Karakterizacija i toksična aktivnost Aggregatibacter actinomycetemcomitans izolata

Periodontal diseases are widely distributed in the world and represent a major oral health problem both in developed and in developing countries. These chronic inflammatory diseases are characterized by the destruction of tooth supporting tissues. It is commonly accepted that dental plaque bacteria are the primary etiologic agents of periodontal disease. More than 700 species have been detected in the oral cavity in different individuals. Approximately 400 of these species have been isolated from different subgingival microenvironments. However, only a few species have been associated with the disease. From these bacteria specifically associated with destructive disease, Aggregatibacter actinomycetemcomitans is considered as one of the bacterial species of etiological importance in periodontitis and has contributed to the initiation and/or progression of destructive forms of periodontitis. The aim of this study was to evaluate the occurance characteristics and toxic activity of A. actinomycetemcomitans in the subgingival biofilm in subjects with periodontal health and disease. Periodontal status of all included subjects was evaluated during the initial screening visit. A fullmouth clinical examination was performed in each patient using a manual probe and the following parameters were recorded at six sites per tooth: probing depth (PD in mm), clinical attachment loss (CAL in mm), bleeding on probing (BOP) and plaque index (PI). Pooled samples of subgingival plaque were taken for culture-based identification of microorganisms and further molecular analysis. Colonies suspected to be A. actinomycetemcomitans were selected for molecular identification using 16S rRNA gene sequencing. Genotyping was performed by polymerase chain reactions specific to the ltx promotor region, serotype-specific and cdt region and by sequencing of 16S rRNA. Cytotoxicity was examined on extravillous trophoblast cell line HTR-8/SVneo...Parodontalna oboljenja su široko rasprostanjena u celom svetu i u velikoj meri narušavaju oralno zdravlje, kako u razvijenim tako i u nerazvijenim zemljama. Parodontopatije su hronična inflamatorna oboljenja potpornog aparata zuba koja usled razaranja dubljih parodontalnih tkiva mogu da rezultiraju gubitkom zuba. Etiologija parodontopatije je polimikrobna po svojoj prirodi. Opisano više od 700 različitih vrsta mikroorganizama koje naseljavaju usnu duplju, od kojih je oko 400 izolovano iz subgingivalne regije. Međutim, samo nekoliko bakterijskih vrsta je dovedeno u vezu sa parodontopatijama, među kojima je i Aggregatibacter actinomycetemcomitans. Ovaj parodontopatogen se smatra veoma važnim u etiologiji parodontopatije, odnosno da doprinosi kako inicijaciji tako i progresiji destrukcije parodontalnih tkiva. Cilj ove studije je bio da se ispita prisustvo, karakteristike i toksična aktivnost bakterije A. actinomycetemcomitans poreklom iz subgingivalnog dentalnog plaka, kod obolelih od parodontopatije kao i kod osoba sa klinički zdravim parodoncijumom. Kod svih ispitanika uključenih u studiju, evidentiran je parodontalni status i nivo oralne higijene verifikacijom kliničkih parametara: dubinom sondiranja (DS u mm), nivoom pripojnog epitla (NPE u mm), krvarenjem na provokaciju (KNP) i plak indeksom (PI). Pulovani uzorci subgingivalnog dentalnog plaka su se koristili za kultivaciju i molekularne analize bakterije A. actinomycetemcomitans. Kultivacijom dobijene kolonije su selektovane u cilju identifikacije bakterije A. actinomycetemcomitans, sekvenciranjem 16S rRNK gena. Korišćene su specifične PCR reakcije u cilju genotipizacije promotorskog operona za leukotoksin, citoletalnog toksina istezanja i serotipizaciju kao i identifikaciju bakterije A. actinomycetemcomitans umnožavanjem dela gena za 16S rRNK. Ispitivan je uticaj bakterije A. actinomycetemcomitans na inhibiciju rasta ekstravilusne trofoblastne ćelijske linije HTR-8/SVneo..

