INTRODUCTION

The oral cavity is an easily accessible site from which to collect biological material for studying and diagnosing systemic and oral diseases, and analyz-ing of microbial communities ¹ . Microbial counts in specimens of saliva and dental plaque have provided information for estimating caries risk in adults and children, showing a positive correlation between caries experience and cariogenic streptococci (CS) counts ² . The methods proposed for counting bacteria include culture in selective media, biochemical tests, immunological and genetic tests with DNA probes, enzyme-linked immunosorbent assay, and polymerase chain reaction (PCR) ³ . Culture in selective media is a useful tool for morphotyping, count-ing colony-forming units (CFU/ml) and obtaining bacterial strains for molecular processes to enable genomic studies. Media such as Mitis Salivarius Agar (MSB) and Tryptic Soy Agar (TSY20B) are often used in studies to demonstrate the correlation between cariogenic streptococci counts and caries lesions ⁴ . Gliosca et al. ⁵ proposed another technique for counting bacteria by means of an adhesion test (AA-MSMG) using the selective culture medium modified Gold’s broth (MSMG-20% sucrose) to evaluate cariogenic risk. This method has satisfac-tory predictive value and is a valid instrument to categorize patient risk. In addition, molecular tech-niques such as real-time polymerase chain reaction (qPCR), due to their high sensitivity, specificity and speed, are also effective for detecting and quantify-ing bacterial species ⁶ .

Regardless of the microbiological analysis methods, saliva sampling needs to be performed using simple, affordable, noninvasive techniques, which are stan-dardized, valid and reproducible.

Some salivary components vary according to the saliva collection method. Therefore, before beginning with a study, a technique must be selected to opti-mize sample collection according to the study ob-jective and/or the biomarker to be analyzed ⁷ , taking into account the type of saliva to be collected (stim-ulated or unstimulated) and the context in which the study is to be performed. Results will thus be comparable to those of other studies.

Saliva samples can be obtained using absorbent or non-absorbent methods. Absorbent methods include collecting samples using different materials such as paper strips, cotton rolls and polystyrene and poly-ethylene swabs. Non-absorbent methods include collecting biological material by spitting or passive drooling into sterile collection containers, and sampling by aspiration with devices such as plastic sy-ringes, among others ^{8, 9} .

There are some studies evaluating the efficiency of saliva collection methods for studying different biomarkers ^{10, 11} ; however, they have not been vali-dated for detecting and identifying microorganisms in children.

The aim of this study is to compare the efficacy of two methods for collecting saliva samples from in-fants under two years old for cariogenic streptococ-ci (CS) count. Microorganisms were counted using two differential selective culture mediums and by molecular detection.

MATERIALS AND METHODS

This study was conducted on 11 infants aged 6 to 28 months who attended an early childhood center in Buenos Aires City. This was part of the project “Horizontal transmission and early colonization of mutans group Streptococci in infants who attend mother-and child educational centers”, approved by the Ethics Committee at the Buenos Aires Uni-versity School of Dentistry (CUDAP: EXP-UBA: 0072332/201 7 N° 012/2018 C.ÉTICA FOUBA). Saliva specimens were collected from each par-ticipant using two different methods: an absorbent method (A), and a non-absorbent method (B). The methods were applied in alternative order on different infants to avoid the possibility of the second method collecting a smaller specimen due to the child being more tired or less cooperative.

In Method (A), saliva samples were collected by swabbing the inner cheek mucosa and floor of the mouth in figure of eight motions with a sterile cotton swab until it was soaked. Then the swab was un-loaded in situ by plating on Petri dishes containing TYSCB (Tryptone Yeast Extract Cystine Sucrose and Bacitracin) culture medium, and then in Ep-pendorf-type tubes containing phosphate-buffered solution (PBS), for transfer. In method (B), saliva samples were collected by aspiration of 1 ml of saliva with a sterile plastic syringe on the floor of the mouth, after stimulation with glove. The content of the plastic syringe was unloaded in Eppendorf-type tubes. The samples were taken to the Microbiological Diagnosis Laboratory at Buenos Aires Universi-ty’s School of Dentistry within 2 hours of sampling.

