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Dissertations in Health Sciences
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THE UNIVERSITY OF EASTERN FINLAND
The aim of this thesis was to assess the possible role of dental and periodontal infections in pathogenesis of intracranial
aneurysm disease. Oral bacterial DNA was detected in most of the ruptured and unruptured intracranial aneurysm tissue samples along with possibly bacterial driven inflammation. Aneurysm patients
had significantly more dental infectious foci than are found in the normal Finnish
population in general. This study clearly demonstrates the need to investigate fur- ther the role of microbiota in the complex
pathobiology of intracranial aneurysms.
MIKKO PYYSALO
DISSERTATIONS | MIKKO PYYSALO | ORAL BACTERIAL FINDINGS IN PATIENTS WITH INTRACRANIAL... | No 5
MIKKO PYYSALO
ORAL BACTERIAL FINDINGS IN PATIENTS
ORAL BACTERIAL FINDINGS IN PATIENTS
WITH INTRACRANIAL ANEURYSMS
Mikko Pyysalo
ORAL BACTERIAL FINDINGS IN PATIENTS WITH INTRACRANIAL ANEURYSMS
Publications of the University of Eastern Finland Dissertations in Health Sciences
No 526
University of Eastern Finland Kuopio
2019
Mikko Pyysalo
ORAL BACTERIAL FINDINGS IN PATIENTS WITH INTRACRANIAL ANEURYSMS
Publications of the University of Eastern Finland Dissertations in Health Sciences
No 526
University of Eastern Finland Kuopio
2019
Series Editors
Professor Tomi Laitinen, M.D., Ph.D.
Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences
Associate professor (Tenure Track) Tarja Kvist, Ph.D.
Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D.
Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences
Associate Professor (Tenure Track) Tarja Malm, Ph.D.
A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences
Lecturer Veli-Pekka Ranta, Ph.D.
School of Pharmacy Faculty of Health Sciences
Distributor:
University of Eastern Finland Kuopio Campus Library
P.O.Box 1627 FI-70211 Kuopio, Finland
www.uef.fi/kirjasto
Name of the printing office PunaMusta 2019
ISBN: 978-952-61-3168-9 (print/nid.) ISBN: 978-952-61-3169-6 (PDF)
ISSNL: 1798-5706 ISSN: 1798-5706 ISSN: 1798-5714 (PDF)
Author’s address: Institute of Dentistry/School of Medicine University of Eastern Finland
KUOPIO FINLAND
Doctoral programme: Doctoral programme of clinical research Supervisors: Adjunct Professor Tanja Pessi, Ph.D.
Faculty of Medicine and Health Technology/Molecule microbiology
University of Tampere TAMPERE
FINLAND
Professor Timo Peltomäki, DDS, Ph.D. Institute of Dentistry/School of Medicine University of Eastern Finland
KUOPIO FINLAND
Reviewers: Professor György Kálmán Sándor, MD, DDS, Ph.D.
Unit of Oral Health Sciences University of Oulu
OULU FINLAND
Docent Pirkko Pussinen, Ph.D. Oral and Maxillofacial diseases University of Helsinki
HELSINKI FINLAND
Opponent: Professor Eija Könönen, Ph.D.
Institute of Dentistry University of Turku TURKU
FINLAND
Series Editors
Professor Tomi Laitinen, M.D., Ph.D.
Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences
Associate professor (Tenure Track) Tarja Kvist, Ph.D.
Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D.
Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences
Associate Professor (Tenure Track) Tarja Malm, Ph.D.
A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences
Lecturer Veli-Pekka Ranta, Ph.D.
School of Pharmacy Faculty of Health Sciences
Distributor:
University of Eastern Finland Kuopio Campus Library
P.O.Box 1627 FI-70211 Kuopio, Finland
www.uef.fi/kirjasto
Name of the printing office PunaMusta 2019
ISBN: 978-952-61-3168-9 (print/nid.) ISBN: 978-952-61-3169-6 (PDF)
ISSNL: 1798-5706 ISSN: 1798-5706 ISSN: 1798-5714 (PDF)
Author’s address: Institute of Dentistry/School of Medicine University of Eastern Finland
KUOPIO FINLAND
Doctoral programme: Doctoral programme of clinical research Supervisors: Adjunct Professor Tanja Pessi, Ph.D.
Faculty of Medicine and Health Technology/Molecule microbiology
University of Tampere TAMPERE
FINLAND
Professor Timo Peltomäki, DDS, Ph.D.
Institute of Dentistry/School of Medicine University of Eastern Finland
KUOPIO FINLAND
Reviewers: Professor György Kálmán Sándor, MD, DDS, Ph.D.
Unit of Oral Health Sciences University of Oulu
OULU FINLAND
Docent Pirkko Pussinen, Ph.D.
Oral and Maxillofacial diseases University of Helsinki
HELSINKI FINLAND
Opponent: Professor Eija Könönen, Ph.D.
Institute of Dentistry University of Turku TURKU
FINLAND
To A, T, C and G
Pyysalo, Mikko
Oral bacterial findings in patients with intracranial aneurysms Kuopio: University of Eastern Finland
Publications of the University of Eastern Finland Dissertations in Health Sciences 526. 2019, 75 p.
ISBN: 978-952-61-3168-9 (print) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-3169-6 (PDF) ISSN: 1798-5714 (PDF)
ABSTRACT
Rupture of an intracranial aneurysm is the most common cause of a subarachnoid haemorrhage, which is a devastating cerebro-vascular event affecting typically working-age people. The prevalence of unruptured intracranial aneurysms remains unknown, but at least one in 20 to 30 adults is likely to carry an asymptomatic aneurysm, out of which approximately 25% will rupture in a lifetime. About 50% of the SAH patients die during the first year from bleeding. Intracranial aneurysm disease is associated with inflammation. Periodontal infection and bacteria are shown to associate with cardiovascular diseases but no studies, in which bacteria have been detected in the saccular intracranial aneurysm tissue, have been conducted.
In this study, a cohort of 70 patients with ruptured or unruptured intracranial aneurysms was collected. Specimens from aneurysm sac tissue were obtained perioperatively after prompt microsurgical clipping of the saccular aneurysm under sterile conditions between June 2010 and June 2014. A cohort of 90 patients undergoing preoperative dental examination due to planned surgical treatment of an intracranial aneurysm were recruited to the study between September 2012 and December 2014. The patients’ teeth were investigated during the acute hospitalization period of aneurysm treatment. Both panoramic tomography and dental cone beam tomography were performed for all the patients. Samples for bacterial DNA analyses were taken from the deepest gingival pocket crevicular fluid of the subpopulation of 60 patients with an unruptured aneurysm using a sterile blotting paper pin. Patients were asked to report their tooth cleaning habits, i.e. how many times a day they cleaned their teeth, and smoking habits. Aneurysm tissue samples were analysed using quantitative polymerase chain reaction (qPCR).
Gingival pocket samples were analysed using qPCR and 16S rRNA gene-based metagenomics.
Oral and pharyngeal bacterial DNA was detected in 70% of ruptured and unruptured intracranial aneurysm tissue samples along with possibly bacterial driven inflammation. The patients with saccular intracranial aneurysms had
To A, T, C and G
Pyysalo, Mikko
Oral bacterial findings in patients with intracranial aneurysms Kuopio: University of Eastern Finland
Publications of the University of Eastern Finland Dissertations in Health Sciences 526. 2019, 75 p.
ISBN: 978-952-61-3168-9 (print) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-3169-6 (PDF) ISSN: 1798-5714 (PDF)
ABSTRACT
Rupture of an intracranial aneurysm is the most common cause of a subarachnoid haemorrhage, which is a devastating cerebro-vascular event affecting typically working-age people. The prevalence of unruptured intracranial aneurysms remains unknown, but at least one in 20 to 30 adults is likely to carry an asymptomatic aneurysm, out of which approximately 25% will rupture in a lifetime. About 50% of the SAH patients die during the first year from bleeding. Intracranial aneurysm disease is associated with inflammation. Periodontal infection and bacteria are shown to associate with cardiovascular diseases but no studies, in which bacteria have been detected in the saccular intracranial aneurysm tissue, have been conducted.