Characterisation and toxic activity of Aggregatibacter actinomycetemcomitans isolates

Parodontalna oboljenja su široko rasprostanjena u celom svetu i u velikoj meri narušavaju oralno zdravlje, kako u razvijenim tako i u nerazvijenim zemljama. Parodontopatije su hronična inflamatorna oboljenja potpornog aparata zuba koja usled razaranja dubljih parodontalnih tkiva mogu da rezultiraju gubitkom zuba. Etiologija parodontopatije je polimikrobna po svojoj prirodi. Opisano više od 700 različitih vrsta mikroorganizama koje naseljavaju usnu duplju, od kojih je oko 400 izolovano iz subgingivalne regije. Međutim, samo nekoliko bakterijskih vrsta je dovedeno u vezu sa parodontopatijama, među kojima je i Aggregatibacter actinomycetemcomitans. Ovaj parodontopatogen se smatra veoma važnim u etiologiji parodontopatije, odnosno da doprinosi kako inicijaciji tako i progresiji destrukcije parodontalnih tkiva. Cilj ove studije je bio da se ispita prisustvo, karakteristike i toksična aktivnost bakterije A. actinomycetemcomitans poreklom iz subgingivalnog dentalnog plaka, kod obolelih od parodontopatije kao i kod osoba sa klinički zdravim parodoncijumom. Kod svih ispitanika uključenih u studiju, evidentiran je parodontalni status i nivo oralne higijene verifikacijom kliničkih parametara: dubinom sondiranja (DS u mm), nivoom pripojnog epitla (NPE u mm), krvarenjem na provokaciju (KNP) i plak indeksom (PI). Pulovani uzorci subgingivalnog dentalnog plaka su se koristili za kultivaciju i molekularne analize bakterije A. actinomycetemcomitans. Kultivacijom dobijene kolonije su selektovane u cilju identifikacije bakterije A. actinomycetemcomitans, sekvenciranjem 16S rRNK gena. Korišćene su specifične PCR reakcije u cilju genotipizacije promotorskog operona za leukotoksin, citoletalnog toksina istezanja i serotipizaciju kao i identifikaciju bakterije A. actinomycetemcomitans umnožavanjem dela gena za 16S rRNK. Ispitivan je uticaj bakterije A. actinomycetemcomitans na inhibiciju rasta ekstravilusne trofoblastne ćelijske linije HTR-8/SVneo...Periodontal diseases are widely distributed in the world and represent a major oral health problem both in developed and in developing countries. These chronic inflammatory diseases are characterized by the destruction of tooth supporting tissues. It is commonly accepted that dental plaque bacteria are the primary etiologic agents of periodontal disease. More than 700 species have been detected in the oral cavity in different individuals. Approximately 400 of these species have been isolated from different subgingival microenvironments. However, only a few species have been associated with the disease. From these bacteria specifically associated with destructive disease, Aggregatibacter actinomycetemcomitans is considered as one of the bacterial species of etiological importance in periodontitis and has contributed to the initiation and/or progression of destructive forms of periodontitis. The aim of this study was to evaluate the occurance characteristics and toxic activity of A. actinomycetemcomitans in the subgingival biofilm in subjects with periodontal health and disease. Periodontal status of all included subjects was evaluated during the initial screening visit. A fullmouth clinical examination was performed in each patient using a manual probe and the following parameters were recorded at six sites per tooth: probing depth (PD in mm), clinical attachment loss (CAL in mm), bleeding on probing (BOP) and plaque index (PI). Pooled samples of subgingival plaque were taken for culture-based identification of microorganisms and further molecular analysis. Colonies suspected to be A. actinomycetemcomitans were selected for molecular identification using 16S rRNA gene sequencing. Genotyping was performed by polymerase chain reactions specific to the ltx promotor region, serotype-specific and cdt region and by sequencing of 16S rRNA. Cytotoxicity was examined on extravillous trophoblast cell line HTR-8/SVneo..

Polymerase chain reaction in the identification of periodontopathogens: A reliable and satisfactory method?

