Physiology of Pseudomonas aeruginosa in biofilms as revealed by transcriptome analysis

Abstract

BackgroundTranscriptome analysis was applied to characterize the physiological activities of Pseudomonas aeruginosa grown for three days in drip-flow biofilm reactors. Conventional applications of transcriptional profiling often compare two paired data sets that differ in a single experimentally controlled variable. In contrast this study obtained the transcriptome of a single biofilm state, ranked transcript signals to make the priorities of the population manifest, and compared ranki ngs for a priori identified physiological marker genes between the biofilm and published data sets.ResultsBiofilms tolerated exposure to antibiotics, harbored steep oxygen concentration gradients, and exhibited stratified and heterogeneous spatial patterns of protein synthetic activity. Transcriptional profiling was performed and the signal intensity of each transcript was ranked to gain insight into the physiological state of the biofilm population. Similar rankings were obtained from data sets published in the GEO database http://www.ncbi.nlm.nih.gov/geo. By comparing the rank of genes selected as markers for particular physiological activities between the biofilm and comparator data sets, it was possible to infer qualitative features of the physiological state of the biofilm bacteria. These biofilms appeared, from their transcriptome, to be glucose nourished, iron replete, oxygen limited, and growing slowly or exhibiting stationary phase character. Genes associated with elaboration of type IV pili were strongly expressed in the biofilm. The biofilm population did not indicate oxidative stress, homoserine lactone mediated quorum sensing, or activation of efflux pumps. Using correlations with transcript ranks, the average specific growth rate of biofilm cells was estimated to be 0.08 h-1.ConclusionsCollectively these data underscore the oxygen-limited, slow-growing nature of the biofilm population and are consistent with antimicrobial tolerance due to low metabolic activity.

The Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection

“…There is often little change in the MIC of individual cells, however growth in populations can lead to an increasing tolerance to antibiotics. The cause of such increased tolerances seen in biofilms has been investigated in a number of different studies, focusing on the extracellular matrix, the involvement of quorum sensing (QS), and the physiological factors observed within the biofilm [19,[26][27][28][29]. The extracellular matrix itself has been shown to obstruct the diffusion of some antimicrobial agents through both chemical and physiological means [27,30,31].…”

“…The matrix however is not selective against antimicrobial agents, and therefore the diffusion of many substances, such as oxygen, metabolites and waste products, can also be altered. Reduced diffusion in combination with high cell densities results in steep chemical gradients from the outer surface towards the central core of an in vitro biofilm [26,32]. This causes a very heterogenic growth pattern throughout in vitro biofilms, which in turn influences regional tolerance towards different types of antibiotic treatments [33,34].…”

“…The absence of such surfaces in surface wounds means the bacterial cells assume a biofilm lifestyle within a self-contained aggregate, devoid of surface attachment [64,65]. There have been a number of in vitro studies that have observed steep chemical gradients (notably oxygen) throughout biofilms, which leads to a heterogenic growth pattern [26,32]. The complex environment inside inflamed mucus seems to govern the growth pattern in biofilms in a different way.…”

Sepsis por Pseudomonas aeruginosa en un lactante previamente sano

“…También se ha descrito un sistema de secreción tipo III, que permite inyectar proteínas efectoras al citoplasma de la célula hospedera, produciendo efectos negativos en la función de la barrera epitelial, asociado al desarrollo de disfunción de macrófagos y neutrófi los 1 . Tiene la capacidad de formar una biopelícula, que lleva a una tolerancia antimicrobiana y disminución de la concentración de oxígeno en su interior, produciendo un crecimiento bacteriano lento en fase estacionaria 2 . La formación de la biopelícula se relaciona con la patogenia de infecciones pulmonares crónicas en pacientes con fi brosis quística y bronquiectasias, así como también a infecciones asociadas a dispositivos como catéteres intravasculares o prótesis, interfi riendo con la llegada del antimicrobiano al sitio y la eliminación bacteriana 2 .…”

“…Tiene la capacidad de formar una biopelícula, que lleva a una tolerancia antimicrobiana y disminución de la concentración de oxígeno en su interior, produciendo un crecimiento bacteriano lento en fase estacionaria 2 . La formación de la biopelícula se relaciona con la patogenia de infecciones pulmonares crónicas en pacientes con fi brosis quística y bronquiectasias, así como también a infecciones asociadas a dispositivos como catéteres intravasculares o prótesis, interfi riendo con la llegada del antimicrobiano al sitio y la eliminación bacteriana 2 .…”