Tracing the interaction of bacteriophage with bacterial biofilms using fluorescent and chromogenic probes

Phages T4 and E79 were fluorescently-labeled with rhodamine isothiocyanate (RITC), fluoroscein isothiccyanate (FITC), and by the addition of 46-diamidino-2-phenylindole (DAPI) to phage-infected host cells ofEscherichia coli andPseudomonas aeruginosa. Comparisons of electron micrographs with scanning confocal laser microscope (SCLM) images indicated that single RITC-labeled phage particles could be visualized. Biofilms of each bacterium were infected by labeled phage. SCLM and epifluorescence microscopy were used to observe adsorption of phage to single-layer surface-attached bacteria and thicker biofilms. The spread of the recombinant T4 phage, YZA1 (containing an rll-LacZ fusion), within alac E. coli biofilm could be detected in the presence of chromogenic and fluorogenic homologs of galactose. Infected cells exhibited blue pigmentation and fluorescence from the cleavage products produced by the phage-encoded -galactosidase activity. Fluorescent antibodies were used to detect nonlabeled progeny phage. Phage T4 infected both surface-attached and surface-associatedE. coli while phage E79 adsorbed toP. aeruginosa cells on the surface of the biofilm, but access to cells deep in biofilms was somewhat restricted. Temperature and nutrient concentration did not affect susceptibility to phage infection, but lower temperature and low nutrients extended the time-to-lysis and slowed the spread of infection within the biofilm.     

Independent functions of viral protein and nucleic acid in growth of bacteriophage

1. Osmotic shock disrupts particles of phage T2 into material containing nearly all the phage sulfur in a form precipitable by antiphage serum, and capable of specific adsorption to bacteria. It releases into solution nearly all the phage DNA in a form not precipitable by antiserum and not adsorbable to bacteria. The sulfur-containing protein of the phage particle evidently makes up a membrane that protects the phage DNA from DNase, comprises the sole or principal antigenic material, and is responsible for attachment of the virus to bacteria. 2. Adsorption of T2 to heat-killed bacteria, and heating or alternate freezing and thawing of infected cells, sensitize the DNA of the adsorbed phage to DNase. These treatments have little or no sensitizing effect on unadsorbed phage. Neither heating nor freezing and thawing releases the phage DNA from infected cells, although other cell constituents can be extracted by these methods. These facts suggest that the phage DNA forms part of an organized intracellular structure throughout the period of phage growth. 3. Adsorption of phage T2 to bacterial debris causes part of the phage DNA to appear in solution, leaving the phage sulfur attached to the debris. Another part of the phage DNA, corresponding roughly to the remaining half of the DNA of the inactivated phage, remains attached to the debris but can be separated from it by DNase. Phage T4 behaves similarly, although the two phages can be shown to attach to different combining sites. The inactivation of phage by bacterial debris is evidently accompanied by the rupture of the viral membrane. 4. Suspensions of infected cells agitated in a Waring blendor release 75 per cent of the phage sulfur and only 15 per cent of the phage phosphorus to the solution as a result of the applied shearing force. The cells remain capable of yielding phage progeny. 5. The facts stated show that most of the phage sulfur remains at the cell surface and most of the phage DNA enters the cell on infection. Whether sulfur-free material other than DNA enters the cell has not been determined. The properties of the sulfur-containing residue identify it as essentially unchanged membranes of the phage particles. All types of evidence show that the passage of phage DNA into the cell occurs in non-nutrient medium under conditions in which other known steps in viral growth do not occur. 6. The phage progeny yielded by bacteria infected with phage labeled with radioactive sulfur contain less than 1 per cent of the parental radioactivity. The progeny of phage particles labeled with radioactive phosphorus contain 30 per cent or more of the parental phosphorus. 7. Phage inactivated by dilute formaldehyde is capable of adsorbing to bacteria, but does not release its DNA to the cell. This shows that the interaction between phage and bacterium resulting in release of the phage DNA from its protective membrane depends on labile components of the phage particle. By contrast, the components of the bacterium essential to this interaction are remarkably stable. The nature of the interaction is otherwise unknown. 8. The sulfur-containing protein of resting phage particles is confined to a protective coat that is responsible for the adsorption to bacteria, and functions as an instrument for the injection of the phage DNA into the cell. This protein probably has no function in the growth of intracellular phage. The DNA has some function. Further chemical inferences should not be drawn from the experiments presented.     

Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates

AIMS: The purpose of this study was to compare the efficacy, in terms of bacterial biofilm penetration and killing, of alkaline hypochlorite (pH 11) and chlorosulfamate (pH 5.5) formulations. METHODS AND RESULTS: Two species biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown by flowing a dilute medium over inclined stainless steel slides for 6 d. Microelectrode technology was used to measure concentration profiles of active chlorine species within the biofilms in response to treatment at a concentration of 1000 mg total chlorine l(-1). Chlorosulfamate formulations penetrated biofilms faster than did hypochlorite. The mean penetration time into approximately 1 mm-thick biofilms for chlorosulfamate (6 min) was only one-eighth as long as for the same concentration of hypochlorite (48 min). Chloride ion penetrated biofilms rapidly (5 min) with an effective diffusion coefficient in the biofilm that was close to the value for chloride in water. Biofilm bacteria were highly resistant to killing by both antimicrobial agents. Biofilms challenged with 1000 mg l(-1) alkaline hypochlorite or chlorosulfamate for 1 h experienced 0.85 and 1.3 log reductions in viable cell numbers, respectively. Similar treatment reduced viable numbers of planktonic bacteria to non-detectable levels (log reduction greater than 6) within 60 s. Aged planktonic and resuspended laboratory biofilm bacteria were just as susceptible to hypochlorite as fresh planktonic cells. CONCLUSION: Chlorosulfamate transport into biofilm was not retarded whereas hypochlorite transport clearly was retarded. Superior penetration by chlorosulfamate was hypothesized to be due to its lower capacity for reaction with constituents of the biofilm. Poor biofilm killing despite direct measurement of effective physical penetration of the antimicrobial agent into the biofilm demonstrates that bacteria in the biofilm are protected by some mechanism other than simple physical shielding by the biofilm matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This study lends support to the theory that the penetration of antimicrobial agents into microbial biofilms is controlled by the reactivity of the antimicrobial agent with biofilm components. The finding that chlorine-based biocides can penetrate, but fail to kill, bacteria in biofilms should motivate the search for other mechanisms of protection from killing by antimicrobial agents in biofilms.     

Diffusion Measurements inside Biofilms by Image-Based Fluorescence Recovery after Photobleaching (FRAP) Analysis with a Commercial Confocal Laser Scanning Microscope