In this study, a cohort of 70 patients with ruptured or unruptured intracranial aneurysms was collected. Specimens from aneurysm sac tissue were obtained perioperatively after prompt microsurgical clipping of the saccular aneurysm under sterile conditions between June 2010 and June 2014. A cohort of 90 patients undergoing preoperative dental examination due to planned surgical treatment of an intracranial aneurysm were recruited to the study between September 2012 and December 2014. The patients’ teeth were investigated during the acute hospitalization period of aneurysm treatment. Both panoramic tomography and dental cone beam tomography were performed for all the patients. Samples for bacterial DNA analyses were taken from the deepest gingival pocket crevicular fluid of the subpopulation of 60 patients with an unruptured aneurysm using a sterile blotting paper pin. Patients were asked to report their tooth cleaning habits, i.e. how many times a day they cleaned their teeth, and smoking habits. Aneurysm tissue samples were analysed using quantitative polymerase chain reaction (qPCR).
Gingival pocket samples were analysed using qPCR and 16S rRNA gene-based metagenomics.
Oral and pharyngeal bacterial DNA was detected in 70% of ruptured and unruptured intracranial aneurysm tissue samples along with possibly bacterial driven inflammation. The patients with saccular intracranial aneurysms had
significantly more dental infectious foci (≥6 mm gingival pockets) than are found in the normal population in general. Fusobacterium nucleatum, which appears to be associated with the progression of periodontitis, was frequently found in the gingival pocket samples from patients with intracranial aneurysms. Active tooth brushing appears to reduce the alpha diversity and the amount of Fusobacterium nucleatum in the gingival pocket microbiome. It appears that oral bacteria might also play a role in the pathogenesis of intracranial aneurysm disease.
Together with previous studies, this study demonstrates the need to investigate further the role of microbiota in the complex pathobiology of intracranial aneurysms.
National Library of Medicine Classification: QW 133, WL 355, WU 242
Medical Subject Headings: Intracranial Aneurysm; Fusobacterium nucleatum; Periodontitis;
Mouth/microbiology; Cardiovascular Diseases
Pyysalo, Mikko
Hammasperäiset bakteerilöydökset aivovaltimopullistumapotilailla Kuopio: Itä-Suomen yliopisto
Publications of the University of Eastern Finland Dissertations in Health Sciences 526. 2019, 75 s.
ISBN: 978-952-61-3168-9 (nid.) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-3169-6 (PDF) ISSN: 1798-5714 (PDF)
TIIVISTELMÄ
Aivovaltimopullistuman puhkeaminen on vaarallisen lukinkalvonalaisen verenvuodon (SAV) yleisin syy. Vuoto ilmaantuu yleisimmin työikäiselle väestölle aiheuttaen huomattavia neurologisia haittoja. Aivovaltimopullistuman esiintyvyydestä ei ole tarkkaa tietoa. Arvioidaan, että vähintään 1/20 -1/30:lla aikuisella on aivovaltimopullistuma. Näistä pullistumista noin 25% puhkeaa elämän aikana. Noin puolet SAV-potilaista kuolee ensimmäisen vuoden aikana vuodosta.
Tulehdusreaktio liittyy pullistuman kehittymiseen ja puhkeamiseen. Parodontiitin on osoitettu olevan sydän- ja verisuonitautien itsenäinen riskitekijä mutta tutkimuksia, jotka käsittelevät suun bakteereiden osuutta aivoverisuonisairauksissa ei ole.
Tässä tutkimuksessa kerättiin kudosnäytteet 70:stä aivovaltimopullistumasta.
Kudosnäytteet otettiin pullistuman leikkaushoidon yhteydessä kesäkuun 2010 ja kesäkuun 2014 välisenä aikana. Toiseen aineistoon tutkittiin 90:n aivovaltimopullistumapotilaan hampaisto kliinisesti ja röntgenologisesti pullistuman hoitoon liittyvän sairaalahoitojakson aikana syyskuun 2012 ja joulukuun 2014 välisenä aikana. Kunkin vuotamatonta pullistumaa sairastavan (n=60) potilaan ientaskusta otettiin bakteerinäyte steriilillä paperinastalla. Potilaita pyydettiin kertomaan tupakoivatko he, ja kuinka usein he harjaavat hampaansa.
Pullistumanäytteet analysoitiin kvantitatiivisellä polymeraasiketjureaktiolla (qPCR) ja ientaskunäytteiden analysointiin käytettiin qPCR:n lisäksi myös 16S rRNA geenin sekvenointiin perustuvaa metagenomista analytiikkaa.
Suun ja nielun bakteereiden DNA:ta löydettiin 70%:sta vuotaneista ja vuotamattomista pullistumanäytteistä. Näytteissä havaittiin myös viitteitä bakteeriperäisestä tulehdusreaktiosta. Pullistumapotilailla oli enemmän tulehduspesäkkeiksi sopivia yli 6 mm syviä ientaskuja kuin normaaliväestöllä.
Fusobacterium nucleatum, joka liittyy parodontiitin kehittymiseen, oli yleisimpiä bakteereita pullistumapotilaiden ientasku- ja pullistumanäytteissä. Aktiivinen hampaiden harjaaminen 2 kertaa päivässä näytti vähentävän bakteereiden monimuotoisuutta sekä Fusobacterium nucleatumin suhteellista osuutta potilaiden
significantly more dental infectious foci (≥6 mm gingival pockets) than are found in the normal population in general. Fusobacterium nucleatum, which appears to be associated with the progression of periodontitis, was frequently found in the gingival pocket samples from patients with intracranial aneurysms. Active tooth brushing appears to reduce the alpha diversity and the amount of Fusobacterium nucleatum in the gingival pocket microbiome. It appears that oral bacteria might also play a role in the pathogenesis of intracranial aneurysm disease.
Together with previous studies, this study demonstrates the need to investigate further the role of microbiota in the complex pathobiology of intracranial aneurysms.
National Library of Medicine Classification: QW 133, WL 355, WU 242
Medical Subject Headings: Intracranial Aneurysm; Fusobacterium nucleatum; Periodontitis;
Mouth/microbiology; Cardiovascular Diseases
Pyysalo, Mikko
Hammasperäiset bakteerilöydökset aivovaltimopullistumapotilailla Kuopio: Itä-Suomen yliopisto
Publications of the University of Eastern Finland Dissertations in Health Sciences 526. 2019, 75 s.
ISBN: 978-952-61-3168-9 (nid.) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-3169-6 (PDF) ISSN: 1798-5714 (PDF)
TIIVISTELMÄ
Aivovaltimopullistuman puhkeaminen on vaarallisen lukinkalvonalaisen verenvuodon (SAV) yleisin syy. Vuoto ilmaantuu yleisimmin työikäiselle väestölle aiheuttaen huomattavia neurologisia haittoja. Aivovaltimopullistuman esiintyvyydestä ei ole tarkkaa tietoa. Arvioidaan, että vähintään 1/20 -1/30:lla aikuisella on aivovaltimopullistuma. Näistä pullistumista noin 25% puhkeaa elämän aikana. Noin puolet SAV-potilaista kuolee ensimmäisen vuoden aikana vuodosta.
Tulehdusreaktio liittyy pullistuman kehittymiseen ja puhkeamiseen. Parodontiitin on osoitettu olevan sydän- ja verisuonitautien itsenäinen riskitekijä mutta tutkimuksia, jotka käsittelevät suun bakteereiden osuutta aivoverisuonisairauksissa ei ole.
Tässä tutkimuksessa kerättiin kudosnäytteet 70:stä aivovaltimopullistumasta.
Kudosnäytteet otettiin pullistuman leikkaushoidon yhteydessä kesäkuun 2010 ja kesäkuun 2014 välisenä aikana. Toiseen aineistoon tutkittiin 90:n aivovaltimopullistumapotilaan hampaisto kliinisesti ja röntgenologisesti pullistuman hoitoon liittyvän sairaalahoitojakson aikana syyskuun 2012 ja joulukuun 2014 välisenä aikana. Kunkin vuotamatonta pullistumaa sairastavan (n=60) potilaan ientaskusta otettiin bakteerinäyte steriilillä paperinastalla. Potilaita pyydettiin kertomaan tupakoivatko he, ja kuinka usein he harjaavat hampaansa.
