sun, W. L., Sun, Y. Z., & Ojcius, D. M. (2005). Association between coinfection of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and and periodontal tissue destruction in chronic periodontitis. Chinese Medical Journal, 118, 915–921. https://scholarlycommons.pacific.edu/dugoni-facarticles/126

This Article is brought to you for free and open access by the Arthur A. Dugoni School of Dentistry at Scholarly Commons. It has been accepted for inclusion in Dugoni School of Dentistry Faculty Articles by an authorized administrator of Scholarly Commons. For more information, please contact [email protected]. Authors L. L. Chen, Y. M. Wu, J. Yan, W. L. Sun, Y. Z. Sun, and David M. Ojcius

This article is available at Scholarly Commons: https://scholarlycommons.pacific.edu/dugoni-facarticles/126

. 916. Chin Med J 2005; 118(11) :915-921 (

strategies. 1 It is widely accepted that periodontitis the nature of the study and the procedures involved, t occurs as a result of infection by subgingival and their consent was obtained. 1 2 , particularly gram-negative anaerobes. • In the earlier epidemiological data, Porphyromonas Sample collection gingiva/is was considered to be responsible for For all the patients, two subgingival plaque samples 3 4 from periodontal pockets with a minimum depth of 3 CP, " whereas Actinobacillus actinomycetem­ comitans was confirmed to be a specific causative mm from two different tooth sites were collected with 2 3 a separate curette for each sample to avoid cross agent of AgP. ' Treponema denticola infection was proved to be closely associated with periodontal contamination. For the healthy population , one diseases such as early onset periodontitis , sample of was collected from each of necrotizing ulcerative and acute the individuals by the same method. The plaque 5 6 samples were placed in 200 1-11 lysis buffer ( 10 pericoronitis. ' Recent basic research as well as mmoi/L Tris-HCI, 1. 0 mmoi/L EDT A, 1. 0% Triton clinical evidence suggested that T. denticola may 10 play an important role in periodontal tissue X-100, pH 8. 0) for PCR assay and stored at 11 5 6 -20't until used. The gingival index ( Gl) and destruction, ' but it has been found occasionally in subgingival samples of CP. 7 However, attachment loss of each pocket was recorded. AL data were classified into 3 grades: ~ 2 mm, 2 mm correlation between coinfection of the three microbes 12 and periodontal tissue destruction is not well to 5 mm, and > 5 mm. The clinical severity of 1 4 periodontitis of the sampled tooth sites was characterized. ' Coinfection of multiple classified into three grades according to Hugoson' s subgingival anaerobes could result in more serious 13 destruction of periodontal tissue in Caucasian CP categories. Mild periodontitis: tooth with 5 8 attachment loss ~ 2 mm, alveolar bone loss ~ 1/3 patients, ' but conflicting conclusions have been reported also. 9 of root length, and no . Moderate periodontitis: tooth with attachment loss between 3 The purpose of this study was to investigate the mm to 5 mm, alveolar bone loss ~ 1/2, slight subgingival infection frequencies of P. gingiva/is, A furcation involvement and slight tooth mobility. actinomycetemitans and T. denticola in Chinese CP Severe periodontitis: tooth with attachment loss > 5 mm, alveolar bone loss > 1/2, obvious furcation patients as measured by a multiple PCR using 13 specific primers derived from the 16SrDNA genes of involvement and obvious tooth mobility. these three microbes, and to evaluate the correlations among the three microbes and Bacteria and growth condition periodontal destruction. P. gingiva/is strain ATCC 33277 , A. actinomycetemitans strain Y4 and T. denticola strain METHODS FM were used as positive controls. P. gingiva/is strain ATCC 33277 was grown in trypticase soy agar Subjects supplemented with haemin ( 5 1-1g/ml) and vitamin Eighty-one untreated CP patients ( 38 men and 43 K1 (1 1-1g/ml), 5% (5 ml/100 ml) sheep blood and women , 27 - 65 years , mean 43 years ) with at menadione ( 1 1-1g/ml) . 14 A. actinomycetemitans least 14 teeth remaining were recruitedin the dental strain Y4 was cultured on TSBV selective agar clinic of the Second Affiliated Hospital, Medical medium. The TSBV medium contains tryptic soy School of Zhejiang University. The patients were medium ( Oxoid ) , 10% ( 10 ml/1 00 ml ) horse diagnosed according to their clinical examination , bacitracin ( 75 IJg/ml) and vancomycin ( 5 results: the average periodontal probing depth 1-1g/ml) . 15 T. denticola strain FM was grown in ( PD) ~ 3 mm, > o. 5 mm new oral spirochete medium with 10% heat and with alveolar bone loss on X-ray examination. inactivated rabbit serum and 10 1-1g/ml Samples from 30 periodontally healthy individuals cocarboxylase. 16 The above anaerobes were ( 12 men and 18 women , 19 - 39 years , mean 32 cultured in an anaerobic chamber at 36't with an years) were also studied. All of the patients and the atmosphere of 85% N2 , 5% C02 10% H2 • E. coli healthy individuals were nonsmokers without any strain DH Sex was used as negative control and systemic disease. Individuals who were under cultured by using MH medium ( Oxoid, England). orthodontic treatment or had antibiotic therapy during the preceding 3 months were excluded. All of the DNA extraction individuals received detailed information concerning Each of the subgingival plaque samples in the lysis 'I!'