Research about the reactional and structural dynamics of biofilms at the molecular level has made great strides, owing to efficient fluorescence imaging methods in terms of spatial resolution and fast acquisition time but also to noninvasive conditions of observation consistent with in situ biofilm studies. In addition to conventional fluorescence intensity imaging, the fluorescence recovery after photobleaching (FRAP) module can now be routinely implemented on commercial confocal laser scanning microscopes (CLSMs). This method allows measuring of local diffusion coefficients in biofilms and could become an alternative to fluorescence correlation spectroscopy (FCS). We present here an image-based FRAP protocol to improve the accuracy of FRAP measurements inside “live” biofilms and the corresponding analysis. An original kymogram representation allows control of the absence of perturbing bacterial movement during image acquisition. FRAP data analysis takes into account molecular diffusion during the bleach phase and uses the image information to extract molecular diffusion coefficients. The fluorescence spatial intensity profile analysis used here for the first time with biofilms is supported both by our own mathematical model and by a previously published one. This approach was validated to FRAP experiments on fluorescent-dextran diffusion inside Lactoccocus lactis and Stenotrophomonas maltophilia biofilms, and the results were compared to previously published FCS measurements.Biofilms are spatially organized populations of microorganisms associated with surfaces in any natural or man-made environment and embedded in a highly hydrated matrix made up of extracellular polymeric substances (EPS). This intercellular matrix constitutes the true interface between the cells and their environment. Convergent evidences suggest a permanent reorganization of the matrix as an adaptive response of the microbial community toward a changing environment (2, 12, 14). In response to external changes, bacteria may metabolize and/or produce a variety of organic exopolymers (polysaccharides, DNA, proteins, etc.) with different physicochemical properties. These EPS may act as a defensive barrier against aggressive environmental parameters (e.g., antimicrobials or predation by bacteriophages, protists, or phagocytes) (6, 8). A deeper understanding of the interrelations between the structure, the reactivity, and the variability of the extracellular polymeric matrix fastening together surface-associated bacteria is of major importance in the comprehension of the biofilm mode of life. For this purpose, the use of specific microelectrodes or ex situ analysis following extraction of polymers has been reported (7, 21). However, these approaches are invasive and poorly resolutive and do not allow dynamic observations of biofilms over time. In recent years, it has been shown that analysis of EPS properties could be greatly improved by using optical-microscopy methods that allow noninvasive in situ observations.Confocal laser scanning microscopy (CLSM), in conjunction with the use of fluorescence reporters, allows direct visualization of the three-dimensional structure of spatiotemporal biofilm and its evolution under environmental stress (e.g., antimicrobials, phages, and protists). Using time lapse imaging, it is possible to track over time the mobility of free molecules in such spatially organized biosystems (16, 19). However, only average diffusion coefficients over the macrostructure are obtained, and the method is not appropriate for fast molecular diffusion. In contrast, fluorescence correlation spectroscopy (FCS) is now a well-established method of characterization of the local and fast diffusion of fluorescently labeled molecules through the depth of a biofilm (4, 10, 11). Early on, FCS was explored by means of homemade equipment by those with specialized knowledge (9, 17). Now the method can be adapted to CLSM but requires dedicated and expensive experimental setup.To access a local resolution similar to that of FCS diffusion processes in conjunction with CLSM convenience, fluorescence recovery after photobleaching (FRAP) appears to be a good technique when the fluorophore concentration is too high for correlation measurements and sufficient for imaging. The basic principle of FRAP is to photobleach a small, spatially confined area by high-intensity laser pulses and then to observe the recovery of fluorescence inside the photobleached area as a function of time. The method has hardly ever been applied to measurements in biofilms (5, 13), and the results present some limitations. In the first approach (13), due to the low-frequency image acquisition of the CLSM setup, a very large biofilm area (800 μm²) was photobleached, leading to average diffusion coefficients over the macrostructure, including water channels and clusters. In contrast, Bryers and Drummond (5) determined local diffusion coefficients in biofilm (with a photobleached surface of ∼80 μm²), using the Axelrod mathematical model (1), which precludes any molecular diffusion during the photobleaching time and is not well adapted for very common mobile molecules (e.g., fluorophores and antibacterial molecules).We present and analyze here an image-based FRAP protocol that can be readily applied by anyone familiar with a CLSM to improve the accuracy of FRAP measurements of the molecular diffusion inside bacterial biofilms. This protocol includes (i) image acquisition of photobleached areas acquired with a commercial CLSM at high frequency, allowing bleach zones smaller than 1 μm²; (ii) an original FRAP analysis used for the first time for measurements in biofilms that takes into account molecular diffusion during the bleach phase, which is based on fluorescence intensity profiles (18) to extract molecular diffusion coefficients; and (iii) a comparison of these results with those obtained by numerical calculation of fluorescence recovery curves, using our own analytical model and the one proposed by Braga et al. (3). This approach was validated by experiments with fluorescent-dextran diffusion inside regular Lactoccocus lactis biofilms and mucoid Stenotrophomonas maltophilia biofilms, and the results were compared to FCS data previously published. However, the proposed protocol may not lead to correct estimation of molecular diffusion coefficients if no consideration of bacterial movements is taken. Indeed, such cellular dynamics may invalidate FRAP analysis and thus indicate a need for using an appropriate visualization tool like kymogram representation. Kymograms are two-dimensional graphs of fluorescence intensity measured along a line (here a straight line drawn on the full width of the images) for each image of a time lapse acquisition. It can thus be used to show fluorescence intensity fluctuations over time along a chosen trajectory and to characterize the motion of structures present in the sample (bacteria in the present study) (15). We show for the first time that kymogram representation is a powerful tool to determine the global trends of biofilm dynamics.     

