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Development of resistance to chlorhexidine diacetate in Pseudomonas aeruginosa and the effect of a 'residual' concentration
Journal of Hospital Infection (2000) 46: 297–303 doi:10.1053/jhin.2000.0851, available online at http://www.idealibrary.com on
Development of resistance to chlorhexidine diacetate in Pseudomonas aeruginosa and the effect of a ‘residual’ concentration Louise Thomas, J.-Y. Maillard, R. J.W. Lambert* and A. D. Russell Pharmaceutical Microbiology Research,Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF and *Unilever Research Colworth, Sharnbrook, Bedfordshire, UK
Summary: Stable resistance in Pseudomonas aeruginosa NCIMB 10421 was obtained by step-wise exposure to gradually increasing concentrations of chlorhexidine diacetate (CHX). Repeated exposure to a proposed ‘residual’ (sub-MIC) concentration of CHX also created stable resistance. Resistance was also developed by a single exposure to the ‘residual’ concentration of CHX, but this was unstable. Similar experiments with Escherichia coli and CHX or cetylpyridinium chloride resulted in no significant increase in resistance. Antibiotic susceptibility profiles of the CHX-resistant P. aeruginosa cultures showed no cross-resistance, although some of the cultures were resistant to benzalkonium chloride. © 2000 The Hospital Infection Society
Keywords: Resistance; chlorhexidine; Pseudomonas aeruginosa; residual concentration.
Introduction Antibiotic resistance in bacteria has been known for some time, but remains a major challenge. Even with knowledge of the mode of action of antibiotics, their bacterial targets, and mechanisms of resistance1 the problem persists. Attention is turning to biocides (antiseptics, disinfectants and preservatives) as a possible cause for bacterial resistance to antibiotics.2,3 Chlorhexidine (CHX) is a bisbiguanide antiseptic, disinfectant and preservative effective against a wide range of bacteria, some fungi and some viruses.4 It is available as the acetate, gluconate and hydrochloride, and is widely used in hand washes, dressings and creams, instrument cleaning solutions, mouthwashes, and in combination with cetrimide as a general disinfectant.3,4 Chlorahexidine acts primarily on the bacterial cell membrane5
Received; revised manuscript accepted Author for correspondence: Professor A. D. Russell, Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, UK. E-mail: [email protected]
0195-6701/00/040297;07 $35.00
causing leakage of intracellular material, respiratory inhibition and cytoplasmic coagulation.6,7 Gram-positive bacteria are more susceptible than Gram-negatives, in particular Pseudomonas aeruginosa and Proteus spp.1 The nature of its action explains why the Gram-negatives, especially P. aeruginosa, are intrinsically resistant, as the outer membrane restricts the access of CHX to its target.2 As bacterial resistance has been reported for a large number of biocides, it is important to investigate the reasons for this.8 When biocides are discussed in terms of antibacterial efficacy the ‘in-use’ concentration is normally considered.9 The mode of use could cause emergence of resistance. For example 4% CHX is usually specified, but after use a residual concentration may be far lower, and below the minimum inhibitory concentration (MIC) for some bacteria, thus exposing the bacterial cells to a selective pressure for resistance. Because of the increasing use of biocides in hospitals, industry and even the home,2 we investigated the effects of sub-MIC concentrations of CHX on Gram-negative bacteria, in particular a strain of © 2000 The Hospital Infection Society
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P. aeruginosa, known to possess intrinsic resistance to CHX.10 The effect of CHX on the susceptibility of CHX-resistant strains to antibiotics was also considered. Materials and methods Bacterial strains Two Gram-negative organisms were studied, namely Pseudomonas aeruginosa NCIMB 10421 and Escherichia coli NCIMB 8879. They were maintained on nutrient agar (NA; Oxoid Ltd, Basingstoke, Hampshire, UK) plates at 37°C. Antibacterials Chlorhexidine diacetate (CHX) was purchased from Sigma (Poole, Dorset, UK); cetylpyridinium chloride (CPC) from BDH Laboratories (Poole, Dorset, UK); benzalkonium chloride (BZK) from ICN Chemicals (Cleveland, Ohio, USA) and cetrimide (Cet) from Thornton & Ross Ltd (Huddersfield, UK). Preparation/sterilization of solutions Solutions of all antibacterials were prepared in distilled water and sterilized by filtration before use. Antibiotics Discs were purchased from Difco (now Beckton Dickenson, Cowley, UK), and Becton Dickinson. The following discs were used: gentamicin 10 g; carbenicillin 100 g; polymyxin B 300 u; ciprofloxacin 5 g; tobramycin 10 g; amikacin 30 g; neomycin 30 g and kanamycin 30 g. Determination of MICs Tubes of 10 mL nutrient broth (NB; Oxoid) containing a range of biocide concentrations were inoculated using 10 L of an overnight NB culture of test strain (containing approximately 1–2109 cfu/mL) to give ca. 1–2106 cfu/mL. The tubes were incubated at 37°C for 48 h in a shaking water bath (Gallenkamp, Fisher Scientific, Loughborough, at 100 rev/min) and examined for growth. The lowest concentration of biocide inhibiting growth was recorded as the MIC. Step-wise development of resistance (Fig. 1a) From the tube above the highest CHX concentration permitting growth, a second series of biocide
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concentrations in NB was inoculated with 10 L of bacterial suspension and incubated as before. This step was repeated every 48 h and the MICs noted. Initial CHX concentrations ranged from 0 to 18 g/ml, and then were subsequently increased to 40, 50, 60 and finally 70 g/mL depending upon the growth of ‘resistant’ micro-organisms. These ‘step-wise’ subcultures in increasing concentrations were continued until no further increase in MIC was observed or the concentrated biocide produced clouding in the broth so that growth could no longer be easily observed. The purity of the cultures was checked by streaking on to selective agar (MacConkey agar (Oxoid) and Pseudomonas agar F (Difco) for E. coli and P. aeruginosa respectively). Pseudomonas aeruginosa cultures were also tested using the BBL® Oxi/ ferm™ Tube II kits (Becton Dickinson). Resistance stability The stability of biocide resistance, was determined by continuous subculture of the resistant strains in biocide-free nutrient broth. Subcultures were performed every 24 h for 15 passages and the MIC determined after 1, 5, 10 and 15 passages. A check of culture purity was performed at each stage. Agar MIC determination of CHX-resistant P. aeruginosa cultures The MIC of CHX for the resistant P. aeruginosa cultures gained at each stage was also determined by the agar dilution method. One microlitre of each broth bacterial suspension with increased MIC was inoculated on to nutrient agar (Oxoid) plates containing increasing concentrations of biocide, using a Denley Multipoint Inoculator (Billinghurst, UK). All plates were incubated at 37°C for 48 h, and the MIC noted. Repeated exposure of bacteria to ‘residual’ concentrations of biocide (Fig. 1b) The effect of a proposed ‘residual’ (sub-MIC) concentration of biocide on sensitive and resistant bacteria was determined. First, resistant cells developed through the step-wise training method were exposed to a ‘residual’ concentration of biocide for five subcultures and subsequent MICs were determined. Ten microlitres of cell suspensions showing increase in resistance at each stage of the step-wise method were inoculated into 10 mL NB (control)
