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Increasing evidence of the damage caused by bacterial biofilms, in both the human body and the environment, is prompting research into new strategies for dealing with them
Bernard Dixon
Even first-year microbiology students learn about iron bacteria attacking water pipes. Both they and professional engineers have also understood that zinc-galvanized steel pipes are totally resistant to corrosion. But they have been mistaken. Investigations in Australia, reported during a recent Society for Applied Microbiology (SfAM) meeting in Brighton, U.K., now highlight a hitherto-unrecognized form of corrosion causing sporadic failures in galvanized steel piping systems. The sources of the problem are biofilms inhabited by aerobic, mainly gram-negative bacteria.
The new findings-indicating the need for improvements in maintenance strategies-have emerged from an intriguing piece of work at Flinders University, Adelaide, and two other centers in Australia. Natalie Bolton and her colleagues have incriminated not only Pseudomonas fluorescens and Bacillus pumilis in the damage but also Afipia spp.
This is the latest addition to a growing compendium of knowledge about the harm caused by microbial biofilms even in locations where past investigators believed their activities were well understood and effectively controlled. In collaboration with coworkers at the Australian Institute for Commercialisation, Melbourne, and OneSteel Market Mills Technology, Newcastle, the Flinders researchers initiated the work following several failures in galvanized piping in different parts of Australia over the past decade.
Bolton and her coworkers removed a length of galvanized pipe from a system in Melbourne experiencing pitting failures and isolated from a corrosion "tubercle" 18 pure cultures, 13 of which they identified by 16S RNA sequencing. When they assessed possible action of the organisms against pieces of metal, they found that the majority significantly increased the corrosion of galvanized steel, but not ungalvanized steel, when attached to the surface as pure culture biofilms. Apparently the first findings of this sort, now published in the Journal of Applied Microbiology (109:239, 2010), the results hold clear lessons for the management of industrial and domestic water systems.
Corrosion of water pipes was only one of a wide range of activities, reported at the SfAM meeting, of the complex microbial communities we term biofilms. Encased in a matrix and attached to surfaces, the constituent organisms differ from their planktonic, free-swimming counterparts in several ways. As well as gene expression and protein production, these include resistance to both antibiotics and immune defences (J. G. Leid, Microbe 4:66, 2009).
Another contributor to the SfAM meeting, Jeremy Webb of Southampton University in the United Kingdom, pointed out that bacteria in biofilms show rapid genetic diversification, which can facilitate their opportunism.He and his colleagues have demonstrated that mutation frequency increases within biofilms, and that mutator strains of Pseudomonas aeruginosa defective in DNA mismatch repair exhibit enhanced microcolony formation. Thus biofilms are important foci for genetic change, and their development may involve intrinsic processes of clonal selection and expansion. Apart from its practical relevance in medicine and environmental microbiology, Webb believes that this model of biofilm growth is analogous to the mutation and selection that occur during the emergence of many eukaryotic cancers.
Adams Roberts of the UCL Eastman Dental Hospital in London reported in Brighton on his studies on oral biofilms, which he described as "potentially an excellent environment for the transfer ofDNAbetween different bacteria." He is especially interested in the role of conjugative transposons, which are able to transfer genes between genera and which usually encode antibiotic resistance.
"In a constant-depth film fermenter we have shown thatDNAcan be transferred between transient bacteria (Bacillus subtilis) to members of an oral biofilm community, demonstrating that oral bacteria can probably acquire genetic information from environmental and/or transient organisms," he said. "We have also found that two of the commonest oral genera (Streptococcus and Veillonella) can act as donors of conjugative transposons to other members of the oral community and may represent a reservoir of transferable antibiotic resistance."
On the basis of investigations on both dental plaque and chronic wound biofilms, Alexander Rickard of Binghamton University in Binghamton, N.Y., proposed that bacteria use autoinducer- 2 (AI-2) to modulate interspecies interactions when juxtaposed with one another in human biofilm communities. Changes in these interactions probably alter the ecology of those communities. One example, based on experiments with biofilms under conditions simulating those of the human mouth, was the promotion by AI-2 of mutualism between the commensal dental plaque organisms Streptococcus oralis and Actinomyces oris. The same signalling molecule also facilitated competition between S. oralis and Streptococcus gordonii.
"While studies of interactions mediated by AI-2 in bacteria common to chronic wounds are still in their infancy, we have discovered that many commensal resident species produce AI-2 while transient pathogenic species do not," Rickard said. "It is possible that these pathogenic species can detect AI-2 from commensals and coordinate their behavior accordingly."
Several presentations at the SfAM meeting reflected progress towards the development of techniques to combat biofilms, from which both the food industry and health care may benefit in future. Ian Connerton and colleagues at the University of Nottingham, United Kingdom, described their work on the effect of bacteriophage on Campylobacter jejuni biofilms. They used phage CP8 or CP30 to treat biofilms of this food-poisoning bacterium on glass surfaces. Counts of the organism declined significantly as a result of the treatment, with evidence that the phage replicated, whereas planktonic cells were only minimally affected. Quantification of the biofilms by means of crystal violet staining and transmission electron microscopy confirmed that the two phages disrupted the biofilms.
The continuing threats posed by biofilms on implants and other medical devices has encouraged Geoff Hanlon of the University of Brighton, United Kingdom, to try to use bacteriophages in a different way. Many different types of antimicrobial surface have been developed in the past, but none have been fully successful. Might it be possible to couple lytic phages chemically onto the materials used to make such devices?
Hanlon and his collaborators decided to test the idea with phage K for Staphylococcus aureus and phage 13 for Pseudomonas aeruginosa, both purified to high titers, and two types of resin bead, TentaGel S-NH2 and TentaGel S-COOH, functionalized on their surfaces with either amino or carboxyl groups. Using two carboxyl-activating coupling compounds, they attached the proteins of the phage capsids to the surfaces of the beads. After copious washing to remove unattached phage, the researchers employed both qualitative and quantitative methods to look for viable phage on the surfaces.
As reported in Brighton, both of the two coupling compounds had attached about 80% of the initial number of phage 13 to the surfaces of the beads, while one of the compounds achieved retention rates of 97 and 61% for phage K. One coupling agent proved to be toxic to phage K but not to phage 13. The phage-coated beads, placed on bacterial lawns, were active against both test bacteria, retaining their viability after being airdried, freeze-dried, and stored at low temperature, room temperature, or body temperature. They also showed significant activity against actively growing bacterial cultures.
Further work is undoubtedly required to establish the stability of the "phage beads" under various environmental conditions. However, these are impressive results-confirming the validity of an approach some people may well have questioned. Finally, an intriguing question from David Harper of Biocontrol Ltd, Sharnbrook, United Kingdom.We have all now become accustomed to the idea that most infections are caused not by free-living planktonic organisms but by those growing as biofilms. Why, then, David asked, are minimal inhibitory concentrations of antibiotics and other antimicrobial substance always reported for planktonic cells?
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