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The presence of oral bacteria in human saliva and the ability to use them as a marker in forensic science, suggests that these streptococci species plays an active role in the oral environment. The primary aim of this investigation was to determine whether Streptococcus bacteria could be used as a quantitatively reliable method to aged human saliva. The experiment is divided into separate sections. The first was a preliminary experiment; identifying the method for the maximum growth of Streptococcus on MSA. Following this an intra individual variation experiment with single sample was conducted to explain and strengthen the method used. However more importantly, the experiment between inter-individual variations between samples and time of delay was investigated. In addition, influence of smoking and gender were also examined since study by Sajith et al 2007 has shown higher levels of Streptococcus in saliva among smoking individuals. Finally, a blind test was conducted to identify the age of an unknown human sample to form a conclusion about the role of Streptococcus bacteria in forensic science
4.1 Validity of methods
The validity of each in vitro test is based on the controls utilized: the presence of the expected colony count (level of 1 x l04 CFUs/ml) on the control plates; the distribution of the bacterial counts throughout each dilution series; and the similarity of the results in the two series of plates used in each test. The growth of bacteria on the positive control plates suggest that Streptococcus can selectively grow in MSA.
The efficiency of the first method used for isolating streptococci cells was assessed using serially diluted saliva samples for up to 10-6 and revealed that after 48 hours the 10-2 plate shows little to no growth. This result prompted the investigation to revise the current method used. Evidence that there is a high bacterial growth on the supernatant was obtained when preincubation of the sample resulted to higher bacterial growth than the diluted palette.
A low or undetectable level of streptococcus growth was resolved by the increased centrifugation of the sample for 10 minutes. In this study, bacteria were seen to grow in greater numbers with higher centrifugation time, which is also consistent with the method used by Konishi et al 2009. This method is the employed to remove mucous components or contamination.
4.2 Variations in sample collection
The fluctuations of the levels of streptococci can possible occur due to the variation in the technique used. The saliva samples analysed throughout these experiment were unstimulated saliva using the passive drooling method. The techniques for collecting saliva has been categorised into stimulated versus unstimulated. The three major salivary glands secrete different levels of saliva. Under stimulated condition the parotid gland increases to more than 50% of total saliva (Humphrey et al 2001). Unstimulated saliva on the other hand contribute to about 25% produced by parotid gland, 60 % produced by submandibular gland, 8 % produced by sublingual glands and finally 8 % produced by minor glands (Dawes 2008). This suggests that the composition of saliva and the amount being produced is greatly affected whether saliva is produced under stimulated or unstimulated conditions. The passive drooling technique which is unstimulated is the most effective technique for collecting saliva sample for it yields a representative combination of saliva secreted from the three glands (Rohleder et al 2009).
Variations in saliva collection do have an effect on the total saliva production, which affects the number of bacteria (REF.)
Furthermore another factor to consider in collecting saliva samples is the effect of antibiotics on saliva production. None of the participants had been treated recently with antibiotics. Previous study found that this often affects the colonisation of exogenous bacteria (REF). Castillo et al. (2006) state that bacterial resistant to antibiotic can interfere to the growth curves of oral streptococci. In this study the minimum inhibitory concentration (MIC) which is define as the lowest antibiotic concentration potentially inhibit bacterial growth was measured. It was found that 0.007mg/l MIC of amoxicillin and penicillin inhibits bacterial growth particularly S. salivarus (Castillo et al 2006). This conclusion is supported by Kuriyama et al. (2002) who found high susceptibility rates (≥77%) of antibiotics against viridans streptococci.
4.3 Physiological and pathological factors affecting human saliva
Variations in Streptococcus in human saliva can be affected by numerous physiological and pathological factors. The inter- and intra-individual variations of the levels of Streptococcus in the saliva have been calculated from experiments of male and female sample. Environmental factors such as dental treatment and diet play a very important role in streptococci growth.
Unstimulated saliva secreted from salivary glands contains high oxygen contents. In the present study, the repeated culturing of unstimulated saliva samples, taken hourly from the same subject yielded comparable results. This was assessed by computing the means and standard deviation. Statistical analysis using Kruskal Walis test did not reveal significant intra individual variations in the average level of streptococci growth (p > 0.05). Intervariation between the hourly collected saliva samples shows no significant difference for the 24 hour and 48 hour incubation period. Therefore there was no significant differences were found on the number of Streptococcus bacteria and time of delay before processing. This suggests that levels of Streptococcus growth of an individual would not vary greatly. This agrees with the study done by Leonor et al 2009 with regards to SSFR.