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(a) Step-wise development of resistance: P. aeruginosa Original P. aeruginosa culture
Stepwise training with CHX
MICs on agar of CHX, BZK, cetrimide and CPC
(b) Repeated exposure of P. aeruginosa to 'residual' concentrations of biocide Original P. aeruginosa culture
Step-wise trained P. aeruginosa (A)
Five exposures to sub-MIC CHX (5 µg/mL)
Five exposures to sub-MIC CHX (5 µg/mL)
Antibiotic susceptibility test (discs)
Broth MIC of CHX and resistance stability examined
Resistance stability examined
(c) Single exposure of P. aeruginosa to 'residual' concentrations of biocide Original P. aeruginosa culture
Single exposure to CHX (1, 2.5 or 5 µg/mL)
Broth MIC of CHX and resistance stability examined
Susceptibility to QACs and antibiotics determined
Susceptibility to QACs and antibiotics determined
Figure 1
Summary of experiments performed with P. aeruginosa NCIMB 10421.
and 10 mL NB with a known concentration of biocide. These cultures were incubated at 37°C for 24 h in a shaking water bath. Bacterial growth obtained in the broth with biocide was then used to inoculate a second set of tubes containing the same ‘residual’ concentration of biocide. This exposure was repeated for five subcultures and the MIC of the fifth subculture in biocide was determined by the broth-dilution method. The same biocide concentration was used at each subculture: 5 g/mL CHX for P. aeruginosa and 0.1 g/mL CHX or g/mL CPC for E. coli. This experiment was repeated with an overnight, 37°C culture of parent strain containing approximately 1–2109 cfu/mL. Cells were exposed to ‘residual’ concentrations of biocide for five subcultures and MICs in NB were determined. The purity of the cultures was checked at each stage of work as previously described. The stability of any increase in MIC and resistance was also performed as before. Single exposure of bacteria to a ‘residual’ concentration of biocide (P. aeruginosa only) (Fig. 1c) Ten microlitres of an overnight culture at 37°C of the P. aeruginosa parent strain containing approximately 1–2109 cfu/mL were used to inoculate a tube of 10 mL NB (control) and 10 mL
NB containing 1, 2.5 or 5 g/mL CHX. The cultures were incubated for 24 h at 37°C in a shaking water bath and growth noted. The broth MIC was determined as before. The stability of any increased resistance and the culture purity were checked as before. Cross-resistance to antibiotics and other cationic biocides (P. aeruginosa only) The MICs of the quaternary ammonium compounds (QACs) CPC, BZK and Cet were determined for each resistant strain of P. aeruginosa gained through both the step-wise training (Fig. 1a) and the ‘residual’ concentration (Fig. 1b,c) methods. One microlitre of an overnight culture was inoculated on to diagnostic sensitivity test (DST) agar (Oxoid) plates, containing increasing biocide concentrations, using the Denley multipoint inoculator. All plates were incubated at 37°C for 48 h and growth recorded. The antibiotic sensitivity/resistance profiles of all CHX-resistant P. aeruginosa cultures were evaluated using the disc diffusion method. A 1/100 dilution of an overnight bacterial culture in NB was swabbed onto 20 mL DST agar. After 3–5 min drying, antibiotic discs were applied to the agar surface, using sterile forceps. The plates were left for 15 min at room temperature for prediffusion and then incubated at 37° for 48 h and read.
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Results Stepwise development of resistance: E. coli The resistance of E. coli NCIMB 8879 was not significantly increased by exposure to gradually increasing concentrations of CHX (Table I). The MIC only increased from 0.2 to 0.7 g/mL after two subcultures and reverted to 0.2 g/mL on the third. The resistance was not stable in the absence of CHX. A two-fold increase in MICs occurred with CPC, but no further increase in resistance developed (Table II). Stepwise development of resistance: P. aeruginosa (Fig. 1a) The resistance of P. aeruginosa NCIMB 10421 to CHX increased after step-wise exposure to increasing concentrations. This increase was slow at first and only manifest after 48 h incubation (Table I). After the 4th subculture growth occurred within 24 h with a further increase in MIC, but after the Table I Increases in MIC of CHX seen in P. aeruginosa and E. coli exposed to gradually increasing concentrations of CHX Number of subcultures (at 48 h intervals) 0 1 2 3 4 5 6
MIC (g/mL) of CHX vs. P. aeruginosa
E. coli
8–10† 14 22 28* 40* 50* 70*
0.2† 0.5 0.7 0.2 –‡ –‡ –‡
sixth subculture MICs reached 70 g/mL at which level the CHX caused turbidity in the broth so precluding further tests. CPC could not be tested as it caused clouding in broth at concentrations of 200 g/mL. Agar MICs for CHX-resistant P. aeruginosa All cultures with increases of CHX MICs in broth were checked with agar MICs (Table III). Broth dilution was the more sensitive test. Repeated exposure to ‘residual’ concentrations of biocide (Fig. 1b) The effects of five exposures to a sub-MIC concentration (5 g/mL) of CHX at 24 h intervals on the parent strain and on resistant derivatives obtained from the stepwise training are seen in Table IV. All MICs were increased, the most significant being that for the parent strain where the increase was seven-fold. Further exposure of the step-wise mutants did not result in cultures becoming CHXsusceptible. Repeated exposure of the E. coli cells to Table III Agar MICs of chlorhexidine for P. aeruginosa cultures gained at each stage of the step-wise training method Number of subcultures (at 48 h intervals)
Nutrient broth MIC (g/mL CHX)
Nutrient agar MIC (g/mL CHX)
8–10* 14 22 28 40 50 70
40* 40 40 80 105 120 145
0 1 2 3 4 5 6 * Original MIC.
