Cytological changes in chlorhexidine-resistant isolates of Pseudomonas stutzeri

Unchalee Tattawasarta, A. C. Hannb, J.-Y. Maillarda, J. R. Furra and A. D. Russella,*

a Welsh School of Pharmacy, and b School of Molecular and Medical Biosciences, Cardiff University, Cardiff CF10 3XF, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive analysis of X-ray (EDAX) have been used to examine chlorhexidine diacetate (CHA)-sensitive and -resistant isolates of Pseudomonas stutzeri and to determine the effects of CHA on the cells. Significant differences were observed in the structure, size and elemental composition of CHA-sensitive and -resistant cells. Treatment with CHA produced considerably greater changes in CHA-sensitive cells, with widespread peeling of the outer membrane, a substantial loss of cytoplasmic electron-dense material and extensive lysis. Cells from the resistant isolates showed no blebbing of the outer membrane and no structural damage. X-ray mapping confirmed the difference in CHA uptake between CHA-sensitive and CHA-resistant cells. It is proposed that changes in the outer membrane form a major mechanism of resistance to CHA in P. stutzeri.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Electron microscopy (EM) is a useful tool for detailed ultrastructural analysis of microorganisms.1 Both scanning electron microscopy (SEM) and transmission electron microscopy (TEM), which provide information about surface and intracellular changes, can be used for studying the effects of antimicrobial agents on microorganisms. EM has previously been used to study the ultrastructural basis of the resistance of Pseudomonas aeruginosa to antiseptics, disinfectants and antibiotics,2 and has been widely used in this laboratory for investigating the effects of antimicrobial agents on a variety of different types of organism.

X-ray microanalysis is a method of elemental analysis at the ultrastructural level and can correlate morphological appearance with chemical composition. The technique exploits the fact that different elements give off X-rays having characteristic energies when a sample is irradiated with an electron beam. The elemental composition of a sample can be determined by analysing the X-ray spectrum.3 An energy-dispersive X-ray analyser offers the advantage that all elements of interest are analysed simultaneously. Energy-dispersive analysis of X-ray (EDAX) is a useful technique for the localization of elements in microbial cells and has previously been used in this laboratory for studying the effects of chlorhexidine on Saccharomyces cerevisiae, P. aeruginosa bacteriophage F116 and Acanthamoeba castellanii.4–6

Stable resistance to chlorhexidine has been demonstrated in some isolates of Pseudomonas stutzeri,7,8 and in this study we have used SEM, TEM and EDAX to better understand the underlying mechanisms involved.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates

Bacterial test isolates (in parentheses, MICs of chlorhexidine in mg/L) were P. stutzeri JM302 (2.5), JM302R (100), NCIMB 10783 (2.5) and 10783R (50). Aqueous cell suspensions were prepared and adjusted to a cell density of c. 2 mg dry wt/mL as described previously.8 In EDAX studies, Elgastat water was used in place of Water for Injections, B.P.

Negative staining, TEM and SEM

Techniques were carried out as described in previous studies.9,10

Effects of chlorhexidine diacetate on P. stutzeri ultrastructure

Chlorhexidine diacetate (CHA) was purchased from Sigma Ltd (Poole, UK). Equal volumes of bacterial cell suspension and a CHA solution were mixed at 20°C to give a final CHA concentration of 100 mg/L. After exposure for 5, 15, 30 and 60 min the suspensions were fixed and prepared for TEM and SEM according to the methods described above.

CHA-treated cells and EDAX analysis

Equal volumes of the cell suspension and CHA solution (final concentration 100 mg/L) were mixed at room temperature. Samples (1 mL) were removed at the required time interval [0 (control), 0.5, 15, 30 and 60 min] and immediately washed twice with 25% w/v polyvinyl pyrrolidone. The cell pellets were harvested by centrifugation at 1500g for 10 min, and used as described previously.4–6

The transmission electron microscope (Philips EM400) with its associated energy-dispersive X-ray detector and multichannel analyser (EDAX 9100/60) were used to analyse the elements in the specimens.3,4–6,11


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
CHA-resistant P. stutzeri: TEM

Cells of CHA-resistant isolates of P. stutzeri differed from those of parent sensitive isolates when negatively stained and examined by TEM (Figure 1a–dGo). Cells of both showed polar flagella; however, the resistant isolates stained with 1% phosphotungstic acid also carried a fibril-like structure on their cell surfaces (Figure 1b and dGo, arrow). This surface structure was found in the form of the electron-dense material around the cells when stained with 1% methylamine tungstate (data not shown). No surface structures were observed in their parent isolates prepared in the same manner (Figure 1a and cGo).



