Bergey’s manual of systematic bacteriology [15] was used as the early standard for identification. It is the leading resource for determining the identity of prokaryotic organisms, emphasizing bacterial species using every characterizing aspect. The advantages of this method are the assessment of living microorganisms, the ability to recognize viable cells in a sample, the easiness of quantitating cells in a piece, and its high sensitivity with appropriate media [16].

The use of molecular techniques for identifying bacteria in periodontology is relatively recent. 16S rRNA sequencing is a molecular biology technique targeting the 16S rRNA gene. This gene is present in all bacteria and contains variable regions that can be used for taxonomic classification. It provides high taxonomic resolution at the genus and species levels, making it suitable for identifying and characterizing individual microbial species within a sample. 16S rRNA sequencing involves PCR amplification of the 16S rRNA gene region, followed by DNA sequencing of the amplicons. The obtained sequences are compared to reference databases to assign taxonomic classifications to the microbes present in a sample.

NGS is a high-throughput sequencing technology that has revolutionized the field of genomics and metagenomics. It enables the rapid and cost-effective sequencing of large volumes of DNA or RNA. This method can be applied to various types of sequencing, including whole-genome sequencing, metagenomic sequencing, transcriptome analysis, and more. It is not limited to 16S rRNA genes and can sequence entire genomes or other genetic elements. NGS generates vast amounts of sequencing data, providing a comprehensive view of the genetic information present in a sample. It allows for a deeper understanding of community microbial diversity and functional potential within a community. NGS can provide both taxonomic information (similar to 16S rRNA sequencing) and functional information by sequencing the entire genetic content of organisms in a sample.

The absence of a dentogingival junction was the principal criterion for diagnosing chronic generalized periodontitis (CGP). The stage of severity was determined based on the depth of periodontal pockets and the degree of destruction of bone tissue. Thus, a mild degree of CGP is characterized by periodontal pockets less than 3 mm deep and the X-ray pattern confirming signs of initial destruction of the inter-dental septa. The depth of periodontal pockets varied from 3 mm to 6 mm in patients with a moderate stage of CGP; I–II degree of pathologic tooth mobility (according to tooth mobility index by Miller scale in Fleszar modification) was frequently revealed during the examination. According to the data obtained by X-ray examination, the destruction of the interdental septa’s cortical plate and bone tissue was equal to 1/2–1/3 of the length of the tooth root.

Periodontal pockets more than 6 mm deep, II–III degree pathological tooth mobility, cortical plate, and bone tissue destruction by more than 1/3 of the tooth root length are typical for the severe stage of CGP. Cortical plate and bone tissue destruction was revealed by X-ray examination.

Smears from periodontal pockets to study the quantitative and qualitative composition of the microflora of this biotope were taken. Our team collected plaque samples with a sterile inoculation needle from periodontal pockets and immediately applied them to nutrient medium and glass plates.

Researchers took samples from the patient’s gingival sulcus in the morning hours on an empty patient’s stomach with a sterile standard ISO (International Organization for Standardization) size 30 paper endodontic pin (DiaDent), 1 cm long, which, after soaking, the tooth was placed in a sterile saline solution and washed thoroughly.

Sugar meat-peptone agar and blood agar were used to isolate aerobic, facultatively anaerobic, and microaerophilic microorganisms [growth mediums (dry), Farmaktiv LLC]. The identification of isolated pure cultures was carried out according to morphological, cultural, and biochemical characteristics following generally accepted methods and the identifier of bacteria by Bergey (1994) [15]. Bergey’s manual of systematic bacteriology is the primary resource for determining the identity of prokaryotic organisms, emphasizing bacterial species, using every characterizing aspect. An isolated pure culture was identified by a complex of morphological, cultural, and biochemical properties (sets MLT00014 STREPTOtest 16_P, MLT00012 STAPHYtest 16_R, Erba Lachema).

The presence of Fusobacterium, Veillonella, Prevotella, and Porphyromonas was determined according to morphological and cultural properties. Smears were taken from periodontal pocket content and stained using Gram and Romanowsky-Giemsa methods, followed by immersion microscopy. To inoculate bacteria, the method of sectoring on dense nutrient media was employed to determine the specific content of each group of microorganisms in 1 g of biomaterial. The number of colony-forming units (CFUs) expressed the degree of microbial colonization. After counting the CFU, the data was converted into decimal logarithms (lg CFU).

The Bioethics Committee of I. Horbachevsky Ternopil National Medical University Ministry of Health of Ukraine approved the study protocols (excerpt from protocol No. 73) on April 3, 2023. All the patient investigations conformed to the principles outlined in the Council of Europe Convention on Human Rights and Biomedicine (April 4, 1997), the World Health Association Helsinki Declaration on Ethical Principles for Scientific Research with Human Participation (1964–2000), and the Order of the Ministry of Health of Ukraine No. 281 of November 1, 2000; Declaration of Helsinki “World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects” (2001), Code of Scientist of Ukraine (2009). Doctors informed every patient about the study’s purposes. Patients signed their “consent of the patient”.

