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Porphyrins and heme in microorganisms: Porphyrin content and its relation to phototherapy and antimicrobial treatments in vivo and in vitro
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.ORCID iD: 0000-0002-5759-4861
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.
Keywords [en]
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: urn:nbn:se:su:diva-149405ISBN: 978-91-7797-079-8 (print)ISBN: 978-91-7797-080-4 (electronic)OAI: oai:DiVA.org:su-149405DiVA, id: diva2:1161518
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
List of papers
1. Determination of porphyrins in oral bacteria by liquid chromatography electrospray ionization tandem mass spectrometry
Open this publication in new window or tab >>Determination of porphyrins in oral bacteria by liquid chromatography electrospray ionization tandem mass spectrometry
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2015 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 407, no 23, p. 7013-7023Article in journal (Refereed) Published
Abstract [en]

Biofilms in the oral cavity can be visualized by fluorescence and a common assumption is that the endogenously produced porphyrins in certain bacteria give rise to this fluorescence. Porphyrin content in oral bacteria has been sparingly investigated, and non-selective detection techniques such as utilizing the Soret fluorescence band of porphyrins are often used. In the present study, a quantitative and selective method for the determination of porphyrins in oral bacteria has been developed and validated using high performance liquid chromatography-tandem mass spectrometry. Lysis of bacteria using Tris-EDTA buffer together with ultrasonication showed high microbial killing efficiency ≥99.98 %, and sample clean-up using C18-solid phase extraction resulted in low matrix effects ≤14 % for all analytes. Using this method, the porphyrin content was determined in the two oral pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as for baker’s yeast, Saccharomyces cerevisiae. Uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, coproporphyrin, and protoporphyrin IX were identified in the investigated microorganisms, and it was shown that the porphyrin profile differs between the two bacteria, as well as for S. cerevisiae. To our knowledge, this is the first time the porphyrin profile has been determined for the bacterium A. actinomycetemcomitans.

Keywords
HPLC/MS/MS, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Saccharomyces cerevisiae, Porphyrins, Oral bacteria
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-135378 (URN)10.1007/s00216-015-8864-2 (DOI)
Funder
Swedish Research Council, K2014-70X -22533-01-3
Available from: 2016-11-08 Created: 2016-11-08 Last updated: 2017-12-04Bibliographically approved
2. Influence of culture conditions on porphyrin production in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis
Open this publication in new window or tab >>Influence of culture conditions on porphyrin production in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis
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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.

Keywords
Porphyrins, Oral bacteria, Dental biofilm, Antimicrobial photodynamic therapy, Phototherapy, Growth conditions
National Category
Analytical Chemistry Cancer and Oncology
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-143480 (URN)10.1016/j.pdpdt.2016.11.001 (DOI)000399507000020 ()27825899 (PubMedID)
Available from: 2017-05-31 Created: 2017-05-31 Last updated: 2017-12-04Bibliographically approved
3. Determination of heme in microorganisms using HPLC-MS/MS and cobalt(III) protoporphyrin IX inhibition of heme acquisition in Escherichia coli
Open this publication in new window or tab >>Determination of heme in microorganisms using HPLC-MS/MS and cobalt(III) protoporphyrin IX inhibition of heme acquisition in Escherichia coli
2017 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 409, no 30, p. 6999-7010Article in journal (Refereed) Published
Abstract [en]

One of the main threats to the achievements in modern medicine is antimicrobial resistance. Molecular targeting of bacterial acquisition mechanisms of heme has been suggested to be an alternative to antibiotics. In the present study, HPLC-MS/MS combined with a simple clean-up based on liquid-liquid extraction has been developed and evaluated for simultaneous determination of heme and porphyrin heme precursors in microorganisms. Experimental design was used to optimize the extraction parameters, to obtain a method with high recovery, low matrix effects, and high precision. The effects of additives in the culture medium on the biosynthesis of heme were studied using Escherichia coli as a model microorganism. 5-Aminolaevulinic acid and hemin increased the heme concentration in E. coli by a factor of 1.5 and 4.5, respectively. Addition of 5-aminolaevulinic acid bypassed the E. coli negative feedback control of heme biosynthesis, which led to high amounts of intracellular porphyrins. The high heme concentration obtained when hemin was used as a culture additive shows that E. coli has an uptake of heme from its surroundings. In contrast, addition of cobalt protoporphyrin IX to the growth medium reduced the amount of heme in E. coli, demonstrating this compound’s ability to mimic real heme and inhibit the heme acquisition mechanisms.

