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An investigation into the antimicrobial mechanisms of action of two contact lens biocides using electron microscopy
Contact Lens & Anterior Eye 28 (2005) 163–168 www.elsevier.com/locate/clae
An investigation into the antimicrobial mechanisms of action of two contact lens biocides using electron microscopy Caroline E. Codling a,1, Anthony C. Hann b, Jean-Yves Maillard a,*, A. Denver Russell a a
Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, UK b Cardiff School of Biosciences, Cardiff University, Cardiff, UK
Abstract Polyquaternium-1 (PQ-1) and myristamidopropyl dimethylamine (MAPD) are biocides used commercially in a contact lens disinfecting solutions. Electron microscopy was used to provide further evidence on the mechanism(s) of action of these agents against a wide range of ocular pathogens including bacteria, fungi and protozoa. Both PQ-1 and MAPD caused multiple forms of damage to the organisms tested, evidenced by structural alterations, blebbing, leakage and cell destruction. The extent of damage and the selectivity against specific type of microorganisms was consistent with the antimicrobial activity of these agents. Although electron microscopy is a powerful tool, it has its limitations when used to examine the mode of action of biocides. Indeed, there was no evidence of gross structural alteration to Acanthamoeba castellani or Aspergillus fumigatus following treatment. # 2005 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved. Keywords: Electron microscopy; Polyquaternium-1; Myristamidopropyl dimethylamine; Contact lens disinfection; Antimicrobial action
1. Introduction Contact lenses are very widely used throughout the world. However, if they are not cleaned and disinfected properly they are susceptible to contamination with microorganisms. These contaminants can be transferred onto the eye when the lenses are worn, potentially resulting in an infection. To prevent contamination of the lenses, disinfecting solutions are used for their cleaning and storage. Opti-Free1 Express1 (Alcon) is a multi-purpose contact lens disinfecting solution, which contains the two biocides polyquaternium-1 (PQ-1, a polymeric quaternary ammonium compound) and myristamidopropyl dimethylamine (MAPD, an amidoamine). It has been found previously that PQ-1 has mainly antibacterial activity, while MAPD has predominantly antifungal and antiprotozoal activity [1,2]. However, there is a paucity of information on the
mechanisms by which they inactivate these organisms. Electron microscopy studies have been used successfully in the past to examine structural damage to cells caused by antimicrobial agents such as chlorhexidine, polyhexamethylene biguanide, tea tree oil and other biocides [3–6]. It is a valuable tool for identifying damage to the cell membranes and cell contents, as well as observing changes in cellular morphology, which could have been caused by exposure to a biocide. In this study, both scanning and transmission electron microscopy (SEM and TEM) were used to identify damage to microorganisms following exposure to PQ-1 and MAPD, providing further information on their mechanism(s) of action.
2. Methods 2.1. Test organisms
* Corresponding author. Tel.: +44 2920 879088; fax: +44 2920 879149. E-mail address: [email protected] (J.-Y. Maillard). 1 Present address: Alimentary Pharmabiotic Centre, Microbiology Department, University College Cork, Cork, Ireland.
