School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK
Received 23 July 2003; returned 13 November 2003; revised 27 November 2003; accepted 28 November 2003
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Abstract |
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Methods and results: Biocide susceptibility data were generated for strains of P. aeruginosa deficient in N-acyl homoserine lactone production, grown planktonically or as biofilm populations using a poloxamer hydrogel construct. Component cells from the biofilm constructs were also tested for their susceptibility. Significant differences in susceptibility were noted between the wild-type strain, a mutant defective in the long chain (C-12) homoserine lactone and a mutant defective in the short chain (C-4) homoserine lactone which could not be related to the biofilm mode of growth. Moreover, differences in susceptibility appeared to be dependent upon the nature of the homoserine lactone deletion and type of biocide rather than the mode of growth.
Conclusions: No general trend exists between homoserine lactone deficiency and biocide susceptibility regardless of mode of growth.
Keywords: biocide susceptibility, N-acyl homoserine lactones, bronopol, chlorhexidine diacetate, cetrimide USP, isothiazolones
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Introduction |
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N-acyl homoserine lactone (HSL)-mediated, quorum sensing has been reported to mediate in biofilm differentiation and the associated changes in susceptibility towards some biocides.10 Quorum-sensing is a cell-density-sensing mechanism that elaborates signal-transduction, thereby facilitating adaptation to the prevailing growth environment.11 Pseudomonas aeruginosa is known to possess at least two quorum-sensing systems. The lasI-lasR system that uses N3-(oxododecanoyl)-homoserine lactone (C12-HSL)12 and the rhlI-rhlR, that uses N-butanoylhomoserine lactone (C4-HSL) as signal molecules.13 Whilst C4-HSL passively diffuses in and out of the cells, C12-HSL accumulates in the cell by diffusion but is actively pumped out by the MexAB-Opr pump.14 Davies et al.10 hypothesized that an abnormal, undifferentiated biofilm formed by a lasI mutant, PA0-JP1, might be sensitive to a biocide, sodium dodecyl sulphate (SDS, 0.2%) that would not otherwise disrupt wild-type biofilms (PA01). Their observations demonstrated that whereas there was no detectable effect on the wild-type biofilm or the lasI mutant biofilm grown in the presence of synthetic 3OC12-HSL, most or all of the bacteria in the lasI mutant biofilm detached from the surface. The abnormal biofilm, therefore, appeared to be sensitive to the detergent biocide SDS. Such broader associations between HSL-mediated biofilm phenotypes and biocide susceptibility have been proposed based on biofilms produced by HSL mutant strains. In no instance has the innate susceptibility of HSL-deficient strains been compared for planktonic culture. The present study was therefore conducted to determine whether such changes in biofilm susceptibility could be attributed to a general loss of the biofilm phenotype or whether other HSL-associated responses could alter biocide susceptibility.
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Materials and methods |
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P. aeruginosa PA01 (HSL wild-type) strain possessing functional lasR and lasI genes15 and mutants PA0-R1 (lasR; C12-HSL),16 PA0-JP1 (lasI; C12-HSL),17 PAN067 (lasB; C4-HSL)11 and PA0-JP2 (lasI, rhlI; C12-HSL, C4-HSL)17 were used throughout the study. Stock cultures for daily use were maintained on Luria broth (LB) plates at 4°C following overnight incubation (30°C), replaced at weekly intervals.
Luria-Bertani broth (LB broth; Difco) was used for all experiments. Growth curves, used to determine the minimal inhibitory concentrations (MICs) were produced in quarter-strength LB broth to ensure minimal interactions between broth components and biocides. This also allowed for growth to be limited through C/N availability rather than oxygen.
Biocides
Stock solutions of the following biocides were prepared in sterile distilled water, filter sterilized and stored either at 4°C or frozen: chlorhexidine diacetate hydrate 98% (Aldrich Chemical Co, Gillingham, UK), cetrimide USP, bronopol (both Sigma Chemical Co.), MIT (N-methyl isothiazolone; Rohm and Haas, Philadelphia, USA), CMIT (4-chloro, N-methyl isothiazolone; Rohm and Haas), and BIT (benzisothiazolone; ICI plc Organics Division, Manchester UK).
MIC determination
Mid-log phase cultures were prepared by inoculating 1 mL of an overnight culture into fresh LB broth (50 mL) in a 250 mL Erlenmeyer flask. The flask was incubated (30°C, 200 osc/min) in a shaking incubator until the culture reached an optical density (470 nm) of 0.6. After a one in two dilution, aliquots (100 µL) were added to equal volumes of biocide solution in 98-well microtitre plates. This gave a concentration of 5 x 106 cfu/mL in quarter strength LB broth. Biocide concentrations were chosen on an incremental scale, between the endpoints of a doubling dilution MIC. Growth was monitored over 24 h at 30°C using an Anthos Microtitre Plate Reader (Model HTIII, Labtec Instruments, Austria). Optical density readings (492 nm) were taken every 30 min following 60 s of high shaking. Appropriate controls were used and four replicate wells selected for each combination of biocide and microorganism.
