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Chloroxylenol- and triclosan-tolerant bacteria from industrial sources—susceptibility to antibiotics and other biocides
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International Biodeterioration & Biodegradation 57 (2006) 51–56 www.elsevier.com/locate/ibiod
Chloroxylenol- and triclosan-tolerant bacteria from industrial sources—susceptibility to antibiotics and other biocides J.C. Leara, J.-Y. Maillarda,, P.W. Dettmarb, P.A. Goddardb, A.D. Russella,{ a
Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, UK b Reckitt Benckiser Healthcare, Dansom Lane, Hull HU8 7DS, UK
Received 28 August 2005; received in revised form 27 October 2005; accepted 4 November 2005 Available online 13 December 2005
Abstract This study aimed to determine the degree of susceptibility of several industrial bacterial isolates tolerant to para-chloro-meta-xylenol, triclosan, three other commonly used biocides, and to a range of clinically significant antibiotics. Minimum inhibitory concentrations (MICs) of benzalkonium chloride, chlorhexidine and phenol were determined and compared with those of standard strains. When elevated MICs were exhibited, suspension efficacy tests were carried out to assess the lethal efficacy of these biocides. Antibiotic sensitivity profiles were initially determined by disk diffusion testing, and antibiotic MICs by a gradient plate method. Biocide MICs were largely similar between industrial and standard strains, although isolates of Acinetobacter johnsonii showed elevated benzalkonium chloride MICs, which were not reflected in terms of lethal effects. Antibiotic sensitivities did not vary greatly between strains. An industrial triclosan-tolerant strain of Citrobacter freundii showed a slightly higher resistance to some antibiotics, but in most cases this was recorded as ‘‘sensitive’’ according to the guidelines used. Chloramphenicol resistance was shown in the triclosan-tolerant strain of A. johnsonii, but also in the sensitive revertant strain, suggesting that this property is intrinsic to the strain and unrelated to triclosan tolerance. This study did not produce evidence suggesting that tolerance to triclosan or para-chloro-meta-xylenol in industrial bacterial isolates promotes the emergence of tolerance to other biocides or increases resistance to antibiotics. r 2005 Elsevier Ltd. All rights reserved. Keywords: Antimicrobial; Resistance; Industrial isolates; Biocides
1. Introduction As usage of biocidal products continues to increase, interest grows in the concept of bacterial resistance to these biocides (Russell, 1999). One particularly important aspect is that of cross- and co-resistance, whereby resistance to a particular biocide may result in a reduced susceptibility to another, and sometimes to chemically unrelated antibiotics. Important antibiotic resistance mechanisms such as impaired uptake (cellular impermeability or drug efflux), drug inactivation and mutation at the target site, can also apply to biocides (Russell, 1999), and it has been suggested that biocides could select for antibiotic-resistant strains if shared targets or common resistance mechanisms were Corresponding author. Tel.: +44 29 2087 9088; fax: +44 29 2087 4149.
E-mail address: [email protected] (J.-Y. Maillard). Deceased.
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0964-8305/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2005.11.002
involved (Suller and Russell, 2000; Gilbert and McBain, 2001). Indeed, various examples of cross-resistance between biocides (Russell et al., 1998; Loughlin et al., 2002) and between biocides and antibiotics (Lambert et al., 2001; Braoudaki and Hilton, 2004) have been reported in the literature. However, it is important to note that other researchers have not been able to demonstrate such a relationship (Lambert, 2004; Gradel et al., 2005). Other issues, such as biocide susceptibility in methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci have also been subjects of conflicting reports (Haines et al., 1997). The concept of linked biocide and antibiotic resistance remains contentious (Russell, 2000), but given the current global problem of antibiotic resistance (Levy, 1998), it is clear that continued studies in this area are essential (Fraise, 2002). Studies on the widely used phenyl ether triclosan have fuelled concerns over linked biocide and antibiotic
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resistance. This compound appears to be a substrate of multidrug efflux pumps such as AcrAB in Escherichia coli and MexAB-OprM in Pseudomonas aeruginosa, and crossresistance between triclosan and antibiotics has been demonstrated in the laboratory (McMurry et al., 1998a; Schweizer, 1998). Furthermore, triclosan inhibits enoyl reductase enzymes involved in bacterial fatty acid synthesis such as FabI in E. coli (McMurry et al., 1998b) and InhA in mycobacteria (Parikh et al., 2000) and there is some evidence that cross-resistance between triclosan and other enoyl-reductase inhibitors such as diazaborines, hexachlorophene and isoniazid could occur (Heath et al., 2000; McMurry et al., 1998b; Parikh et al., 2000). In a previous study by this research group (Lear et al., 2002), bacterial strains were collected from sites of likely triclosan and/or para-chloro-meta-xylenol exposure in two biocidal product manufacturing facilities, and identified. The aim of this study was to determine the industrial strains’ susceptibility profile to other commonly used biocides and to clinically relevant antibiotics. Any relationship in susceptibility to these agents could then be considered.
shown by streaking samples on to plain tryptone soya agar plates. Similarly, solutions of the water-soluble biocides (benzalkonium chloride, chlorhexidine and phenol) were prepared at 20 or 100 final concentration in distilled water and sterilised by filtration through 0.2 mm membrane filters (Sartorius, Go¨ttingen, Germany). Antibiotic disks of amikacin (30 mg), carbenicillin (100 mg), ceftazidime (30 mg), cefuroxime (30 mg), chloramphenicol (30 mg), ciprofloxacin (5 mg), erythromycin (15 mg), gentamicin (10 mg), kanamycin (30 mg), neomycin (30 mg), piperacillin (100 mg), polymyxin B (300 mg), tetracycline (30 mg) and tobramycin (10 mg) were purchased from Becton Dickinson (Oxford, UK). Antibiotic powders were obtained from Sigma.
2.2. Bacterial isolates The characterisation profiles of the triclosan-tolerant industrial isolates of Acinetobacter johnsonii, Citrobacter freundii, and para-chloro-metaxylenol-tolerant strains of P. aeruginosa and P. stutzeri are presented in Table 1. An additional A. johnsonii strain which lost its triclosan tolerance following 15 subcultures in the absence of the biocide (referred to as the triclosan-sensitive revertant; Table 1) was also examined. All organisms were studied together with their corresponding standard strains obtained from the National Collection of Industrial and Marine Bacteria (NCIMB; Aberdeen, UK; Table 1). These strains were selected according to details supplied by NCIMB, and were the type strains of each species with the exception of P. aeruginosa NCIMB 10421, which was the strain recommended for disinfectant testing according to the Standard BS EN 1276 (CEN, 1997).