Aggregatibacter actinomycetemcomitans is considered one of the bacterial species of etiological importance in periodontitis. The aim of this study was to evaluate the serotype of A. actinomycetemcomitans in the subgingival biofilm in subjects with periodontal health and disease. Pooled samples of subgingival plaque were taken for culture-based identification of microorganisms. Colonies suspected to be A. actinomycetemcomitans were selected for molecular identification using either multiplex or conventional PCR in serotype-specific genotyping and 16S rRNA gene sequencing. In silico analysis showed that most selected colonies belong to the genus Campylobacter, although positive signals for serotypes of A. actinomycetemcomitans were obtained with these samples. Identification of A. actinomycetemcomitans by conventional PCR for 16S rRNA with one species-specific and one universal primer was inconclusive because an almost identical signal with Campylobacter gracilis was obtained. Although PCR-based methods for the identification of A. actinomycetemcomitans are more rapid, sequencing should not be omitted. [Projekat Ministarstva nauke Republike Srbije, br. 41008 and br. 173048

Polymerase chain reaction in the identification of periodontopathogens: A reliable and satisfactory method?

Aggregatibacter actinomycetemcomitans is considered one of the bacterial species of etiological importance in periodontitis. The aim of this study was to evaluate the serotype of A. actinomycetemcomitans in the subgingival biofilm in subjects with periodontal health and disease. Pooled samples of subgingival plaque were taken for culture-based identification of microorganisms. Colonies suspected to be A. actinomycetemcomitans were selected for molecular identification using either multiplex or conventional PCR in serotype-specific genotyping and 16S rRNA gene sequencing. In silico analysis showed that most selected colonies belong to the genus Campylobacter, although positive signals for serotypes of A. actinomycetemcomitans were obtained with these samples. Identification of A. actinomycetemcomitans by conventional PCR for 16S rRNA with one species-specific and one universal primer was inconclusive because an almost identical signal with Campylobacter gracilis was obtained. Although PCR-based methods for the identification of A. actinomycetemcomitans are more rapid, sequencing should not be omitted

Multiple Major and Minor Anomalies Associated With Klippel-Feil Syndrome: A Case Report

Klippel-Feil syndrome is defined as congenital fusion of two or more cervical vertebrae. In this article, we report a 55-year-old male patient with one-year history of neck pain, headaches, and one episode of syncope after a severe trauma. X-rays and magnetic resonance imaging of cervical spine revealed fused vertebral bodies of C2-C5. The major anomalies associated with Klippel-Feil syndrome (small stature, thoracic kyphoscoliosis, lumbar scoliosis, restricted opening mouth, and bilateral sensorineural hearing loss) as well as multiple minor anomalies (mild face asymmetry, high arched palate, rhinoscoliosis, high nasal bridge, inclined septi nasi, and thin upper lip) were detected. This is a rare case describing the anomalies of the nose in Klippel-Feil syndrome patients. Our patient had no central cord impairment following a severe trauma

