Biofilms are surface-attached microbial communities with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts. One of the best-known of these biofilm-specific properties is the development of antibiotic resistance that can be up to 1,000-fold greater than planktonic cells. We report a genetic determinant of this high-level resistance in the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa. We have identified a mutant of P. aeruginosa that, while still capable of forming biofilms with the characteristic P. aeruginosa architecture, does not develop high-level biofilm-specific resistance to three different classes of antibiotics. The locus identified in our screen, ndvB, is required for the synthesis of periplasmic glucans. Our discovery that these periplasmic glucans interact physically with tobramycin suggests that these glucose polymers may prevent antibiotics from reaching their sites of action by sequestering these antimicrobial agents in the periplasm. Our results indicate that biofilms themselves are not simply a diffusion barrier to these antibiotics, but rather that bacteria within these microbial communities employ distinct mechanisms to resist the action of antimicrobial agents.

In this study, stratified patterns of protein synthesis and growth were demonstrated in Pseudomonas aeruginosa biofilms. Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs. One construct carried an isopropyl-␤-Dthiogalactopyranoside (IPTG)-inducible gfpmut2 gene encoding a stable GFP. The second construct carried a GFP derivative, gfp-AGA, encoding an unstable GFP under the control of the growth-rate-dependent rrnBp 1 promoter. Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen. The zone of active GFP expression was approximately 60 m wide in colony biofilms and 30 m wide in flow cell biofilms. The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy. Cell elongation was localized at the air interface of the biofilm. The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm. Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 m into the biofilm. P. aeruginosa was incapable of anaerobic growth in the medium used for this investigation. These results show that while mature P. aeruginosa biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing.Biofilms are communities of microorganisms that are associated with surfaces. Cell clusters in biofilms are known to be characterized by gradients in the concentrations of oxygen, nutrients, and metabolic waste products. It is widely recognized that such chemical heterogeneity in microbial biofilms can lead to microorganisms in the biofilm exhibiting different rates of growth and metabolic activity. Even pure cultures of microorganisms growing in biofilms experience these gradients and may exist in a range of metabolic states. The variety of growth states that can be represented in a biofilm, even for a single species, surely contributes to the special ecology and antimicrobial tolerance manifested by biofilms. Given the fundamental significance of growth status, it is surprising that there have been few investigations in which the growth patterns in biofilms have been visualized.Several studies have investigated spatial patterns of cellular activity inside biofilms, using approaches such as staining with nucleic acid dyes that differentially indicate DNA and RNA (17), hybridization to 16S rRNA with fluorescently tagged oligonucleotide probes (9), the induction of alkaline phosphatase followed by staining with a fluorogenic phosphatase substrate (8,19), and green fluorescent protein (GFP) expression from a growth-rate-dependent promoter (11). These previous investigations have revealed gradients in metabolic activity in biofilms.The purpose ...

It has long been suspected that microbial biofilms harbor cells in a variety of activity states, but there have been few direct experimental visualizations of this physiological heterogeneity. Spatial patterns of DNA replication and protein synthetic activity were imaged and quantified in staphylococcal biofilms using immunofluorescent detection of pulse-labeled DNA and also an inducible green fluorescent protein (GFP) construct. Stratified patterns of DNA synthetic and protein synthetic activity were observed in all three biofilm systems to which the techniques were applied. In a colony biofilm system, the dimensions of the zone of anabolism at the air interface ranged from 16 to 38 m and corresponded with the depth of oxygen penetration measured with a microelectrode. A second zone of activity was observed along the nutrient interface of the biofilm. Much of the biofilm was anabolically inactive. Since dead cells constituted only 10% of the biofilm population, most of the inactive cells in the biofilm were still viable. Collectively, these results suggest that staphylococcal biofilms contain cells in at least four distinct states: growing aerobically, growing fermentatively, dead, and dormant. The variety of activity states represented in a biofilm may contribute to the special ecology and tolerance to antimicrobial agents of biofilms.

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Gene expression in biofilms is dependent on bacterial responses to the local environmental conditions. Most techniques for studying bacterial gene expression in biofilms characterize average values across the entire population. Here, we describe the use of laser capture microdissection microscopy (LCMM) combined with multiplex quantitative real-time reverse transcriptase PCR (qRT-PCR) to isolate and quantify RNA transcripts from small groups of cells at spatially resolved sites within biofilms. The approach was first tested and analytical parameters were determined for Pseudomonas aeruginosa containing an isopropyl-␤-D-thiogalactopyranoside-inducible gene for the green fluorescent protein (gfp). The results show that the amounts of gfp mRNA were greatest in the top zones of the biofilms, and that gfp mRNA levels correlated with the zone of active green fluorescent protein fluorescence. The method then was used to quantify transcripts from wild-type P. aeruginosa biofilms for a housekeeping gene, acpP; the 16S rRNA; and two genes regulated by quorum sensing, phzA1 and aprA. The results demonstrated that the amount of acpP mRNA was greatest in the top 30 m of the biofilm, with little or no mRNA for this gene at the base of the biofilms. In contrast, 16S rRNA amounts were relatively uniform throughout biofilm strata. Using this strategy, the RNA amounts of individual genes were determined, and therefore the results are dependent on both gene expression and the half-life of the transcripts. Therefore, the uniform amount of rRNA throughout the biofilms likely is due to the stability of the rRNA within ribosomes. The levels of aprA mRNA showed stratification, with the largest amounts in the upper 30-m zone of these biofilms. The results demonstrate that mRNA levels for individual genes are not uniformly distributed throughout biofilms but may vary by orders of magnitude over small distances. The LCMM/qRT-PCR technique can be used to resolve and quantify this RNA variability at high spatial resolution.