Bacteriophage ecology in Escherichia coli and Pseudomonas aeruginosa mixed-biofilm communities

Phage therapy is being reexamined as a strategy for bacterial control in medical and other environments. As microorganisms often live in mixed populations, we examined the effect of Escherichia coli bacteriophage λW60 and Pseudomonas aeruginosa bacteriophage PB-1 infection on the viability of monoculture and mixed-species biofilm and planktonic cultures. In mixed-species biofilm communities, E. coli and P. aeruginosa maintained stable cell populations in the presence of one or both phages. In contrast, E. coli planktonic populations were severely depleted in coculture in the presence of λW60. Both E. coli and P. aeruginosa developed phage resistance in planktonic culture; however, reduced resistance was observed in biofilm communities. Increased phage titers and reduced resistance in biofilms suggest that phage can replicate on susceptible cells in biofilms. Infectious phage could be released from mixed-culture biofilms upon treatment with Tween 20 but not upon treatment with chloroform. Tween 20 and chloroform treatments had no effect on phage associated with planktonic cells, suggesting that planktonic phage were not cell or matrix associated. Transmission electron microscopy showed bacteriophage particles to be enmeshed in the extracellular polymeric substance component of biofilms and that this substance could be removed by Tween 20 treatment. Overall, this study demonstrates how mixed-culture biofilms can maintain a reservoir of viable phage and bacterial populations in the environment.     

Size effects on diffusion processes within agarose gels

To investigate diffusion processes in agarose gel, nanoparticles with sizes in the range between 1 and 140 nm have been tested by means of fluorescence correlation spectroscopy. Understanding the diffusion properties in agarose gels is interesting, because such gels are good models for microbial biofilms and cells cytoplasm. The fluorescence correlation spectroscopy technique is very useful for such investigations due to its high sensitivity and selectivity, its excellent spatial resolution compared to the pore size of the gel, and its ability to probe a wide range of sizes of diffusing nanoparticles. The largest hydrodynamic radius (R(c)) of trapped particles that displayed local mobility was estimated to be 70 nm for a 1.5% agarose gel. The results showed that diffusion of particles in agarose gel is anomalous, with a diverging fractal dimension of diffusion when the large particles become entrapped in the pores of the gel. The latter situation occurs when the reduced size (R(A)/R(c)) of the diffusing particle, A, is >0.4. Variations of the fractal exponent of diffusion (d(w)) with the reduced particle size were in agreement with three-dimensional Monte Carlo simulations in porous media. Nonetheless, a systematic offset of d(w) was observed in real systems and was attributed to weak nonelastic interactions between the diffusing particles and polymer fibers, which was not considered in the Monte Carlo simulations.     

Diffusion of bacteriophages through artificial biofilm models

The simple two-chamber diffusion method was improved to study the diffusion properties of bacteriophage (phage) T4 through a model biofilm agarose gel membrane (AGM) embedded with dead host Escherichia coli K12 cells. The apparent diffusion coefficient (D(app) ) of phage T4 was calculated to be 2.4 × 10(-12) m(2) /s in 0.5% AGM, which was lower than the coefficient of 4.2 × 10(-12) m(2) /s in 0.5% AGM without host cells. The phage adsorption process by dead host cells slowed the apparent phage diffusion. The Langmuir adsorption equation was used to simulate phage adsorption under different multiplicity of infections (MOIs); the maximum adsorbed phage MOI was calculated to be 417 PFU/CFU, and the Langmuir adsorption constant K(L) was 6.9 × 10(-4) CFU/PFU. To evaluate the effects of phage proliferation on diffusion, a simple syringe-based biofilm model was developed. The phage was added into this homogenous biofilm model when the host cells were in an exponential growth phase, and the apparent diffusion coefficient was greatly enhanced. We concluded that D(app) of phages through biofilms could be distinctly affected by phage adsorption and proliferation, and that the idea of D(app) and these methods can be used to study diffusion properties through real biofilms.     