Procena periodontalnog zdravlja hospitalizovanih bolesnika sa shizofrenijom

Background/Aim. Many studies on oral health of psychiatric inpatients reported schizophrenia as the most common psychiatric disorder among their sample population. The available evidence suggests the higher prevalence and severity of periodontal disease among the psychiatric inpatients. The aim of this study was to evaluate periodontal health among the inpatients with schizophrenia and to consider possible risk factors for their current periodontal diseases. Methods. This cross-sectional study comprised 190 inpatients with schizophrenia at the Clinic for Psychiatric Disorders "Dr Laza Lazarević" in Belgrade, and 190 mentally healthy patients at the Clinic for Periodontology and Oral Medicine, Faculty of Dental Medicine, University in Belgrade. The Community Periodontal Index for Treatment Needs (CPITN) and sociodemographic characteristics were registered in both groups as well as the characteristics of the primary disease among the inpatients with schizophrenia. Results. The patients in the study group had significantly higher scores of the CPITN (2.24 ± 0.98) than the patients in the control group (1.21 ± 1.10). Most of the patients in the study group had supra, or subgingival calculi (46.8%), in contrast to the control group patients, who had in most cases gingival bleeding (45.8%). The periodontal pockets where detected in 35.8% of schizophrenic inpatients. The linear regression analysis showed that the gender and age were statistically significant predictors of the CPITN value among the inpatients with schizophrenia. Conclusion. The results of this study generally indicate the need for continuous research of psychiatric patients' oral health, in order to determine the modes of its improvement. Similar studies should elucidate significance of psychiatric patients' periodontal health and sensitize psychiatrists and psychiatric nurses to the oral problems of their patients.Uvod/Cilj. Mnogobrojna istraživanja oralnog zdravlju hospitalizovanih psihijatrijskih bolesnika pokazala su da je shizofrenija najčešći psihijatrijski poremećaj među njima. Dostupni podaci ukazuju na veliku prevalenciju i težinu periodontopatije kod bolesnika sa psihijatrijskim poremećajima. Cilj ove studije bio je da se proceni periodontalno zdravlje hospitalizovanih osoba sa shizofrenijom, kao i da se ukaže na moguće faktore rizika od oboljenja potpornog aparata zuba tih bolesnika. Metode. Studijom preseka obuhvaćeno je 190 osoba sa shizofrenijom, hospitalizovanih u Klinici za psihijatrijske bolesti "Dr Laza Lazarević" u Beogradu i 190 mentalno zdravih osoba, pacijenata Klinike za parodontologiju i oralnu medicinu Stomatološkog fakultet, Univerziteta u Beogradu. U obe grupe ispitanika registrovane su vrednosti Zajednički periodontni indeks potreba tretmana - Community Periodontal Index for Treatment Needs (CPITN), kao i sociodemografska obeležja, dok su karakteristike primarne bolesti beležene u studijskoj grupi. Rezultati. Ispitanici studijske grupe su imali znatno veće vrednosti CPITN (2,24 ± 0,98) u odnosu na ispitanike kontrolne grupe (1,21 ± 1,10). Kod većine ispitanika studijske grupe registrovano je prisustvo supra i subgingivalnog kamenca (46,8%), za razliku od ispitanika kontrolne grupe koji su češće imali gingivalno krvarenje (45,8%). Periodontalni džepovi su registrovani kod 35,8% hospitalizovanih osoba sa shizofrenijom. Linearna regresiona analiza pokazala je da su pol i starost ispitanika bili statistički značajni prediktori vrednosti CPITN hospitalizovanih osoba sa shizofrenijom. Zaključak. Rezultati ovog istraživanja ukazuju na postojanje potrebe za kontinuiranim istraživanjem stanja oralnog zdravlja psihijatrijskih bolesnika, kako bi se odredili načini za njegovo unapređenje. Slične studije bi trebalo da razjasne značaj peridodontalnog zdravlja psihijatrijskih bolesnika, kako bi se psihijatrima i medicinskim sestrama ukazalo na postojanje problema oralnog zdravlja njihovih bolesnika

Efekat hlorheksidina na ekspresiju receptor aktivatora NF-kb liganda (RANKL) i osteoprotežerina (OPG) kod ljudi i kućnih ljubimaca