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Figure 1. Transmission electron micrographs of negatively stained cells of CHA-resistant isolates and their parent sensitive isolates of P. stutzeri stained with 1% phosphotungstic acid. (a) JM302; (b) JM302R, showing densely stained cell ‘wall’ and fibril-like structure around the cell; (c) NCIMB 10783; (d) NCIMB 10783R. Magnification in each case x12,500. Bars represent 1 µm.

 
Cells of CHA-resistant isolates were larger than those of parent isolates, e.g. cells of JM302 were 1.65 ± 0.21 µm (S.D.) in length and 0.43 ± 0.05 µm in width, with corresponding values of 2.17 ± 0.21 µm and 0.83 ± 0.05 µm for JM302R. These results were statistically different (Student's t test, P = 0.95).

TEM of thin sections of P. stutzeri revealed that the flagella and fibril-like structure usually found on the surface by negative staining (Figure 1Go) had been lost. However, the extracellular structure in the form of granular materials was present on some cells of isolate 10783R (data not shown). There was a marked difference in cell structure between sensitive and resistant isolates. The cell envelope of JM302R was wavy, thicker and more densely stained than its parent sensitive isolate. The cell envelope of NCIMB 10783R did not show any difference from its parent isolate; however, amorphous material, which did not appear in the sensitive isolate, was observed outside the cell.

CHA-resistant P. stutzeri: SEM

The results of SEM studies (Figure 2a–dGo) demonstrated the rough surface of cells of resistant isolates compared with those of sensitive isolates. Amorphous material was observed outside the resistant cells (Figure 2dGo, arrow).



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Figure 2. Scanning electron micrographs of CHA-resistant and parent isolates of P. stutzeri. (a) JM302; (b) JM302R, showing a rough cell ‘wall’; (c) NCIMB 10783; (d) NCIMB 10783R showing amorphous material around the cells. Magnification in all cases x20,000. Bars represent 1 µm.

 
Effect of CHA on cell structures

Exposure of the CHA-sensitive isolate JM302 to CHA (100 mg/L) produced progressive cellular damage, the extent depending on the period of treatment (Figure 3a–dGo). After exposure to CHA for 5 min (Figure 3aGo), numerous small blebs of the outer membrane were seen, with an occasional larger ballooning showing evidence of inner membrane breakage, which resulted in cytoplasmic leakage (Figure 3aGo, arrow). There was no apparent cytological lysis and the cytoplasm still contained electron-dense material.



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Figure 3. Transmission electron micrographs of P. stutzeri isolate JM302 treated with CHA 100 mg/L: (a) 5 min, blebbing of the outer membrane; arrow shows breakage of the inner membrane resulting in cytoplasmic leakage; (b) 15 min, cytoplasmic swelling; (c) 30 min, cell lysis; (d) 60 min, ghost cells. Magnification in all cases x40,000. Bars represent 0.1 µm.

 
After exposure for 15 min (Figure 3bGo), cells had lost their electron-dense material and large blebs on the outer membrane were seen. Cytoplasmic swelling was observed at the area where the outer membrane appeared to be ruptured. Although the cytoplasmic outgrowth was lightly stained and contained filamentous materials, cytological lysis was not apparent.

Following exposure for 30 and 60 min (Figure 3c and dGo), the cells showed widespread peeling of the outer membrane. Most cells underwent extensive lysis and, as a result, lost a substantial amount of cytoplasmic electron-dense material. Many cells exposed for 60 min became ghost cells with complete extraction of cytoplasmic contents (Figure 3dGo, arrow).

The results obtained from a parallel experiment using the resistant isolate (JM302R) showed no blebbing of the outer membrane and no structural damage when the cells were exposed to CHA at 100 mg/L (Figure 4a–dGo). However, in comparison with untreated JM302R, a decreased outer membrane thickness of treated cells was observed. After exposure for 60 min, the outer membrane of some treated cells showed blisters (Figure 4dGo).



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Figure 4. Transmission electron micrographs of P. stutzeri isolate JM302R treated for up to 60 min with CHA 100 mg/L. Cells have well-defined cell walls. Cytoplasm is intact and contains electron-dense material. No cell lysis was seen: (a) 5 min; (b) 15 min; (c) 30 min; (d) 60 min, arrow shows a blister of the outer membrane. Magnification in all cases x40,000. Bars represent 0.1 µm.

 
Elemental distribution in P. stutzeri

The EDAX spectra of P. stutzeri isolates demonstrated the presence of Mg, P, Cl, K and Ca. A silicon (Si) peak may originate from the absorption of X-rays in the inactive region of the detector or from Si-containing contamination products in the microscope. Elemental concentrations are provided in Tables I and IIGoGo. Changes in Cl concentrations after exposure of JM302 and JM302R to CHA (100 mg/L) are depicted in Table IGo. As untreated P. stutzeri cells contained Cl, the Cl concentration cannot represent the amount of CHA taken up by the cells. However, after cell treatment with CHA the Cl concentration increased, indicating uptake of the antibacterial agent.


