Relationship between Gram negative enteric rods, Aggregatibacter actinomycetemcomitans, and clinical parameters in periodontal disease

Abstract

Background:

The association between Gram negative enteric rods and Aggregatibacter actinomycetemcomitans in periodontal diseases has received little attention in the literature. The objective of this study was to explore the relationship between these organisms and clinical parameters of periodontal disease.

Materials and Methods:

Clinical parameters and occurrence of Gram-negative enteric rods and A. actinomycetemcomitans were examined in 76 patients with chronic periodontitis. Chi-square and Mann-Whitney tests were used to determine differences in clinical variables versus the presence or absence of both microorganisms. Correlation among both organisms and clinical data were determined using Spearman rank correlation coefficient.

Results:

Gram-negative enteric rods and A. actinomycetemcomitans were detected in 20 (26.3%) and 18 (23.7%) individuals, respectively. A total of 14 (18.4%) patients harbored both microorganisms studied. There were significantly positive correlations between enteric rods and presence of A. actinomycetemcomitans (r=0.652, P<0.0001). Both microorganisms were significant and positively correlated with probing depth (PD), clinical attachment level, and bleeding on probing (P<0.0001). The mean PD (mm) of the sampled sites was significantly deeper in patients with presence of A. actinomycetemcomitans and Gram-negative enteric rods.

Conclusion:

The results of the present study suggest a strong positive correlation between Gram-negative enteric rods and A. actinomycetemcomitans in the population studied. This finding must be taken into account when considering the best therapeutic approach, including the utilization of antimicrobials. The adverse clinical outcomes observed in presence of these microorganisms could have implications in the pathogenesis of periodontal disease and a possible impact on outcomes after treatment.

INTRODUCTION

Periodontitis, a chronic inflammatory disease, begins with a microbial infection, followed by a host-mediated destruction of soft tissue caused by hyperactivated or primed leukocytes and the generation of cytokines, eicosanoids, and matrix metalloproteinases that cause clinically significant connective tissue and bone destruction.[1] Aggregatibacter actinomycetemcomitans is implicated in the pathogenesis of periodontitis.[2] On the other hand, the role of Gram-negative enteric rods in the pathogenesis of periodontal disease is unknown, but some investigators have suggested that these organisms may have an impact on progression and treatment of periodontal disease.[3] As pointed out by Slots et al.[3] enteric rods have been shown to persist after periodontal debridement and surgery and have been implicated as key pathogens in cases of refractory periodontitis.[3–7]

These microorganisms were detected at higher frequency and in higher proportions in patients with failing implants.[5] These bacteria are able to produce virulence factors and tissue destruction.[2,8] Examples of virulence factors include enterotoxins and endotoxins produced by Gram negative enteric rods[8] and leukotoxins produced by A. actinomycetemcomitans.[2] Enteric rods have shown the capacity to invade human tissue,[8] while A. actinomycetemcomitans has demonstrated the ability to invade human oral epithelial cells in cell culture.[9] Invasiveness and ubiquitous intraoral distribution may be the main reasons for the reported observation of rather poor results after conventional, merely mechanical treatment of periodontal infections.[10]

To our knowledge, there are no investigations that study the interaction among enteric rods and A. actinomycetemcomitans. Geographical differences in the presence of these microorganisms could impact clinical parameters and periodontal treatment protocols, which may enable the establishment of specific therapeutic strategies. The aim of this study was to investigate the relationships between these organisms and clinical parameters of patients with chronic periodontitis.

MATERIALS AND METHODS

Subjects

Seventy-six systemically healthy Colombian subjects (45 women and 31 men), aged 27 to 66 years (46±8.08 years), who attended the dental clinics of the University of Antioquia were invited to participate in this study between October 2008 and March 2009. Informed and written consent was obtained from each participant. The study design was approved by the Ethical Committee on Human Research of the University Investigation Department of the University of Antioquia, according to the Declaration of Helsinki on experimentation involving human subjects. Patients with a diagnosis of chronic periodontitis were considered candidates for the study. The diagnosis of chronic periodontitis was made based on criteria defined at the workshop sponsored by the American Academy of Periodontology.[11] Exclusion criteria included diabetes, cardiovascular disease, or any other systemic disease that could alter the course of periodontal disease. Pregnancy, recent (six months) consumption of systemic antimicrobials or anti-inflammatory drugs, periodontal therapy during the last six months, and subjects regularly using chlorhexidine mouthwash also served as exclusion criteria.

Clinical evaluation

Medical history and clinical examination were conducted for each patient and a full-mouth periapical X-ray status was performed. One of the authors (CA) performed all clinical examinations. The presence or absence of bleeding on probing (BOP), plaque, and suppuration were registered. The presence of plaque was recorded dichotomously, as the presence or absence of plaque according to detection of plaque deposits determined by running the tip of a periodontal probe along the tooth surface at the gingival margin of each site. BOP was designated as positive if bleeding occurred within 20 s after periodontal probing. Probing depth (PD) and clinical attachment level (CAL) were measured at all approximal, buccal, and lingual surfaces to the nearest millimeter by a calibrated standard probe.^* Location of gingival margin (GM): The distance between the gingival margin and a fixed reference point on the tooth (cement enamel junction (CEJ) or the margin of a restoration). A negative value was given when the gingival margin was located coronal to the CEJ. PD was recorded from the GM to the base of the pocket. CAL was calculated as PD+GM.

