Biofilms as complex differentiated communities.

Prokaryotic biofilms that predominate in a diverse range of ecosystems are often composed of highly structured multispecies communities. Within these communities metabolic activities are integrated, and developmental sequences, not unlike those of multicellular organisms, can be detected. These structural adaptations and interrelationships are made possible by the expression of sets of genes that result in phenotypes that differ profoundly from those of planktonically grown cells of the same species. Molecular and microscopic evidence suggest the existence of a succession of de facto biofilm phenotypes. We submit that complex cell-cell interactions within prokaryotic communities are an ancient characteristic, the development of which was facilitated by the localization of cells at surfaces. In addition to spatial localization, surfaces may have provided the protective niche in which attached cells could create a localized homeostatic environment. In a holistic sense both biofilm and planktonic phenotypes may be viewed as integrated components of prokaryote life.

Gene expression in Pseudomonas aeruginosa: evidence of iron override effects on quorum sensing and biofilm-specific gene regulation.

Prior studies established that the Pseudomonas aeruginosa oxidative stress response is influenced by iron availability, whereas more recent evidence demonstrated that it was also controlled by quorum sensing (QS) regulatory circuitry. In the present study, sodA (encoding manganese-cofactored superoxide dismutase [Mn-SOD]) and Mn-SOD were used as a reporter gene and endogenous reporter enzyme, respectively, to reexamine control mechanisms that govern the oxidative stress response and to better understand how QS and a nutrient stress response interact or overlap in this bacterium. In cells grown in Trypticase soy broth (TSB), Mn-SOD was found in wild-type stationary-phase planktonic cells but not in a lasI or lasR mutant. However, Mn-SOD activity was completely suppressed in the wild-type strain when TSB was supplemented with iron. Reporter gene studies indicated that sodA transcription could be variably induced in iron-starved cells of all three strains, depending on growth stage. Iron starvation induction of sodA was greatest in the wild-type strain and least in the lasR mutant and was maximal in stationary-phase cells. Reporter experiments in the wild-type strain showed increased lasI::lacZ transcription in response to iron limitation, whereas the expression level in the las mutants was minimal and iron starvation induction of lasI::lacZ did not occur. Studies comparing Mn-SOD activity in P. aeruginosa biofilms and planktonic cultures were also initiated. In wild-type biofilms, Mn-SOD was not detected until after 6 days, although in iron-limited wild-type biofilms Mn-SOD was detected within the initial 24 h of biofilm establishment and formation. Unlike planktonic bacteria, Mn-SOD was constitutive in the lasI and lasR mutant biofilms but could be suppressed if the growth medium was amended with 25 microM ferric chloride. This study demonstrated that (i) the nutritional status of the cell must be taken into account when one is evaluating QS-based gene expression; (ii) in the biofilm mode of growth, QS may also have negative regulatory functions; (iii) QS-based gene regulation models based on studies with planktonic cells must be modified in order to explain biofilm gene expression behavior; and (iv) gene expression in biofilms is dynamic.

Biodegradation of naphthenic acids by microbial populations indigenous to oil sands tailings.

Organic acids, similar in structure to naphthenic acids, have been associated with the acute toxicity of tailings produced by the oil sands industry in northeastern Alberta, Canada. Bacterial cultures enriched from oil sands tailings were found to utilize as their sole carbon source both a commercial mixture of naphthenic acids and a mixture of organic acids extracted from oil sands tailings. Gas chromatographic analysis of both the commercial naphthenic acids and the extracted organic acids revealed an unresolved "hump" formed by the presence of many overlapping peaks. Microbial activity directed against the commercial mixture of naphthenic acids converted approximately 50% of organic carbon into CO2 and resulted in a reduction in many of the gas chromatographic peaks associated with this mixture. Acute toxicity testing utilizing the Microtox test revealed a complete absence of detectable toxicity following the biodegradation of the naphthenic acids. Microbial activity mineralized approximately 20% of the organic carbon present in the extracted organic acids mixture, although there was no indication of a reduction in any gas chromatographic peaks with biodegradation. Microbial attack on the organic acids mixture reduced acute toxicity to approximately one half of the original level. Respirometric measurements of microbial activity within microcosms containing oil sands tailings were used to provide further evidence that the indigenous microbial community could biodegrade naphthenic acids and components within the extracted organic acids mixture.

Observations of the ultrastructure of infected kidney stones.

Struvite stones are formed as the result of urinary tract infection by urease-producing bacteria. Ultrastructural examination of calculi removed from a patient revealed bacteria incorporated throughout the stone matrix. Exopolysaccharide stained by ruthenium red was associated with most of the bacteria, but it represented only a small portion of the organic matrix in the stone. Localised deposits of calcium and phosphorus, components of carbonate-apatite, and magnesium, a struvite component, were detected in close proximity to the cells. Histochemical examinations revealed that several of the gram-negative bacteria within the stone matrix possessed high levels of urease activity. We propose that bacterial slime production, intimately involved in the initiation of stone matrix deposition, is less prominent in mature stones because of the increased incorporation of host-derived mucoproteins and mucopolysaccharides.

