Characterization of Staphylococcus aureus isolated from human dental infection

Staphylococcus aureus is an opportunistic pathogen causing dental infection and systemic infections in human body. This organism decreases susceptibility to several types of antibiotics every day and becomes more resistant which is a growing sense of concern in this era. Considering this fact, the study was attempted to characterize the S. aureus from human dental infection and to determine the antibiogram profile of isolates. Sixty four (64) samples were collected from the patients with dental infection who visited different dental clinics and hospitals in Mymensingh, Bangladesh for treatment. Isolation and identification of S. aureus were conducted by using cultural, morphological, and biochemical characteristics. Polymerase chain reaction was performed for final confirmation of S. aureus followed by detection of methicillin resistant S. aureus (MRSA) targeting mecA and mecC genes. Antibiotic susceptibility test of isolated bacteria was tested against seven antibiotics by disk diffusion methods. Forty isolates among 64 samples were found positive for S. aureus based on cultural characteristics. Among them 30 isolates were found positive in coagulase test. Depending on the result of coagulase test, all the 30 isolates were subjected to antibiotic sensitivity test and among them 25 were 100% resistant to penicillin, ampicillin and amoxicillin. All the 25 isolates were subjected to polymerase chain reaction (PCR) to identify methicillin resistant gene mecA and mecC. Eight isolates were positive for mecA gene while no isolates were positive for mecC. The present findings conclude that S. aureus is prevalent in dental infections and contain methicillin resistant genes.


INTRODUCTION
Human oral cavity acts as a growth medium for pathogenic microorganisms due to its moistures, temperature and nutrient content such as lipid, carbohydrate and protein (Mohapatra et al., 2012).There are several types of dental infections which occur in a patient's oral cavity such as tooth decay, periodontal disease, dental ache, dental plaque, dentin hypersensitivity, dental abscess, dental calculus, *Corresponding author.E-mail: sukumar.saha@bau.edu.bd.Tel: +8809167401-6/63214.Fax: +8809161510.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License hyperdontia, malocclusion, acid erosion, acute necrotizing ulcerative gingivitis, dental fluorosis, tooth impaction, etc. Staphylococcus aureus is a putative pathogen of many oral diseases, such as oral mucositis, periodontitis, peri-implantitis, endodontic infections and even dental caries (Gibson et al., 2000;Heitz-Mayfield and Lang, 2010;Poeschl et al., 2011;Passariello et al., 2012).
S. aureus is a Gram-positive, non-motile, non-spore forming grape like clusters and the most important coagulase positive pathogen from staphylococci due to combination of toxic mediated virulence, invasiveness and antibiotic resistance (Loir et al., 2003).Some strains of S. aureus have developed drug resistance (Faden, 2018).
Methicillin-resistant S. aureus (MRSA) (Rajaduraipandi et al., 2006) is the strains of S. aureus that obtained resistance to beta-lactam antibiotics, which incorporates such as penicillin, amoxicillin, ampicillin, methicillin, oxacillin, cephalosporins, etc. (David and Daum, 2010).The propensity of S. aureus to acquire antibiotic resistance has prompted worldwide dissemination of clone expressing various antimicrobial resistances.Several hospital and nonhospital bacterial diseases are caused due to MRSA strains and sometimes lead to death (Bannerman and Peacock, 2007;Moussa et al., 2011;Peters et al., 2013;Faden, 2018).
Contaminations due to S. aureus, including the MRSA strains has long been common in Bangladesh.Because, indiscriminate use of antibiotics being a typical practice, hospital environments are not sufficiently clean as well as congestion of patients and attendants support spread of the infectious agents including S. aureus (Khan et al., 2007).The possible presence of S. aureus is particularly important in dental caries because of its increased resistance (Vellappally et al., 2017).So it is very logical to check the status of microbial resistance against commonly used antibiotics for the treatment of dental infections occurred by S. aureus.Considering the fact that the present study aimed to characterize S. aureus in human dental infections collected from the patients of different clinics and hospital of Mymensingh district, Bangladesh.

Sample collection
A total of 64 samples of human dental infection were collected from different private clinics and Mymensingh Medical College Hospital (MMCH), Mymensingh during the period from January to March, 2018.The samples were collected directly by rubbing the infected teeth with sterile cotton buds and immediately placed them into nutrient broth and transferred to the laboratory within 1 to 2 h.

Isolation and identification of bacteria
Each nutrient broth containing samples were incubated at 37°C for 4 h for enrichment.Samples were then streaked on Staphylococcus species specific growth media mannitol salt agar (HiMedia, India) and incubated at 37°C for 48 h to get single colonies.The suspected colonies were further identified as S. aureus after culturing onto mannitol salt agar (MSA) and 5% sheep blood agar.Gram's staining and biochemical test including coagulase and catalase tests were performed for further confirmation of S. aureus (Cheesbrough, 1985).

Antimicrobial susceptibility test
Antibiotic susceptibility test was conducted using disk diffusion method or Kirby-Bauer (Bauer et al., 1966).All the coagulase positive isolates of S. aureus were subjected to antibiotic sensitive test against seven antibiotics namely penicillin (10 µg), ampicillin (20 µg), amoxicillin (30 µg), ciprofloxacin (5 µg), tetracycline (30 µg), erythromycin (15 µg) and cephradine (30 µg) (Himedia, India) which were generally prescribed by physicians in the study areas.The zone of inhibition produced by S. aureus against each antibiotic was measured and interpreted as resistant, intermediate resistant and susceptible according to standards of Clinical Laboratory and Standards Institute (CLSI, 2016).

