TY - JOUR
AU - PhD, Takahiko Oho, DDS,
AB - ABSTRACT Introduction: In the Japan Ground Self-Defense Force (JGSDF), personnel periodically perform intensive training that mimics the conditions seen in battle and during natural disasters. Military training involves intensive, stressful conditions, and changes in immune responses have been found in personnel following training. Good oral condition is important for military personnel to fulfill their duties; however, they have difficulty performing daily oral care under training conditions. In this study, we investigated the impact of a 7-day field training on the oral health status of JGSDF personnel by comparing their oral condition before and just after training. Materials and Methods: The participants were 59 male and 3 female JGSDF personnel undergoing a 7-day field training. All personnel provided informed written consent to participate, and this study was approved by the ethics committee of the Kagoshima University Graduate School of Medical and Dental Sciences. Oral health behaviors before and during the training period were surveyed using a self-administered questionnaire. Dental caries was assessed before training in terms of decayed, missing and filled teeth (DMFT), and periodontal condition was examined before and immediately after training using the community periodontal index (CPI). The presence of eight species of bacteria in dental plaque, including commensal streptococci that are early colonizers on the tooth surface, cariogenic bacteria, and periodontopathic bacteria, was determined using real-time polymerase chain reaction. We also assessed antibacterial factors and a stress marker in saliva samples. Sample collection was performed before and just after training. In addition to difference analysis between groups, logistic regression analysis was performed to examine the association between each health behavior and periodontal deterioration. Results: The frequency of toothbrushing decreased, and snacking increased during the training period. Thirty-five personnel (56.5%) showed an increase in individual CPI code, and 57 personnel (91.9%) showed deterioration in the CPI code in 1 or more sextants after training (Figure 1). Toothbrushing frequency was significantly associated with CPI deterioration; the odds ratio in subjects who did not brush their teeth was 7.51 compared to those who brushed at least once during the training period. Severe periodontal deterioration was observed in the high-DMFT group (Figure 2), and toothbrushing frequency during the training period decreased more in this group compared to the low-DMFT group. The percentages of Streptococcus sanguinis and Streptococcus gordonii increased significantly after the training period suggesting dental plaque maturation, and an increase in S. sanguinis was associated with toothbrushing frequency. The lactoferrin concentration in saliva increased significantly after training. Conclusions: We demonstrated periodontal deterioration in JGSDF personnel after a 7-day training. Behavioral changes, especially discontinuation of regular toothbrushing, fostered dental plaque maturation, resulting in inflammatory changes in participants' periodontal condition. The results indicate the importance of performing toothbrushing at least once over a 7-day training period for prevention of periodontal deterioration. The regimen could be applicable to evacuees from disasters because they are under conditions of stress that may limit oral hygiene activity. INTRODUCTION In the Japan Ground Self-Defense Force (JGSDF), personnel periodically perform intensive training that mimics the conditions seen in battle and during natural disasters. The training programs for military personnel may be very strict, involving not only long-term heavy physical activity but also exposure to psychological stressors, sleep deprivation, shifts in circadian rhythm, and exposure to extreme hot and cold environments.1 Such challenges have an impact on a participant's health condition. Many reports have demonstrated the impacts of stressful conditions on the immune responses of human subjects.2,–4 Military training involves intensive stressful conditions; thus, decreased immunoglobulin A5 and increased levels of interleukin-66, toll-like receptor 4, and tumor necrosis factor-α7 have been found in personnel following training. Military personnel have difficulty in performing daily oral care to maintain good oral condition under the conditions associated with training.8 Škec9 examined oral hygiene in army recruits and emphasized regular checkups for combat readiness. Furthermore, the incidence of severe oral conditions, including acute necrotizing ulcerative gingivitis, has been reported during rescue activities.10 Good oral condition is important for the personnel to fulfill their duties. To our knowledge, no studies have examined the effects of a 7-day physically and psychologically stressful training on the oral condition of military personnel by comparing participants' oral condition before and just after training. More than 700 species of bacteria are present in the oral cavity as biofilm constituents, competing with each other in their own community. The pathogenicity of these oral bacteria contributes to infectious diseases, including dental caries and periodontal diseases in the oral cavity. In addition, many investigators have shown that oral bacteria are associated with general health impairment, including