CC BY
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Section 2. Biomedical science
Section 2. Biomedical science
Kabanova Arina Akexandrovna, Vitebsk State Medical University, Assistant professor, the Faculty of Stomatology E-mail: arinakabanova@mail.ru
Pohodenko-Chudakova Irina Olegovna, Belorussian State Medical University, Head of Oral Surgery Chair, the Faculty of Stomatology
E-mail: ip-c@yandex.ru
The ability to form a biofilm by odontogenic infectious agents obtained from patients with odontogenic pyoinflammatory processes of various prevalence
Financial support: Grant — Belarusian Republican Foundation for Fundamental Research, contract M14M-093 from 23.05.2014
Abstract: The purpose of the study was to investigate the biofilm formation by bacteria agents causing odontogenic infections. 117 patients with pyoinflammatory diseases of the maxillofacial region were examined. During the study it was revealed that the odontogenic infection pathogens were able to form the microbial biofilm in a varying degree, P aeruginosa had the strongest biofilm formation ability and S. epidermidis had the least biofilm formation ability.
Keywords: bacteria, biofilm, odontogenic infections.
Introduction
The issue of pathogenesis, diagnostics, treatment and prevention of odontogenic pyoinflammatory diseases of the maxillofacial area are still of current importance in modern medicine [1, 242-243; 2, 28-34; 3, 199-206]. The increase of progressive phlegmon cases, frequently complicated by contact mediastenit, thrombosis of the cavernous sinus of the dura mater, brain abscess sepsis was determined in the Republic of Belarus in recent years [4, 107-111; 5, 36-40].
Infectious process is the manifestation of the interaction of human organism and microorganisms. Currently, the main part of microbiologists admitted that the majority of microorganisms in natural and artificial environments exist in the form of structured, attached to the surface communities — biofilms (BF) [6, 1714-1724; 7, 847-867].
Biofilm is a microbial community characterized by cells attached to a surface or to each other
and enclosed in a synthetic matrix of extracellular polymeric substances. This community demonstrates changes in phenotype growth parameters and specific genes expression [8, 167-193]. The characteristic feature of all biofilms is their striking resistance to physical and biochemical influences, including antibiotic resistance [9, 135-138]. In spite ofthe fact that the resistance is recognized for many years, its biological basis is still not fully explained. Factor, which partly explains the resistance of phenotype includes high cell density and physical displacement of antibiotic. The physiological changes may occur within a biofilm, including general stress response, closing key metabolic processes and the induction of protective mechanisms [10, 34-39].
The population of cells in the biofilm is heterogeneous and includes fast- and slow-growing bacteria. A number of them are resistant to the antibiotic due to the expression of enzyme inactivation, the rest don’t express the similar systems. Macroorganism
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Theabilitytoformabiofilmbyodontogenicinfectiousagentsobtainedfrompatientswithodontogenicpyoinflammatoryprocessesofvariousprevalence
resistance depends on the interaction between the whole cell population and treatment directed against the multicellular community. Formation of biofilms is a complex process consisting of several stages such as cell adhesion on the surface and redistribution of cell mass; active cell dividing to create cell clusters; formation of the exopolymeric slimy matrix, the distribution of biofilm cells in the environment. Initial microbial cell attach to the surface of the substrate is carried out by the action of electrostatic, hydrophobic forces, van der Waals forces, non-specific adhesion. Fimbriae and flagella play an important role in cell adhesion and aggregation of gramnegative microorganisms [11, 1855-1863]. It is revealed, that the level of adhesion with the subsequent formation of biofilms is mostly expressed in materials such as latex, silicone, polyvinyl chloride. Adherence to polyurethane, stainless steel and titanium are less evident. All above-listed materials are widely used in medical practice. Thus it is an additional factor of biofilm formation which leads to the development of infections with clinical resistance to antimicrobial therapy [12, 1567-1572].
It is revealed by using confocal laser microscopy, scanning electron microscopy that biofilms are complex three dimensional structures. After the adhesion microorganisms begin to proliferate rapidly to form multicellular layers and abundantly synthesize exopolymer matrix components [13, 1454-1465]. The matrix is chemically multivendor and varies in different microorganisms [14, 157-168]. The extracellular layer contains up to 40-95 % polysaccharides [15, 2119-2130]. The concentration of other chemical components varies greatly. Proteins can amount to 60 %, lipids to 40 %, and nucleic acids to 1-20 %. These compounds are in a hydrated condition because water forms 80-90 % of the biofilm [16, 495].
Cells within the matrix are arranged in a certain way. The structure of multicellular clusters is represented as formations that resemble pillars “cemented” in exopolysaccharide layer thus allowing to maintain the concentration of nutrients necessary for the growth of the population and cell protection. Matrix is divided by canals filled with water, and also has cavities and voids. Nutrients and oxygen are transported through the canals from the external to
the internal parts of the biofilm and simultaneously metabolites are removed from the cells. Bacterial cells in biofilms have a complex organization with a specific polymorph cytoarchitectonics. The cells with strongly altered morphology and dead cells are revealed [17, 157-168].