These consisted of Acanthamoeba castellanii (Neff strain) trophozoites and cysts, Aspergillus fumigatus (ATCC 10894), Candida albicans (ATCC 10231), Pseudomonas
1367-0484/$ – see front matter # 2005 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.clae.2005.08.002
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aeruginosa (ATCC 15442), Serratia marcescens (ATCC 13880) and Staphylococcus aureus (ATCC 6538). 2.2. Chemicals and culture media Tryptone soya broth (TSB), Sabouraud liquid medium (SLM) and potato dextrose agar (PDA) were purchased from Oxoid (Basingstoke, UK). Peptone–yeast–glucose broth (PYG) included 0.75% proteose peptone, 0.75% yeast extract (Oxoid) and 1.5% glucose (Sigma, Poole, UK). PBST consisted of phosphate buffered saline and 0.05% Tween 80 (Sigma). Disinfecting solution vehicle (DSV; buffered saline), PQ-1 and MAPD were supplied by Alcon Research Ltd. (Texas, USA). All chemicals for electron microscopy sample preparation were purchased from Agar Scientific (Stanstead, UK). 2.3. Exposure of organisms to biocides Bacteria were grown overnight at 37 8C in TSB and C. albicans was grown overnight in SLM at 37 8C. A. fumigatus was grown on PDA slopes for 3–5 days at 30 8C and the spores were harvested in PBST. A. castellanii was grown in 50 mL PYG at 30 8C for 3 days to produce trophozoites [7]. All organisms were washed in DSV and adjusted to approximately 1010 cfu mL 1 (or 108 cells mL 1 for A. castellanii). Two concentrations of PQ-1 (within the range 0–1000 mg mL 1 depending upon the microorganisms tested) and MAPD (within the range 0–250 mg mL 1 depending upon the microorganisms tested) were prepared fresh in 1.8 mL of DSV, to which 0.2 mL of cells were added. The concentrations were chosen so that one was lower than the MIC, and one was higher. These concentrations were higher than those used in Opti-Free Express, which are lower than the MICs for all of the microorganisms tested. However, these concentrations were the most relevant for the contact times tested. Bacteria were exposed to the biocides for 5 min at 25 8C, C. albicans and A. fumigatus for 30 min, and A. castellanii for 3 h. These exposure times were used in a previous study [1] and are similar to those used in the ISO 14729 standard for contact lens disinfectant testing, with the exception of A. castellanii [1,8]. 2.4. Preparation of samples for electron microscopy Different fixation protocols were required for the different microorganisms due to intrinsic physiological variations, for example in their cell wall properties. Untreated cells and cells exposed to PQ-1 and MAPD were processed for electron microscopy according to the following methods which were based on existing protocols [4,5,9]: (1) Bacterial samples: Cells were fixed for 1 h in 2% (v/v) glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.4), washed thoroughly in the same buffer, post-fixed
for 1 h in 1% (w/v) osmium tetroxide and dehydrated in a graded series of alcohol concentrations (50, 70, 80, 90, 95%, and four cycles of 100% ethanol for 10 min each). (2) Fungal samples: Cells were fixed overnight in a mixture of aldehydes (2%, v/v, glutaraldehyde and 8%, v/v paraformaldehyde in 0.1 M cacodylate buffer containing 1% sucrose and 6 mM CaCl2, pH 7.4), washed with the same buffer, post-fixed for 2 h in 1%, w/v, osmium tetroxide, block stained for 45 min with 0.5%, w/v, uranyl acetate and gradually dehydrated in a series of alcohol concentrations (as above). (3) A. castellanii samples: Cells were fixed for 1 h in 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer containing 1% sucrose and 6 mM CaCl2, washed in the same buffer, post-fixed in 1% osmium tetroxide, block stained in 0.5% (w/v) uranyl acetate and dehydrated (as above). For scanning electron microscopy, the dehydrated samples were critical point-dried and then coated with gold before viewing using a Philips XL20 scanning electron microscope operated at an accelerating voltage of 25–30 kV. For transmission electron microscopy, the dehydrated samples were embedded in resin, thin-sectioned and counterstained with lead citrate before examination using a Philips TEM 208 transmission electron microscope operated at an accelerating voltage of 80 kV.
3. Results All electron micrographs are representative of at least three fields of observation. Control cells of S. marcescens are shown in Fig. 1A. PQ-1 produced disruption of S. marcescens cell membrane as exemplified by Fig. 1B. There are large portions of the membrane, which are severely damaged and in some cases leakage of intracellular materials was observed. In addition, cytoplasmic coagulation was observed, evidenced by dark areas in the cells (Fig. 1B). In the SEM picture (Fig. 1D), blebs are clearly visible on the outer membrane, confirming membrane damage. MAPD also induced severe membrane damage as indicated by the general appearance of the cell membrane and by leakage of cytoplasmic contents (Fig. 1C). Similar results were obtained with P. aeruginosa. PQ-1 produced severe membrane damage (Fig. 2B) when compared to untreated cells (Fig. 2A). Cytoplasmic coagulation was also observed (Fig. 2B). MAPD also induced membrane damage (Fig. 2C) exemplified by abundant blebbing (Fig. 2D), which probably resulted in the formation of empty vesicles as observed in Fig. 2C. Cytoplasmic coagulation was also clearly visible (Fig. 2C). PQ-1 also produced membrane damage in S. aureus (Fig. 3B), although gross alteration was less evident with the Gram-positive microorganism when compared to the untreated cell (Fig. 3A). Nevertheless, evidence of blebbing
C.E. Codling et al. / Contact Lens & Anterior Eye 28 (2005) 163–168
Fig. 1. S. marcescens: (A) control, untreated (TEM); (B) PQ-1 50 mg mL 1 (TEM); (C) MAPD 100 mg mL 1 (TEM); (D) PQ-1 200 mg mL bar = 300 nm. Key to arrows: B, blebbing; CC, cytoplasmic coagulation; L, leakage of material; MD, membrane damage.
