A quantitative real-time RT-PCR assay for mature <it>C. albicans </it>biofilms
<p>Abstract</p> <p>Background</p> <p>Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetraz...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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BMC
2011-05-01
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| Series: | BMC Microbiology |
| Online Access: | http://www.biomedcentral.com/1471-2180/11/93 |
| _version_ | 1818355478646751232 |
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| author | Dongari-Bagtzoglou Anna Kashleva Helena Thompson Angela Xie Zhihong |
| author_facet | Dongari-Bagtzoglou Anna Kashleva Helena Thompson Angela Xie Zhihong |
| author_sort | Dongari-Bagtzoglou Anna |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high <it>C. albicans </it>cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR) that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells.</p> <p>Results</p> <p>The XTT and qRT-PCR assays were in good agreement when biofilm changes were measured in planktonic cultures or in early biofilms which contain lower cell densities. However, the real-time qRT-PCR assay could also accurately quantify small-medium size changes in mature biofilms caused by mechanical biomass reduction, antifungal drugs or immune effector cells, that were not accurately quantifiable with the XTT assay.</p> <p>Conclusions</p> <p>We conclude that the qRT-PCR assay is more accurate than the XTT assay when measuring small-medium size effects of anti-fungal agents against mature biofilms. This assay is also more appropriate when mature biofilm susceptibility to anti-fungal agents is tested on complex biological surfaces, such as organotypic cultures.</p> |
| first_indexed | 2024-12-13T19:41:58Z |
| format | Article |
| id | doaj.art-a149c97aa84743dfb4761389f9e3ed0a |
| institution | Directory Open Access Journal |
| issn | 1471-2180 |
| language | English |
| last_indexed | 2024-12-13T19:41:58Z |
| publishDate | 2011-05-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Microbiology |
| spelling | doaj.art-a149c97aa84743dfb4761389f9e3ed0a2022-12-21T23:33:40ZengBMCBMC Microbiology1471-21802011-05-011119310.1186/1471-2180-11-93A quantitative real-time RT-PCR assay for mature <it>C. albicans </it>biofilmsDongari-Bagtzoglou AnnaKashleva HelenaThompson AngelaXie Zhihong<p>Abstract</p> <p>Background</p> <p>Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high <it>C. albicans </it>cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR) that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells.</p> <p>Results</p> <p>The XTT and qRT-PCR assays were in good agreement when biofilm changes were measured in planktonic cultures or in early biofilms which contain lower cell densities. However, the real-time qRT-PCR assay could also accurately quantify small-medium size changes in mature biofilms caused by mechanical biomass reduction, antifungal drugs or immune effector cells, that were not accurately quantifiable with the XTT assay.</p> <p>Conclusions</p> <p>We conclude that the qRT-PCR assay is more accurate than the XTT assay when measuring small-medium size effects of anti-fungal agents against mature biofilms. This assay is also more appropriate when mature biofilm susceptibility to anti-fungal agents is tested on complex biological surfaces, such as organotypic cultures.</p>http://www.biomedcentral.com/1471-2180/11/93 |
| spellingShingle | Dongari-Bagtzoglou Anna Kashleva Helena Thompson Angela Xie Zhihong A quantitative real-time RT-PCR assay for mature <it>C. albicans </it>biofilms BMC Microbiology |
| title | A quantitative real-time RT-PCR assay for mature <it>C. albicans </it>biofilms |
| title_full | A quantitative real-time RT-PCR assay for mature <it>C. albicans </it>biofilms |
| title_fullStr | A quantitative real-time RT-PCR assay for mature <it>C. albicans </it>biofilms |
| title_full_unstemmed | A quantitative real-time RT-PCR assay for mature <it>C. albicans </it>biofilms |
| title_short | A quantitative real-time RT-PCR assay for mature <it>C. albicans </it>biofilms |
| title_sort | quantitative real time rt pcr assay for mature it c albicans it biofilms |
| url | http://www.biomedcentral.com/1471-2180/11/93 |
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