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Influence of culture conditions on porphyrin production in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
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Number of Authors: 6
2017 (English)In: Photodiagnosis and Photodynamic Therapy, ISSN 1572-1000, E-ISSN 1873-1597, Vol. 17, p. 115-123Article in journal (Refereed) Published
Abstract [en]

Background: Increasing antibiotic resistance among pathogens has raised the demands for new treatment methods such as antimicrobial photodynamic therapy (aPDT) and phototherapy (PT). Experiments for investigating the effects of these methods are often performed in vitro, but the procedures for cultivation of microbes vary between different studies. The aim of this study has been to elucidate how the profile of endogenously produced porphyrins differs by changing the variables of bacteria culturing conditions.

Methods: Two oral pathogens, Aggregatibacter actinomycetemcomitans and Porphyromonasgingivalis, were selected as model organisms. The contents of porphyrins and heme in the bacteria were analysed with liquid chromatography-tandem mass spectrometry when bacteria was cultivated for different lengths of time (3-9 days), upon passaging as well as when growth medium were supplemented with or without horse blood.

Results: Both porphyrin and heme content in A. actinomycetemcomitans are highly affected by the age of the culture, and that the porphyrin profiles changes during cultivation. When cultivated colonies of A. actinomycetemcomitans were passaged onto a new, fresh growth medium a large change in porphyrin content occurred. Additional porphyrins were detected; uroporphyrin and 7-carboxylporphyrin, and the total porphyrin content increased up to 28 times, When P. gingivalis was grown on blood containing medium higher concentrations of protoporphyrin IX (2.5 times) and heme (5.4 times) were quantified compared to bacteria grown without blood.

Conclusions: This study demonstrate that there is a need for more standardized culturing protocols when performing aPDT and PT experiments in vitro to avoid large variations in porphyrin profiles and concentrations, the aPDT/PT target compounds, depending on the culturing conditions.

Place, publisher, year, edition, pages
2017. Vol. 17, p. 115-123
Keyword [en]
Porphyrins, Oral bacteria, Dental biofilm, Antimicrobial photodynamic therapy, Phototherapy, Growth conditions
National Category
Analytical Chemistry Cancer and Oncology
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-143480DOI: 10.1016/j.pdpdt.2016.11.001ISI: 000399507000020PubMedID: 27825899OAI: oai:DiVA.org:su-143480DiVA, id: diva2:1104046
Available from: 2017-05-31 Created: 2017-05-31 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Porphyrins and heme in microorganisms: Porphyrin content and its relation to phototherapy and antimicrobial treatments in vivo and in vitro
Open this publication in new window or tab >>Porphyrins and heme in microorganisms: Porphyrin content and its relation to phototherapy and antimicrobial treatments in vivo and in vitro
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

One of the greatest threats to human health is increasing antimicrobial resistance among pathogens, and finding alternatives for treatment of bacterial infections is of highest importance together with a more controlled use of antibiotics. Porphyrins and heme have both been shown to be a promising class of compounds for inactivation of bacteria; porphyrins by their excellent properties to act as a photosensitizer, and heme by its importance as an iron source during a bacterial infection in vertebrates.

This thesis describes the development of analytical methods for the identification and determination of porphyrins and heme using liquid chromatography coupled to tandem mass spectrometry. Subsequently, these developed methods were applied to bacterial samples to investigate different culture conditions and additives effect to the intracellular porphyrin and heme composition. Singlet oxygen production of three naturally occurring porphyrins have been determined together with the photosensitivity for blue light and the porphyrin content in E. coli. Toothbrushes equipped with a LED, emitting light with a wavelength of 450 nm, were used in an eight week randomized clinical trial to investigate any positive periodontal effect of blue light.

Porphyrin and heme content in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were highly affected by the different cultivation conditions. The culture age of A. actinomycetemcomitans affected the porphyrin profile, while only small changes were observed for P. gingivalis during growth. A large change of the porphyrin profile could be observed when the bacteria were passaged onto a new growth medium. Additional porphyrins were detected and the total porphyrin content increased up to 28 times. These findings highlight the need for more standardized cultivation procedures when performing in vitro experiments.

Heme content in Escherichia coli was affected when different additives related to biosynthesis of heme were added to the growth medium. The uptake of heme could be reduced with 52% when a compound that chemically looks similar to heme was added to the growth medium. Since heme acquisition is important for many pathogens, this could be a promising target for antimicrobial drugs.

E. coli showed no sensitivity for 405 nm light using light doses up to 172.8 J/cm2 and only low concentrations of porphyrins could be quantified. By adding a porphyrin precursor to E. coli the intracellular concentration of porphyrins increased remarkably and a light dose of 57.6 J/cm2 reduced the bacterial number with > 5 log10 steps. This shows that E. coli can be killed due to their endogenous porphyrins.

In the clinical study we could see a weak trend that the 450 nm LED toothbrush possessed a phototherapeutic effect for three clinical indices. All indices were decreased in the intervention group, but there were no statistically significant difference compared to the control group. However, four inflammation markers were significantly decreased in the intervention group while only one decreased significantly in the control group.

In conclusion, this thesis has shown that porphyrins and heme are produced endogenously in microorganisms and that the porphyrin profiles vary depending on culture conditions and different additives. Furthermore, porphyrins may be used as endogenous photosensitizers to inactivate bacteria, but more research is necessary to determine if there is a specific porphyrin that contributes more to the photosensitivity.

Place, publisher, year, edition, pages
Stockholm: Department of environmental science and analytical chemistry, Stockholm university, 2017
Keyword
Porphyrins, heme, phototherapy, antimicrobial resistance, singlet oxygen, photosensitizer, bacteria, HPLC-MS/MS, oral bacteria, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Escherichia coli
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-149405 (URN)978-91-7797-079-8 (ISBN)978-91-7797-080-4 (ISBN)
Public defence
2018-01-19, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Available from: 2017-12-21 Created: 2017-11-30 Last updated: 2017-12-20Bibliographically approved

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