Bacterial biofilm in chronic lesions of hidradenitis suppurativa.

BACKGROUND: Chronic nonhealing or recurrent inflammatory lesions, reminiscent of infection but recalcitrant to antibiotic therapy, generally characterize biofilm-driven diseases. Chronic lesions of hidradenitis suppurativa (HS) exhibit several characteristics, which are compatible with well-known biofilm infections. OBJECTIVES: To determine and quantify the potential presence of bacterial aggregates in chronic HS lesions. METHODS: In 42 consecutive patients with HS suffering from chronic lesions, biopsies were obtained from lesional as well as from perilesional skin. Samples were investigated using peptide nucleic acid-fluorescence in situ hybridization in combination with confocal laser scanning microscopy. In addition, corresponding histopathological analysis on haematoxylin and eosin slides was performed. RESULTS: Biofilms were seen in 67% of the samples of chronic lesions and in 75% of the perilesional samples. The mean diameter of aggregates in lesional skin was significantly greater than in perilesional skin (P = 0·01). Large biofilms (aggregates > 50 µm in diameter) were found in 42% of lesional samples and in only 5% of the perilesional samples (P = 0·009). The majority of the large biofilms were situated in sinus tracts (63%) or in the infundibulum (37%). The majority of the sinus tract samples (73%) contained active bacterial cells, which were associated with inflammation. CONCLUSIONS: This study suggests that biofilm formation is associated with inflammation of chronic HS lesions. The aggregates most likely occur as a secondary event, possibly due to predisposing local anatomical changes such as sinus tracts (tunnels), keratinous detritus and dilated hair follicles.

Microbial motility involvement in biofilm structure formation--a 3D modelling study.

A computational model explaining formation of mushroom-like biofilm colonies is proposed in this study. The biofilm model combines for the first time cell growth with twitching motility in a three-dimensional individual-based approach. Model simulations describe the tendency of motile cells to form flat biofilms spreading out on the substratum, in contrast with the immotile variants that form only round colonies. These computational results are in good qualitative agreement with the experimental data obtained from Pseudomonas aeruginosa biofilms grown in flowcells. Simulations reveal that motile cells can possess a serious ecological advantage by becoming less affected by mass transfer limitations. Twitching motility alone appears to be insufficient to generate mushroom-like biofilm structures with caps on stalks. Rather, a substrate limitation-induced detachment of motile cells followed by reattachment could explain this intriguing effect leading to higher-level biofilm structure.

DNase1L2 suppresses biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus.

BACKGROUND: The formation of biofilms, which is an important step in bacterial colonization, can be inhibited by deoxyribonuclease (DNase)-mediated breakdown of extracellular DNA. We have recently demonstrated that epidermal keratinocytes strongly express DNase1-like 2 (DNase1L2) in a differentiation-associated manner. OBJECTIVES: To determine whether enzymatically active DNase1L2 is present in human stratum corneum and whether it is able to suppress bacterial biofilm formation. METHODS: DNase1L2 was extracted from normal human stratum corneum, immunocaptured and incubated with plasmid DNA. DNA hydrolysis was monitored by gel electrophoresis and ethidium bromide staining. The effect of DNase1L2 on biofilm formation was assayed by cultivation of Pseudomonas aeruginosa and Staphylococcus aureus in the presence or absence of purified recombinant DNase1L2 in microtitre plates and subsequent quantification of biofilm-forming bacteria by crystal violet staining. RESULTS: DNase1L2 was found to be present in an enzymatically active form in the stratum corneum of human skin. In an in vitro assay, purified recombinant DNase1L2 efficiently suppressed the formation of biofilms by P. aeruginosa and S. aureus. CONCLUSIONS: Our data suggest that DNase1L2 is a novel component of the innate antimicrobial defence of the epidermis.

Development and dynamics of Pseudomonas sp. biofilms.

Pseudomonas sp. strain B13 and Pseudomonas putida OUS82 were genetically tagged with the green fluorescent protein and the Discosoma sp. red fluorescent protein, and the development and dynamics occurring in flow chamber-grown two-colored monospecies or mixed-species biofilms were investigated by the use of confocal scanning laser microscopy. Separate red or green fluorescent microcolonies were formed initially, suggesting that the initial small microcolonies were formed simply by growth of substratum attached cells and not by cell aggregation. Red fluorescent microcolonies containing a few green fluorescent cells and green fluorescent microcolonies containing a few red fluorescent cells were frequently observed in both monospecies and two-species biofilms, suggesting that the bacteria moved between the microcolonies. Rapid movement of P. putida OUS82 bacteria inside microcolonies was observed before a transition from compact microcolonies to loose irregularly shaped protruding structures occurred. Experiments involving a nonflagellated P. putida OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven. The results are discussed in relation to the prevailing hypothesis that biofilm bacteria are in a physiological state different from planktonic bacteria.

