1 Welsh School of Pharmacy, Cardiff University, Cardiff; 2 School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK
Received 30 October 2002; returned 22 January 2003; revised 10 February 2003; accepted 25 February 2003
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Abstract |
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Methods: Three aspects were investigated: the lethal effects of the biocides on the organisms, the leakage of K+ from treated cells, and the lysis of spheroplasts derived from the cells.
Results: PQ-1 was found to have predominantly antibacterial activity, and induced K+ leakage from the bacteria and C. albicans. It also caused lysis of spheroplasts of S. marcescens, but not those of C. albicans. MAPD was active against all of the organisms, but showed higher activity against the fungi and amoeba. It induced K+ leakage from A. fumigatus and C. albicans, and like PQ-1, lysed the spheroplasts of S. marcescens but not C. albicans.
Conclusions: The two biocides have different spectra of antimicrobial activity. PQ-1 has mainly antibacterial activity, whereas MAPD was active against all of the test organisms, particularly the fungi.
Keywords: biocides, potassium leakage, spheroplast lysis
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Introduction |
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Potential targets for PQ-1 are the bacterial cytoplasmic membrane and fungal plasma membrane, since these membranes are a common target for QACs.4 K+ leakage is an ideal indicator of damage to the membranes of both types of organism. When the membrane is damaged, these ions leak out of the cells very rapidly, and are easily detected by atomic absorption spectrophotometry. Another method to assess the membrane as a target for the biocides is spheroplast lysis. Spheroplasts treated with the biocides will lyse very rapidly if the membrane is attacked. This is seen as a decrease in the absorbance of the suspension, and visible effects can also be seen using phase-contrast microscopy.5
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Materials and methods |
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These consisted of Acanthamoeba castellanii (Neff strain) trophozoites and cysts, Aspergillus fumigatus (ATCC 10894), Candida albicans (ATCC 10231), Pseudomonas aeruginosa (ATCC 15442), Serratia marcescens (ATCC 13880) and Staphylococcus aureus (ATCC 6538).
Chemicals and culture media
Tryptone soya broth (TSB), tryptone soya agar (TSA), Sabouraud liquid medium (SLM), Sabouraud dextrose agar (SDA) and potato dextrose agar (PDA) were purchased from Oxoid (Basingstoke, UK). PBST consisted of phosphate-buffered saline and 0.05% Tween 80 (Sigma, Poole, UK). Peptone-yeastglucose broth (PYG) included 0.75% proteose peptone, 0.75% yeast extract (Oxoid) and 1.5% glucose (Sigma). The neutralizer consisted of 0.75% azolectin and 5% Tween 80 (Sigma). Disinfecting solution vehicle (DSV), PQ-1 and MAPD were supplied by Alcon Research Ltd. All other chemicals were purchased from Sigma.
Lethal effects
Bacteria were grown overnight at 37°C in 10 mL TSB, and C. albicans was grown overnight at 37°C in 10 mL SLM. A. fumigatus was grown on PDA slopes for 35 days at 30°C and the spores were harvested in PBST. A. castellanii was grown in 50 mL PYG at 30°C for 3 days (trophozoites) or 100 mL PYG with 50 mM MgCl2 for 10 days (cysts). All of the organisms were washed in DSV, and adjusted to approximately 108 cfu/mL (106 cells/mL for A. castellanii). Subsequently, 0.1 mL of cells was added to 0.9 mL PQ-1 or MAPD of the desired final concentration equilibrated at 20°C, mixed and held at 20°C for 5 min (bacteria), 30 min (C. albicans and A. fumigatus) or 3 h (A. castellanii). Then, 0.1 mL was removed and added to 0.9 mL neutralizer, mixed and left for 10 min, followed by serial dilution in 0.9 mL DSV. These were plated onto TSA (bacteria), SDA (C. albicans) and PDA (A. fumigatus). After incubation overnight (bacteria and C. albicans), for 48 h (A. fumigatus) or 710 days (A. castellanii) at 30 or 37°C depending on the organism, colonies were counted and log viability reductions calculated. A. castellanii was enumerated using the plaque assay method.6
Potassium leakage
Bacteria were grown overnight at 37°C on TSA plates, harvested by washing with polished water (Elgastat UHP filtered deionized water), and double washed in polished water. C. albicans was grown overnight on SDA at 37°C, and harvested and washed as for the bacteria. A. fumigatus was grown on PDA slopes for 35 days at 30°C, the spores were harvested and double washed in polished water. A. castellanii was grown in PYG at 30°C for 3 days (trophozoites) or PYG with 50 mM MgCl2 for 10 days (cysts), and the cells were then double washed in polished water. All of the organisms were adjusted to 2 mg dry weight of cells/mL in polished water. PQ-1 and MAPD solutions were prepared at double the required concentration so that the addition of an equal volume of cell suspension produced a final concentration of 1 mg dry weight of cells/mL. The organisms were exposed to PQ-1 and MAPD at 20°C and 5 mL samples were removed at intervals. These were filtered using 0.2 µm pore Minisart filters to remove the cells and the filtrates analysed for K+ content using atomic absorption spectrophotometry (Instrumentation Laboratory aa/ae spectrophotometer 457). The negative controls were cells exposed to polished water with no PQ-1 or MAPD, and positive controls were cells lysed by boiling in polished water for 20 min.
