Effect of Zoocin A on Growth of the Biofilm Producing Cariogenic Oral Bacteria

Zoocin A has a potential antibacterial properties and its use as an anti-cariogenic agent needs to be explored Objective: Dental caries is an infectious disease, caused mainly by mutans streptococci (MS). The aim of the present study is to evaluate the antimicrobial effect of zoocin A on the biofilm producing cariogenic oral bacteria compared with antibiotics. Methods: The samples were collected from dental caries and plaques of 130 cases referring to eight government dental clinics of HamedanIran for treatment. The isolated bacteria were identified on the basis of morphological, biochemical and molecular methods. The antimicrobial effects of the zoocin A and antibiotics were compared. Results: Zoocin A showed varying degrees of inhibition on the most common oral biofilm producing bacteria we isolated which were identified as S. mutans, S. mitis, S. sanguinis, S. gordonii, Lactobacillus gasseri, and Granulicatella adiacens by PCR using16S rRNA gene sequence. Minimum inhibitory concentration (MIC) of 2.2 mg-l and 3.2 mg-l was observed against S. mutans and S. gordonii respectively. Conclusion: In Hamedan, S.mitis and S. mutans are dominant species in decayed teeth of cases referring to government dental clinics. Isolated bacteria showed varying sensitivity to different antibiotics. Zoocin A inhibited the growth of some Streptococcal species like S. mutans and S. gordonii. Antimicrobial property of Zoocin A against cariogenic agents collected from high risk caries patients is equal to antibiotics tested in this study.


INTRODUCTION
Dental decay is a common preventable, oral infectious disease associated with increased consumption of sugar and carbohydrates. 1 Dental caries has been known since recorded history, but was not an important health problem until sucrose became a major component of the human diet. 1 Tooth decay is the result of irreversible solubilization of tooth mineral by acid produced by certain bacteria that reside in dental plaques. 1 In 1890s, Miller was the first to relate bacteria with dental decay. 2 The mouth is colonized by various bacterial species, but only some species participate in dental decay. Streptococci form 20% of the oral bacteria that participate in biofilm formation. 3 Streptococcus mutans is the main etiologic agent of dental decay. 1 It is a gram positive, non-motile, facultative anaerobic coccus which can metabolize carbohydrates. 4 Lactobacilli are associated with progression of the decay. 1 They are gram positive, facultative anaerobic or microaerophilic, rod shaped, non-spore forming bacteria. 5 Tooth decay is a controllable infectious disease. Rigorous debridement of teeth surfaces is the standard treatment. 1 Short-term use of antimicrobial agents, especially antibiotics is beneficial. 1 Antibiotics are constantly in use for treating dental caries and have been used for inhibition of biofilm formation. 6 However, certain bacteria have developed mechanisms for resisting antibiotics. 7 The emergence of resistant bacteria is occurring rapidly worldwide, endangering the efficacy of antibiotics. 8 The number of multidrug resistant pathogens is increasing, which is a serious problem. Thus, developing a new generation of antibacterial agents to treat infectious diseases is becoming important. 9 On the other hand, development of new generation of antibiotics by the pharmaceutical industry has been stalled due to various obstacles including economic obstacle. 8 Many new antibacterial substances have the potential to replace the antibiotics. 9 Bacteriocins are proteinaceous toxins found in almost all bacteria. They often inhibit the growth of related organisms. 10 A bacteriocin -like inhibitory substance called zoocin A is an extracellular enzyme secreted by Streptococcus equi subsp. zooepidermicus 4881 which hydrolyzes peptidoglycan cross bridges of susceptible Streptococci, most notably Streptococcus mutans. Thus, it acts as a potential antibacterial agent for reducing the population of the competitors, suggesting its use as an anti-cariogenic agent. 7 The aim of the present study is to evaluate the antimicrobial effect of Zoocin A on the biofilm producing cariogenic oral bacteria isolated from study cases compared with antibiotics.

Study Cases
130 cases referring to eight government dental clinics of Hamedan-Iran were randomly selected. Oral cavity and teeth of every case was checked by the dentist. A questionnaire was filled which contained questions regarding their demographic features, oral cavity hygiene and teeth conditions. To achieve an overall view of oral and teeth health conditions of the participants, questions scored zero to 10. 10 indicates excellent oral and teeth health conditions and zero shows poor oral and teeth conditions, requiring emergency treatment. The oral hygiene questions were evaluated on the basis of IDC-M version10. 11 Written informed consent was obtained from every participant. Data were analyzed using SPSS software. Students' T-test and Chi square test were some of the statistical tests used for analysis of the data.