2. Materials and methods 2.1. Chemicals
2.3. Susceptibility to other biocides—minimum inhibitory concentrations (MICS)
Chlorhexidine diacetate and dimethylsulfoxide were purchased from Sigma (Poole, UK), the quaternary ammonium compound benzalkonium chloride from ICN Biochemicals (Cleveland, USA) and phenol from BDH Chemicals (Poole, UK). Triclosan and para-chloro-meta-xylenol were kindly supplied by Ciba Specialty Chemicals, (Basel, Switzerland) and Reckitt Benckiser (Hull, UK), respectively. Solutions of triclosan and para-chloro-meta-xylenol were prepared in dimethylsulfoxide at 20 or 100 the desired final concentrations according to the range being tested. Such solutions were always sterile, as
Plates containing test concentrations of biocides were prepared by the addition of 1.0 or 0.2 ml of the appropriate solutions to 19.0 or 19.8 ml volumes of molten extra-strength tryptone soya agar (Oxoid, Basingstoke, UK), mixed well, poured and allowed to set. In the case of triclosan and para-chloro-meta-xylenol, control experiments demonstrated that the final concentrations of dimethylsulfoxide (1% or 5%) were not inhibitory to the strains tested. Plates were inoculated, incubated and colonies enumerated as described by Lear et al. (2002). The lowest concentration inhibiting bacterial growth was taken as the MIC, and values were compared with
Table 1 Triclosan, para-chloro-meta-xylenol, chlorhexidine, benzalkonium chloride and phenol MIC values for strains of A. johnsonii, C. freundii, P. aeruginosa and P. stutzeri Straina
MIC (mg ml 1)b Triclosan
PCMX
BZK
CHX
Phenol
Triclosan-tolerant, revertant and industrial strains A. johnsonii S (NCIMB 12460) 4–5 A. johnsonii RS 4–5 A. johnsonii IS 4100c C. freundii SS (NCIMB 11490) 4100c C. freundii IS 4100c
80–85 NT 80–85 165–170 180–185
30–40 130–140 120–130 120–130 180–190
2.0–2.2 2.8–3.0 2.4–2.6 4–5 8–11
1100–1200 1100–1200 1100–1200 1500–1600 1500–1600
Para-chloro-meta-xylenol-tolerant and industrial strains P. aeruginosa SS (NCIMB 10421) 4100c P. aeruginosa IS 4100c P. stutzeri SS (NCIMB 11358) o5 P. stutzeri IS o5
41000c 41000c 200–220 465–470
330–340 300–310 180–190 180–190
60–85 35–45 3–5 5–6
900–1000 900–1000 1400–1500 1400–1500
Source site of isolation, incubation temperature and stability of tolerance are described in Lear et al. (2002). Note that C. freundii SS and IS, were tolerant to the lethal effect of triclosan and P. aeruginosa SS and IS to that of para-chloro-meta-xylenol Lear et al. (2002). a SS, standard strain; RS, revertant strain; IS, industrial triclosan- or para-chloro-meta-xylenol-tolerant strain. b PCMX: para-chloro-meta-xylenol; BZK: benzalkonium chloride; CHX: chlorhexidine diacetate. c 4: MIC value quoted as this was the highest biocide concentration achievable in agar; NT: Not tested.
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2.4. Susceptibility to other biocides—lethal effects The lethal effect of biocides was investigated with a suspension test as described by Lear et al. (2002) when significant MIC differences were demonstrated between the industrial and standard or revertant strains. The neutralizer (5% Tween 80, 1.5% lecithin; Sigma) used to quench the activity of the biocides was tested as follows: 1 ml biocide solution at 10 final concentration was added to 8 ml neutraliser, followed by 1 ml bacterial suspension. Following a 5-min contact period at 22 1C, surviving bacteria were serially diluted and counted as described above. The use of distilled water instead of biocide solution enabled log reductions in cfu ml 1 to be calculated. The potential toxicity of the neutraliser was assessed using the following protocol: 1 ml bacterial suspension was added to 9 ml extrastrength (i.e. 10/9th) neutraliser and, following a contact time of 5 min at 22 1C, survivors were serially diluted and counted as described above. For the calculation of the reduction in bacterial concentration, 9 ml distilled water was used instead of the neutraliser. The toxicity of 1% or 5% dimethylsulfoxide was also assessed using this protocol.
2.5. Susceptibility to antibiotics—disk diffusion method Antibiotic sensitivity profiles were determined using a method based upon the Comparative Method of the British Society for Antimicrobial Chemotherapy (BSAC, 1991). Overnight tryptone soya broth suspensions adjusted to 1 109 cfu ml 1 (1 108 cfu ml 1 for A. johnsonii strains) were swabbed on to diagnostic sensitivity testing agar (Oxoid, Basingstoke, UK) using the ‘‘wet swab’’ method (BSAC, 1991). Undiluted suspensions were used, as dilutions did not produce acceptable semi-confluent growth. Antibiotic disks were immediately placed on the agar with a mechanical dispenser (Becton Dickinson). The plates were then incubated at 22 1C for 48 h or 37 1C for 24 h as appropriate, and diameters of inhibition zones measured and interpreted according to established criteria (BSAC, 1991).
2.6. Susceptibility to antibiotics—gradient plate MICs If industrial triclosan- or para-chloro-meta-xylenol-tolerant strains exhibited ‘‘intermediate’’ or ‘‘resistant’’ results compared with standard or revertant strains in disk diffusion testing, antibiotic MICs were determined on diagnostic sensitivity testing agar using the gradient plate technique (Szybalski, 1952). Chloramphenicol stock solutions were prepared in absolute ethanol. A 0.2 ml aliquot was added to 19.8 ml of molten extrastrength diagnostic sensitivity testing agar. A control experiment established that 1% ethanol (the highest concentration used) was noninhibitory. All other antibiotics dissolved satisfactorily in distilled water and stock solutions were sterilised by filtration through 0.2 mm membrane filters (Sartorius). Gradient plates were prepared by pouring 20 ml diagnostic sensitivity testing agar containing the maximum final antibiotic concentration into a Petri dish, and allowing the agar to set with the plate set at an angle. Once set, the plates were returned to the normal level position and a further 20 ml of plain diagnostic sensitivity testing agar was added to each plate. A diffusion period of 2 h allowed an antibiotic concentration gradient to form across the plate. A 10-mL aliquot of an overnight tryptone soya broth suspension was then streaked along the antibiotic gradient with a sterile swab. Following incubation at 22 1C for 48 h or 37 1C for 24 h as required, the length of confluent growth along the concentration gradient was measured. MIC values of standard, revertant and industrial tolerant strains were compared with National Committee for Clinical Laboratory Standards (NCCLS) interpretive standards for dilution testing (Jorgensen et al., 1999) and MIC thresholds denoting sensitivity (Martindale, 1995).
2.7. Statistical analysis Five replicates were conducted for all experiments except biocide MIC determinations, where there were three replicates. Results of the suspension tests and antibiotic MICs were compared using two sample t-tests (TT), Mann–Whitney U-test (MWU) or one-way ANOVA as appropriate with Minitabs Release 13 software (Minitabs, PA, USA).