Spatial Organization of Microbial Biofilm Communities.

The application of advanced microscopy and molecular and electrochemical high-resolution methods has provided insights into the structural organization and function of biofilm communities. It appears that cellular properties such as growth differentiation, chemotaxis, and cell-to-cell signaling enable biofilm communities to organize structurally in response to the external conditions and the activities of the different biofilm members. Thereby resource utilization becomes optimized, and processes which require syntrophic relationships or special micro-environments become facilitated.

In situ reverse transcription-PCR for monitoring gene expression in individual Methanosarcina mazei S-6 cells.

An in situ reverse transcription-PCR protocol for detecting specific mRNA in Methanosarcina mazei S-6 is described. This method allowed us to detect heat shock-induced increases in the intracellular levels of the transcript of the universal stress gene dnaK. The cell walls of paraformaldehyde-fixed cells were permeabilized by a thermal cycling procedure or by lysozyme treatment, and the cellular DNA was removed with DNase. The cells were subjected to a seminested reverse transcription-PCR protocol in which a digoxigenin-labeled primer was used. Detection of the reporter molecule was based on the 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate-Fast Red detection system and binding of anti-digoxigenin-alkaline phosphatase conjugate. Fluorescence in permeabilized cells increased after a heat shock compared to fluorescence in non-heat-shocked cells, and the increase corresponded to an increase in the level of the dnaK transcript.

Assessment of flhDC mRNA levels in Serratia liquefaciens swarm cells.

We reported previously that artificial overexpression of the flhDC operon in liquid-grown Serratia liquefaciens resulted in the formation of filamentous, multinucleated, and hyperflagellated cells that were indistinguishable from surface-induced swarm cells (L. Eberl, G. Christiansen, S. Molin, and M. Givskov, J. Bacteriol. 178:554-559, 1996). In the present report we show by means of reporter gene measurements, Northern analysis, and in situ reverse transcription-PCR that the amount of flhDC mRNA in surface-grown swarm cells does not exceed the maximum level found in nondifferentiated, vegetative cells. This suggests that surface-induced S. liquefaciens swarm cell differentiation, although dependent on flhDC gene expression, does not occur through elevated flhDC mRNA levels.

Role of commensal relationships on the spatial structure of a surface-attached microbial consortium.

A flow cell-grown model consortium consisting of two organisms, Burkholderia sp. LB400 and Pseudomonas sp. B13(FR1), was studied. These bacteria have the potential to interact metabolically because Pseudomonas sp. B13(FR1) can metabolize chlorobenzoate produced by Burkholderia sp. LB400 when grown on chlorobiphenyl. The expected metabolic interactions in the consortium were demonstrated by high performance liquid chromatography (HPLC) analysis. The spatial structure of the consortium was studied by fluorescent in situ rRNA hybridization and scanning confocal laser microscopy. When the consortium was fed with medium containing a low concentration of chlorobiphenyl, microcolonies consisting of associated Burkholderia sp. LB400 and Pseudomonas sp. B13(FR1) bacteria were formed, and separate Pseudomonas sp. B13(FR1) microcolonies were evidently not formed. When the consortium was fed citrate, which can be metabolized by both species, the two species formed separate microcolonies. The structure development In the consortium was studied online using a gfp-tagged Pseudomonas sp. B13(FR1) derivative. After a shift In carbon source from citrate to a low concentration of chlorobiphenyl, movement of the Pseudomonas sp. B13(FR1) bacteria led to a change in the spatial structure of the consortium from the unassociated form towards the associated form within a few days. Experiments Involving a gfp-based Pseudomonas sp. B13(FR1) growth activity reporter strain Indicated that chlorobenzoate supporting growth of Pseudomonas sp. B13(FR1) is located close to the Burkholderia sp. LB400 microcolonies in chlorobiphenyl-grown consortia.

Physiological states of individual Salmonella typhimurium cells monitored by in situ reverse transcription-PCR.

