D.G. Allison

Biofilms in vitro and in vivo: do singular mechanisms imply cross-resistance?

Microbial biofilm has become inexorably linked with mans failure to control them by antibiotic and biocide regimes that are effective against suspended bacteria. This failure relates to a localized concentration of biofilm bacteria, and their extracellular products (exopolymers and extracellular enzymes), that moderates the access of the treatment agent and starves the more deeply placed cells. Biofilms, therefore, typically present gradients of physiology and concentration for the imposed treatment agent, which enables the less susceptible clones to survive. Such clones might include efflux mutants in addition to genotypes with modifications in single gene products. Clonal expansion following subeffective treatment would, in the case of many antibiotics, lead to the emergence of a resistant population. This tends not to occur for biocidal treatments where the active agent exhibits multiple pharmacological activity towards a number of specific cellular targets. Whilst resistance development towards biocidal agents is highly unlikely, subeffective exposure will lead to the selection of less susceptible clones, modified either in efflux or in their most susceptible target. The latter might also confer resistance to antibiotics where the target is shared. Thus, recent reports have demonstrated that sublethal concentrations of the antibacterial and antifungal agent triclosan can select for resistant mutants in Escherichia coli and that this agent specifically targets the enzyme enoyl reductase that is involved in lipid biosynthesis. Triclosan may, therefore, select for mutants in a target that is shared with the anti-E. coli diazaborine compounds and the antituberculosis drug isoniazid. Although triclosan may be a uniquely specific biocide, sublethal concentrations of less specific antimicrobial agents may also select for mutations within their most sensitive targets, some of which might be common to therapeutic agents. Sublethal treatment with chemical antimicrobial agents has also been demonstrated to induce the expression of multidrug efflux pumps and efflux mutants. Whilst efflux does not confer protection against use concentrations of biocidal products it is sufficient to confer protection against therapeutic doses of many antibiotics. It has, therefore, been widely speculated that biocide misuse may have an insidious effect, contributing to the evolution and persistence of drug resistance within microbial communities. Whilst such notions are supported by laboratory studies that utilize pure cultures, recent evidence has strongly refuted such linkage within the general environment where complex, multispecies biofilms predominate and where biocidal products are routinely deployed. In such situations the competition, for nutrients and space, between community members of disparate sensitivities far outweighs any potential benefits bestowed by the changes in an individuals antimicrobial susceptibility.

The biofilm matrix

The extracellular matrix is a complex and extremely important component of all biofilms, providing architectural structure and mechanical stability to the attached population. The matrix is composed of cells, water and secreted/released extracellular macromolecules. In addition, a range of enzymic and regulatory activities can be found within the matrix. Together, these different components and activities are likely to interact and in so doing create a series of local environments within the matrix which co-exist as a functional consortium. The matrix architecture is also subject to a number of extrinsic factors, including fluctuations in nutrient and gaseous levels and fluid shear. Together, these intrinsic and extrinsic factors combine to produce a dynamic, heterogeneous microenvironment for the attached and enveloped cells.

Assessment of resistance towards biocides following the attachment of micro-organisms to, and growth on, surfaces

Aims: To develop a rapid method for the assessment of biocidal activity directed towards intact biofilms.

Clonal variation in maximum specific growth rate and susceptibility towards antimicrobials

Aims: To examine associations between growth rate within bacterial populations and survival patterns following treatment with antimicrobial agents.

A role for rhamnolipid in biofilm dispersion

Biofilms are often considered as localized zones of high cell density. Quorum sensing provides a means for control of population processes and has been implicated in the regulation of biofilm activities. We present a role for quorum sensing in programmed detachment and dispersal processes. Biofilms of Pseudomonas aeruginosa PAO1 and its isogenic homoserine lactone (HSL) mutant P. aeruginosa PAO-JP2 were grown in batch culture on glass substrata; differences were found in the rate and extent of formation of biofilm. Climax communities were observed for PAO1 at 24 h. These were later accompanied by foaming, a drop in the surface tension of culture media and dispersal of the biofilm, after which no subsequent biofilm accretion occurred. PAO-JP2 cultures reformed biofilm post-detachment and did not foam. Prevention of biofilm reformation in the wild type was related to some component excreted into the culture medium. Rhamnolipid, a biosurfactant regulated by quorum sensing, was detected in PAO1 cultures. When rhamnolipid was added to freshly inoculated substrata, biofilm formation was inhibited. At 20 h, PAO1 biofilms were transferred to medium with added rhamnolipid: biofilm was relatively unaffected. Biofilm events were also studied in medium supplemented with N -butyryl- L -homoserine lactone, which is involved in the regulation of rhamnolipid synthesis. Both strains exhibited similar trends of rapid biofilm formation and dramatic changes in the rate and extent of biofilm accretion. In both cases, there was premature foaming, lowered surface tension and elevated rhamnolipid levels. A role for HSLs in maintenance of biofilm and events leading to dispersion of cells is proposed. This role would encompass dispersion but not necessarily detachment of cells from biofilm and supports a new function for rhamnolipid in pathogenesis, whereby rhamnolipid would promote the dissemination of cells from a nidus of infection.