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Table I. Chlorine concentration in P. stutzeri after treatment with chlorhexidine 100 mg/L at 20°C
 

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Table II. Concentrations of phosphorus, magnesium and calcium in P. stutzeri after treatment with chlorhexidine 100 mg/L at 20°C
 
The results of X-ray mapping confirmed the difference in CHA uptake between CHA-sensitive and -resistant isolates. X-ray dots of Cl in CHA-treated JM302 increased with time and were evenly distributed over the entire cell (data not shown). By contrast, these dots in CHA-exposed JM302R were less dense and difficult to distinguish from the background signal at 0.5 and 15 min exposure, but can be seen to increase after 30 and 60 min (Figure 5Go).



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Figure 5. Image and X-ray maps of Cl in P. stutzeri JM302R treated with CHA 100 mg/L at 20°C, showing localization of chlorine in the cell. (a) Control (untreated) (x18,750); (b) 15 min (x18,750); (c) 30 min (x18,750); (d) 60 min (x31,250). Bar represents 0.1 µm.

 
Concentrations of various other elements in CHA-treated cells are detailed in Table IIGo. As the period of contact increased so the concentrations of P, Mg and Ca decreased, followed by an increase after 60 min.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Both TEM and SEM provide cytological evidence to help explain both the mechanism of action of, and cellular resistance to, CHA. Ultrastructural differences were observed in cells of CHA-sensitive isolates of P. stutzeri and those trained to high-level resistance to the bisbiguanide (Figures 1 and 2GoGo). Cell envelopes of resistant cells stained more intensely than sensitive cells, implying greater absorption of staining materials. The resistant cells had rough surfaces, with external amorphous material (Figure 2dGo). This may be exopolysaccharide in nature, having reacted with chemicals during EM preparation. The conventional method of producing thin-sectioned preparations caused a slight loss of surface structure. It is noteworthy, however, that the subcellular structure of P. stutzeri cells has been well preserved using the preparation method described here. There was no evidence of cytoplasmic extraction or of cell envelope disruption in any of the samples examined.

The results presented here suggest that CHA effects outer membrane alterations. The acquisition by stepwise training of CHA resistance is associated with alterations in the architecture of the outer membrane, as no ultrastructural damage was observed in CHA-resistant cells exposed to concentrations of CHA that produce extensive damage in sensitive cells (Figures 3 and 4GoGo). Polymyxin resistance in P. aeruginosa has been linked to both ultrastructural and chemical alterations in the outer membrane so that uptake of the antibiotic to its target site, the underlying cytoplasmic membrane, is reduced.12–14 Ultrastructural changes in benzalkonium-treated P. aeruginosa have also been described.15

EDAX is a useful tool for studying the distribution of elements within bacterial cells (Figure 5Go, Tables I and IIGoGo), yeast cells4 and bacteriophages,5 and of the effects of antimicrobial agents thereon. Elements of interest in this investigation were Mg and Ca (both having a role in maintaining the structural organization of the outer membrane16–18), P (primarily associated with structural elements such as lipids, proteins and polysaccharides19) and Cl, used previously as a marker for CHA.4,5 Concentrations of Mg, Ca and P were significantly lower in the CHA-resistant isolate, JM302R, than in the sensitive isolate JM302; whereas the Cl concentrations were not significantly different.

P was located entirely within the bacterial cell (data not shown). Because the concentrations of Mg, Ca and Cl were much lower (Table IGo), the X-ray maps of these elements could not distinguish their localization from the background signal. Using X-ray microanalysis, Chang et al.19 reported that P and Mg were located in the cytoplasm and the cell envelope whereas Ca was found predominantly in the cell envelope of Escherichia coli B cells.

The Cl concentration in CHA-treated cells of JM302 decreased over a 30 min contact period and then rose at 60 min (Table IGo). This may equate with the well-known biphasic leakage phenomenon that occurs with this chemical agent.20 By contrast, the Cl content of CHA-treated JM302R remained constant for the first 15 min and then increased (Table IGo). This suggests that the biphasic phenomenon does not apply with the resistant isolate; this is not borne out when the P, Mg and Ca contents are considered (Table IIGo), although the effects of CHA on the resistant isolate are, as expected, much less. P release from CHA-treated E. coli has been described by Rye & Wiseman.21

In conclusion, the reduced uptake of CHA by the resistant isolate coincided with smaller amounts of Mg, Ca and P in the cell. A major mechanism of CHA resistance in P. stutzeri is likely to be linked to changes in the binding site(s) available at the outer membrane.


    Notes
 
* Corresponding author. Tel: +44-1222-875812; Fax: +44-1222-874149; E-mail: russellD2{at}cardiff.ac.uk

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    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 1 April 1999; returned 26 August 1999; revised 16 September 1999; accepted 11 October 1999