Multilayer topography affects the metabolism and the physiological activity of cells. Peripheral layers are highly aerated in comparison with the central parts where favorable conditions for anaerobes are generated. A reduced metabolism of microorganisms in the biofilm leads to antibiotic resistance, because antibacterials are most effective against metabolic active cells [18, 130-138].
The purpose of the study was to investigate the biofilm formation of the bacteria causing odontogenic infections.
Material and methods
Clinical methods
A complex examination of 172 patients with pyo-inflammatory diseases of the maxillofacial region was performed. All patients underwent stationary treatment in the Department of Maxillofacial Surgery of the «Vitebsk Regional Clinical Hospital» from 2012 to 2014.
Patients were divided into four groups: group 1 (n = 40) — patients with acute suppurative odontogenic periostitis of the jaw; group 2 (n = 82) — patients with odontogenic jaw osteomyelitis, complicated by the odontogenic phlegmon of one cellular space; group 3 (n = 32) — patients with odontogenic jaw osteomyelitis, complicated by the odontogenic phlegmon of more than one cellular spaces; group 4 (n = 18) — patients with odontogenic phlegmon of the mouth floor.
The diagnosis — acute odontogenic inflammatory process, age over 18 years, voluntary informed consent to participation in the study became the criteria for patient inclusion in the study. Exclusion criteria were: age less than 18 years, pregnancy, concomitant diseases, lack of voluntary informed consent. The average age of patients in group 1 was 39.2 ± 14.5 years, group 2 - 39 ± 14 years, group 3 - 34.5 ± 11 years, group 4 - 34.2 ± 10.5 years.
Therapeutic activities for patients with pyoin-flammatory processes of the maxillofacial region were complex and included surgical treatment and
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Section 2. Biomedical science
pharmacotherapy. Surgical treatment of purulent focus had been performed on the day of hospitalization. Antibiotic therapy, analgesic therapy, desensitization, detoxification were administered.
Hospitalization duration and illness duration before hospitalization were studied.
During the surgery the wound discharge samples were received for further microbiological study. After aspiration the content of the abscess was placed in a transport system with a growth medium of Carrie Blair.
Microbiological methods
Blood agar was used for various types of streptococci detection. Staphylococci were isolated on the yolk-salt agar, Saburo medium was used to detect fungi, Endo medium or Levin medium was used to detect coliform bacteria, seeding of Proteus group microbes was performed by the Shushkevich method. For the biofilm formation we used modified G. D. Christensen’s method [19, 996-1006]. In the polystyrene plate we prepared microorganism suspension in Mueller-Hinton broth at a density of 0.5 optical density units determined by densitometer. This density conforms 1.5 x 108 CFU/ml microorganism concentration. 150 microliters of the derived suspension of bacteria strain were placed in the 12 wells of a polystyrene plate. Wells with 150 ml. of Mueller-Hinton broth were used as a negative control. The plate was incubated in the thermostat at 37 °C for 24 hours. The wells were washed by 150 ml. of distilled water four times using an automatic washer. Consequently, all forms of planktonic bacteria were removed from the wells and the biofilm left.
Formed in the polystyrene plate biofilm was fixed in the wells by adding 160 ml. 2.5 % glutaral-dehyde solution (exposure for 5 minutes) into each well. The plate was washed four times using 200 ml. of distilled water per well per cycle, and 180 ml. of 0.25 % crystal violet solution was added for 5 minutes, after that the plate was washed and dried for 10 minutes.
200 ml. of 33 % acetic acid solution was added to all wells. The exposure time at room temperature amounted to 10 minutes. The plate was placed into a multichannel spectrophotometer in order to determine the optical density (OD) of the wells using 620 nm. wavelength. According to the received data the average value of eight wells was estimated
and the ability of a microorganism biofilm formation in vitro was evaluated.
The lack ofthe ability to form biofilm was detected when the density averaged 0.12 optical density points (ODP), medium ability — at 0.12-0.24 points, strong ability — at more than 0.24 points.
Statistical methods
The obtained data were statistically analyzed using the software package «Statistica» (Version 10-Index, StatSoft Inc., USA) and «Excel». Before using the methods of descriptive statistics the type of quantitative traits distribution was determined using the Shapiro-Wilk test. Arithmetic mean (M) and standard deviation (S) for signs with a normal distribution were calculated. If distributing feature was different from normal the median (Me), the lower 25th (LQ) and upper 75th quartile (UQ) were calculated. Correlation analysis was implemented by nonparametric Spearman method. The value of correlation coefficient r = 0.7-0.99 pointed to a strong correlation, r = 0.3-0.69 — medium strength correlation, r = 0-0.29 — weak correlation. Differences were considered statistically significant when p < 0.05 [20, 496].
