Characterization of upper respiratory tract isolates from children with recurrent tonsillitis

To analyze the profile of airway Upper Respiratory Tract isolates from children with recurrent tonsillitis. Samples were taken using nasal cavity, oropharynx and nasopharynx swabs from30 children called the test group before tonsillectomy. Counting, isolation, identification and sequencing of the 16S rRNA, biofilm production and antimicrobial sensitivity investigation were performed. Staphylococcus aureus (S. aureus) was the only microorganism recovered in 36.6% of patients, being more present in the oropharynx and with greater resistance to Research, Society and Development, v. 10, n. 15, e363101523181, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i15.23181 2 erythromycin 95%, penicillin 85% and cefoxitin 85%. All isolates were formed by biofilm, 20% formed by strongly adherent biofilm. S. aureus resistant and biofilm formed, were isolated in the three studied sites, suggesting that this species contributes to recurrent tonsillitis. Impact of the study: The ability of S. aureus to acquire resistance and its other associated factors such as biofilm formation, can make this microorganism recover more easily in the microbiota of patients, taking into account the other microorganisms present there after the use of antibacterial.


Introduction
The microbiota of a site is defined by the presence of microorganisms and their metabolites present in the different sites of the host (Thursby and Juge2017). Each site has its own microbiota, with specific species (Coyte et al., 2015). The airway Upper Respiratory Tract (URT) is divided into two parts, the upper and the lower and, each of these sites has a specific microbiota, the URT microbiota is distinct from each other and has patterns of colonization and succession. Since early childhood, URT has been colonized by bacterial species of different genera, such as Streptococcus spp., Haemophilus spp., Coagulase negative Staphylococcus, Streptococcus viridans, Haemophilusinfluenzae, Streptococcus pneumoniae, Moraxella catarrhalis and Staphylococcus spp. (Round and Mazmanian 2009;Johnston and Douglas, 2018). Respiratory tract infections are described when they affect the respiratory tract, both upper and lower, in which there is obstruction of the air passage, both at the nasal and pulmonary levels (World Health Organization 2014) being a major concern worldwide, as they are responsible for high morbidity and mortality, especially in children and the elderly (de Steenhuijsen Piters et al., 2015). Although frequent, when infection occurs in URT, they usually do not cause death, but significantly interfere with the individual's quality of life, in addition to generating costs for the country's health (World Health Organization 2014).
The Department of Information Technology of the Unified Health System (DATASUS) recorded that during 10 years (2009 to 2019), 13,030,947 patients were hospitalized due to respiratory diseases. Of this, 553,511 were individuals affected by chronic diseases of the tonsils and / or adenoids. Children are the patients most affected by tonsillitis and may have acute or chronic illness (Brazil 2020).
Palatine tonsils are aggregates of lymphoid tissue, located in the oropharynx (Lima et al. 2015), in certain circumstances, infections called tonsillitis can occur (Georgalas et al., 2014). In addition to viral conditions, tonsillitis can also develop as a result of opportunistic bacterial infections, caused by bacteria from the microbiota itself: ß-hemolytic Streptococcusof group A, Staphylococcus aureus(S. aureus) and Haemophilusinfluenzae (Wang et al., 2017).
Repetitive tonsillitis can occur due to the predominance of Gram-positive bacteria, which produce biofilm, among which is S. aureus (Torrettaet al. 2016). In addition to treatment failure due to the presence of resistant bacteria and, the acquisition of resistance to antibacterials (Nejashmikjet al. 2017;Cavalcanti et al., 2019).
A study conducted in Belgium showed that the nasal cavity of 20% of the population is persistently colonized byS.
aureus, considered one of the main sites for the spread of the bacteria in URT (Sivaraman et al., 2009). It is believed that 30% of individuals who develop infection with S. aureus in the nasopharynx, are due to bacterial migration from one site to another (Jiménez-Truque et al., 2016). Migration can also reach the oropharynx and can be detected on the surface or in the nucleus of tonsils (Pereira et al., 2008).
Understanding the colonization dynamics of the most incident bacteria, transmission routes, risk factors for infection progression and conditions that promote the appearance of bacterial resistance to antibacterials; will allow the optimization of strategies to effectively control the bacteria present in recurrent tonsillitis in children, contributing to significant improvement in the quality of life of affected patients. Thus, the objectives of this study were to analyze the profile of URT isolated from children with recurrent tonsillitis.

