The efficacy of anti-microbial agents against Actinomyces species isolated from patients with dental caries

INTRODUCTION

Destruction of teeth is a serious problem in oral and general health of the human being, and dental caries is one of the major defects of dental hard tissue [1]. It is a chronic bacterial disease that affects dental structures caused by a huge number of bacterial communities that inhabit teeth surface, forming a biofilm of attached dental plaque. The first habitats are acidogenic bacteria, such as Streptococcus mutans and Lactobacilli, which find shelter in dental groves and fissures, and may be attached to smooth surface of inter-proximal areas as well as buccal and lingual regions. When optimum prevention methods are not followed, these biofilms are nourished by fermentable glucose and sugars derived from patient diet, forming a byproduct of lactic acids that causes dissolution of dental enamel, dentin, and even cementum by de-mineralization of these surfaces, followed by destruction of the weakened surfaces by bacterial enzymes and toxins [2, 3]. It is a multi-episodic disease characterized by de-mineralization and re-mineralization from salivary buffering actions and minerals gained from food and prophylactic procedures [4]. The disease progresses when the compensating re-mineralization factors are no longer able to overcome destructive actions of the lesion microbiota [5]. Clinical feature of these invasions is the formation of white cavities and lesions that later undergo discoloration (brown or black). When getting deep into the tooth surface, this discoloration is caused due to staining from bacterial products or dietary origin [6], and when these lesions penetrate even deeper in dentine, pain and sensitivity are experienced by patients during consumption of sugary diet or drinking cold beverages. These symptoms can be more serious when the lesion is located closer to dental pulp or invade it causing severe pain and irreversible inflammation of the pulp. This condition may be acute or chronic, and if left untreated, it may cause the loss of pulp vitality and inflammation of the periapical and periodontal tissues with formation of abscess, while in cases of severe destruction, it may lead to loss of teeth and endangering para-oral structures and mid-facial spaces [7].
Studies on dental plaque reveal the presence of seve­ral species contributing to starting of adhesion and progression of the biofilm. The second largest contributing type of micro-organisms are anaerobic, Gram-positive (+ve) bacteria, called Actinomyces species [8-10], which have been demonstrated to be one of the pathogens to cause biofilms formation on tooth surfaces in patients of all ages [11]. Detection of Actinomyces species was also seen in root surface caries. Recent studies suggest that it has a major role in dental cavities with association of Streptococci and Lactobacilli, especially Actinomyces naeslundii [12]. The seriousness of actinomycotic infection gets more severe when these species penetrate into host tissues or skin causing a severe injury, called actinomycosis, that is a persistent infectious disease and its abortifacient agent, formerly known as Streptothrix israeli, have long been recognized [13].
The management of Actinomyces spp. usually entails prolonged courses of anti-microbials. Surgical intervention may also be necessary in advanced cases, depending on areas of infection (e.g., epidural infections, brain abscesses), in patients with large hemoptysis, or in cases where intensive abscesses and fistulous tracts are found [14]. Actinomyces spp. were found sensitive to a large variety of anti-microbial agents, such as penicillin, ampicillin, amoxicillin, and erythromycin [15, 16]. However, challenges with accurate Actinomyces genus identification and classification changes make it difficult to interpret multiple findings [17]. As our understanding of the illnesses caused by Actinomyces species advances, the knowledge of precisely recognized Actinomyces susceptibility may become increasingly significant [18]. For culturing of Actinomyces species, blood agar (BA) can be used with some nutritional requirements [19]. Gene sequencing based on the standard of 16S rRNA was introduced in the 80s of the past century, providing a reliable methodology for identification and isolation of studied bacterial species [20, 21], which is the metho­dology used in the current research. In order to identify Actinomyces spp., with quick and specifical isolation of these types of species in dental caries, and trying to investigate the effect of several antibiotic agents against them, the current study was conducted.

OBJECTIVES

The objective of this research was to isolate Actinomyces species from dental caries using polymerase chain reaction technology, and to examine the in vitro antibiotic resistance of isolates using a specific 16S rRNA primer to investigate the effect of several anti-bacterial agents, such as amikacin, gentamicin, ciproflo­xacin, amoxicillin, ceftriaxone, cefixime, vancomycin, and erythromycin against Actinomyces species.

