Salivary Cortisol Associated with Increasing Mutans streptococci Count in Drug Abuser

Yassin, Bilal A.; Al-Mizraqchi, Abbas S.

doi:10.30699/ijmm.17.3.318

year 17, Issue 3 (May - June 2023) Iran J Med Microbiol 2023, 17(3): 318-323 | Back to browse issues page

‎ 10.30699/ijmm.17.3.318

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Yassin B A, Al-Mizraqchi A S. Salivary Cortisol Associated with Increasing Mutans streptococci Count in Drug Abuser. Iran J Med Microbiol 2023; 17 (3) :318-323
URL: http://ijmm.ir/article-1-2077-en.html

Salivary Cortisol Associated with Increasing Mutans streptococci Count in Drug Abuser

Bilal A. Yassin

¹, Abbas S. Al-Mizraqchi²

1- Department of Basic Sciences, College of Dentistry, University of Baghdad, Baghdad, Iraq , bilotb@gmail.com
2- Medical Microbiology, Department of Basic Sciences, College of Dentistry, University of Baghdad, Baghdad, Iraq

Keywords: Mutans Streptococci, Salivary Cortisol, Drug Abuser

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Introduction

Addiction is a chronic brain disease that can have negative effects on both physical and social well-being, as individuals cannot control their need for drugs. The brains of addicted individuals have altered responses compared to non-addicted individuals (1, 2). Addiction is considered as a brain disorder because it involves functional changes to brain circuits involved in reward, stress, and self-control. These changes can persist for a long time, even after withdrawal (3). Addiction is similar to other life-threatening diseases, such as heart disease, by disrupting organs’ normal, healthy functioning with harmful severe effects. However, in many cases, addiction can be prevented and treated; otherwise, if left untreated, it can result in fatal consequences (4, 5).
The cortisol hormone is normally secreted in response to increased stress. The biological response to stress includes the interaction of two systems: the hypothalamic-pituitary-adrenal axis (HPA) and the sympathetic nervous system (SNS) or sympathetic adreno-medullary system (SAM). The latter is linked with the production of epinephrine and norepinephrine, which rapidly initiate a fight or flight response (6, 7). A cascade of neurochemicals is produced by the HPA, which is activated more slowly. When a stressful event is experienced, the hypothalamus is stimulated to produce Corticotropin-releasing hormone (CRH; previously known as corticotropin-releasing factor), which stimulates the release of adrenocorticotropic hormone (ACTH) into the circulation from the pituitary gland. Circulating ACTH stimulates the adrenal cortex to secrete the glucocorticoid hormone i.e., cortisol (8, 9). Unbound serum cortisol enters the saliva via intracellular mechanisms. Due to the partial conversion of cortisol to cortisone during passage through the salivary glands, the absolute level of free cortisol in the saliva is estimated between 10% to 35% lower than the level in the blood (10, 11). The use of salivary biomarkers has gained increased popularity over the past decade in psychological and biomedical research since collecting salivary samples is non-invasive and convenient. Nowadays, salivary cortisol measurement is widely used as an alternative to measuring cortisol levels in plasma or serum. In addition, the adrenal cortex is expected to increase plasma glucocorticoid levels in response to the stress of venipuncture for blood collection (10, 12), which may bias the results.
Mutans streptococci are classified into five biotypes and nine serotypes (a-h and k). Oral streptococcus mutans (serotype c, e, f, k) and streptococcus sorbinus (serotype d, g) are the predominant species isolated from human saliva and dental plaque (13, 14). The oral cavity of an individual can be colonized by one or by multiple clonal types of Mutans streptococci (15, 16). Mutans streptococci have all the requirements for being a caries-inducing bacteria. Therefore, oral cavity of individuals heavily colonized by Mutans streptococci were thought to be at high risk for caries (17) automatically.
Saliva is one of the innate immune system components that counteracts pathogenic microorganisms both mechanically by its flow and immunologically by range of immunoglobulins (Ig) and enzymes. Increasing levels of unbound salivary cortisol may reduce the level of these protective proteins. Even when drug abusers stop consuming addictive substances, They may persistently experience elevated cortisol levels. Thus, we hypothesized that increasing systemic cortisol might compromise immune elements of saliva e.g., IgA; hence, paving the way for increasing Mutans streptococci count. Therefore, this study aimed to investigate this potential relationship in drug abusers.