Methodology
This study deals with an experimental research (Köche, 2016;Pereira et al., 2018) for its methodological basement.

Obtaining of the samples
It is a laboratory research, the study was a cross-sectional observation from January to December of 2019, submitted to and approved by the Ethics Committee of the Clinical Hospital of the Federal University of Goiás (CEP/HC/UFG), under number 84908818.3.00005083.
To obtain the test samples, children who already had surgical indication for tonsil removal were selected who attended the Otorhinolaryngology Clinic of the Federal University of Goiás Hospital. The number of children chosen was based on the flow of surgeries that occurred in this clinic for tonsil removal per year. The sample number of was thirty patients. The samples were collected in three different sites of the URT, nasal cavity, oropharynx and nasopharynx; the samples were obtained before the surgical procedure of tonsillectomy, however, with one patient already sedated. As the children in the test group had undergone recent antibiotic therapy due to recurrent infections, four children who had not used antibacterial for ninety days and had surgical indication for adenoid hypertrophy only, with no infection present for a control group, were selected. Both samples were processed in the same way. Research, Society andDevelopment, v. 10, n. 15, e363101523181, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i15.23181 4 The clinical specimen of the oropharynx was obtained with a swab, after opening the oral cavity, using a mouth opener, next to one of the anterior tonsillar pillars. The material of the nasal cavity was obtained with a swab, close to the inferior nasal concha, with the aid of a nasal speculum, to prevent contact with the nasal vestibule. In the nasopharynx, the sample was collected with a swab, through the mouth, moving the palate away. The swabs, duly identified, were introduced into Stuart transport medium, later; they were stored at room temperature and transported immediately for processing.

Sample processing and standard plate counting
For processing, the swabs containing the clinical specimens were inserted in 3mL of brain heart broth (BHI). A 10 1 dilution was performed, transferring an aliquot of 1 mL of BHI with the sample, to a test tube containing 9 mL of peptone water and subsequently homogenized.
An aliquot of 100µL of each dilution was sown, containing salt mannitol agar, GC (gonococcal) base agar, chocolate agar and blood agar. Samples shown on mannitol agar were incubated in aerobiosis at 37 °C for 24 to 48 hours and samples in the other culture media were incubated in CO2 at 37 °C for 24 to 48 hours.
After growth, morphocolonial characterization, counting and isolation of colonies on nutrient agar was carried out, with subsequent incubation. Then, the morphotintorial characterization and the 3% potassium hydroxide test (3% KOH) were performed.

Identification of the most incident URT bacteria
After the morphotintorial analysis, the bacteria were subjected to biochemical tests for identification (Table 1)  Plaque counting was performed to estimate the number of bacteria in the original sample at each site studied. The calculation used was to take the number of colonies on the plate and multiply by the sample dilution index = number of bacteria / mL (Tortora et al. 2012).

Antibacterial sensitivity test
Antibiogram tests were performed using the disk diffusion method. The choice of antibacterials was based on recommendations by the Institute of Clinical and Laboratory Standards (CLSI 2019).
At first, a bacterial suspension in 0.85% saline solution was performed until turbidity, corresponding to the 0.5 MacFarland scale. A swab was embedded in this suspension and the sample was seeded on the surface of Mueller Hunton agar.
Paper disks containing the selected antibiotics were placed on the agar surface. Subsequently, the plates were incubated at 37 °C for 18 hours. For the reading of the tests, the diameter of the inhibition halos was measured and the results were interpreted according to the CLSI (2019). To evaluate the activity of β-lactamase resistance, mediated by the blaZ gene, the measure of the penicillin halo was considered. The isolates were indicative of enzymatic activity, with the formation of an inhibitory halo ≤ 29 mm in the penicillin disc, with bounded edges. Resistance to oxacillin mediated by the mecA gene was also evaluated, considering the measurement of the cefoxitin halo. Isolates with inhibition halo ≤ 21 mm were considered resistant to oxacillin / cefoxitin.

