ORIGINAL ARTICLE

Subgingival microbiological profile of periodontitis patients in Dominican Republic

Perfil microbiológico subgingival de pacientes con periodontitis en República Dominicana

 

James R. Collins¹, Sofía Chinea³, Rosanna J. Cuello¹, Alba P. Florian¹, Patricia Palma², Nagore Ambrosio³, María J. Marín³, Elena Figuero³, David Herrera³

¹ Pontificia Universidad Católica Madre y Maestra (PUCMM), Escuela de Odontología, Departamento de Periodoncia, Santo Domingo, República Dominicana,
² Universidad de Chile, Escuela de Odontología, Departamento de Patología y Medicina Oral, Santiago, Chile,
³ Universidad Complutense de Madrid, Facultad de Odontología, Grupo de investigación ETEP (Etiología y Terapéutica de las Enfermedades Periodontales), Departmento de Especialidades Clínicas Odontológicas, Madrid, Spain,

Received: February 2019; Accepted: March 2019.

 

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ABSTRACT

Several studies have tried to associate the presence of different pathogens with the onset and progression ofperiodontitis, reporting a wide variety of results from different populations and environments. The aim of this study was to determine the main periodontal pathogens present in the subgingival biofilm of Dominican patients with periodontitis, by using specific microbiological culturing techniques. Periodontitis patients were selected after a full-mouth periodontal evaluation, and assigned to different periodontitis groups based on percentage of affected locations. Subgingival samples were collected and analyzed by means of specific culture techniques. Anaerobic counts, frequency of detection and proportions of target pathogens were calculated. Variables were analyzed by means of Student's T-test or chi-square test. Twenty-nine subjects were recruited, of whom 17 were diagnosed with generalized periodontitis (GenP) and 12 with localized periodontitis (LocP). The most prevalent bacterial species in both groups was Prevotella intermedia (94.1% in GenP and 91.7% in LocP), followed by Porphyromonas gingivalis (88.2% in GenP and 83.3% in LocP). Total microbiota in subgingival samples was 1.3 x10⁷ colony-forming units (CFU)/mL (standard deviation, SD=1.5 x10⁷) and 9.6x10^s CFU/mL (SD=1.1 x10⁷) in GenP and LocP subjects, respectively, though differences were not statistically significant (p=0.222). The highest counts were observed for P gingivalis in both groups, with mean concentration 2.5x10^s CFU/mL (6.1x10^s) in GenP and 2.9x10^s CFU/mL (5x10^s) in LocP, with no statistically significant difference (p=0.879). These results suggest that relevant periodontal pathogens are found with diversity and abundance in the subgingival microbiota of adult Dominican patients with periodontitis.

Key words: Microbiology; Culture techniques; Dominican Republic; Reriodontitis.

RESUMEN

Varios estudios han tratado de asociar la presencia de diferentes patógenos con el inicio y la progresión de la periodontitis, mostrando una gran variedad de resultados en diferentes poblaciones y entornos. El objetivo del presente estudio fue determinar los principales patógenos periodontales presentes en la biopelícula subgingival de pacientes dominicanos con periodontitis, utilizando técnicas específicas de cultivo microbiológico. Los pacientes con periodontitis se seleccionaron después de una evaluación periodontal de boca completa y se asignaron a diferentes grupos de periodontitis según el porcentaje de localizaciones afectadas. Las muestras subgingivales fueron recolectadas y analizadas mediante técnicas de cultivo específicas. Se calcularon los recuentos anaeróbicos, la frecuencia de detección y las proporciones de los patógenos seleccionados. Las variables se analizaron mediante la prueba T de Student o la prueba de chi-cuadrado. Se reclutaron veintinueve sujetos, 17 diagnosticados como periodontitis generalizada (GenP) 12 con periodontitis localizada (LocP). La especie bacteriana más prevalente en ambos grupos fue Prevotella intermedia (94.1% y 91.7%, respectivamente) y seguida de Porphyromonas gingivalis (88.2% y 83.3%, respectivamente). La microbiota total en muestras subgingivales fue 1.3 x10⁷ unidades formadoras de colonias (CFU)/mL (desviación estándar, SD=1.5 x10⁷) y 9.6x10⁶ CFU / mL (SD=1.1 x10⁷) en sujetos GenP y LocP, respectivamente, pero no hubo diferencias estadísticamente significativas (p=0.222). Los recuentos más altos se observaron para P. gingivalis en ambos grupos, con una concentración media de 2.5x10⁶ CFU/mL (6.1x10⁶) en GenP y 2.9x10⁶ CFU/mL (5x10⁶) en LocP, sin diferencias estadísticamente significativas (p=0.879). Estos resultados sugieren que se encuentran patógenos periodontales relevantes con diversidad y abundancia en la microbiota subgingival de pacientes adultos dominicanos con periodontitis.