Statistical analysis and testing of hypotheses for the resulting findings were performed by means of parametric criterion Z-test [17, 18].

Microbiological studies have shown that various microorganisms inhabit the gingival sulcus and periodontal pockets in chronic catarrhal gingivitis and CGP in patients with primary hypothyroidism. In patients with hypothyroidism, 536 strains of microorganisms that were either aerobic, facultative aerobic, or anaerobic were identified.

The degree of general insemination of the studied microbial oral ecosystem of patients with primary hypothyroidism and periodontal disease was on average 4.4 lg CFU/mL ± 1.2 lg CFU/mL, ranging from 2.7 lg CFU/g ± 0.2 lg CFU/g (non-pathogenic Neisseria) to 6.8 lg CFU/g ± 0.2 lg CFU/g (oral streptococci, Table 2). In this study, the sample mean is denoted as M and the standard error as m.

Microorganisms’ colonization density of periodontal pockets has reached clinically significant indicators. Half of the patients have in periodontal pockets potentially pathogenic Gram-negative bacteria belonging to the following genera: Veillonella (in 66.7%), Fusobacterium (68.3%), Spirochaeta (75.0%); pathogenic β-hemolytic streptococci (51.7%); non-pathogenic Neisseria (55.0%). Gram-negative Enterobacteriaceae were in some patients, in particular strains of Escherichia coli and Klebsiella (35.0% and 15.0%, respectively). The density of their colonization of periodontal pockets was 2.3 lg CFU/mL ± 0.1 lg CFU/mL and 1.2 lg CFU/mL ± 0.3 lg CFU/mL, respectively.

The studies showed that the composition of the microflora of periodontal pockets in patients with thyroid hypofunction was poly-microbial and represented by various associations of microorganisms. Most of the associations consisted of 5 or more species of bacteria (in 78.3% of patients). In 15% of cases, associations with 3–5 types of microorganisms, only four patients (6.7%) have two-component associations.

Microbiomes of periodontal pockets are prevalent in species of anaerobic and facultative anaerobic microorganisms. Most patients with hypothyroidism have the leading “biomarker bacteria” and periodontopathogenic microorganisms: Prevotella, Porphyromonas, and Spirochaeta.

An α-hemolytic streptococci was isolated in almost all patients (93.3%). The density of their colonization of the periodontal ecological niche in average of 6.8 lg CFU/mL ± 0.2 lg CFU/mL is less than standard data. Only 68.3% of patients have lactobacilli cultures; their density was equal to 3.4 lg CFU/mL ± 0.2 lg CFU/mL.

It indicates the influence of systemic disease on the development of dysbiotic processes in the oral cavity. Porphyromonas strains, which belong to Gram-negative non-spore-forming anaerobes, were found in most patients with hypothyroidism (83.3% of patients) and coagulase-positive and coagulase-negative staphylococci in 75.0% and 78.3% of cases, respectively.

Analysis of the results of microbiological studies of patients with gastroduodenal pathology associated with Helicobacter pylori showed that the microflora of the pockets was poly-associative. Potentially pathogenic and pathogenic microorganisms were found in all patients. This microflora represents associations of various species of the Streptococcus genus and Staphylococcus genus, as well as lactic acid bacteria, fungi, and anaerobic microorganisms. On average, associations containing 3 to 6 types of microorganisms were inoculated from the sample. Patients with gastroduodenal pathology have aerobic-anaerobic associations in 40% of cases, aerobic-anaerobic-fungal in 48%, and anaerobic-fungal associations in 12% of observations. Streptococcus spp. represented the main part of the aerobic microflora. The dominant species was Staphylococcus haemolyticus (77.3%). Analysis of the species composition of the anaerobic microflora of the periodontal pockets showed significant inoculation with Gram-negative microorganisms (Bacteroides forsitus, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis) (Figure 1). Peptostreptococcus anaerobius was detected in 35% cases in the whole spectrum of Gram-positive anaerobic microorganisms. The analysis of the quantitative composition of the microflora of the periodontal pockets showed that contamination by the previously mentioned types of bacteria in the main group averaged from 1 × 10⁵ to 1 × 10⁸ CFU/mL. Microscopic studies revealed significant microbial contamination of periodontal pockets of patients with gastric and duodenal ulcers. There was a shift in the microbial spectrum toward an increase in Gram-negative rods, associated with a decrease in Gram-positive rods and Gram-negative cocci. During bacteriological studies, staphylococci and streptococci were most often identified in diagnostically significant concentrations among facultative anaerobic microorganisms and among obligate anaerobes—Fusobacterium spp., Peptostreptococcus spp., Prevotella spp., Porphyromonas gingivalis. In high concentrations from 3 × 10⁵ CFU/mL to 2 × 10⁸ CFU/mL, Fusobacterium was isolated in 77.2% of patients. The dominance of these microorganisms in the microbial associations of periodontal pockets is characteristic. A high frequency of detection (67%) of Candida fungi with a colonization rate of 1 × 10²–1 × 10⁴ CFU/mL was determined.

