Pseudomonas aeruginosa cells adapted to benzalkonium chloride show resistance to other membrane-active agents but not to clinically relevant antibiotics

M. F. Loughlina, M. V. Jonesb and P. A. Lamberta,*

a Microbiology Research Group, School of Life and Health Sciences, Aston University, Birmingham B4 7ET; b Unilever Research, Port Sunlight, Merseyside, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 Acknowledgements
 References
 
Our objective was to determine whether strains of Pseudomonas aeruginosa can adapt to growth in increasing concentrations of the disinfectant benzalkonium chloride (BKC), and whether co-resistance to clinically relevant antimicrobial agents occurs. Attempts were made to determine what phenotypic alterations accompanied resistance and whether these explained the mechanism of resistance. Strains were serially passaged in increasing concentrations of BKC in static nutrient broth cultures. Serotyping and genotyping were used to determine purity of the cultures. Two strains were examined for cross-resistance to other disinfectants and antibiotics by broth dilution MIC determination. Alterations in outer membrane proteins and lipopolysaccharide (LPS) expressed were examined by SDS–PAGE. Cell surface hydrophobicity and charge, uptake of disinfectant and proportion of specific fatty acid content of outer and cytoplasmic membranes were determined. Two P. aeruginosa strains showed a stable increase in resistance to BKC. Co-resistance to other quaternary ammonium compounds was observed in both strains; chloramphenicol and polymyxin B resistance were observed in one and a reduction in resistance to tobramycin observed in the other. However, no increased resistance to other biocides (chlorhexidine, triclosan, thymol) or antibiotics (ceftazidime, imipenem, ciprofloxacin, tobramycin) was detected. Characteristics accompanying resistance included alterations in outer membrane proteins, uptake of BKC, cell surface charge and hydrophobicity, and fatty acid content of the cytoplasmic membrane, although no evidence was found for alterations in LPS. Each of the two strains had different alterations in phenotype, indicating that such adaptation is unique to each strain of P. aeruginosa and does not result from a single mechanism shared by the whole species.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 Acknowledgements
 References
 
Pseudomonas aeruginosa is an important nosocomial pathogen due to its ubiquitous presence wherever there is water, the devastating effect upon patients of infection, usually as a result of an excessive immune response, and the organism's high resistance to both host defences and antibacterial agents in general. Resistance to host defences is key in infection of the cystic fibrosis lung, where the organism survives the massive immune response produced by the host, whereas the lung tissue fares far worse, leading eventually to complete respiratory failure and death.1

P. aeruginosa has long been regarded as an antibiotic-resistant organism, its low permeability outer membrane preventing access of many agents to their sites of action.2 More recently the presence of constitutive and enhanceable efflux mechanisms removing a huge range of antimicrobial agents from the cell is considered just as important a factor of resistance, especially if coupled with enzymic mechanisms of resistance.3–5 Resistance to disinfectants is similarly a long recorded aspect of this organism, it having been isolated in stock solutions of most commonly used biocidal agents.6 Adaptation of Pseudomonas species to disinfectants by serial passage in increasing concentrations of the biocide is also well documented,7,8 although in Gram-negative bacteria no clearly elucidated links with resistance to antibiotics have yet been observed.9

This study aimed to generate stable resistance to the disinfectant benzalkonium chloride (BKC) by serial passage and to determine whether co-resistance to clinically relevant antibiotics was produced. In addition, associated changes in the cell membrane and surface characteristics were examined to determine possible mechanisms of resistance.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 Acknowledgements
 References
 
Chemicals used

All chemicals used in the experiments were of the highest quality available and purchased from Sigma-Aldrich. Antimicrobial agents were prepared and stored according to standard protocols.10

Bacteria

A group of 16 strains of P. aeruginosa was used. Fourteen were obtained by routine screening of hospital environments (strain codes CL7, CL8, CL9, CL10, CL11, CL12, CL13, CL14, CL16, 17TS, OO14, OO72, 9766 and 9481) whereas two were laboratory strains, PAO1 (ATCC 15692) and ATCC 15442.