The saliva samples were vortexed and seeded in two differential selective culture mediums for colo-ny-forming unit count (CFU/ml).

The samples taken using method A were plated in TYSCB (in situ), and 100ul of the elute in PBS was seeded in modified Gold’s broth (MSMG) ⁵ . Equiv-alent aliquots of the samples taken using method B were seeded in TYSCB and MSMG.

Cultures were incubated under anaerobiosis (GasPack - Mitsubishi®) for 48 hours at 36 °C ± 1 °C. After incubation, calibrated personnel (Kappa >0.75) ob-served the cultures under stereoscopic microscope (“Arcano” ST30-L binocular stereo microscope) at 50X magnification. Counts were performed consider-ing the morphological characteristics of the colonies described for cariogenic streptococci under the study conditions. On TYSCB, counts were performed up to a maximum 300 characteristic colonies per plate, and values higher than this were not recorded. Adhered colonies applying the AA-MSMG were counted in 3 areas of 1cm ² , using a grid covering the entire contact the area of the culture bottle.

Molecular processing to determine cariogenic streptococci (Streptococcus mutans - Streptococcus so-brinus) was performed using species-specific prim-ers for real-time polymerase chain reaction (qPCR) method. The genomic material from the samples from methods A and B was obtained by following the instructions provided by the manufacturer of the commercial kit (Presto™ Mini gDNA Bacteria Kit, Geneaid). DNA integrity was quantified and evalu-ated by spectrometry (CYTATION 3 Cell Imaging reader, Biotek). The DNA samples employed had values between 1.7 and 2.0 (ratio 260/280 nm) and their concentrations were normalized at 20 ng/mi-croliter.

Detection and quantification of Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. so-brinus) were performed by qPCR in a CFX96TM Real Time System thermocycler (Bio-Rad Laboratories, Inc.). Species-specific primers were employed, using as a target the gene encoding the following glycotransferase enzymes: gtfB, for S. mutans and gtfT S. sobrinus ¹² . The reactions were performed in triplicate, using Sso Advanced Universal SYBR Green Supermix (Bio-Rad Laboratories, Inc.) in a final volume of 10 microliters.

Statistical processing consisted of calculating the mean rank for each collection method and each culture medium, and comparing the results by two-way analysis of variance by ranks (Friedman’s test). For the molecular technique, the percentages of positive results for S. mutans and S. sobrinus for each collec-tion method were calculated, and differences were analyzed using McNemar’s test.

RESULTS

Seven females and four males took part in this test. Average age was 14.9 months (SD±4.67).

Mean rank for the CFU/ml count for AA-MSMG was 1.83 for method A, and 1.17 for method B (Fig. 1), with statistically significant differences (p=0.021).

Mean rank for the count on cultures on selective TYSCB medium was 1.54 for method A, and 1.46 for method B, without statistically significant dif-ference (p=0.705) (Table 1), though recovery was greater in method A (Fig. 2).

Fig.1 Mean rank of CFU/ml according to a sampling method. AA-MSMG: Culture on Gold Broth modified with 20% of sucrose (A): absorbent method, (B): non-absorbent method. 

Table 1 Cariogenic streptococcus count in TYSCB for collection methods A and B 

Fig.2 Culture on Tryptone Yeast Extract Cystine Sucrose and Bacitracin (TYSCB) and Gold Broth modified with 20% sucrose (AA-MSMG). Method A = absorbent. Method B = non-absorbent. 

With qPCR, positive results for S. sobrinus and S. mutans were 36.4% and 45.5%, respectively, for method A; and 75% and 41.7%, respectively, for method B. For qPCR, no significant difference was found between sampling methods (S. sobrinus: p=0.21; S. mutans: p=0.941) (Table 2).

Table 2 Detection of cariogenic streptococcus by qPCR using collection methods A and B 

DISCUSSION

Microbiological studies on saliva specimens are needed to establish cariogenic risk level and identi-fy microorganisms that may be associated with the dental caries process. However, taking samples from infants may involve difficulties related to children’s cooperation or unfavorable perception of sampling methods by aspiration with syringes.