Results
Illness duration before hospitalization in the group 1 was 3.05 ± 1.8 days, in the group 2 - 3.85 ± 2.2 days, in the group 3 - 4.12 ± 2.2 days, in the group 4 -4.22 ± 2.6 days.
Hospitalization duration in the group 1 was 6.1 ± 2.5 days, in the group 2 - 9.14 ± 2.8 days, in the group 3 - 11.4 ± 5 days, in the group 4 - 13.7 ± 5 days.
As a result of microbiological researches of the wound exudate different microorganisms were detected. Among them S. epidermidis — 62 isolates (36 %), S.pyogenes — 18 (10 %), S. aureus — 6 (3 %), P. aeruginosa — 9 (5 %), K. pneumonia — 1 (0.6 %), E. coli — 1 (0.6 %), C. albicans — 3 (0.2 %) were found. Microorganisms were not revealed in the wound exudate of 71 patients (40 %).
In the group 1 (patients with acute suppurative odontogenic periostitis of the jaw): S. epidermidis — 18 isolates (45 %), S. pyogenes — 3 (7.5 %),
S.aureus — 2 (5 %), P aeruginosa — 3 (7.5 %) were detected. Microorganisms were not revealed in the wound exudate of 13 patients (32.5 %).
In the group 2 (patients with odontogenic osteomyelitis of the jaw, complicated by the odontogenic
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phlegmon of one cellular space): S. epidermidis — 26 isolates (32 %), S. pyogenes — 10 (12 %), P. aeruginosa — 4 (5 %), E.coli — 1 (1 %), C. albicans — 2 (3 %), K. pneumonia — 1 (1 %) were detected. Microorganisms were not revealed in the wound exudate of 38patients (46 %).
In the group 3 (patients with odontogenic osteomyelitis of the jaw, complicated by the odontogenic phlegmon of two cellular spaces); S. epider-midis — 11 isolates (34 %), S.aureus — 3 (10 %), C. albicans — 1 (3 %) were detected. Microorganisms were not revealed in the wound exudate of 17 patients (53 %).
In the group 4 (patients with odontogenic phlegmon of the floor of the mouth); S. epidermi-dis — 7 isolate (40 %), S. aureus — 1 (5 %), S.pyogenes — 5 (30 %), P aeruginosa — 2 (10 %) were detected. Microorganisms were not revealed in the wound exudate of 3 patients (15 %).
Bacterial sensitivity to antimicrobial agents is presented in figures 1, 2, 3, 4.
The highest sensitivity of the detected S.aureus isolates was revealed to amikacin and vancomycin — 100 %, sensitivity to ofloxacin and gentamycin was 83 %, to ciprofloxacin — 66 %. The lowest sensitivity was determined to oxacillin (33.3 %), ce-fazolin (50 %), lincomicin (50 %), amoxycillin + cla-vulanic acid (50 %).
The highest sensitivity of the detected S. epidermi-dis isolates was revealed to vancomycin - 100 %, sensitivity to amikacin was 95 %, to ciprofloxacin 94 %, to ceftriaxone - 90 %, to ofloxacin - 88 %, to gentamycin and oxacillin - 85 %, to amoxycillin + clavulanic acid - 80 %, to lincomicin - 78 %, to cefazolin - 70 %.
The highest sensitivity of the detected S. pyogenes isolates was revealed to ceftriaxone — 100 %, to ciprofloxacin — 94 %, to lincomicin — 85 %, to ofloxacin — 82 %, to azithromycin — 78 %, to ce-fazolin — 75 %, to ampicillin — 60 %.
The highest sensitivity of the detected P. aeruginosa isolates was revealed to ceftriaxone — 100 %, to amikacin — 75 %, to ofloxacin — 72 %, sensitivity to ciprofloxacin was 62.5 %, to cefepime — 50 %. Resistance of P. aeruginosa to lincomycin defined in 100 % of studies.
The selected isolates of odontogenic infection pathogens are the most sensitive to amikacin,
vancomycin, ceftriaxone, the highest resistance to lincomycin and cefazolin. Particulary P. aeruginosa was resistant to lincomycin in 100 % of cases.
The researches revealed a different ability of pathogens odontogenic infections to form a biofilm in 96-well plate that is presented in table 1.
Table 1. - The ability of odontogenic infection pathogens to form a biofilm
Pathogen OD of biofilm (М ± a)
P aeruginosa 0.27 ± 0.03
S. aureus 0.22 ± 0.05
S. pyogenes 0.16 ± 0.05
S. epidermidis 0.14 ± 0.04
According to the table 1, the highest ability to form a biofilm in vitro has P aeruginosa, the lowest — S. epidermidis.