Starvation and nutrient resuscitation of Klebsiella pneumoniae isolated from oil well waters.

Klebsiella pneumoniae isolated from oil well waters reduced in size in response to nutrient starvation. The cells remained viable during starvation and later were able to grow rapidly when stimulated by nutrients. The heterotrophic potential, culture absorbance and extracellular polysaccharide production decreased during cell starvation whereas an initial increase in colony-forming units was observed on agar plates. Transmission electron microscopy (TEM) after 24 d revealed that the cells had changed to small rods or cocci between 0.5 by 0.25 micron and 0.87 by 0.55 micron. When transferred to half-strength brain heart infusion medium, TEM showed cell division and rod-shaped cells after 45 min and full resuscitation within 4 h. Cell response was much slower in sodium citrate medium and resuscitation took 8 h.

Influence of culture conditions on expression of the mucoid mode of growth of Pseudomonas aeruginosa.

Five strains of Pseudomonas aeruginosa that are routinely distinguished by diagnostic laboratories on the basis of their colony morphology on agar media were grown in different media to assess the effects of culture conditions on mucoid growth, which we define as the copious production of exopolysaccharide. On brain heart infusion agar, only two of these strains (mucoid and gelatinous) grew as slimy mucoid colonies. None of the five strains produced a mucoid pattern of growth in Mueller-Hinton broth, in which all grew as turbid, nonmucoid, homogeneous suspensions of bacterial cells. When Mueller-Hinton broth was supplemented with Mg2+, all of the strains produced some mucoid aggregated growth, but growth in a modified version of the chemically defined medium of Vogel and Bonner, with elevated levels of Mg2+ and gluconate, produced patently mucoid growth in all strains. This mucoid growth in a liquid medium takes the form of large, coherent, slimy aggregates within the medium and of a "collar" of adherent microcolonies at the air-medium interface. Direct observations by light and electron microscopy showed the submerged aggregates and the adherent microcolonies to be composed of bacterial cells enmeshed in a copious exopolysaccharide matrix. When agar was added to the supplemented medium of Vogel and Bonner, only the mucoid and gelatinous strains produced slimy mucoid colonies on its surface. We conclude that both chemical and physical factors affect exopolysaccharide production by these clinical strains of P. aeruginosa and that the colony morphology on a single agar medium is an insufficient criterion for the designation of a given isolate as being mucoid or nonmucoid.

The use of specific antibodies to demonstrate the glycocalyx and spatial relationships of a K99-, F41- enterotoxigenic strain of Escherichia coli colonizing the ileum of colostrum-deprived calves.

Electron microscopy was used to study the interaction between the glycocalyx of enterotoxigenic Escherichia coli strain 210 (09:K30+;K99-;F41-:H-) and the glycocalyx of epithelial cells in then ileum of experimentally infected newborn colostrum-deprived calves. Fixation of tissues in anti-K30 antibody and ruthenium red was used to stabilize the bacterial glycocalyx so that the spatial relationship between the bacteria and the intestinal epithelial cells could be characterized. When strain 210 was grown in vitro and reacted with anti-K30 antibody prior to staining with ruthenium red, the extensive glycocalyx could be clearly visualized surrounding the bacterial cells. By negative staining, an unidentified pilus was also seen. Sections of ileum from infected calves, which were not fixed in antibody nor stained with ruthenium red, revealed attached bacteria which were surrounded by an electron-translucent zone and no visible bacterial glycocalyx. When ruthenium red staining was used, the bacterial glycocalyx partially collapsed during the dehydration steps of fixation, but could be seen as either a fibrous capsule or an electron-dense accretion on the bacterial cell surface. When ileal tissue was reacted for one hour in anti-K30 antibody before staining with ruthenium red, the bacterial glycocalyx was seen as a discrete electron-dense structure up to 1.0microm thick which was in intimate contact with the glycocalyx of the epithelial cells. The importance of the bacterial exopolysaccharide to microcolony formation on the villi could be clearly visualized.

Use of specific antibody to demonstrate glycocalyx, K99 pili, and the spatial relationships of K99+ enterotoxigenic Escherichia coli in the ileum of colostrum-fed calves.

The attachment of enterotoxigenic Escherichia coli (ETEC) strain B44 (O9:K30:K99:F41:H-) to the ileal epithelium of newborn colostrum-fed calves was studied by electron microscopy. Stabilization of the bacterial glycocalyx (K30) and pili (K99) by fixation of tissue sections in specific antibody and staining with ruthenium red were used so that the bacterial surface structures could be clearly visualized and their spatial relationship to the intestinal brush border defined. When sections of ileum from infected calves were neither fixed in antibody nor stained with ruthenium red, the ETEC cells colonizing the small intestine were separated from each other and from the brush border by an electron-translucent halo; neither the glycocalyx nor the pili could be clearly resolved. When ruthenium red staining was used, the halo was partially filled by a net of electron-dense fibers composed of pili and condensed glycocalyx which extended to the brush border. Tissue sections reacted with anti-K30 antibody before staining with ruthenium red revealed microcolonies of ETEC surrounded by a discrete electron-dense glycocalyx 0.3 to 1.0 micrometers thick and in tight contact with the epithelial cell surface. When ileal tissue was treated with K99 antibody, the K99 pili were visible as discrete fibers extending from the bacterial cell surface through the glycocalyx. We discuss the role of these cell surface components in pathogenic adhesion and in the formation of protected microcolonies at the surface of the infected ileal epithelium.