Elevated Lytic Phage Production as a Consequence of Particle Colonization by a Marine <Emphasis Type="Italic">Flavobacterium</Emphasis> (<Emphasis Type="Italic">Cellulophaga</Emphasis> sp.)

Bacteria growing on marine particles generally have higher densities and cell-specific activities than free-living bacteria. Since rapidity of phage adsorption is dependent on host density, while infection productivity is a function of host physiological status, we hypothesized that marine particles are sites of elevated phage production. In the present study, organic-matter-rich agarose beads and a marine phage-host pair (Cellulophaga sp., PhiS(M)) were used as a model system to examine whether bacterial colonization of particles increases phage production. While no production of phages was observed in plain seawater, the presence of beads enhanced attachment and growth of bacteria, as well as phage production. This was observed because of extensive lysis of bacteria in the presence of beads and a subsequent increase in phage abundance both on beads and in the surrounding water. After 12 h, extensive phage lysis reduced the density of attached bacteria; however, after 32 h, bacterial abundance increased again. Reexposure to phages and analyses of bacterial isolates suggested that this regrowth on particles was by phage-resistant clones. The present demonstration of elevated lytic phage production associated with model particles illustrates not only that a marine phage has the ability to successfully infect and lyse surface-attached bacteria but also that acquisition of resistance may affect temporal phage-host dynamics on particles. These findings from a model system may have relevance to the distribution of phage production in environments rich in particulate matter (e.g., in coastal areas or during phytoplankton blooms) where a significant part of phage production may be directly linked to these nutrient-rich "hot spots."     

Anisotropic nutrient transport in three-dimensional single species bacterial biofilms

The ability for a biofilm to grow and function is critically dependent on the nutrient availability, and this in turn is dependent on the structure of the biofilm. This relationship is therefore an important factor influencing biofilm maturation. Nutrient transport in bacterial biofilms is complex; however, mathematical models that describe the transport of particles within biofilms have made three simplifying assumptions: the effective diffusion coefficient (EDC) is constant, the EDC is that of water, and/or the EDC is isotropic. Using a Monte Carlo simulation, we determined the EDC, both parallel to and perpendicular to the substratum, within 131 real, single species, three-dimensional biofilms that were constructed from confocal laser scanning microscopy images. Our study showed that diffusion within bacterial biofilms was anisotropic and depth dependent. The heterogeneous distribution of bacteria varied between and within species, reducing the rate of diffusion of particles via steric hindrance. In biofilms with low porosity, the EDCs for nutrient transport perpendicular to the substratum were significantly lower than the EDCs for nutrient transport parallel to the substratum. Here, we propose a reaction-diffusion model to describe the nutrient concentration within a bacterial biofilm that accounts for the depth dependence of the EDC.     

In situ measurements of viral particles diffusion inside mucoid biofilms

Fluorescence correlation spectroscopy (FCS) under two-photon excitation was used successfully to characterize the diffusion properties of model virus particles (bacteriophages) in bacterial biofilm of Stenotrophonas maltophilia. The results are compared to those obtained with fluorescent latex beads used as a reference. The FCS data clearly demonstrated the possibility for viral particles to penetrate inside the exopolymeric matrix of mucoid biofilms, and hence to benefit from its protective effect toward antimicrobials (antibiotics and biocides). Microbial biofilms should hence be considered as potential reservoirs of pathogenic viruses, and are probably responsible for numerous persistent viral infections.     