cardiovascular diseases, pneumonia, and abscess formation.11,–13 The human immune system defends against bacterial attack to maintain healthy body function. However, conditions of excessive physical or psychological stress can result in dysfunction of these mechanisms of homeostasis. The oral cavity is at risk of damage by stressful conditions resulting from persistent bacterial attack. This study was performed to clarify the impact of a 7-day training on oral health status among JGSDF personnel. Oral health behaviors before training and during the training period were examined using a self-administered questionnaire. We examined periodontal condition and determined bacterial contents in dental plaque samples, antimicrobial factors, and a stress marker in saliva samples before and just after the 7-day training period. Changes in bacterial content as well as antimicrobial factors and stress markers during the training period provide useful information for developing new strategies for maintaining healthy oral function in personnel. The presence of eight species of bacteria in dental plaque, including commensal streptococci that are early colonizers on the tooth surface, cariogenic bacteria, and periodontopathic bacteria, was determined using real-time polymerase chain reaction (PCR). These data were used to examine the impact of changes in oral health behavior on periodontal condition and changes in oral bacterial content and salivary components. MATERIALS AND METHODS Participants Fifty-nine male and three female JGSDF personnel undergoing 7-day field training in Japan, participated in this study. In field training, personnel continuously act under the conditions of a disaster, an emergency event, or combat, day and night, over a long period. The subjects ranged in age from 20 to 53 years (34.7 ± 8.8 years, mean ± SD). All personnel were in good general health before training, and no significant impairments occurred in their health condition during the training period. All personnel provided informed written consent to participation after receiving sufficient explanation of the study. We instructed the personnel to perform oral hygiene practices in the same way as they did during previous training periods. This study was approved by the ethics committee of the Kagoshima University Graduate School of Medical and Dental Sciences (No. 284, 446, 557). Questionnaires for Oral Health Behavior After training had finished, the personnel were asked to complete a self-administered questionnaire about oral health behaviors before training and during the training period. The questionnaire assessed the frequency of toothbrushing, mouthwash rinsing, gum chewing, and snacking. Oral Examination Dental caries and periodontal status were examined to evaluate oral health status. Dental caries status, including decayed, missing, and filled teeth (DMFT),14 was evaluated before training. Periodontal status was evaluated before and immediately after training using the community periodontal index (CPI) according to World Health Organization (WHO) criteria.15 Briefly, an examiner assessed three indicators of periodontal status using WHO CPI probes: gingival bleeding, calculus, and periodontal pockets for 10 index teeth, categorized in sextants. Each sextant was assigned a code number that indicated the condition of most seriously affected site in that sextant according to the following criteria: code 0 = healthy; code 1 = bleeding observed after probing the gingival sulcus; code 2 = calculus detected during probing; code 3 = periodontal pocket (4–5 mm); code 4 = periodontal pocket (6 mm or deeper). The highest code across sextants for each subject was defined as the “individual CPI code.” A single dentist (K.Y.) performed oral examinations throughout the survey, thus eliminating interexaminer variability. Sample Collection Plaque and saliva samples were collected before and immediately after training. Supragingival plaque samples were collected from sound buccal surfaces of maxillary first molars using sterile dental explorers. Samples were suspended in 0.5 mL of sterile phosphate buffered saline (pH 7.0) and stored at −30°C until further analysis. Stimulated whole saliva was collected by chewing a piece of gum with no taste. After chewing the gum and swallowing saliva for the first 30 seconds, participants expectorated whole saliva into a tube on ice for 2 minutes. Samples were then stored at −30°C until further analysis. Bacterial Quantification Using Real-Time PCR Chromosomal DNA was extracted from collected plaque samples according to a method described previously.16 Briefly, collected plaque suspension was centrifuged, and the resultant precipitates were resuspended in 100 μL of lysis buffer (20 mM Tris/HCl, 2 mM ethylenediaminetetraacetic acid, 1% Triton X-100; pH 8.0). The lysate was boiled at 100°C for 10 minutes, and chromosomal DNA was obtained by centrifugation. The primers used for the quantification of the bacteria are shown in Table I. For real-time PCR, 20 μL of a mixture containing 3 μL of lysed sample, 10 pmol of each primer, and 10 μL of Fast SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, Massachusetts) was placed in each well of a 48-well plate. The samples were amplified using the StepOne Real-time PCR System (Thermo Fisher Scientific). Amplification consisted of an initial denaturation step at 95°C for 20 seconds, followed by 45 cycles at 95°C for 3 seconds and 60°C for 30 seconds. The