was observed (Fig. 3B). When treated with MAPD there was also abundant blebbing as observed by SEM (Fig. 3D) but not by TEM (Fig. 3C). However, the presence of ghost cells suggested that leakage of the cell cytoplasmic contents occurred as a result of gross cell alterations (Fig. 3C).
165
1
(SEM); (A–C)
Untreated C. albicans cells showed a very well defined structure (Fig. 4A). Exposure to MAPD produced an interesting alteration as it appeared to ‘‘dissolve’’ the plasma membrane, leaving a space between the cell wall and cytoplasm (Fig. 4B). However, the SEM picture did not
Fig. 2. P. aeruginosa: (A) control, untreated (TEM); (B) PQ-1 25 mg mL 1 (TEM); (C) MAPD 50 mg mL 1 (TEM); (D) MAPD 100 mg mL 1 (SEM); (A–C) bar = 300 nm. Key to arrows: B, blebbing; CC, cytoplasmic coagulation; L, leakage of material; MD, membrane damage; V, empty vesicles.
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Fig. 3. S. aureus: (A) control, untreated (TEM); (B) PQ-1 5 mg mL 1 (TEM); (C) MAPD 25 mg mL 1 (TEM); (D) MAPD 50 mg mL bar = 300 nm. Key to arrows: B, blebbing; GC, ghost cell; L, leakage of material; MD, membrane damage.
show any structural damage (Fig. 4D). PQ-1 did not produce any visible alteration to the yeast (Fig. 4C). Neither biocide appeared to have any structural effect on A. fumigatus (data not shown).
Fig. 4. C. albicans: (A) control, untreated (TEM); (B) MAPD 10 mg mL bar = 1 mm. Key to arrows: PM, plasma membrane not visible.
1
1
(SEM); (A–C)
Finally, there was some evidence of precipitation of cell contents around the cell membrane and nuclear membrane in A. castellanii trophozoites treated with PQ-1 (Fig. 5C and D) when compared to untreated cells (Fig. 5A and B). MAPD
(TEM); (C) PQ-1 1000 mg mL
1
(TEM); (D) MAPD 10 mg mL
1
(SEM); (A–C)
C.E. Codling et al. / Contact Lens & Anterior Eye 28 (2005) 163–168
Fig. 5. A. castellanii trophozoites: (A) control, untreated (SEM); (B) control, untreated (TEM); (C) PQ-1 40 mg mL (A–D) bar = 5 mm. Key to arrows: PP, precipitation.
did not induce any morphological alteration of the trophozoites (data not shown).