3. Results 3.1. Susceptibility to other biocides—MICs Agar MIC values of benzalkonium chloride, chlorhexidine and phenol for all strains were fairly variable (Table 1), with some industrial strains, such as the industrial triclosan-tolerant C. freundii, showing slightly elevated values compared with their standard counterparts and others appearing marginally more sensitive than their standard strains, e.g. industrial para-chloro-meta-xylenoltolerant P. aeruginosa. However, the industrial triclosantolerant and revertant sensitive strains of A. johnsonii exhibited considerably higher MICs for benzalkonium chloride than the standard strain (Table 1). 3.2. Susceptibility to other biocides—lethal effects Control experiments established that the neutralisers used in this study were not toxic to the test strains and effectively quenched biocidal activity (data not shown). Suspension tests were conducted with benzalkonium chloride and strains of A. johnsonii only (Fig. 1). They were significantly lower than the standard strain when exposed to benzalkonium chloride at 5 mg ml 1. Although the log reductions in bacterial number of the industrial and revertant strains were not significantly different from each other. However, such difference in inactivation was not shown at higher concentrations. The log reductions of the three strains following exposure to benzalkonium chloride at 10 mg ml 1 were not significantly different and at 20 mg ml 1 a44 log reduction was observed in all strains. 5.00
Log Reduction (cfu ml-1) ± SD
published data (Hammond et al., 1987; Ranganathan, 1996; Kibbe, 2000; Weller, 2000).
53
>4
4.00
3.00
2.00
1.00
0.00 55
10
20
Concentration of BZK (µg ml-1)
Fig. 1. Lethal effects of BZK against A. johnsonii. Standard strain (J); revertant strain (&) and industrial triclosan-tolerant strain (m).
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3.3. Susceptibility to antibiotics—disk diffusion method
B, tetracycline and tobramycin were employed for C. freundii strains, and chloramphenicol and P. stutzeri and A. johnsonii strains, since these were the organisms and antibiotics where ‘‘intermediate’’ or ‘‘resistant’’ results were obtained when compared with the standard strains (Table 2). Amikacin, ceftazidime and ciprofloxacin (for C. freundii) were not tested as antibiotic powders were unobtainable at the time of study. For all antibiotics tested the industrial triclosan-tolerant strain of C. freundii exhibited higher MIC values than the standard strain (Table 3). With the exception of tetracycline these differences were all significant. However, although higher, these MIC values were all designated ‘‘sensitive’’ under the listed guidelines (Martindale, 1995; Jorgensen et al., 1999). The chloramphenicol MIC of the industrial para-chlorometa-xylenol-tolerant strain of P. stutzeri was significantly higher than that of the standard strain (Table 4). However, both MIC values were high, and both fell into the ‘‘resistant’’ category for both sets of guidelines consulted. MICs of chloramphenicol against A. johnsonii strains were significantly lower for the standard strain than for the industrial triclosan-tolerant strain, and interestingly, the
Largely, the antibiotic susceptibility profiles did not differ between standard strains and their industrial triclosan- or para-chloro-meta-xylenol-tolerant counterparts, each producing a ‘‘sensitive’’ result (Table 2). Where resistance did occur it was also nearly always demonstrated by both standard and industrial strains, namely with C. freundii to erythromycin, P. aeruginosa to chloramphenicol, erythromycin and kanamycin, and P. stutzeri to cefuroxime (Table 2). However, industrial and revertant strains of A. johnsonii showed intermediate resistance to chloramphenicol when compared with the standard strain, and the industrial strain of C. freundii was intermediately resistant to several of the antibiotics and resistant to polymyxin B. The industrial strain of P. stutzeri was also resistant to chloramphenicol (Table 2). 3.4. Susceptibility to antibiotics—gradient plate MICs Gradient plates for carbenicillin, cefuroxime, gentamicin, kanamycin, neomycin, piperacillin, polymyxin Table 2 Antibiotic sensitivity profiles of bacterial strains Straina
Susceptibility (Sensitive (S), intermediate (I) or resistant (R) to antibiotic AN
CB
CAZ
CXM
C
CIP
E
GM
K
N
PIP
PB
TE
NN
Triclosan-tolerant, revertant and standard strains A. johnsonii SS S S S A. johnsonii RS S S S A. johnsonii IS S S S C. freundii SS S S S C. freundii IS I I I
S S S S I
S I I S S
S S S S I
S S S R R
S S S S I
S S S S I
S S S S I
S S S S I
S S S S R
S S S S I
S S S S I
Para-chloro-meta-xylenol-tolerant and standard P. aeruginosa SS S S P. aeruginosa IS S S P. stutzeri SS S S P. stutzeri IS S S
R R R R
R R S R
S S S S
R R S S
S S S S
R R S S
S S S S
S S S S
S S S S
S S S S
S S S S
strains S S S S
AN, amikacin; CB, carbenicillin; CAZ, ceftazidime; CXM, cefuroxime; C, chloramphenicol; CIP, ciprofloxacin; E, erythromycin; GM, gentamicin; K, kanamycin; N, neomycin; PIP, piperacillin; PB, polymyxin B; TE, tetracycline; NN, tobramycin. Note: Antibiotic sensitivity profiles were determined using a method based upon the Comparative Method of the British Society for Antimicrobial Chemotherapy (1991). a SS, standard strain; RS, revertant strain; IS, industrial triclosan- or para-chloro-meta-xylenol-tolerant strain.
Table 3 Gradient plate antibiotic MICs for C. freundii standard strain NCIMB 11490 and its industrial triclosan-tolerant isolate MIC (7 SD) mg ml
Standard strain Triclosan-tolerant isolate
1
CB
CXM
GM
K
N
PIP
PB
TE
NN
3.96 (0.38) 4.82 (0.20)
1.88 (0.10) 3.15 (0.23)
1.33 (0.08) 2.15 (0.10)
3.11 (0.07) 4.01 (0.27)
3.19 (0.28) 4.13 (0.13)
2.78 (0.14) 3.35 (0.57)
1.11 (0.13) 1.91 (0.31)
2.67 (0.14) 2.73 (0.10)
1.08 (0.14) 1.54 (0.10)
CB, carbenicillin; CXM, cefuroxime; GM, gentamicin; K, kanamaycin; N, neomycin; PIP, piperacillin; PB, polymyxin B; TE, tetracycline; NN, tobramycin.