Pullistumanäytteet analysoitiin kvantitatiivisellä polymeraasiketjureaktiolla (qPCR) ja ientaskunäytteiden analysointiin käytettiin qPCR:n lisäksi myös 16S rRNA geenin sekvenointiin perustuvaa metagenomista analytiikkaa.
Suun ja nielun bakteereiden DNA:ta löydettiin 70%:sta vuotaneista ja vuotamattomista pullistumanäytteistä. Näytteissä havaittiin myös viitteitä bakteeriperäisestä tulehdusreaktiosta. Pullistumapotilailla oli enemmän tulehduspesäkkeiksi sopivia yli 6 mm syviä ientaskuja kuin normaaliväestöllä.
Fusobacterium nucleatum, joka liittyy parodontiitin kehittymiseen, oli yleisimpiä bakteereita pullistumapotilaiden ientasku- ja pullistumanäytteissä. Aktiivinen hampaiden harjaaminen 2 kertaa päivässä näytti vähentävän bakteereiden monimuotoisuutta sekä Fusobacterium nucleatumin suhteellista osuutta potilaiden
ientaskuissa. Näiden tulosten pohjalta näyttää siltä, että suun bakteereilla saattaa olla osuutta aivovaltimopullistumataudissa. Yhdessä aiempien tutkimusten kanssa tämän tutkimuksen tulokset toimivat kiinnostavana avauksena kohti uusia tutkimuksia bakteereiden osuudesta aivovaltimopullistumataudissa.
Yleinen suomalainen asiasanasto: kallonsisäinen aneurysma; parodontiitti; bakteerit; suu;
sydän- ja verisuonitaudit
ACKNOWLEDGEMENTS
This study was carried out at the Departments of oral- and maxillofacial diseases and neurosurgery in Tampere University Hospital between 2010–2017 and was conducted in co-operation with University of Tampere Department of molecular microbiology.
First, I express my warmest gratitude to my main supervisor, docent Tanja Pessi for her devotion to this project. Her guidance towards modern microbiological methods made this thesis possible. In addition to gigabytes of emails and whatsapps, hours of phone calls, she was there whenever I asked to “meet me tomorrow at half past two”.
Likewise, I feel priviledged to have professor Timo Peltomäki being my second supervisor. He was there whenever I knocked his door. I know, I had a bunch of new ideas concerning my work. Together we dumped the crazy ones. He kept my feet on the ground.
I am also grateful for docent Pirkko Pussinen and professor György Kálmán Sándor for their work as reviewers of this thesis. I came up with an idea for next research project while meeting with docent Pussinen on a warm summer day in Helsinki. Both reviewers gave very constructive comments and helped me to improve the quality of the thesis.
Furthermore, I express my warm gratitude to my co-authors, professor emeritus Juha Öhman, professor emeritus Pekka Karhunen, professor Terho Lehtimäki, professor Niku Oksala, PhD Liisa Pyysalo, DDS Jorma Järnstedt, MD Jenni Hiltunen, M.Sc Pashupati Mishra and M.Sc Kati Sundström. Professor Öhman encouraged us to contact professor Karhunen and keep on going. They made this study possible.
I feel honored that professor Eija Könönen agreed to act as opponent during the public dissertation.
I feel privileged to have such a good friend and colleaque as Anttiveikko Koivumäki in my life. We have spent memorable moments in operation theatre discussing about biology behind maxillofacial diseases and human behaviour. But still, it is different to talk the talk than walk the walk. Thanks for everything, mate.
I am happy to have bosses like Eeva Torppa-Saarinen and Jukka Kuusisto, whose attitude towards research is more than positive. Thank you for all the possibilities to suddenly run away to see research patients. Thank you Anne Riipinen-Luhtalampi, Piitu Oksanen and Anne Simi for arranging things ready for me to suddenly disappear and coming back to treat patients.
I thank Seppo Kaskenmäki, Svante Hellsten, Harry Kneckt and Jan Hallquisth for providing gear and James Marshall Hendrix for giving the example how to use them for the times I process study protocols in my head. I am honoured to have met professors Hirotoshi Sano and Juha Hernesniemi who both encouraged me to keep on going with the aneurysm research.
ientaskuissa. Näiden tulosten pohjalta näyttää siltä, että suun bakteereilla saattaa olla osuutta aivovaltimopullistumataudissa. Yhdessä aiempien tutkimusten kanssa tämän tutkimuksen tulokset toimivat kiinnostavana avauksena kohti uusia tutkimuksia bakteereiden osuudesta aivovaltimopullistumataudissa.
Yleinen suomalainen asiasanasto: kallonsisäinen aneurysma; parodontiitti; bakteerit; suu;
sydän- ja verisuonitaudit
ACKNOWLEDGEMENTS
This study was carried out at the Departments of oral- and maxillofacial diseases and neurosurgery in Tampere University Hospital between 2010–2017 and was conducted in co-operation with University of Tampere Department of molecular microbiology.
First, I express my warmest gratitude to my main supervisor, docent Tanja Pessi for her devotion to this project. Her guidance towards modern microbiological methods made this thesis possible. In addition to gigabytes of emails and whatsapps, hours of phone calls, she was there whenever I asked to “meet me tomorrow at half past two”.
Likewise, I feel priviledged to have professor Timo Peltomäki being my second supervisor. He was there whenever I knocked his door. I know, I had a bunch of new ideas concerning my work. Together we dumped the crazy ones. He kept my feet on the ground.
I am also grateful for docent Pirkko Pussinen and professor György Kálmán Sándor for their work as reviewers of this thesis. I came up with an idea for next research project while meeting with docent Pussinen on a warm summer day in Helsinki. Both reviewers gave very constructive comments and helped me to improve the quality of the thesis.
Furthermore, I express my warm gratitude to my co-authors, professor emeritus Juha Öhman, professor emeritus Pekka Karhunen, professor Terho Lehtimäki, professor Niku Oksala, PhD Liisa Pyysalo, DDS Jorma Järnstedt, MD Jenni Hiltunen, M.Sc Pashupati Mishra and M.Sc Kati Sundström. Professor Öhman encouraged us to contact professor Karhunen and keep on going. They made this study possible.
I feel honored that professor Eija Könönen agreed to act as opponent during the public dissertation.
I feel privileged to have such a good friend and colleaque as Anttiveikko Koivumäki in my life. We have spent memorable moments in operation theatre discussing about biology behind maxillofacial diseases and human behaviour. But still, it is different to talk the talk than walk the walk. Thanks for everything, mate.
I am happy to have bosses like Eeva Torppa-Saarinen and Jukka Kuusisto, whose attitude towards research is more than positive. Thank you for all the possibilities to suddenly run away to see research patients. Thank you Anne Riipinen-Luhtalampi, Piitu Oksanen and Anne Simi for arranging things ready for me to suddenly disappear and coming back to treat patients.
I thank Seppo Kaskenmäki, Svante Hellsten, Harry Kneckt and Jan Hallquisth for providing gear and James Marshall Hendrix for giving the example how to use them for the times I process study protocols in my head. I am honoured to have met professors Hirotoshi Sano and Juha Hernesniemi who both encouraged me to keep on going with the aneurysm research.
I deeply thank my family from the bottom of my heart for all their love and support. My parents Hilkka and Lasse and my sister Kirsi have provided all the support and mushroom pizza I needed to complete this thesis. Lotta and Mikael: I need apologize for all the inconvenience this project may have caused to you. Finally, I owe my deepest gratitude to my wife Liisa for love and support. Together we are stronger.
This study was financially supported by personal grants from the Hilda Kauhanen foundation, Petri Honkanen foundation and Orion research foundation, which are gratefully acknowledged.
Tampereen Takahuhdissa, 31. Heinäkuuta 2019 Mikko Pyysalo
LIST OF ORIGINAL PUBLICATIONS
This dissertation is based on the following original publications:
I Pyysalo MJ, Pyysalo LM, Pessi T, Karhunen PJ, Öhman JE.