Effect of pH
The main point to consider in this study is that S. salivarius and S.mitis only make up <1% of the components that contributes to dental plaque. The main cariogenic bacteria responsible are the S.mutants.
Viridans streptococci have species-specific interaction around areas of the oral cavity and pharynx. Both S.salivarius and S. mitis is the predominant species on the dorsum of the tongue where as S.mutants adhere around teeth and enamel.
S.mutants creates a negative interaction (amensalism) between organisms, creating low pH environment toxic environment for other species (Houte et al 1996). Low pH is the results of fermentation of carbohydrates which contributes to the acidity of tooth decay. With respect to the method use for collecting saliva samples, Dasayanke et al (1995) has reported a higher number of S.mutants on stimulated saliva than unstimuated therefore validating the method use in this experiment.
Saliva has a direct effect on plaque acidity. According to ... pH of unstimulated saliva (resting) is more acidic then stimulated saliva. Stimulated saliva is being produced most of the time and has an approximate pH range of 6-7, suitable for microbial growth. The pH as low as 5.1 is said to be critical pH for the dissolution of enamel (ref). The pH drop is due to acids produced by bacteria fermenting carbohydrates. Urea is also one of the components present in saliva and in cervical fluid. Urea is associated with the production of nitrogen source for bacterial growth which is predominately found on surfaces where salivary access is greatest. Among the genus streptococci, S. salivarius produce high urease concentration. The major role of urea in the oral cavity is pH control, protecting organisms against acid damage. The survival rates of S. salivarius in the presence of different urea concentration were studied in vitro by Ywan et al 2000. Result from the study shows the following: the production of ammonia enhances the survival of acid sensitive species such as S. Streptococcus and also inhibiting the progression of tooth decay producing less cariogenic micro flora. Ammonia can neutralise plaque acid, promoting a suitable oral environment on other microbes. Dental plaque is uncommon on areas such as tongue, lower incisors and where the teeth are constantly bathed with the buffers and concentrated calcium ions of saliva. Neutral or even alkali saliva
This process of remineralisation takes longer in an acid environment, but is rapid if the fluid next to the enamel is neutral or even alkali. Caries is therefore uncommon in those parts of the mouth near the outflow of salivary glands, like the lower incisors, where the teeth are constantly bathed with the buffers and concentrated calcium ions of saliva. If the total outflow of saliva can be increased, there is a greater chance of protection of all the teeth in the arch.
Saliva is generally saturated with respect to calcium and phosphate which help in remineralizing
Saliva (factor 1) may
Affect the local exposure of plaque to dietary carbohydrate
(factor 2) and may have a direct effect on plaque acidity (e.g.,
flow rate, buffering effect). These interactions are translated
in changes in plaque's microbial composition (factor 3) and,
hence, its pH-lowering and cariogenic potential (factors 4
and 5). A relatively low local salivary exposure, then, is
associated with a relatively high exposure of plaque to
carbohydrate, which, in turn, leads to an enrichment of
plaque with highly-acid-tolerant Lactobacillus and MS
(capable of acidogenesis at a low pH) and an increase in
plaque's pH-lowering and cariogenic potential.
Most study in the literature with respect to genus streptococci was made about the main contributing agents of plaque formation which is the S. mutants. A six year longitudinal study regarding stimulated salivary flow rate (SSFR) on children was conducted by Leonor et al 2009. SSFR on adults and in children showed a wide inter and intra individual variations. Adults SSFR are reported to have 0.7 to 3 ml/min, while children SSFR is approximately between 0.1 to 6 ml/min. One of the possible explanations is the increase size of salivary glands
Previous studies found that one ml of saliva can contain as many as 4 x 10-7 bacterial cells (Takada et al 2006).
Saliva comes in contact into the surfaces in the mouth especially supraginggival surface of the teeth. Studies shows Streptococcus can be detected 2-4 hours after brushing and believe to grow rapidly with doubling times of between 1-3 hours. The double time of bacterial growth promotes cell density dependent causing a dramatic exponential increase of growth.
It has been found that many of early microbial colonizers of human dental plaque are of great importance in the succession stages of biofilm formation and its overall effect on the oral health of the host. The host provides the main source of nutrients needed for growth and reproduction. In addition dietary intake by the host can also act as a source of nutrients for colonising microbes.