Expression of the multiple antibiotic resistance operon (mar) during growth of Escherichia coli as a biofilm

The multiple antibiotic resistance (mar) operon is a global regulator controlling the expression of various genes in Escherichia coli which constitutes the mar regulon. Upregulation of mar leads to a multi‐drug resistant phenotype, which includes resistance towards structurally unrelated antibiotics, organic solvents and the disinfectant pine oil. Biofilms also display similar decreases in susceptibility to antimicrobial agents. A marOII‐lacZ fusion strain (SPC105) of E. coli was used to monitor mar expression under various growth conditions including batch, continuous and biofilm culture. In chemically‐defined media (CDM), mar expression was maximal in mid‐log and declined in the stationary phase. Conversely, in rich media (Luria‐Bertani broth), minimal expression in mid‐log was followed by an increase in the stationary phase. In continuous culture, expression was inversely related to specific growth rate (μ = 0.05–0.4 h−1). LacZ expression by the marOII‐lacZ fusion was generally low within the total biofilm population and equivalent to that of stationary phase cultures grown in batch culture. When the expression of mar in CDM batch culture was compared with that in biofilm populations, β‐galactosidase activity was generally higher throughout batch culture than in the attached population. Overall, these results suggest that while mar expression will be greatest within the depths of a biofilm where growth rates are suppressed, its probable induction within biofilms cannot explain the elevated levels of antibiotic resistance observed.

The use of poloxamer hydrogels for the assessment of biofilm susceptibility towards biocide treatments

P. GILBERT, M.V. JONES, D.G. ALLISON, S. HEYS, T. MAIRA AND P. WOOD. 1998. Poloxamer F127 is a non‐toxic, di‐block copolymer of polyoxyethylene and polyoxypropylene. Aqueous solutions (30% w/v) show thermoreversible gelation, being liquid at temperatures < 15 °C and robust gels at temperatures > 15 °C. Chilled poloxamer (30% in tryptone soya broth) was mixed with an inoculum of Pseudomonas aeruginosa (104 cfu ml−1) and placed as 100 μl drops onto separate glass cover‐slips. These were placed into sealed Petri dishes containing moistened cotton wool and incubated at 35 °C. Viable counts could be performed on the poloxamer gels by transfer of the coverslips to diluents at < 15 °C. Growth curves in the gels and in liquid batch cultures were indistinguishable from one another with stationary phase cell densities, being approximately 5 times 1010 cfu ml−1 in each at 16 h. SDS‐PAGE of cell envelope preparations showed the poloxamer‐grown cells to exhibit a biofilm rather than planktonic phenotype. Susceptibility towards various concentrations of chlorhexidine, iodine and hydrogen peroxide was assessed for 10 min at 35 °C for suspensions of broth‐grown cells and for incubated poloxamer‐gels (1 and 16 h). The gels were immersed in biocide, on their glass supports, before transfer to neutralizer at 10 °C where dissolution was complete within 5 min. Further serial dilutions and plate counts were made. While modest decreases in susceptibility towards all biocides were associated with incorporation of the inoculum with the gel (1 h incubation), substantial changes were noted after prolonged incubation and adaptation to a biofilm phenotype (16 h incubation). The gel populations mimic the localized high cell densities observed in biofilms and will also be subject to the same nutrient and chemical gradients as found within natural biofilms. Thermoreversible gelation enables complete recovery of the test inoculum without further trauma. They therefore provide an effective model for assessing biofilm susceptibility towards biocides and would be suitable for screening programmes.

Immigration and emigration of Burkholderia cepacia and Pseudomonas aeruginosa between and within mixed biofilm communities.

Aims:  To investigate the dynamics of binary culture biofilm formation through use of both the Sorbarod model of biofilm growth and the constant depth film fermenter (CDFF).

Performance evaluation of disinfectant formulations using poloxamer- hydrogel biofilm-constructs

G. WIRTANEN, S. SALO, D.G. ALLISON, T. MATTILA‐SANDHOLM AND P. GILBERT. 1998. Poloxamer F127 is a di‐block co‐polymer of polyoxyethylene and polyoxypropylene. Aqueous solutions show thermo‐reversible gelation, being liquid at temperatures < 15 °C and robust gels at temperatures > 15 °C. Chilled poloxamer solutions (30% w/v) were inoculated with approximately 10+−5 cfu ml−1 of stationary phase cultures of Pseudomonas aeruginosa, Ps. fluorescens, Pantoea agglomerans, Micrococcus luteus, Staphylococcus epidermidis, Bacillus subtilis or Listeria innocua. Drops (200 μl) of the inoculated poloxamers were placed on stainless steel coupons held in Petri dishes containing moistened cotton wool and incubated at 30 °C for 5 h. All strains grew well giving between 106–7 cfu ml−1 at 5–6 h. The cultured gels were readily applied to tests of biocide effectiveness as the stainless steel coupons could be removed and flooded with biocide solution for fixed exposure times. Provided that the temperature of the biocide solutions was > 15 °C, the integrity of the gels could be maintained during exposure. After exposure, the gels and their supports were removed to separate tubes containing neutralizer solution (< 15 °C). The gels rapidly dispersed within 5 min to ensure a complete recovery of the sample population. Biofilm‐constructs and cell suspensions (107 cfu ml−1) were exposed to four commercial disinfectant formulations, based on hypochlorite, alcohol, hydrogen peroxide and a tenside, at recommended use levels. Cell suspensions, in the presence of bovine serum albumen (BSA; 0·03% w/v), were subject to a > 5‐log kill within 5 min while the killing effected against the biofilm‐constructs varied between 0·4 and 2‐log reductions. The results indicate a high degree of reproducibility between replicate samples, with patterns of susceptibility varying both as a function of organism, biocide type and concentration. The experiments strongly support the view that poloxamer‐constructs are suitable for application in trials and testing of disinfectant formulations.

The influence of green fluorescent protein incorporation on bacterial physiology: a note of caution

Aims:  To investigate the effect of green flourescent protein (GFP) incorporation on bacterial physiology.