MATERIAL AND METHODS

SAMPLES COLLECTED AND METHODOLOGY

This cross-sectional survey study was conducted from March, 2021 to June, 2021 (three-month period). Eighty samples from patients with dental caries who visited a dental facility at the University of Babylon in Hilla City, Iraq, were collected and tested for the current investigation. Samples were obtained from each case by excavation of infected dentin from large carious lesions using a sterile spoon excavator. Each patient signed a consent form before being enrolled in the study and before sample collection. Samples were carefully gathered to prevent contamination. Part of the obtained specimens was instantly inoculated into a blood agar media at the patient’s chairside. The remaining part of the sample was transferred to the Department of Microbiology for additional examination after being plated onto blood, MacConkey, mannitol, and nutrient agar media, and incubated for 24 hours at 37oC under aerobic and anaerobic conditions. Gram stain, colony morphology, and biochemical tests were performed to diagnose bacterial isolates. In addition, Vitek-2 Compact system (BioMérieux, Marcy-l’Étoile, France) was used for investigation of bacterial species with 16S rRNA technique. Each isolate underwent cultural, morphological, and biochemical testing to help with identification [22, 23]. Actinomyces spp. isolates were screened and identified by utilizing Vitek-2 Compact system. This form of identification is phenotypic, and relies on biochemical processes to identify the isolates. There were 64 wells on Vitek-2 card, each holding a separate fluo­rescent biochemical experiment. Phosphatase, urea, nitrate, and actidione assays were performed on 20 of the 64 samples for carbohydrate assimilation, followed by automatic filling and sealing of the cards; then, the cards were transported to incubator (35oC) and procedures were all handled by Vitek-2 machine. According to a specific algorithmic system, each output report was de-coded, and the acquired findings were identified by ID-GP (identification of Gram-positive bacteria data-bank). The corresponding supporting software automatically suggested ID results from these systems. When no ID or poor discrimination was indicated, the test was repeated and new results were considered in the analysis of data. After being plated into culture media, each strain was cultured at 37oC overnight, according to the manufacturer’s instructions. Identification of Actinomyces spp. was done by specific 16S rRNA gene, as shown in Table 1, which presents the PCR settings and primer sequence utilized in the study [24].

EXTRACTING DNA FROM BACTERIAL ISOLATE

Using a genomic DNA purification kit and additional genomic DNA supplement, genomic DNA was isolated from each sample using Geneaid (Geneaid, USA) and observed with a UV-transmitting light source. Then, the sediments of bacterial cells were transported into a phosphate buffer solution and spun in micro-centrifuge at 14,000-16,000 rpm speed for one minute. Supernatant was then discarded with proteinase K, and 20 µl distilled water was added, followed by 10 minutes of incubation at 60oC, with three minutes interval of inversion. Actinomyces species were identified molecularly using the recovered DNA in a procedure described by the Institute of Clinical and Laboratory Standards [25].

TESTS TO DETERMINE ANTIBIOTIC SENSITIVITY (DISK DIFFUSION TEST) (TABLE 2)

The test was carried out using an isolated bacterial organism from a pure culture. Five isolated Actinomyces colonies were grown in blood agar mixed with 5 ml of nutrient broth, and used as an inoculum after incubation for 2 hours to produce a moderate turbidity bacterial suspension similar to cloudiness in ready-made McFarland tube standard. The inoculum was transmitted to a Muller-Hinton agar medium with sterile swabs. To find cells expressing hetero-resistance, antibiotic discs were placed onto these growth media using a flamed forceps, followed by incubation for 18 hours at 37oC. A transparent ruler was used to assess antibiotic inhibition areas, and the size of the zone was compared with the reference zone [25].
The technology of polymerase chain reaction (PCR) were applied to compare DNA strands with provided templates, and the specific sequence was multiplied into billions of copies (amplicon). In this study, Actinomyces meyeri and A. viscosus were detected by 16S rRNA genes, as shown in Table 1. All 18 (100%) A. meyeri samples identified with Vitek-2 system provided positive results for specific 16S rRNA gene at 519 bp (Figure 3), and all 19 (100%) A. viscosus samples identified by Vitek-2 system provided positive results for specific 16S rRNA gene at 787 bp, as shown in Figure 4.
All of the identified A. meyeri and A. viscosus were tested using disc diffusion method in Kirby-Bauer disc, to examine the in vitro antibiotic susceptibility of isolates that were isolated from dental caries using specific 16S rRNA primer. The utilization of selective antibiotic tests, such as amikacin, gentamicin, ciprofloxacin, amo­xicillin, ceftria­xone, cefixime, vancomycin, and erythro­mycin demonstrated their impact on A. meyeri and A. viscosus isolates. The results corresponded to CLSI, 2023 [26], and are shown in Table 4 and Figures 5 and 6. Descriptive statistics were abbreviated, and the results were analyzed using SPSS software, version 19.0 (IBM Corp., Armonk, NY, USA).

RESULTS

After examining 80 specimens, 72 (90%) produced positive cultures, whereas 8 (10%) produced negative cultures (Figure 1, Table 3). The results showed that 44 (68%) out of 72 positive cultures on various growth media were categorized as Gram-positive, and 28 (32%) were Gram-negative (Figure 2, Table 3). In total, 18 (40.9%) bacterial isolates were associated with A. meyeri, while 19 (43.1%) isolates were related to A. viscosus, as shown in Table 3. In all the isolates, antibiotic sensitivity of all the anti­biotics employed in this study against A. meyeri was 18 (100%) for amoxicillin, 16 (88.8%) for ciprofloxacin, 14 (77.7%) for ceftriaxone, 13 (72.2%) for amikacin, 12 (66.6%) for gentamicin, 11 (61.11%) for cefixime, 7 (38.9%) for erythromycin, and 3 (16.6%) for vancomycin. Amoxicillin showed the greatest rate of antibiotic sensitivity compared with other antibiotics employed in this study against A. meyeri.
In all the isolates, antibiotic sensitivity of all the anti­biotics employed in this study against A. viscosus was 19 (100%) for amoxicillin, 17 (89.4%) for ciprofloxacin, 14 (73.6%) for amikacin, 13 (68.4%) for ceftriaxone, 12 (63.15%) for gentamicin, 11 (57.8%) for cefixime, 7 (36.8%) for erythromycin, and 4 (21.02%) for vancomycin. The highest rate of antibiotic sensitivity was associated with amoxicillin among all the antibiotics employed in this study against A. viscosus.