Materials and Methods

The subjects

The study design was a case-control consisting of a total of 86 male subjects, aged between 17 and 30 years. The study was conducted between March 2022, and May 2022, after obtaining ethical approval from the Ethics Committee, College of Dentistry, University of Baghdad (Ref# 508, Project# 580822). Samples were collected from patients who were admitted to Ibn Rushd psychiatric hospital, Iraq. The participants were divided into two groups: the cases group (n= 43), which included withdrawn drug abusers and the control group (n= 43), who were clinically healthy and non-drug users. Drug abusers were defined as previously described (18). Before conducting the study, all participants or their guardians for those < 17 years, had to sign a consent form.

Inclusion Criteria

The participant included in this study should have no signs and symptoms of any chronic diseases or immune deficiency diseases. All participants should not take any medication on the day of sample collection that might alter the biomarker or microorganisms under study. Each volunteer smokes less than 10 cigarettes per day and does not drink alcohol. In addition, patients who were previously consuming cocaine, amphetamine and other derivative were also included.

Exclusion Criteria

Exclusion criteria included participants aged less than 17 years, uncooperative, fearful, anxious, or unconscious patients, participants smoking more than 10 cigarettes or another type of smoking such as vape, and participants using other types of drugs not consistent with our study criteria.
Each patient was confirmed as a drug abuser by the laboratory of Ibn Rushd psychiatric hospital by taking a urine sample and analyzing it with a multidrug line cup urine test, which was also conducted for controls to confirm the negative result. Consent was obtained from all participants before conducting the study.

Salivary sample collection

Unstimulated saliva was collected in the morning between 8 to 10 am. Each participant was asked to refrain from eating or drinking for one hour before saliva collection. The participant was then instructed to put the collection tube near the lip and drool the saliva by tilting the head forward.
After collecting about 3 to 5 ml of saliva, collection tubes were numbered and placed in a cool box with ice to transfer it to the laboratory to be cultured in less than one hour. Salivary samples were centrifuged at 3000 rpm for 10 min, and the supernatant was isolated in Eppendorf tubes, which were stored in a deep freeze at -20ºC for chemical analysis later (19).

Identification of Mutans streptococci

Culturing, isolation and identification of bacteria including colony morphology, Gram stain, catalase and fermentation test were done according to methods previously described (20, 21).

Determination of salivary cortisol level

In the first incubation step, 10 μL of the sample was incubated with a cortisol-specific biotinylated antibody and a ruthenium complex labeled cortisol derivative. Depending on the concentration of the analyte in the sample and the formation of the respective immune complex, the labeled antibody binding site is occupied in part with the sample analyte and in part with the ruth enylated hapten.
In the second incubation step, streptavidin-coated microparticles were added, and the complex was bound to the solid phase via the interaction of biotin and streptavidin.
The reaction mixture was then aspirated into the measuring cell, where the microparticles were magnetically captured onto the surface of the electrode. Unbound substances were removed with ProCell/ProCell M. Application of a voltage to the electrode then induced chemiluminescent emission which is measured by a photomultiplier.
Results were determined using a calibration curve generated by 2-point calibration and a master curve provided via the reagent barcode Or e-barcod.

Statistical analysis

Data analysis was performed using SPSS software (version 26). The data were normally distributed, as tested by Shapiro-Wilk test. Independent t-test were used to compare the means of variables between the study groups. Liner regression was used to estimate the relation between salivary levels of cortisol and Mutans in the study sample.

Results

Salivary Cortisol

Salivary levels of cortisol in drug abusers and control groups are shown in Table 1. The mean of salivary cortisol level among drug abusers (24.61 ±6.77 nmol/L) was significantly higher (P = 0.0001) than in the control group (10.56 ±5.19 nmol/L). Higher levels of salivary Mutans were also detected in drug abusers (101.47 ± 27.49) than in the control group (30.69 ±10.49), as shown in Table 1.

Table 1. Mean of salivary levels of cortisol and Mutans in the study sample groups.