The dynamic antimicrobial action of chlorine, a quaternary ammonium compound, glutaraldehyde, and nisin within biofilm cell clusters of Staphylococcus epidermidis was investigated using time-lapse confocal scanning laser microscopy. The technique allowed for the simultaneous imaging of changes in biofilm structure and disruption of cellular membrane integrity through the loss of an unbound fluorophore loaded into bacterial cells prior to antimicrobial challenge. Each of the four antimicrobial agents produced distinct spatial and temporal patterns of fluorescence loss. The antimicrobial action of chlorine was localized around the periphery of biofilm cell clusters. Chlorine was the only antimicrobial agent that caused any biofilm removal. Treatment with the quaternary ammonium compound caused membrane permeabilization that started at the periphery of cell clusters, then migrated steadily inward. A secondary pattern superimposed on the penetration dynamic suggested a subpopulation of less-susceptible cells. These bacteria lost fluorescence much more slowly than the majority of the population. Nisin caused a rapid and uniform loss of green fluorescence from all parts of the biofilm without any removal of biofilm. Glutaraldehyde caused no biofilm removal and also no loss of membrane integrity. Measurements of biocide penetration and action time at the center of cell clusters yielded 46 min for 10 mg liter ؊1 chlorine, 21 min for 50 mg liter ؊1 chlorine, 25 min for the quaternary ammonium compound, and 4 min for nisin. These results underscore the distinction between biofilm removal and killing and reinforce the critical role of biocide reactivity in determining the rate of biofilm penetration.The action of a biocide or antibiotic against microorganisms in biofilms varies in time and space. There is insight to be gained into the phenomena important in this process by watching, through a microscope, the antimicrobial attack. Here we describe the application of a recently developed technique for visualizing antimicrobial action (29) to biofilms formed by Staphylococcus epidermidis. We describe distinct behaviors for the four antimicrobial agents examined, which were chlorine, glutaraldehyde, a quaternary ammonium compound (QAC), and an antimicrobial peptide, nisin.S. epidermidis, a commensal resident of the skin and an opportunistic pathogen, is a common culprit in nosocomial infections (13,15,31). In particular, this microorganism is known to form biofilms on indwelling devices such as catheters, prosthetic joints, and contact lenses. There is therefore interest in understanding the efficacy of biocides against biofilms formed by this organism in such applications as control of contamination on hospital countertops and in catheter lock solutions, skin disinfectants, and contact lens storage case disinfection.Numerous evaluations of biocide activity against S. epidermidis biofilms have been reported for such agents as chlorhexidine, hydrogen peroxide, povidone-iodine, alcohols, and chlorine (4,6,10,17,19,21,32). These ...

The transient diffusion of fluorescent tracers into biofilm cell clusters of Staphylococcus epidermidis was visualized by time lapse confocal scanning laser microscopy. Rhodamine B diffused into the center of cell clusters that were 200 to 600 m in diameter within a few minutes. The apparent effective diffusion coefficient calculated from these data averaged 3.7 ؋ 10 ؊7 cm 2 s ؊1 or 11% of the value in pure water. Fluorescein diffused into biofilm more rapidly, with a diffusion coefficient that averaged 1.6 ؋ 10 ؊6 cm 2 s ؊1 , or 32% of the value in water. This study provides direct, visual confirmation that solutes the size of many antibiotics and biocides can diffuse rapidly into biofilms.One long-standing explanation for the antibiotic tolerance of microorganisms in biofilms is that the biofilm matrix constitutes a barrier to effective penetration of antimicrobial agents. If an antibiotic simply does not reach cells in the interior of a cluster, the failure of the agent to kill these cells is easy to understand. On the other hand, if the antibiotic can be shown to penetrate throughout the biofilm, then one must turn to biological explanations for antibiotic tolerance.The objective of this study was to directly visualize the penetration of an antibiotic-sized tracer molecule into the interior of biofilm cell clusters, noninvasively and in real time. This was accomplished by using fluorescent dyes that were imaged by confocal scanning laser microscopy. Quantitative image analysis was performed to extract numerical values of the effective diffusion coefficient in the biofilm. MATERIALS AND METHODSBacteria and media. Staphylococcus epidermidis strain RP62A (ATCC 35984) was grown on tryptic soy broth (TSB) at 37°C. Full strength TSB was used to grow shake flask cultures that provided the inoculum for biofilm experiments. Biofilms were grown on 1/10 strength TSB.Biofilm reactor. Biofilms were grown in glass capillary tubes (Friedrich and Dimmock, Millville, N.J.) under continuous-flow conditions. Glass tubes were used rather than a more clinically relevant material because glass provided an optically clear substratum for microscopy. In addition, these capillary tubes had a square cross-section, which facilitated microscopic observation of the biofilm through the capillary walls. The nominal inside dimension of the tube was 0.9 mm, and it was approximately 10 cm long. Autoclaved 1/10 strength TSB was delivered to the capillary by gravity feed from a 5-liter carboy. The head difference from the feed carboy to the waste outlet was approximately 1.5 m. The flow rate of medium, which was monitored by counting drops passing through a flow break between the feed carboy and capillary, was between 120 and 180 ml h Ϫ1 . This flow rate corresponds to a Reynolds number of 37 to 56 based on the hydraulic radius of the clean tube. The medium carboy and the reactor itself were placed inside separate 37°C incubators stacked on top of each other. The reactor was inoculated by injecting 1 to 2 ml of overnight culture into a septum j...