The medium ability to biofilm formation was revealed at 16 % of S.aureus isolates, 84 % had strong ability to biofilm formation, that indicates the ability of S.aureus to form biofilm in varying degrees. 34 % of S. epidermidis isolates demonstrated inability to form biofilm, 27 % had medium ability, 39 % had strong ability to biofilm formation. Ability to biofilm formation of S. pyogenes: 20 % of isolates had lack of the ability, 30 % had medium ability, 50 % had strong ability to biofilm formation. Selected isolates of P aeruginosa demonstrated a strong ability to form biofilm in 100 % of cases.
In the group of patients with acute suppurative odontogenic periostitis of the jaw was identified following pathogens ability to form a biofilm. Isolates of S.aureus demonstrated a strong ability to form biofilm in 100 % of cases. 45 % of isolates S. epidermidis had lack of the ability, 45 % had medium ability, 10 % had strong ability to form biofilm. Isolates of P aeruginosa demonstrated a strong ability to biofilm formation in 100 % of cases, and isolates of S. pyogenes had lack of the ability in 100 % cases.
In the group of patients with odontogenic osteomyelitis of the jaw, complicated by the odontogenic phlegmon of one cellular space was identified following pathogens ability to form a biofilm. S. epidermidis had lack of the biofilm formation ability in 17 % of cases, medium — 53 %, strong — 30 %. S. pyogenes demonstrated lack of the biofilm formation ability in 25 % of cases, medium — 37.5 %, strong — 37.5 %. Strong ability to biofilm
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Section 2. Biomedical science
formation was determined at P. aeruginosa isolates in 100 % of cases.
In the group of patients with odontogenic osteomyelitis of the jaw, complicated by the odontogenic phlegmon of two cellular space S.aureus isolates had medium biofilm formation ability in 33 % of cases, strong in 66 % of cases. S. epidermidis isolates demonstrated lack of the biofilm formation ability in 17 % of cases, in 17 % — medium, in 66 % — strong ability.
In the group of patients with odontogenic phlegmon of the floor of the mouth all identified pathogenic microorganisms demonstrated strong biofilm formation ability in 100 % of cases.
Discussion
According to the received results, we think that the inability to detect the microorganism in the material obtained during the surgical treatment of suppurative focus, can be explained by the presence of anaerobic infection in the focus of inflammation.
The highest resistance of pathogens odontogenic infections to these antibiotics may explain the frequent use of lincomycin and cefazolin as empiric antibacterial therapy for the development of odontogenic inflammatory processes.
Thus, increase in biofilm formation ability by S. epidermidis и S. pyogenes as the spread of inflammation
was revealed. At the same time the strong biofilm formation ability ofP. aeruginosa isolates was not related to the severity of inflammation.
Weak positive correlation between illness duration before hospitalization and the intensity of the inflammatory process (r = 0.195) was revealed. Hospitalization duration was in a direct positive correlation with the intensity of the inflammatory process (r = 0.57). The severity ofbiofilm formation ability was in a direct positive correlation with the intensity of the inflammatory process too (r = 0.313).
Conclusion
Thus, in the course of the study was revealed that pathogens of odontogenic infections able to form microbial biofilm in a varying degree. P aeruginosa had the strongest biofilm formation ability and S. epidermidis had the least biofilm formation ability. The severity of biofilm formation ability was in a direct positive correlation with the intensity of the inflammatory process. Further study of the etiology of the odontogenic inflammatory diseases should be performed with taking into account the ability of pathogens to form microbial biofilms.
Conflict of interest
The authors declare they have no conflict of interest.
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Kytikova Oxana Yr’evna, PhD, Vladivostok Branch of FGBNU "Far Eastern Scientific Center of Physiology and Pathology of Respiration", Research Institute of Medical Climatology and Rehabilitation Treatment, Vladivostok E-mail: kytikova@yandex.ru Gvozdenko Tatyana Aleksandrovna, MD, Research Institute of Medical Climatology and Rehabilitation Treatment, Vladivostok E-mail: vfdnz@mail.ru Vitkina Tatyana Isaakovna, MD, Research Institute of Medical Climatology and Rehabilitation Treatment, Vladivostok E-mail: vfdnz@mail.ru
DNA damage induced by a ozone in peripheral blood lymphocytes of elderly patients with chronic obstructive pulmonary disease
Abstract: We have assessed DNA damage by using various therapeutic concentrations of ozone in the peripheral blood lymphocytes of elderly patients with chronic obstructive pulmonary disease (in vitro). The results of this work demonstrate that ozone induces DNA damage. It was also noticed, that there is a clear dose-dependent increase in DNA damage.
Keywords: DNA damage, ozone, chronic obstructive pulmonary disease, elderly patients.
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