Use of the Malthus-AT system to assess the efficacy of permeabilizing agents on the activity of antibacterial agents against Pseudomonas aeruginosa

Letters in Applied Microbiology 1998, 26, 422–426

Use of the Malthus-AT system to assess the efficacy ofpermeabilizing agents on the activity of antibacterial agentsagainst Pseudomonas aeruginosa

H.M. Ayres, D.N. Payne 1, J.R. Furr and A.D. RussellWelsh School of Pharmacy, University of Wales Cardiff, Cardiff and 1Reckitt and Colman Products Ltd, Hull, UK

1754/98: received 9 February 1998 and accepted 3 March 1998

H.M. AYRES, D.N. PAYNE, J.R. FURR AND A.D. RUSSELL. 1998. The Malthus-AT systemprovided a satisfactory method for examining the effects of permeabilizing agents on theactivity of sub-inhibitory concentrations of antibacterial agents against Pseudomonasaeruginosa G48. Under this system, disodium edetate potentiated the activity ofchlorhexidine diacetate (CHA), cetylpyridinium chloride, para-chlorometaxylenol andtriclosan. Nitrilotriacetic acid enhanced the activity of some of the antibacterialstested, whereas sodium polyphosphate markedly reduced the efficacy of CHA.

INTRODUCTION

Minute changes in conductivity can be readily detected whenbacteria grow in appropriate culture media (Firstenberg-Edenand Eden 1984 ; Gibson and Clark 1987). As the organismsgrow, they utilize nutrients within the media metabolizingsubstrates of low to products of high conductivity. Suchchanges in conductivity are proportional to the number ofviable organisms present (Newsom 1978 ; Richards et al.1978). A suitable method for measuring changes in con-duction is by means of the Malthus-AT system.

Permeabilizing agents are defined as chemical agents thatincrease the permeability of the outer membrane (OM) ofGram-negative bacteria (Russell and Chopra 1996). Severalpermeabilizers are known (Vaara 1992) and in a previousreport, it was demonstrated that various compounds couldincrease the effects of lysozyme against a strain of Pseudo-monas aeruginosa as a consequence of their action on the OMof this organism (Ayres et al. 1993). In this paper, the use ofthe Malthus system for assessing the effects of permeabilizerson the activity of some antibacterial agents against the samestrain is reported.

MATERIALS AND METHODS

Test organisms

Pseudomonas aeruginosa G48 was grown overnight at 37 °C in100 ml Nutrient broth (Oxoid, Basingstoke, UK) in a shaking

Correspondence to : Professor A.D. Russell, Welsh School of Pharmacy,University of Wales Cardiff, Cardiff CF1 3XF, UK.

© 1998 The Society for Applied Microbiology

water-bath. The resulting growth was triple-washed at 4000 gfor 10 min and resuspended in sterile saline (0·9% w/v) ;this suspension was then diluted in saline to give approx-imately 107 cfu ml−1.

Special Peptone Yeast Extract medium

Special Peptone Yeast Extract (SPYE) medium was used inthe Malthus analyser. This contained Special peptone(Oxoid) 30 g, Yeast extract (Oxoid) 2·5 g and glass-distilledwater to 1 litre.

Chemicals

Chlorhexidine diacetate (CHA) was purchased from ZenecaPharmaceuticals (Macclesfield, UK), triclosan from Ciba-Geigy, cetylpyridinium chloride (CPC) and disodium edetate(EDTA) from BDH (Poole, UK), the trisodium salt of nitrilo-triacetic acid (NTA), the trisodium salt of citric acid dihy-drate, disodium tartrate and benzalkonium chloride (BZK ;Sigma Code B6295) from Sigma, and sodium polyphospate(SPP) from Aldrich.

Chloroxylenol (p-chloro-m-xylenol, PCMX) was used asthe formulation Dettol, containing 4·8% w/v PCMX andprovided by Reckitt and Colman (Hull, UK). Triclosan wasdissolved in a few drops of 95% v/v ethanol and then madeup to volume with sterile distilled water. Preliminary experi-ments demonstrated that the small amount of ethanol had noeffect on cell viability.