.'1' !iii lmp :111 !,~15-921 Chinese Medical ]oumal2005; 118(11) :915-921 • 917 • .•:r-----'IIi 1• '!l~lved buffer was boiled for 10 minutes, and 10 1-11 of the samples , each reaction was repeated twice using 10 i111.1 ' supernatant was directly used as template in PCR. the same sample. If the two PCR results were not !·li Cultured P. gingiva/is strain ATCC 33277 , A. consistent, a third time reaction was carried out. ~1 1 d1 I actinomycetemitans strain Y4, T. denticola strain llr. pies FM, and E coli strain DH So: were suspended in Detection of PCR Products . H of 3 0. 01 moi/L PBS (pH 8. 0). Genomic DNAs of the Ten tJI of each reaction product mixed with 10 1-11 of 2 11~ with bacterial strains , which would be used as controls in x loading buffer was fractionated on 2% agarose gel lj~ross PCR, were obtained by the phenol chloroform stained with 1 IJg/ml ethidium bromide, using a 100 ,,,,Iii one method. bp DNA ladder ( Sangon , Canada ) as a size !!Ph of marker. ,11:iftque PCR primers and amplification /i(10 A multiple PCR assay was developed to detect the Statistical analysis ll[riton 16SrDNA genes of P. gingiva/is, A. Chi-square test by using SPSS9. 0 software was !i1d at actinomycetemitans and T. denticola in the performed for statistical analysis. and ·!•IIIi subgingival plaque samples. PCR amplification was Ill AL carried out in a volume of 100 1-11 containing 10 1-11 of il! RESULTS ;,!''mm the template, 10 1-11 PCR buffer (20 mmoi/L Tris-CI, of 50 mmoi/L KCI, pH 8. 4) and 5 U Taq polymerase Detection of P. gingiva/is 16SrDNA, A. ( Sangon , East Markham Ontario L3 R 2 RS , actinomycetemitans 16SrDNA and T. denticola 's Canada ) , 0. 25 mmoi/L of each dNTP, 2. 5 16SrDNA in healthy individuals by multiple PCR mmoi/L MgCI , and 25 pmoi/L primers specific for 2 By using multiple PCR, the detection results of all the 16SrDNA genes of P. gingiva/is, A. actino­ the three microbes in the plaque or sulcus samples mycetemitans 16SrDNA and T. denticola. Primers could be simultaneously obtained. In addition, clear specific for P. gingiva/is 16SrDNA gene were: 5 '­ and exact amplification fragments representative for AGG CAG CTT GCC ATA CTG CG-3' (sense) , one, two or all of the three microbes could be 5 '-ACT GTT AGC AAC TAC CGA TGT -3 ' 7 shown ( Fig. ) . In the 30 samples from 30 ' >5 ( antisense ) . Primers specific for A. actino­ periodontally healthy individuals, 3 ( 10. 0% ) of the ' ·on mycetemitans 16SrDNA gene were: 5' -ATG CCA samples were P. gingiva/is 16SrDNA positive, 2 AA T TGA CGT T AA AT -3' (sense) , 5' -AAA CCC ATC TCT GAG TTC TTC TTC-3' (antisense). 17 ( 6. 7% ) samples were A. actinomycetemitans Primers specific for T. denticola 16SrDNA gene 16SrDNA positive, and 1 ( 3. 3%) sample was T. denticola 16SrDNA positive. Furthermore, only one A. were: 5' -TAA TAC CGA ATG TGC TCA TTT ACA T-3' (sense), 5' -TCA AAG AAG CAT TCC CTC sulcus sample was found to be positive for both TTC TTC TTA-3 ' ( antisense ) . 7 Expected sizes 16SrDNAs of P. gingiva/is and A. actino­ of the target fragments amplified from the 16SrDNA mycetemitans. genes of P. gingiva/is, A. actinomycetemitans and T. denticola were 404 bp, 557 bp and 316 bp, Detection of P. gingiva/is 16SrDNA, A. 7 17 respectively. ' The PCR program includes an actinomycetemitans 16SrDNA and T. denticola initial denaturation step at 94 'C for five minutes 16SrDNA in the subgingival plaque samples by followed by 35 cycles of denaturation at 94 'C for one multiple PCR minute, primer annealing at 54 'C for 1 minute and The positive rates of P. gingiva/is 16SrDNA, A. extension at 72'C for 1. 5 minutes, and then a final actinomycetemitans 16SrDNA and T. denticola step at 72'C for seven minutes. Ten tJI of distilled 16SrDNA in 162 subgingival plaque samples from water was added instead of 10 1-11 template in the the patients were 84. 6%, 83. 3% and 88. 3%, PCR as blank control, and 10 tJI DNA template of respectively (Tables 1 & 2) . Compared with £. coli DH So: was used as negative control. Ten 1-11 samples from periodontal healthy individuals, the of mixed DNA templates of P. gingiva/is strain positive rates of 16SrDNAs from the three ATCC33277 , A. actinomycetemitans strain Y4 and anaerobes in subgingival plaque samples from the T. denticola strain FM was used as positive patients were significantly higher ( x2 ~72. 789, P < controls. To guarantee the reproducibility of PCR 0. 01). Using 16SrDNA as a genetic marker for the reaction, we repeated each reaction three times in bacteria in the 162 subgingival plaque samples , 11 0 our trial test to set up PCR conditions using the samples were positive for the three anaerobes, one same DNA templates. When used in clinical sample was negative for all three anaerobes , and . 918. Chin Med J 2005; 118(11) :915-921

