Distribution of Porphyromonas gingivalis fimA and mfa1 fimbrial genotypes in subgingival plaques

Background Strains of periodontal disease-associated bacterium Porphyromonas gingivalis have different pathogenicity, which can be attributed to clonal genetic diversity. P. gingivalis typically expresses two types of fimbriae, FimA and Mfa1, which comprise six (I, Ib, II, III, IV, and V) and two (mfa53 and mfa70) genotypes, respectively. This study was conducted to investigate the distribution of the two fimbrial genotypes of P. gingivalis in clinical specimens. Methods Subgingival plaques were collected from 100 participants during periodontal maintenance therapy and examined for P. gingivalis fimbrial genotypes by direct polymerase chain reaction and/or DNA sequencing. We also analyzed the relationship between fimbrial genotypes and clinical parameters of periodontitis recorded at the first medical examination. Results Both fimbrial types could be detected in 63 out of 100 samples; among them, fimA genotype II was found in 33 samples (52.4%), in which the mfa70 genotype was 1.75 times more prevalent than mfa53. The total detection rate of fimA genotypes I and Ib was 38.1%; in these samples, the two mfa1 genotypes were observed at a comparable frequency. In two samples positive for fimA III (3.2%), only mfa53 was detected, whereas in four samples positive for fimA IV (6.3%), the two mfa1 genotypes were equally represented, and none of fimA V-positive samples defined the mfa1 genotype. No associations were found between clinical parameters and fimbrial subtype combinations. Discussion Both P. gingivalis fimbrial types were detected at various ratios in subgingival plaques, and a tendency for fimA and mfa1 genotype combinations was observed. However, there was no association between P. gingivalis fimbrial genotypes and periodontitis severity.


INTRODUCTION
Periodontal diseases are developed because of colonization of the subgingival area by multiple bacterial species (Page & Kornman, 1997). Socransky et al. (1998) have determined that three bacterial species, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, are mostly responsible for the development and advancement In contrast to fimA, there are few clinical data regarding mfa1 genotypes. Recently, we found that the mfa1 gene had at least two variants (Nagano et al., 2015), encoding proteins with molecular weights about 70 kDa (67 (Arai, Hamada & Umemoto, 2000;Hamada et al., 1996) or 75 kDa (Park et al., 2005)) and 53 kDa (Arai, Hamada & Umemoto, 2000;Nagano et al., 2015), hereafter called Mfa 70 and Mfa 53 (mfa 70 and mfa 53 , respectively, for the genes). In this study, we investigated the distribution of P. gingivalis mfa1 as well as fimA genotypes in clinical specimens.

MATERIALS AND METHODS Participants
A total of 100 patients, who visited Aichi Gakuin University Dental Hospital at Nagoya, Japan, for periodontal treatment from September 2016 to March 2017, participated in this study. The study was approved by the institutional review board (Aichi Gakuin University, School of Dentistry, Ethics Committee, approval numbers 460 and 478), and written informed consent was obtained from all participants.

Clinical oral examination, and consolidation and maintenance treatments
Among the 100 participants, 81 could be examined for clinicopathological parameters of periodontitis at the first visit. Clinical oral examination was performed according to the guidelines published by The Japanese Society of Periodontology (2015). Probing pocket depth (PD) and bleeding on probing (BOP) analyzed in six sites per tooth (buccal-mesial, mid-buccal, buccal-distal, lingual-mesial, mid-lingual, and lingual-distal) for all remaining teeth. The PD and BOP values were utilized to calculate periodontal inflamed surface area (PISA) and periodontal epithelial surface area (PESA), which reflect the surface area of bleeding epithelium and total pocket epithelium (in mm 2 ), respectively, using a free spreadsheet (downloaded from www.parsprototo.info) (Nesse et al., 2008(Nesse et al., , 2009. Consolidation and maintenance treatments mainly consisted of professional scaling and cleaning. Patients visited the hospital for consultation every 1-6 months.

Collection of subgingival plaques
Subgingival plaque samples were collected by a sterile hand scaler and transferred in either one ml of sterile reduced transfer fluid (RTF) consisting of 0.01% dithiothreitol in PBS, pH 7.4 or one ml of distilled water. The samples were immediately placed at 4 C and analyzed within 4 h.