MALTHUS, PERMEABILIZERS AND BIOCIDES 423

Malthus system

The Malthus system measures conductance ; every 6 min, a10 kHz sine wave signal is applied to each cell and theamplitude of the return signal is measured and recorded.Initially, a baseline is established of three consecutive readingsof ³0·6 microsiemens, after which the computer plots chan-ges in conductance as growth occurs with time. These graphsresemble typical sigmoidal growth curves.

Test procedure

Cell suspension (0·5 ml) was added to 9·5 ml of test solutionin distilled water, of the final desired concentration at 20 °C.After contact for 5 min, 1 ml was added to 9 ml of ESTneutralizing solution and mixed well. A 1 ml volume wasthen transferred to a sterile Malthus cell containing 2 mlsterile SPYE medium. Each cell consisted of a glass tube withtwo platinum electrodes immersed in the cell suspension. Upto eight cells were attached to a board, which held the cellsin a water-bath maintained at a constant temperature of 30 °Cto avoid unwanted changes in conductivity, and also to pro-vide an electrical connection to the Malthus analyser.

Conductance across the electrodes was measured every 6min over a period of 40 h. This information was collectedand computer analysed.

All tests were run twice through the Malthus system andfor each Malthus run, all tests were duplicated, giving aminimum of four values for each test. Experiments wereundertaken with suspensions treated with antibacterial agentin the presence and absence of a permeabilizer or with per-meabilizer alone. Untreated cells served as a control.

Growth was compared by differences in detection times(DTs), defined as the time from inoculation for pre-selectedchange in the electrical parameter to take place (Gibson 1989).In this study, the DT was determined by three consecutiveincreases of 0·8 microsiemens or greater and occurred whenthe culture reached a certain threshold, usually 105–106 cfuml−1. Detection time correlates well with the number ofviable cells present and the higher the inoculum, the shorterthe DT.

Neutralizing system

The neutralizing solution used was the European SuspensionTest (EST) fluid containing phosphate buffer (50 mmol l−1)pH 7·2 10 ml, L-histidine 1 g, sodium thiosulphate 5 g,lecithin 3 g, Tween-80 30 ml, distilled water 900 ml. Theneutralizing ability of the EST fluid was established by theaddition of first, the biocide and then the cell suspension ;DTs were then measured as before.

© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 422–426

RESULTS

Control and permeabilizer-treated suspensions

The size of the inoculum was calculated to give a DT forcontrol (untreated) cultures of about 10 h. In addition, theinoculum size was always determined by a viable countingmethod so that the numbers of cells present in the originalcell suspensions were known. Little variation was observedand results from different Malthus runs could therefore becompared.

The permeabilizers tested had no effect on the pattern ofconductivity changes found in control cultures, with DTvalues of a comparable nature.

Suitability of the neutralizer system

The EST neutralizing solution was effective in quenchingthe activity of biocides because the DTs of cells exposed tobiocide plus this solution were not significantly different fromthe DTs of control (untreated) cells.

Antibacterial plus permeabilizer

Sub-minimum inhibitory concentrations of antibacterialagents were used to give DTs of about 20 h. Both potentiationand antagonism could then be demonstrated, potentiation(antagonism) occurring when the DT for the antibacterialplus permeabilizer was greater than (less than) the DT forthe antibacterial used alone.

No DT was recorded for the tests in which no growthoccurred over a period of 40 h. When no value was obtained,the results could not be analysed numerically.

Chlorhexidine diacetate plus permeabilizer. A suitable con-centration of CHA was found to be 90 mg ml−1, which gavea mean DT of 20·2 h and a narrow range of values (Table 1).EDTA, at the higher concentration studied (10 mmol l−1),produced a delay in DT and of the other agents, NTA, citrateand tartrate all potentiated the activity of CHA. As expected,SPP gave an antagonistic response (Table 1).

Cetylpyridinium chloride plus permeabilizer. The results ofequivalent experiments are provided in Table 2. Cetyl-pyridinium chloride alone (100 mg ml−1) produced a meanDT of 16·2 h. EDTA (10 mmol l−1) potentiated activity tosome extent, but the most marked enhancement was seenwith NTA. This permeabilizer also produced greatest potent-iation of the activity of another quaternary ammonium com-pound, BZK (data not shown).