ARTICLE IN PRESS J.C. Lear et al. / International Biodeterioration & Biodegradation 57 (2006) 51–56 Table 4 Gradient plate MICs of chloramphenicol for P. stutzeri standard strain NCIMB 11358 and its para-chloro-meta-xylenol-tolerant isolate, and A. johnsonii standard strain NCIMB 12460, its revertant and industrial triclosan-tolerant isolates MIC mg ml 1 (7 SD) Choramphenicol P. stutzeri Standard strain Para-chloro-meta-xylenol-tolerant isolate A. johnsonii Standard strain Revertant Industrial triclosan-tolerant isolates
77.63 (13.66) 109.80 (4.47) 4.97 (2.10) 33.90 (12.74) 32.10 (2.88)
triclosan-sensitive revertant strain (Table 4). There was no significant difference between the latter two strains. 4. Discussion Most triclosan- and para-chloro-meta-xylenol-tolerant strains did not appear to show increased tolerance to benzalkonium chloride, chlorhexidine or phenol, and MICs of these agents correlated well with published data (Hammond et al., 1987; Ranganathan, 1996; Kibbe, 2000; Weller, 2000). Similarly, there was little evidence of crossresistance when antibiotic sensitivity profiles were investigated, with largely ‘‘sensitive’’ results for all strains. Nonetheless, some cases of reduced susceptibility were observed. The industrial strain of C. freundii showed higher levels of resistance to all the antibiotics tested, both in disk diffusion and MIC testing, although these largely significantly higher MICs were all within threshold values for sensitivity. Higher MIC values were also noted for benzalkonium chloride and chlorhexidine when biocides were tested (Table 1). Reduced susceptibility to such a variety of agents with differing modes of action suggests a non-specific mechanism of resistance such as increased impermeability due to outer membrane adaptation (Nikaido, 1994; Tattawasart et al., 2000). The industrial triclosan-tolerant strain of A. johnsonii showed an elevated benzalkonium chloride MIC in comparison with the standard strain, but this property was also shown by the triclosan-sensitive revertant. This pattern of results was also shown for chloramphenicol, both in disk diffusion and MIC testing. This revertant strain was originally produced from the industrial isolate, having lost tolerance to triclosan after repeated subculture in the absence of the biocide (Lear et al., 2002). Benzalkonium chloride tolerance and chloramphenicol resistance were evidently retained in the absence of selective pressure from triclosan. This would suggest that the reduced susceptibilities may be intrinsic features of the industrial and revertant strains, and not ‘‘linked’’ to
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triclosan tolerance as such. Furthermore, the elevated benzalkonium chloride MICs were not reflected in terms of sensitivity to the lethal effect of the biocide, except where the very low concentration of 5 mg ml 1 was used. In addition, a concentration of 20 mg ml 1 produced a44 log reduction in concentration within 5 min and therefore the tolerance of these isolates to benzalkonium chloride, in terms of MIC only, is unlikely to be of significance in practice. Differences between effective concentrations reported with suspension tests or in agar for a specific biocide have been discussed previously (Lear et al., 2002). In disk diffusion testing, the industrial para-chloro-metaxylenol-tolerant strain of P. stutzeri showed a ‘‘resistant’’ response to chloramphenicol, compared with ‘‘sensitive’’ for the standard strain. Although a higher chloramphenicol MIC was also observed later, both MICs were categorised as ‘‘resistant’’ under the guidelines used. It must be borne in mind that different methodologies and resistance thresholds may produce such differences in results, but again the MIC result suggests that chloramphenicol resistance is an intrinsic feature of both strains and is unrelated to triclosan tolerance. Hence, the only possible association observed, although not linked to triclosan tolerance, was that between benzalkonium chloride and chloramphenicol in the A. johnsonii strains. Such crossresistance has previously been documented in P. aeruginosa (Loughlin et al., 2002) and E. coli (Langsrud et al., 2004). Resistance to chloramphenicol has been associated with a loss of outer membrane porin proteins in Gram-negative bacteria (Toro et al., 1990), and the resulting lack of permeability or uptake could confer some degree of resistance to biocides. Drug expulsion by efflux has also been linked to chloramphenicol and benzalkonium chloride cross-resistance in E. coli (Langsrud et al., 2004). In summary, the triclosan and para-chloro-meta-xylenoltolerant industrial strains of bacteria investigated in this study did not demonstrate relevant tolerance in practice to other biocides or cross-resistance to the antibiotics tested. Where some decrease in susceptibility was demonstrated it appeared to be innate to the microorganisms, relatively insignificant and/or not ‘‘linked’’ with previously observed biocide tolerance per se. Given the debate over such issues (Schweizer, 1999; Russell, 2000; Fraise, 2002), these are interesting and significant results. Instances of linked biocide and antibiotic resistance (Sanchez et al., 2005), and the lack of it (Reynaldo et al., 2004), continue to appear in the literature. However, there is currently no evidence that the phenomenon has caused a real problem in practice (Gilbert and McBain, 2003). Nonetheless, it is clear that continued investigation of biocide and antibiotic resistance is essential in order to monitor this phenomenon (Fraise, 2002). Acknowledgements The authors would like to thank Reckitt Benckiser Healthcare, Hull, UK for their professional and financial
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support provided for this study, including a research studentship to JCL. The authors want to acknowledge the contribution of Prof. AD Russell to this manuscript. Sadly Prof. AD Russell passed away in September 2004. References Braoudaki, M., Hilton, A.C., 2004. Adaptive resistance to biocides in Salmonella enterica and Escherichia coli 0157 and cross-resistance to antimicrobial agents. Journal of Clinical Microbiology 42, 73–78. British Society for Antimicrobial Chemotherapy (BSAC), 1991. A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27 (Suppl.), 1–50. Comite´ Europe´en de Normalisation, 1997. Chemical disinfectants and antiseptics—quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas–test method (phase 2, step 1), BS EN 1276. British Standards Institution, London, UK. Fraise, A.P., 2002. Biocide abuse and antimicrobial resistance—a cause for concern? Journal of Antimicrobial Chemotherapy 49, 11–12. Gilbert, P., McBain, A.J., 2001. Biocide usage in the domestic setting and concern about antibacterial and antibiotic resistance. Journal of Infection 43, 85–91. Gilbert, P., McBain, A.J., 2003. Potential impact of increased use of biocides in consumer products on prevalence of antibiotic resistance. Clinical Microbiology Review 16, 189–208. Gradel, K.O., Randall, L., Sayers, A.R., Davies, R.H., 2005. Possible associations between Salmonella persistence in poultry houses and resistance to commonly used disinfectants and a putative role of mar. Veterinary Microbiology 107, 127–138. Haines, K.A., Klein, D.A., McDonnell, G., Pretzer, D., 1997. Could antibiotic-resistant pathogens be cross-resistant to hard surface disinfectants? American Journal of Infection Control 25, 439–441. Hammond, S.A., Morgan, J.R., Russell, A.D., 1987. Comparative susceptibility of hospital isolates of Gram-negative bacteria to antiseptics and disinfectants. Journal of Hospital Infection 9, 255–264. Heath, R.J., Roland, G.E., Rock, C.O., 2000. Inhibition of the Staphylococcus aureus NADPH-dependent enoyl-acyl carrier reductase by triclosan and hexachlorophene. Journal of Biological Chemistry 275, 