1. Introduction
Upper respiratory tract (URT) infections include inflammation of the nose (rhinitis), sinuses (sinusitis), middle ear (otitis media), pharynx (pharyngitis), tonsils (tonsillitis) and larynx (laryngitis). The microbiota of different regions of URT consists of a large number of diverse microorganisms. The most commonly isolated bacteria belong to the genera
Streptococcus,
Neisseria,
Haemophilus,
Moraxella,
Staphylococcus,
Corynebacterium,
Propionibacterium,
Prevotella, and
Porphyromonas [
1,
2,
3]. Among them, the most common causes of infections are:
Streptococcus pneumoniae,
S. pyogenes,
Haemophilus influenzae,
Moraxella catarrhalis, and
Staphylococcus aureus [
3,
4,
5,
6,
7]. The imbalance in the URT microbiota is associated with the increase in the number of invasive pathogenic bacteria, leading to inflammation and infection. Infection and development of the disease occurs after colonization of the mucous membrane of the URT by pathogenic bacteria [
2]. Colonization by different bacteria is a continuous process on the mucous membranes of the nose and throat in both children and adults.
Waldeyer’s lymphatic ring is a barrier to the penetration of pathogens into the respiratory and digestive tract. The Waldeyer’s ring includes: the pharyngeal tonsil (
tonsilla pharyngea) or adenoid, which is attached to the upper wall of the nasal part of the pharynx; tubal tonsils (
tonsilla tubaria) located in the immediate vicinity of the pharyngeal openings of the Eustachian tubes; palatine tonsils (
tonsilla palatina) located in the oral part of the pharynx, oropharynx, on the side walls between the anterior and posterior palatal arches and the lingual tonsil (
tonsilla linqualis) located on the last third of the tongue [
8]. It has a role in humoral (synthesis of a large number of immunoglobulins) and cellular immunity (activation of T and B lymphocytes) [
9]. The palatine tonsils play a role in initiating an immune response against antigens that enter the body through the oral cavity, while the adenoid protects the nasopharyngeal mucosa from airborne pathogens. These structures have the greatest immune activity in children aged 3–10 years. The palatine tonsils are known to produce five isotypes of immunoglobulins (IgA, IgM, IgE, IgD, and IgG), of which IgA is considered the most important because secretory IgA (SIgA) is a key component of the URT mucosal immune system [
10]. The tonsils and URT are protected from microbiological, allergological and other agents as long as the tonsils’ epithelium is intact. When the epithelium is disrupted, the function of the tonsils is disturbed, and lesions appear which are often filled with purulent contents. In diseased tonsils, the infection can spread lymphatically to regional lymph nodes. Chronic tonsillitis (CT) is a long-term infection that occurs as a result of multiple repeated infections of the tonsils. The pathogenesis of the disease is a consequence of several repeated episodes of acute tonsillitis or as a result of a persistent infection that leads to chronic inflammation that is long-lasting and slowly progressing. Inflammation can affect all lymphatic structures belonging to the Waldeyer’s ring. The palatine tonsils and adenoids are most often affected by inflammation [
11]. There are several ways to categorize this infection: (a) CT can be of bacterial, viral and fungal etiology, depending on the infectious agent underlying the chronic inflammation; (b) according to macroscopic and microscopic characteristics, CT can be hypertrophic or atrophic; and (c) in relation to age, CT can be divided into CT in children and CT in adults. Tonsillitis is the third most common among ENT (ear, nose, and throat) infections in the general population, after rhinopharyngitis and otitis. In France, about nine million cases of tonsillitis are diagnosed each year, while in Spain that number is four million [
12]. The prevalence of CT is up to 11.7% in United States, while the number of tonsillectomies annually increases. Recurrent throat infections or sleep-disordered breathing are indications for more than 530,000 tonsillectomies which are performed in children and adolescents in the US every year. Children with recurrent tonsillitis have significantly worse health status and physical functioning compared to healthy children of the same age [
13,
14].