4. Discussion These results support previous findings that PQ-1 and MAPD have different spectra of antimicrobial activity [1]. In this study, it was found that PQ-1 caused damage to the bacterial cytoplasmic membrane, with evidence of leakage of cytoplasmic contents through the ruptured membrane. Previous studies of the leakage of K+ ions from PQ-1 treated
167
1
(SEM); (D) PQ-1 500 mg mL
1
(TEM);
bacteria also suggested that the cytoplasmic membrane was damaged by this biocide [1]. PQ-1 is a quaternary ammonium compound and these biocides are known to act on the bacterial cytoplasmic membrane and to cause coagulation of the cytoplasm [10,11]. This was clearly exemplified in Figs. 1B and 2B and to some extent in Fig. 3B. Membrane damage in the form of blebs on the cell surface was also observed in Figs. 1D and 3B. These results confirm that PQ-1 interacts primarily at the bacterial cytoplasmic membrane level. These observations also supported our finding that several genes associated with the cell membrane are important in the action of PQ-1 against S. marcescens [12]. However, PQ-1 produced
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no visible effects on A. fumigatus or C. albicans. This was expected since these organisms are less susceptible to the quaternary ammonium compound (QAC) [1]. It was interesting to note that A. castellanii suffered some precipitation of cell content after treatment with PQ-1 (Fig. 5D). This was unexpected since the QAC is not necessarily active against protozoa [1,2]. MAPD also induced damage to the bacterial cytoplasmic membrane. This was exemplified by the formation of blebs on the cell surface (Figs. 1C, 2D and 3D), and the leakage of cellular material through broken areas of the membrane (Figs. 1C and 2C), or by the appearance of ghost cells (Fig. 3C). However, this is in contrast with previous results, which indicated that MAPD did not induce potassium leakage from bacteria [1]. A possible explanation is that the formation of blebs is an indicator of gross membrane damage but not necessarily associated with leakage of cytoplasmic material. In addition, the number of ghost cells resulting from exposure to MAPD might be too few to provide a reading of potassium leakage as measured with atomic absorption spectrophotometry. Indeed, this methodology involves the use of a very high number of cells (i.e. 1 mg dry weight of cells mL 1). Fig. 4B suggested that MAPD severely damages the plasma membrane of C. albicans, to the extent of dissolving it. This concurred with previous potassium leakage results, which suggested that MAPD damaged the plasma membrane and induced leakage from the cells [1]. There was no visible effect on A. fumigatus even though it is sensitive to MAPD and previous results have shown that this biocide induced some potassium leakage, suggesting damage to the plasma membrane [1]. A. castellanii did not show any visible damage after treatment with MAPD (data not shown). MAPD was shown to be very active against this microorganism although no potassium leakage was observed [1,13]. Our result confirmed that the lethal action of MAPD against A. castellanii trophozoites is not through gross structural alterations. Overall, there was a good correlation between the gross structural damage caused by PQ-1 and MAPD and the observed lethality of these agents as measured in a suspension test [1]. Although electron microscopy provides useful information on the mechanisms of action of biocides, it has its limitation and therefore it must be used alongside other methods to determine the antimicrobial mechanisms of action of biocides. For example, no alteration to protozoa was observed as a result of MAPD exposure. Likewise, no damage was observed to A. fumigatus although these biocides are fungicidal. Nevertheless, through this study, a
better understanding of the interaction of contact lens biocides with ocular pathogens was obtained.
Acknowledgements We would like to thank Alcon Research Ltd (Fort Worth, Texas, USA) for a research studentship (to C.E.C.). The authors would also like to acknowledge the contribution of Prof. A. Denver Russell who sadly passed away in September 2004.