The connection between ruptured cerebral aneurysms and odontogenic bacteria. Journal of Neurology, Neurosurgery and Psychiatry. 84(11):1214- 1218, 2013
II Pyysalo MJ, Pyysalo LM, Pessi T, Karhunen PJ, Lehtimäki T, Oksala N, Öhman JE. Bacterial DNA findings in ruptured and unruptured intracranial
aneurysms. Acta Odontologica Scandinavica. 74(4):315-320, 2016
III Pyysalo MJ, Pyysalo LM, Hiltunen J, Järnstedt J, Helminen M, Karhunen PJ, Pessi T. The dental infections in patients undergoing preoperative dental examination before surgical treatment of saccular intracranial aneurysm.
BMC Research Notes. 20;11(1):600, 2018
IV Pyysalo MJ, Mishra PP, Sundström K, Lehtimäki T, Karhunen PJ, Pessi T.
Increased tooth brushing frequency is associated with reduced gingival pocket bacterial diversity in patients with intracranial aneurysms.
PeerJ. Jan 25;7:e6316, 2019
The publications were adapted with the permission of the copyright owners.
I deeply thank my family from the bottom of my heart for all their love and support. My parents Hilkka and Lasse and my sister Kirsi have provided all the support and mushroom pizza I needed to complete this thesis. Lotta and Mikael: I need apologize for all the inconvenience this project may have caused to you. Finally, I owe my deepest gratitude to my wife Liisa for love and support. Together we are stronger.
This study was financially supported by personal grants from the Hilda Kauhanen foundation, Petri Honkanen foundation and Orion research foundation, which are gratefully acknowledged.
Tampereen Takahuhdissa, 31. Heinäkuuta 2019 Mikko Pyysalo
LIST OF ORIGINAL PUBLICATIONS
This dissertation is based on the following original publications:
I Pyysalo MJ, Pyysalo LM, Pessi T, Karhunen PJ, Öhman JE.
The connection between ruptured cerebral aneurysms and odontogenic bacteria. Journal of Neurology, Neurosurgery and Psychiatry. 84(11):1214- 1218, 2013
II Pyysalo MJ, Pyysalo LM, Pessi T, Karhunen PJ, Lehtimäki T, Oksala N, Öhman JE. Bacterial DNA findings in ruptured and unruptured intracranial
aneurysms. Acta Odontologica Scandinavica. 74(4):315-320, 2016
III Pyysalo MJ, Pyysalo LM, Hiltunen J, Järnstedt J, Helminen M, Karhunen PJ, Pessi T. The dental infections in patients undergoing preoperative dental examination before surgical treatment of saccular intracranial aneurysm.
BMC Research Notes. 20;11(1):600, 2018
IV Pyysalo MJ, Mishra PP, Sundström K, Lehtimäki T, Karhunen PJ, Pessi T.
Increased tooth brushing frequency is associated with reduced gingival pocket bacterial diversity in patients with intracranial aneurysms.
PeerJ. Jan 25;7:e6316, 2019
The publications were adapted with the permission of the copyright owners.
CONTENTS
ABSTRACT ... 7
TIIVISTELMÄ ... 9
ACKNOWLEDGEMENTS ... 11
1 INTRODUCTION ... 19
2 REVIEW OF THE LITERATURE ... 21
2.1 Periodontal microbiology ... 21
2.1.1 Porphyromonas gingivalis ... 21
2.1.2 Fusobacterium nucleatum ... 22
2.1.3 The Streptococcus mitis group ... 22
2.1.4 Prevotella intermedia ... 23
2.1.5 Aggregatibacter actinomycetemcomitans ... 23
2.1.6 Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mitis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans in cardiovascular diseases ... 24
2.2 Development of periodontitis ... 26
2.2.1 Healthy periodontium ... 26
2.2.2 Periodontitis ... 26
2.3 Cardiovascular and cerebrovascular diseases and periodontitis ... 28
2.3.1 General considerations ... 28
2.3.2 Intracranial aneurysms ... 28
2.4 Low-grade systemic inflammation in periodontitis and cardiovascular diseases ... 30
2.5 Molecular analysis of bacteria ... 31
2.5.1 Next generation sequencing ... 31
2.6 Tools for data analysis ... 34
2.6.1 Pipelines and softwares ... 34
3 AIMS OF THE STUDY ... 37
4 MATERIALS AND METHODS ... 39
4.1 Patients (Studies I-IV) ... 39
4.2 Autopsy cases (Study I) ... 40
4.3 Dental examinations and interviews (Study III, IV) ... 40
4.4 Samples (I-IV) ... 41
4.4.1 Aneurysm samples ... 41
4.4.2 Gingival pocket samples ... 41
4.4.3 Control samples and data ... 41
4.5 Ethical issues ... 42
4.6 Immunohistochemical studies: staining of receptors recognising bacteria .. 42
4.7 Detection of bacteria using PCR (Studies I-III) ... 42
4.8 16S rRNA gene amplification ... 43
4.9 Data analyses... 44
CONTENTS
ABSTRACT ... 7
TIIVISTELMÄ ... 9
ACKNOWLEDGEMENTS ... 11
1 INTRODUCTION ... 19
2 REVIEW OF THE LITERATURE ... 21
2.1 Periodontal microbiology ... 21
2.1.1 Porphyromonas gingivalis ... 21
2.1.2 Fusobacterium nucleatum ... 22
2.1.3 The Streptococcus mitis group ... 22
2.1.4 Prevotella intermedia ... 23
2.1.5 Aggregatibacter actinomycetemcomitans ... 23
2.1.6 Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mitis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans in cardiovascular diseases ... 24
2.2 Development of periodontitis ... 26
2.2.1 Healthy periodontium ... 26
2.2.2 Periodontitis ... 26
2.3 Cardiovascular and cerebrovascular diseases and periodontitis ... 28
2.3.1 General considerations ... 28
2.3.2 Intracranial aneurysms ... 28
2.4 Low-grade systemic inflammation in periodontitis and cardiovascular diseases ... 30
2.5 Molecular analysis of bacteria ... 31
2.5.1 Next generation sequencing ... 31
2.6 Tools for data analysis ... 34
2.6.1 Pipelines and softwares ... 34
3 AIMS OF THE STUDY ... 37
4 MATERIALS AND METHODS ... 39
4.1 Patients (Studies I-IV) ... 39
4.2 Autopsy cases (Study I) ... 40
4.3 Dental examinations and interviews (Study III, IV) ... 40
4.4 Samples (I-IV) ... 41
4.4.1 Aneurysm samples ... 41
4.4.2 Gingival pocket samples ... 41
4.4.3 Control samples and data ... 41
4.5 Ethical issues ... 42
4.6 Immunohistochemical studies: staining of receptors recognising bacteria .. 42
4.7 Detection of bacteria using PCR (Studies I-III) ... 42
4.8 16S rRNA gene amplification ... 43
4.9 Data analyses... 44
5 RESULTS ... 45
5.1 Study I ... 45
5.1.1 Patient and aneurysm characteristics ... 45
5.1.2 Molecular microbiological and immunohistochemical findings in aneurysm walls ... 46
5.2 Study II ... 48
5.2.1 Patient characteristics ... 48
5.2.2 Molecular microbiological findings in aneurysm walls ... 50
5.3 Study III ... 54
5.3.1 Clinical findings ... 54
5.3.2 Bacterial analyses ... 56
5.4 Study IV ... 56
5.4.1 Overall bacterial content of the gingival pockets ... 56
5.4.2 The association of clinical parameters with alpha diversity ... 59
6 DISCUSSION ... 65
6.1 General considerations ... 65
6.2 Periodontitis in patients with cerebro-cardiovascular diseases ... 66
6.3 Shared genetic factors of periodontitis and cardiovascular diseases ... 67
6.4 Limitations of the studies ... 68
6.5 The risk of sample contamination ... 70
6.6 The use of molecular detection of bacteria in clinical practice and future considerations ... 70
7 CONCLUSIONS ... 71
REFERENCES ... 73 ORIGINAL PUBLICATIONS
ABBREVIATIONS
Arg Arginin
ATCC American type culture collection
CD Cluster of differentiation COX Cyklo-oxygenase CRP C-reactive protein CVDs Cardiovascular diseases
DAA Differential analysis of abundance
DAB Diaminobenzidine DNA Deoxyribonucleic acid GCF Gingival crevicular fluid HAEC Human aortal endothelial cell HDL High-density lipoprotein HOMD Human oral microbiome