Biofilm assay
The biofilm assay was performed using microtiter plates according to the method of Tendolkaret al. (2004), with adaptations.
After the growth of bacteria on nutrient agar, the strains were inoculated in 4 mL of Trypticase soy broth (TSB), plus 1% glucose and incubated in aerobiosis for 18 hours at 37 ºC. A 0.5 mL aliquot of the TSB broth with bacterial growth was transferred to another tube containing 4.5 mL of TSB -10 1 dilution. A total of 200 μL of the dilution was transferred to a polystyrene microplate, U-bottom, with 96 wells.
The tests were performed in triplicate. For the negative control, 200 μL of the TSB medium without inoculum were used, also in triplicate. For the positive control, strain of Pseudomonas aeruginosa PA01 was prepared, also in triplicate, according to the methodology performed for the samples of S. aureus to be tested. The plates were incubated at intervals of 24 hours and 48 hours, at 37 ºC.
After the incubation period, samples were aspirated from each well, using a pipette with individual tips for each sample, so that there was no contamination. Then, with the aid of a pipette, each well was washed three times using phosphate buffered saline (PBS) solution, pH 7,2.
The plates were dried at room temperature for one hour. After drying, 200 μL of methanol were deposited in each well and incubated for 15 minutes, to assist in fixation, subsequently the methanol was removed.

Amplification of the encoding gene for the 16S rRNA subunit
The molecular identification of bacterial isolates started with the extraction of genomic DNA according to the methodology described by Van Soolingen and collaborators (1994). The integrity and purity of the DNA were evaluated in agarose gel at 1 % and the concentration of DNA determined in NanoVue Plus UV-Vis (GE Healthcare®) spectrophotometer.
The genetic material was kept in a freezer at -4 ºC until preparation for sequencing. Then the amplification of the coding region for the 16S subunit was performed rRNA by PCR with the use of 27F universal primers (

Sequencing the 16S rRNA coding region and analyzing the results
Sequencing was performed using primers 27F, 1541R, 926F (5′-AAACTYAAAKGAATTGACGG3′), 530F (5'-TGACTGACTGAGTGCCAGCMGCCGCGG-3'), 519R (5'-GTNTTACNGCGGCKGCT-GNGGCGNGG-3'), the sequencer used was ABI 3500 from Applied Biosystems® at Center for Biological Resources and Genomic Biology (CREBIO). The sequences obtained were analyzed using the Codon Code Aligner software (Codon Code Corporation) and compared for homology, with the GenBank NCBI database using BLASTn. The sequencesobtainedweredepositedat Gene Bank NCBI (Table2). The dendogram was obtained in the MegaX software from a set of DNA sequences by means of a computational method based on distance matrices. The distances were calculated through the Jukes and Cantor model and after calculating the distances and filling in the matrix the Neighbor-Joining algorithm were used to infer the similarity relations between the sequences. ClustalW alignments were also performed in the MegaX software.

Results and Discussion
Among a total of 30 children, 56.6% (17/30), were male. The age of the patients who participated in the research ranged from four to 12 years, with a mean of 8.18 and a standard deviation of 2.6. The age and sex variables did not show any correlation, this is an indication of sample homogeneity.  Table 3.
According to the results obtained in this study, S. aureus was the most prevalent microorganism and its colonization was mainly in the oropharynx (8/11), followed by the nasal cavity (7/11) and nasopharynx (5/11). From these results, the other tests were developed with samples of S. aureus.

Colony count of S. aureus at the evaluated sites
For the colony forming unit (CFU) count at each evaluated site, a higher concentration of S. aureus can be seen in the oropharynx, followed by the nasal cavity (Table 4).

Antibacterial sensitivity test
For the antibiogram test, 20 strains of S. aureus from the different sites surveyed were analyzed. According to the results (Figure 1)

Inducible resistance to clindamycin in clinical isolates of S. aureus
In the antibiogram test, it was possible to detect the presence of inducible resistance to clindamycin by the D-test in (30%) (6/20) of the tested S. aureus.

Capacity of S. aureus to form biofilm
In the test to detect biofilm production, 100% (20/20) of S. aureus samples were positive for biofilm formation, with 80% (16/20) presenting moderate form and 20% (4/20) presenting strong form to biofilm. The anatomical sites were distributed equally in the biofilm formation; there was not a site that formed in a prevalent way.