References [1] Codling CE, Maillard J-Y, Russell AD. Aspects of the antimicrobial mechanisms of action of a polyquaternium and an amidoamine. J Antimicrob Chemother 2003;51:1153–8. [2] Rosenthal RA, McAnally CL, McNamee LS, Buck SL, Schlitzer RL, Stone RP. Broad spectrum antimicrobial activity of a new multipurpose disinfecting solution. CLAO J 2000;26:120–6. [3] Maillard J-Y, Hann AC, Beggs TS, Day MJ, Hudson RA, Russell AD. Electronmicroscopic investigation of the effects of biocides on Pseudomonas aeruginosa PAO bacteriophage F116. J Med Microbiol 1995;42:415–20. [4] Khunkitti W, Hann AC, Lloyd D, Furr JR, Russell AD. Biguanideinduced changes in Acanthamoeba castellanii: an electron microscopic study. J Appl Microbiol 1998;84:53–62. [5] Tattawasart U, Hann AC, Maillard J-Y, Furr JR, Russell AD. Cytological changes in chlorhexidine-resistant isolates of Pseudomonas stutzeri. J Antimicrob Chemother 2000;45:145–52. [6] Carson CF, Mee BJ, Riley TV. Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy. Antimicrob Agents Chemother 2002;46:1914–20. [7] Khunkitti W, Lloyd D, Furr JR, Russell AD. Acanthamoeba castellanii: growth, encystment, excystment and biocide susceptibility. J Infect 1998;36:43–8. [8] ISO 14729:2001. Ophthalmic optics – contact lens care products – Microbiological requirements and test methods for products and regimens for hygienic management of contact lenses. Geneva: ISO, 2001. [9] Thomas DG, Hann AC, Day MJ, Wilson JM, Russell AD. Structural changes induced by mupirocin in Staphylococcus aureus cells. Int J Antimicrobiol Ag 1999;13:9–14. [10] McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev 1999;12:147–79. [11] Maillard J-Y. Bacterial target sites for biocide action. J Appl Microbiol 2002;92:16–27. [12] Codling CE, Jones BV, Mahenthiralingam E, Russell AD, Maillard JY. Identification of genes involved in the resistance of Serratia marcescens to polyquaternium-1. J Antimicrob Chemother 2004;54:370–5. [13] Hughes R, Kilvington S. Comparison of hydrogen peroxide contact lens disinfection systems and solutions against Acanthamoeba polyphaga. Antimicrob Agents Chemother 2001;45:2038–43.
An investigation into the antimicrobial mechanisms of action of two contact lens biocides using electron microscopy Caroline E. Codling a,1, Anthony C. Hann b, Jean-Yves Maillard a,*, A. Denver Russell a a
Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, UK b Cardiff School of Biosciences, Cardiff University, Cardiff, UK
Abstract Polyquaternium-1 (PQ-1) and myristamidopropyl dimethylamine (MAPD) are biocides used commercially in a contact lens disinfecting solutions. Electron microscopy was used to provide further evidence on the mechanism(s) of action of these agents against a wide range of ocular pathogens including bacteria, fungi and protozoa. Both PQ-1 and MAPD caused multiple forms of damage to the organisms tested, evidenced by structural alterations, blebbing, leakage and cell destruction. The extent of damage and the selectivity against specific type of microorganisms was consistent with the antimicrobial activity of these agents. Although electron microscopy is a powerful tool, it has its limitations when used to examine the mode of action of biocides. Indeed, there was no evidence of gross structural alteration to Acanthamoeba castellani or Aspergillus fumigatus following treatment. # 2005 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved. Keywords: Electron microscopy; Polyquaternium-1; Myristamidopropyl dimethylamine; Contact lens disinfection; Antimicrobial action
1. Introduction Contact lenses are very widely used throughout the world. However, if they are not cleaned and disinfected properly they are susceptible to contamination with microorganisms. These contaminants can be transferred onto the eye when the lenses are worn, potentially resulting in an infection. To prevent contamination of the lenses, disinfecting solutions are used for their cleaning and storage. Opti-Free1 Express1 (Alcon) is a multi-purpose contact lens disinfecting solution, which contains the two biocides polyquaternium-1 (PQ-1, a polymeric quaternary ammonium compound) and myristamidopropyl dimethylamine (MAPD, an amidoamine). It has been found previously that PQ-1 has mainly antibacterial activity, while MAPD has predominantly antifungal and antiprotozoal activity [1,2]. However, there is a paucity of information on the
mechanisms by which they inactivate these organisms. Electron microscopy studies have been used successfully in the past to examine structural damage to cells caused by antimicrobial agents such as chlorhexidine, polyhexamethylene biguanide, tea tree oil and other biocides [3–6]. It is a valuable tool for identifying damage to the cell membranes and cell contents, as well as observing changes in cellular morphology, which could have been caused by exposure to a biocide. In this study, both scanning and transmission electron microscopy (SEM and TEM) were used to identify damage to microorganisms following exposure to PQ-1 and MAPD, providing further information on their mechanism(s) of action.
2. Methods 2.1. Test organisms
* Corresponding author. Tel.: +44 2920 879088; fax: +44 2920 879149. E-mail address: [email protected] (J.-Y. Maillard). 1 Present address: Alimentary Pharmabiotic Centre, Microbiology Department, University College Cork, Cork, Ireland.