Sample collection
Cases were asked to rinse their mouth with sterile distilled water prior to sample collection. Sterile swabs were used to collect samples from dental caries and plaques. Used swabs were inserted in sterile test tubes containing 5 ml sterile phosphate buffer saline and were transported to the laboratory in ice box within 3 hours.

Isolation of the bacteria
In the laboratory, collected samples were cultured on Brain and Heart infusion agar (Sigma Aldrich), Columbia agar (Sigma, Aldrich), blood agar (Oxoid) containing 5% de-fibrinated sheep blood, Trypticase AzolectinTween (TAT) (Hi Media), Hewitt agar (Sigma Aldrich), and Luria Bertani broth (LB) (Hi Media) incubated at 37˚C for 24 -48 hours under aerobic and anaerobic conditions. To maintain anaerobic conditions we used both glass jar as well as anaerobic incubator (Anaerobic Incubator BJPX-G Series, Biobase).

Biofilm formation
Ability of the isolated bacteria to form biofilm was estimated both by dyed test tube and microtiter plate methods. 12 Experiments for biofilm formation were repeated thrice. The density of the stained biofilm of the bacteria sticking to the wall of the glass tubes, dyed by crystal violet, indicated the biofilm producing ability of the bacteria. In microtiter procedure, control and samples were placed in alternate wells of a microtiter plate.

Antibiotic sensitivity of the isolated strains
Isolated bacteria were cultured on Muller-Hinton agar. Antibiogram test was performed by Kirby-Bauer disc diffusion method using Vancomycin, Tetracycline and Erythromycin Difco discs. 13 Whonet 5.6 software was used to measure the zone of inhibition of tested antibiotics.

Effect of Zoocin A on the isolated strains
Effect of the Zoocin A was studied by well diffusion and disc diffusion methods on isolated strains using Muller-Hinton agar. In the well diffusion method, 20μl Zoocin A solution (Sigma Aldrich) was poured in 6x3 mm well cut on solid Muller-Hinton agar on which each one of the isolated bacteria were grown and covered by soft agar gel. Effect of Zoocin A on the isolated strains were measured after 24 hours incubation at 30˚C. Distilled water was used as control. In disk diffusion method, 6mm disks impregnated with 2-3μg Zoocin A were placed on agar plates on which isolated bacteria were grown and incubated for 24 hours at 30˚C. Zone of inhibition on each isolate was measured. Participants' teeth defects were categorized on the basis of ICD-M10, a subclass of WHO classification version 10 (ICD10). 11 Pain was the main reason for 85.4% cases to visit the dentist which is grouped under K02.1. Based on the 10 score system, in the 3/4 th of the cases oral and dental health was in good condition. Maximum distribution i.e. 21.5% belonged to score 8. Demographic features of the cases is presented in Table 1. all gram positive bacteria with negative oxidase and nitrate reduction tests and were non-motile. Figure 1a shows the dyed test tubes containing these isolates and Figure 1b shows attachment of the biofilm to the bottom of the wells of microtiter plate.

Bacterial isolation and identification
In microtiter procedure, control and samples were placed in alternate wells of a microtiter plate ( Figure  1b). Control wells were colored pale yellow and wells containing cases' samples colored dark blue. Color appearance on the wall of the tube observed by naked eye was qualitative way of biofilm formation.

Molecular identification of the isolated species
The isolated species capable of forming biofilm in vitro were identified by PCR using 16srRNA gene.    (Table 4). PCR was carried out using GeneAmp PCR system 9700.
Comparison was made with sequences present in the Human Oral Microbiom Database (HOMD) employing its software. Sequences with less than 90% similarity were deleted from the study. After DNA preparation and phylogenic analysis of the isolated species was carried out and were compared with HOMD, employing the HOMD online software). Abundance of bacteria belonging to phylum Firmicutes and Actinobacteria which were found almost in all samples, followed by comparatively lesser distribution of Proteobacteria, Fusobacteria and Bacteroidetes (present only in one sample) are shown in Table 3. & Dendogram 1 & 2. Dominant bacteria belonged to the Phylum Firmicutes with redundancy of S.mitis, and S.mutans followed by Lactobacilli indicating that reduction of pH attracts   acidophilic bacteria. Table 4 shows 10 common oral bacteria along with the sequences used and their code number in HOMD database.