Table 1. Detection rates of the 16SrDNA genes of P. gingiva/is, A. actinomycetemitans and T. denticola in subgingival plaque samples with different gingival index ( Gl) samples P. gingiva/is 16SrDNA A. actinomycetemitans16SrDNA T. denticola 16SrDNA Gl ( n) negative positive negative positive negative positive 31 6 25 (80. 6%) 7 24 (77. 4%) 7 24 (77. 4%) 2 77 15 62 (80. 5%) 19 58 (75. 3%) 6 71 (92. 2%) 3 54 4 50 (92. 6%) 53 (98.1%) 6 48 (88. 9%) Total 162 25 137 (84. 6%) 27 135 (83.8%) 19 143 (88.3%) Pvalue 0. 136 0.002 0.095

Table 2. Detection rates of the 16SrDNA genes of P. gingiva/is, A. actinomycetemitans and T. denticola in subgingival plaque samples with attachment loss ( AL) samples P. gingivalis16SrDNA A. actinomycetemitans16SrDNA T. denticola 16SrDNA AL (mm) ( n) negative positive negative positive negative positive

~2 74 20 54 (73. 0%) 20 54 (73. 0%) 13 61 (82. 4%) 2-5 54 3 51 (94. 4%) 5 49 (90. 7%) 6 48 (88. 9%) >5 34 2 32 (94. 1%) 2 32 (94.1%) 0 34 (100%) Total 162 25 137 (84. 6%) 27 135 (83.3%) 19 143 (88. 3%) Pvalue 0. 001 0.005 0.031 the remaining 51 samples were positive for either 2 4 6 7 8 9 10 11 one or two anaerobes (Table 3).