Biofilm is considered the main microbiological factor that leads to chronic infections because it protects bacteria from the host’s defensive response and leads to the emergence of resistance to the applied therapy [
15]. The tonsillar tissue and adenoids are predisposed to biofilm formation due to cryptic tissue structure and direct, repeated exposure to respiratory bacterial pathogens [
16].
S. aureus in the form of biofilm can lead to the occurrence of chronic infections in the URT, including CT [
17,
18]. The nasal and oral cavity may act as the starting points for the spread of
S. aureus infection to other sites in the body and for the development of systemic infections [
19]. Except
S. aureus, several other respiratory pathogens, such as
Haemophilus influenzae,
Klebsiella pneumoniae and
Streptococcus pneumoniae, can persists predominantly intracellularly in the form of biofilm in adenoids and palatine tonsils [
2,
16,
20].
Given the importance of the biofilm in infection establishment, the emergence of resistant strains and difficulties in eradicating them, the aim of this study was to identify bacteria that cause CT, determine their sensitivity to commercial antibiotics, to examine their ability to form biofilm as well as to histologically confirm infection and the presence of bacteria in the tissue itself.
3. Discussion
This is the first study conducted in Serbia that examined the microbiological profile of the tonsillar tissue core. Chronic tonsillitis has a negative impact on quality of life. Patients most often complain on the frequent inflammation of the throat and purulent angina, recurrent peritonsillar abscesses, tingling, burning, and a constant feeling of a foreign body in the throat, bad breath, difficulty breathing and/or swallowing, persistent chronic infection of the nose, paranasal cavities, etc. The symptoms are especially pronounced in children, where often lead to chronic snoring or even suffocation during sleep. These symptoms are accompanied by poor nutrition and development of the child. Nasopharyngeal obstruction disrupts the ventilation of the eardrum and can lead to the development of secretory otitis with consequent deafness [
21]. Based on data conducted in our study (
Table 2), the most common symptom was sore throat (
Table 2). Many authors found that the most common symptoms for surgical removal of tonsillar tissue were: airway obstruction [
22,
23], sleep apnea [
24] and in other cases sore throat, fever, pharyngitis, stomatitis, and tonsillar tumor [
25]. Haidara et al., in a one-year follow-up of 315 patients, showed that the most common symptom of CT was swallowing pain—odynophagia (88, 63%), while other incidence-based symptoms were as follows: fever (86, 27%), snoring (38, 43%), ear pain (37, 65%), excessive salivation (20.39%), difficulty swallowing (15, 25%), sleep apnea syndrome (14, 12%), and shortness of breath (10, 20%) [
12].
According to Hibbert et al. [
26], in 55 (94%) of the cases with acute tonsillitis children were prescribed an antibiotic, which is in accordance with our data obtained by filling out the questionnaire (
Table 2). In support of the fact that in recent years there has been a higher number of resistant strains due to excessive use of antibiotics, Cavalcanti et al. [
15] reported that 83.6% of
Staphylococcus aureus isolates from tonsil tissue was resistant to penicillin and 13.6% to amoxicillin with clavulanic acid. Additionally, the present study demonstrated that
S. aureus isolates were resistant to penicillin (
Table 5). The ineffectiveness of penicillin has led to increased use of other antibiotics such as β-lactam inhibitors and cephalosporins [
15,
27]. Antibiotic resistance can be explained by the impossibility of antibiotic penetration and action in the tonsil core, (especially if the bacterial cells are covered with an biofilm extracellular matrix), the resistance of strains to typical antibiotic treatments due to the constant use of antibiotics due to recurrent infections and the prevalence of biofilm formation [
28]. Additionally, one of key factors to antibiotic resistance against penicillin group of antibiotics is beta-lactamase production. These enzymes are produced by Gram (+) bacteria extracellularly, and by Gram (-) bacteria in the periplasmic space. Β-lactamases can inactivate almost all β-lactam antibiotics by binding covalently to their carbonyl moiety and hydrolyzing the β-lactam ring [
29].