The possibility of using levels of specific mRNAs in individual bacteria as indicators of single-cell physiology was investigated. Estimates of the numbers of groEL and tsf mRNAs per cell in Salmonella typhimurium cells in different physiological states were obtained by Northern analysis. The average number of groEL mRNAs per cell was estimated to be 22 in fast-growing cultures and 197 in heat-shocked cultures. The average number of tsf mRNAs per cell was estimated to be 37 in fast-growing cultures, 4 in slow-growing cultures, and 0 in nongrowing cultures. The potential of mRNA-targeted in situ reverse transcription (RT)-PCR to monitor quantitatively different levels of groEL and tsf mRNA in individual cells and thus monitor both specific gene induction and general growth activity was assessed. Neither groEL nor tsf mRNA was present in stationary-phase cells, but it was shown that stationary-phase cells contain other RNA species at high levels, which may provide a possibility for monitoring directly stationary-phase individual cells by the use of in situ RT-PCR. The outcome of the in situ RT-PCR analyses indicated that a population of fast-growing cells is heterogeneous with respect to groEL mRNA single-cell contents, suggesting a cell-cycle-controlled expression of groEL in S. typhimurium, whereas a fast-growing culture is homogeneous with respect to tsf mRNA single-cell contents, suggesting that the level of tsf mRNA is relatively constant during the cell cycle.

Inhibition of Escherichia coli precursor-16S rRNA processing by mouse intestinal contents.

The correlation between ribosome content and growth rate found in many bacterial species has proved useful for estimating the growth activity of individual cells by quantitative in situ rRNA hybridization. However, in dynamic environments, the stability of mature ribosomal RNA causes problems in using cellular rRNA contents for direct monitoring of bacterial growth activity in situ. In a recent paper, Cangelosi and Brabant suggested monitoring the content of precursors in rRNA synthesis (pre-rRNAs) as an alternative approach. These are rapidly broken down after the cessation of bacterial growth. We have applied fluorescence in situ hybridization of pre-16S rRNA to Escherichia coil cells growing in vitro in extracts from two different compartments of the mouse intestine: the caecal mucus layer, where E. coli grew rapidly, and the contents of the caecum, which supported much slower bacterial growth. The amounts of 23S rRNA and pre-16S rRNA measured for E. coil growing in intestinal mucus corresponded to that expected for bacteria with the observed growth rate. In contrast, the slow-growing E. coli cells present in intestinal contents turned out to have an approximately ninefold higher content of pre-16S rRNA than cultures of the same strain growing rapidly in rich media. We present results suggesting that the mouse intestinal contents contain an agent that inhibits the growth of E. coli by disturbing its ability to process pre-16S rRNA.

Non-genetic population heterogeneity studied by in situ polymerase chain reaction.

Expression of a lac operon in Salmonella typhimurium single cells was monitored using lac mRNA targeting in situ reverse transcription-polymerase chain reaction (RT-PCR). It is demonstrated that suboptimal induction of the lac operon in a culture of S. typhimuriuml/F'lac+ cells generates a subpopulation in which transcription of the lac operon occurs and another subpopulation in which transcription of the lac operon is repressed, whereas suboptimal induction of the lac operon in a culture of S. typhimuriuml/F'lacY cells generates a population with uniform levels of lac mRNA. The outcome of the single-cell lac mRNA detection assay was compared with the outcome of a single-cell beta-galactosidase assay. In cultures grown under different suboptimal lac induction conditions, the fraction of cells in which transcription of the lac operon occurred was concurrent with the fraction of cells showing beta-galactosidase activity. Besides supporting the hypothesis that the lactose permease has a role in generating non-genetic heterogeneity in suboptimally induced cultures of Lac+ cells, these results demonstrate the usefulness of in situ RT-PCR for the study of non-genetic population heterogeneities.

Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR.

An in situ PCR protocol by which we can monitor the presence or absence of lac mRNA in individual cells of a Salmonella typhimurium F' lac+ strain has been developed. In this protocol, fixed cells are permeabilized with lysozyme and subjected to a seminested reverse transcriptase PCR using reporter molecule-labeled primers, and subsequently, intracellular reporter molecules are detected microscopically at the individual-cell level by use of a horseradish peroxidase-conjugated antifluorescein antibody assay. In order to determine the sensitivity of the in situ PCR assay, the ability to detect lac mRNA in suboptimally isopropyl-beta-D-thiogalactopyranoside-induced cells was investigated. By use of a single-cell beta-galactosidase assay, it was confirmed that homogeneous suboptimally induced cultures of S. typhimurium F' lacY cells could be established, and the number of functional lac mRNAs in individual cells was estimated from standard population level beta-galactosidase assays. Cells estimated to contain a single lac mRNA were detected as containing lac mRNA by the in situ PCR method. Conclusively, we demonstrate the potential of in situ PCR for detection of even poorly expressed mRNA in individual bacterial cells.