Inter variation was assessed by separately computing the mean and standard deviation across all 4 samples of unstimulated saliva at each time of delay.
The amount of saliva in the mouth is not constant and varies within a
person over time (Dawes, 1972) and between individuals (Dawes, 1987;
All plates at 10-2 showed more than 40 000 colony-forming units per milliliter (more than 40 colony counted on the plate) of the suspension, confirming the accuracy and usefulness of this method for similar studies (Petti et al 1997)
Smoker non smoker
The greatest volume of saliva is produced before, during and after meals, reaching its maximum peak at around 12 a.m., and falls considerably at night, while sleeping (2).
Detection of streptococci from female samples
Very little is known concerning the role of Streptococcus bacteria in the aging of saliva in forensics. The results of the present study show that number of streptococci colonies varied considerably from each samples. There was a variation in the numbers of colonies at different incubation period from a single sample.
Differences of the number of streptococci colonies by the time of delay from different samples have also been noted.
Streptococci are Gram-positive spherical or ovoid bacteria, which are <2 μm in diameter. They are catalase-negative, facultatively anaerobic, non-motile, and do not produce spores or gas.
Morphology identification was not carried out for growth on the culture medium because a selective media was used and thus, all bacteria were included in the colony-forming unit count.
According to previous studies43, 44 every colony counted on the plate corresponds to about 1 x l04 CFU/ml of saliva; thus, the limits of detectable salivary Streptococcus concentration range between 1 x l04 and 3 x l06 CFU/ml. However, this limitation is shared with many other rapid methods43- 45
The control group consistently (15 from 15 counts at each analysis) produced bacterial counts greater than 40 000 colony-forming units mL
Confirming the accuracy and usefulness of this model for similar studies.
Streptococcus salivarius is found on the dorsal surface of the tongue (16) and is one of the earliest colonizers of the oral cavity in infants. It has been shown to preferentially attach to keratinized oral epithelial cells in vitro, reflecting its preference for the tongue surface (23). Other oral streptococci preferentially attach to different sites in the buccal cavity
3.4 Comparison of growth curves of male and female samples
The numbers of differences between males and females have been observed regarding the number of Streptococci.
Saliva is supersaturated with calcium and phosphate ions, its function as re mineralizing agent, replacing any of these ions lost from the enamel surface as a result of acid dissolution.
Samples A and B shows similar trend of decrease on the levels of Streptococcus growth. The slight increase sample B however at 4 hours before processing shows a small exponential phase on which sample A does not exhibit. Sample C exhibit a rapid increase of exponential phase for the 1.5 hour time of delay before processing.
Thus, tests for the production of P-D-galactosidase and P-D-glucosidase gave different results depending upon the substrates (naphthol or nitrophenol derivatives) used. It follows, therefore, that the glycosidase activities detected with 4-methylumbelliferone derivatives may not necessarily agree with those demonstrable with other types of synthetic glycosidase substrates.
Found that filled teeth may be related to both high and low mutans streptococcal levels: micro-organism distribution varied considerably among different restorations, even in the same subject;
consequently, in order to evaluate the chance of recurrent
decay, Wallman and Krasse32 argued that microbial
analysis of saliva samples had certain limitations
and correctly proposed to analyse restoration margins
directly. On the other hand, Wright et aZ.36 in a longitudinal
study, found that salivary mutans streptococci
significantly decreased after restorative caries treatment
and slightly increased in the post-restorative phase.
These authors, however, made some saliva samplings
during the last restorative treatment examination, when
mutans streptococci may still persist in saliva in large
numbers, and, in the statistical analysis, they associated
the results of these samples with the results of other
samples, made some days after the last conservative
treatment, and considered them as belonging to the
post-restorative phase. This may explain the statistically
non-significant increase in micro-organisms found in
the post-restorative phase by these authors.
Chemical and physical properties of different
mutans may colonize them and salivary micro-organism
concentration would, consequently, be high.
Affect of smoking
Comparison of growth curves between smoker and non smoker
The initial increase in Streptococcus activity of sample C after 1.5 hour evidently shows the logarithmic phase of the Streptococcus activity in saliva. The initial growth can be caused by several mechanisms including competition for substrates such as dextrose and saccharose (Martin et al 2006). This competitive interaction between Streptococcus and members of the resident microflora results in the fluctuating number of colonies. Furthermore certain strains of Streptococcus have synergetic and antagonistic interactions within the oral flora (Shelburne et al 2005)
The increase after 4 hours in sample D is due to the growth of infecting bacteria as a result of the availability of fermentable carbohydrates at this stage less bacteria is available to interfere with each other's growth (Takashi et al 2005).