database
IA Intracranial aneurysm Ig Immunoglobulin IL Interleukin
LBP Lipopolysaccharide binding protein
LDL Low-density lipoprotein
LITA Left internal thoracic artery Lys Lysin
MMP Matrix metalloproteinase NGS Next-generation sequencing NLR Nucleotide binding domain
and leucin-rich-repeat- containing receptor
OTU Operative taxonomic unit PCoA Principal component analysis PCR Polymerase chain reaction pH Hydrogen ion concentration PMNs Polymorphonuclear
leukocytes
qPCR Quantitative polymerase chain reaction
rRNA Ribosomal ribonucleic acid RT-PCR Real-time polymerase chain
reaction
SAH Subarachnoid haemorrhage SD Standard deviation
TLR Toll-like receptor TNF Tumour necrosis factor
5 RESULTS ... 45
5.1 Study I ... 45
5.1.1 Patient and aneurysm characteristics ... 45
5.1.2 Molecular microbiological and immunohistochemical findings in aneurysm walls ... 46
5.2 Study II ... 48
5.2.1 Patient characteristics ... 48
5.2.2 Molecular microbiological findings in aneurysm walls ... 50
5.3 Study III ... 54
5.3.1 Clinical findings ... 54
5.3.2 Bacterial analyses ... 56
5.4 Study IV ... 56
5.4.1 Overall bacterial content of the gingival pockets ... 56
5.4.2 The association of clinical parameters with alpha diversity ... 59
6 DISCUSSION ... 65
6.1 General considerations ... 65
6.2 Periodontitis in patients with cerebro-cardiovascular diseases ... 66
6.3 Shared genetic factors of periodontitis and cardiovascular diseases ... 67
6.4 Limitations of the studies ... 68
6.5 The risk of sample contamination ... 70
6.6 The use of molecular detection of bacteria in clinical practice and future considerations ... 70
7 CONCLUSIONS ... 71
REFERENCES ... 73 ORIGINAL PUBLICATIONS
ABBREVIATIONS
Arg Arginin
ATCC American type culture collection
CD Cluster of differentiation COX Cyklo-oxygenase CRP C-reactive protein CVDs Cardiovascular diseases
DAA Differential analysis of abundance
DAB Diaminobenzidine DNA Deoxyribonucleic acid GCF Gingival crevicular fluid HAEC Human aortal endothelial cell HDL High-density lipoprotein HOMD Human oral microbiome
database
IA Intracranial aneurysm Ig Immunoglobulin IL Interleukin
LBP Lipopolysaccharide binding protein
LDL Low-density lipoprotein
LITA Left internal thoracic artery Lys Lysin
MMP Matrix metalloproteinase NGS Next-generation sequencing NLR Nucleotide binding domain
and leucin-rich-repeat- containing receptor
OTU Operative taxonomic unit PCoA Principal component analysis PCR Polymerase chain reaction pH Hydrogen ion concentration PMNs Polymorphonuclear
leukocytes
qPCR Quantitative polymerase chain reaction
rRNA Ribosomal ribonucleic acid RT-PCR Real-time polymerase chain
reaction
SAH Subarachnoid haemorrhage SD Standard deviation
TLR Toll-like receptor TNF Tumour necrosis factor
tPA Tissue plasminogen activator UIA Unruptured intracranial
aneurysm
WHO World health organization
1 INTRODUCTION
In the modern research era, multicellular organisms, including Homo sapiens, cannot be considered as discrete individuals according to classical definitions of the term.
The human is a holobiont consisting of host cells and symbiotic micro-organisms such as bacteria, archaea, fungi and viruses. (Rosenberg and Zilber-Rosenberg, 2016) The human body consists of approximately 3.8·1013 microbial cells and 3.0·1013 human cells, of which only 0.3 x 1013 are nucleated (Sender, Fuchs and Milo, 2016).
When a new human holobiont is born, its microbes are obtained from the mother´s vaginal (Wampach et al., 2018) and possibly placental (Aagaard et al., 2014; Collado et al., 2016) microbiotas. The development of the microbiota of a newborn may have started before birth (Collado et al., 2016).
The oral cavity is a main route for microbes to enter the human body, and functions as a natural course for passage to the respiratory and gastrointestinal tracts.
It is a moist cavity, lined by mucosa, with a relatively stable temperature (from 34 to 36°C). Hydrogen ion concentration (pH) remains close to neutrality in most areas of the cavity and thus the environment supports the growth of a wide spectrum of microorganisms. (Marcotte and Lavoie, 1998)
Over 750 bacterial species have been isolated from human oral cavity (Dewhirst et al., 2010). Of all these isolates, only 57% have been officially named at the species level, 13% are unnamed but can be cultivated and 30% are known only as uncultivated phylotypes (Dewhirst et al., 2010). Infectious diseases of the oral cavity are driven by dysbiosis (Sanz et al., 2017). Dental caries is no longer thought to be caused by a single organism, Streptococcus mutans, but to occur as a result of disturbances in dental plaque ecology and gene expression (He et al., 2018). In endodontic infections the apical part of the root canal system drives the selection of a more diverse and more anaerobic bacterial community than the coronal part (Özok et al., 2012). Periodontitis is initially caused by a disturbance in the host response to supragingival biofilm infection leading to, and modified by, systemic low-grade inflammation (Graves, Corrêa and Silva, 2019), which is associated closely with cerebro- and cardiovascular diseases (Schenkein and Loos, 2013). Periodontal bacteria or their DNA have been detected in the blood vessels of cardiovascular patients (Figuero et al., 2011), although there are only two studies in which the bacterial content of celebral arteries in intracranial aneurysm were assessed (Cagli et al., 2003; Aboukais et al., 2019). The aim of this study was based on the finding that there are reports linking periodontitis to cardiovascular diseases (via systemic low- grade inflammation) and cardiovascular diseases to cerebrovascular diseases (Pyysalo et al., 2013; Schenkein and Loos, 2013). As a first group, we conducted a study based on quantitative polymerase chain reaction (qPCR) in order to detect oral bacterial DNA from ruptured and unruptured intracranial aneurysm tissue samples.
In addition, we performed immunohistochemical analyses to detect possible bacteria-driven inflammatory reactions from the aneurysm tissue samples. The
tPA Tissue plasminogen activator UIA Unruptured intracranial
aneurysm
WHO World health organization
1 INTRODUCTION
In the modern research era, multicellular organisms, including Homo sapiens, cannot be considered as discrete individuals according to classical definitions of the term.
The human is a holobiont consisting of host cells and symbiotic micro-organisms such as bacteria, archaea, fungi and viruses. (Rosenberg and Zilber-Rosenberg, 2016) The human body consists of approximately 3.8·1013 microbial cells and 3.0·1013 human cells, of which only 0.3 x 1013 are nucleated (Sender, Fuchs and Milo, 2016).
When a new human holobiont is born, its microbes are obtained from the mother´s vaginal (Wampach et al., 2018) and possibly placental (Aagaard et al., 2014; Collado et al., 2016) microbiotas. The development of the microbiota of a newborn may have started before birth (Collado et al., 2016).
The oral cavity is a main route for microbes to enter the human body, and functions as a natural course for passage to the respiratory and gastrointestinal tracts.