Effects of stress treatments on the detection of Salmonella typhimurium by in situ hybridization.

In order to assess the usefulness of quantitative in situ rRNA hybridization as an indicator of the physiological state of bacteria, we have used this method to measure the cellular contents of 16 S and 23 S rRNA in Salmonella typhimurium subjected to a number of different stress treatments. The contents of rRNA in S. typhimurium decreased when the bacteria were subjected to carbon starvation, heat stress, and osmotic stress prior to the hybridization, whereas no decrease in the intracellular contents of rRNA was observed when the bacteria were subjected to cold stress, acetic acid or ethanol treatment prior to the hybridization. We must conclude, that the content of 16 S rRNA and 23 S rRNA cannot be used as the sole indicator of the physiological state or viability of food borne pathogens. Viable as well as non-viable food borne bacteria will be detected when methods based on detection of rRNA are used.

Plasmid stability: comments on the dimer catastrophe hypothesis.

Using a derivative of the plasmid pBR322 we have tested the dimer catastrophe hypothesis of plasmid instability. Most of the theory was confirmed by our observations, but our data suggest that some of the quantitative aspects need modification. In a recF strain of Escherichia coli we estimated the difference in loss rate between the plasmid in the monomeric and the dimeric state to be a factor of 13-14 and the difference in the loss rate between the plasmid in the monomeric and the trimeric state to be a factor of 14-50. We were able to confirm that plasmid oligomers were heterogeneously distributed within a rec+ population, but we were unable to detect any pronounced difference in the level of growth inhibition exerted by the plasmid when in the monomeric, dimeric, or trimeric state. This leaves open the question as to whether runaway plasmid multimerization was prevented (i) by a small correlation between the inhibition of growth and the 'multimeric status' of the plasmid, (ii) by intramolecular homologous recombination, or (iii) whether the process of runaway multimerization is too slow to be recognized within the duration of the experiments, i.e. 200 generations of growth.

Role of ribosome degradation in the death of heat-stressed Salmonella typhimurium.

Heat treatment of Salmonella typhimurium results in cell death, which coincides with a significant reduction of the cellular content of 16S ribosomal RNA. It is suggested that the degradation of ribosomal RNA is a direct cause of cell death. This conclusion is based on the observation of carbon-starved and magnesium-supplemented cells, which survive heat treatment much better, and which also maintain stable levels of ribosomal RNA.

A statistical analysis of the formation of plasmid-free cells in populations of Escherichia coli.

By methods analogous to those used in the classical statistical analysis of bacterial mutation, we have analyzed the formation of plasmid-free cells in populations of Escherichia coli harboring pBR322-derived plasmids. Application of fluctuation tests and papilla analysis suggested that there is a high variance in the probability that a plasmid-containing cell will produce a plasmid-free daughter cell. Apparently a subpopulation of plasmid-containing cells gives rise to progeny that produces plasmid-free cells with a high and unpredictable rate. This finding raises the question of whether plasmid maintenance can be adequately described by the conventional mathematical models.

Fluctuation analysis of mutations to nalidixic acid resistance in Escherichia coli.

Mutations of Escherichia coli from sensitivity to nalidixic acid resistance were studied by fluctuation analysis. The mutant distributions in replicate cultures were not significantly affected either by the age of the carbon-starved preculture used for inocula or by the inoculum size. The data from 23 fluctuation tests (48 cultures each) were pooled. The mean number of mutations per culture was estimated to be 0.71 from the fraction of cultures without mutants or 0.74 and 0.77 by maximum-likelihood estimation based on the two models under consideration. When the pooled data were compared with the theoretical expectations, the fits were unsatisfactory (P < 0.005). The lack of fit was caused mainly by too high a frequency of cultures with between 17 and 32 mutants and too high a frequency of cultures with more than 128 mutants. Possible reasons for the lack of fit and its implications with respect to estimation of mutation rates from fluctuation tests are discussed.