Preparation of spheroplasts
S. marcescens: an overnight TSB culture grown at 37°C was used to inoculate 50 mL TSB containing 0.3 M sucrose, 0.1 M MgSO4 and 100 µg/mL cefoxitin. After 4 h incubation at 37°C in a shaking water bath, a sample was checked microscopically for the presence of spheroplasts. The spheroplasts were then washed by centrifugation at 2000 rpm (IEC Centra MP4R) and concentrated five-fold in 0.5 M sucrose.
P. aeruginosa: a 4 h culture grown in TSB at 37°C was washed by centrifugation at 2000 rpm and concentrated 10-fold in water. This was then used to inoculate a flask containing 0.2 mg/mL EDTA, 0.033 M Tris (pH 7.4), 0.5 M sucrose and 10 µg/mL lysozyme. This was shaken at 20°C for 1 h and then checked microscopically for the presence of spheroplasts.
C. albicans: a 4 h culture grown in SLM at 37°C was washed and concentrated 10-fold in 1 M sorbitol. The cells were finally resuspended in a buffer containing 1.4 M sorbitol, 40 mM HEPES (pH 7.5), 0.5 mM MgCl2 and a trace of 2-mercaptoethanol. The suspension was shaken at 20°C for 15 min and then 5 mg/mL lyticase was added to lyse the cells. This was shaken for 45 min at 20°C and then a sample was checked microscopically for the presence of spheroplasts (adapted from Adams & Gross7).
S. aureus: various methods were tested for the preparation of spheroplasts, using lysostaphin, cefoxitin and penicillin.
In each case, the spheroplasts were exposed to PQ-1 and MAPD, and the absorbance (550 nm) was recorded at time intervals over 30 min. Samples were also checked microscopically for lysis of the spheroplasts. PQ-1 and MAPD were prepared at double the required concentration (5 mL volume), and 2 mL washed spheroplasts and 3 mL 1 M sucrose (S. marcescens) or 3 mL 2 M sorbitol (C. albicans) were added to achieve the desired final concentrations of PQ-1 and MAPD.
All of the experiments were carried out in triplicate and mean results given.
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Results |
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Large differences in the activity of PQ-1 and MAPD were observed against the various organisms (Figure 1). A. fumigatus and C. albicans were found to be resistant to PQ-1, but were quite susceptible to MAPD. The former was more active against the three bacterial species than against the other organisms. Of these, S. aureus was the most, and S. marcescens the least, susceptible. MAPD showed a broader spectrum of activity, and was active against all of the organisms. It had high activity against A. castellanii at very low concentrations, the two fungi at intermediate concentrations, and the bacteria at higher concentrations.
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As with the lethal effects results, there were clear differences in the amount of K+ released from the various organisms (Figure 2). K+ release peaked after 12 min and then usually remained level or decreased slightly. This indicated that there was very rapid damage to the membrane caused by the biocides. Figure 3 shows possible damage to the cytoplasmic membrane, the amount of K+ released increased with the concentration of biocide used. Figure 4 is an example of a situation where the biocide did not cause significant membrane damage. In this case the K+ level released remained roughly the same with each concentration of biocide used. MAPD showed possible membrane activity against S. aureus, C. albicans and A. fumigatus, and PQ-1 against P. aeruginosa, S. marcescens, S. aureus and C. albicans. Neither biocide induced K+ leakage from A. castellanii trophozoites or cysts (data not shown).