Bently et al (cited by petri et al 1997) found that filled teeth may be related to both high and low Streptococci levels, microorganisms distribution varied considerably among the samples.
From the results of the experiments the number of streptococci recoverable varied both with the time of delay and individual saliva samples. Relationship between the bacterium and the host may vary with the health conditions of the host organism (Todar, 2005, Palmer et al 2001). Overall, these relationships determine if the bacteria lives symbiotically, thrives off nutrients at the host's expense, or causes the host disease.
The frequency of which teeth are exposed to cariogenic (acidic) environments affects the likelihood of caries development. After meals or snacks, the bacteria in the mouth metabolize sugar, resulting in an acidic by-product which decreases pH. As time progresses, the pH returns to normal due to the buffering capacity of saliva and the dissolved mineral content of tooth surfaces. During every exposure to the acidic environment, portions of the inorganic mineral content at the surface of teeth dissolves and can remain dissolved for two hours. Since teeth are vulnerable during these acidic periods, the development of dental caries relies heavily on the frequency of acid exposure.
In an attempt to understand these variations
3.6 Blind testing
The effect of time on the growth of Streptococcus
The numbers of Streptococcus cells in cultures were seen to decrease in a trend as the exposure time was increased (see graph figure…). The Streptococcus cells were found to exist in culture with concentrations up to 10-2 dilution factor. No streptococci were observed to grow in culture with concentrations higher than 10-2 dilution factor.
Growth may be defined as an increase in size or mass. A bacterial cell increases in size and then divides into two. Growth in a bacterial population is generally taken to mean the increase in mass and subsequent increase in numbers through replication, however a distinction can be made between the increasing sizes (growth) and increasing the number of cells (multiplication).
An increase in bacterial numbers will be a discontinuous process whereas growth as an increase in mass will be a continuous process.
Bacteria indigenous to the oral cavity interact with a variety of salivary proteins and glycoproteins. Some of these interactions are believed to mediate binding of oral bacteria to the hard and soft surfaces in the oral cavity, and others seem to play a role in their elimination. The enzyme a-amylase, which hydrolyzes a-1,4-glucan bonds in polysaccharides containing three or more 1,4-linked glucose residues, is one of the major components of human saliva,
This study did not examine many samples. However it was noted that intraindividual variation Streptococcus bacteria in saliva does not vary considerably than inter individuals. However, further work is required to determine if this is always the case.
Streptococcus is detected on all saliva samples and it would therefore be unlikely to obtain false positive results from such samples.
Approximately 90% of saliva is produced by the salivary glands (parotid, sub-mandibular and sublingual) which are located outside the oral cavity. Oral cavity is formed from the cheeks, the soft and hard plates, and the tongue.
Saliva contains a large number of approximately 108CFU/ml of a variety of microbes. Saliva not only contains fluid produced by salivary glands, but also
The tongue is the major
source of many salivary bacteria and also a potential reservoir
of putative periodontal pathogens . The processes
of chewing and daily mechanical oral cleansing,
including toothbrushing and interdental cleaning, dislodge
dental plaque and mucosal membrane biofilm,
which disperse and increase the salivary bacterial load
for variable periods of time . Bacteria in saliva may
be transferred to and colonize new sites, or they may be
destroyed or eliminated from the oral cavity in various
ways, such as nonspecific and/or specific oral defenses,
swallowing, spitting, or antimicrobial components of
dentifrices [22, 27] and mouthwashes .
Factors affecting bacterial growth:
Bacterial environment is controlled by environmental conditions, and to grow and replicate bacteria needs carbohydrates. Carbohydrates levels are frequently elevated by the intake of dietary sucrose.
In concordance with previous findings (15)
An extensive variations was observed on smokers.
Effect of smoking and non smoking to Streptococcus
Zonuz et al 2008
Later study show the increased risk of respiratory tract infection and also with oral colonization of potentially pathogenic microbes associated with smoking.
Metabolites of chemicals in tobacco smoke have been found in the follicular fluid of smokers Nicotine derived nitrosamino-ketone is found in higher concentrations in cervical mucus of smokers than non-smokers.
For example, a high saliva count of mutans
streptococci indicates the presence of a strong cariogenic
challenge to the teeth