* These cultures were found to be stable after 15 subcultures in CHX-free nutrient broth and used for further experimentation; † Original MIC; ‡ Not done (see text).
Table IV MICs of P. aeruginosa cultures following repeated exposure to CHX (5g/mL) Culture number
Table II Increases in MIC of CPC seen in E. coli exposed to gradually increasing concentrations of CPC Number of subcultures (at 48 h intervals) 0 1 2
MIC (g/mL) of CPC
1–1.2† 2.0* 1.6–1.8
* This culture was found to be stable after 15 subcultures in CPC-free nutrient broth. † Original MIC.
1* 2 3 4 5
Original MIC (g/mL CHX) before multiple exposure to CHX (5g/mL) 8–10 28† 40† 50† 70†
MIC (g/ml CHX) after five subcultures in CHX (5g/ml) 70‡ 70‡ 70‡ 70‡ 70‡
* Standard parent strain; † Cultures from step-wise training method, trained to higher MIC than standard parent strain; ‡ These cultures were found to be stable after 15 subcultures in CHX-free nutrient broth.
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0.1 g/mL CHX or 0.5 g/mL CPC had no effect on the MIC. Single exposure of P. aeruginosa to a ‘residual’ concentration of biocide (Fig. 1c) The MIC of CHX for the parent strain after a single exposure to various sub-MIC concentrations was increased in a concentration-dependent manner (Fig. 2). Furthermore the resistance generated by exposure to 1 g/mL CHX was stable after 15 subcultures. Cross-resistance of CHX-resistant P. aeruginosa to antibiotics and cationic biocides There was no cross-resistance of the CHX-resistant strains to any of the antibiotics tested (not shown). Table V shows the susceptibility of the CHXresistant strains from stepwise cultures to QACs. Cultures 1–4, 9 had no increase of resistance to BZK, and culture 5 was in fact more susceptible than the parent strain. Cultures 6–8 had a slightly increased resistance, probably due to further treatment with CHX following the stepwise procedure. Table V also shows the results of exposing the CHX-resistant cultures from the ‘residual’ concentration method to QACs. Tests on cetrimide and CPC were difficult as the MICs were already very high. All these cultures were resistant to BZK with MICs of 275–350 g/mL, which compared with 200–230 g/mL for the parent. The cross resistance appeared to depend on the CHX concentration used to generate resistance, and the length of
48 h MIC of CHX (µg/mL)
70 60 50 40 30 20 10 0
1 2.5 5 Concentration (µg/mL) of CHX to which culture exposed
Figure 2 MICs in broth at 37°C of P. aeruginosa following single exposure to ‘residual’ concentrations of CHX of 1, 2.5 and 5 g/mL. , before exposure to CHX; , after exposure to CHX.
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Table V MICs of quaternary ammonium compounds for the CHXresistant P. aeruginosa cultures Culture number
MIC of CHX (g/mL)
Cetrimide
8–10 28 40 50 70 70 70 70 70 70 35–45 60–65 65–70
850 850 850 850 480 850 850 850 850 850 850 850 850
1 2 3 4 5 6 7 8 9 10 11 12 13
48 h MIC (g/mL) of CPC 980 980 980 980 900–980 980 980 980 920–980 980 980 980 980
BZK 200–230 220–240 230–240 220–230 160–190 230–250 250–270 270 210 350 280 275 350
Key of cultures: 1, standard parent strain; 2–5, cultures from step-wise training method; 6, culture 2 following repeated exposure to CHX (5 g/mL); 7, culture 3 following repeated exposure to CHX (5 g/mL); 8, culture 4 following repeated exposure of CHX (5 g/mL); 9, culture 5 following repeated exposure to CHX (5 g/mL); 10, standard parent strain following repeated exposure to CHX (5 g/ml); 11, standard parent strain following single exposure to CHX (1 g/mL); 12, standard parent strain following single exposure to CHX (2.5 g/mL); 13, standard parent strain following single exposure to CHX (5 g/mL).