Antibiotic sensitivity of the isolates
The inhibition zone measured using Who net 5.6 software showed mean diameter of inhibition zone obtained in case of erythromycin (15μg) was 22.80 ± 6.00 mm, for vancomycin (30μg) it was 9.0 ± 00.13 while for tetracycline (30ug) the diameter was 22.3 ± 15mm. Based on the diameter of inhibition zone of tested antibiotics, erythromycin and tetracycline were both effective against isolated bacteria but vancomycin did not inhibit their growth.

Effect of Zoocin A on the isolated species
Zoocin A inhibited the growth of biofilm producing bacteria with maximum effect on some Streptococci particularaly S. mutans and S. gordonii. MIC of 2.2 and 3.2 μg were effective against S. mutans and S. gordonii  Table 5 shows the effect of Zoocin A on the isolated bacteria. Zoocin A at lower concentration than antibiotics inhibits the growth of Streptococcus mutans and S. gordonii.

DISCUSSION
After Miller stated that oral biofilm producing bacteria are responsible for dental decay, various researches worked on the identification of oral bacteria isolated from tooth decay, based on their culture and phenotypic characteristics. 14,15 The advances in bacterial isolation techniques, led researches to the identification of bacteria by molecular procedures. 14,16 Samples for identification of oral bacteria were collected from dental cavities and biofilms; According to previous researches, biofilm formed on the teeth surfaces attracts more colony forming bacteria. 10,17 Similar o revious study, this adopted 16SrRNA as a reliable method for identification of the isolated bacteria and to differentiate between the MS species. 14 In the present study, identification of the bacteria isolated from oral cavity of the participants was carried out by all available procedures. Comparing the efficacy of the different techniques for bacterial identification, our findings indicate a precise and accurate identification by molecular procedure. Among the diagnostic techniques employed in this study, it is evident that at present, 16SrRNA is the most precise way of bacterial identification. 18 14,17,[20][21][22] . According to them a diversity of bacterial species, primarily grampositive species, are involved in caries formation and bacterial profile changes with the progression of disease. They believe that caries is the result of a shift in the balance of the resident microflora driven by changes in local environmental conditions like acidic conditions produced by species that live in decayed tooth which supports the "ecological plaque hypothesis" . Loesche also believes S.mutans was involved with the initiation of decay, whereas the lactobacilli were associated with the progression of the lesion. 1 We noticed that some of the isolated bacteria were common with bacteria reported by other researchers in different parts of the world while some species were different indicating that not only bacterial population differs in different stages of tooth decay 1 but also it is influenced by different epidemiological factors including race or geographical distribution of the population. 23 Amongst those who had their own permanent teeth, decayed teeth per person in the elderly was 1.2 which was comparatively but non-significantly less than the average number of decayed teeth per person in the  24 In contrast to previous study that reported all species they isolated were susceptible to the tested antibiotics 15 , we isolated resistant species as well. In the present study we came across vancomycin resistant Streptococci which is rarely reported. 25,26 The present controversy leads to the conclusion that antibiotic susceptibility is not a constant and fixed bacterial specificity and inspection of antibiotic susceptibility is required for prophylaxis and treatment. 15 In Hmedan, S.mitis, and S. mutans are dominant species in decayed teeth of the cases referring to governmental clinics and isolated bacteria are sensitive to tetracyclin and erythromycin but resistant to vancomycin. 25,26 This study also proved that zoocin A like antibiotics has antibacterial activity with maximum inhibitory effect on S. mutans and S. gordonii. Based on the findings of the present study, tooth decay starts with colonization of tooth with Mutans streptococci which are affected by zoocin A. Zoocin A by inhibiting the growth of Mutans streptococci which are the initiating agents in tooth decay can stop tooth decay even at lower concentrations compared to antibiotics. Further clinical researches may lead to extrapolation of our findings; protection and prevention of dental decay by zoocin A rather than antibiotics will be feasible in near future. The findings of this study are in accordance with Lester and Simmond's findings. 27 Akesson et al have the same opinion and believe much of the potential for use of such agents as enzybiotics stems from their ability to target related bacteria. 28

CONCLUSION
At present, MS bacteria are known as etiological agents of dental caries of which S. mutans is the leading cause of tooth decay and the most cariogenic streptococcus. By eliminating the established MS populations from the oral cavity, the chance of caries development reduces. Many new antibacterial substances have been found to replace the old antibiotics; however, finding and identifying new antimicrobial substances is a difficult task. Zoocin A is a bacteriocin which is able to inhibit the growth of S. mutans and S. gordonii, the main tooth decaying agents. Thus, it may be a potential