Microbial sampling

Microbial sampling on periodontitis patients was performed on pockets ≥5 mm. The deepest six pockets were selected for sampling. After removing supragingival plaque with curets and isolating the area with cotton pellets, the paper points were inserted into each periodontal pocket for 20 seconds. The paper points^§ were transferred to a tube with Viability Medium Göteborg Anaerobically (VMGA) III medium.[12] All samples were labeled properly and processed within four hours after sampling. The samples were analyzed using microbial culture techniques for the presence of periodontopathic bacteria according to slots.[13] Briefly, most samples were processed at room temperature (25°C) and incubated in CO₂ and anaerobic culture systems. Brucella blood agar medium was incubated at 35°C in an anaerobic jar for seven days. The Trypticicase Soy Serum Bacitracin Vancomycin agar medium was incubated in 10% CO₂ at 37°C for four days. Presumptive identification was performed according to the methods described[13,14] and using a commercial identification micromethod system^¶ for A. actinomycetemcomitans. Total viable counts (TVC) were defined as the total number of colony-forming units obtained on non-selective media plates. Species found on selective media were enumerated and their percentage of TVC was calculated.

Isolation of Gram-negative enteric rods by culture: After placement for 20 s, the paper points were pooled into a vial containing 2.0 ml of VMGA III transport medium.[12] The sample vials were maintained at room temperature, transferred to the laboratory, and processed within 4 h after sampling. After the vials were placed in an incubator for 30 min at 37°C, bacterial plaque was mechanically dispersed with a test tube mixer at the maximal setting for 60 s. Serial 10-fold dilutions were prepared in pepton water, and aliquots were plated on MacConkey agar. The plates were incubated aerobically at 37°C for 24 h. Each isolate was characterized according to colonial and cellular morphology and Gram-stain characteristics. Gram-negative enteric rods were speciated using a standardized biochemical test.^# Total viable counts were defined as the total number of colony forming units obtained on non selective media plates. Species found on selective media were enumerated and presented as counts ×10⁵.

Each patient provided a pooled subgingival plaque sample. Equal numbers of isolates were used from each subject.

Statistical analysis

Data were entered into an Excel^** database and were proofed for entry errors. The database was subsequently locked, imported into Statistical Package for Social Sciences (SPSS) for Windows,^†† formatted, and analyzed. Indicators of descriptive statistics were used, such as frequencies, percentage, average, variance, and standard deviation. The presence of A. actinomycetemcomitans and Gram-negative enteric rods-positive individuals were described as the percentage of individuals with at least one infected pocket. The Chi-square test was used to assess differences between BOP versus the presence or absence of A. actinomycetemcomitans and Gram-negative enteric rods. PD and CAL differences and the presence or absence of A. actinomycetemcomitans and Gram-negative enteric rods were determined by the Mann-Whitney test. Association among A. actinomycetemcomitans and Gram-negative enteric rods was expressed through a non-parametric correlation coefficient (Spearman rank). Only sites presenting concomitantly CAL and PD of 4 mm or more at baseline were considered in the analyses of CAL, PD, and BOP. The significance level was set at 0.05 for all tests.

RESULTS

A total of 45 women (59.2%) and 31 men (40.8%) with chronic periodontitis were studied (age: 46±8.08 years, of whom 21.05% [16 subjects]) were current smokers. Table 1 describes the clinical characteristics of the patients.

Table 1.

Clinical data at sampled sites: Probing depth, clinical attachment level, percentage (%) of sites with bleeding on probing, percentage (%) of sites with plaque, percentage (%) of sites with suppuration

Among 76 patients examined, Gram-negative enteric rods, and A. actinomycetemcomitans were detected in 20 (26.3%) and 18 (23.7%) individuals, respectively. A total of 14 (18.4%) patients harbored both microorganisms studied.

Gram-negative enteric rods in periodontal pockets was highly significant and positively correlated with presence of A. actinomycetemcomitans (r=0.652, P<0.0001) and also both organisms were highly significant and positively correlated with PD, CAL, and BOP [Table 2].

Table 2.

Correlations among Gram negative enteric rods and A. actinomycetemcomitans with probing depth, clinical attachment level, percentage of sites with bleeding on probing

Patients with presence or absence of A. actinomycetemcomitans and Gram-negative enteric rods showed significantly different clinical conditions, as assessed by the clinical parameters studied (P<0.001) [Table 3]. The mean PD (mm) of the sampled sites was significantly deeper in patients with presence of A. actinomycetemcomitans and Gram-negative enteric rods, when compared with patients with absence of both microorganisms. Similar results were found for the CAL data. The proportion of sites with BOP was significantly higher in patients with presence of A. actinomycetemcomitans and Gram-negative enteric rods, when compared with patients without these bacteria.