424 H.M. AYRES ET AL.

Table 1 Effect of permeabilizers onactivity of chlorhexidinediacetate (CHA, 90 mg ml−1)against Pseudomonasaeruginosa

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Mean Range of Number of testsNumber detection time values in which no

Permeabilizer* of tests (h) (h) growth detected—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––None (CHA alone) 9 20·2 18·2–22·2 0EDTA

1 mmol l−1 4 23·5 22·6–NG† 210 mmol l−1 6 32·0 26·3–NG 3

NTA0·005 4 –‡ – 4

Citrate0·5 4 – – 41·0 4 – – 4

Tartrate0·5 4 – – 41·0 4 26·7 23·8–NG 1

SPP0·0625 4 10·9 10·6–11·2 00·125 4 12·3 11·2–13·3 00·5 4 14·5 13·1–15·9 0

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

* Concentrations are mmol l−1 (EDTA) or % w/v (all others).† NG, No growth detected.‡ No value obtained (no growth obtained in any Malthus cell).

Table 2 Effect of permeabilizers onactivity of cetylpyridinium chloride(CPC, 100 mg ml−1) against Pseudomonasaeruginosa

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––


Permeabilizer* of tests (h) (h) growth detected—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––None (CPC alone) 7 16·2 13·7–18·6 0EDTA

1 mmol l−1 6 16·2 13·5–17·2 010 mmol l−1 6 22·8 20·8–24·0 0

NTA0·005 4 –‡ – 4

Citrate0·1 4 16·0 15·5–16·5 00·25 6 18·9 13·7–23·5 00·5 3 18·6 17·1–20·1 01·0 4 15·6 13·0–18·1 0

Tartrate0·1 4 19·0 17·6–20·3 00·25 4 17·3 16·8–18·2 00·5 4 13·2 11·7–14·8 01·0 2 10·4 10·2–10·6 0

SPP0·125 4 16·5 15·4–17·7 00·5 4 21·8 21·2–23·1 0

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

* Concentrations are mmol l−1 (EDTA) or % w/v (all others).‡ No value obtained (no growth obtained in any Malthus cell).


MALTHUS, PERMEABILIZERS AND BIOCIDES 425

Table 3 Effect of permeabilizers onactivity of triclosan (25 mg ml−1)against Pseudomonas aeruginosa

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––


Permeabilizer* of tests (h) (h) growth detected—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––None (triclosan alone) 10 23·2 20·4–28·3 0EDTA

1 mmol l−1 4 26·9 25·8–27·9 010 mmol l−1 4 –‡ – 4

NTA0·005 6 16·4 13·1–18·7 0

Citrate0·25 4 17·4 15·9–18·9 00·5 4 19·6 17·9–21·2 01·0 4 26·7 26·3–27·0 0

Tartrate0·5 4 11·8 9·0–13·1 01·0 4 10·2 10·8–15·0 0

SPP0·125 4 25·0 23·7–26·2 00·25 4 23·0 21·3–24·6 00·5 2 24·2 23·3–25·0 0

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

* Concentrations are mmol l−1 (EDTA) or % w/v (all others).‡ No value obtained (no growth obtained in any Malthus cell).

Triclosan plus permeabilizer. Triclosan (25 mg ml−1) gave amean DT of 23·9 h (Table 3). EDTA, at the higher con-centration (10 mmol l−1) produced significant potentiation,while tartrate was clearly antagonistic.

Chloroxylenol plus permeabilizer. Potentiation of PCMXwas seen with 10 mmol l−1 EDTA (Table 4), but produced nopotentiation and variable results were obtained with citrate.

DISCUSSION

In the experiments described here, Ps. aeruginosa cells wereexposed to biocide alone, permeabilizer alone or biocide pluspermeabilizer, then to a neutralizing solution to stop thereaction before being transferred to a recovery medium(SPYE broth) for detection of cell growth in the Malthusapparatus. The size of the control inoculum, i.e. of cellswhich had not been exposed to any biocide, permeabilizer orcombination, had been set to give a detection time of about10 h. Permeabilizers themselves had no effect on the DTvalues.

Preliminary experiments were undertaken with biocidesalone to determine concentrations that would give DTs ofabout 20 h when treated cells were subsequently incubatedin recovery medium. It was thus an easy matter to calculate


whether the presence of a permeabilizer in the pre-treatmentsystem enhanced or reduced the activity of a biocide.