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S. marcescens: spheroplasts were stable in 0.3 M sucrose (control) since the absorbance remained level, whereas the water control showed very rapid lysis (Figure 5a and b). Within 1 min the absorbance had fallen to less than 0.2 from a starting point of 0.54, and then remained level. MAPD produced some lysis, although less than the water control. There was slightly more lysis at 10 µg/mL MAPD than at 5 µg/mL, but the lysis was not as rapid as with water, since the absorbance continued to decrease for 10 min before levelling. Phase-contrast microscopy substantiated these results (data not shown), showing lysis of the spheroplasts. PQ-1 caused less lysis than water, but the extent of the lysis did not seem to correlate with the concentrations tested. Phase-contrast microscopy showed damage to the spheroplasts produced by PQ-1, but seemed to be similar for all of the concentrations tested (data not shown).
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P. aeruginosa: the spheroplasts formed proved extremely difficult to wash since they formed a sticky, loose pellet, and no reliable data could be produced.
S. aureus: various methods were tested for the preparation of spheroplasts, but no reliable data could be produced.
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Discussion |
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The results show that there are large differences in the activity of PQ-1 and MAPD against the various organisms. This was expected because the former has predominantly antibacterial activity, whereas the latter has mainly antifungal and anti-amoebal activity.2 A. fumigatus and C. albicans were found to be resistant to PQ-1, which was more active on the three bacterial species, although S. marcescens was less susceptible than P. aeruginosa and S. aureus. MAPD had a broader spectrum of activity, and was active against all of the organisms, although the bacteria were the least susceptible. These results substantiate previous findings that PQ-1 is antibacterial and MAPD is antifungal and antiprotozoal.2 Because the mechanisms of action of PQ-1 and MAPD were being studied, suspension tests with planktonic cultures were undertaken. Sessile cells in the form of biofilms may be more prevalent in practice and the findings in this paper could therefore over-estimate the efficacy of the two biocides.
Potassium leakage
Potential targets for PQ-1 are the cytoplasmic membrane of the bacteria and the plasma membrane of the fungi, since these are common targets for QACs.3 K+ leakage is an ideal indicator of membrane damage as it leaks out of the cells very rapidly, and can be easily detected by atomic absorption spectrophotometry.8
It can be seen from the results that there are large differences between the organisms in the amount of K+ released after treatment. This is not due to different levels of damage caused by PQ-1 and MAPD, but probably mainly due to intrinsic differences between the organisms. For example, an amoeba has a very different physiology from a bacterial cell and it would thus be expected that the different types of organism contain different levels of potassium. This was confirmed in the positive controls (cells lysed by boiling, data not shown). Our results indicate that membranes are also potential target sites for PQ-1. MAPD induced possible plasma membrane damage to A. fumigatus and C. albicans. However, neither biocide induced potassium leakage from A. castellanii. This was probably due to the presence of a cell wall, especially in the cyst form of the organism, that is very resistant to damage.
Spheroplasts
Another method to confirm the bacterial cytoplasmic and yeast plasma membranes as targets for biocidal action is the lysis of spheroplasts.5,9 Spheroplasts are more sensitive than normal cells since the cell wall is incomplete, so when treated with the biocides they will lyse very rapidly if the membrane is attacked. This is seen as a decrease in the absorbance of the suspension, and also visible effects can be observed using phase-contrast microscopy.
S. marcescens: rapid lysis was seen after treatment with both PQ-1 and MAPD. However, there did not seem to be any difference in the appearance of spheroplasts treated with different concentrations of PQ-1. Also the absorbance results showed no correlation between extent of lysis and concentration. This suggests that PQ-1 does have a lytic effect on spheroplasts of S. marcescens, but it may be that only a very low concentration is required, or that it only has a limited effect and does not lyse the spheroplasts further even at very high concentrations.
C. albicans: neither PQ-1 nor MAPD induced lysis of the spheroplasts since all of the readings were similar to the sorbitol control. These results were substantiated by phase- contrast microscopy pictures, which showed that MAPD induced some lysis, but there were no visible effects caused by PQ-1 (data not shown).
P. aeruginosa and S. aureus: several problems were encountered with these organisms so no reliable data could be produced.
In conclusion, it was found that PQ-1 and MAPD have different spectra of antimicrobial activity. PQ-1 showed predominantly antibacterial activity, whereas MAPD was active against all of the organisms, but showed higher activity against the fungi and amoebae. PQ-1 induced possible cytoplasmic membrane damage to P. aeruginosa, S. marcescens and S. aureus, and plasma membrane damage to C. albicans. MAPD induced possible plasma membrane damage to A. fumigatus and C. albicans. Both PQ-1 and MAPD induced lysis of spheroplasts of S. marcescens, but not those of C. albicans. Damage to the membrane was sufficient to cause K+ leakage but this injury was not always sufficient to lyse spheroplasts.
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Acknowledgements |
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Footnotes |
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References |
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