Table 3. Distribution of the multiple PCR detection results for the 16SrDNA genes of P. gingiva/is ( Pg), A. actinomy­ cetemitans ( Aa) and T. denticola ( Td) and its relation with severity of periodontitis severity of periodontitis Detection results samples severe mild moderate Fig. Detection by multiple PCR of the P. gingiva/is, A. Pg+Aa+Td+ 110 32 36 42 actinomycetemitans and T. denticola 16SrDNAs in Pg +Aa+Td- • 14 8 6 0 subgingival plaque samples. Lane 1 : 100 bp marker; Pg+Aa-Td+ * 12 4 5 3 Lane 2: positive control ( mixed DNA templates from P. Pg -Aa + Td + * 8 2 1 5 gingiva/is strain ATCC33277 , A. actinomycetemitans Pg +Aa -Td- . 0 0 strain Y4 and T. denticola strain FM) ; Lane 3: a Pg -Aa- Td + * 13 13 0 0 positive sample for P. gingiva/is, A. actinomycetemitans and T. denticola; Lane 4 : a Pg-Aa+Td-* 3 2 0 positive sample for P. gingiva/is positive and A. Pg-Aa-Td- 1 0 0 actinomycetemitans but negative for T. denticola; Lane Total 162 63 49 50 5 : a positive sample for P. gingiva/is positive and T. Pvalue 0.001 denticola but negative for A. actinomycetemitans; Lane + : positive; - : negative; • : data in these rows were combined. 6 : a positive sample for A. actinomycetemitans and T. denticola but negative for P. gingiva/is; Lane 7 : a Infection of P. gingiva/is, A. actino- positive sample for P. gingiva/is but negative for A. mycetemitans and T. denticola in the patients actinomycetemitans and T. denticola; Lane 8 : a Two samples were taken from two different teeth of positive sample for A. actinomycetemitans but negative for T. denticola and P. gingiva/is; Lane 9: a positive each patients. Only one patient had one sample sample for T. denticola but negative for P. gingiva/is positive for all three anaerobes, but another was and A. actinomycetemitans; Lane 10: negative control negative for all three. In 80 out of the 81 patients, (DNA template from E coli strain DH5a); and Lane there was at least one of the two subgingival plaque 11 : blank control. samples positive for one of the three microbes. Coinfection of the three microbes was found in both other had one infected with only one anaerobe. One samples from 39 patients and in one of the two patient had two samples both positive for two samples from 31 out of the 80 patients. Six of the anaerobes, while the other three patients had both remaining 10 infected patients had one sample samples positive for only one anaerobe. The infected with two of the three anaerobes while the consistency between two samples from the same Chinese Medical ]oumal2005; 118(1 1) :915-921 • 919.