percentages of specific bacteria were calculated on the basis of the comparative Ct (ΔΔCt) method using the following equation: % = 1/2(Ct target − Ct total) × 100.16 TABLE I Oligonucleotide Primers Species  Designation of Primers  Sequence  Amplicon Size (bp)  Target  Reference  S. mutans  Smut3368-F  5′-GCCTACAGCTCAGAGATGCTATTCT-3′  114  gtfB  16  Smut3481-R  5′-GCCATACACCACTCATGAATTGA-3′  S. sobrinus  Ssob287-F  5′-TTCAAAGCCAAGACCAAGCTAGT-3′  88  gtfT  16  Ssob374-R  5′-CCAGCCTGAGATTCAGCTTGT-3′  S. sanguinis  tnpA-F  5′-CAAAATTGTTGCAAATCCAAAGG-3′  74  tnpA  47  tnpA-R  5′-GCTATCGCTCCCTGTCTTTGA-3′  S. gordonii  gtfG-F  5′-CGGATGATGCTAATCAAGTGACC-3′  177  gtfG  48  gtfG-R  5′-GTTAGCTGTTGGATTGGTTGCC-3′  S. oralis  gtfR-F  5′-ACCAGCAGATACGAAAGAAGCAT-3′  235  gtfR  48  gtfR-R  5′-AGGTTCGGGCAAGCGATCTTTCT-3′  P. gingivalis  Pg1198-F  5′-TACCCATCGTCGCCTTGGT-3′  126  16S rRNA  49  Pg1323-R  5′-CGGACTAAAACCGCATACACTTG-3′  A. actinomycetemcomitans  Aa1956-F  5′-CAGCATCTGCGATCCCTGTA-3′  147  IktA  49  Aa2102-R  5′-TCAGCCCTTTGTCTTTCCTAGGT-3′  F. nucleatum  619-F  5′-CGCAGAAGGTGAAAGTCCTGTAT-3′  101  16S rRNA  50  719-R  5′-TGGTCCTCACTGATTCACACAGA-3′  Universal  Uni152-F  5′-CGCTAGTAATCGTGGATCAGAATG-3′  69  16S rRNA  49  Uni220-R  5′-TGTGACGGGCGGTGTGTA-3′  Species  Designation of Primers  Sequence  Amplicon Size (bp)  Target  Reference  S. mutans  Smut3368-F  5′-GCCTACAGCTCAGAGATGCTATTCT-3′  114  gtfB  16  Smut3481-R  5′-GCCATACACCACTCATGAATTGA-3′  S. sobrinus  Ssob287-F  5′-TTCAAAGCCAAGACCAAGCTAGT-3′  88  gtfT  16  Ssob374-R  5′-CCAGCCTGAGATTCAGCTTGT-3′  S. sanguinis  tnpA-F  5′-CAAAATTGTTGCAAATCCAAAGG-3′  74  tnpA  47  tnpA-R  5′-GCTATCGCTCCCTGTCTTTGA-3′  S. gordonii  gtfG-F  5′-CGGATGATGCTAATCAAGTGACC-3′  177  gtfG  48  gtfG-R  5′-GTTAGCTGTTGGATTGGTTGCC-3′  S. oralis  gtfR-F  5′-ACCAGCAGATACGAAAGAAGCAT-3′  235  gtfR  48  gtfR-R  5′-AGGTTCGGGCAAGCGATCTTTCT-3′  P. gingivalis  Pg1198-F  5′-TACCCATCGTCGCCTTGGT-3′  126  16S rRNA  49  Pg1323-R  5′-CGGACTAAAACCGCATACACTTG-3′  A. actinomycetemcomitans  Aa1956-F  5′-CAGCATCTGCGATCCCTGTA-3′  147  IktA  49  Aa2102-R  5′-TCAGCCCTTTGTCTTTCCTAGGT-3′  F. nucleatum  619-F  5′-CGCAGAAGGTGAAAGTCCTGTAT-3′  101  16S rRNA  50  719-R  5′-TGGTCCTCACTGATTCACACAGA-3′  Universal  Uni152-F  5′-CGCTAGTAATCGTGGATCAGAATG-3′  69  16S rRNA  49  Uni220-R  5′-TGTGACGGGCGGTGTGTA-3′  View Large TABLE I Oligonucleotide Primers Species  Designation of Primers  Sequence  Amplicon Size (bp)  Target  Reference  S. mutans  Smut3368-F  5′-GCCTACAGCTCAGAGATGCTATTCT-3′  114  gtfB  16  Smut3481-R  5′-GCCATACACCACTCATGAATTGA-3′  S. sobrinus  Ssob287-F  5′-TTCAAAGCCAAGACCAAGCTAGT-3′  88  gtfT  16  Ssob374-R  5′-CCAGCCTGAGATTCAGCTTGT-3′  S. sanguinis  tnpA-F  5′-CAAAATTGTTGCAAATCCAAAGG-3′  74  tnpA  47  tnpA-R  5′-GCTATCGCTCCCTGTCTTTGA-3′  S. gordonii  gtfG-F  5′-CGGATGATGCTAATCAAGTGACC-3′  177  gtfG  48  gtfG-R  5′-GTTAGCTGTTGGATTGGTTGCC-3′  S. oralis  gtfR-F  5′-ACCAGCAGATACGAAAGAAGCAT-3′  235  gtfR  48  gtfR-R  5′-AGGTTCGGGCAAGCGATCTTTCT-3′  P. gingivalis  Pg1198-F  5′-TACCCATCGTCGCCTTGGT-3′  126  16S rRNA  49  Pg1323-R  5′-CGGACTAAAACCGCATACACTTG-3′  A. actinomycetemcomitans  Aa1956-F  5′-CAGCATCTGCGATCCCTGTA-3′  147  IktA  49  Aa2102-R  5′-TCAGCCCTTTGTCTTTCCTAGGT-3′  F. nucleatum  619-F  5′-CGCAGAAGGTGAAAGTCCTGTAT-3′  101  16S rRNA  50  719-R  5′-TGGTCCTCACTGATTCACACAGA-3′  Universal  Uni152-F  5′-CGCTAGTAATCGTGGATCAGAATG-3′  69  16S rRNA  49  Uni220-R  5′-TGTGACGGGCGGTGTGTA-3′  Species  Designation of Primers  Sequence  Amplicon Size (bp)  Target  Reference  S. mutans  Smut3368-F  5′-GCCTACAGCTCAGAGATGCTATTCT-3′  114  gtfB  16  Smut3481-R  5′-GCCATACACCACTCATGAATTGA-3′  S. sobrinus  Ssob287-F  5′-TTCAAAGCCAAGACCAAGCTAGT-3′  88  gtfT  16  Ssob374-R  5′-CCAGCCTGAGATTCAGCTTGT-3′  S. sanguinis  tnpA-F  5′-CAAAATTGTTGCAAATCCAAAGG-3′  74  tnpA  47  tnpA-R  5′-GCTATCGCTCCCTGTCTTTGA-3′  S. gordonii  gtfG-F  5′-CGGATGATGCTAATCAAGTGACC-3′  177  gtfG  48  gtfG-R  5′-GTTAGCTGTTGGATTGGTTGCC-3′  S. oralis  gtfR-F  5′-ACCAGCAGATACGAAAGAAGCAT-3′  235  gtfR  48  gtfR-R  5′-AGGTTCGGGCAAGCGATCTTTCT-3′  P. gingivalis  Pg1198-F  5′-TACCCATCGTCGCCTTGGT-3′  126  16S rRNA  49  Pg1323-R  5′-CGGACTAAAACCGCATACACTTG-3′  A. actinomycetemcomitans  Aa1956-F  5′-CAGCATCTGCGATCCCTGTA-3′  147  IktA  49  Aa2102-R  5′-TCAGCCCTTTGTCTTTCCTAGGT-3′  F. nucleatum  619-F  5′-CGCAGAAGGTGAAAGTCCTGTAT-3′  101  16S rRNA  50  719-R  5′-TGGTCCTCACTGATTCACACAGA-3′  Universal  Uni152-F  5′-CGCTAGTAATCGTGGATCAGAATG-3′  69  16S rRNA  49  Uni220-R  5′-TGTGACGGGCGGTGTGTA-3′  View Large Measurement of Salivary Components Lactoferrin Sandwich enzyme-linked immunosorbent assay (ELISA) was performed to determine lactoferrin concentration following the method of Franco et al.17 Maxisorp microtitration plates (Nunc, Roskilde, Denmark) were coated with 100 μL per well of goat anti-human lactoferrin antibody (1 μg/mL, Betyl Laboratories Inc., Montgomery, Texas) in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and incubated overnight at 4°C. After the plates were washed three times with 300 μL per well of phosphate buffered saline containing 0.05% Tween 20 (PBST, pH 7.4), residual protein binding sites were blocked with 100 μL of 1% (wt/vol) bovine serum albumin in PBST at 37°C for 1.5 hours. The wells were washed with PBST, and samples and human milk lactoferrin standards (AbD Serotec, Hercules, California) from 1.56 to 50 ng/mL were added to the wells (100 μL each) and incubated for 1.5 hours. The plates were then washed with PBST and incubated with 