Palabras clave: Microbiología; Técnicas de cultivo; República Dominicana; Periodontitis.

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INTRODUCTION

Periodontal diseases are a group of diseases of infectious origin that occur with the inflammation of the tissues supporting the teeth. They are currently highly prevalent worldwide¹², and represent one of the main factors leading to tooth loss^3-6.

The primary etiological factor of periodontitis is the presence of bacteria organized in biofilms, which develop as interactive communities of microorganisms. The relationships between the bacteria embedded in a biofilm can be symbiotic, when there is a beneficial relationship among the bacteria that make up the biofilm and between them and the host, or dysbiotic, when there is a change in the community of microorganisms that leads to the development of pathology⁷.

Several studies, mainly based on microbiological culturing for analysis of the samples, have tried to associate the presence of different pathogens with the diagnosis of periodontitis, reporting a wide variety of results from different populations and environments^8-11. One of these studies by Sanz et al. (2000)¹¹ compared patients with periodontitis in Spain and the Netherlands, finding significant differences between the microbiological profiles. Aggregatibacter actinomycetemcomitans was found to be more prevalent in Dutch than in Spanish patients (23% versus 3%), whereas Porphyromonas gingivalis was found to be more prevalent in Spanish than in Dutch patients (65% versus 37%). These differences may be explained by patients' genetic profiles or as a result of the difference in the use of antibiotics between these two countries¹². In Latin America, the "red complex" of bacteria (P gingivalis, Tannerella forsythia and Treponema denticola) is found in high levels in patients with periodontitis ^13,14,9,10. In Brazil, black-pigmented bacteria of the species Porphyromonas was detected in patients with periodontitis (89.4%), gingivitis (30%) and healthy patients (8%)¹³. In another study of the Brazilian population, Porphyromonas has prevalence of 74% in patients with periodontitis¹⁴.

In the Dominican Republic to date, only two studies have been conducted analyzing subgingival microflora of Dominican patients with periodontitis, and they report different results. Slots et al.¹⁵ performed the first study in which direct microscopic examination revealed that nonmotile organisms and cocci made up 85% of total microorganisms, while spirochetes only accounted for 3%. Non-selective culturing showed 53% Gram-negative organisms, 15% Fusobacterium nucleatum, 7% black-pigmented anaerobes and 10% Parvimonas micra. In contrast, a recent study using polymerase chain reaction (PCR) found prevalence of "red complex" bacteria, with approximately 90%, in patients with periodontitis, especially T. forsythia, differing from reports published for other Latin American countries¹⁶.

The primary aim of the current study was to determine the main periodontal pathogens present in the subgingival biofilm of patients with periodontitis in the Dominican Republic, by using specific microbiological culturing techniques. The secondary aim was to compare these pathogens between patients with generalized and localized periodontitis.

MATERIALS AND METHODS Study design

This cross-sectional study was approved by the institutional ethical committee of Pontificia Universidad Católica Madre y Maestra (PUCMM). All participants signed written informed consent. Study procedures were conducted according to the Declaration of Helsinki, the UNESCO Universal Declaration and the requirements of the Dominican Republic legislation.

Participants

Patients at the PUCMM dental clinic (Santo Domingo, Dominican Republic) who met the inclusion criteria were invited to participate in the study. All subjects provided informed consent. The screening period lasted from January 2014 to August 2015.

Inclusion criteria were: (1) age 18 years or older; (2) non-smokers; (3) at least 15 teeth present; (4) had not received periodontal treatment in the 12 months prior to the study; (5) were free of systemic diseases that could affect the tissue response (such as diabetes or immune diseases); (6) presented periodontitis, defined as the presence of at least 3 interproximal non-adjacent sites with probing pocket depth (PPD) of 4 mm or greater and (7) radiographic evidence of alveolar bone loss. Exclusion criteria were: (1) pregnant women and (2) having taken antibiotics and/or anti-inflammatory drugs in the previous month.