Generation of resistance

Cells were statically incubated in nutrient broth containing BKC at one-half of the lowest MIC determined for each strain, a passage concentration of 0.00078% (w/v) BKC. The bacteria were serially passaged by transferring c. 1 x 105 cells from 16 h culture to fresh media containing the same concentration of BKC and incubated at 37°C for a further 16 h. This was repeated for a total of 5 days and the strains' MIC for BKC re-determined. Any strains that had shown an increase in MIC were then inoculated into media containing twice the original concentration of BKC and a sample of the cell culture frozen at -70°C in nutrient broth containing 10% (w/v) glycerol and termed passage 1 (P1). This procedure continued until cells reached a MIC of 0.05% (w/v), which is the highest concentration of BKC to remain in solution in nutrient broth. Two of the strains (OO14 and PAO1) were further passaged in BKC up to 0.025% (w/v) BKC, that concentration termed passage 6 (P6). Passage of strains OO14 and PAO1 produced frozen samples of adapted cells from non-adapted wild-type (WT) cells at P1 [0.00078% (w/v) BKC], P2 [0.0015% (w/v) BKC], P3 [0.003% (w/v) BKC], P4 [0.006% (w/v) BKC], P5 [0.0125% (w/v) BKC] and P6 (0.025%) levels of passage.

Determining culture purity

At each passage stage, cells from strains OO14 and PAO1 were plated on nutrient agar and their Habs serotype determined. In addition, pulsed-field gel electrophoresis was undertaken on the chromosomal DNA of cells from each passage following digestion with the restriction enzyme DraI (Boehringer Mannheim). Conditions for the preparation and digestion of samples and subsequent electrophoresis followed those of Livesley et al.,11 and used the CHEF DRIII apparatus (Bio-Rad, Hemel Hempstead, UK).

MICs

The MIC of BKC and each of the other antimicrobial agents was determined by the broth dilution method using an inoculum of 1 x 105 cells. In cases where the disinfectant used produced a cloudy precipitate at higher concentrations, a minimum bactericidal concentration was determined by plating out 100 µL of cells from tubes with such precipitate on to nutrient agar and incubating for 24 h at 37°C. This was carried out for strains OO14 and PAO1.

In addition the cells showing the highest resistance to BKC in strains OO14 and PAO1 were passaged through nutrient broth containing no disinfectant for the same period of time as that which generated such resistance and the MIC for each strain after each passage determined.

Outer membrane protein and lipopolysaccharide (LPS) visualization

Outer membrane proteins from each passage of strains PAO1 and OO14 were isolated by preferential solubilization with N-lauroyl sarcosine, and visualized by Coomassie staining after SDS–PAGE analysis. Bands were then analysed with Phoretix 1D Advanced gel analysis software (version 4.01; Non-linear Dynamics, Newcastle upon Tyne, UK). Outer membrane samples treated with proteinase K were run on 12% (w/v) acrylamide gels containing urea at a final concentration of 4 M and were visualized using the method of Tsai & Frasch.12 Identification of outer membrane proteins was achieved by comparison of molecular weights with those determined by Hancock et al.13

LPS quantification

Quantification of LPS was achieved by a colorimetric assay that detects the presence of 2-keto-3-deoxyoctonate (KDO) in sarcosine-derived outer membrane samples.14

Fatty acid isolation and analysis

Non-detergent separation of outer and cytoplasmic membranes was achieved by passage through a French pressure cell, and subsequent refrigerated centrifugation through a stepped sucrose gradient. Samples were then derivatized to methyl esters and examined by gas chromatography to determine the fatty acids present.15

Whole cell lipids

Whole cell lipids extracted by the Bligh & Dyer method16 were separated by thin-layer chromatography and visualized with molybdenum blue reagent.17 The relative proportions of each lipid were determined using Phoretix software.