100 μL of horseradish peroxidase-conjugated goat anti-human lactoferrin antibody (Betyl Laboratories Inc.) diluted 1:10,000 in PBST for 1.5 hours at 37°C. Then, the plates were washed with PBST and incubated with 100 μL of ABTS substrate solution per well for 60 minutes at 37°C. The absorbance at 405 nm was determined using a microplate reader (Model 680, Bio-Rad Laboratories, Hercules, California). The lactoferrin concentration was determined using the standard curve, and interpolation was performed (r2 = 0.99). LL37 Sandwich ELISA was performed for measurement of LL37. Briefly, microtitration plates were coated with anti-LL37 IgG antibody18 (10 μg/mL) in coating buffer. After blocking, samples and human LL37 standards19 from 62.5 to 1,000 ng/mL were added to the wells. LL37 was detected using biotinylated anti-LL37 IgG antibody (0.9 μg/mL), followed by reaction with alkaline phosphatase-conjugated streptavidin (Vector Laboratories, Burlingame, California). Color development was performed using p-nitrophenyl-phosphate in diethanolamine buffer, and absorbance at 405 nm was determined. The LL37 concentration was determined using the standard curve. Lysozyme Lysozyme concentration was determined by dot blot assay. Samples and human recombinant lysozyme standards (Wako Pure Chemical Industries Ltd., Osaka, Japan) from 31.2 to 2,000 ng/mL in 100 μL of Tris buffered saline (20 mM Tris, 0.15 M NaCl, pH 7.2) were blotted onto nitrocellulose membranes using a Bio-Dot microfiltration apparatus (Bio-Rad Laboratories). The membranes were blocked with 3% skimmed milk in Tris buffered saline containing 0.1% Triton-X 100 (TBST) for 1.5 hours at room temperature. After washing with TBST, the membranes were incubated with rabbit anti-human lysozyme antibody (Nordic Immunological Laboratory, Eindhoven, The Netherlands) diluted 1:1,000 in TBST for 1.5 hours at room temperature. The membranes were washed with TBST and incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Zymed Laboratories, San Francisco, California) in TBST for 1.5 hours at room temperature. Colorimetric development was performed using a BCIP/NBT tablet (Sigma Chemical Co., St. Louis, Missouri), and the lysozyme concentration was quantified using Image J software (http://imagej.nih.gov/ij/). α-Amylase The intensity of physical or psychological stress was monitored by measuring the level of α-amylase activity using a commercial kit (α-Amylase Measuring Kit; Kikkoman, Tokyo, Japan). In the assay, α-amylase in the saliva sample reacts with the substrate, yielding 2-chloro-4-nitrophenol, which is yellow in color and can be detected using a spectrophotometer at 405 nm. Statistical Analysis The differences between groups were analyzed using the Student t-test, Wilcoxon signed rank test, Mann–Whitney U test, or χ2 test as appropriate. Logistic regression analysis was performed, with CPI change after training as the dependent variable. We compared the CPI code in each sextant for each subject before with that after training, and the number of sextants exhibiting an increase in CPI code was calculated. The participants were divided into two groups according to the number of CPI sextants that deteriorated (no/slight deterioration = 0–2, severe deterioration = 3–5). The independent variables were behavior frequencies during the training period, which were categorized as follows; toothbrushing (0, ≥1), mouthwash rinsing (0, ≥1), gum chewing (0, ≥1), and snacking (≥1 time per 2 days, <1 time per 2 days). Multiple regression analysis was performed to gauge the effect of each health behavior on the bacterial percentage. All statistical analyses were performed using SPSS version 20 (IBM SPSS Japan, Tokyo, Japan). In all analyses, p < 0.05 was taken to indicate statistical significance. RESULTS Oral Health Behavior and Oral Health Status The frequencies of oral health behaviors before and during the training period were compared. All personnel performed toothbrushing every day before training, but the percentage decreased to 6.5 during training, and 40.3% of personnel did not brush their teeth at all during the training period. The number of personnel who snacked every day or every 2 days increased markedly during the training period. There were significant differences between the frequencies of toothbrushing and snacking before and those after training (p < 0.05, Wilcoxon signed rank test). Periodontal condition was examined using CPI. Changes in individual CPI code between before and after training are shown in Table II. Thirty-five personnel (56.5%) showed an increase in individual CPI code after training; 24 from code 0, 6 from code 1, 4 from code 2, and 1 from code 3. A significant difference in code distribution between before and after training was observed (p < 0.05, Wilcoxon signed rank test). Next, we counted the number of CPI sextants that deteriorated after training in each subject (Fig. 1). Fifty-seven individuals (91.9%) showed deterioration of the CPI code in 1 or more sextants after training, and 16 (25.8%) showed deterioration in two sextants. TABLE II Change in CPI Individual Code Before and After Training CPI Individual Code Before Training  CPI Individual Code After Training  Total  0  1  2  3  4  0  2a  20  0  4  0  26  1  0  10  0  6  0  16  2  0  0  5  4  0  9  3  0  0  0  10  1  11  Total  2  30  5  24  1  62  CPI Individual Code Before Training  CPI Individual Code After Training  Total  0  1  2  3  4  0  2a  20  0  4  0  26  1  0  