Clinical outcomes

A full-mouth clinical examination was performed on each patient and the following parameters were recorded at six sites per tooth using a North

Carolina periodontal probe (Hu-Friedy, Chicago, IL, USA): (1) Plaque index (PlI) in percentage of sites, following O'Leary¹⁷; PPD; recession (REC) and clinical attachment loss (CAL) in millimeters; and bleeding on probing (BOP) as present/absent 30 seconds after probing, following Ainamo & Bay¹⁸. Based on their periodontal information (proportions of affected sites), patients were assigned to the generalized or localized periodontitis groups.

Microbiological sampling

In each patient, four sampling sites were selected, one in each quadrant, choosing those most accessible and with deepest probing depth and bleeding on probing¹⁹. At the selected sites, supragingival plaque was removed and the sites were dried with sterile cotton roles and air. Two consecutive sterile paper points (#30, Maillefer, Ballaigues, Switzerland) were inserted as deep as possible into the pocket, and left in place for 10 seconds. The paper points were transferred to a vial containing 2 ml of reduced transport fluid²⁰, and pooled with all the other paper points. The vial was kept at 4 °C and sent to the laboratory at the Complutense University of Madrid (Spain) and processed within 24-36 hours.

Culture analyses

Vials were vortexed (30 seconds), serially diluted in phosphate-buffered saline, and plated on two different media: (1) blood agar medium (Blood Agar Base II®, Oxoid, Basingstoke, United Kingdom) supplemented with 5% horse blood, haemin (5 mg/l) and menadione (1 mg/l), and (2) Dentaid-1 medium²¹. After 4-14 days of anaerobic incubation (80% N₂, 10% CO₂ and 10% H₂), the plates were examined for the identification of A. actinomycetemcomitans, P gingivalis, P intermedia, T. forsythia, P micra, Campylobacter rectus, F. nucleatum, Capnocytophaga spp. and Eikenella corrodens, based on different microbiological procedures: colony morphology, Gram-staining, catalase test, N-benzoyl-dL-arginine-2-napthylamide, indole and alpha -glucosidase activity; and standard biochemical test (RapIDTM ANA II System; Remel, Lenexa, KS, USA). Bacterial counts were expressed in colony-forming units (CFU) per mL of the original sample and total anaerobic counts were calculated, as well as counts of the detected periodontal pathogens. In addition to the quantitative microbiological data, the frequency of detection and proportions for each bacterial species were calculated.

Statistical analysis

Sample size calculation could not be performed. A convenience sample of 29 subjects was therefore selected based on previous microbiological studies^8-11. The primary outcome variable was total anaerobic count (CFU/mL). Secondary outcome variables included all other microbiological variables, including frequency of detection of target pathogens, counts of each study pathogen, proportions of flora of each pathogen, and all clinical variables (PPD, BOP, REC, PlI, CAL). A subject-level analysis was performed for each study parameter. Data were expressed as means and standard deviation (SD), prevalence and proportions (%) for all variables. Total anaerobic counts and counts of each study pathogen were log transformed to fit a normal distribution. After evaluating the normality of the distribution (assessed by the Shapiro Wilk test), differences between different periodontal diagnosis groups were compared by Student's T or MannWhitney U-test for quantitative variables, and chi-square or Fisher tests for categorical variables. The level of statistical significance was set at p<0.05. A statistical software package IBM® SPSS Statistics 25.0 (IBM Corporation, Armonk, NY, USA) was used for data analysis.

RESULTS

Patient sample description

Twenty-nine patients were recruited, of whom 17 were diagnosed with generalized periodontitis (GenP) and 12 with localized periodontitis (LocP). Table 1 shows patient demographics. No statistically significant difference was found between groups. Mean age was 42.3 and 46.2 years, and percentage of males was 29.4% and 58.4% in the GenP and LocP groups, respectively.

Table 1: Demographic data for GenP and LocP periodontitis groups.

Periodontal outcome variables

Table 2 shows clinical outcome variables. PPD and BOP values were significantly higher in GenP patients than in LocP patients (p<0.05). Mean PPD was 2.9 mm (SD=0.6) in the GenP versus 2.1 mm (SD=0.2) in LocP subjects. Similarly, BOP percentages were significantly higher in the GenP group (60.7% [SD=26.4%] versus 33.0% [SD=17.1%]). Table 3 shows the frequency distribution of PPD.