Isolation and identification of black-pigmented bacteria
The samples collected in RTF were thoroughly suspended, serially diluted, and aliquots spread on blood agar consisting of Brucella HK agar (Kyokuto Pharmaceutical Industrial Co., Ltd, Tokyo, Japan), 5% laked rabbit blood, and 100 mg/ml kanamycin, as anaerobic bacteria, including P. gingivalis, are typically kanamycin resistant (Jousimies-Somer et al., 2002). Plates were cultured at 37 C under anaerobic conditions for a week, and the emerged black-pigmented colonies were streaked on fresh plates to ensure isolation of single clones, which were then subjected to species identification. For this, genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) and analyzed for 16S rRNA-encoding genes by polymerase chain reaction (PCR) using primers (5′-GAAGAGTTTGATCMTGG CTCAGATTG-3′ and 5′-TACGGYTACCTTGTTACGACTTCAC-3′) slightly modified from the universal primers 27F and 1492R (Frank et al., 2008). PCR products were subjected to DNA sequencing by a dye-terminator method and sequencing reads were analyzed by the BLAST search (https://blast.ncbi.nlm.nih.gov/). Bacterial species were identified if samples showed the lowest expectation (E) value (i.e., the highest probability) in the list of BLAST results. Most of E values were 0, whereas the highest was 3 Â 10 -66 , i.e., were sufficiently low to identify bacterial species.

Genotyping of fimA and mfa1
FimA genotypes were determined by PCR, sequencing, and BLAST analysis. Plaque samples in RTF or water were directly used as PCR templates. Primers for PCR (5′-AGCTTGTAACAAAGACAACGAGGCAG-3′ and 5′-GAGAATGAATACGGGGAG TGGAGCG-3′) were designed for common fimA regions based on fimA sequencing data for 84 P. gingivalis strains . PCR-amplified fragments of a predicted size (around 1.2 kb) were sequenced by the dye-terminator method and the fimA genotype was determined by BLAST analysis.

Statistical analysis
The data were expressed as the mean ± SEM. Differences between groups were analyzed by the nonparametric Kruskal-Wallis H test, and were considered statistically significant at P < 0.01. The Chi square test is used to determine if there is a relationship in the genotype distribution (P < 0.01).

Isolation of P. gingivalis
The first 73 dental plaque samples were collected in RTF to isolate P. gingivalis by a culture method. Although black-pigmented colonies were obtained from the majority of samples, 16S rDNA sequencing analysis showed that they were mostly formed by Prevotella species, and the isolation rate of P. gingivalis was only 5.5%. Therefore, we decided to examine fimbrial genotypes by direct PCR; in addition, the collection solution was changed to water, did not affect experimental results, but slightly improved detectability.

Distribution of fimA and mfa1 genotypes
The distribution of fimbrial fimA and mfa1 genotypes is summarized in Table 1. Among the 100 samples, both fimbrial types were detected in 63 and a single type in 15 samples, whereas 22 had no fimbrial genes. FimA genotype II was the most prevalent and detected in 33 of the 63 samples positive for both fimbrial genes (52.4%), followed by genotypes Ib and I detected in 27.0% and 11.1% samples, respectively, whereas the frequency of the other fimA genotypes was low. Although there was no statistically significant difference in combination of the fimA and mfa1 genotypes, the following tendency was observed. The mfa 53 and mfa 70 genotypes were detected at comparable frequencies (44.4% and 55.6%, respectively) and each of them showed almost the same frequency in samples positive for fimA genotypes I, Ib, and IV. However, the prevalence of mfa 70 was 1.75 times higher than that of mfa 53 in genotype-II positive samples, whereas only mfa 53 was detected in the two genotype III-positive samples, and no mfa1 genes were found in genotype V-positive samples.

Relationship between clinical parameters and fimbrial genotypes
We also examined the association of the fimbrial genotypes with clinical characteristics of periodontitis (maximal and mean PD values, and BOP, PISA, and PESA values) ( Table 2). However, no statistically significant differences in periodontitis severity were observed depending on the fimbrial genotypes.