Periodontal disease is a chronic, multi-factorial disease of the tissues supporting the teeth. Periodontitis in companion animals is an almost identical disease to that in humans in terms of disease course and clinical presentation. Receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG) are bioactive molecules that control bone resorption. This study aims to evaluate the effect of Chlorhexidine (CXD) on the RANKL and OPG expressions in gingival crevicular fluid (GCF) collected from subjects with chronic periodontitis. GCF was obtained from subjects with chronic periodontitis.10 subjects (CXD1) rinsed the mouth with 0.12% CXD, 10 subjects (CXD2) utilized 0.20% CXD and the last 10 (PL) used Placebo solution for 7 days. RANKL and OPG concentrations in GCF were measured by enzyme-linked immunosorbent assays ELISA at baseline and after 7 days. Periodontal clinical variables: clinical attachment loss (CAL), probing pocket depth (PPD), papilla-bleeding index (PBI) were evaluated in all groups. After 7 days in CXD1 and CXD2 group RANKL/OPG ratio exhibited a significant decrease (p lt 0.05) in contrast to the PL group where results showed similar values of RANKL/OPG ratio at baseline and after the observation period. RANKL/OPG ratio was positively correlated with PPD, CAL and PBI before and after the observation period in both Chlorhexidine (CXD1, CXD2) groups. In an existing inflammatory response, chlorhexidine reduced the level of periodontal inflammation, which leads to reduction of RANKL/OPG relative ratio. Decrease of RANKL/OPG ratio will apparently induce maintenance of alveolar bone and slow down periodontal tissue breakdown.Parodontopatije su hronična, multikauzalna oboljenja potpornog aparata zuba. Parodontalna oboljenja koja srećemo kod kućnih ljubimaca su prema toku i kliničkoj slici skoro identična onima koje se javljaju kod ljudi. RANKL i osteoprotegerin (OPG) su bioaktivni molekuli koji kontrolišu koštanu resorpciju. Cilj ove studije je evaluacija efekata hlorheksidina na ekspresiju RANKL-a i OPG-a u gingivalnoj tečnosti (GT) uzetoj od pacijenata sa hroničnom parodontopatijom. 10 pacijenata (CXD1) su ispirali usta sa 0.12% CXD, 10 pacijenata (CXD2) su koristili 0.20% CXD i poslednjih 10 pacijenata (PL) su koristili placebo rastvor 7 dana. RANKL i OPG koncentracije u GT su merene ELISA testom na početku i posle sedam dana. Parodontalni klinički parametri CAL, PPD i PBI su evaluirani u svim grupama. Posle 7 dana u CXD1 i CXD2 grupi RANKL/OPG odnos je pokazao signifikantno smanjenje (p lt 0.05) u poređenju sa PL grupom gde su zabaleženi slični rezultati na početku i nakon opservacionog perioda. RANKL/OPG odnos je pokazao pozitivnu korelaciju sa vrednostima PPD-a, CAL-a i PBI-a pre i nakon observacionog perioda u obe eksperimentalne grupe (CXD1, CXD2). U prisutnom inflamatornom odgovoru hlorheksidin je redukovao nivo inflamacije, što je uslovilo redukciju RANKL/OPG odnosa. Rezultati istraživanja dokazuju da koncentracija hlorheksidina ne utiče statistički značajno na smanjenje RANKL/OPG odnosa

Melatonin Action in Type 2 Diabetic Parotid Gland and Dental Pulp: In Vitro and Bioinformatic Findings

Type 2 diabetes mellitus (T2DM) is associated with functional deterioration of the salivary gland and dental pulp, related to oxidative stress. The aim was to integrate experimental and bioinformatic findings to analyze the cellular mechanism of melatonin (MEL) action in the human parotid gland and dental pulp in diabetes. Human parotid gland tissue was obtained from 16 non-diabetic and 16 diabetic participants, as well as human dental pulp from 15 non-diabetic and 15 diabetic participants. In human non-diabetic and diabetic parotid gland cells (hPGCs) as well as in dental pulp cells (hDPCs), cultured in hyper- and normoglycemic conditions, glial cell linederived neurotrophic factor (GDNF), MEL, inducible nitric oxide synthase (iNOS) protein expression, and superoxide dismutase (SOD) activity were measured by enzyme-linked immunosorbent assay (ELISA) and spectrophotometrically. Bioinformatic analysis was performed using ShinyGO (v.0.75) application. Diabetic participants had increased GDNF and decreased MEL in parotid (p < 0.01) and dental pulp (p < 0.05) tissues, associated with increased iNOS and SOD activity. Normoglycemic hDPCs and non-diabetic hPGCs treated with 0.1 mM MEL had increased GDNF (p < 0.05), while hyperglycemic hDPCs treated with 1 mM MEL showed a decrease in up-regulated GDNF (p < 0.05). Enrichment analyses showed interference with stress and ATF/CREB signaling. MEL induced the stress-protective mechanism in hyperglycemic hDPCs and diabetic hPGCs, suggesting MEL could be beneficial for diabetes-associated disturbances in oral tissues