Uptake/binding of BKC

Adapted cells from strains OO14 and PAO1 were treated with 0.003% (w/v) BKC for 10 min at 25°C and then removed by centrifugation for 10 min at 10 000g, leaving unbound BKC in the supernatant. Using the colorimetric assay devised by Scott,18 it was possible to determine how much BKC was left in the supernatant and so calculate how much had bound to or been taken up by the cells.

Hydrophobicity

Cell surface hydrophobicity of OO14 and PAO1 adapted cells was determined by the microbial adhesion to hydrocarbon (MATH) assay using n-hexadecane as the hydrocarbon phase.19

Cell surface charge

Determination of cell surface charge was achieved by particle microelectrophoresis using the Zetamaster Particle Electrophoresis Analyser (Malvern Instruments). Stationary phase cells were suspended in 10 mM KCl at a concentration of 2 x 107 cells/mL.

Permeability assay using N-phenyl naphthalene (NPN)

The hydrophobic probe NPN normally has restricted access to the outer membrane of P. aeruginosa, an access that is enhanced by the action of the membrane-active agent BKC and the outer membrane permeabilizer EDTA. NPN fluoresces strongly in a hydrophobic environment such as that found within the outer membrane. Approximately 5 x 107 adapted cells of strains OO14 and PAO1 were pre-treated for 30 min with either EDTA (400 mg/L) or BKC (0.003%, w/v). NPN was then added to the cells at a final concentration of 10 µM. The uptake of NPN into such treated cells was determined by measuring the fluorescence emitted at 420 nm after excitation at 350 nm using a Perkin Elmer spectrophotometer.

Plasmid preparation

Plasmid preparation and quantification from WT and passage 5 OO14 cells were achieved using Wizard Maxipreps DNA purification kit and standard molecular techniques.20


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 Acknowledgements
 References
 
Generation of resistance and purity of culture

Increases in resistance to BKC were observed in almost all strains examined, often to a concentration in excess of 0.05% (w/v) (Table 1Go). Resistance to BKC in OO14 and PAO1 continued while grown in concentrations up to 0.025% (w/v) and was retained through 5 weeks of passage in disinfectant-free media (data not shown). Properties of these two strains were investigated further. Their serotypes remained the same throughout the adaptation process and through the passage in disinfectant-free media. Similarly the pulsed-field profile for both strains remained unchanged throughout the experiment.


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Table 1. MIC for strains of P. aeruginosa passaged in increasing concentrations of BKC
 
Co-resistance to other antimicrobial agents

Both strain PAO1 and strain OO14 cells were tested at each stage of adaptation to BKC for co-resistance to other antibacterial agents (Table 2Go). In PAO1 an increase in resistance was observed for the membrane-active disinfectants cetylpyridinium chloride and cetrimide, the antibiotic chloramphenicol and the membrane-active antibiotic polymyxin B. No significant changes were observed for the phenolic disinfectants triclosan and thymol, the membrane-active disinfectants dodecyl trimethyl-ammonium bromide and chlorhexidine, or the antibiotics imipenem, ciprofloxacin and tobramycin. A reduction in resistance to ceftazidime was observed.


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Table 2. MIC values of antibacterial agents (units shown) for cells of P. aeruginosa strains OO14 and PAO1 adapted to BKC
 
Strain OO14 showed increases in resistance to the membrane-active disinfectants cetrimide and cetylpyridinium chloride on serial passage. No alteration in resistance was observed for the disinfectants thymol, dodecyl trimethyl-ammonium bromide, triclosan, chlorhexidine or the antibiotics polymyxin B, ceftazidime, ciprofloxacin, chloramphenicol or imipenem. Resistance to tobramycin decreased in a stepwise fashion as the cells became more resistant to BKC.