Bacteriophage and associated polysaccharide depolymerases – novel tools for study of bacterial biofilms

Bacteriophage for three representative strains of Gram-negative biofilm bacteria have proved to be of widespread occurrence. Lytic bacteriophage have been isolated from local sewage for the bacterium 1·15, an exopolysaccharide (EPS)-producing pseudomonad found originally as a component of biofilms in a local river, and for two Enterobacter agglomerans strains from industrial biofilms. Representative examples of all three bacteriophage possess a relatively low burst size and on solid media, exhibit very large plaques surrounded by a wide halo (5–20 mm) indicative of polysaccharide depolymerase action. The bacteriophage are thus similar to other viruses for EPS-producing bacteria in inducing the synthesis of enzymes degrading the polymers which occlude the bacterial cell surface. In each preparation, the polysaccharase activity was associated both with sedimented phage particles and with the supernate of bacterial lysates. The enzymes have been partially purified and used to prepare polysaccharide digests in which the major products from each polysaccharide are the presumed repeat units of the polymers or oligomers of these. The soluble phage enzymes each degrade their substrate by acting as endo -glycanohydrolases. The phage and their associated enzymes thus provide very useful highly specific tools for studies of biofilms incorporating the bacterial host strains. Their potential applications in studies on bacterial biofilms are discussed.     

Nucleic acid economy in bacteria infected with bacteriophage T2

1. During the first 10 minutes of viral growth following infection of E. coli by phage T2 in broth, a pool of DNA is built up that contains phosphorus later to be incorporated into phage. This pool receives phosphorus from, but does not contain, the bacterial DNA. 2. After 10 minutes, DNA synthesis and phage maturation keep pace in such a way that the amount of precursor DNA increases moderately for a time and then remains constant. 3. The pool so described is defined in terms of the kinetics of transport of phosphorus from its origins in the culture medium, the bacterial DNA, and the DNA of the parental phage, to the viral progeny. The most interesting parameter of this system is the size of the precursor pool, which measures 10^–9 to 2 x 10^–9 µg. DNA-P (50 to 100 phage particle equivalents) per bacterium. 4. Neither the precursor nor the intracellular phage population exchanges phosphorus with the phosphate in the medium. More interestingly, the phosphorus in mature phage does not exchange with phosphorus in the precursor, showing that maturation is an irreversible process. 5. Maturation is also a remarkably efficient process. About 90 per cent of labeled phosphorus introduced early into the precursor pool is later incorporated into phage. 6. Viral DNA is synthesized at the rate of about 1.5 x 10^–10 µg. DNA-P (7 or 8 phage particles) per bacterium per minute. This is somewhat faster than bacterial DNA is formed, but considerably slower than RNA is formed, in uninfected bacteria. 7. The transport of phosphorus from medium to viral precursor DNA takes an average of 8 or 9 minutes, and from precursor to phage an additional 7 or 8 minutes. 8. Metabolically active RNA has been detected in infected bacteria.     

Prophage spontaneous activation promotes DNA release enhancing biofilm formation in Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is able to form biofilms in vivo and previous studies propose that pneumococcal biofilms play a relevant role both in colonization and infection. Additionally, pneumococci recovered from human infections are characterized by a high prevalence of lysogenic bacteriophages (phages) residing quiescently in their host chromosome. We investigated a possible link between lysogeny and biofilm formation. Considering that extracellular DNA (eDNA) is a key factor in the biofilm matrix, we reasoned that prophage spontaneous activation with the consequent bacterial host lysis could provide a source of eDNA, enhancing pneumococcal biofilm development. Monitoring biofilm growth of lysogenic and non-lysogenic pneumococcal strains indicated that phage-infected bacteria are more proficient at forming biofilms, that is their biofilms are characterized by a higher biomass and cell viability. The presence of phage particles throughout the lysogenic strains biofilm development implicated prophage spontaneous induction in this effect. Analysis of lysogens deficient for phage lysin and the bacterial major autolysin revealed that the absence of either lytic activity impaired biofilm development and the addition of DNA restored the ability of mutant strains to form robust biofilms. These findings establish that limited phage-mediated host lysis of a fraction of the bacterial population, due to spontaneous phage induction, constitutes an important source of eDNA for the S. pneumoniae biofilm matrix and that this localized release of eDNA favors biofilm formation by the remaining bacterial population.     