Our study compared a suction sampling method and an absorbent sampling method, finding that they provided similar results for colony counts and ge-netic material. The absorbent method enabled great-er detection of CFU/ml in the AA-MSMG culture technique.

The absence of significant differences between methods when the count was performed on TYSCB may be explained by the fact that the count param-eters established for this method did not assign an exact numerical value when counts were higher than 300 colonies, which was taken as maximum value. We assume that the differences observed could only be attributable to the intrinsic characteristics of each method, because the samples were taken simultane-ously under similar conditions.

This result was in agreement with other reports ^{14, 15} which used swabbing to collect specimens from in-fants aged 0 to 30 months and 0 to 6 months. However, Motisuki et al. ¹³ , for stimulated saliva simples in a population of children aged 5 to 13 years, found a significantly lower CS CFU/ml count when samples were collected by swabbing than when col-lected by methods using dental biofilm or drooling into sterile collector tubes. This difference may be explained by the fact that our swabbing method ap-plied motion in the infant’s oral cavity, which may have caused detachment of microorganisms in the oral cavity during the procedure.

Some studies have compared the ability to detect specific bacteria in unstimulated and stimulated saliva samples collected using different techniques and cultured in different mediums under the hypothesis that the bacterial component in saliva varies accord-ing to type of sample collected.

One study on 3-year-olds ¹⁶ determined the presence of S. mutans in unstimulated saliva samples collect-ed by soaking a cotton swab under the tongue compared to samples taken by moving a swab around in the oral cavity. It found that presence of S. mutans was higher in oral swab samples than in unstimu-lated saliva samples. This agrees with the results of our study, which found that the oral swab technique produced higher CS recovery.

Saliva is increasingly being used as a biological material in which to study the oral microbiome. It is therefore necessary to create a protocol for col-lection methods, considering potential interference factors. A recent study by Omori ¹⁷ reports finding similar percentages of relative abundance of streptococci for the drool method and the swab method, recommending the cotton swab method to study the microbiome in subjects who cannot produce saliva or have difficulty in spitting.

There is evidence that stimulated saliva samples could be used as a substitute for unstimulated saliva for oral microbiota studies and that bacterial profile would not vary significantly according to type of saliva specimen used ¹⁸ . Another study ¹⁹ comparing the composition of oral microbiota between stimulated saliva (with paraffin block) and unstimulated saliva (paper points) found significant differences, with stimulated saliva containing an estimated number of species three times higher than unstimulated saliva. Different authors have suggested that some absor-bent collection devices can introduce bias and errors in the subsequent data analysis, mainly in relation to immunoglobulin assays and studies focusing on certain steroid hormones ^20-22 . Our study found that swabbing did not alter the detection of cario-genic streptococci identified by molecular analysis (qPCR), in agreement with other authors ^{23, 24} who established that microbial profiles in saliva are min-imally affected by the collection method.

Taking saliva samples in young children is chal-lenging as a result of situations such as the time involved in obtaining specimen volumes large enough for subsequent processing and analysis, compliance with protocols prior to sampling (fluid, food and medication intake, and mouthwash before sampling), sleep cycles, and the child’s predisposition to the practice. For example, Granger et al. ²⁵ reported difficulties in collecting saliva from children aged 6 to 15 months related to ethnicity and the socioeco-nomic level of the families.

Although the aim of the current study was not to establish association between sampling and aspects related to family income level, it is worth noting that the early childhood center where it was conducted is attended by children from low-income families. The center provides meals (breakfast, lunch and af-ternoon snack), sleep time (30 to 60 minutes to rest) and play activities. These situations justified the performance of this study to identify the most appro-priate method for collecting samples in the context. In developed countries today, there are available commercial devices that are easy to use, cause mini-mum discomfort to participants, and obtain adequate quantities of specimens for subsequent processing. However, in developing countries, where financial and bureaucratic limitations hamper the purchase of complex devices, the standardization of protocols with low-cost supplies offers alternatives for con-ducting microbiological studies.

This study shows that the oral swab method for collecting saliva samples is more effective in terms of recovering microorganisms, and does not alter CS detection by molecular methods. It thus provides preliminary evidence contributing to the develop-ment of protocols and methods for obtaining saliva specimens from infants.