Introduction
Sulphate-reducing bacteria (SRB) are strict anaerobes, with an optimal temperature range of between 25 and 44°C and a pH between 5.5 and 9.0. There are currently over 20 well know generas such as Desulfovibrio, Desulfomonas, Desulfotomaculum, Desulfolobus, Desulfobacter, Desulfococus, Desulfosarcina, among others (1). These fastidious microorganisms can be found in environments such as freshwater and salt marshes or in the human body, mainly in the intestinal microbiota, where the species Desulfovibrio desulfuricans is frequently detected (2, 3).
In a pioneering work, Sefer and Cãlinescu (4),(1969) reported the presence of SRB associated with Streptococcus sp. in seven teeth extracted due to extensive tooth decay. Furthermore they reported the presence of Desulfovibrio sp. in carious dentine scrapings. Only after almost three decades, studies on the presence of SRB in the oral cavity resumed, with a study describing the occurrence of SRB in the buccal mucosa, tongue, saliva, subgingivial and supragingivial biofilms (5-9).
As well as tissue samples from periodontal pockets, Boopathy et al. (6), (2002) identified the presence of SRB in 9 to 17 patients with periodontitis, identifying Desulfovibrio spp. among the samples tested. Other studies indicate a greater number of SRB in subgingivial biofilms when compared to the posterior and anterior tongue, the buccal mucosa, vestibular mucosa and the supragingivial biofilm (7-9).
Several studies indicate a strong association between periodontal status and the presence of SRB in the oral microbiota, with Desulfovibrio fairfieldensis and Desulfovibrio desulfuricans species being reported as causative agents of periodontitis, without a true understanding of their role in bone loss (7, 10-13). Such bacterial species can form a more aggressive complex in periodontal diseases when associated with other periodontal pathogens such as T. denticola, T. forsythus, P. gengivalis and metallogenic bacteria (9-14).
The objective of this study was to detect for the presence of SRB and evaluate the possible association between SRB and cultivable facultative bacteria of oral sites with different periodontal conditions.
Material and Methods
The study was carried out on 9 samples from different oral sites in 8 patients (in the patients number 4, two samples were collected from two different oral sites) (Table 1). Material was collected using a modified Postgate E culture medium, indicated for the growth and isolation of SRB (1), composed of the following (g/Litre of distilled water): KH2PO4 (0.5); NH4Cl (1.0); Na2SO4 (1.0); CaCl2.2H2O (0.67); MgCl2.6H2O (1.68); sodium lactate (7.0); yeast extract (1.0); ascorbic acid (0.1); agar-agar (1.9); NaCl (5.0); rezasurina (4.0 mL); FeSO4.7H2O (0.5). In addition, a reducing solution for anaerobic bacteria was used as a transport solution for facultative bacteria, consisting of the following (g/Litre of distilled water): sodium thioglycolate (0.124); ascorbic acid (0.1); NaCl (5.0); and rezasurina (4.0 ml). The pH of the medium was adjusted to 7.6 with NaOH.
N = no; M = masculine; F = feminine
Identifying SRB and isolating facultative bacteria
Immediately after collection, each sample was placed in separate tubes containing reducing solution for anaerobic bacteria, shaken for 10 seconds and 1.0 ml of this solution was inoculated in modified Postgate E medium.
Subsequently, samples in reducing solution were transferred, in 1.0 ml aliquots, to nutrient broth culture medium, and the striation method using plate count medium was performed. After verifying the isolation of colonies, incubated from 24 to 48 hrs, they were again inoculated in nutrient broth medium for subsequent biomolecular identification of the species present.
Samples inoculated in separate tubes containing modified Postgate E medium were incubated for 28 days at 30°C. The media was macroscopically inspected, observing discoloration due to the formation of black precipitates dispersed throughout the culture medium when positive for the presence of SRB. Then, after 28 days, the media with black color confirmed the presence of SRB. The precipitates were representative of the binding of the iron ion present in modified Postgate E culture medium with the reduction of sulphate by SRB to form iron sulphide (1). Samples negative for the presence of SRB developed a pink or white-transparent coloration, indicating the growth of other anaerobic bacteria that do not reduce sulphate.
Biomolecular identification of facultative bacteria
The isolation of facultative bacteria was possible only for samples from patients numbers 5, 7 and 8, where the protocol was carried out to identify these microorganisms. The MOBio UltraClean Microbial DNA® kit was used to extract DNA from the sample. The extracted DNA was visualized by electrophoresis of a 1% agarose gel stained by SYBR Safe®. After the extraction step, the 16S RNA gene (1500bp) was amplified by PCR using the universal primers for bacteria: SAdir (5’-AGAGTTTGATCATGGCTCAGA-3’) and S17rev (5’-GTTACCTTGTTACGACTT-3’). Amplification was performed in a GeneAmp PCR System 9700® thermocycler (Applied Biosystems) and included an inital denaturation cycle (94°C for 5 minutes); 30 intermediate denaturation cycles (94°C for 30 seconds), annealment (55°C for 30 seconds) and an extension cycle (72°C for 30 seconds) and one final extension cycle (72°C for 5 minutes).
All PCR products were analysed by agarose gel (1%) electrophoresis, stained by SYBR Safe and then purified with the UltraClean® PCR Clean-up® kit (MOBio). After this step, the quantity and purity of the PCR products was determined by optical density spectrophotometry (NanoDrop® ND-1000 UV-Vis - Thermo Scientific).