DISCUSSION

Actinomyces species are available in different phenotypical and genotypical types. The cultivation of these types of species is considered difficult [27]. Furthermore, identification and differentiation of these anaero­bic micro-organisms in clinical specimens are incredibly troublesome due to their complex transportation and growth requirements as well as their meticulous and delayed growth habits [28]. Initial efforts were to isolate A. naeslundii and A. viscosus types using catalase activity [29, 30]. The advancement of PCR technologies made it possible to identify different types of Actinomyces species, such as A. bovis, A. viscosus, A. naeslundii, A. odontolyticus, and A. israeli using 16S rRNA gene analysis procedure [31]. The development of molecu­lar techniques corresponding to PCR and 16S rRNA improved the detection and distinction of closely related eubacteria species and enabled gene sequencing in a variety of ways [13]. A PCR methodology is extremely sensitive and may be adjusted to detect relatively few germs [32]. This ability of PCR is very helpful for distinguishing Actinomyces spp., since these organisms are available in low quantity in healthy individuals and not simply cultivated in wellness, which is attributable to their difficult and slow-growing nature [33]. Another characteristic of PCR is that it measures DNA rather than living organisms [34]. On the other hand, culture ways might not discover all micro-organisms, since they are not feasible and capable of growing into colonies [35]. Therefore, the diagnostic value of PCR is considered important once the micro-organisms are expected to be found in decay specimens [36]. The literature have investigated distinctly Actinomyces spp. In most studies on dental caries focused on complications related to these type of species in dental suppurative infections following some surgical procedures, it was confirmed that A. meyeri and A. viscosus may be found in dental caries [37]. Additionally, it was found to be part of the mixed flora in infections caused by Eubacteria species [38]. The aggregations of these types of micro-organisms are known clinically to form sulphur granules in abscesses and may complicate oral surgical treatments [39]. Because of this, current treatment plans include drugs that are effective against Actinomyces-associated lactamase producers, similar to Staphylococci, Enterobacter, and Gram-negative anaerobes [40].
The anti-microbial susceptibilities of numerous Acti­nomyces species include those of newer medications, such as augmentin [41]. A few individuals did not responded to amoxicillin at all or only in a limited degree, but they did responded favorably to long-term, high-dose treatment [42]. Rarely, treatment of chronic oral infections may last up to three weeks [43], strengthening the effect of amoxicillin using clavulanic acid. Also augmentin was found helpful, increasing the dosage of amoxicillin that is required in advanced chronic oral cases to increase tissue content of the drug, especially in mixed infections with enteric bacteria, such as Ente­robacter spp. [44]. In oral actinomycosis, the latter is typically true. Addition of antiprotozoal or clindamycin for strict anaerobes may be required to fight more severe zoonosis illnesses [45]. When mixed with β-lacta­mase inhibitors, amoxicillin was found to be powerful against Actinomyces spp. These data are in agreement with the results of a research [16]. In a clinical study, blood cultures and 16S rRNA sequencing techniques were applied to isolate Actinomyces species, specifically A. viscosus, from cellulitis infections that healed by intravenous administration of ampicillin. It was concluded that actinomycosis infections are easy to manage using a first-line treatment powered by penicillin and amoxicillin [46]. Additional data confirmed the effectiveness of penicillin [47]. In cases of bacteremia, subjects were treated by β-lactam antibiotics or cephalosporins in periods of 3 days to 6 months [48]. In antibiotic sensitivity tests, Actinomyces colonies were isolated from dental plaque samples adjacent to dental caries lesions with a percentage of 36.2%. All Actinomyces isolates were sensitive to penicillin and showed the highest inhibition among several types of antibiotics, with vancomycin reported as the lowest [12]. For patients allergic to aminopenicillins, tetracyclines and cephalosporins are suggested with a much lower clinical efficacy compared with penicillin and β-lactamase inhibitors [49].

CONCLUSIONS

The majority of antibiotics utilized in the current study against A. meyeri and A. viscosus showed a good rate of sensitivity. The highest rate of antibiotic sensitivity was associated with amoxicillin in all isolates, followed by ciprofloxacin, and then to a lesser degree, other antibio­tics used in this study, including amikacin, ceftriaxone, gentamicin, cefixime, erythromycin, and vancomycin.
Moreover, the detection of Actinomyces spp. with PCR analysis appeared to be extreme accurate when used for presentation of antibiotics. Additionally, according to the findings of the current study, amoxicillin should be considered as the first choice antibiotic agent against Actinomyces species.

CONFLICT OF INTERESTS

The authors declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article.

References