Identifikacija parodontopatogenih mikroorganizama PCR tehnikom

INTRODUCTION Periodontitis is an inflammatory disease of the supporting tissues of teeth and is a major cause of tooth loss in adults. The onset and progression of periodontal disease is attributed to the presence of elevated levels of a consortium of pathogenic bacteria. Gram negative bacteria, mainly strict anaerobes, play the major role. OBJECTIVE The present study aimed to assess the presence of the main types of microorganisms involved in the aetiopathogenesis of periodontal disease: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Treponema denticola, Tanerella forsythia and Prevotella intermedia in different samples collected from the oral cavity of 90 patients diagnosed with periodontitis. METHOD Bacterial DNA detection was performed in diverse biological materials, namely in dental plaque, gingival tissue and saliva, by means of multiplex PCR, a technique that allows simultaneous identification of two different bacterial genomes. RESULTS In the dental plaque of the periodontitis patients, Treponema denticola dominated. In the gingival tissue, Tannerella forsythia and Treponema denticola were the microbiota most frequently detected, whilst in saliva Treponema denticola and Eikenella corrodens were found with the highest percentage. CONCLUSION The identification of microorganisms by multiplex PCR is specific and sensitive. Rapid and precise assessment of different types of periodontopathogens is extremely important for early detection of the infection and consequently for the prevention and treatment of periodontal disease. In everyday clinical practice, for routine bacterial evaluation in patients with periodontal disease, the dental plaque is the most suitable biological material, because it is the richest in periodontal bacteria.Uvod Epidemiološki podaci iz čitavog sveta ukazuju na veliku rasprostranjenost gingivitisa i parodontopatije, oboljenja potpornog aparata zuba. U etiopatogenezi oboljenja parodoncijuma ključnu ulogu igraju različiti rodovi Gram-negativnih bakterija, ponajviše striktnih anaeroba. Cilj rada Cilj rada je bio da se ispita postojanje genoma glavnih parodontopatogenih mikroorganizama Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Treponema denticola, Tanerella forsythia i Prevotella intermedia u različitim uzorcima poreklom iz usne duplje pacijenata s klinički dijagnostikovanom parodontopatijom. Metod rada Kao biološki materijal u kojem je dokazivano postojanje DNK mikroorganizama korišćeni su zubni plak, tkivo zapaljene gingive i pljuvačka. Za otkrivanje bakterijskog genoma primenjena je multipleks tehnika reakcije lančanog umnožavanja (engl. polymerase chain reaction PCR), odnosno simultana amplifikacija gena dve različite bakterije. Rezultati S manjom ili većom učestalošću, u svim ispitanim uzorcima utvrđeno je postojanje parodontopatogenih mikroorganizama. U zubnom plaku osoba obolelih od parodontopatije najčešći je bio genom vrste Treponema denticola. U tkivu parodoncijuma otkriveno je u najvećem procentu postojanje genoma vrsta Tannerella forsythia i Treponema denticola, što je odlika hroničnog oblika parodontopatije, a u pljuvački ispitanika dominirale su Treponema denticola i Eikinella corrodens. Najmanje ukupno postojanje bakterija je zapaženo u pljuvački. Zaključak Primenjeni metod PCR ima veliku osetljivost i specifičnost. Brzo i precizno otkrivanje mikroorganizama je veoma važno za pravovremeno dijagnostikovanje infekcije, a samim tim i za prevenciju i lečenje parodontopatija. U svakodnevnoj kliničkoj praksi optimalan biološki materijal za dokazivanje parodontopatogena kod osoba obolelih od parodontopatije je zubni plak, koji se smatra pouzdanim pokazateljem zastupljenosti pojedinih bakterija u obolelom parodoncijumu