MIC values were determined by relating mid-log phase growth rate to biocide concentration and extrapolating to give the concentration required for 100% growth inhibition. In this manner, a more precise MIC value was determined than is possible by the more traditional tube-dilution endpoint method.
Biocide susceptibility of planktonic cultures
Aliquots (200 µL) of 24 h liquid culture, grown in quarter strength LB broth, were transferred to sterile Eppendorf tubes and centrifuged in a benchtop Microfuge (13 000 r.p.m., 10 min). The supernatants were discarded and cell pellets resuspended in pre-warmed biocide (200 µL, 37°C, 10 min) before transfer to chilled sterile neutralizer (900 µL, 20 min). Most-probable-number (MPN) counts were carried out with LB broth (see below).
Biocide susceptibility of biofilm populations
Poloxamer F127 hydrogels are di-block co-polymers of polyoxyethylene and polyoxypropylene that demonstrate thermo-reversible gelation properties. At temperatures below 15°C they are liquid and fully miscible with water but firm gels at temperatures in excess of 15°C. They allow for high cell densities, equivalent to the biofilm mode of growth, to be cultured within the gels at 30°C and subsequently exposed to a biocide.18 Accordingly, flakes of poloxamer (40%) were added to quarter strength LB broth and refrigerated (4°C) overnight for hydration to occur. The dissolved poloxamer solutions were then autoclaved and returned to the refrigerator until required. Before use, sterile, chilled poloxamer (3 mL) was inoculated with 1:100 dilution of an overnight culture (300 µL) in fresh quarter strength LB broth to give 104105 cfu/mL. This was mixed well and drops (200 µL) of the poloxamer gel carefully placed onto sterile glass supports and incubated for 24 h (static incubator, 30°C) in a fully hydrated chamber. Following incubation, the glass supports (in triplicate) were transferred to pre-warmed biocide (37°C, 10 min) before removing to an appropriate chilled neutralizer solution (1800 µL, 20 min). The poloxamer hydrogel was dispersed in the chilled solution, thereby releasing the cells, after which an MPN count was carried out.
Biocide susceptibility of component biofilm cells
Following incubation (24 h), the poloxamer hydrogel constructs were transferred together with their glass supports to chilled sterile saline (5 min). The dispersed hydrogel solution was then transferred to a sterile Eppendorf tube and treated as per the planktonic cells above.
Neutralization of biocides
Chlorhexidine diacetate and cetrimide USP were neutralized using a lecithin (1% w/v: Sigma)Tween 80 (2% w/v; BDH, Poole, UK) solution, whereas bronopol was neutralized using sodium thioglycolate (Sigma) at an equimolar concentration to the highest bronopol concentration used.19 The antimicrobial effects of the other biocides were arrested by dilution (>1:100).
MPN count
MPNs were carried out using the wells (98) of sterile microtitre plates. Five replicates were used for each sample with 1:10 serial dilutions in quarter strength LB broth. Growth of the microorganism in the broth caused a visible change in turbidity after incubation (48 h) permitting a well to be scored either positive or negative for growth. The first serial dilution of the test sample was into the neutralizer solution and from this the serial dilutions were continued in the LB broth solution. Since the neutralizer was not a specific growth substrate for the microorganism, the presence or absence of growth could not be monitored with a turbidity change. Instead, five replicate droplets (30 µL) were placed onto a nutrient agar plate divided into five sections, with any colony formation in each section scored as positive growth. On the basis of probability theory it is possible to calculate, from the numbers of positive and negative samples taken from each microtitre plate well receiving a certain amount of inoculum, the most probable number of microorganisms in that quantity of inoculum. The MPN is then obtained by multiplying the result by the appropriate dilution factor. A table of MPN values using five tubes per dilution at a dilution ratio of 1:10 is provided by Alexander.20 Each experiment was carried out in duplicate and repeated on a separate occasion to give quadruplicate test samples for statistical analysis.
Statistical analysis
A Students t-test and one-way ANOVA were carried out using Microsoft Excel to evaluate statistical significance.
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Results and discussion |
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Initially, MIC values were determined for P. aeruginosa PA01, PA0-R1 and PAN067 to a range of biocides when grown as planktonic cultures (Table 1). Unexpectedly, the MICs of the HSL-deficient strains were not comparable to those of the wild-type for all biocides. The MICs were similar for chlorhexidine diacetate and bronopol but markedly different for the isothiazolones. Interestingly, no significant difference was demonstrated between the wild-type and P. aeruginosa PAN067 for cetrimide USP, but was observed with strain PA0-R1. P. aeruginosa PA0-R1 was generally the least susceptible strain. As shown in Figure 1 it possessed a similar susceptibility to the wild-type strain for the membrane interactive biocides, chlorhexidine diacetate and cetrimide USP, but was significantly decreased for the isothiazolones. This was particularly apparent for CMIT, where PA0-R1 was over 150% less sensitive to the biocide compared to the wild-type, P. aeruginosa PA01. P. aeruginosa PAN067 was significantly more susceptible to all biocides except chlorhexidine diacetate and bronopol.