Oropharyngoscopic status showed that bilateral hypertrophy of the palatine tonsils was present in 82.35% of operated cases [
12], which is consistent with the present study (
Table 2). The data suggest that a number of pathogenic microorganisms isolated from infected tonsils may influence the development of tonsillar hypertrophy [
30]. Torretta et al. [
17], showed that there is a significant association between bacteria isolated from tonsil tissue that can form biofilm and tonsil hypertrophy.
Recurrent infection results in CT, where tonsillar hypertrophy leads to respiratory obstruction [
5]. In the present study, the most frequent bacteria were
S. aureus and
S. oralis (
Table 3). Our results showed prevalence of
S. oralis in children, which can be explained by the outbreaks of tonsillitis which is common in children of all ages, especially in the school-aged children in which is crowding common [
31]. Additionally,
S. oralis is known to be the first to colonize enamel, often interacting with periodontal pathogens such as
Porphyromonas gingivalis which is the causative agent of chronic periodontitis [
32]. Repeated and untreated Streptoccocal infection can lead to the colonization of some other pathogenic bacteria such as
S. aureus in adults (
Table 3). Additionally, in immunocompromised patients, it can cause bacterial endocarditis, respiratory diseases, and can also cause streptococcal septicemia [
33]. Jeong et al. [
34] identified
S. aureus (30.3%),
Haemophilus influenzae (15.5%), and
S. pyogenes (14.4%) as the most commonly isolated bacteria in patients with CT. Microbiological analysis of palatine tonsil and adenoid tissues, showed that species of the genera
Bacterioides and
Streptococcus are present in the crypts of the tonsils,
H. influenzae was observed in the tissue itself, while species of the genera
Fusobacteria,
Pseudomonas, and
Burkholderia were isolated from surface tissue layers. These results suggest that hyperplasia of the adenoids and palatine tonsils is probably the result of the presence of several species of pathogenic bacteria, which changes the previous perception that adenotonsillitis is a consequence of infection caused by a single bacterial species that colonizes only the tissue surface [
35]. In accordance with the obtained results (
Figure 1 and
Figure 2), a number of other studies in which the tissue of the palatine tonsils was analyzed, it was shown that the most common bacterial species in the tissues is
S. aureus [
18,
27,
36]. Galli et al. [
36] has shown that all isolated strains from palatine tonsil tissue have the ability to form a biofilm, whereas in the case of adenoid tissue 60% of species had the ability to form a biofilm [
36], while in the present study majority of
S. aureus isolates have strong biofilm-producing capacity (
Table 4). Other authors showed that except of
S. aureus-positive tissue samples, the most common isolates were:
Streptococcus anginosus,
Streptococcus constellatus,
S. viridans,
H. influenzae, and
H. parainfluenzae [
14,
32]. Several studies have identified
S. aureus as the main cause of tonsillitis, or as a likely co-pathogen with a prevalence of 83% [
17,
18,
37].
The presence of
S. aureus in the tonsillar tissue even after the inflammatory process may be related to its ability to form a biofilm (
Figure 3). The presence of biofilm explains the inactivity of antibiotics and thus the recurrence of infection [
15]. Chole and Faddis [
38] confirmed, by histological analysis, the presence of bacteria in the form of biofilm in the crypts of palatine tonsil tissue. Dense accumulations of G (+) bacteria were observed in the crypts, while inflammatory cells were seen on the periphery of these bacterial colonies. Bacterial colonies were detected by electron microscopy as an amorphous mass immersed in the extracellular matrix, which proved the authors’ presence of biofilm. Bacteria in the biofilm, within the tonsil tissue, are protected from the host’s defensive response and from the action of antibiotics and can synthesize various endotoxins without obstacles. Endotoxins in the crypts of the tonsils lead to chronic inflammation. Additionally, when local environmental conditions are favorable, bacteria in the biofilm become motile, resulting in re-infection [
39]. Immunohistological staining of the palatine tonsil section from which
S. aureus was isolated clearly showed an association between
S. aureus-specific staining and histological signs of tonsillitis (loose epithelium, tissue necrosis, presence of exudates) [
18].