Claus Sternberg

Quantification of biofilm structures by the novel computer program COMSTAT

The structural organization of four microbial communities was analysed by a novel computer program, COMSTAT, which comprises ten features for quantifying three-dimensional biofilm image stacks. Monospecies biofilms of each of the four bacteria, Pseudomonas: putida, P. aureofaciens, P. fluorescens and P. aeruginosa, tagged with the green fluorescent protein (GFP) were grown in flow chambers with a defined minimal medium as substrate. Analysis by the COMSTAT program of four variables describing biofilm structure - mean thickness, roughness, substratum coverage and surface to volume ratio - showed that the four Pseudomonas: strains represent different modes of biofilm growth. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies, which were eventually succeeded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum, and formed flat, uniform biofilms. P. aureofaciens resembled P. aeruginosa, but had a stronger tendency to form micro-colonies. Finally, the biofilm structures of P. fluorescens had a phenotype intermediate between those of P. putida and P. aureofaciens. Analysis of biofilms of P. aureofaciens growing on 0.03 mM, 0.1 mM or 0.5 mM citrate minimal media showed that mean biofilm thickness increased with increasing citrate concentration. Moreover, biofilm roughness increased with lower citrate concentrations, whereas surface to volume ratio increased with higher citrate concentrations.

Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung

The leading cause of mortality in patients with cystic fibrosis (CF) is respiratory failure due in large part to chronic lung infection with Pseudomonas aeruginosa strains that undergo mucoid conversion, display a biofilm mode of growth in vivo and resist the infiltration of polymorphonuclear leukocytes (PMNs), which release free oxygen radicals such as H2O2. The mucoid phenotype among the strains infecting CF patients indicates overproduction of a linear polysaccharide called alginate. To mimic the inflammatory environment of the CF lung, P. aeruginosa PAO1, a typical non-mucoid strain, was grown in a biofilm. This was treated with low levels of H2O2, as if released by the PMNs, and the formation of mucoid variants was observed. These mucoid variants had mutations in mucA, which encodes an anti-sigma factor; this leads to the deregulation of an alternative sigma factor (sigma22, AlgT or AlgU) required for expression of the alginate biosynthetic operon. All of the mucoid variants tested showed the same mutation, the mucA22 allele, a common allele seen in CF isolates. The mucoid mucA22 variants, when compared to the smooth parent strain PA01, (i) produced 2-6-fold higher levels of alginate, (ii) exhibited no detectable differences in growth rate, (iii) showed an unaltered LPS profile, (iv) were approximately 72% reduced in the amount of inducible-beta-lactamase and (v) secreted little or no LasA protease and only showed 44% elastase activity. A characteristic approximately 54 kDa protein associated with alginate overproducing strains was identified as AlgE (Alg76) by N-terminal sequence analysis. Thus, the common phenotype of the mucoid variants, which included a genetically engineered mucA22 mutant, suggested that the only mutation incurred as a result of H2O2 treatment was in mucA. When a P. aeruginosa biofilm was repeatedly exposed to activated PMNs in vitro, mucoid variants were also observed, mimicking in vivo observations. Thus, PMNs and their oxygen by-products may cause P. aeruginosa to undergo the typical adaptation to the intractable mu- coid form in the CF lung. These findings indicate that gene activation in bacteria by toxic oxygen radicals, similar to that found in plants and mammalian cells, may serve as a defence mechanism for the bacteria. This suggests that mucoid conversion is a response to oxygen radical exposure and that this response is a mechanism of defence by the bacteria. This is the first report to show that PMNs and their oxygen radicals can cause this phenotypic and genotypic change which is so typical of the intractable form of P. aeruginosa in the CF lung. These findings may provide a basis for the development of anti-oxidant and anti-inflammatory therapy for the early stages of infection in CF patients.

Involvement of N‐acyl‐l‐homoserine lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens

Several bacterial species possess the ability to differentiate into highly motile swarmer cells capable of rapid surface colonization. In Serratia liquefaciens, we demonstrate that initiation of swarmer‐cell differentiation involves diffusible signal molecules that are released into the growth medium. Using high‐performance liquid chromatography (HPLC), high resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, we identified N‐butanoyl‐l‐homoserine lactone (BHL) and N‐hexanoyl‐l‐homoserine lactone (HHL) in cell‐free Serratia culture supernatants. BHL and HHL are present in a ratio of approximately 10:1 and their structures were unequivocally confirmed by chemical synthesis. The swrlswarmer initiation) gene, the predicted translation product of which exhibits substantial homology to the Luxl family of putative Nacyl homoserine lactone (AHL) synthases is responsible for directing synthesis of both BHL and HHL. In an swrl mutant, swarming motility is abolished but can be restored by the addition of an exogenous AHL. These results add swarming motility to the rapidly expanding list of phenotypes known to be controlled through quorum sensing.

Molecular tools for study of biofilm physiology.

Publisher Summary This chapter describes methods for the handling and analysis of microbial behavior of organisms in biofilm communities at both microscopic and macroscopic levels. Only methods and reporter systems that can be applied without disturbing the spatial organization of the organisms in the biofilm are presented. The in situ methods described in this chapter can be used for more than just identifying or tracing cells or genes in biofilms. By combining promoters that respond to specific environmental signals with appropriate marker genes, it may be possible to tag specific organisms and use these as monitor systems to estimate local chemical composition directly in the biofilms. Changes in environmental conditions will also have significant effects on the physiological state of the organisms. Such shifting conditions may result in several responses, such as altered growth rates, stress response, starvation, or even cell death. Most of these responses can be visualized directly using specific promoter–reporter fusions. The ribosome number is a reliable indicator of growth rate in bacteria growing in balanced growth and has been used as a standard for growth rates in biofilm-embedded bacteria as well.