It is a moist cavity, lined by mucosa, with a relatively stable temperature (from 34 to 36°C). Hydrogen ion concentration (pH) remains close to neutrality in most areas of the cavity and thus the environment supports the growth of a wide spectrum of microorganisms. (Marcotte and Lavoie, 1998)
Over 750 bacterial species have been isolated from human oral cavity (Dewhirst et al., 2010). Of all these isolates, only 57% have been officially named at the species level, 13% are unnamed but can be cultivated and 30% are known only as uncultivated phylotypes (Dewhirst et al., 2010). Infectious diseases of the oral cavity are driven by dysbiosis (Sanz et al., 2017). Dental caries is no longer thought to be caused by a single organism, Streptococcus mutans, but to occur as a result of disturbances in dental plaque ecology and gene expression (He et al., 2018). In endodontic infections the apical part of the root canal system drives the selection of a more diverse and more anaerobic bacterial community than the coronal part (Özok et al., 2012). Periodontitis is initially caused by a disturbance in the host response to supragingival biofilm infection leading to, and modified by, systemic low-grade inflammation (Graves, Corrêa and Silva, 2019), which is associated closely with cerebro- and cardiovascular diseases (Schenkein and Loos, 2013). Periodontal bacteria or their DNA have been detected in the blood vessels of cardiovascular patients (Figuero et al., 2011), although there are only two studies in which the bacterial content of celebral arteries in intracranial aneurysm were assessed (Cagli et al., 2003; Aboukais et al., 2019). The aim of this study was based on the finding that there are reports linking periodontitis to cardiovascular diseases (via systemic low- grade inflammation) and cardiovascular diseases to cerebrovascular diseases (Pyysalo et al., 2013; Schenkein and Loos, 2013). As a first group, we conducted a study based on quantitative polymerase chain reaction (qPCR) in order to detect oral bacterial DNA from ruptured and unruptured intracranial aneurysm tissue samples.
In addition, we performed immunohistochemical analyses to detect possible bacteria-driven inflammatory reactions from the aneurysm tissue samples. The
dental infectious burden, as well as the bacterial content of the gingival pockets of the patients with intracranial aneurysms, were assessed using clinical examination and molecular analyses methods: qPCR and 16S rRNA gene sequencing.
2 REVIEW OF THE LITERATURE
2.1 PERIODONTAL MICROBIOLOGY
The microbiota (née flora) of the healthy oral cavity is highly diverse and both site- and subject-specific (Aas et al., 2005). Microbial communities form multispecies biofilms which generally exist in harmony with the host. Biofilm-host interaction gives important benefits which contribute to overall health and well-being. Within these biofilms, microorganisms live in close proximity with each another, which results in a wide range of either synergistic or antagonistic interactions. Both composition and function of the microbiome are highly influenced by the oral environment. Changes in local conditions can (in part) determine whether the relationship between the biofilm and the host is symbiotic or potentially damaging (dysbiotic). (Marsh 2003, Roberts and Darveau 2015)
In general, superficial keratinized and non-keratinized mucosa (buccal mucosa, keratinized gingiva and hard palate) consist mainly of firmicutes, followed in decreasing order of relative abundance by proteobacteria, bacteroidetes and either actinobacteria or fusobacteria, whereas in deeper sites, as in gingival pockets, the abundance of firmicutes decreases dramatically and that of actinobacteria increases (Segata et al., 2012).
When this study was planned, only limited number of studies regarding oral bacterial presence in atherosclerotic plaques or vascular samples was available.
Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans had been isolated from the vascular samples (Han et al., 2004;
Saito et al., 2008; Kozarov et al., 2005) Since streptococcae are predominant in the supragingival area (Kolenbrander and London, 1993) and Prevotella intermedia is known to enhance the progression of periodontitis (Maeda et al., 1998) bacteria chosen to deal in this review are Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mitis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans.
2.1.1 Porphyromonas gingivalis
Porphyromonas gingivalis is a non-motile, asaccharolytic, obligately anaerobic Gram- negative rod. It forms black-pigmented colonies on blood agar plates and has an absolute requirement for iron and amino acids for growth and energy production (How, Song and Chan, 2016). It produces several virulence factors, the expressions of which are regulated by the external environment of the pathogen. These virulence factors include enzymes (hyaluronidase, chondroitin sulfatase), capsule, fimbriae, lipopolysaccharide, exopolysaccharide, outer membrane proteins, gelatinase, collagenase, trypsin-like protease and aminopeptidase (How et al., 2016). Two widely studied peptidases, gingipains K and R, are responsible for 85% of the proteolytic
dental infectious burden, as well as the bacterial content of the gingival pockets of the patients with intracranial aneurysms, were assessed using clinical examination and molecular analyses methods: qPCR and 16S rRNA gene sequencing.
2 REVIEW OF THE LITERATURE
2.1 PERIODONTAL MICROBIOLOGY
The microbiota (née flora) of the healthy oral cavity is highly diverse and both site- and subject-specific (Aas et al., 2005). Microbial communities form multispecies biofilms which generally exist in harmony with the host. Biofilm-host interaction gives important benefits which contribute to overall health and well-being. Within these biofilms, microorganisms live in close proximity with each another, which results in a wide range of either synergistic or antagonistic interactions. Both composition and function of the microbiome are highly influenced by the oral environment. Changes in local conditions can (in part) determine whether the relationship between the biofilm and the host is symbiotic or potentially damaging (dysbiotic). (Marsh 2003, Roberts and Darveau 2015)
In general, superficial keratinized and non-keratinized mucosa (buccal mucosa, keratinized gingiva and hard palate) consist mainly of firmicutes, followed in decreasing order of relative abundance by proteobacteria, bacteroidetes and either actinobacteria or fusobacteria, whereas in deeper sites, as in gingival pockets, the abundance of firmicutes decreases dramatically and that of actinobacteria increases (Segata et al., 2012).
When this study was planned, only limited number of studies regarding oral bacterial presence in atherosclerotic plaques or vascular samples was available.
Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans had been isolated from the vascular samples (Han et al., 2004;
Saito et al., 2008; Kozarov et al., 2005) Since streptococcae are predominant in the supragingival area (Kolenbrander and London, 1993) and Prevotella intermedia is known to enhance the progression of periodontitis (Maeda et al., 1998) bacteria chosen to deal in this review are Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mitis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans.
2.1.1 Porphyromonas gingivalis
Porphyromonas gingivalis is a non-motile, asaccharolytic, obligately anaerobic Gram- negative rod. It forms black-pigmented colonies on blood agar plates and has an absolute requirement for iron and amino acids for growth and energy production (How, Song and Chan, 2016). It produces several virulence factors, the expressions of which are regulated by the external environment of the pathogen. These virulence factors include enzymes (hyaluronidase, chondroitin sulfatase), capsule, fimbriae, lipopolysaccharide, exopolysaccharide, outer membrane proteins, gelatinase, collagenase, trypsin-like protease and aminopeptidase (How et al., 2016). Two widely studied peptidases, gingipains K and R, are responsible for 85% of the proteolytic
activity of P. gingivalis at the site of infection: Gingipain R cleaves bonds after arginines and Gingipain K cleaves after lysines (de Diego et al., 2014). In a susceptible host, these virulence factors cause bone resorption, destruction of periodontal tissues, inhibition of host protective mechanisms and induce cytokine production (How et al. 2016).
P. gingivalis is referred to as a “keystone” pathogen: its presence shifts the microbiota in the periodontal pocket to a dysbiotic one (Hajishengallis, Darveau and Curtis, 2012). The count of P. gingivalis correlates with pocket depth and bleeding on probing (Oliveira et al., 2016).
2.1.2 Fusobacterium nucleatum
Fusobacterium nucleatum is a Gram-negative, anaerobic rod of the mammalian mouth and digestive tract (Strauss et al., 2008). It is a crucial microorganism in dental biofilms because of its coordinator role. It coaggregates with both late and early dental biofilm colonizers, working like a bridge between different bacteria.
