Influence of culture conditions on porphyrin production in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis.
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 Porphyromonas gingivalis, 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.
Department of Environmental Science and Analytical Chemistry, Division of Analytical and Toxicological Chemistry, Stockholm University, S-106 91 Stockholm, Sweden.
Jonas
J
Fyrestam
Department of Dental Medicine, Karolinska Institutet, S-141 04 Huddinge, Sweden.
Nadja
N
Bjurshammar
Department of Aquatic Sciences and Assessment, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden.
Elin
E
Paulsson
Department of Environmental Science and Analytical Chemistry, Division of Analytical and Toxicological Chemistry, Stockholm University, S-106 91 Stockholm, Sweden.
Nesrine
N
Mansouri
Department of Dental Medicine, Karolinska Institutet, S-141 04 Huddinge, Sweden.
Annsofi
A
Johannsen
Department of Environmental Science and Analytical Chemistry, Division of Analytical and Toxicological Chemistry, Stockholm University, S-106 91 Stockholm, Sweden. Electronic address: conny.ostman@aces.su.se.
Conny
C
Östman
D016976:Aggregatibacter actinomycetemcomitans / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D006418:Heme / Q000096:biosynthesis; D006801:Humans; D011166:Porphyrins / Q000096:biosynthesis*; D016966:Porphyromonas gingivalis / Q000378:metabolism*
D011166:Porphyrins; D006418:Heme
Antimicrobial photodynamic therapy; Dental biofilm; Growth conditions; Oral bacteria; Phototherapy; Porphyrins