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The susceptibilities of total biofilms, component cells dispersed from biofilms and planktonic equivalents to chlorhexidine diacetate (x5 MIC), cetrimide USP (x1.5 MIC) and bronopol (x250 MIC) were assessed. The biocide concentrations used were based on their ability to give sufficient kill in 24 h cultures of the wild-type strain. Results (Figure 2) are expressed as the log percentage survival relative to controls that had been exposed to distilled water.
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Similar observations were made with cetrimide USP (Figure 2b). Biofilms were more resistant to cetrimide than either the planktonic cells or the component biofilm cells. There was, however, no significant difference in response amongst the four strains tested. The component biofilm cells of PA0-JP1 and PA0-JP2 were very susceptible to cetrimide exposure compared to either the wild-type or PA0-R1. There was also variation between the strains in response when grown as planktonic cells that were unrelated to the response of the biofilm-grown cells. For both chlorhexidine diacetate and cetrimide USP, the response of the biofilms was similar, providing almost total protection with no significant difference in susceptibility between the wild-type and the mutant strains. This was in contrast to the planktonic cells and component biofilm cells that were significantly more susceptible to the biocides than the complete biofilm. This could either suggest that HSL deletion does not affect susceptibility to these two biocides or that the biofilm matrix acted as a reaction-diffusion limitation of penetration of these agents.4,5 It is unlikely, therefore, that HSLs play a role in the response of P. aeruginosa biofilms to these membrane active biocides.
With bronopol, significant differences in response were observed between the planktonic cells and the component biofilm cells that were not reflected in the complete biofilm (Figure 2c). For biofilm populations, there was no significant difference in susceptibility between the wild-type or the C12-HSL mutants, whereas PA0-JP2 was most susceptible. Strain PA0-JP1 was generally the least susceptible organism for each mode of cell preparation. These differences may suggest a role for HSLs in resistance to bronopol.
Bronopol possesses two different action mechanisms.22 Under aerobic conditions, bronopol catalytically oxidizes thiol-containing materials to produce reactive free oxygen radicals which are directly responsible for the bactericidal activity. Catalytic oxidation of thiols in the presence of excess thiols leads to the creation of an anoxic state. Under such conditions, a slower anoxic reaction with thiols consumes the bronopol and slows growth. Consumption of bronopol by the anoxic route leads to the eventual removal of bronopol from treated suspensions and the resumption of growth.22 The results presented therefore suggest that the redox state and hence the availability of glutathione (GSH) and glutathione disulphide (GSSG) are affected by the HSL signals, the knockout mutants experiencing an oxygen stress which is exacerbated by growth as a biofilm and which is retained by cells dispersed from the biofilm. The consumptive reaction dominates and results in increased levels of survival.
In P. aeruginosa, the las system is able to regulate the rhl system.23 Since the main HSL of the las system, C12-HSL, is missing in PA0-JP1, this may result in a loss of the hierarchical control of the rhl system. Under such circumstances, it is possible that the rhl system will become up-regulated, leading to the expression of genes under its control. One of these genes is rpoS, a general stress response regulator that controls the expression of genes that are known to confer resistance to a variety of stresses, including antibiotic susceptibility, in some Gram-negative bacteria.24 Thus, enhanced expression of rpoS may account for the reduced susceptibility of PA0-JP1 to the electrophilic biocide, bronopol, through possibly increasing the amount of reductive thiols available for interaction.
Other antimicrobial agents are known to act in this manner, i.e. through a reactive consumption route, notably the organomercurials and isothiazolones. We are not aware, however, of any antibiotics that are primarily thiol-interactive.
The studies reported here have demonstrated that whilst intact biofilms were generally less susceptible to the biocides tested than their planktonic counterparts, this was not necessarily related to HSL deficiency. Furthermore, biofilm component cells were generally more susceptible than the intact biofilm, a feature observed previously25 and suggestive of a community-specific resistance phenomenon. Significant differences were however observed between the relative susceptibilities of the HSL mutant strains and the wild-type P. aeruginosa when grown planktonically. Such differences were not reflected in the biofilm.
In Gram-negative bacteria, HSLs are known to be associated with aspects of biofilm formation, including biofilm differentiation, maintenance and detachment.10,26,27 Indeed, biofilm-associated disease states such as P. aeruginosa and Burkholderia cepacia infections of the cystic fibrotic lung have been well characterized with respect to the production and involvement of HSLs.28,29 A major problem arising from such infections is the high intrinsic resistance against antibiotics and biocides impeding effective medical treatment. In this respect, HSLs present a very attractive proposition as novel therapeutic targets. It is clear from this study and others that HSLs affect more than just biofilm formation, affecting a whole panoply of cell physiologies both within and between different Gram-negative species. As such, the extent to which HSLs can be used as novel therapeutic targets or the extent to which HSL agonists such as the furanones30 might potentiate antibiotic susceptibility is still open to debate.
Regardless, caution should be exerted when attempting to relate planktonic susceptibility to that of a biofilm. Moreover, our study indicates that there is no definite trend between HSL deficiency and biocide susceptibility.
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Acknowledgements |
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Footnotes |
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References |
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