In Situ Growth Rates and Biofilm Development of Pseudomonas aeruginosa Populations in Chronic Lung Infections

The growth dynamics of bacterial pathogens within infected hosts are a fundamental but poorly understood feature of most infections. We have focused on the in situ distribution and growth characteristics of two prevailing and transmissible Pseudomonas aeruginosa clones that have caused chronic lung infections in cystic fibrosis (CF) patients for more than 20 years. We used fluorescence in situ hybridization (FISH) directly on sputum specimens to examine the spatial distribution of the infecting P. aeruginosa cells. Mucoid variants were present in sputum as cell clusters surrounded by an extracellular matrix, whereas nonmucoid variants were present mainly as dispersed cells. To obtain estimates of the growth rates of P. aeruginosa in CF lungs, we used quantitative FISH to indirectly measure growth rates of bacteria in sputum samples (reflecting the in vivo lung conditions). The concentration of rRNA in bacteria isolated from sputa was measured and correlated with the rRNA contents of the same bacteria growing in vitro at defined rates. The results showed that most cells were actively growing with doubling times of between 100 and 200 min, with some growing even faster. Only a small stationary-phase subpopulation seemed to be present in sputa. This was found for both mucoid and nonmucoid variants despite their different organizations in sputum. The results suggest that the bacterial population may be confronted with selection forces that favor optimized growth activities. This scenario constitutes a new perspective on the adaptation and evolution of P. aeruginosa during chronic infections in CF patients in particular and on long-term infections in general.

Bacterial plasmid conjugation on semi-solid surfaces monitored with the green fluorescent protein (GFP) from Aequorea victoria as a marker

Horizontal transfer of the TOL plasmid was examined in Pseudomonas putida (Pp) KT2442 micro-colonies on semi-solid agar surfaces. Horizontal gene transfer is usually studied in large populations where all information is based on average estimates of the transfer events in the entire population. We have used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a plasmid marker, in combination with single-cell observations. This provided hitherto unknown details on the distribution of cells active in conjugation. In the present study, donor cells containing the gfp gene expressed from the bacteriophage T7 phi 10 promoter on the TOL plasmid, and recipient cells expressing the corresponding phage RNA polymerase allowed us to monitor the occurrence of ex-conjugants as green fluorescent cells upon illumination with blue light (470-490 nm). Further, the recipients were labeled with the luxAB genes to distinguish micro-colonies of donor cells from recipient cells. We conclude that conjugal plasmid transfer in Pp KT2442 cells on semi-solid surfaces occurs mainly during a short period of time after the initial contact of donors and recipients, indicating that spread of the TOL plasmid is limited in static, but viable cultures.

MODERN MICROSCOPY IN BIOFILM RESEARCH : CONFOCAL MICROSCOPY AND OTHER APPROACHES

Microscopy is the only technique whereby bacterial biofilms can be studied at the single-cell level in situ. Our understanding of biofilm structure, physiology and control hinges on the application of confocal scanning laser microscopy and other advanced microscopic techniques. Gene expression in four dimensions (x,y,z,t), interspecies interactions, and the role of exopolymer are being defined.

Insight into the microbial multicellular lifestyle via flow‐cell technology and confocal microscopy

Biofilms are agglomerates of microorganisms surrounded by a self‐produced extracellular matrix. During the last 10 years, there has been an increasing recognition of biofilms as a highly significant topic in microbiology with relevance for a variety of areas in our society including the environment, industry, and human health. Accordingly a number of biofilm model systems, molecular tools, microscopic techniques, and image analysis programs have been employed for the study of biofilms under controlled and reproducible conditions. Studies using confocal laser scanning microscopy (CLSM) of biofilms formed in flow‐chamber experimental systems by genetically color‐coded bacteria have provided detailed knowledge about biofilm developmental processes, cell differentiations, spatial organization, and function of laboratory‐grown biofilms, in some cases down to the single cell level. In addition, the molecular mechanisms underlying the increased tolerance that biofilm cells often display towards antibiotic treatment are beginning to be unravelled.

Microfluidic dissolved oxygen gradient generator biochip as a useful tool in bacterial biofilm studies

A microfluidic chip for generation of gradients of dissolved oxygen was designed, fabricated and tested. The novel way of active oxygen depletion through a gas permeable membrane was applied. Numerical simulations for generation of O(2) gradients were correlated with measured oxygen concentrations. The developed microsystem was used to study growth patterns of the bacterium Pseudomonas aeruginosa in medium with different oxygen concentrations. The results showed that attachment of Pseudomonas aeruginosa to the substrate changed with oxygen concentration. This demonstrates that the device can be used for studies requiring controlled oxygen levels and for future studies of microaerobic and anaerobic conditions.

Growing and analyzing biofilms in flow cells.

The setup of flow cell systems for the study of microbial biofilms and methods for the analysis of structural biofilm development are described in this unit. Use of flow cells allows direct microscopic investigation of biofilm development. The biofilms in flow cells develop under hydrodynamic conditions, and the environment can be carefully controlled and easily changed. The protocols in this unit include construction of the flow cell and the bubble trap, assembly and sterilization of the flow cell system, inoculation of the flow cells, running of the system, image capture and analysis, and disassembly and cleaning of the system. In addition, embedding and fluorescent in situ hybridization of flow cell-grown biofilms are addressed.