(Kolenbrander et al., 2002) Invasion and adherence are the most important mechanisms for F.nucleatum to colonize, disseminate, escape from host defense, and to induce host responses (Han 2015). Adhesin (and invasin) FadA has been identified to bind and to invade host cells being the main virulence factor identified from F.
nucleatum (Xu et al., 2007; Han 2015). F.nucleatum creates favourable conditions for other periodontal bacteria (together with Prevotella intermedia). The mechanisms behind this include promoting neutral pH and a capnophilic environment, which induces increased gingival crevicular fluid (GCF) formation and colonization of another more proteolytic but acid-intolerant bacterium, P. gingivalis. (Takahashi, 2005; Huang, Li and Gregory, 2011) The dependence of P.gingivalis on F.nucleatum has been shown in one cell culture study, in which The human gingival epithelial cell line (Ca9-22) and Human Aorta Endothelial Cell (HAEC) were infected by F. nucleatum and P. gingivalis: Coincubation of P. gingivalis ATCC 33277 with F. nucleatum significantly increased P. gingivalis invasion to host cells, resulting in a 2–20-fold increase in invasion efficiencies (Saito et al., 2008). In another in vitro biofilm study, the presence of Actinomyces naeslundii was shown to be important for the growth of F. nucleatum (Periasamy et al., 2009). Intestinal F. nucleatum is associated with colorectal cancer, and reducing it in patients with colorectal cancer may improve their response to chemotherapy and decrease cancer recurrence (Yu et al., 2017).
2.1.3 The Streptococcus mitis group
Normally, streptococci (together with actinomyces) are predominant in the supragingival area in healthy periodontium as a part of the natural bacterial flora.
They can adhere to the saliva-coated tooth surface by attachment between adhesins and receptors (Kolenbrander and London, 1993). The genus Streptococcus is the most abundant in the salivary microbiome (Nasidze et al., 2009). The Streptococcus mitis group includes 13 species of bacteria. They are Gram-positive microbes sharing
common virulence traits and similar strategies with other bacteria from the family Streptococcaceae to escape from the oral niche and to establish a distant infection in other parts of the host, typically infective endocarditis, meningitis, bacteremia/sepsis or toxic shock-like syndrome (Sitkiewicz, 2018). The virulence factors of S. mitis include capsule, pili, lipoteichoic acid, fibronectin-, laminin-, fibrinogen-, and collagen-binding proteins (Nasidze et al., 2009; Sitkiewicz, 2018).
2.1.4 Prevotella intermedia
Prevotella intermedia, a periodontitis-associated member of the orange complex (F.
nucleatum, P. intermedia, Prevotella nigrescens, Parviomonas micra, Eubacterium nodatum and various Camplylobacter species), is among the most frequently encountered species in subgingival plaque (Kamma et al., 2004). It is an obligately anaerobic, Gram negative, moderately saccaharolytic short rod (0.5 by 2 micrometers) (Chen et al., 2010). The main virulence factors are proteases. P.intermedia encodes beta-lactamases and multidrug/efflux transporters providing resistance to antibiotics. Adhesion, competing with surrounding microbes and horizontal gene transfer are the main drive of the evolution of pathogen. (Ruan et.al., 2015) It has been suggested that P.
intermedia may increase the activity of degradative enzymes (such as alkaline phosphatase, esterase, esterase-lipase and chymotrypsin) under certain conditions and enhance the progression of periodontitis (Maeda et al., 1998). Since P. intermedia has a highly dynamic genome and it can take up various exogenous factors through horizontal gene transfer, it has been suggested that P. intermedia and P. nigrescens may serve as “crucial substances” in subgingival plaque, and that P. intermedia along with other members of the “orange complex” could reflect changes in microbial and environmental dynamics in the subgingival microbiome (Zhang et al., 2017).
2.1.5 Aggregatibacter actinomycetemcomitans
Aggregatibacter actinomycetemcomitans is a Gram-negative, facultative anaerobic bacillus that causes periodontal diseases such as localized aggressive periodontitis (née juvenile periodontitis) (Gholizadeh et al., 2017). Its presence is linked to aggressive periodontitis in adolescents (Fine et al., 2007). Virulence factors for A.
actinomycetemcomitans are endotoxin (as in other Gram-negative bacteria; causes a general pro-inflammatory host response), cytolethal distending toxin (Cdt) and a leukotoxin (LtxA) (Åberg et al., 2014). In the local environment, its presence is linked to periodontal bone loss (Fine et al., 2007). Systemically, the lipopolysaccharide of A.
actinomycetemcomitans is responsible for inducing low-grade systemic inflammation via pro-inflammatory mediators such as interleukins (IL) -1β, −6, −8, and tumour necrosis factor (TNF) -α (Bodet, Chandad and Grenier, 2006). As a result of leukotoxin A production, A.actinomycetemcomitans has substantial pro-inflammatory effects on human brain endothelial cells (Dietmann et al., 2013), and it helps A.
actinomycetemcomitans to evade the host immune system by killing neutrophils, lymphocytes, and monocytes (Sampathkumar et al., 2017).
activity of P. gingivalis at the site of infection: Gingipain R cleaves bonds after arginines and Gingipain K cleaves after lysines (de Diego et al., 2014). In a susceptible host, these virulence factors cause bone resorption, destruction of periodontal tissues, inhibition of host protective mechanisms and induce cytokine production (How et al. 2016).
P. gingivalis is referred to as a “keystone” pathogen: its presence shifts the microbiota in the periodontal pocket to a dysbiotic one (Hajishengallis, Darveau and Curtis, 2012). The count of P. gingivalis correlates with pocket depth and bleeding on probing (Oliveira et al., 2016).
2.1.2 Fusobacterium nucleatum
Fusobacterium nucleatum is a Gram-negative, anaerobic rod of the mammalian mouth and digestive tract (Strauss et al., 2008). It is a crucial microorganism in dental biofilms because of its coordinator role. It coaggregates with both late and early dental biofilm colonizers, working like a bridge between different bacteria.
(Kolenbrander et al., 2002) Invasion and adherence are the most important mechanisms for F.nucleatum to colonize, disseminate, escape from host defense, and to induce host responses (Han 2015). Adhesin (and invasin) FadA has been identified to bind and to invade host cells being the main virulence factor identified from F.
nucleatum (Xu et al., 2007; Han 2015). F.nucleatum creates favourable conditions for other periodontal bacteria (together with Prevotella intermedia). The mechanisms behind this include promoting neutral pH and a capnophilic environment, which induces increased gingival crevicular fluid (GCF) formation and colonization of another more proteolytic but acid-intolerant bacterium, P. gingivalis. (Takahashi, 2005; Huang, Li and Gregory, 2011) The dependence of P.gingivalis on F.nucleatum has been shown in one cell culture study, in which The human gingival epithelial cell line (Ca9-22) and Human Aorta Endothelial Cell (HAEC) were infected by F. nucleatum and P. gingivalis: Coincubation of P. gingivalis ATCC 33277 with F. nucleatum significantly increased P. gingivalis invasion to host cells, resulting in a 2–20-fold increase in invasion efficiencies (Saito et al., 2008). In another in vitro biofilm study, the presence of Actinomyces naeslundii was shown to be important for the growth of F. nucleatum (Periasamy et al., 2009). Intestinal F. nucleatum is associated with colorectal cancer, and reducing it in patients with colorectal cancer may improve their response to chemotherapy and decrease cancer recurrence (Yu et al., 2017).
2.1.3 The Streptococcus mitis group
Normally, streptococci (together with actinomyces) are predominant in the supragingival area in healthy periodontium as a part of the natural bacterial flora.
They can adhere to the saliva-coated tooth surface by attachment between adhesins and receptors (Kolenbrander and London, 1993). The genus Streptococcus is the most abundant in the salivary microbiome (Nasidze et al., 2009). The Streptococcus mitis group includes 13 species of bacteria. They are Gram-positive microbes sharing
common virulence traits and similar strategies with other bacteria from the family Streptococcaceae to escape from the oral niche and to establish a distant infection in other parts of the host, typically infective endocarditis, meningitis, bacteremia/sepsis or toxic shock-like syndrome (Sitkiewicz, 2018). The virulence factors of S. mitis include capsule, pili, lipoteichoic acid, fibronectin-, laminin-, fibrinogen-, and collagen-binding proteins (Nasidze et al., 2009; Sitkiewicz, 2018).