10  0  6  0  16  2  0  0  5  4  0  9  3  0  0  0  10  1  11  Total  2  30  5  24  1  62  a Values represent number of personnel. View Large TABLE II Change in CPI Individual Code Before and After Training CPI Individual Code Before Training  CPI Individual Code After Training  Total  0  1  2  3  4  0  2a  20  0  4  0  26  1  0  10  0  6  0  16  2  0  0  5  4  0  9  3  0  0  0  10  1  11  Total  2  30  5  24  1  62  CPI Individual Code Before Training  CPI Individual Code After Training  Total  0  1  2  3  4  0  2a  20  0  4  0  26  1  0  10  0  6  0  16  2  0  0  5  4  0  9  3  0  0  0  10  1  11  Total  2  30  5  24  1  62  a Values represent number of personnel. View Large FIGURE 1 View largeDownload slide Distribution of the number of CPI sextants exhibiting deterioration after training. FIGURE 1 View largeDownload slide Distribution of the number of CPI sextants exhibiting deterioration after training. The effects of oral health behaviors during the training period on periodontal condition were examined. We divided personnel into two groups (no/slight deterioration, severe deterioration) according to the number of CPI sextants exhibiting deterioration. The relationship between oral health behavior and CPI deterioration is shown in Table III. There was a significant difference in toothbrushing frequency between the two groups. However, there were no differences in the frequency of mouthwash rinsing, gum chewing, or snacking between the two groups. Logistic regression analysis indicated that only toothbrushing frequency was significantly associated with CPI deterioration; the odds ratio in personnel who did not brush their teeth was 7.51 relative to those who did brush at least once during the training period (p = 0.002; 95% CI = 2.04–27.65). TABLE III Relationship Between Oral Health Behavior During Training Period and CPI Deterioration Behavior  CPI Deterioration  p Valuea  No/Slight  Severe  Toothbrushing  0.004   At Least Once  23b  14      Not Doing  6  19     Mouthwash Rinsing  0.696   At Least Once  4  3      Not Doing  25  30     Gum Chewing  0.798   At Least Once  11  14      Not Doing  18  19     Snacking  0.409   Once or More/2 Days  22  21      Less Than Once/2 Days  7  12     Behavior  CPI Deterioration  p Valuea  No/Slight  Severe  Toothbrushing  0.004   At Least Once  23b  14      Not Doing  6  19     Mouthwash Rinsing  0.696   At Least Once  4  3      Not Doing  25  30     Gum Chewing  0.798   At Least Once  11  14      Not Doing  18  19     Snacking  0.409   Once or More/2 Days  22  21      Less Than Once/2 Days  7  12     a Determined by χ2 test. b Values represent number of personnel. View Large TABLE III Relationship Between Oral Health Behavior During Training Period and CPI Deterioration Behavior  CPI Deterioration  p Valuea  No/Slight  Severe  Toothbrushing  0.004   At Least Once  23b  14      Not Doing  6  19     Mouthwash Rinsing  0.696   At Least Once  4  3      Not Doing  25  30     Gum Chewing  0.798   At Least Once  11  14      Not Doing  18  19     Snacking  0.409   Once or More/2 Days  22  21      Less Than Once/2 Days  7  12     Behavior  CPI Deterioration  p Valuea  No/Slight  Severe  Toothbrushing  0.004   At Least Once  23b  14      Not Doing  6  19     Mouthwash Rinsing  0.696   At Least Once  4  3      Not Doing  25  30     Gum Chewing  0.798   At Least Once  11  14      Not Doing  18  19     Snacking  0.409   Once or More/2 Days  22  21      Less Than Once/2 Days  7  12     a Determined by χ2 test. b Values represent number of personnel. View Large Effects of Dental Caries Experience on Periodontal Condition and Oral Health Behavior We examined the association of dental caries experience with the change in periodontal condition during training. The number of DMFT of all personnel examined was 10.5 ± 6.1 (mean ± SD); subjects were divided into two groups according to the mean value of DMFT: 5.3 ± 3.5 for the low-DMFT group (n = 30) and 15.4 ± 3.3 for the high-DMFT group (n = 32). The distributions for all groups according to the number of CPI sextants exhibiting deterioration after training are shown in Figure 2. The personnel in the low-DMFT group mainly showed slight deterioration in CPI, whereas the personnel in the high-DMFT group showed severe deterioration in CPI. The difference in distributions of the number of CPI sextants exhibiting deterioration between the two groups was significant. None of the personnel in the high-DMFT group brushed their teeth every day (Table IV). The percentage of personnel who did not perform toothbrushing during the training period in the high-DMFT group was approximately double that in the low-DMFT group. The difference in toothbrushing frequency between the two groups was significant. FIGURE 2 View largeDownload slide Distribution of low-DMFT and high-DMFT groups according to the number of CPI sextants exhibiting deterioration after training. Open bar, low-DMFT group; closed bar, high-DMFT group. *p < 0.05 between two groups, as determined by Mann–Whitney U test. FIGURE 2 View largeDownload slide Distribution of low-DMFT and high-DMFT groups according to the number of CPI sextants exhibiting deterioration after training. Open bar, low-DMFT group; closed bar, high-DMFT group. *p < 0.05 between two groups, as determined by Mann–Whitney U test. TABLE IV Relationship Between Toothbrushing Frequency During Training Period and Dental Caries Experience Toothbrushing Frequency  Low DMFT  High DMFT*  Every Day  4 (13.3)a  0 (0)  Once per 2 Days  6 (20.0)  6 (18.8)  Once per 3 or 4 Days  9 (30.0)  7 (21.9)  Once per 5 to 7 Days  3 (10.0)  2 (6.3)  Not Doing  8 (26.7)  17 (53.1)  Toothbrushing Frequency  Low DMFT  High DMFT*  Every Day  4 (13.3)a  0 (0)  Once per 2 Days  6 (20.0)  6 (18.8)  Once per 3 or 4 Days  9 (30.0)  7 (21.9)  Once per 5 to 7 Days  3 (10.0)  2 (6.3)  Not Doing  8 (26.7)  17 (53.1)  * p < 0.05 between two groups, as determined by Mann–Whitney U test. a Values represent number of personnel (%). View Large TABLE IV Relationship Between Toothbrushing Frequency During Training Period and Dental Caries Experience Toothbrushing Frequency  Low DMFT  High DMFT*  Every Day  4 (13.3)a  0 (0)  Once per 2 Days  6 (20.0)  6 (18.8)  Once per 3 or 4 Days  9 (30.0)  7 (21.9)  Once per 5 to 7 Days  3 (10.0)  2 (6.3)  Not Doing  8 (26.7)  17 (53.1)  Toothbrushing Frequency  Low DMFT  High DMFT*  Every Day  4 (13.3)a  0 (0)  Once per 2 Days  6 (20.0)  6 (18.8)  Once per 3 or 4 Days  9 (30.0)  7 (21.9)  Once per 5 to 7 Days  3 (10.0)  2 (6.3)  Not Doing  8 (26.7)  17 (53.1)  * p < 0.05 between two groups, as determined by Mann–Whitney U test. a Values represent number of personnel (%). View Large Relationship Between Oral Health Behaviors and Bacterial Content in Dental Plaque The percentages of bacteria in dental plaque before and after training are shown in Table V. The percentages of Streptococcus sanguinis and Streptococcus gordonii increased significantly, whereas those of Streptococcus mutans, Streptococcus sobrinus, and Aggregatibacter actinomycetemcomitans decreased significantly following training. We examined the association of oral health behaviors during the training period with the change in plaque bacterial content. Personnel who did not brush their teeth or rinse with mouthwash showed significant increases in the S. sanguinis percentage (Table VI). Multiple regression analysis indicated that toothbrushing frequency was significantly associated with S. sanguinis percentage after training (standardized partial regression coefficient β-weight = 0.281, p = 0.041). TABLE V Percentage of Bacteria in Dental Plaque Before and After Training Bacterium  Before (%)  After (%)  S. mutans  0.016 ± 0.052a  0.004 ± 0.021*  S. sobrinus  0.0008 ± 0.0018  0.0002 ± 0.0010*  S. sanguinis  0.056 ± 0.114  0.148 ± 0.258*  S. gordonii  0.189 ± 0.421  0.307 ± 0.556*  S. oralis  0.124 ± 0.245  0.202 ± 0.333  P. gingivalis  0.016 ± 0.083  0.039 ± 0.109  A. actinomycetemcomitans  0.00008 ± 0.00030  0.00002 ± 0.00004*  F. nucleatum  0.151 ± 0.306  0.173 ± 0.236  Bacterium  Before (%)  After (%)  S. mutans  0.016 ± 0.052a  0.004 ± 0.021*  S. sobrinus  0.0008 ± 0.0018  0.0002 ± 0.0010*  S. sanguinis  0.056 ± 0.114  0.148 ± 0.258*  S. gordonii  0.189 ± 0.421  0.307 ± 0.556*  S. oralis  0.124 ± 0.245  0.202 ± 0.333  P. gingivalis  0.016 ± 0.083  0.039 ± 0.109  A. actinomycetemcomitans  0.00008 ± 0.00030  0.00002 ± 0.00004*  F. nucleatum  0.151 ± 0.306  0.173 ± 0.236  * p < 0.05 between before and after training, as determined by Wilcoxon signed rank test. a Values represent mean ± SD. View Large TABLE V Percentage of Bacteria in Dental Plaque Before and After Training Bacterium  Before (%)  After (%)  S. mutans  0.016 ± 0.052a  0.004 ± 0.021*  S. sobrinus  0.0008 ± 0.0018  0.0002 ± 0.0010*  S. sanguinis  0.056 ± 0.114  0.148 ± 0.258*  S. gordonii  0.189 ± 0.421  0.307 ± 0.556*  S. oralis  0.124 ± 0.245  0.202 ± 0.333  P. gingivalis  0.016 ± 0.083  0.039 ± 0.109  A. actinomycetemcomitans  0.00008 ± 0.00030  0.00002 ± 0.00004*  F. nucleatum  0.151 ± 0.306  0.173 ± 0.236  Bacterium  Before (%)  After (%)  S. mutans  0.016 ± 0.052a  0.004 ± 0.021*  S. sobrinus  0.0008 ± 0.0018  0.0002 ± 0.0010*  S. sanguinis  0.056 ± 0.114  0.148 ± 0.258*  S. gordonii  0.189 ± 0.421  0.307 ± 0.556*  S. oralis  0.124 ± 0.245  0.202 ± 0.333  P. gingivalis  0.016 ± 0.083  0.039 ± 0.109  A. actinomycetemcomitans  0.00008 ± 0.00030  0.00002 ± 0.00004*  F. nucleatum  0.151 ± 0.306  0.173 ± 0.236  * p < 0.05 between before and after training, as determined by Wilcoxon signed rank test. a Values represent mean ± SD. View Large TABLE VI Effects of Oral Health Behavior During Training Period on the Change of S. sanguinis Percentage in Dental Plaque Behavior  Before (%)  After (%)  Toothbrushing   At Least Once  0.052 ± 0.090a  0.089 ± 0.137   Not Doing  0.062 ± 0.144  0.235 ± 0.359*  Mouthwash Rinsing   At Least Once  0.037 ± 0.057  0.042 ± 0.049   Not Doing  0.059 ± 0.119  0.161 ± 0.271*  Behavior  Before (%)  After (%)  Toothbrushing   At Least Once  0.052 ± 0.090a  0.089 ± 0.137   Not Doing  0.062 ± 0.144  0.235 ± 0.359*  Mouthwash Rinsing   At Least Once  0.037 ± 0.057  0.042 ± 0.049   Not Doing  0.059 ± 0.119  0.161 ± 0.271*  * p < 0.05 between before and after training, as determined by Wilcoxon signed rank test. a Values represent mean ± SD. View Large TABLE VI Effects of Oral Health Behavior During Training Period on the Change of S. sanguinis Percentage in Dental Plaque Behavior  Before (%)  After (%)  Toothbrushing   At Least Once  0.052 ± 0.090a  0.089 ± 0.137   Not Doing  0.062 ± 0.144  0.235 ± 0.359*  Mouthwash Rinsing   At Least Once  0.037 ± 0.057  0.042 ± 0.049   Not Doing  0.059 ± 0.119  0.161 ± 0.271*  Behavior  Before (%)  After (%)  Toothbrushing   At Least Once  0.052 ± 0.090a  0.089 ± 0.137   Not Doing  0.062 ± 0.144  0.235 ± 0.359*  Mouthwash Rinsing   At Least Once  0.037 ± 0.057  0.042 ± 0.049   Not Doing  0.059 ± 0.119  0.161 ± 0.271*  * p < 0.05 between before and after training, as determined by Wilcoxon signed rank test. a Values represent mean ± SD. View Large Salivary Components Salivary contents of lactoferrin, LL37, and lysozyme were measured to examine the effects of training. Lactoferrin concentration (mean ± SD μg/mL) increased significantly after training (22.5 ± 27.6) compared with baseline (16.7 ± 19.4), whereas LL37 and lysozyme showed no changes (p < 0.05, Wilcoxon signed rank test). α-amylase activity was measured to examine stress levels induced by training, but no significant changes were observed after training. DISCUSSION This study was performed to examine the impact of changes in oral health behavior on periodontal condition and changes in oral bacterial contents and salivary components among JGSDF personnel after a 7-day training. Before training, all of the JGSDF personnel brushed their teeth every day, and 41.9% showed no gingival bleeding (CPI = 0). With regard to the daily oral hygiene routine (i.e., before training) and oral health status, Škec9 reported that 97.4% of Croatian recruits had habitually brushed their teeth every day, and approximately 36% showed no gingival bleeding. Senna et al20 also reported that 40.95% of Italian call-up soldiers had healthy periodontal condition. The toothbrushing frequency and periodontal condition of the JGSDF personnel before training in this study were compatible with these previous reports. The frequencies of toothbrushing and snacking changed during the training period. Toothbrushing frequency decreased during the training period, and similar results were reported in JGSDF personnel.8 Personnel were engaged in persistent field training, and it seemed to be difficult to perform toothbrushing regularly. The frequency of snacking increased during the training period. Personnel had to take meals at irregular times during the training period, and they may have tried to supplement their energy by frequent snacking. These changes in behavior seemed to result in impairment of oral condition. In fact, the periodontal condition of participants deteriorated during the training period, and the distribution of individual CPI codes shifted, indicating deterioration. To elucidate the cause of periodontal deterioration, the relationship between oral health behaviors and periodontal condition was examined. Twenty-three of 37 personnel who brushed their teeth at least once during the training period showed no/slight deterioration in CPI, whereas 19 of 25 personnel who did not brush during the training period showed severe deterioration. Logistic regression analysis revealed a significant association between CPI deterioration and toothbrushing frequency (odds ratio = 7.51). Löe et al21 reported that the Gingival Index increased in healthy persons after 7 days of interrupted oral hygiene. Our results are consistent with their results and indicate that toothbrushing is an important behavior to prevent periodontal deterioration during the training period. Regarding the change in periodontal condition after a 7-day training, major changes were observed in the gingiva, but no change was observed in calculus deposition. The use of the Gingival Index22 may have more accurately measured gingival changes seen after 7 days of poor or no oral hygiene. To examine the effects of dental plaque bacteria on gingival condition, changes in bacterial content during the training period were examined. Dental plaque is composed of many bacterial species, and the microbial interactions in the communities have been investigated.23,–25 Dental plaque formation is initiated by the attachment of early colonizers, including S. sanguinis, Streptococcus oralis, and S. gordonii, on salivary pellicle-coated enamel surfaces.26 Late colonizers, including periodontopathic bacteria such as Porphyromonas gingivalis, A. actinomycetemcomitans, and Treponema denticola bind to previously bound bacteria, and sequential binding occurs by bridging with the next coaggregating partner cells.23 Between early and late colonizers, Fusobacterium nucleatum, the most numerous Gram-negative species in healthy sites, acts as a “bridge” by coaggregating with both types of colonizer.27 Cariogenic streptococci, including S. mutans and S. sobrinus, are not abundant in the initial colonizing community on the tooth surface. If sucrose, as a carbohydrate substrate, is introduced into the community, the bacteria produce exopolysaccharides, such as water-insoluble glucan, which act to accumulate many species forming a cariogenic biofilm.28 Therefore, we selected eight specific bacteria described in Table I. The percentages of two species, S. sanguinis and S. gordonii, increased after training. Socransky et al29 examined bacterial species present at various times during plaque development on the tooth surface and demonstrated that S. sanguinis was consistently present, becoming predominant at days 1 to 2 and there after. Takeshita et al30 examined the assembly process of dental plaque microbiota on hydroxyapatite disks in young adults and found an increase in the relative abundance of S. gordonii on day 7 compared to that on day 1. Our results are consistent with these previous studies. Streptococcus sanguinis and S. gordonii are known to produce hydrogen peroxide as an antibacterial agent against other bacterial species.31,32 The increase in these two streptococci seems to be the result of their high capacity to produce hydrogen peroxide. On