The microflora of periodontal pockets of patients with colitis was dominated by cocci, which accounted for 49.8% of all cultured microorganisms. Gram-positive facultative anaerobic cocci belonging to the genera Staphylococcus and Streptococcus were dominant (Figure 2). In addition, the spectrum of microbiocenosis included anaerobic gram-negative rods, which made up 35.0% of the microbial community, Candida fungi (11.0%), and Spirochaeta (7.0%). The number of lactobacilli decreased (6.0%), which indicates dysbiosis of the periodontal pockets. Enterobacterium, such as Escherichia coli and Klebsiella spp., occupied the smallest segment in the microbiocenosis of the periodontal pockets (2.7%), which belonged to invasive microflora.

The degree of total microbial colonization of the gingival sulcus of patients with catarrhal gingivitis was 5.62 lg CFU/mL ± 2.32 lg CFU/mL. The periodontal pocket microorganism colonization density was 6.17 lg CFU/mL ± 2.56 lg CFU/mL, which is a few times higher than in patients with chronic gingivitis (Figure 3).

The microbiological studies of samples from patients with chronic pancreatitis showed that various microorganisms inhabit periodontal pockets and gingival sulcus (Table 3). The degree of its total colonization ranged between 7.23 lg CFU/mL ± 0.09 lg CFU/mL in the case of chronic catarrhal gingivitis and 7.37 lg CFU/mL ± 0.15 lg CFU/mL in the case of CGP. Microorganisms in periodontal pathology were inoculated from gingival and periodontal pockets with approximately the same inoculation density. Most microorganisms were isolated within the same order, except for Escherichia coli. Their number was a few times higher in patients with CGP than in those with chronic catarrhal gingivitis (2.19 lg CFU/mL ± 0.13 lg CFU/mL vs. 1.83 lg CFU/mL ± 0.10 lg CFU/mL, respectively). α-hemolytic streptococci, Spirochaeta, Fusobacterium, and Veillonella, which formed the main normal microflora of gingival and periodontal pockets, dominated this biotope. Staphylococci (78.6%) and lactobacilli (73.8%) were patients’ second most frequently detected bacteria. In patients with CGP, the frequency of detection of coagulase-positive staphylococci was approximately the same (77.8%), while the frequency of coagulase-negative staphylococci was slightly lower (66.7%). Lactobacilli, which also belong to the normal microflora of the oral cavity and gum pockets, were cultured in periodontitis with a frequency almost 20% lower than in gingivitis. Saprophytic Neisseria and Corynebacterium were also detected less often in the second case. The frequency of detection of β-hemolytic streptococci, which, like staphylococci, are considered pyogenic cocci and play a significant role in the development of the inflammatory process, has increased significantly (from 26.2% in chronic catarrhal gingivitis to 72.2 % in CGP). At the same time, Escherichia coli, Klebsiella, and Candida fungi were cultured more frequently from the contents of gingival sulcus and periodontal pockets. Microbiological studies of the microbiocenosis of periodontal pockets of patients with CGP and gastrointestinal tract pathology revealed that the frequency of anaerobic microorganisms’ detection was higher than in patients with generalized periodontitis without concomitant systemic pathology, i.e., Helicobacter pylori (7.0 times), Bacteroides forsythias (4.2 times), Fusobacterium nucleatum (5.2 times), Prevotella intermedia, and Porphyromonas gingivalis (5.0 times).

According to the research results, we can assume that a significant prevalence of periodontal diseases and lesions of the hard tissues of teeth in patients with associated systemic pathology indicates the direct influence of these concomitant diseases on the state of the patient’s dental health.

The degree of colonization by microorganisms in these pathologies differed slightly. At the same time, the frequency of detection of specific populations of opportunistic bacteria (i.e., β-hemolytic streptococci, Escherichia coli, Klebsiella, Candida fungi) increased in CGP compared to chronic catarrhal gingivitis.

The microbiological study of periodontal and gingival pockets of patients with periodontal pathology accompanied by systemic diseases indicates the need for a differentiated approach to antibacterial therapy.

It is important to emphasize that systemic diseases, disorders, or conditions do not cause periodontitis but instead predispose, accelerate, or otherwise increase disease progression.