patient was 53. 1% ( 43/81). (Table 3 ) . Due to the small number of cases infected with one and/or two anaerobes, no Association between clinical signs and positive statistical differences could be drawn among the rates of P. gingiva/is 16SrDNA, A. actino­ infection rates and the different state of severity of mycetemitans 16SrDNA and T. dent/cola the disease. 16SrDNA DISCUSSION No correlation could be found between the positive rates of P. gingiva/is and T. dentico/a and G I ( l = In the previous reports, PCR assay was used for 3. 997 , P = 0. 136 ; = 4. 699 , P = 0. 095 ; ex ' = l rapid clinical diagnosis as a routine method to detect 0. 05 ) . However, the positive rates of A. the 16SrDNA of P. gingiva/is, A. actino­ actinomycetemitans seemed to be related with Gl mycetemitans or T. denticola in subgingival plaque (l = 12. 870 , P = 0. 002, ex' = 0. 0167) . It was samples. In this study, we established a new more significant when Gl was 3, compared to the Gl multiple PCR assay to detect simultaneously the =1 or 2 =9. 925, P=O. 002; =12. 781, P= 7 10 Cl l 16SrDNA genes of the three microbes. " In 0. 000; ex' =0. 0167) (Table 1 ). repeated multiple PCR in the same clinical samples, the reproducibility was found to be reliable. All the The detection rates of P. gingiva/is, A results in this study indicate that multiple PCR can Actinomycetemcomitans and T. denticola varied be used as a diagnostic method for the three 2 with different AL degrees ( x = 14. 035, P = 0. 001 ; microbes in clinical samples. l =10. 699, P=O. 005; l =6. 974, P=O. 031 ;ex' = 0. 05 ) . It showed that the positive rates of P. For each of the positive detection rates of the three gingiva/is and A. actinomycetemitans in middle or microbes, significantly more patients were infected deep pockets with AL 2 mm -5 mm or > 5 mm were with P. gingiva/is ( 84. 6%), A. actino- much higher than in shallow ones with AL ~ 2 mm mycetemitans ( 83. 3%) and T. dentico/a 2 cx = 9. 764, P = o. 002; l = 6. 421 , P = o. 011 ; ( 88. 3% ) , respectively, than the periodontally and l = 6. 271 , P = 0. 012; l = 6. 421 P = 0. 011 ; healthy individuals, indicating the three microbes ex' = 0. 0167) (Table 2) . However, T. denticola were the prevalent bacteria in CP patients. A. was found more frequently in deep pockets ( AL > 5 actinomycetemitans was generally believed to be a 2 2 3 mm) than in shallow pockets ( AL ~ 2 mm) ( x = specific pathogen only associated with AgP. • 6. 790, P = 0. 009; ex' = 0. 0167) . No differences However, in this study, a high infection rate of A. could be found between detection rates of T. actinomycetemitans in the CP patients was found. denticola in moderate ( 2 mm < AL ~ 5 mm) and In our previous study, serum antibody against A. shallow pockets ( AL ~ 2 mm) ( l = 4. 054, P = actinomycetemitans in approximate 30% of Chinese 0. 044; ex' =0. 05) (Table 2). CP patients had been demonstrated. Sirinian as well as Umeda pointed out that distribution of A. Association between severity degree of actinomycetemitans in different ethnic groups were periodontitis and positive rates of P. gingiva/is, distinct and Asians may have an increased risk for 18 20 A. actlnomycetemltans and T. denticola harbouring this microbe in periodontal pockets. - Compared to the frequencies between cases These data indicated that the real role of A. infected with one and/ or two microbes , cases actinomycetemitans in CP in different ethnic infected with all three microbes in sites with different populations remains to be determined. degrees of severity of periodontitis showed a statistically significant difference ( l = 13. 725, P = Previous data reported that presence of P. gingiva/is 0. 001; ex' = 0. 05). The coinfection rates of the and T. denticola in periodontal pockets was related 6 21 three anaerobes in sites with severe periodontitis with high Gl scores, ' while A. was significantly higher than in those with mild actinomycetemitans' s association with Gl was disease Cl =12.951, P=O.OOO; ex' =0.0167). tenuous. 22 However, our data showed that no However, no statistically significant differences of association between infection of P. gingiva/is or T. the coinfection rates between severe and moderate denticola and Gl could be found, but infection of A. periodontitis and between moderate and mild actinomycetemitans was closely associated with Gl periodontitis could be found ( x2 = 2. 263, P = (Table 1 ) . Besides, some reports indicated that P. 0. 133; x2 = 4. 492, p = 0. 034; ex' = 0. 0167) gingiva/is and T. dentico/a were frequently detected I . 920. Chin Med I 2005; 118(11) :915-921 c

in middle ( >4 mm) or deep ( >6 mm) periodontal progressive periodontitis. Med Oral 2000 ;5: 151-158. 3 24 pockets / • whereas the presence of A. 4. Griffen AL, Becker MR, Lyons SR, et al. Prevalence actinomycetemitans in deep pocket was only of Porphyromonas gingiva/is and periodontal health 2 23 25 status. J Clin Microbial 1998 ;36 :3239-3242. occasional. • We found that A. 5. Chan EC, Mclaughlin R. and virulence of actinomycetemitans, like P. gingiva/is and T. oral spirochetes. Oral Microbiol lmmunol 2000; 15:1-9. denticola, was more frequently detectable in deep 6. Takeuchi Y, Umeda M, Sakamoto M, et al. 2 pockets than in shallow ones (Table 2). Since Gl , Treponema denticola, and and AL are important clinical indicators showing Porphyromonas gingiva/is are associated with severity gingival and periodontal tissue of periodontal tissue destruction. 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