Sequencing of the PCR products was carried out by the Sector of DNA Sequencing, in a MEGABACE 1000 automatic sequencer. The resulting sequence electropherograms were analysed by the program Chromas Lite, version 2.01 (McCarthy, 1996, www.tecnelysium.com.au). The DNA sequences obtained were compared to those already in Genbank (www.ncbi.nlm.nih.gov).
The search for sequence similarity of the 16S ribosomal RNA gene of the isolated samples was carried out using BLAST (Basic Alignment Search Tool, http://genome.eerie.fr/bin/blast-guess.cgi.) revealing the bacterial species isolated.
Results
Of the eight patients included in this study, only three were positive for the presence of SRB (Table 2). In the first patient (number 2), the sample was isolated from a root fragment; in the second (number 4), from a root fragment and a healthy tooth with vertical bone loss and with a mobility degree of 3; and in the third (number 8), a healthy tooth extracted for orthodontic purposes. In the final patient sample, Lactobacillus casei, a cultivable facultative bacterial species was identified. Other facultative bacterial species were identified in patient 5 (Kurthia Gibsonii) and patient 7 (Pseudomonas aeruginosa) (Table 2).
(-) Negative; (+) Positive; FAB = Facultative Anaerobic Bacteria
Discussion
Bisson-Boutelliez et al. (11), (2010) reported that the invasive capacity of Desulfovibrio spp. in oral epithelial cells induced the secretion of cytokines, IL-6 and IL-8 by these cells. According to the author, such evidence contributes to the initialization and perpetuation of periodontal disease. In addition, the author also related the accumulation of sulphate, as a final metabolic product of SRB in periodontal pockets, as responsible for causing cellular damage to cells (7). This is demonstrated by the isolation of sulphate-reducing bacteria in 10 subgingivial plaque samples from various patients with periodontitis and no less than one periodontal pocket in 64% of patients with periodontitis (7, 8).
In the current study, of the nine samples, four were positive for the presence of SRB (66.67%), indicating that this bacterial group may be a part of the normal oral microbiota while also potentially being associated with periodontal disease. This was observed in one patient where SRB was found in two distinct sites, tooth 35 and 31. However the sample from tooth 31 had severe periodontal destruction, with vertical bone loss, presenting the possibility that SRB has a role in the pathogenesis of periodontal disease. The same was similar for two other samples analyzed from root fragments (patients numbers 2 and 4), where clinically these teeth presented different degrees of periodontal involvement. These findings are in accordance with Langendijk et al. (7), (2000), who reported a higher prevalence of SRB in patients with periodontal pockets and bleeding upon probing and in patients with various angular bone defects, furcation and endodontic complications, observing a direct relationship between the pocket depth and the presence of SRB. Willis et al. (5), (1999) identified a greater prevalence of SRB in saliva when compared with sub- and supragingivial biofilms, the anterior and posterior tongue and the oral mucosa.
In sites without direct contact with the oral environment, SRB was not isolated from the endodontic lesion biopsy or the root canal paper cone, which highlights the need for oral microbiota exposure for potential colonisation by SRB.
One of the important characteristics of SRB is its ability to associate with different types of surfaces where it constructs a cooperative consortium with other types of bacteria to form a biofilm (15, 16). It may be suggested that once present in the oral microbiota, different oral sites can be colonized by SRB, as demonstrated by patient 4, with the colonization of SRB in distant sites (tooth 35 and 31).
The Lactobacillus spp., which is strongly associated with dental caries is present in the oral microbiota, dental biofilm and the dentine surface and changes the level of lactic acid in these microenvironments (17), while the species Lactobacillus casei can be found in supragingivial interproximal plaque (17) and dental biofilms (18). The reduction of pH in the microenvironment caused by Lactobacillus spp. results in the metabolic inhibition and increase of bacteria present in the oral microbiota of this microenvironment (19). However, the current study identified SRB and Lactobacillus casei in the same sample, which may indicate a possibility symbiotic relationship between these microorganisms. In association with aerobic bacteria, SRB benefits the formation of an anaerobic microenvironment in the biofilm caused by the metabolism of the aerobic bacteria present, where oxygen consumption will ensure a reduction in the necessary conditions for the growth of SRB, leading to maximum metabolic activity (16). Lactobacillus casei is a facultative bacteria that produces lactic acid (18) which can be used by the SRB as a source of organic carbon lactate (1), which is a salt of lactic acid. Langendijk et al. (8), (2001) reported the oxidation of lactate in the presence of sulphate in ten SRB strains isolated from the oral microbiota.
Many intestinal bacteria produce lactate which stimulates the production of sulphate by the SRB present in the colon (20). Newton et al. (21), (1998) evaluated the effect of Desulfovibrio desulfuricans on intestinal bacterial populations in continuous cultures, among these, Lactobacillus acidophilus. This study noted a great reduction of lactate, which is an important electron donor for Desulfovibrio sp., as well as a significant reduction in the population of Lactobacillus acidophilus species.
The findings here need to be considered with caution because the small number of samples. In this way, it could not be able to establish associations of SRB with other cultivable bacteria. Therefore, it is necessary to increase the number of samples for future effective conclusions.
In the current study it was possible to demonstrate a higher prevalence of SRB in saliva samples from patients with gastritis and periodontal diseases (22). The methodology applied was more selective in the detection of the site of action of SRB. The presence of SRB in different dental tissues with distinct periodontal features demonstrates that new studies need to be developed in order to determine the true role of SRB in the oral microbiota. In addition, it was possible to verify the presence of Lactobacillus casei together with SRB in one sample.
