P. Gilbert

Cationic antiseptics: diversity of action under a common epithet

Cationic antimicrobials have been in general use within clinical and domestic settings for over half a century. Recently, the use of antiseptics and disinfectants has been questioned in such settings because of the possibility that chronic exposure of the environment to such agents might select for less susceptible strains towards these agents and towards third party antibiotics. Whilst no supportive evidence has emerged from retrospective field studies of high use environments such debate has tempered new applications for these molecules. In the clinic, use of antiseptics, together with products, such as dressings, catheters and sutures, which are impregnated with biocides has increased. Prominent amongst these biocides are the cationics. Much of the research pertaining to the mechanisms of action of cationic antibacterials was conducted in the 1960s and 1970s and has not been subject to extensive review. Analysis of available publications suggest that monoquaternary ammonium compounds (QAC, cetrimide, benzalkonium chloride), biquaternaries and bisbiguanides (Chlorhexidine, Barquat), and polymeric biguanides (Vantocil, Cosmocil) whilst having similarities in action mechanism, differ substantially in the nature of their interaction with cell envelopes. This has profound implications in terms of cross-resistance where changes in susceptibility towards QAC is not reflected in changes towards other cationics. This review examines action mechanisms for these agents and highlights key differences that render them distinct categories of antibacterial agent.

Protozoan grazing and its impact upon population dynamics in biofilm communities

Aims:u2002 To determine the impact of protozoan grazing on the population dynamics of a multispecies bacterial biofilm community.

Development and characterization of a simple perfused oral microcosm

Aims:u2002 To validate perfused, inline, filter‐based fermentation systems (multiple Sorbarod devices, MSD) for their ability to maintain stable oral bacterial communities. MSD enable replicate (nu2003=u20035) microcosm biofilms (BF) to be established and sampled, together with their perfusates (PA, cells in eluted medium).

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.

Effects of chronic triclosan exposure upon the antimicrobial susceptibility of 40 ex-situ environmental and human isolates.

Background:u2002 Triclosan (TCS) exposure of Escherichia coli selects for tolerant clones, mutated in their enoyl–acyl carrier protein reductase (FabI). It has been inferred that this phenomenon is widespread amongst bacterial genera and might be associated with resistance to third party agents.

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.

Individual microflora beget unique oral microcosms.

Aims:u2002 To examine the efficacy of the multiple Sorbarod device (MSD) for the reproduction of inter‐individual variations in oral microbiotas. The MSD supports sessile growth on parallel cellulose filters, perfused with artificial saliva. This enables biofilms (BF) to be grown and sampled, together with released cells in eluted medium (perfusates, PAs).

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

Aims:u2002 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).