These consisted of Acanthamoeba castellanii (Neff strain) trophozoites and cysts, Aspergillus fumigatus (ATCC 10894), Candida albicans (ATCC 10231), Pseudomonas
1367-0484/$ – see front matter # 2005 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.clae.2005.08.002
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aeruginosa (ATCC 15442), Serratia marcescens (ATCC 13880) and Staphylococcus aureus (ATCC 6538). 2.2. Chemicals and culture media Tryptone soya broth (TSB), Sabouraud liquid medium (SLM) and potato dextrose agar (PDA) were purchased from Oxoid (Basingstoke, UK). Peptone–yeast–glucose broth (PYG) included 0.75% proteose peptone, 0.75% yeast extract (Oxoid) and 1.5% glucose (Sigma, Poole, UK). PBST consisted of phosphate buffered saline and 0.05% Tween 80 (Sigma). Disinfecting solution vehicle (DSV; buffered saline), PQ-1 and MAPD were supplied by Alcon Research Ltd. (Texas, USA). All chemicals for electron microscopy sample preparation were purchased from Agar Scientific (Stanstead, UK). 2.3. Exposure of organisms to biocides Bacteria were grown overnight at 37 8C in TSB and C. albicans was grown overnight in SLM at 37 8C. A. fumigatus was grown on PDA slopes for 3–5 days at 30 8C and the spores were harvested in PBST. A. castellanii was grown in 50 mL PYG at 30 8C for 3 days to produce trophozoites [7]. All organisms were washed in DSV and adjusted to approximately 1010 cfu mL 1 (or 108 cells mL 1 for A. castellanii). Two concentrations of PQ-1 (within the range 0–1000 mg mL 1 depending upon the microorganisms tested) and MAPD (within the range 0–250 mg mL 1 depending upon the microorganisms tested) were prepared fresh in 1.8 mL of DSV, to which 0.2 mL of cells were added. The concentrations were chosen so that one was lower than the MIC, and one was higher. These concentrations were higher than those used in Opti-Free Express, which are lower than the MICs for all of the microorganisms tested. However, these concentrations were the most relevant for the contact times tested. Bacteria were exposed to the biocides for 5 min at 25 8C, C. albicans and A. fumigatus for 30 min, and A. castellanii for 3 h. These exposure times were used in a previous study [1] and are similar to those used in the ISO 14729 standard for contact lens disinfectant testing, with the exception of A. castellanii [1,8]. 2.4. Preparation of samples for electron microscopy Different fixation protocols were required for the different microorganisms due to intrinsic physiological variations, for example in their cell wall properties. Untreated cells and cells exposed to PQ-1 and MAPD were processed for electron microscopy according to the following methods which were based on existing protocols [4,5,9]: (1) Bacterial samples: Cells were fixed for 1 h in 2% (v/v) glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.4), washed thoroughly in the same buffer, post-fixed
for 1 h in 1% (w/v) osmium tetroxide and dehydrated in a graded series of alcohol concentrations (50, 70, 80, 90, 95%, and four cycles of 100% ethanol for 10 min each). (2) Fungal samples: Cells were fixed overnight in a mixture of aldehydes (2%, v/v, glutaraldehyde and 8%, v/v paraformaldehyde in 0.1 M cacodylate buffer containing 1% sucrose and 6 mM CaCl2, pH 7.4), washed with the same buffer, post-fixed for 2 h in 1%, w/v, osmium tetroxide, block stained for 45 min with 0.5%, w/v, uranyl acetate and gradually dehydrated in a series of alcohol concentrations (as above). (3) A. castellanii samples: Cells were fixed for 1 h in 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer containing 1% sucrose and 6 mM CaCl2, washed in the same buffer, post-fixed in 1% osmium tetroxide, block stained in 0.5% (w/v) uranyl acetate and dehydrated (as above). For scanning electron microscopy, the dehydrated samples were critical point-dried and then coated with gold before viewing using a Philips XL20 scanning electron microscope operated at an accelerating voltage of 25–30 kV. For transmission electron microscopy, the dehydrated samples were embedded in resin, thin-sectioned and counterstained with lead citrate before examination using a Philips TEM 208 transmission electron microscope operated at an accelerating voltage of 80 kV.