4654–4659. Jorgensen, J.H., Turnidge, J.D., Washington, J.A., 1999. Antibacterial susceptibility tests: dilution and disk diffusion methods. In: Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., Yolken, R.H. (Eds.), Manual of Clinical Microbiology, seventh ed. ASM Press, Washington, DC, pp. 1526–1543. Kibbe, A.H., 2000. Benzalkonium chloride. In: Kibbe, A.H. (Ed.), Handbook of Pharmaceutical Excipients, third ed. Pharmaceutical Press, London, pp. 33–35. Lambert, R.J.W., 2004. Comparative analysis of antibiotic and antimicrobial biocide susceptibility data in clinical isolates of methicillinsensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa between 1989 and 2000. Journal of Applied Microbiology 97, 699–711. Lambert, R.J.W., Joynson, J., Forbes, B., 2001. The relationships and susceptibilities of some industrial, laboratory and clinical isolates of Pseudomonas aeruginosa to some antibiotics and biocides. Journal of Applied Microbiology 91, 972–984. Langsrud, S., Sundheim, G., Holck, A.L., 2004. Cross-resistance to antibiotics of Escherichia coli adapted to benzalkonium chloride or exposed to stress inducers. Journal of Applied Microbiology 96, 201–208. Lear, J.C., Maillard, J.-Y., Dettmar, P.W., Goddard, P.A., Russell, A.D., 2002. Chloroxylenol- and triclosan-tolerant bacteria from
industrial sources. Industrial Microbiology and Biotechnology 29, 238–242. Levy, S.B., 1998. The challenge of antibiotic resistance. Scientific American March, 46–53. Loughlin, M.F., Jones, M.V., Lambert, P.A., 2002. Pseudomonas aeruginosa cells adapted to benzalkonium chloride show resistance to other membrane-active agents but not to clinically relevant antibiotics. Journal of Antimicrobial Chemotherapy 49, 631–639. Martindale, W., 1995. Antibacterial agents. In: Reynolds, J.E.F., Parfitt, K., Parsons, A.V., Sweetman, S.C. (Eds.), Martindale, The Extra Pharmacopoeia. Pharmaceutical Press, London, pp. 79–224. McMurry, L.M., Oethinger, M., Levy, S.B., 1998a. Overexpression of marA, soxS or acrAB produces resistance to triclosan and laboratory and clinical strains of Escherichia coli. FEMS Microbiology Letters 166, 305–309. McMurry, L.M., Oethinger, M., Levy, S.B., 1998b. Triclosan targets lipid synthesis. Nature 394, 531–532. Nikaido, H., 1994. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264, 382–388. Parikh, S.L., Xiao, G., Tonge, P.J., 2000. Inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis, by triclosan and isoniazid. Biochemistry 39, 7645–7650. Ranganathan, N.S., 1996. Chlorhexidine. In: Ascenzi, J.M. (Ed.), Handbook of Disinfectants and Antiseptics. Dekker Inc., New York, pp. 235–264. Reynaldo, M.B., Flores, M.B., Viegas Caetano, J.A., Margarinos, M.C., 2004. Efficacy of biocides against hospital isolates of Staphylococcus sensitive and resistant to methicillin, in the province of Buenos Aires, Argentina. Pan-American Journal of Public Health 16, 187–192. Russell, A.D., 1999. Bacterial resistance to disinfectants: present knowledge and future problems. Journal of Hospital Infection 43 (Suppl.), 57–68. Russell, A.D., 2000. Do biocides select for antibiotic resistance? Journal of Pharmacy and Pharmacology 52, 227–233. Russell, A.D., Tattawasart, U., Maillard, J.-Y., Furr, J.R., 1998. Possible link between bacterial resistance and use of antibiotics and biocides. Antimicrobial Agents and Chemotherapy 42, 2151. Sanchez, P., Moreno, E., Martinez, J.L., 2005. The biocide triclosan selects Stenotrophomonas maltophilia mutants that overproduce the SmeDEF multidrug efflux pump. Antimicrobial Agents and Chemotherapy 49, 781–782. Schweizer, H.P., 1998. Intrinsic resistance to inhibitors of fatty acid biosynthesis in Pseudomonas aeruginosa is due to efflux: application of a novel technique for generation of unmarked chromosomal mutants for the study of efflux systems. Antimicrobial Agents and Chemotherapy 42, 394–398. Schweizer, H.P., 1999. Use of antiseptics and multidrug resistance. ASM News 65, 124. Suller, M.T.E., Russell, A.D., 2000. Triclosan and antibiotic resistance in Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 46, 11–18. Szybalski, W., 1952. Microbial selection. I. Gradient plate technique for study of bacterial resistance. Science 116, 46–48. Tattawasart, U., Maillard, J.-Y., Furr, J.R., Russell, A.D., 2000. Outer membrane changes in Pseudomonas stutzeri strains resistant to chlorhexidine diacetate and cetylpyridinium chloride. International Journal of Antimicrobial Agents 16, 233–238. Toro, C.S., Lobos, S.R., Calde´ron, I., Rodriguez, M., Mora, G.C., 1990. Clinical isolates of a porinless Salmonella typhi resistant to high levels of chloramphenicol. Antimicrobial Agents and Chemotherapy 34, 1715–1719. Weller, P.J., 2000. Chlorhexidine. In: Kibbe, A.H. (Ed.), Handbook of Pharmaceutical Excipients, third ed. Pharmaceutical Press, London, pp. 121–125.
International Biodeterioration & Biodegradation 57 (2006) 51–56 www.elsevier.com/locate/ibiod
Chloroxylenol- and triclosan-tolerant bacteria from industrial sources—susceptibility to antibiotics and other biocides J.C. Leara, J.-Y. Maillarda,, P.W. Dettmarb, P.A. Goddardb, A.D. Russella,{ a
Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, UK b Reckitt Benckiser Healthcare, Dansom Lane, Hull HU8 7DS, UK
Received 28 August 2005; received in revised form 27 October 2005; accepted 4 November 2005 Available online 13 December 2005
Abstract This study aimed to determine the degree of susceptibility of several industrial bacterial isolates tolerant to para-chloro-meta-xylenol, triclosan, three other commonly used biocides, and to a range of clinically significant antibiotics. Minimum inhibitory concentrations (MICs) of benzalkonium chloride, chlorhexidine and phenol were determined and compared with those of standard strains. When elevated MICs were exhibited, suspension efficacy tests were carried out to assess the lethal efficacy of these biocides. Antibiotic sensitivity profiles were initially determined by disk diffusion testing, and antibiotic MICs by a gradient plate method. Biocide MICs were largely similar between industrial and standard strains, although isolates of Acinetobacter johnsonii showed elevated benzalkonium chloride MICs, which were not reflected in terms of lethal effects. Antibiotic sensitivities did not vary greatly between strains. An industrial triclosan-tolerant strain of Citrobacter freundii showed a slightly higher resistance to some antibiotics, but in most cases this was recorded as ‘‘sensitive’’ according to the guidelines used. Chloramphenicol resistance was shown in the triclosan-tolerant strain of A. johnsonii, but also in the sensitive revertant strain, suggesting that this property is intrinsic to the strain and unrelated to triclosan tolerance. This study did not produce evidence suggesting that tolerance to triclosan or para-chloro-meta-xylenol in industrial bacterial isolates promotes the emergence of tolerance to other biocides or increases resistance to antibiotics. r 2005 Elsevier Ltd. All rights reserved. Keywords: Antimicrobial; Resistance; Industrial isolates; Biocides
1. Introduction As usage of biocidal products continues to increase, interest grows in the concept of bacterial resistance to these biocides (Russell, 1999). One particularly important aspect is that of cross- and co-resistance, whereby resistance to a particular biocide may result in a reduced susceptibility to another, and sometimes to chemically unrelated antibiotics. Important antibiotic resistance mechanisms such as impaired uptake (cellular impermeability or drug efflux), drug inactivation and mutation at the target site, can also apply to biocides (Russell, 1999), and it has been suggested that biocides could select for antibiotic-resistant strains if shared targets or common resistance mechanisms were Corresponding author. Tel.: +44 29 2087 9088; fax: +44 29 2087 4149.