Molecular characterization of S. aureus
For final confirmation, all the coagulase positive isolates of S. aureus were subjected to molecular test as methods described by Stuhlmeier and Stuhlmeier (2003).Boiling method was followed for genomic DNA extraction (Dashti et al., 2009).Furthermore, penicillin and penicillin like (ampicillin and amoxicillin) antibiotic resistant isolates of S. aureus were subjected polymerase chain reaction (PCR) for detection of mecA and mecC genes using previously published primers (Lee, 2003;Stegger et al., 2012).The list of primers used in the study is shown in Table 1.

RESULTS AND DISCUSSION
Out of 64 samples of dental infection, 40 (62.5%)samples were found positive for S. aureus by observing cultural and morphological characteristics (Table 2).S. aureus growth in MSA appeared as golden yellow pigmented colonies and Gram's staining exhibited Gram positive, cocci shaped and arranged in grape like cluster under microscope.These findings are similar to the findings of Ray and Ryan (2003).The percentage of S. aureus isolated from human dental infection is more frequent as the findings of Ohara-Nemoto et al. (2008) reported 46.4%.McCormack et al. (2015) and Kim and Lee (2015) reported 18 and 36.6% S. aureus in human oral and perioral infections that is comparatively lower than the current results.
All the 40 isolates which were assumed positive for S. aureus were subjected to catalase and coagulase test where all of the isolates found positive for catalase test and 30 of them for coagulase test.Bubble formation in catalase and curd like clot formation in coagulase test indicated that the samples were positive for S. aureus indicating their ability to breakdown the hydrogenperoxide to release free oxygen and plasma coagulation by activation of prothrombin (Karmakar et al., 2016).Of the 40 isolates of S. aureus, 30 of them created βhemolytic character in blood agar.The findings of the present study were higher than the findings of Karmakar et al. (2016) who reported 40% of β-hemolysis in blood agar.
Dental patients usually take antibiotics primarily to treat postoperative and secondary infections.All 30 coagulase positive isolates of S. aureus were subjected to antibiotic susceptibility test and the results revealed that 93.33% isolates were found resistant to penicillin followed by 90% to amoxicillin, 83.33% to ampicillin and 33.33% to erythromycin where 80% sensitive to cephradine, 63.33% to ciprofloxacin, and 60% to erythromycin (53.33%) (Table 3).Kim and Lee (2015) reported that S. aureus isolated from the periodontal patients showed 88% (36/41) isolates resistant to penicillin and ampicillin which is comparable to the present study.They also observed the susceptibility rate and found 98% isolates susceptible to cefotetan, 95% to ciprofloxacin and 90% to tetracycline and erythromycin that is comparatively higher than the current study.Similar antimicrobial susceptibility results were reported by previous authors (Rajaduraipandi et al., 2006;Khan et al., 2007;Kim, 2012;Naeem et al., 2012).The higher resistant rate to commonly used antibiotics indicates indiscriminate or haphazard use that may have effect on treatment cost, poor prognosis as well as enhance the bacterial infection and growth virulent pathogens.
Coagulase positive isolates of S. aureus were finally confirmed by molecular test using polymerase chain reaction (PCR) and all them were found to be positive (Figure 1) which showed the relevance of the statement of Ohara-Nemoto et al. (2008).A total of 25 isolates found resistant to penicillin, ampicillin and amoxicillin which were further conducted to molecular characterization to detect mecA and mecC genes (Table 4).Out of 25 isolates, 8 were found positive for mecA and no isolates found mecC positive (Figures 2 and 3).It is a new gene which is emerging in the world especially in European countries and spreading among human and animals throughout the world which follows the statement of Paterson et al. (2014) but not for Bangladesh.The prevalence of mecA gene was 12.5% which was higher than the findings of Ayepola et al. (2015) who reported 2.4%.Smith et al. (2003) reported 6% MRSA positive isolates in oral infection.Another study conducted by Renvert et al. (2008) in Sweden observed similar finding connected to patients of periodontal infections.According to Kurita et al. (2006), dental patients are not only responsible for spreading MRSA, health professional may transmit this pathogen through their instruments.There are on established guideline for controlling MRSA but Centers for Disease Control and Prevention (CDC) recommends some standard precautionary measure that   might help to reduce the spread of MRSA in dental patients (Harte, 2010).The present results revealed that mecA positive but not mecC positive isolates of S. aureus are associated with dental infection in Mymensingh city of Bangladesh.

Conclusion
Prevalence of S. aureus in dental patients is quite high and showed resistance to commonly used antibiotics as well as carried MRSA.Despite these results, the sample size of this study is not sufficient and study period was too short to uncover actual picture of MRSA involved in dental infection in Mymensingh, Bangladesh.Large scale studies could be done both in hospitalized patients and in community to identify prevalence of MRSA, genome analysis, identification of toxin gene and other antibiotic resistant gene.Regular brushing of teeth, keeping up oral cleanliness and consulting with dental doctors to check up the teeth once in a month should be taken to maintain a distance from dental infections.

Table 2 .
Cultural and biochemical characterization of S. aureus.

Table 3 .
Molecular characterization of S. aureus.