3. Results All electron micrographs are representative of at least three fields of observation. Control cells of S. marcescens are shown in Fig. 1A. PQ-1 produced disruption of S. marcescens cell membrane as exemplified by Fig. 1B. There are large portions of the membrane, which are severely damaged and in some cases leakage of intracellular materials was observed. In addition, cytoplasmic coagulation was observed, evidenced by dark areas in the cells (Fig. 1B). In the SEM picture (Fig. 1D), blebs are clearly visible on the outer membrane, confirming membrane damage. MAPD also induced severe membrane damage as indicated by the general appearance of the cell membrane and by leakage of cytoplasmic contents (Fig. 1C). Similar results were obtained with P. aeruginosa. PQ-1 produced severe membrane damage (Fig. 2B) when compared to untreated cells (Fig. 2A). Cytoplasmic coagulation was also observed (Fig. 2B). MAPD also induced membrane damage (Fig. 2C) exemplified by abundant blebbing (Fig. 2D), which probably resulted in the formation of empty vesicles as observed in Fig. 2C. Cytoplasmic coagulation was also clearly visible (Fig. 2C). PQ-1 also produced membrane damage in S. aureus (Fig. 3B), although gross alteration was less evident with the Gram-positive microorganism when compared to the untreated cell (Fig. 3A). Nevertheless, evidence of blebbing
C.E. Codling et al. / Contact Lens & Anterior Eye 28 (2005) 163–168
Fig. 1. S. marcescens: (A) control, untreated (TEM); (B) PQ-1 50 mg mL 1 (TEM); (C) MAPD 100 mg mL 1 (TEM); (D) PQ-1 200 mg mL bar = 300 nm. Key to arrows: B, blebbing; CC, cytoplasmic coagulation; L, leakage of material; MD, membrane damage.
was observed (Fig. 3B). When treated with MAPD there was also abundant blebbing as observed by SEM (Fig. 3D) but not by TEM (Fig. 3C). However, the presence of ghost cells suggested that leakage of the cell cytoplasmic contents occurred as a result of gross cell alterations (Fig. 3C).
165
1
(SEM); (A–C)
Untreated C. albicans cells showed a very well defined structure (Fig. 4A). Exposure to MAPD produced an interesting alteration as it appeared to ‘‘dissolve’’ the plasma membrane, leaving a space between the cell wall and cytoplasm (Fig. 4B). However, the SEM picture did not
Fig. 2. P. aeruginosa: (A) control, untreated (TEM); (B) PQ-1 25 mg mL 1 (TEM); (C) MAPD 50 mg mL 1 (TEM); (D) MAPD 100 mg mL 1 (SEM); (A–C) bar = 300 nm. Key to arrows: B, blebbing; CC, cytoplasmic coagulation; L, leakage of material; MD, membrane damage; V, empty vesicles.
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C.E. Codling et al. / Contact Lens & Anterior Eye 28 (2005) 163–168
Fig. 3. S. aureus: (A) control, untreated (TEM); (B) PQ-1 5 mg mL 1 (TEM); (C) MAPD 25 mg mL 1 (TEM); (D) MAPD 50 mg mL bar = 300 nm. Key to arrows: B, blebbing; GC, ghost cell; L, leakage of material; MD, membrane damage.
show any structural damage (Fig. 4D). PQ-1 did not produce any visible alteration to the yeast (Fig. 4C). Neither biocide appeared to have any structural effect on A. fumigatus (data not shown).
Fig. 4. C. albicans: (A) control, untreated (TEM); (B) MAPD 10 mg mL bar = 1 mm. Key to arrows: PM, plasma membrane not visible.