the other hand, the percentage of S. mutans, S. sobrinus, and A. actinomycetemcomitans decreased significantly after training. These bacteria may have been affected by interaction among antimicrobial agents released by the dominant bacteria. It is also possible that the percentages of these bacteria decreased relative to the increments in other dominant bacteria. Regarding the effects of oral health behaviors, an increase in S. sanguinis percentage was observed after discontinuation of toothbrushing or mouthwash rinsing, and toothbrushing frequency was significantly associated with S. sanguinis percentage after training. Taken together, the results indicate that plaque maturation occurred following discontinuation of toothbrushing and induced periodontal inflammation during the training period. Among the salivary components, the concentration of lactoferrin, an ion-binding protein found in secreted fluids including saliva, milk, tears, etc.,33 increased after training. Lactoferrin is released by neutrophil activation, and the molecule has been investigated in studies on inflammatory diseases.34,35 With regard to periodontal disease, increases in lactoferrin concentration in the gingival crevicular fluid (GCF) of gingivitis patients36 and in the saliva of periodontitis patients37 have been reported. The increase in lactoferrin level found in this study seemed to be caused by the inflammatory changes in periodontal tissues during the training period. On the other hand, LL37 and lysozyme concentrations showed no changes after training. LL37 is an antimicrobial peptide and lysozyme is a defense protein, and both antimicrobial agents are present in saliva and GCF.38,39 Correlations between these antimicrobial agents and periodontal diseases have been reported. Surna et al40 demonstrated an increase in lysozyme concentration in GCF, but not in saliva, of gingivitis and periodontitis patients, findings that are compatible with our results. Regarding the change in LL37 concentration in periodontal disease patients, an increase in GCF has been reported,41 but no reports have shown a change in saliva. Not only inflammation but also physical stressors have been reported to induce LL37 and lysozyme.42,43 To clarify the level of stress, we measured salivary α-amylase activity as a stress marker. In numerous previous studies, salivary α-amylase has been an effective stress marker for the autonomic/sympathetic nervous system44; the α-amylase response to a stressor occurs more rapidly than changes in salivary cortisol.45 The participants showed no changes in the α-amylase level after training. The training intensity may have been insufficient to elevate the α-amylase level, or these personnel may have adapted to such a load through periodic training activity. In any case, the stress level induced by this training may have not been sufficient to elevate LL37 and lysozyme. Periodontal deterioration during the training period was associated with dental caries experience, and severe periodontal deterioration was observed in the high-DMFT group. Toothbrushing frequency during the training period decreased more in the high-DMFT than in the low-DMFT group. DMFT, the accumulated number of teeth affected by dental caries, reflects a subject's total caries experience including past and present caries.14 Both dental caries and periodontal disease result from the prolonged presence of pathogenic plaque bacteria, which affect the teeth and periodontal tissues; these can be controlled by mechanical and chemical plaque control regimens. It is thought that personnel in the high-DMFT group did not have proper plaque control habits in childhood and adolescence, and such habits could have resulted in a decline in toothbrushing frequency during the stressful training period. It is also possible that the high-DMFT group may have had an increased plaque Retention Index.22 The results suggest that guidance to maintain continuous toothbrushing is necessary for personnel with a high experience of dental caries. In conclusion, we demonstrated periodontal deterioration in JGSDF personnel after a 7-day training. Behavioral changes, especially discontinuation of regular toothbrushing, fostered dental plaque maturation, resulting in the inflammatory changes seen in participants' periodontal condition and the elevation of salivary lactoferrin. It would be of interest to compare these results with measures of oral health status in other professionals who also work under special circumstances, including police officers and fire fighters. Our results indicate the importance of performing toothbrushing at least once during a 7-day training period to prevent periodontal deterioration. We think that periodontal deterioration would increase during deployments of greater than 7 days because anaerobic bacteria would predominate in the accumulated plaque bacterial community leading to severe periodontal deterioration.46 Therefore, it is necessary to examine the appropriate frequency of toothbrushing for training periods exceeding 7 days. 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TI - Impact of a 7-Day Field Training on Oral Health Condition in Japan Ground Self-Defense Force Personnel
JF - Military Medicine
DO - 10.7205/MILMED-D-16-00383
DA - 2017-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/impact-of-a-7-day-field-training-on-oral-health-condition-in-japan-dfWP4snUJk
SP - e1869
EP - e1877
VL - 182
IS - 7
DP - DeepDyve
ER -