exposure to it. The cultures exposed to 5 g/mL CHX were the most resistant with BZK MICs of 350 g/mL. Discussion Failure to develop CHX resistance in E. coli by stepwise training was also reported by Fitzgerald et al.11 Although others have claimed that resistance could be generated in E. coli,12 our results are consistent with those of previous workers.13 The rapid increase in CHX resistance in P. aeruginosa following exposure to gradually increasing concentrations of CHX (Fig. 1a, Table I) was also shown by Tattawasart et al.14 although, unlike ours, their resistance was unstable. Nicoletti et al.12 also found an unstable resistance, but the MICs concerned were far greater than those found here. Our CHX-resistant strains were still susceptible to antibiotics, as were those described by Tattawasart et al.14 Furthermore our resistant cultures had no increase in resistance to the QACs (CPC, cetrimide, BZK). It was difficult to estimate MICs of cetrimide and CPC for the CHX-resistant P. aeruginosa cultures as the parent was already very resistant (Table V). Culture 5, however, was more susceptible to both
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cetrimide and BZK than the parent, and the MIC of CPC for Culture 9 was lower. The reasons for these susceptibilities are as yet unclear. Chlorhexidine and the QACs are surface-active agents with similar, but not identical modes of antibacterial action and resistance to one might be thought to imply resistance to all. However for our isolates this was not the case, and in-depth efflux and permeability studies are under way to explore this aspect further. A possible explanation for increased resistance to CHX in P. aeruginosa after step-wise exposure could be physiological adaptation of the envelope to prevent entry of the biocide. This might be due to an increase in cell envelope components such as lipopolysaccharide or phospholipid caused by progressive mutations. Changes of intra-cellular biochemical processes could also account for the resistance. Joynson et al.15 developed step-wise resistance in P. aeruginosa to BZK, but this was unstable resistance to aminoglycosides. It was concurrent with a change in the cell envelope. Exposure of P. aeruginosa (NCIMB 10421) to another QAC also resulted in increased resistance.16 This was associated with reduced susceptibility to CHX, and related to changes in the outer membrane fatty acids. Nonetheless this was not thought to be the only mechanism for the resistance. In our study, a seven-fold increase in MIC of CHX for the parent strain was achieved by repeated exposure to a ‘residual’ or sub-MIC concentration (Fig. 1b, Table IV). Significant resistance was also obtained by a single exposure, the level being concentration-dependent (Fig. 2). However, only the culture obtained after exposure to the lowest concentration of CHX had a stable resistance. Again these ‘residual’ concentration mutants had no increase in antibiotic resistance, but did have cross-resistance to BZK (Table V), which appeared to depend on the concentration of CHX to which the cells had been exposed (higher concentrations giving more resistance). The mechanism of resistance in these mutants probably differs from that in the step-wise mutants, in which there was no cross-resistance to BZK. It could be due to switching of an efflux system. This is more likely to explain resistance after repeated exposure than single exposure, as the resistance following the former was stable. Resistance appeared to depend on the duration of exposure to the antibacterial. Cultures from the stepwise training and the repeated exposure to ‘residual’ concentrations had a stable resistance,
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unlike those obtained from a single exposure to the ‘residual’ concentration (except that exposed to the lowest concentration). This might indicate the presence of different resistance mechanisms, that had been selected as a result of different exposure conditions (i.e. exposure times). Pseudomonas aeruginosa is known to possess efflux pump systems,17 which extrude drugs having the potential to damage the cell. The presence of multi-drug resistance determinants conferring resistance to antibiotics, antiseptics, disinfectants, preservatives, and even intercalating dyes is well documented for Staphylococcus aureus.17–20 Greater attention is being devoted to similar efflux systems in Gram-negative bacteria, but these seem to confer resistance to antibiotics more than biocides.17,21–23 If the efflux pumps in S. aureus have such a wide substrate specificity, it is possible that at least one of those present in P. aeruginosa could confer resistance to biocides as well as antibiotics. Very little information is available of the effects of low concentrations of biocides on bacteria. Irizarry et al.24 claimed that residual levels of disinfectant on surfaces might provide a selective advantage for MRSA strains. This claim has, in part, been confirmed for P. aeruginosa and CHX in this study with a partial resistance to another biocide (BZK) being observed, but without antibiotic resistance. The significance of these findings is as yet unclear, as the in-use CHX concentration is far higher than those at which resistance was obtained. However, it might be possible to train pseudomonads in agar instead of broth to become resistant to higher concentrations of CHX, although the degree of sensitivity might be reduced (Table III). This work does, however, bring to light the possible implications of exposure to low (‘residual’) concentrations of biocide. A study on the use of QACs for disinfection and the occurrence of resistance to BZK found that 30% of strains tested could grow in 200 g/mL BZK, which is the lowest recommended in-use concentration of this commonly-used agent.25 In our study, although the resistance to CHX is below the in-use concentration, the cross-resistance to BZK (275–350 g/mL) is well above the recommended in-use concentration of this biocide. Acknowledgement The authors wish to thank Unitever Research, Colworth and the BBSRC for providing a CASE Industrial Studentship (to LT) for this work.