Table 3.

Comparison of clinical data at sampled sites: PD, CAL, percentage of sites with BOP

DISCUSSION

In this study, we investigated the relationships between A. actinomycetemcomitans and Gram-negative enteric rods and clinical parameters from patients with untreated chronic periodontitis. Information from the present study may have therapeutic implications for the treatment of non-oral infections caused by oral pathogens. Dissemination of periodontal pathogens to other body sites frequently occurs and may cause serious diseases.[15,16]

This study identified Gram negative enteric rods in 20 (26.31%) of 76 patients. Our previous paper[17] reported four species of Gram-negative enteric rods in subgingival plaque in 20 (26.31%) of 76 patients: K. pneumoniae occurred in twelve patients, Pseudomonas aeruginosa in four patients, and three other species were recovered with lower prevalence.

Their prevalence in the periodontal pocket varies around the world, ranging from 14% in the United States to 92% in Sudan.[18,19] In Latin America, similar frequencies to those encountered in our study were reported among Brazilians[20,21] and Colombians.[22,24] These bacteria are often recovered from the subgingival microbiota of patients considered to be clinically refractory to mechanical and conventional antibiotic periodontal therapies.[3–8] Additionally, they exhibit less susceptibility to chlorhexidine[25] and in vitro resistance to the majority of adjunctive antibiotics used to treat periodontitis.[7,17,23,26]

In the present investigation, A. actinomycetemcomitans was observed in 18 (23.7%) individuals. Their frequency in chronic periodontitis varied between 15% and 40% in America, Europe, and Australia.[27–29] Our values are similar to the frequencies reported, using culture techniques (26.6%; 26.3%) and polymerase chain reaction (PCR) (23.6%) in South Americans populations.[24,30,31] A. actinomycetemcomitans has ability to survive and colonize periodontal pockets probably related to its large scope of virulence factors that include proteolytic activity, capacity of modulate immune response, evasion of phagocytosis, ability to evade, and damage the immune system and produce periodontal breakdown.[32] Some previously published studies even demonstrated that A. actinomycetemcomitans is a pathogen which is able to invade periodontal tissues, but evade mechanical-chemical therapies.[33,34]

To the best of our knowledge, there are no studies on the association of periodontal Gram-negative enteric rods and A. actinomycetemcomitans and relating these microorganisms with clinical parameters. In this study, a significantly positive correlation between Gram-negative enteric rods and A. actinomycetemcomitans was observed (P<0.0001). Slots et al.[7] found that A. actinomycetemcomitans was isolated from approximately one-fifth of the patients, whose cultures were positive for subgingival Gram negative enteric rods. In this regard, Botero et al.[23] noted that colonies of enteric rods are bigger in size indicating that these organisms could colonize the periodontal pockets in high proportions. On the other hand, PCR detection does not take into consideration whether the sample is viable, and thus may yield to a higher frequency.[23] D’Ercole et al.,[35] recently compared conventional culture methods and multiplex PCR for the detection of periodontopathogenic bacteria and observed that for both methods, there was a good degree of accuracy in the determination of A. actinomycetemcomitans. As with Botero et al.[23] the present study reports on the occurrence of the microorganisms detected based on culture techniques because it allows us later to work with the cultured microorganisms.

Clinical parameters studied were significantly increased in presence of A. actinomycetemcomitans and Gram-negative enteric rods, when compared with patients with absence of both microorganisms [Tables 2 and 3]. This evidence indicated that A. actinomycetemcomitans and Gram-negative enteric rods are closely associated with the process of periodontal breakdown and both microorganisms may be involved in the course of tissue destruction such as pocket deepening or active attachment loss. However, the differences in disease severity (probing PD) and smoking habits among the subjects, detected in the present study, could have had an impact on the microbiological results, because both factors have been shown to have an influence on the subgingival microflora,[22] but their particular impact on the present population could not be assessed.

Enteric rods and A. actinomycetemcomitans share certain aspects that could explain its biological plausibility associated with adverse periodontal parameters. These bacteria produce aggressive virulence factors and tissue destruction.[2,8] Invasiveness and ubiquitous intraoral distribution may be the main reasons for the reported observation of deep pocket.

The present investigation confirms that the presence of Gram negative enteric rods and A. actinomycetemcomitans is related to adverse periodontal conditions. These results could impact periodontal treatment and should be taken into account in the mechanical and antimicrobial treatment of periodontal disease in Latin American populations. The study of the subgingival microbiota in a particular country becomes pertinent to identify its possible impact on outcomes after treatment.[22] A larger investigation would be more appropriate to study correlations between Gram-negative enteric rods and A. actinomycetemcomitans further. Differences in host response, oral hygiene habits, oral health care access, and microbial composition may help explain these differences in the clinical expression of periodontitis in the population studied.[36] More exhaustive investigations addressing the relationship between periodontitis and environmental, economic, and genetic variables are needed in Latin America.[22]