Group	Cortisol (nmol/L)	*Mutans (CFUml)10^-5**
Group	Mean ± SD	Mean ± SD
Drug abuser	24.61 ± 6.77	101.47 ± 27.49
Control	10.56 ±5.19	30.69 ±10.49
P-value*	0.0001	0.0001

* Independent T test; significance was considered at P < 0.05

A positive association was found between the salivary levels of cortisol and Mutans in the study sample (R = 0.801). The increase in the salivary cortisol level could be accounted for by an elevation of salivary Mutans in approximately 64% (R2= 0.64.2) of the sample, with a prediction value of 3.863, as shown in Table 2.

Table 2. Liner regression between salivary levels of cortisol (predictor) and mutans (dependent variable)

	Mean ±SD	R^c	R²	Unstandardized B		P value
				constant	predictor
Cortisol^a	16.57 ±8.54	0.801	0.642	1.961	3.863	< 0.0001
Mutans^b	66.08 ±41.17

a: predictor; b: dependent variable; c: Pearson correlation test

Discussion

Salivary cortisol can be used as a biomarker of adrenocortical function, even though its levels correspond to only 50-60% of plasma cortisol concentrations (22-24). The saliva collection procedure is considered non-invasive and comfortable; therefore, salivary cortisol levels were selected as the biomarker of choice for the present study (25, 26).
The results of the present study showed that the mean of the salivary cortisol level among the drug-abuser was higher than that of the control group, with a significant difference between the two groups. This finding is consistent with results reported by other studies (27, 28). The salivary cortisol concentration significantly reduced when patient received therapy in contrast to untreated patients. Psychomotor stimulants, such as cocaine and amphetamine, as well as natural rewards, such as sex and food, cause a release of dopamine in the nucleus accumbens, regardless of their mechanism of action (29, 30).
The current study also demonstrated a positive and significant relation between the salivary cortisol level and the viable count of Mutans streptococci. Notably, no available data was found from previous studies concerning the relationship between the salivary cortisol level and the viable count of Mutans streptococci in drug-abuser except one study (19) that highlighted such a relationship in children suffering from early childhood caries. The relation between salivary cortisol level and Mutans streptococci in this study might be explained through several possible reasons. For instance, higher cortisol levels might suppress oral immunity and induce the proliferation of cariogenic bacteria, leading to an increased incidence of dental caries (31, 32). Additionally, an increase in salivary cortisol may affect microbial colonization processes such as adhesion and co-adhesion (33, 34). Basal salivary cortisol secretion was positively associated with dental caries; from a theoretical perspective, salivary cortisol could suppress mucosal immunity against cariogenic bacteria (35). Further, during stress, the circulating cortisol level increases the production of acid which provide a suitable medium for these bacteria. Furthermore, atrophic changes in the major salivary glands caused by corticosteroids may lead to a decrease in the quantity, volume, and composition of the saliva. Meanwhile, decreasing the salivary secretion contributes to reducing the mechanical clearance of cariogenic bacteria (36, 37).
Limitations of the current study included the lack of measuring oral hygiene/periodontal and caries activity indices. The addictive substances used by the subjects were not specified, and all subjects who had withdrawn from drug addiction were included. Measuring plasma cortisol levels would give a better picture of this hormone for the participants. Investigating the viable count of individual serotype is more precise than measuring Mutans streptococci group. Furthermore, Lactobacilli, another important food-fermenting lactic acid bacteria, was not included. Despite these limitations, to the best of our knowledge, this study is one of the few that investigated the relationship between Mutans streptococci and salivary cortisol in patients withdrawn from addictive substances. However, further larger-scale studies are advised to confirm the current findings, which is derived from this pilot study, before generalizing the results.

Conclusion

The data from the current study indicate that a higher concentration of salivary cortisol is associated with an increasing count of Mutans streptococci in patients who have quit consuming addictive drugs compared to control subjects. These results suggest including oral hygiene care as a part of rehabilitation programs of drug addiction.

Author Contributions

ASA conceived and designed the study, conducted research, provided research materials, and collected and organized data. BAY analyzed and interpreted data. BAY wrote the initial and final draft of the article and provided logistic support. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript.

Acknowledgment

None.

Funding

No funding.

Ethical Approval

The Ethics

Availability of Data and Materials

Ethical approval was obtained from the Ethics Committee College of Dentistry, University of Baghdad (Ref# 508, Project# 580822).

Conflicts of Interest

No conflicts of interest.

Type of Study: Original Research Article | Subject: Medical Bacteriology
Received: 2023/03/28 | Accepted: 2023/04/29 | ePublished: 2023/06/26

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