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References 1. Russell AD, Chopra I. Understanding Antibacterial Action and Resistance, 2nd edn. Hertfordshire: Ellis Horwood 1996; 28–206. 2. McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action and resistance. Clin Microbiol Rev 1999; 12: 147–179. 3. Levy SB. The challenge of antibiotic resistance. Sci Am 1998; 278: 32–39. 4. Martindale: The Extra Pharmacopoeia. Chlorhexidine acetate, gluconate and hydrochloride, 31st edition. London: The Pharmaceutical Press 1996; 1124–1126. 5. The Pharmaceutical Codex: Principles And Practice of Pharmaceutics. Chlorhexidine and other diguanides, 12th edition. Lund W (Ed). London: Pharmaceutical Press 1994; 579–580. 6. Denyer SP. Mechanisms of action of antibacterial biocides. Int Biodeterior Biodegrad 1995; 36: 227–245. 7. Denyer SP, Stewart GSAB. Mechanisms of action of disinfectants. Int Biodeterior Biodegrad 1998; 41: 261–268. 8. Chapman JS. Characterising bacterial resistance to preservatives and disinfectants. Int Biodeterior Biodegrad 1998; 41: 241–245. 9. Reybrouck G. Evaluation of the antibacterial and antifungal activity of disinfectants. In: Russell AD, Hugo WB, Ayliffe GAJ (Eds). Principles and Practice of Disinfection, Preservation and Sterilization, 2nd edn. Oxford: Blackwell Scientific Publications 1992; 114–133. 10. Russell AD. Bacterial resistance to disinfectants: present knowledge and future problems. J Hosp Infect 1999; 43 (supplement): S57–S68. 11. Fitzgerald KA, Davies A, Russell AD. Sensitivity and resistance of Escherichia coli and Staphylococcus aureus to chlorhexidine. Lett Appl Microbiol 1992; 14: 33–36. 12. Nicoletti G, Boghossian V, Gurevitch F, Borland R, Morgenroth P. The antimicrobial activity in vitro of chlorhexidine, a mixture of isothiazolinones (‘Kathon’ CG) and cetyltrimethylammonium bromide (CTAB). J Hosp Infect 1993; 23: 87–111. 13. Walker EM, Lowes JA. An investigation into in vitro methods for the detection of chlorhexidine resistance. J Hosp Infect 1985; 6: 389–397. 14. Tattawasart U, Maillard J-Y, Furr JR, Russell AD. Development of resistance to chlorhexidine diacetate and cetylpyridinium chloride in Pseudomonas stutzeri
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and changes in antibiotic susceptibility. J Hosp Infect 1999; 42: 219–229. Joynson JA, Forbes B, Lambert R. Resistance of Pseudomonas aeruginosa following repeated exposure to sublethal concentrations of benzalkonium chloride. J Pharm Pharmacol 1999; 51: 30. Méchin L, Dubois-Brissonnet F, Heyd B, Leveau JY. Adaptation of Pseudomonas aeruginosa ATCC 15442 to didecyldimethylammonium bromide induces changes in membrane fatty acid composition and in resistance of cells. J Appl Microbiol 1999; 86: 859–866. Paulsen IT, Brown MH, Skurray RA. Proton-dependent multidrug efflux systems. Microbiol Rev 1996; 60: 575–608. Littlejohn TG, Paulsen IT, Gillespie MT et al. Substrate specificity and energetics of antiseptic and disinfectant resistance in Staphylococcus aureus. FEMS Microbiol Lett 1992; 95: 259–266. Heir E, Sundheim G, Holck AL. The qacG gene on plasmid pST94 confers resistance to quaternary ammonium compounds in staphylococci isolated from the food industry. J Appl Microbiol 1999; 86: 378–388. Muñoz-Bellido JL, Alonso Manzanares MA, Martínez Andrés JA et al. Efflux pump-mediated quinolone resistance in Staphylococcus aureus strains wild type for gyrA, gyrB, grlA, and norA. Antimicrob Agents Chemother 1999; 43: 354–356. Poole K, Krebes K, McNally C, Neshat S. Multiple antibiotic resistance in Pseudomonas aeruginosa: Evidence for involvement of an efflux operon. J Bacteriol 1993; 175: 7363–7372. Masuda N, Gotoh N, Ishii C, Sakagawa E, Ohya S, Nishino T. Interplay between chromosomal -lactamase and the MexAB-OprM efflux system in intrinsic resistance to -Lactams in Pseudomonas aeruginosa. Antimicrob Agents Chemother 1999; 43: 400–402. Ziha-Zarifi I, Llanes C, Köhler T, Pechere J-C, Plesiat P. In vivo emergence of multidrug-resistance mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM. Antimicrob Agents Chemother 1999; 43: 287–291. Irizarry L, Merlin T, Rupp J et al. Reduced susceptibility of methicillin-resistant Staphylococcus aureus to cetylpyridinium chloride and chlorhexidine. Chemotherapy 1996; 42: 248–252. Sundheim G, Langsrud S, Heir E, Holck AL. Bacterial resistance to disinfectants containing quaternary ammonium compounds. Int Biodeterior Biodegrad 1998; 41: 235–239.
Development of resistance to chlorhexidine diacetate in Pseudomonas aeruginosa and the effect of a ‘residual’ concentration Louise Thomas, J.-Y. Maillard, R. J.W. Lambert* and A. D. Russell Pharmaceutical Microbiology Research,Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF and *Unilever Research Colworth, Sharnbrook, Bedfordshire, UK
Summary: Stable resistance in Pseudomonas aeruginosa NCIMB 10421 was obtained by step-wise exposure to gradually increasing concentrations of chlorhexidine diacetate (CHX). Repeated exposure to a proposed ‘residual’ (sub-MIC) concentration of CHX also created stable resistance. Resistance was also developed by a single exposure to the ‘residual’ concentration of CHX, but this was unstable. Similar experiments with Escherichia coli and CHX or cetylpyridinium chloride resulted in no significant increase in resistance. Antibiotic susceptibility profiles of the CHX-resistant P. aeruginosa cultures showed no cross-resistance, although some of the cultures were resistant to benzalkonium chloride. © 2000 The Hospital Infection Society
Keywords: Resistance; chlorhexidine; Pseudomonas aeruginosa; residual concentration.
Introduction Antibiotic resistance in bacteria has been known for some time, but remains a major challenge. Even with knowledge of the mode of action of antibiotics, their bacterial targets, and mechanisms of resistance1 the problem persists. Attention is turning to biocides (antiseptics, disinfectants and preservatives) as a possible cause for bacterial resistance to antibiotics.2,3 Chlorhexidine (CHX) is a bisbiguanide antiseptic, disinfectant and preservative effective against a wide range of bacteria, some fungi and some viruses.4 It is available as the acetate, gluconate and hydrochloride, and is widely used in hand washes, dressings and creams, instrument cleaning solutions, mouthwashes, and in combination with cetrimide as a general disinfectant.3,4 Chlorahexidine acts primarily on the bacterial cell membrane5
Received; revised manuscript accepted Author for correspondence: Professor A. D. Russell, Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, UK. E-mail: [email protected]
0195-6701/00/040297;07 $35.00
causing leakage of intracellular material, respiratory inhibition and cytoplasmic coagulation.6,7 Gram-positive bacteria are more susceptible than Gram-negatives, in particular Pseudomonas aeruginosa and Proteus spp.1 The nature of its action explains why the Gram-negatives, especially P. aeruginosa, are intrinsically resistant, as the outer membrane restricts the access of CHX to its target.2 As bacterial resistance has been reported for a large number of biocides, it is important to investigate the reasons for this.8 When biocides are discussed in terms of antibacterial efficacy the ‘in-use’ concentration is normally considered.9 The mode of use could cause emergence of resistance. For example 4% CHX is usually specified, but after use a residual concentration may be far lower, and below the minimum inhibitory concentration (MIC) for some bacteria, thus exposing the bacterial cells to a selective pressure for resistance. Because of the increasing use of biocides in hospitals, industry and even the home,2 we investigated the effects of sub-MIC concentrations of CHX on Gram-negative bacteria, in particular a strain of © 2000 The Hospital Infection Society