E-mail address: [email protected] (J.-Y. Maillard). Deceased.
{
0964-8305/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2005.11.002
involved (Suller and Russell, 2000; Gilbert and McBain, 2001). Indeed, various examples of cross-resistance between biocides (Russell et al., 1998; Loughlin et al., 2002) and between biocides and antibiotics (Lambert et al., 2001; Braoudaki and Hilton, 2004) have been reported in the literature. However, it is important to note that other researchers have not been able to demonstrate such a relationship (Lambert, 2004; Gradel et al., 2005). Other issues, such as biocide susceptibility in methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci have also been subjects of conflicting reports (Haines et al., 1997). The concept of linked biocide and antibiotic resistance remains contentious (Russell, 2000), but given the current global problem of antibiotic resistance (Levy, 1998), it is clear that continued studies in this area are essential (Fraise, 2002). Studies on the widely used phenyl ether triclosan have fuelled concerns over linked biocide and antibiotic
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resistance. This compound appears to be a substrate of multidrug efflux pumps such as AcrAB in Escherichia coli and MexAB-OprM in Pseudomonas aeruginosa, and crossresistance between triclosan and antibiotics has been demonstrated in the laboratory (McMurry et al., 1998a; Schweizer, 1998). Furthermore, triclosan inhibits enoyl reductase enzymes involved in bacterial fatty acid synthesis such as FabI in E. coli (McMurry et al., 1998b) and InhA in mycobacteria (Parikh et al., 2000) and there is some evidence that cross-resistance between triclosan and other enoyl-reductase inhibitors such as diazaborines, hexachlorophene and isoniazid could occur (Heath et al., 2000; McMurry et al., 1998b; Parikh et al., 2000). In a previous study by this research group (Lear et al., 2002), bacterial strains were collected from sites of likely triclosan and/or para-chloro-meta-xylenol exposure in two biocidal product manufacturing facilities, and identified. The aim of this study was to determine the industrial strains’ susceptibility profile to other commonly used biocides and to clinically relevant antibiotics. Any relationship in susceptibility to these agents could then be considered.
shown by streaking samples on to plain tryptone soya agar plates. Similarly, solutions of the water-soluble biocides (benzalkonium chloride, chlorhexidine and phenol) were prepared at 20 or 100 final concentration in distilled water and sterilised by filtration through 0.2 mm membrane filters (Sartorius, Go¨ttingen, Germany). Antibiotic disks of amikacin (30 mg), carbenicillin (100 mg), ceftazidime (30 mg), cefuroxime (30 mg), chloramphenicol (30 mg), ciprofloxacin (5 mg), erythromycin (15 mg), gentamicin (10 mg), kanamycin (30 mg), neomycin (30 mg), piperacillin (100 mg), polymyxin B (300 mg), tetracycline (30 mg) and tobramycin (10 mg) were purchased from Becton Dickinson (Oxford, UK). Antibiotic powders were obtained from Sigma.
2.2. Bacterial isolates The characterisation profiles of the triclosan-tolerant industrial isolates of Acinetobacter johnsonii, Citrobacter freundii, and para-chloro-metaxylenol-tolerant strains of P. aeruginosa and P. stutzeri are presented in Table 1. An additional A. johnsonii strain which lost its triclosan tolerance following 15 subcultures in the absence of the biocide (referred to as the triclosan-sensitive revertant; Table 1) was also examined. All organisms were studied together with their corresponding standard strains obtained from the National Collection of Industrial and Marine Bacteria (NCIMB; Aberdeen, UK; Table 1). These strains were selected according to details supplied by NCIMB, and were the type strains of each species with the exception of P. aeruginosa NCIMB 10421, which was the strain recommended for disinfectant testing according to the Standard BS EN 1276 (CEN, 1997).
2. Materials and methods 2.1. Chemicals
2.3. Susceptibility to other biocides—minimum inhibitory concentrations (MICS)
Chlorhexidine diacetate and dimethylsulfoxide were purchased from Sigma (Poole, UK), the quaternary ammonium compound benzalkonium chloride from ICN Biochemicals (Cleveland, USA) and phenol from BDH Chemicals (Poole, UK). Triclosan and para-chloro-meta-xylenol were kindly supplied by Ciba Specialty Chemicals, (Basel, Switzerland) and Reckitt Benckiser (Hull, UK), respectively. Solutions of triclosan and para-chloro-meta-xylenol were prepared in dimethylsulfoxide at 20 or 100 the desired final concentrations according to the range being tested. Such solutions were always sterile, as
Plates containing test concentrations of biocides were prepared by the addition of 1.0 or 0.2 ml of the appropriate solutions to 19.0 or 19.8 ml volumes of molten extra-strength tryptone soya agar (Oxoid, Basingstoke, UK), mixed well, poured and allowed to set. In the case of triclosan and para-chloro-meta-xylenol, control experiments demonstrated that the final concentrations of dimethylsulfoxide (1% or 5%) were not inhibitory to the strains tested. Plates were inoculated, incubated and colonies enumerated as described by Lear et al. (2002). The lowest concentration inhibiting bacterial growth was taken as the MIC, and values were compared with
Table 1 Triclosan, para-chloro-meta-xylenol, chlorhexidine, benzalkonium chloride and phenol MIC values for strains of A. johnsonii, C. freundii, P. aeruginosa and P. stutzeri Straina
MIC (mg ml 1)b Triclosan
PCMX
BZK
CHX
Phenol
Triclosan-tolerant, revertant and industrial strains A. johnsonii S (NCIMB 12460) 4–5 A. johnsonii RS 4–5 A. johnsonii IS 4100c C. freundii SS (NCIMB 11490) 4100c C. freundii IS 4100c
80–85 NT 80–85 165–170 180–185
30–40 130–140 120–130 120–130 180–190
2.0–2.2 2.8–3.0 2.4–2.6 4–5 8–11
1100–1200 1100–1200 1100–1200 1500–1600 1500–1600
Para-chloro-meta-xylenol-tolerant and industrial strains P. aeruginosa SS (NCIMB 10421) 4100c P. aeruginosa IS 4100c P. stutzeri SS (NCIMB 11358) o5 P. stutzeri IS o5
41000c 41000c 200–220 465–470
330–340 300–310 180–190 180–190
60–85 35–45 3–5 5–6
900–1000 900–1000 1400–1500 1400–1500
Source site of isolation, incubation temperature and stability of tolerance are described in Lear et al. (2002). Note that C. freundii SS and IS, were tolerant to the lethal effect of triclosan and P. aeruginosa SS and IS to that of para-chloro-meta-xylenol Lear et al. (2002). a SS, standard strain; RS, revertant strain; IS, industrial triclosan- or para-chloro-meta-xylenol-tolerant strain. b PCMX: para-chloro-meta-xylenol; BZK: benzalkonium chloride; CHX: chlorhexidine diacetate. c 4: MIC value quoted as this was the highest biocide concentration achievable in agar; NT: Not tested.