EDTA potentiated the activity of various agents as hadbeen observed in studies undertaken using other experimentalsystems (Haque and Russell 1974a,b ; Dankert and Schut1976 ; Russell and Furr 1977). However, the enhanced effectoccasionally found for NTA, e.g. with CHA (Table 1) orCPC (Table 2) was surprising. As expected in view of theknown interaction of CHA with phosphates (Longworth1971), SPP reduced the activity of the biguanide (Table 1).However, its general potentiating role in the procedure usedin this paper was much less than would have been predictedfrom the rapid method devised previously (Ayres et al. 1993).

The Malthus system provided a useful, comparativelyrapid method of studying several tests simultaneously. Itenables monitoring of growing cultures to be undertakenwithout the need to remove samples at regular intervals, andoverall results were generally more consistent than thoseobtained by means of a viable counting technique.

ACKNOWLEDGEMENT

The authors thank Reckitt and Colman Products, Hull, andBBSRC for a CASE research studentship (to H.M.A.).

426 H.M. AYRES ET AL.

Table 4 Effect of permeabilizerson activity of p-chloro-m-xylenol(PCMX, 96 mg ml−1) againstPseudomonas aeruginosa

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––


Permeabilizer* of tests (h) (h) growth detected—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––None (PCMX alone) 13 24·4 22·7–27·4 0EDTA

1 mmol l−1 6 28·6 24·4–NG† 110 mmol l−1 6 31·3 25·1–NG 3

NTA0·005 4 19·9 18·6–22·1 0

Citrate0·5 8 24·5 20·0–NG 31·0 8 25·9 22·7–NG 2

Tartrate0·25 4 23·2 22·8–23·6 00·5 4 20·7 17·5–22·7 0

SPP0·125 4 20·3 17·7–23·6 00·5 4 20·5 17·5–22·7 0

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

* Concentrations are mmol l−1 (EDTA) or % w/v (all others).† NG, No growth detected.

REFERENCES

Ayres, H. (1995) Role of permeabilizers in increasing the sensitivityof Pseudomonas aeruginosa to biocides. Ph.D. Thesis, Universityof Wales.

Ayres, H.M., Furr, J.R. and Russell, A.D. (1993) A rapid method ofevaluating permeabilizing activity against Pseudomonas aeruginosa.Letters in Applied Microbiology 17, 149–151.

Dankert, J. and Shut, I.K. (1976) The antibacterial activity of chlor-oxylenol in combination with ethylenediamine tetraacetic acid.Journal of Hygiene, Cambridge 76, 11–22.

Firstenberg-Eden, R. and Eden, G. (1984) Estimation of microbiallevels. In Impedence Microbiology ed. Firstenberg-Eden, R. andEden, G. pp. 73–98. Letchworth : Research Studies Press.

Gibson, D.M. (1989) Optimization of automated electrometricmethods. In Rapid Microbiological Methods for Foods, Beveragesand Pharmaceuticals ed. Stannard, C.J., Petitt, S.B. and Skinner,


F.A. Society for Applied Bacteriology Technical Series No. 25.pp. 87–99. Oxford : Blackwell Scientific Publications.

Haque, H. and Russell, A.D. (1974a) Effect of ethylenediaminetetraacetic acid and related chelating agents on whole cells ofGram-negative bacteria. Antimicrobial Agents and Chemotherapy5, 447–452.

Haque, H. and Russell, A.D. (1974b) Effect of chelating agents onthe susceptibility of some strains of Gram-negative bacteria tosome antibacterial agents. Antimicrobial Agents and Chemotherapy6, 200–206.

Longworth, A.R. (1971) Chlorhexidine. In Inhibition and Destructionof the Microbial Cell ed. Hugo, W.B. pp. 95–106. London: Aca-demic Press.

Russell, A.D. and Furr, J.R. (1977) The antibacterial activity of anew chloroxylenol preparation containing ethylenediamine tetra-acetic acid. Journal of Applied Bacteriology 43, 253–260.

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Use of the Malthus-AT system to assess the efficacy of permeabilizing agents on the activity of antibacterial agents against Pseudomonas aeruginosa