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P. aeruginosa, known to possess intrinsic resistance to CHX.10 The effect of CHX on the susceptibility of CHX-resistant strains to antibiotics was also considered. Materials and methods Bacterial strains Two Gram-negative organisms were studied, namely Pseudomonas aeruginosa NCIMB 10421 and Escherichia coli NCIMB 8879. They were maintained on nutrient agar (NA; Oxoid Ltd, Basingstoke, Hampshire, UK) plates at 37°C. Antibacterials Chlorhexidine diacetate (CHX) was purchased from Sigma (Poole, Dorset, UK); cetylpyridinium chloride (CPC) from BDH Laboratories (Poole, Dorset, UK); benzalkonium chloride (BZK) from ICN Chemicals (Cleveland, Ohio, USA) and cetrimide (Cet) from Thornton & Ross Ltd (Huddersfield, UK). Preparation/sterilization of solutions Solutions of all antibacterials were prepared in distilled water and sterilized by filtration before use. Antibiotics Discs were purchased from Difco (now Beckton Dickenson, Cowley, UK), and Becton Dickinson. The following discs were used: gentamicin 10 g; carbenicillin 100 g; polymyxin B 300 u; ciprofloxacin 5 g; tobramycin 10 g; amikacin 30 g; neomycin 30 g and kanamycin 30 g. Determination of MICs Tubes of 10 mL nutrient broth (NB; Oxoid) containing a range of biocide concentrations were inoculated using 10 L of an overnight NB culture of test strain (containing approximately 1–2109 cfu/mL) to give ca. 1–2106 cfu/mL. The tubes were incubated at 37°C for 48 h in a shaking water bath (Gallenkamp, Fisher Scientific, Loughborough, at 100 rev/min) and examined for growth. The lowest concentration of biocide inhibiting growth was recorded as the MIC. Step-wise development of resistance (Fig. 1a) From the tube above the highest CHX concentration permitting growth, a second series of biocide
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concentrations in NB was inoculated with 10 L of bacterial suspension and incubated as before. This step was repeated every 48 h and the MICs noted. Initial CHX concentrations ranged from 0 to 18 g/ml, and then were subsequently increased to 40, 50, 60 and finally 70 g/mL depending upon the growth of ‘resistant’ micro-organisms. These ‘step-wise’ subcultures in increasing concentrations were continued until no further increase in MIC was observed or the concentrated biocide produced clouding in the broth so that growth could no longer be easily observed. The purity of the cultures was checked by streaking on to selective agar (MacConkey agar (Oxoid) and Pseudomonas agar F (Difco) for E. coli and P. aeruginosa respectively). Pseudomonas aeruginosa cultures were also tested using the BBL® Oxi/ ferm™ Tube II kits (Becton Dickinson). Resistance stability The stability of biocide resistance, was determined by continuous subculture of the resistant strains in biocide-free nutrient broth. Subcultures were performed every 24 h for 15 passages and the MIC determined after 1, 5, 10 and 15 passages. A check of culture purity was performed at each stage. Agar MIC determination of CHX-resistant P. aeruginosa cultures The MIC of CHX for the resistant P. aeruginosa cultures gained at each stage was also determined by the agar dilution method. One microlitre of each broth bacterial suspension with increased MIC was inoculated on to nutrient agar (Oxoid) plates containing increasing concentrations of biocide, using a Denley Multipoint Inoculator (Billinghurst, UK). All plates were incubated at 37°C for 48 h, and the MIC noted. Repeated exposure of bacteria to ‘residual’ concentrations of biocide (Fig. 1b) The effect of a proposed ‘residual’ (sub-MIC) concentration of biocide on sensitive and resistant bacteria was determined. First, resistant cells developed through the step-wise training method were exposed to a ‘residual’ concentration of biocide for five subcultures and subsequent MICs were determined. Ten microlitres of cell suspensions showing increase in resistance at each stage of the step-wise method were inoculated into 10 mL NB (control)
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(a) Step-wise development of resistance: P. aeruginosa Original P. aeruginosa culture
Stepwise training with CHX
MICs on agar of CHX, BZK, cetrimide and CPC
(b) Repeated exposure of P. aeruginosa to 'residual' concentrations of biocide Original P. aeruginosa culture
Step-wise trained P. aeruginosa (A)
Five exposures to sub-MIC CHX (5 µg/mL)
Five exposures to sub-MIC CHX (5 µg/mL)
Antibiotic susceptibility test (discs)
Broth MIC of CHX and resistance stability examined
Resistance stability examined
(c) Single exposure of P. aeruginosa to 'residual' concentrations of biocide Original P. aeruginosa culture
Single exposure to CHX (1, 2.5 or 5 µg/mL)
Broth MIC of CHX and resistance stability examined
Susceptibility to QACs and antibiotics determined
Susceptibility to QACs and antibiotics determined
Figure 1
Summary of experiments performed with P. aeruginosa NCIMB 10421.
and 10 mL NB with a known concentration of biocide. These cultures were incubated at 37°C for 24 h in a shaking water bath. Bacterial growth obtained in the broth with biocide was then used to inoculate a second set of tubes containing the same ‘residual’ concentration of biocide. This exposure was repeated for five subcultures and the MIC of the fifth subculture in biocide was determined by the broth-dilution method. The same biocide concentration was used at each subculture: 5 g/mL CHX for P. aeruginosa and 0.1 g/mL CHX or g/mL CPC for E. coli. This experiment was repeated with an overnight, 37°C culture of parent strain containing approximately 1–2109 cfu/mL. Cells were exposed to ‘residual’ concentrations of biocide for five subcultures and MICs in NB were determined. The purity of the cultures was checked at each stage of work as previously described. The stability of any increase in MIC and resistance was also performed as before. Single exposure of bacteria to a ‘residual’ concentration of biocide (P. aeruginosa only) (Fig. 1c) Ten microlitres of an overnight culture at 37°C of the P. aeruginosa parent strain containing approximately 1–2109 cfu/mL were used to inoculate a tube of 10 mL NB (control) and 10 mL
NB containing 1, 2.5 or 5 g/mL CHX. The cultures were incubated for 24 h at 37°C in a shaking water bath and growth noted. The broth MIC was determined as before. The stability of any increased resistance and the culture purity were checked as before. Cross-resistance to antibiotics and other cationic biocides (P. aeruginosa only) The MICs of the quaternary ammonium compounds (QACs) CPC, BZK and Cet were determined for each resistant strain of P. aeruginosa gained through both the step-wise training (Fig. 1a) and the ‘residual’ concentration (Fig. 1b,c) methods. One microlitre of an overnight culture was inoculated on to diagnostic sensitivity test (DST) agar (Oxoid) plates, containing increasing biocide concentrations, using the Denley multipoint inoculator. All plates were incubated at 37°C for 48 h and growth recorded. The antibiotic sensitivity/resistance profiles of all CHX-resistant P. aeruginosa cultures were evaluated using the disc diffusion method. A 1/100 dilution of an overnight bacterial culture in NB was swabbed onto 20 mL DST agar. After 3–5 min drying, antibiotic discs were applied to the agar surface, using sterile forceps. The plates were left for 15 min at room temperature for prediffusion and then incubated at 37° for 48 h and read.