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2.4. Susceptibility to other biocides—lethal effects The lethal effect of biocides was investigated with a suspension test as described by Lear et al. (2002) when significant MIC differences were demonstrated between the industrial and standard or revertant strains. The neutralizer (5% Tween 80, 1.5% lecithin; Sigma) used to quench the activity of the biocides was tested as follows: 1 ml biocide solution at 10 final concentration was added to 8 ml neutraliser, followed by 1 ml bacterial suspension. Following a 5-min contact period at 22 1C, surviving bacteria were serially diluted and counted as described above. The use of distilled water instead of biocide solution enabled log reductions in cfu ml 1 to be calculated. The potential toxicity of the neutraliser was assessed using the following protocol: 1 ml bacterial suspension was added to 9 ml extrastrength (i.e. 10/9th) neutraliser and, following a contact time of 5 min at 22 1C, survivors were serially diluted and counted as described above. For the calculation of the reduction in bacterial concentration, 9 ml distilled water was used instead of the neutraliser. The toxicity of 1% or 5% dimethylsulfoxide was also assessed using this protocol.
2.5. Susceptibility to antibiotics—disk diffusion method Antibiotic sensitivity profiles were determined using a method based upon the Comparative Method of the British Society for Antimicrobial Chemotherapy (BSAC, 1991). Overnight tryptone soya broth suspensions adjusted to 1 109 cfu ml 1 (1 108 cfu ml 1 for A. johnsonii strains) were swabbed on to diagnostic sensitivity testing agar (Oxoid, Basingstoke, UK) using the ‘‘wet swab’’ method (BSAC, 1991). Undiluted suspensions were used, as dilutions did not produce acceptable semi-confluent growth. Antibiotic disks were immediately placed on the agar with a mechanical dispenser (Becton Dickinson). The plates were then incubated at 22 1C for 48 h or 37 1C for 24 h as appropriate, and diameters of inhibition zones measured and interpreted according to established criteria (BSAC, 1991).
2.6. Susceptibility to antibiotics—gradient plate MICs If industrial triclosan- or para-chloro-meta-xylenol-tolerant strains exhibited ‘‘intermediate’’ or ‘‘resistant’’ results compared with standard or revertant strains in disk diffusion testing, antibiotic MICs were determined on diagnostic sensitivity testing agar using the gradient plate technique (Szybalski, 1952). Chloramphenicol stock solutions were prepared in absolute ethanol. A 0.2 ml aliquot was added to 19.8 ml of molten extrastrength diagnostic sensitivity testing agar. A control experiment established that 1% ethanol (the highest concentration used) was noninhibitory. All other antibiotics dissolved satisfactorily in distilled water and stock solutions were sterilised by filtration through 0.2 mm membrane filters (Sartorius). Gradient plates were prepared by pouring 20 ml diagnostic sensitivity testing agar containing the maximum final antibiotic concentration into a Petri dish, and allowing the agar to set with the plate set at an angle. Once set, the plates were returned to the normal level position and a further 20 ml of plain diagnostic sensitivity testing agar was added to each plate. A diffusion period of 2 h allowed an antibiotic concentration gradient to form across the plate. A 10-mL aliquot of an overnight tryptone soya broth suspension was then streaked along the antibiotic gradient with a sterile swab. Following incubation at 22 1C for 48 h or 37 1C for 24 h as required, the length of confluent growth along the concentration gradient was measured. MIC values of standard, revertant and industrial tolerant strains were compared with National Committee for Clinical Laboratory Standards (NCCLS) interpretive standards for dilution testing (Jorgensen et al., 1999) and MIC thresholds denoting sensitivity (Martindale, 1995).
2.7. Statistical analysis Five replicates were conducted for all experiments except biocide MIC determinations, where there were three replicates. Results of the suspension tests and antibiotic MICs were compared using two sample t-tests (TT), Mann–Whitney U-test (MWU) or one-way ANOVA as appropriate with Minitabs Release 13 software (Minitabs, PA, USA).
3. Results 3.1. Susceptibility to other biocides—MICs Agar MIC values of benzalkonium chloride, chlorhexidine and phenol for all strains were fairly variable (Table 1), with some industrial strains, such as the industrial triclosan-tolerant C. freundii, showing slightly elevated values compared with their standard counterparts and others appearing marginally more sensitive than their standard strains, e.g. industrial para-chloro-meta-xylenoltolerant P. aeruginosa. However, the industrial triclosantolerant and revertant sensitive strains of A. johnsonii exhibited considerably higher MICs for benzalkonium chloride than the standard strain (Table 1). 3.2. Susceptibility to other biocides—lethal effects Control experiments established that the neutralisers used in this study were not toxic to the test strains and effectively quenched biocidal activity (data not shown). Suspension tests were conducted with benzalkonium chloride and strains of A. johnsonii only (Fig. 1). They were significantly lower than the standard strain when exposed to benzalkonium chloride at 5 mg ml 1. Although the log reductions in bacterial number of the industrial and revertant strains were not significantly different from each other. However, such difference in inactivation was not shown at higher concentrations. The log reductions of the three strains following exposure to benzalkonium chloride at 10 mg ml 1 were not significantly different and at 20 mg ml 1 a44 log reduction was observed in all strains. 5.00
Log Reduction (cfu ml-1) ± SD
published data (Hammond et al., 1987; Ranganathan, 1996; Kibbe, 2000; Weller, 2000).
53
>4
4.00
3.00
2.00
1.00
0.00 55
10
20
Concentration of BZK (µg ml-1)
Fig. 1. Lethal effects of BZK against A. johnsonii. Standard strain (J); revertant strain (&) and industrial triclosan-tolerant strain (m).