Table 2: Mean values and standard deviation (SD) for clinical variables.

Table 3: Frequency distribution of probing depth in both groups.

Table 4 shows data from the sampling locations. Plaque was 100%, BOP 85.7% and 62.5%, and mean PPD 5.8 and 5.0 mm in GenP and LocP, respectively. Statistically significant differences were found for PD values between groups (p=0.037).

Table 4: Mean values and standard deviation (SD) of clinical variables at sites selected for microbial sampling.

Subgingival samples

Table 5 shows data on detection of pathogens from subgingival samples, including their frequency of detection, mean concentrations and proportions. The most prevalent bacterial species in GenP subjects were P intermedia (94.1%) and F. nucleatum (94.1%) followed by P gingivalis (88.2%). In LocP patients P. intermedia (91.7%) and P. gingivalis (83.3%) were the most prevalent bacteria, followed by F. nucleatum (58.3%). Statistically significant differences were only found for F. nucleatum (p=0.019).

Table 5: Frequency of detection (%), mean counts (in colony forming units, CFU/mL) and mean proportions of microbiota (%) of periodontal pathogens in subgingival samples.

Total microbiota counts in subgingival samples were 1.3 x10⁷ CFU/mL (SD=1.5 x10⁷) and 9.6x10⁶ CFU/mL (SD=1.1 x10⁷) in GenP and LocP subjects, respectively, but differences were not statistically significant (p=0.222). The highest counts were observed for P. gingivalis in both groups, with a mean concentration of 2.5x10⁶ CFU/mL (6.1x10⁶) in GenP groups and 2.9x10⁶ CFU/mL (5x10⁶) in LocP, followed by P. intermedia [with mean concentration 7.2x10⁵ CFU/mL (1.4x10⁶) in GenP groups and 2.7x10⁵ CFU/mL (4.1x10⁵) in LocP] and T. forsythia [with mean concentration 4.5x10⁵ CFU/ mL (8.4x10⁵) in GenP groups and 3.1x10⁵ CFU/mL (5.9x10⁵) in LocP]. Statistically significant differences were not found between groups for any individual bacteria counts. In terms of proportions, similar mean values were found between groups without statistically significant differences between them.

DISCUSSION

In the present study, subgingival samples from 29 Dominican patients with periodontitis were analyzed using microbiological culturing in specific non-selective and selective media. The bacterial species with highest frequency of detection were P intermedia, P gingivalis and F. nucleatum (93.1%, 86.2% and 79.3%, respectively). No difference was detected when localized and generalized periodontitis patients were compared. There are few previous studies on the Dominican population using cultures. The results reported by Slots et al.¹⁵ are consistent with ours (P gingivalis, F. nucleatum and P. intermedia as the most prevalent species), but the frequencies of detection are different, almost certainly attributable to the use of different transport media [reduced transport fluid (RTF) or Viability

Medium, Göteborg, Anaerobically prepared (VMGA) III]. Collins et al.¹⁶ studied gingivitis, periodontitis and healthy participants from the Dominican Republic using specific PCR, finding frequencies of 93.3% for P. gingivalis, 53.3% for P. intermedia and proportions greater than 80% for other species such as F. nucleatum, P micra and E. corrodens. They detected these three species in a greater number of patients compared to the current study, possibly due to the lower detection limits of molecular techniques compared to culture.

In the general population, the reported prevalence (86.2%) of P. gingivalis agrees with the current evidence, with this species being one of the most frequently detected species in subgingival samples in patients with periodontitis. In Spain, this bacterial species was found in 64.5%¹¹ and 77.8%¹⁰ of the population. In Chile and Colombia, its prevalence has been reported as 83.8% and 65.9%, respectively. All these studies used similar methodologies and the populations analyzed had similar clinical features. In all cases, the samples were processed between 24 and 48 hours after being taken.