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Results Stepwise development of resistance: E. coli The resistance of E. coli NCIMB 8879 was not significantly increased by exposure to gradually increasing concentrations of CHX (Table I). The MIC only increased from 0.2 to 0.7 g/mL after two subcultures and reverted to 0.2 g/mL on the third. The resistance was not stable in the absence of CHX. A two-fold increase in MICs occurred with CPC, but no further increase in resistance developed (Table II). Stepwise development of resistance: P. aeruginosa (Fig. 1a) The resistance of P. aeruginosa NCIMB 10421 to CHX increased after step-wise exposure to increasing concentrations. This increase was slow at first and only manifest after 48 h incubation (Table I). After the 4th subculture growth occurred within 24 h with a further increase in MIC, but after the Table I Increases in MIC of CHX seen in P. aeruginosa and E. coli exposed to gradually increasing concentrations of CHX Number of subcultures (at 48 h intervals) 0 1 2 3 4 5 6
MIC (g/mL) of CHX vs. P. aeruginosa
E. coli
8–10† 14 22 28* 40* 50* 70*
0.2† 0.5 0.7 0.2 –‡ –‡ –‡
sixth subculture MICs reached 70 g/mL at which level the CHX caused turbidity in the broth so precluding further tests. CPC could not be tested as it caused clouding in broth at concentrations of 200 g/mL. Agar MICs for CHX-resistant P. aeruginosa All cultures with increases of CHX MICs in broth were checked with agar MICs (Table III). Broth dilution was the more sensitive test. Repeated exposure to ‘residual’ concentrations of biocide (Fig. 1b) The effects of five exposures to a sub-MIC concentration (5 g/mL) of CHX at 24 h intervals on the parent strain and on resistant derivatives obtained from the stepwise training are seen in Table IV. All MICs were increased, the most significant being that for the parent strain where the increase was seven-fold. Further exposure of the step-wise mutants did not result in cultures becoming CHXsusceptible. Repeated exposure of the E. coli cells to Table III Agar MICs of chlorhexidine for P. aeruginosa cultures gained at each stage of the step-wise training method Number of subcultures (at 48 h intervals)
Nutrient broth MIC (g/mL CHX)
Nutrient agar MIC (g/mL CHX)
8–10* 14 22 28 40 50 70
40* 40 40 80 105 120 145
0 1 2 3 4 5 6 * Original MIC.
* These cultures were found to be stable after 15 subcultures in CHX-free nutrient broth and used for further experimentation; † Original MIC; ‡ Not done (see text).
Table IV MICs of P. aeruginosa cultures following repeated exposure to CHX (5g/mL) Culture number
Table II Increases in MIC of CPC seen in E. coli exposed to gradually increasing concentrations of CPC Number of subcultures (at 48 h intervals) 0 1 2
MIC (g/mL) of CPC
1–1.2† 2.0* 1.6–1.8
* This culture was found to be stable after 15 subcultures in CPC-free nutrient broth. † Original MIC.
1* 2 3 4 5
Original MIC (g/mL CHX) before multiple exposure to CHX (5g/mL) 8–10 28† 40† 50† 70†
MIC (g/ml CHX) after five subcultures in CHX (5g/ml) 70‡ 70‡ 70‡ 70‡ 70‡
* Standard parent strain; † Cultures from step-wise training method, trained to higher MIC than standard parent strain; ‡ These cultures were found to be stable after 15 subcultures in CHX-free nutrient broth.
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0.1 g/mL CHX or 0.5 g/mL CPC had no effect on the MIC. Single exposure of P. aeruginosa to a ‘residual’ concentration of biocide (Fig. 1c) The MIC of CHX for the parent strain after a single exposure to various sub-MIC concentrations was increased in a concentration-dependent manner (Fig. 2). Furthermore the resistance generated by exposure to 1 g/mL CHX was stable after 15 subcultures. Cross-resistance of CHX-resistant P. aeruginosa to antibiotics and cationic biocides There was no cross-resistance of the CHX-resistant strains to any of the antibiotics tested (not shown). Table V shows the susceptibility of the CHXresistant strains from stepwise cultures to QACs. Cultures 1–4, 9 had no increase of resistance to BZK, and culture 5 was in fact more susceptible than the parent strain. Cultures 6–8 had a slightly increased resistance, probably due to further treatment with CHX following the stepwise procedure. Table V also shows the results of exposing the CHX-resistant cultures from the ‘residual’ concentration method to QACs. Tests on cetrimide and CPC were difficult as the MICs were already very high. All these cultures were resistant to BZK with MICs of 275–350 g/mL, which compared with 200–230 g/mL for the parent. The cross resistance appeared to depend on the CHX concentration used to generate resistance, and the length of
48 h MIC of CHX (µg/mL)
70 60 50 40 30 20 10 0
1 2.5 5 Concentration (µg/mL) of CHX to which culture exposed
Figure 2 MICs in broth at 37°C of P. aeruginosa following single exposure to ‘residual’ concentrations of CHX of 1, 2.5 and 5 g/mL. , before exposure to CHX; , after exposure to CHX.