2.1.4 Prevotella intermedia
Prevotella intermedia, a periodontitis-associated member of the orange complex (F.
nucleatum, P. intermedia, Prevotella nigrescens, Parviomonas micra, Eubacterium nodatum and various Camplylobacter species), is among the most frequently encountered species in subgingival plaque (Kamma et al., 2004). It is an obligately anaerobic, Gram negative, moderately saccaharolytic short rod (0.5 by 2 micrometers) (Chen et al., 2010). The main virulence factors are proteases. P.intermedia encodes beta-lactamases and multidrug/efflux transporters providing resistance to antibiotics. Adhesion, competing with surrounding microbes and horizontal gene transfer are the main drive of the evolution of pathogen. (Ruan et.al., 2015) It has been suggested that P.
intermedia may increase the activity of degradative enzymes (such as alkaline phosphatase, esterase, esterase-lipase and chymotrypsin) under certain conditions and enhance the progression of periodontitis (Maeda et al., 1998). Since P. intermedia has a highly dynamic genome and it can take up various exogenous factors through horizontal gene transfer, it has been suggested that P. intermedia and P. nigrescens may serve as “crucial substances” in subgingival plaque, and that P. intermedia along with other members of the “orange complex” could reflect changes in microbial and environmental dynamics in the subgingival microbiome (Zhang et al., 2017).
2.1.5 Aggregatibacter actinomycetemcomitans
Aggregatibacter actinomycetemcomitans is a Gram-negative, facultative anaerobic bacillus that causes periodontal diseases such as localized aggressive periodontitis (née juvenile periodontitis) (Gholizadeh et al., 2017). Its presence is linked to aggressive periodontitis in adolescents (Fine et al., 2007). Virulence factors for A.
actinomycetemcomitans are endotoxin (as in other Gram-negative bacteria; causes a general pro-inflammatory host response), cytolethal distending toxin (Cdt) and a leukotoxin (LtxA) (Åberg et al., 2014). In the local environment, its presence is linked to periodontal bone loss (Fine et al., 2007). Systemically, the lipopolysaccharide of A.
actinomycetemcomitans is responsible for inducing low-grade systemic inflammation via pro-inflammatory mediators such as interleukins (IL) -1β, −6, −8, and tumour necrosis factor (TNF) -α (Bodet, Chandad and Grenier, 2006). As a result of leukotoxin A production, A.actinomycetemcomitans has substantial pro-inflammatory effects on human brain endothelial cells (Dietmann et al., 2013), and it helps A.
actinomycetemcomitans to evade the host immune system by killing neutrophils, lymphocytes, and monocytes (Sampathkumar et al., 2017).
2.1.6 Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mitis, Prevotella intermedia and Aggregatibacter
actinomycetemcomitans in cardiovascular diseases
Cardiovascular diseases (CVDs) are a group of disorders of the heart and blood vessels and they include: coronary heart disease, peripheral arterial disease, cerebrovascular disease, rheumatic heart disease, congenital heart disease, pulmonary embolism and deep vein thrombosis. CVDs are the number one cause of death globally: more people die annually from CVDs than from any other cause.
There is an association between chronic periodontitis and CVDs (Li et al., 2017).
Abilities of bacteria to invade vascular tissue and intravascular plaque
The ability of F. nucleatum to invade blood vessels has been demonstrated in cell culture studies and animal models (Han et al., 2004; Saito et al., 2008; Velsko et al., 2015; Chukkapalli et al., 2017). In humans, the presence of P. gingivalis, A.
actinomycetemcomitans and F. nucleatum was assessed in atheromatous plaque samples obtained from endarterectomies (n=42) using nested PCR: 78.6% of the samples were positive for P. gingivalis DNA, 66.7% for A. actinomycetemcomitans and 50.0% for F. nucleatum DNA. The simultaneous presence of various bacterial species within the same specimen was a common observation. (Figuero et al., 2011) The presence of A. actinomycetemcomitans (together with P. gingivalis) has been confirmed within the coronary artery plaque by cell culture invasion assays and immunofluorescence microscopy together with positive PCR findings (Kozarov et al., 2005). The bacterial DNA of S. mitis has been found in thrombus aspirates (Pessi et al., 2013) and atherosclerotic plaque (Eberhard et al., 2017). Cocci and rods were seen in scanning electron microscopy or in transmission electron microscopy together with positive next-generation sequencing (ngs) results (Pessi et al., 2013; Armingohar et al., 2014). It has been suggested that high blood pressure at the aortic valve may prevent the adhesion and proliferation of bacterial colonies (Raffaelli et al., 2010).
PCR was used in order to detect periodontal pathogens (Tannerella forshytia, P.
gingivalis, A. actinomycetemcomitans, P. intermedia, F. nucleatum, Campylobacter rectus, Eikenella corrodens and Treponema denticola), but neither 19 aortic valve specimens nor the blood samples were positive for the genoma of these pathogens (Raffaelli et al., 2010).
Abilities of bacteria to invade myocardium
The presence of A. actinomycetemcomitans, P. gingivalis, Tannerella forsythia, T.
denticola, P. intermedia, Parvimonas micra, F. nucleatum, Campylobacter rectus, Eubacterium nodatum, Eikenella corrodens and Capnocytophaga species in adult human myocardium has been confirmed, with an immunohistochemically detected impact on atrial and myocardial tissue bacterial recognition molecules, such as CD14, CD68
and lipopolysaccharide binding protein (LBP) (Ziebolz et al., 2018). In children with congenital heart disease, the presence of A. actinomycetemcomitans in myocardial tissue was negative, although 8% of saliva samples were positive (Bozdogan et al., 2016).
Porphyromonas gingivalis in cardiovascular diseases
The presence of P. gingivalis in the gingival pockets has been associated with uncontrolled diabetes and cardiovascular diseases (Norio Aoyama, Suzuki, Kobayashi, et al., 2018). The presence of P. gingivalis has been detected at the sites of atherosclerotic disease in humans by cell culture invasion assays and immunofluorescence microscopy (Kozarov et al. 2005) and in several PCR or 16S rRNA gene-based studies (Mahendra et al., 2015; Szulc et al., 2015; Mahalakshmi, Krishnan and Arumugam, 2017; Mougeot et al., 2017; Atarbashi-Moghadam et al., 2018). It has been shown that P. gingivalis has the ability to invade host cells, providing an escape from the action of the immune system or therapeutic agents.
This characteristic is thought to be a key virulence factor, along with the presence of a bacterial capsule, lipopolysaccharide, and secretion of proteolytic enzymes (gingipains) (Bostanci and Belibasakis, 2012). Gingipains have been shown to be able to cause neurodegeneration and amyloid accumulation, linked to Alzheimer´s disease, in mice (Ilievski et al., 2018) and in humans (Dominy et al., 2019). In a cell culture model, the modulative action of P. gingivalis on the cytokine response in the cells was demonstrated: It disrupts the adhesion activity and the viability through the action of Arg-gingipain and Lys-gingipain and thereby may contribute to the pathogenesis of cardiovascular diseases (Baba et al., 2002). In addition, it has been shown that P. gingivalis can induce high-density lipoprotein (HDL) oxidation, weakening the atheroprotective function of HDL and even making it proatherogenic (H.-J. Kim et al., 2018). The ability of P. gingivalis and F. nucleatum (together with T.
denticola and Tannerella forsythia) to directly and actively invade the aortic adventitial layer of integrin beta 6 -/- mice has been shown by fluorescence in an in situ hybridization study. Along with invasion, alterations in Toll-like receptor (TLR) and nucleotide-binding domain and leucine-rich-repeat-containing receptor (NLR) gene expressions were observed. (Velsko et al., 2015) The invasive properties of P.
gingivalis are partly dependent on other bacteria, as was shown in a cell culture study by Saito and colleagues (Saito et al., 2008). Animal studies have shown the impact of P. gingivalis on cardiac hypertrophy via oxidative stress (Sato et al., 2016) and cardiac rupture after myocardial infarct, as well as its ability to invade the ischemic myocardium and promote cardiomyocyte apoptosis (Shiheido et al., 2016).
In conclusion, there is strong evidence that all these 5 bacteria (F. nucleatum, P.
gingivalis, A. actinomycetemcomitans, S. mitis and P. intermedia) are cabable of invading the human vascular wall.