In our study, P intermedia, a Gram-negative bacillus, was also found in a high percentage of cases, having been recovered in the cultures from 93.1% of the subjects. These findings are similar to those from Spain and Chile, where the occurrence of P. intermedia was 97.2% and 72.5%, respectively. However, in a Colombian population the frequency was lower, at 19.4%¹⁰. These differences could be attributed to the different lifestyles of people living in each geographical location or to the clinical presentations of periodontitis²²²³. On the other hand, differences could also be explained, not only by genetic differences²⁴ , but also by epigenetic differences, which could be related to variations in the abundance and distribution of microbial species in populations²⁵. It should be highlighted that T. forsythia was detected in more than 50% of the patients in the present study, but in 90% of the subjects in the PCR-based study¹⁶. The greater frequency of detection using molecular methodologies evidences the greater sensitivity of the PCR technique^26-28, though it should also be considered that detection and abundance of this anaerobic species, which is difficult to culture, could have been underestimated due to the time elapsed between sampling and processing. However, although T. forsythia may be partly underestimated, its detection by culture highlights its high frequency in Dominican patients compared to other populations, with reported frequencies of 16.2%, 39.0% and 36.1%, in Chile, Colombia and Spain, respectively.

A. actinomycetemcomitans has been associated by various authors, such as Jardim et al.²⁹ or Sulugodu et al.³⁰, with a higher rate of progression in periodontitis. In the present study, its occurrence was 11.8% and 8.3% for GenP and LocP, respectively, which was low compared to other studies, such as Mullally et al.³¹, which reported a detection frequency of 19%, and higher in patients with LocP.

It should be considered that results for bacterial abundance may be a direct consequence of the sampling and processing protocol. The volume of transport medium in which the sample was placed, and from which dilutions are subsequently prepared for inoculation into the culture media, as well as the number of sites from which the sample was collected, will reflect the total counts of viable cells present in these samples. Both these factors may make comparisons between studies difficult. However, in the present study, the most abundant species in relative terms were P gingivalis and P intermedia, followed by F. nucleatum and T.

forsythia, similarly to Slots et al.¹⁵, with the exception of T. forsythia, which was not evaluated. The advantages of detecting bacteria by means of culture techniques are that it enables determination of number of viable cells in a sample, detection of species that could be present unusually, analysis of susceptibility to antimicrobials and characterization of the microbiota associated with oral diseases³². Microbiological culturing techniques are fundamental and basic diagnostic methods widely used as research tools in molecular biology. In addition, these methods are still considered the "gold standard" methods of reference in periodontal microbiology, to which other microbiological identification procedures are compared³³. However, culturing can be affected by the handling and processing of the sample, especially in the current study, in which sampling and analysis were performed in different countries. These aspects, along with the relatively small sample size, should be considered as potential limitations of the study. In the present study, periodontal diagnosis was initially based on the 1999 classification of periodontal diseases³⁴. In the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions, the terms "aggressive" and "chronic" have disappeared, and the condition "periodontitis" should be now categorized with a multidimensional system of stages (I, II, III, IV) and grades (A, B, C). The stages will capture the severity of the disease and the anticipated complexity of the therapy, while the grades consider the identification of risk factors that may impact general health and the progression of the disease^35-37. This new approach may allow the development of meticulous treatment strategies according to the specific needs of each patient (precision medicine). With regard to the extension and distribution of periodontitis, minor differences are expected with the new classification. To determine a case of LocP or GenP, the presence of <30% or >30%, respectively, of affected sites was used in the 1999 classification³⁴, while with the 2018 approach, LocP is described as <30% of the teeth involved and >30% for GenP.

The strength of this study is that samples were analyzed in an experienced laboratory, which has already processed samples from distinct geographical populations (Spain, The Netherlands, Chile, Colombia) in different international research projects. This enables comparison of the results of the present study (and population) to previous studies which followed identical methodology and were analyzed in the same laboratory^10,11. It was possible to detect and identify nine bacterial species that are part of the subgingival microbiota and that are closely associated with the dysbiotic phenomena that trigger periodontal diseases. Therefore, this study constitutes an approximation of the bacterial composition in both diversity and abundance of the subgingival microbiota of patients with periodontitis in the Dominican Republic.

CONCLUSION

Within the limitations of the present study, the subgingival microbial composition in patients with periodontitis from Dominican Republic showed a high frequency of detection of P. intermedia, P. gingivalis and F. nucleatum. Our data suggest that periodontal pathogens in the subgingival microbiota of adult Dominican patients with periodontitis have overall similar diversity and abundance to those in other geographical populations, but with higher frequency of detection of T forsythia.

CORRESPONDENCE

Dra. Elena Figuero. Departamento de Especialidades Clínicas Odontológicas Facultad de Odontología. Universidad Complutense de Madrid Plaza Ramón y Cajal s/n (Ciudad Universitaria). 28040 Madrid, España
elfiguer@ucm.es

FUNDING

None

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