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Table V MICs of quaternary ammonium compounds for the CHXresistant P. aeruginosa cultures Culture number
MIC of CHX (g/mL)
Cetrimide
8–10 28 40 50 70 70 70 70 70 70 35–45 60–65 65–70
850 850 850 850 480 850 850 850 850 850 850 850 850
1 2 3 4 5 6 7 8 9 10 11 12 13
48 h MIC (g/mL) of CPC 980 980 980 980 900–980 980 980 980 920–980 980 980 980 980
BZK 200–230 220–240 230–240 220–230 160–190 230–250 250–270 270 210 350 280 275 350
Key of cultures: 1, standard parent strain; 2–5, cultures from step-wise training method; 6, culture 2 following repeated exposure to CHX (5 g/mL); 7, culture 3 following repeated exposure to CHX (5 g/mL); 8, culture 4 following repeated exposure of CHX (5 g/mL); 9, culture 5 following repeated exposure to CHX (5 g/mL); 10, standard parent strain following repeated exposure to CHX (5 g/ml); 11, standard parent strain following single exposure to CHX (1 g/mL); 12, standard parent strain following single exposure to CHX (2.5 g/mL); 13, standard parent strain following single exposure to CHX (5 g/mL).
exposure to it. The cultures exposed to 5 g/mL CHX were the most resistant with BZK MICs of 350 g/mL. Discussion Failure to develop CHX resistance in E. coli by stepwise training was also reported by Fitzgerald et al.11 Although others have claimed that resistance could be generated in E. coli,12 our results are consistent with those of previous workers.13 The rapid increase in CHX resistance in P. aeruginosa following exposure to gradually increasing concentrations of CHX (Fig. 1a, Table I) was also shown by Tattawasart et al.14 although, unlike ours, their resistance was unstable. Nicoletti et al.12 also found an unstable resistance, but the MICs concerned were far greater than those found here. Our CHX-resistant strains were still susceptible to antibiotics, as were those described by Tattawasart et al.14 Furthermore our resistant cultures had no increase in resistance to the QACs (CPC, cetrimide, BZK). It was difficult to estimate MICs of cetrimide and CPC for the CHX-resistant P. aeruginosa cultures as the parent was already very resistant (Table V). Culture 5, however, was more susceptible to both
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cetrimide and BZK than the parent, and the MIC of CPC for Culture 9 was lower. The reasons for these susceptibilities are as yet unclear. Chlorhexidine and the QACs are surface-active agents with similar, but not identical modes of antibacterial action and resistance to one might be thought to imply resistance to all. However for our isolates this was not the case, and in-depth efflux and permeability studies are under way to explore this aspect further. A possible explanation for increased resistance to CHX in P. aeruginosa after step-wise exposure could be physiological adaptation of the envelope to prevent entry of the biocide. This might be due to an increase in cell envelope components such as lipopolysaccharide or phospholipid caused by progressive mutations. Changes of intra-cellular biochemical processes could also account for the resistance. Joynson et al.15 developed step-wise resistance in P. aeruginosa to BZK, but this was unstable resistance to aminoglycosides. It was concurrent with a change in the cell envelope. Exposure of P. aeruginosa (NCIMB 10421) to another QAC also resulted in increased resistance.16 This was associated with reduced susceptibility to CHX, and related to changes in the outer membrane fatty acids. Nonetheless this was not thought to be the only mechanism for the resistance. In our study, a seven-fold increase in MIC of CHX for the parent strain was achieved by repeated exposure to a ‘residual’ or sub-MIC concentration (Fig. 1b, Table IV). Significant resistance was also obtained by a single exposure, the level being concentration-dependent (Fig. 2). However, only the culture obtained after exposure to the lowest concentration of CHX had a stable resistance. Again these ‘residual’ concentration mutants had no increase in antibiotic resistance, but did have cross-resistance to BZK (Table V), which appeared to depend on the concentration of CHX to which the cells had been exposed (higher concentrations giving more resistance). The mechanism of resistance in these mutants probably differs from that in the step-wise mutants, in which there was no cross-resistance to BZK. It could be due to switching of an efflux system. This is more likely to explain resistance after repeated exposure than single exposure, as the resistance following the former was stable. Resistance appeared to depend on the duration of exposure to the antibacterial. Cultures from the stepwise training and the repeated exposure to ‘residual’ concentrations had a stable resistance,
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unlike those obtained from a single exposure to the ‘residual’ concentration (except that exposed to the lowest concentration). This might indicate the presence of different resistance mechanisms, that had been selected as a result of different exposure conditions (i.e. exposure times). Pseudomonas aeruginosa is known to possess efflux pump systems,17 which extrude drugs having the potential to damage the cell. The presence of multi-drug resistance determinants conferring resistance to antibiotics, antiseptics, disinfectants, preservatives, and even intercalating dyes is well documented for Staphylococcus aureus.17–20 Greater attention is being devoted to similar efflux systems in Gram-negative bacteria, but these seem to confer resistance to antibiotics more than biocides.17,21–23 If the efflux pumps in S. aureus have such a wide substrate specificity, it is possible that at least one of those present in P. aeruginosa could confer resistance to biocides as well as antibiotics. Very little information is available of the effects of low concentrations of biocides on bacteria. Irizarry et al.24 claimed that residual levels of disinfectant on surfaces might provide a selective advantage for MRSA strains. This claim has, in part, been confirmed for P. aeruginosa and CHX in this study with a partial resistance to another biocide (BZK) being observed, but without antibiotic resistance. The significance of these findings is as yet unclear, as the in-use CHX concentration is far higher than those at which resistance was obtained. However, it might be possible to train pseudomonads in agar instead of broth to become resistant to higher concentrations of CHX, although the degree of sensitivity might be reduced (Table III). This work does, however, bring to light the possible implications of exposure to low (‘residual’) concentrations of biocide. A study on the use of QACs for disinfection and the occurrence of resistance to BZK found that 30% of strains tested could grow in 200 g/mL BZK, which is the lowest recommended in-use concentration of this commonly-used agent.25 In our study, although the resistance to CHX is below the in-use concentration, the cross-resistance to BZK (275–350 g/mL) is well above the recommended in-use concentration of this biocide. Acknowledgement The authors wish to thank Unitever Research, Colworth and the BBSRC for providing a CASE Industrial Studentship (to LT) for this work.
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