The Society for Applied Microbiology's 75th Anniversary Conference attracted participants as diverse and interdependent as the polymicrobial systems they are studying
While medical microbiologists in particular still love to isolate organisms in pure culture, most of their kindred now recognize the limitations and artificiality of this simplistic craft. The greater reality is, of course, the complex world of polymicrobial communities such as biofilms. Scrutiny of these heterogeneous populations is now providing both deeper insights into the sophistication of microbial life and pointers towards possible avenues for cooperation and control.
Many examples emerged during the Society for Applied Microbiology (SfAM) meeting held recently in Edinburgh, Scotland. They came from domains as disparate as the dynamics of the human intestinal flora and the bioremediation of contaminated soil.
Biofilms can be a significant force driving microbial evolution too, as illustrated by one of the SfAM contributions. It came from Soren Molin and coworkers at the BioCentrum DTU in Lyngby, Denmark. They have established, as a model consortium, a biofilm consisting of just two organisms. These were Acinetobacter sp. C6, which was able to mineralize benzyl alcohol, and Pseudomonas putida KT2440, which could mineralize benzoate but not benzyl alcohol because it had been cured of the TOL plasmid.
Grown together in a flow-cell and supplied with benzyl alcohol as the sole source of energy and available carbon, the bacterial duo established a commensal relationship. Acinetobacter oxidized the substrate to benzoate, some of which was excreted (due to a bottleneck in the degradation pathway) and was then used by the pseudomonad as its carbon and energy source.
However, this was not the full story. “Despite the apparent commensality of the association, a niche developed in the structured film, in which the wild-type cells of KT2440 could not persist,” Molin reported. “Reproducibly, variants appeared, displaying a number of phenotypic changes, including the capacity to occupy the free niche and exploit efficiently the released benzoate from C6.” Genetic analysis confirmed that there were alterations in the composition of the cell surface of the variants. Moreover, the selective forces resulting in the emergence of the variants appeared to be restricted to the biofilm. They were present only rarely, if at all, in homogeneous suspended cultures.
In the early years of research on bacterial biofilms, they have sometimes been portrayed as passive occupants of inactive surfaces--whether oil rigs or shellfish in the oceans, or tissues or implants in the body. Another SfAM contributor, Jeremy Webb of the University of Southampton, argued that this view was seriously incomplete. One of his examples was the subtle interactions between the seaweed Delisea pulchra and its epiphytic biofilms. Far from remaining impervious to the presence of the bacteria, the alga partially determines their composition and abundance by releasing furanones which interfere with bacterial signalling.
Webb also described how biofilms undergo intrinsic ontogenic effects such as regulated differentiation and cell death which lead much of the structure to disperse and slough away. One such mechanism is the internal production of reactive oxygen (ROI) or nitrogen intermediates (RNI). “We have demonstrated that nitric oxide (NO), used widely as a signalling molecule in biological systems, induces dispersal and dissolution of P. aeruginosa biofilms at low, sublethal concentrations,” Webb said. “Analogous to apoptosis in eukaryotes, this induction of programmed cell death by ROIs or RNIs followed by dispersal and biofilm sloughing appears widespread among bacteria.”
These findings help to demonstrate how polymicrobial communities are modulated in the natural world, rather than simply being tolerated on the surfaces they occupy. However, they also indicate potential methods of dealing with unwanted biofilms on artefacts ranging from surgical implants to manmade structures in the sea. Several different applications of the furanones synthesized by D. pulchra, for example, are now being developed commercially. More broadly, Webb believes that, since nature modulates but does not totally eliminate biofilms, we should seek to control them by nonbiocidal strategies.
Cary Lambert of Nottingham University described work on another resident of polymicrobial biofilms, the gram-negative bacterium Bdellovibrio. A predator of other gram-negative bacteria, including pathogens of plants, humans, and other animals, it occurs in the soil and in marine and other aquatic environments. Vigorous flexing of its sheathed flagellum has allowed it to win the distinction of holding the world speed record for swimming by bacteria. Bellovibrio uses type IV pili to locate and enter its prey, by penetrating their outer layers, growing within the periplasm and lysing the cell (see Microbe, August 2006, p. 353).
Lambert's talk was titled “Living together while being eaten” because Bdellovibrio never destroys a population of prey entirely, even when let loose under laboratory conditions against inferior numbers of bacteria. Despite this limitation, which mathematical modelling is helping to explain, there are hopes of harnessing the organism as an alternative to antibiotics to combat gram-negative pathogens found in ulcers and burn wounds.
The effect of the use and abuse of antimicrobial substances over the last century on the nature of polymicrobial communities was discussed by Peter Gilbert of the University of Manchester. He used the results of microcosm studies to contrast the vigorous emergence of antibiotic-resistant bacteria with the absence of anything like the same trend for agents such as biguanides, triclosan, and quaternary ammonium compounds.
Products containing chemicals of this sort, he pointed out, are invariably deployed in situations with stable polymicrobial populations, such as soil or the skin, or on aesthetically clean, hard surfaces, whose resident/transient species have a broad susceptibility profile. “Exposure to antibacterials will therefore kill/inhibit the growth of some strains and reduce the growth efficiency of others, yet leave many others unaffected,” Gilbert said. “Whilst partially inhibited strains will be subject to a selection pressure towards less susceptible phenotypes, this will incur a fitness-cost and a temporary loss of competitive efficiency.
“Climax communities generally resist the influx of new species since adventitious arrivees must be able either to out-compete residents in terms of nutrient utilisation and/or occupy vacant functional niches. During sublethal exposure to antibacterials, colonisation resistance is lost and adapted strains must re-compete for their position in the community. Invariably this battle is lost, with the effects of antibacterial use being a clonal expansion of pre-existing less susceptible strains with displacement of the susceptible ones.”
Several papers highlighted new insights, coming from ribosomal sequence analysis, into the bacterial diversity of the human colon. In turn, this is helping researchers to predict the effects of dietary additives such as probiotics, prebiotics, and nondigestible carbohydrates. Emma Woodmansey of Smith and Nephew Research Centre, York, United Kingdom, reported promising results from feeding trials designed to counteract adverse changes in the gut population. For example, as we age our colon harbors rising numbers of facultative anaerobes and falling numbers of anaerobic lactobacilli and bifidobacteria. These shifts may result in increased putrefaction and greater susceptibility to disease.
There were also warnings in Edinburgh that bioremediation efforts often prove to be disappointing because specialized scavengers, developed in the laboratory, have to function not in isolation but in the ecological networks of which they become a part. A team based at the University of Perugia, Italy, described one project in which they inoculated Botryosphaeria rhodina in a site heavily contaminated with aromatic hydrocarbons, and achieved superior results when the fungus worked in cooperation with the autochthonous microflora.
Whether applied to pristine, contaminated or bioremediated soil, or to the human gut in health or disease, the central message of the SfAM conference may be summarized in the same words. While much can be learned about the behavior of specific isolates under laboratory conditions, understanding and modifying polymicrobial communities in the real world pose rather more formidable challenges.