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High-level fluorescence labelling of Gram-positive pathogens

Abstract number: P1855

Aymanns S., Mauerer S., van Zandbergen G., Wolz C., Spellerberg B.

Objectives: Fluorescence labelling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. To facilitate fluorescence labelling of streptococci and related genera we wanted to improve green fluorescent protein (GFP) expression in these species.

Methods: We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbours a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene promoter of Streptococcus agalactiae and was designated pBSU101.

Results: Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10–50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labelling in different Gram-positive pathogens, numerous species were transformed. We successfully labelled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, S. anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration.

Conclusion: The plasmid pBSU101 harbouring the egfp gene under the control of the CAMP factor promoter of S. agalactiae results in high-level fluorescence in numerous streptococcal species and related genera. It represents a versatile novel vector for in vitro and in vivo studies of bacterial pathogenesis.

Session Details

Date: 10/04/2010
Time: 00:00-00:00
Session name: Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Vienna, Austria, 10 - 13 April 2010
Presentation type:
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