Outer membrane protein alterations

The outer membrane proteins of adapted cells of PAO1 and OO14 were examined (Figures 1 and 2GoGo, respectively). The proportion of a protein band of c. 25 kDa increased from undetectable in the WT (non-adapted) cells to 14% of the outer membrane protein in the most resistant cells of PAO1. OO14 showed an increase in the proportion of a 44 kDa outer membrane protein from 4% in WT cells to 14% in the most resistant cells. OO14 showed no change in proportion of the 25 kDa protein, however.



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Figure 1. Outer membrane proteins of P. aeruginosa strain PAO1 passaged in increasing concentrations of BKC. *, position of 25 kDa protein. Ladder contains aprotinin (6.5 kDa), lysozyme (16.5 kDa), ß-lactoglobulin A (25 kDa), triosephosphate isomerase (32.5 kDa), aldolase (47.5 kDa), glutamic dehydrogenase (62 kDa), MBP–paramyosin (83 kDa) and MBP–ß-galactosidase (175 kDa). WT, wild-type, non-passaged PAO1 cells; P1, cells adapted to 0.00078% (w/v) BKC; P2, cells adapted to 0.0015% (w/v) BKC; P3, cells adapted to 0.003% (w/v) BKC; P4, cells adapted to 0.006% (w/v) BKC; P5, cells adapted to 0.0125% (w/v) BKC; P6, cells adapted to 0.025% (w/v) BKC.

 


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Figure 2. Outer membrane proteins of P. aeruginosa strain OO14 passaged in increasing concentrations of BKC. P4 of OO14 was non-recoverable from frozen culture. Lanes between ladder and WT contain samples prepared at lower denaturing temperatures. For details of abbreviations (ladder, WT, P1, etc.) see legend to Figure 1Go.

 
LPS content

There appeared to be little alteration in the banding pattern of LPS from strain OO14 adapted cells on SDS– PAGE. It was not possible to visualize LPS isolated from the outer membrane of PAO1 by silver staining due to the lack of periodate-sensitive sugars, as reported previously.21 Determination of the amount of LPS present in PAO1 and OO14 cells was achieved by measuring the quantity of KDO present. In both cases little discernible trend was observed, except for a reduction in concentration of KDO in those most resistant cells that had been grown in disinfectant-free media (Figure 3Go).



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Figure 3. Quantity (µg) of KDO present in 2 mg (dry weight) samples of outer membrane material isolated from P. aeruginosa strains PAO1 ({square}) and OO14 ({blacksquare}) cells adapted to BKC (error bars show S.E.M.). P4 of OO14 was non-recoverable from frozen culture.

 
Fatty acid content of outer and cytoplasmic membranes

Analysis of the fatty acids in the outer membrane of PAO1 showed no clear increase or decrease in any of the fatty acids present. The fatty acids of the cytoplasmic membrane of PAO1 showed a stepwise increase in the proportions of 14:0 and 16:0 and a decrease in the proportion of an unknown fatty acid eluting between 2-OH 10:0 and 12:0 (Table 3Go). Further examination of the unknown fatty acid by electron impact mass spectrometry revealed that it is represented by two peaks with a mass charge ratio of 205 and 220.


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Table 3. Proportion of fatty acids in cytoplasmic membrane of P. aeruginosa strain PAO1 cells adapted to BKC
 
Cells of strain OO14 exhibited no trend in alteration of proportion of any of the fatty acids present.

Whole cell lipids

No alteration was observed in the proportions of whole cell lipids of PAO1 cells adapted to BKC (10% diphosphatidylglycerol, 50% phosphatidylethanolamine, 20% phosphatidylglycerol, 10% phosphatidylcholine and 10% lyso phosphatidylethanolamine). However, as OO14 cells became more resistant to BKC during passage the proportion of phosphatidylcholine dropped from 20% to 6%.