DISCUSSION
In this study, we first attempted to isolate P. gingivalis from dental plaque samples by a culture method, because we thought that analysis of chromosomal DNA purified from isolated bacterial clones by PCR would provide unequivocal fimbrial genotyping results. However, P. gingivalis was rarely isolated by the culture method. On the other hand, direct PCR detected either fimA or mfa1 in 78% samples, indicating that P. gingivalis was present with high frequency in patients receiving periodontal maintenance therapy, although its proportion among dental plaque bacteria was low.
FimA genotypes have been determined by PCR using genotype-specific primers Nakagawa et al., 2002b); in addition, restriction enzyme digestion is used to discriminate genotypes I and Ib (Nakagawa et al., 2002b), which, however, may not be necessary for the analysis of the entire fimA gene, because genotypes I and Ib cannot be clearly discriminated ( Fig. S1 and Nagano et al., 2013). Furthermore, immunological analysis did not detect any differences in antigenicity between FimA I and Ib fimbriae Nakagawa et al., 2002b). Therefore, we do not discuss differences between genotypes I and Ib here. In contrast, genotype II (and possibly IV) could be further divided into two or more groups (Fig. S1 and Nagano et al., 2013). Regarding mfa1, two genotypes are currently known: mfa 53 and mfa 70 . However, in 12% of fimA-positive specimens, mfa1 was not detected, suggesting that existence of additional mfa1 genotypes. Therefore, reclassification of fimA and mfa1 genotypes would be needed in the future.
In this study, we observed that in samples positive for fimA genotype II, mfa 70 genotype was detected 1.75 times more frequently compared to mfa 53 , and in the previous study, where we analyzed 84 P. gingivalis strains stocked in our laboratory, the frequency of mfa 70 detection among fimA II strains was 3.6 times higher than that of mfa 53 (Nagano et al., 2015). These findings indicate that mfa 70 is the major mfa1 genotype in P. gingivalis strains positive for fimA II. On the other hand, in this study, the two mfa1 genotypes had almost the same detection rate in samples positive for fimA I (including I and Ib), whereas our previous results indicate that mfa 70 detection frequency was 2.3 times higher compared to that of mfa 53 in fimA-I strains (Nagano et al., 2015). Among fimA IV-positive samples, the detection rate of each mfa1 genotype was the same in this study, and in our previous study, mfa 53 and mfa 70 genotypes were detected in four and two samples, respectively (Nagano et al., 2015). Taken together, these results suggest that strains with fimA genotypes I and IV tend to have either the same frequency of mfa1 genotypes or slightly higher prevalence of mfa 70 . Although we found only two genotype III-positive samples in this study, both had mfa 53 , which was consistent with our earlier findings that 12 out of 13 genotype-III strains carried mfa 53 (Nagano et al., 2015). In this study, mfa1 was not detected in genotype V-positive samples, which, however, were all found mfa 53 -positive in our previous study (Nagano et al., 2015). These results indicate that genotype-III and -V strains almost exclusively carry mfa 53 . Thus, there is a tendency for correlation between the two fimbrial types in P. gingivalis: fimA II strains preferably carry mfa 70 , whereas fimA I/IV strains may have both mfa1 genotypes in equal proportions, and fimA III/V strains mostly carry mfa 53 . However, the reason for such correlations is unknown because there is a wide distance between the two genetic loci. There are polymorphisms in other P. gingivalis genes (Enersen, 2011). Thus, the ragA gene, which encodes a major outer membrane protein and is located downstream of the mfa1 gene, exhibits four genetic variants (Hall et al., 2005;Liu et al., 2013); besides, genetic variability has also been reported for capsular antigens (Laine, Appelmelk & Van Winkelhoff, 1996;Laine, Appelmelk & Van Winkelhoff, 1997). In future studies, it will be interesting to find out whether these genetic polymorphisms are correlated with those in the fimA and mfa1 genes.
We did not observe statistically significant associations between clinical parameters of periodontitis and the distribution of fimbrial genotypes. However, there was a time lapse between periodontal examination and sample collection, and it was possible that P. gingivalis clones were replaced during that interval; still, the chances for such clonal change may be low because it is reported that P. gingivalis showed high clonal stability (Valenza et al., 2009;Van Winkelhoff, Rijnsburger & Van Der Velden, 2008). In addition, we would like to note that most similar studies have the same problems which are inherent to this type of clinical research, because generally the treatment for chronic periodontitis takes a long time. Therefore, it is necessary to develop an appropriate study design for examining the relationship between bacterial genotypes and clinical symptoms of periodontitis.

CONCLUSIONS
There was a tendency in the distribution of fimbrial genotypes fimA (I-V) and mfa1 (mfa 53 and mfa 70 ) among patients with periodontitis. However, we did not observe any associations between fimbrial genotypes and the severity of the disease.