A scanning electron microscopy study of the invasion of leaflets of a bloat-safe and a bloat-causing legume by rumen microorganisms.

A newly developed technique using ruthenium red to detect foci of bacterial digestion in mounts of whole leaflets that had been incubated with rumen bacteria was used to compare the digestion of alfalfa, a bloat-causing legume, and sainfoin, a bloat-safe legume. When whole leaflets were suspended in an artificial rumen medium and inoculated with rumen bacteria, massive bacterial adhesion and proliferation were noted at the stomata of alfalfa leaflets after 6 h of incubation, whereas only a few isolated bacteria adhered near the stomata of sainfoin leaflets After 22 h of incubation, the epidermal layers of alfalfa leaflets had peeled away in many areas, revealing an extensive bacterial invasion of the underlying mesophyll tissue in which large bacterial microcolonies had formed in intercellular spaces, and in intracellular spaces in several areas where plant cell walls had broken down. After 22 h of incubation, the surface of sainfoin leaflets resembled that of alfalfa leaflets at 6 h, with bacterial microcolonies adhering to the area surrounding the stomata, but without sloughing of the epidermis. Uninoculated control leaflets of both species showed no surface alteration but part of their normal bacterial flora had proliferated to form microcolonies on the surface after 22 h incubation. Dry matter loss due to leaching or bacterial digestion when whole leaflets of legumes were suspended in an artificial rumen medium, alone or with rumen bacteria, was significantly higher in the bloat-causing group. Values of leaching and of bacterial digestion were positively correlated. We conclude that reported differences in plant anatomy, and in cell wall chemistry, produce distinct rates or organic nutrient release from legume leaflets, and that these same differences produce an equally distinct susceptibility of leaflets to bacterial invasion, plant cell rupture, and the consequent release of intracellular plant components. The rate of release of organic nutrients from legume leaflets may be important in the etiology of foamy pasture bloat. This technique of in vitro digestion of whole leaflets followed by ruthenium red staining shows some promise of providing a rapid and qualitative test to distinguish, within a species, cultivars that may differ in their bloat-related characteristics.

In vitro digestion of bloat-safe and bloat-causing legumes by rumen microorganisms: gas and foam production.

Leaves of three bloat-safe legumes -- birdsfoot trefoil (Lotus corniculatus L.), sainfoin (Onobrychis viciaefolia Scop.), and cicer milkvetch (Astralagus cicer L.) -- and of three bloat-causing legumes -- alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.), and white clover (Trifolium repens L.) -- were incubated with strained rumen fluid or with mixed rumen fluid and solids. Gas released was measured during the early period (0 to 22 h) of this in vitro digestion. Gas volume was greater with a 1:1 (wt/vol) mixture of solid and fluid rumen contents than with rumen fluid alone. It was greater with whole and chewed leaves from the bloat-causing legumes than with whole leaves from the bloat-safe legumes. However, when leaves were homogenized, volumes of gas from bloat-causing and bloat-safe legumes were similar. More gas was released from homogenized leaves than from the same weight of whole leaves. The amount of foam produced on chewed herbage and homogenized leaves of bloat-causing legumes was greater than on those of bloat-safe legumes. These results are consistent with the rate of disintegration and digestion of legumes by rumen bacteria being an important determinant in pasture bloat. Measurement of gas produced early in in vitro digestion may provide a useful bioassay for evaluating the bloat-causing potential of legumes in breeding selections if variability of the method can be reduced.

Comparison of the cell envelope structure of a lipopolysaccharide-defective (heptose-deficient) strain and a smooth strain of Salmonella typhimurium.

The cell envelope structure of Salmonella typhimurium LT2, which has a heptose-deficient lipopolysaccharide (LPS), is significantly different from that of an isogenic strain with a normal LPS. The rough strain, when examined by freeze-etching, lacks most surface structures that are routinely present in the smooth strain (surface particles and flagella) and has few transmemberane studs in the cytoplasmic membrane (those present are generally found in aggregates), and the outer membrane cleavage is substantially stronger than that of the smooth strain. These envelope differences were independent of both growth temperature and culture age. Examination of ultrathin sections indicated that the rough strain has an outer membrane which forms a much more defined double-track artifact than the smooth strain. The addition of MgCl2 to the growth medium of the rough strain decreased the extent of outer membrane cleavage, and flagella became evident in freeze-etched preparations. The presence of supplemental MgCl2 in the growth medium, which resulted in these morphological changes in the rough strain, also produced growth at a previously restrictive temperature and a decrease in the leakage of periplasmic enzymes. The smooth strain was unaltered morphologically or physiologically by MgCl2 under identical conditions. It is suggested that the outer membrane of the rough strain is more planar.