Uptake/binding of BKC to P. aeruginosa cells

PAO1 cells showed no trend in binding of BKC as they became more resistant to the disinfectant, whereas strain OO14 cells showed a reduction in BKC removed from the media by cells as their resistance to BKC increased (Figure 4Go), although P3 showed the same value as the WT cells.



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Figure 4. Proportion (%) of BKC removed from media by PAO1 ({square}) and OO14 ({blacksquare}) P. aeruginosa strain cells adapted to BKC (error bars show s.e.m.). P4 of OO14 was non-recoverable from frozen culture.

 
Hydrophobicity and cell surface charge

Strain PAO1 cells adapted to BKC showed no trend in alteration in cell surface hydrophobicity as measured by the MATH assay. As resistance to BKC increased in strain OO14 cells, however, they showed a gradual increase in hydrophobicity reflected by a higher percentage of cells moving to the hydrophobic phase of the MATH assay (Figure 5Go).



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Figure 5. Proportion (%) of P. aeruginosa strain PAO1 ({square}) and OO14 ({blacksquare}) cells adapted to BKC that moved from aqueous to hydrophobic phase (error bars show s.e.m.). P4 of OO14 was non-recoverable from frozen culture.

 
Cells from strain OO14 showed no discernible trend in change of electrophoretic cell surface charge, although cells from strain PAO1 showed a slight increase in negativity as they became more resistant to BKC (Figure 6Go).



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Figure 6. Zeta potential (mV) of P. aeruginosa strain PAO1 ({square}) and OO14 ({blacksquare}) cells adapted to BKC (error bars show s.e.m.). P4 of OO14 was non-recoverable from frozen culture.

 
Resistance of outer membrane to permeabilization by BKC and EDTA

As strain PAO1 cells became more resistant to BKC they showed a reduction in the amount of NPN incorporated into the outer membrane when treated with either EDTA or BKC. This reduction was reflected in a reduction in fluorescence observed (Figure 7Go). Strain OO14 cells showed little alteration in the susceptibility of the cells to the permeabilizing action of EDTA or BKC, even if cells were shown to be more resistant to the biocidal action of the disinfectant.



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Figure 7. Fold-increase in uptake of NPN into P. aeruginosa strain PAO1 cells, adapted to BKC, as a result of permeabilizing action of 400 µg/mL EDTA ({square}) and 0.003% (w/v) BKC ({blacksquare}) determined by increase in fluorescence (error bars show S.E.M.).

 
Plasmid preparation

Plasmid DNA in strain OO14 of c. 9300 bp in size was reduced in quantity three-fold between WT and most BKC-adapted cells.


    Discussion and conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 Acknowledgements
 References
 
Range of resistance

The ability of P. aeruginosa to survive in increasing concentrations of cationic biocides such as chlorhexidine or BKC is well known. Going back to 1971 with work by Adair et al.22 showing that BKC-resistant cells had cross-resistance to other quaternary ammonium compounds (QACs), there has been ample evidence that adaptive resistance, stable or otherwise, can lead to a reduction in susceptibility to other disinfectant agents.

However, the range of co-resistance seems specific to both the strain adapted and the biocide used in the adaptation. Strain PAO1 showed resistance to all QACs tested, which indicates an alteration in a common site of action. However, it showed no resistance to chlorhexidine, which is unusual, since work by Russell et al.23 has shown that chlorhexidine-adapted Pseudomonas stutzeri cells showed cross-resistance to QACs. OO14 cells showed a far more limited range of cross-resistance, only to cetrimide and cetylpyridium chloride. Considering that the treatment given to each strain of cells was, as far as possible, identical, it would appear that each strain has the potential to develop a unique spectrum of resistance when placed under the same environmental pressures. The only cross-resistances with antibiotics occurred in PAO1 against polymyxin B and chloramphenicol. The membrane-active nature of this antibiotic would lend credence to the suggestion that changes due to BKC resistance would lead to resistance to membrane-active antibiotics, as shown by Russell et al.23 in chlorhexidine-treated cells of P. stutzeri. However, these resistances were not shared by OO14, and later work by Adair et al.24 indicated that BKC-resistant cells were more susceptible to polymixin B than non-adapted cells. Indeed the fact that many strains cannot produce the stable resistance to BKC exhibited here indicates that often the ‘unique' mechanism of resistance to the biocide is too damaging to other aspects of bacterial metabolism for the cells to survive very long. This is mirrored in observations from this work, that as OO14 and PAO1 strains became adapted to BKC, they became far more difficult to recover from frozen culture, indicating a reduction in cold tolerance.