1
1
(SEM); (A–C)
Finally, there was some evidence of precipitation of cell contents around the cell membrane and nuclear membrane in A. castellanii trophozoites treated with PQ-1 (Fig. 5C and D) when compared to untreated cells (Fig. 5A and B). MAPD
(TEM); (C) PQ-1 1000 mg mL
1
(TEM); (D) MAPD 10 mg mL
1
(SEM); (A–C)
C.E. Codling et al. / Contact Lens & Anterior Eye 28 (2005) 163–168
Fig. 5. A. castellanii trophozoites: (A) control, untreated (SEM); (B) control, untreated (TEM); (C) PQ-1 40 mg mL (A–D) bar = 5 mm. Key to arrows: PP, precipitation.
did not induce any morphological alteration of the trophozoites (data not shown).
4. Discussion These results support previous findings that PQ-1 and MAPD have different spectra of antimicrobial activity [1]. In this study, it was found that PQ-1 caused damage to the bacterial cytoplasmic membrane, with evidence of leakage of cytoplasmic contents through the ruptured membrane. Previous studies of the leakage of K+ ions from PQ-1 treated
167
1
(SEM); (D) PQ-1 500 mg mL
1
(TEM);
bacteria also suggested that the cytoplasmic membrane was damaged by this biocide [1]. PQ-1 is a quaternary ammonium compound and these biocides are known to act on the bacterial cytoplasmic membrane and to cause coagulation of the cytoplasm [10,11]. This was clearly exemplified in Figs. 1B and 2B and to some extent in Fig. 3B. Membrane damage in the form of blebs on the cell surface was also observed in Figs. 1D and 3B. These results confirm that PQ-1 interacts primarily at the bacterial cytoplasmic membrane level. These observations also supported our finding that several genes associated with the cell membrane are important in the action of PQ-1 against S. marcescens [12]. However, PQ-1 produced
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no visible effects on A. fumigatus or C. albicans. This was expected since these organisms are less susceptible to the quaternary ammonium compound (QAC) [1]. It was interesting to note that A. castellanii suffered some precipitation of cell content after treatment with PQ-1 (Fig. 5D). This was unexpected since the QAC is not necessarily active against protozoa [1,2]. MAPD also induced damage to the bacterial cytoplasmic membrane. This was exemplified by the formation of blebs on the cell surface (Figs. 1C, 2D and 3D), and the leakage of cellular material through broken areas of the membrane (Figs. 1C and 2C), or by the appearance of ghost cells (Fig. 3C). However, this is in contrast with previous results, which indicated that MAPD did not induce potassium leakage from bacteria [1]. A possible explanation is that the formation of blebs is an indicator of gross membrane damage but not necessarily associated with leakage of cytoplasmic material. In addition, the number of ghost cells resulting from exposure to MAPD might be too few to provide a reading of potassium leakage as measured with atomic absorption spectrophotometry. Indeed, this methodology involves the use of a very high number of cells (i.e. 1 mg dry weight of cells mL 1). Fig. 4B suggested that MAPD severely damages the plasma membrane of C. albicans, to the extent of dissolving it. This concurred with previous potassium leakage results, which suggested that MAPD damaged the plasma membrane and induced leakage from the cells [1]. There was no visible effect on A. fumigatus even though it is sensitive to MAPD and previous results have shown that this biocide induced some potassium leakage, suggesting damage to the plasma membrane [1]. A. castellanii did not show any visible damage after treatment with MAPD (data not shown). MAPD was shown to be very active against this microorganism although no potassium leakage was observed [1,13]. Our result confirmed that the lethal action of MAPD against A. castellanii trophozoites is not through gross structural alterations. Overall, there was a good correlation between the gross structural damage caused by PQ-1 and MAPD and the observed lethality of these agents as measured in a suspension test [1]. Although electron microscopy provides useful information on the mechanisms of action of biocides, it has its limitation and therefore it must be used alongside other methods to determine the antimicrobial mechanisms of action of biocides. For example, no alteration to protozoa was observed as a result of MAPD exposure. Likewise, no damage was observed to A. fumigatus although these biocides are fungicidal. Nevertheless, through this study, a
better understanding of the interaction of contact lens biocides with ocular pathogens was obtained.
Acknowledgements We would like to thank Alcon Research Ltd (Fort Worth, Texas, USA) for a research studentship (to C.E.C.). The authors would also like to acknowledge the contribution of Prof. A. Denver Russell who sadly passed away in September 2004.
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