Significance of Bacteriophages for Controlling Bacterioplankton Growth in a Mesotrophic Lake

Bacterium-specific viruses have attracted much interest in aquatic microbial ecology because they have been shown to be about 10 times more abundant than planktonic bacteria. So far most of the studies of interactions of planktonic bacteria and viruses have been done in marine environments, and very little is known about these interactions in lakes. Therefore, we studied phage proliferation in Lake Constance, a large mesotrophic lake in Germany. We enumerated bacteria and quantified the fraction of bacteria with mature intracellular phage particles and the number of free viruses by transmission electron microscopy. Between the end of March and early August 1992, peaks of bacterial abundance were followed in 1 to 2 weeks by peaks in the fraction of bacteria containing visible phage particles (0 to 1.7%) and in the number of free viruses (1 x 10(sup7) to 4 x 10(sup7) ml(sup-1)). We estimated that 1 to 17% +/- 12% of all bacteria were phage infected, implying that phage-induced mortality was <34% +/- 24% of total mortality. A direct comparison between phage-induced mortality, the net decrease of bacterial numbers, and bacterial growth rates indicated that phage-induced mortality accounted for <11% of total bacterial mortality during the phytoplankton spring bloom and 18 to 21% following the bloom. Estimated burst sizes ranged from 21 to 121 phages. Phage production rates of 0.5 x 10(sup6) to 2.5 x 10(sup6) ml(sup-1) day(sup-1) accounted for 70 to 380% of the observed net increase rates of free phages, implying high rates of simultaneous phage decay. The cyclic dynamics between bacteria and phages and the varying size structure of the intracellular mature phage particles suggested that phage infection was important in structuring the bacterial host assemblage during the study period.     

The effectiveness of antibiotics on bacteria in biofilms

Bacteria can form different types of communities, united by common notion: biofilms. The aim of the present study was to determine the capacity of different antibiotics to penetrate into biofilms and act on unrelated bacteria. The study revealed that the formation of barriers between the community and the environment on artificial biofilms occurred in all strains of unrelated Gram-positive and Gram-negative bacteria used in this investigation. The capacity of antibiotics to penetrate into biofilms varied in different strains of the same species. For certain antibiotics similarity in their penetrating capacity was found to exist with respect to biofilms of unrelated bacteria. The penetration of antibiotics into mixed biofilms depended on the strain which determined its minimal value, so that the protection of one microorganism by another was thus observed. The method for the evaluation of the effectiveness of antibiotic penetration into bacterial biofilms, suitable for use in bacteriological laboratories, is proposed.     

Susceptibility of Staphylococcus epidermidis planktonic cells and biofilms to the lytic action of staphylococcus bacteriophage K

AIMS: To evaluate differences in biofilm or planktonic bacteria susceptibility to be killed by the polyvalent antistaphylococcus bacteriophage K. METHODS AND RESULTS: In this study, the ability of phage K to infect and kill several clinical isolates of Staphylococcus epidermidis was tested. Strains were grown in suspension or as biofilms to compare the susceptibility of both phenotypes to the phage lytic action. Most strains (10/11) were susceptible to phage K, and phage K was also effective in reducing biofilm biomass after 24 h of challenging. Biofilm cells were killed at a lower rate than the log-phase planktonic bacteria but at similar rate as stationary phase planktonic bacteria. CONCLUSIONS: Staphylococcus epidermidis biofilms and stationary growth phase planktonic bacteria are more resistant to phage K lysis than the exponential phase planktonic bacteria. SIGNIFICANCE OF STUDY: This study shows the differences in Staph. epidermidis susceptibility to be killed by bacteriophage K, when grown in biofilm or planktonic phenotypes.     