Keywords
Heme acquisition, Heme analysis, Escherichia coli, HPLC-MS/MS, Antimicrobial resistance, Porphyrins
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-149378 (URN)10.1007/s00216-017-0610-5 (DOI)000417149000006 ()29043383 (PubMedID)
Available from: 2017-11-29 Created: 2017-11-29 Last updated: 2017-12-27Bibliographically approved
4. Escherichia coli is killed by 405 nm blue light due to its endogenous porphyrins induced by 5-aminolevulinic acid
Open this publication in new window or tab >>Escherichia coli is killed by 405 nm blue light due to its endogenous porphyrins induced by 5-aminolevulinic acid
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Antimicrobial phototherapy without the use of an exogenous photosensitizer has been suggested to be a complement to antibiotics.  A commonly accepted hypothesis is that bacteria are producing endogenous porphyrins that may act as photosensitizers. To our best knowledge there are no studies linking the production of singlet oxygen for naturally occurring porphyrins to the bacterial porphyrin content, and the photosensitivity of bacteria. In the present study, we determined the quantum yield of singlet oxygen for three porphyrins commonly detected in bacteria. Porphyrin content in E. coli was determined by HPLC-MS/MS before and after administration of 5-aminolaevulinic acid (5-ALA) to the cultivation broth. 5-aminolaevulinic acid is a porphyrin precursor and will induce high amounts of intracellular porphyrins. Cultures of E. coli grown with and without 5-ALA were illuminated with 405 nm light at different light doses. Relative to the amount, uroporphyrin demonstrated the highest quantum yield of singlet oxygen, followed by coproporphyrin and protoporphyrin IX. E. coli was analyzed for porphyrin content and only low amounts of coproporphyrin I, coproporphyrin III and protoporphyrin IX could be detected. In addition, E. coli showed no sensitivity for 405 nm light at the highest dose (172.8 J/cm2). However, when E. coli was grown in 5 mM 5-ALA for 48 h, the intracellular content of porphyrins increased remarkably. Uroporphyrin was the most abundant porphyrin with 48% of total porphyrin content. Addition of 5-ALA also made E. coli more sensitive for blue light. A light dose of 4.8 J/cm2 reduced viable E. coli with 3 log10 steps and at a light dose of 57.6 J/cm2 the killing efficiency was higher than the level to work as an disinfectant (>5 log10 steps). These results shows that E. coli is be killed by light due to its endogenously produced porphyrins and that uroporphyrin could play an important part in these mechanisms.  

National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-149379 (URN)
Available from: 2017-11-29 Created: 2017-11-29 Last updated: 2017-12-04Bibliographically approved
5. The Effect of Local Phototherapy on Gingival Inflammation - A Randomized Controlled Study
Open this publication in new window or tab >>The Effect of Local Phototherapy on Gingival Inflammation - A Randomized Controlled Study
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Aim: To investigate if a toothbrush with incorporated 450 nm LEDs used in daily oral care can reduce dental plaque and gingival inflammation.

Materials and methods: An 8-week single blinded randomized clinical trial comparing toothbrushes with and without 450 nm blue LED light emission including 48 subjects. Clinical parameters of interest were plaque index (PI), gingival index (GI) and bleeding on probing (BOP). Analyzes were also made on Interleukin-1β (IL), IL-6, IL -8, and Tumor necrosis factor alpha.

Results: A significant reduction in PI, GI and BOP was detected within the groups from baseline to follow-up visit, but no statistical differences between subjects using a toothbrush with 450 nm blue light and a toothbrush without light. Significant decreases in the levels of both MMP-8 and TIMP-1 in saliva, and IL-1b and IL-8 in GCF for the blue light toothbrush groups.

Conclusion: A toothbrush with 450 nm LEDs did not yield any statistical significant adjunctive effect to tooth brushing with regard to reduction in gingival inflammation. A larger decrease in PI for the blue light toothbrush compared to control was found at a significance level of p=0.058. To get an optimal phototherapeutic effect we suggest that a wavelength of 405 nm should be used in future studies.

National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-149380 (URN)
Available from: 2017-11-29 Created: 2017-11-29 Last updated: 2017-12-04Bibliographically approved

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