Mechanisms of resistance

In a similar fashion to the differences in resistance spectra, alterations in phenotype associated with resistance appear to be specific to the strain examined. PAO1 showed an increase in the proportion of the 25 kDa outer membrane protein. A protein of this size, OprG, has been associated with alterations in the LPS in P. aeruginosa and is upregulated in low Mg2+ conditions.13 However, expression of OprG has not been associated with resistance to any antibacterial agents. It indicates, however, that low Mg2+ conditions are also associated with resistance to the membrane-active antibiotic polymyxin B, often accompanied by increased expression of the 20 kDa outer membrane protein OprH.25 However, no such increase in expression was observed in either of the strains examined, indicating that whereas co-resistance to polymyxin B occurred in adapted PAO1 cells it was not due to alteration in expression of outer membrane proteins, but was more likely to be as a result of other changes in the membrane structure that will be discussed shortly. In strain OO14 cells there was an increase in expression of an outer membrane protein of 44 kDa in size. The putative porin OprE is of that size, but is unlikely to be associated with resistance since such resistance would logically be a decrease in expression of the protein as has been reported for OprD and resistance to imipenem,13 a carbapenem that utilizes the OprD porin for entrance into the cell.

Examination of the outer and cytoplasmic membranes showed alterations in the proportion of fatty acids and phospholipids present. The increase in 16:0 observed in the cytoplasmic membrane of PAO1 cells is unusual, as most previous work reports a decrease in membrane 16:0,7,28 but does not differentiate between outer and cytoplasmic membranes. One possible explanation can be seen in the early work of Anderes et al.,27 which determined that resistance to BKC was accompanied by increases in the proportion of both 16:0 and 18:1 in both of what were termed phospholipid and free fatty acid fractions. However, if the cells were harvested from BKC-rich media these alterations were reversed. Jones et al.7 harvested their cells from such media, which may explain the apparently contradictory nature of their results when compared with those of this work. The decrease in proportion of an unknown fatty acid with a retention time just greater than that of 16:0 has no explanation, although resistance to solvents such as toluene in strains of Pseudomonas putida has been associated with the trans isomerization of cis-oleic acid, C16:1, C-9.28,29 If the unknown fatty acid in question were unsaturated in nature then a reduction in its proportion would result in a rigidification of the membrane in question. Such a rigidification has been suggested as a mechanism for resistance to membrane-active agents and may explain the increase in susceptibility to cold observed as the cells became resistant to BKC.

Certainly the proportion of 16:0 in outer and cytoplasmic membranes appears to play a role in many aspects of P. aeruginosa adaptation to the environment. In P. aeruginosa found in the cystic fibrosis lung it has been noted that there is a degree of substitution by 16:0 on to the lipid A portion of LPS isolated from such cells.30 This substitution also seems to confer an increased resistance to polymyxin B and antibacterial peptides and can be mimicked by growing the cells in a low Mg2+ media. It may be that there is a wider role for the substitution of 16:0 and similar fatty acids in phospholipid membranes as a response to a range of environmental pressures, biocides being just one of them.