Morphogenesis of Type 2 Parainfluenza Virus Examined by Light and Electron Microscopy

The development of type 2 parainfluenza virus in HeLa and stable human amnion cells was examined by use of antisera labeled with fluorescein and ferritin. Serum containing antibody predominantly to soluble viral antigen gave specific fluorescence which was first detectable in small cytoplasmic foci 8 to 10 hr after initiation of infection. By 20 to 24 hr, when the production of infective virus and hemagglutinin was maximal, large perinuclear aggregates of fluorescence were observed which corresponded in distribution and time of appearance to the eosinophilic inclusions seen in similar preparations stained with azure eosin. The inclusions, examined by electron microscopy, were composed of fibrils, presumably viral ribonucleoprotein, which specifically bound the antibody labeled with ferritin. With antiserum to concentrated virus, on the other hand, specific fluorescence was most marked at the surface of infected cells. Foci of fluorescence at the surface represented segments of membrane which had become differentiated morphologically and antigenically to resemble the viral envelope. These were the sites where mature virions appeared. The latter exhibited marked pleomorphism; in some instances, particles were formed which lacked recognizable internal fibrils but which possessed an enclosing membrane bearing viral antigen. Filamentous forms showing an organized internal structure were also observed at the cell surface, but were never encountered in negatively stained preparations. No clear relationship between these filaments and the spherical or oval forms could be established. In negatively stained preparations, nucleocapsid released by rupture of viral particles was similar in appearance to that reported for other paramyxoviruses. It seems probable that this component has a helical configuration.     

Characterization of the diffusive properties of biofilms using pulsed field gradient-nuclear magnetic resonance

The mobility of water in intact biofilms was measured with pulsed field gradient nuclear magnetic resonance (PFG-NMR) and used to characterise their diffusive properties. The results obtained with several well-defined systems, viz. pure water, agar, and agar containing inert particles or active bacteria were compared to glucose diffusion coefficients measured with micro-electrodes and those calculated utilising theoretical diffusion models. A good correspondence was observed indicating that PFG-NMR should also enable the measurement of diffusion coefficients in heterogeneous biological systems. Diffusion coefficients of several types of natural biofilms were measured as well and these results were related to the physical biofilm characteristics. The values had a high accuracy and reflected the properties of a sample of ca. 100 biofilms, while non-uniformity or non-geometrical shapes did not negatively influence the results. The monitored PFG-NMR signal contains supplementary information on e.g. cell fraction or spatial organisation but quantitative analysis was not yet possible. Copyright 1998 John Wiley & Sons, Inc.     

Bacteriophage evolution given spatial constraint

Spatial structure can impede mixing, diffusion, and motility. In microbiology laboratories, spatial structure is commonly achieved via formation of agar gels, within which bacteriophage (phage) replication results in localized clearings called plaques. Developing a better understanding of phage plaque formation is relevant because of the ubiquity of phage plaquing in the laboratory; because plaque size has been employed as a measure of phage fitness; because many bacteria exist within environments that display significant spatial structure (e.g., biofilms, soils, sediments, and in or on plant or animal tissues); and because spatial structure could impede phage exploitation of bacterial communities. There is, however, a relative dearth of experimentation and analysis considering phage plaque formation from the perspective of selection acting on individual phage growth parameters-latent period, burst size, and adsorption rate. Here we consider the impact of these parameters on rates of plaque wavefront velocity (rates of radial plaque enlargement), especially as functions of existing phage and environmental properties. We do so based on analyses of published equations which predict plaque enlargement rates. These indicate that greater wavefront velocities should be associated with (i) latent period reductions, (ii) larger burst sizes, or (iii) faster virion binding to bacteria. We suggest, however, that deviations could occur, respectively, (i) if virion adsorption is "slow" or if burst sizes are large, (ii) if burst sizes are already large, or (iii) if virion binding rates are already fast, bacterial densities are especially high, or burst sizes are large. Higher initial lawn bacterial densities could also contribute to faster plaque expansion, but only if adsorption is otherwise slow or burst sizes are large. By contrast, faster virion diffusion is always expected to result in greater plaque wavefront velocities. Overall, we provide a snapshot of how phage populations may respond evolutionarily to selection for more-rapid propagation during spatially constrained growth.