Sequencing the 16S rRNA
We did the sequencing of S. aureus using the gene 16S rRNA. After confirming the S. aureus by sequencing, the sequence was analysed by NCBI and deposited on Gene Bank. On Table 5 are shown twosequences from genbank that presented higher identity with these study samples. The alignments (Figure 2A) corroborate with the results found in the dendogram, showing that the sequences that belong to the branches in common have a strong homology, this homology being represented by the *, characterizing that they have the same evolutionary ancestry.
Next to the branches ( Figure 2B) is the percentage of replicated trees in which the associated taxons were grouped in the bootstrap test (500 replicas).
Based on the method used (Jukes and Cantor) this dendrogram was drawn in scale, so the length of the branches is in the same units of the evolutionary distances, so it is observed that the sequences obtained from the samples RF18 and RF15; OR5 and CN6; RF2 and CN7; OR6 and CN3; OR18 and OR16 are in very similar lengths of branches evidencing close evolutionary relations between such samples.Clades 93 and 96 showed a common evolutionary relationship.

Discussion
A research carried out in Hungary, evaluated the presence of the same pathogens from the present study in healthy children, a total of 580 children were evaluated with a surface swab, 210 (36.20%) children were positive for at least one of the investigated microorganisms. Our control group has similar results as the number of children investigated in Hungary, according with the patient's history (Kovács et al., 2020).
The group A Streptococcus pyogenes is described as being responsible for tonsillitis in pediatric patients, being cited by many authors as the most important cause of infection (Sidell and Shapiro 2012;Pontin et al. 2016). A study carried out in Russia, with 54 pediatric patients undergoing tonsillectomy, for treatment of recurrent tonsillitis, identified the presence of S.
Another study carried out in Poland, with 71 pediatric patients, submitted to tonsillectomy for the treatment of recurrent tonsillitis, and demonstrated the presence of S. pyogenes in 8.3% (Katkowska et al., 2017).
It is suggested that the decision to diagnose recurrent tonsillitis, be based on the patient's history and physical examination, that is, without a laboratory examination, it can not be said which bacteria is responsible for recurrent tonsillitis.
In the present study, we did not have access to laboratory tests previously performed (Marinho, 2010).
In the present study, S. aureus was prevalent in this study, (36.6%) of patients. Similar data were found by Gul et al. The children volunteers of this study were surgically indicated, due recurrent tonsillitis. The predominant isolation of S. aureus corroborates the results published by Cavalcanti et al. (2019), who described the presence of S. aureus in patients with recurrent tonsillitis, suggesting that this bacterial species may be part of the etiology of recurrent tonsillitis.
A study carried out with children from New Zealand, demonstrated that S. aureus was present in a greater proportion in the nasal cavity regions (39%) and in the oropharynx (32%) (Berry et al., 2015). Similar data were isolated in this study,  The biofilm presence in cases of S. aureus infection is described as a factor that makes the treatment difficult (Brackman et al., 2013). All isolates in the present study showed the ability to form biofilm, ranging from strong to moderate.
This finding confirms the results of other studies, which also correlate the involvement of biofilm-forming S. aureus in cases of recurrent tonsillitis (Torrettaet al. 2013;Torrettaet al. 2015).
Amoxicillin is used as the first choice for the treatment of recurrent tonsillitis. In these situations, antibacterial resistance is mainly related to the use of β-lactams. Since 1970, there have been reports of therapeutic failures using amoxicillin. Therapeutic failures are justified by the bacterium producing the enzyme β-lactamase that inactivates the antibacterial action. This resistance mechanism is common to S. aureus (Pichichero, 1995;Katkowskaet al.,2017).
In the present study, patients used amoxicillin, amoxicillin + clavulanate, ampicillin + sulbactam and acetyl cefuroxime, all belonging to the class of β-lactams. The S. aureus isolated, showed greater resistance to erythromycin, penicillin, cefotaxime and ciprofloxacin. These drugs are macrolides, β-lactams and quinolones classes and have different mechanisms of action. The resistance mechanisms of S. aureus are efflux pump, modification of the target site and production of enzymes (Foster, 2017;Haaber et al., 2017;Vestergaard et al.,2019).

Conclusion
We conclude that S. aureus can be isolated from any of the studied sites, suggesting that the three sites should be used for the collection of clinical specimens in case of investigation. Antimicrobial resistance and the ability to form biofilms suggest that S. aureus contributes to recurrent tonsillitis. On the other hand, the chemical and physical resistance found in this study, may have contributed to bacteria recovering after the use of antibacterials, colonizing patients' microbiota more quickly compared to other colonizing species of the three anatomical sites.
Suggestions for future work is study a big number of the samples, to have a good perception of the microbiota that are located on tonsile