When examining the less specific changes in the proportions of whole cell lipids it was noted that there was a reduction in phosphatidylcholine as cells of strain OO14 became more resistant to BKC. There has been no work examining alterations in the phospholipids of P. aeruginosa in response to BKC adaptation, but research examining lipids associated with polymyxin B resistance makes no mention of a reduction in phosphatidylcholine. Instead researchers observed reduction in phosphatidylethanolamine and phosphatidylglycerol and an increase in diphosphatidylglycerol.31–33 This perhaps indicates that because this alteration is only observed in strain OO14 cells, cells without increased resistance to polymyxin B, it may be that phosphatidylcholine is only important in resistance to BKC and other QACs.

Cell surface hydrophobicity and charge are notoriously susceptible to environmental changes and this should be taken into account before lending too much significance to alterations or trends observed. However, in the work of Jones et al.,7 cells adapted to QACs were found to have increases in cell surface hydrophobicity when examined by the MATH assay supporting the results observed for strain OO14 cells adapted to BKC in this work.

An explanation could involve the increase in hydrophobicity resulting from a reduction in the negatively charged (hydrophilic) binding sites for the positive head group of the disinfectant. This may also explain the results observed for the binding and/or uptake of BKC by OO14 cells. As OO14 cells become more adapted to BKC they ‘lose' or mask their binding sites for the disinfectant. These binding sites are negatively charged and so detract from the hydrophobicity of the cell, so as they are lost or masked the cell increases in hydrophobicity.

The cell surface charge in PAO1 cells increased (in negativity) very slightly as they became more resistant to BKC. Such a small reduction is likely to be as a result of a change in some outer membrane structure associated with resistance rather than the actual cause of such a drop in sensitivity.

The action of EDTA in permeabilizing the outer membranes of P. aeruginosa cell walls is well known,34 and resistance is usually a result of increased expression of the outer membrane protein OprH and associated with both polymyxin B and aminoglycoside resistance.25 PAO1 cells proved more resistant to the action of both BKC and EDTA as they became adapted to the disinfectant, whereas OO14 cells showed no such resistance. Neither strain showed any alteration in the expression of OprH. Whereas the action of BKC upon cell membranes is not completely understood, EDTA is thought to act by chelating Mg2+ ions from between adjacent LPS molecules in the outer membrane, resistance arising due to the replacement of such ions with the non-chelatable OprH protein. However, the only changes associated with LPS in PAO1 cells not occurring in OO14 cells was the increase in expression of a 25 kDa outer membrane protein of the same size as OprG, a protein associated with changes in LPS, but not associated with any previous resistance phenotype.

In OO14 cells there was a noticeable reduction in resistance to tobramycin (six-fold) as cells became adapted to BKC. This seemed unusual as aminoglycosides are thought to have membrane permeabilizing properties that aid their own uptake into the cell.35 However, the presence of plasmid DNA that reduced in quantity as the cells reduced their resistance to tobramycin indicates the presence of a resistance plasmid containing an aminoglycoside modifying enzyme.36

In conclusion, it appears that resistance to BKC is acquired readily and by passage in sub-MIC concentrations by P. aeruginosa strains, retained even in the absence of the disinfectant, and there is cross-resistance with the membrane-active antibiotic polymyxin B. However, this cross-resistance does not occur with all strains so adapted, and resistance to other antibiotics is not observed. Any cross-resistance to other antimicrobial agents would appear to be as a result of a general decrease in permeability of the outer and possibly the cytoplasmic membranes; however, such changes offer little protection against non-membrane-active antibiotics and are unlikely to represent a serious obstacle to therapy of patients if organisms with these changes are acquired nosocomially.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 Acknowledgements
 References
 
This work was supported by a BBSRC grant and CASE award in conjunction with Unilever plc.


    Notes
 
* Corresponding author. Tel: +44-121-3593611 ext. 4471; E-mail: P.A.lambert{at}aston.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 Acknowledgements
 References
 
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Received 30 July 2001; returned 19 September 2001; revised 8 November 2001; accepted 2 January 2002