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54
3.3. Susceptibility to antibiotics—disk diffusion method
B, tetracycline and tobramycin were employed for C. freundii strains, and chloramphenicol and P. stutzeri and A. johnsonii strains, since these were the organisms and antibiotics where ‘‘intermediate’’ or ‘‘resistant’’ results were obtained when compared with the standard strains (Table 2). Amikacin, ceftazidime and ciprofloxacin (for C. freundii) were not tested as antibiotic powders were unobtainable at the time of study. For all antibiotics tested the industrial triclosan-tolerant strain of C. freundii exhibited higher MIC values than the standard strain (Table 3). With the exception of tetracycline these differences were all significant. However, although higher, these MIC values were all designated ‘‘sensitive’’ under the listed guidelines (Martindale, 1995; Jorgensen et al., 1999). The chloramphenicol MIC of the industrial para-chlorometa-xylenol-tolerant strain of P. stutzeri was significantly higher than that of the standard strain (Table 4). However, both MIC values were high, and both fell into the ‘‘resistant’’ category for both sets of guidelines consulted. MICs of chloramphenicol against A. johnsonii strains were significantly lower for the standard strain than for the industrial triclosan-tolerant strain, and interestingly, the
Largely, the antibiotic susceptibility profiles did not differ between standard strains and their industrial triclosan- or para-chloro-meta-xylenol-tolerant counterparts, each producing a ‘‘sensitive’’ result (Table 2). Where resistance did occur it was also nearly always demonstrated by both standard and industrial strains, namely with C. freundii to erythromycin, P. aeruginosa to chloramphenicol, erythromycin and kanamycin, and P. stutzeri to cefuroxime (Table 2). However, industrial and revertant strains of A. johnsonii showed intermediate resistance to chloramphenicol when compared with the standard strain, and the industrial strain of C. freundii was intermediately resistant to several of the antibiotics and resistant to polymyxin B. The industrial strain of P. stutzeri was also resistant to chloramphenicol (Table 2). 3.4. Susceptibility to antibiotics—gradient plate MICs Gradient plates for carbenicillin, cefuroxime, gentamicin, kanamycin, neomycin, piperacillin, polymyxin Table 2 Antibiotic sensitivity profiles of bacterial strains Straina
Susceptibility (Sensitive (S), intermediate (I) or resistant (R) to antibiotic AN
CB
CAZ
CXM
C
CIP
E
GM
K
N
PIP
PB
TE
NN
Triclosan-tolerant, revertant and standard strains A. johnsonii SS S S S A. johnsonii RS S S S A. johnsonii IS S S S C. freundii SS S S S C. freundii IS I I I
S S S S I
S I I S S
S S S S I
S S S R R
S S S S I
S S S S I
S S S S I
S S S S I
S S S S R
S S S S I
S S S S I
Para-chloro-meta-xylenol-tolerant and standard P. aeruginosa SS S S P. aeruginosa IS S S P. stutzeri SS S S P. stutzeri IS S S
R R R R
R R S R
S S S S
R R S S
S S S S
R R S S
S S S S
S S S S
S S S S
S S S S
S S S S
strains S S S S
AN, amikacin; CB, carbenicillin; CAZ, ceftazidime; CXM, cefuroxime; C, chloramphenicol; CIP, ciprofloxacin; E, erythromycin; GM, gentamicin; K, kanamycin; N, neomycin; PIP, piperacillin; PB, polymyxin B; TE, tetracycline; NN, tobramycin. Note: Antibiotic sensitivity profiles were determined using a method based upon the Comparative Method of the British Society for Antimicrobial Chemotherapy (1991). a SS, standard strain; RS, revertant strain; IS, industrial triclosan- or para-chloro-meta-xylenol-tolerant strain.
Table 3 Gradient plate antibiotic MICs for C. freundii standard strain NCIMB 11490 and its industrial triclosan-tolerant isolate MIC (7 SD) mg ml
Standard strain Triclosan-tolerant isolate
1
CB
CXM
GM
K
N
PIP
PB
TE
NN
3.96 (0.38) 4.82 (0.20)
1.88 (0.10) 3.15 (0.23)
1.33 (0.08) 2.15 (0.10)
3.11 (0.07) 4.01 (0.27)
3.19 (0.28) 4.13 (0.13)
2.78 (0.14) 3.35 (0.57)
1.11 (0.13) 1.91 (0.31)
2.67 (0.14) 2.73 (0.10)
1.08 (0.14) 1.54 (0.10)
CB, carbenicillin; CXM, cefuroxime; GM, gentamicin; K, kanamaycin; N, neomycin; PIP, piperacillin; PB, polymyxin B; TE, tetracycline; NN, tobramycin.
ARTICLE IN PRESS J.C. Lear et al. / International Biodeterioration & Biodegradation 57 (2006) 51–56 Table 4 Gradient plate MICs of chloramphenicol for P. stutzeri standard strain NCIMB 11358 and its para-chloro-meta-xylenol-tolerant isolate, and A. johnsonii standard strain NCIMB 12460, its revertant and industrial triclosan-tolerant isolates MIC mg ml 1 (7 SD) Choramphenicol P. stutzeri Standard strain Para-chloro-meta-xylenol-tolerant isolate A. johnsonii Standard strain Revertant Industrial triclosan-tolerant isolates
77.63 (13.66) 109.80 (4.47) 4.97 (2.10) 33.90 (12.74) 32.10 (2.88)
triclosan-sensitive revertant strain (Table 4). There was no significant difference between the latter two strains. 4. Discussion Most triclosan- and para-chloro-meta-xylenol-tolerant strains did not appear to show increased tolerance to benzalkonium chloride, chlorhexidine or phenol, and MICs of these agents correlated well with published data (Hammond et al., 1987; Ranganathan, 1996; Kibbe, 2000; Weller, 2000). Similarly, there was little evidence of crossresistance when antibiotic sensitivity profiles were investigated, with largely ‘‘sensitive’’ results for all strains. Nonetheless, some cases of reduced susceptibility were observed. The industrial strain of C. freundii showed higher levels of resistance to all the antibiotics tested, both in disk diffusion and MIC testing, although these largely significantly higher MICs were all within threshold values for sensitivity. Higher MIC values were also noted for benzalkonium chloride and chlorhexidine when biocides were tested (Table 1). Reduced susceptibility to such a variety of agents with differing modes of action suggests a non-specific mechanism of resistance such as increased impermeability due to outer membrane adaptation (Nikaido, 1994; Tattawasart et al., 2000). The industrial triclosan-tolerant strain of A. johnsonii showed an elevated benzalkonium chloride MIC in comparison with the standard strain, but this property was also shown by the triclosan-sensitive revertant. This pattern of results was also shown for chloramphenicol, both in disk diffusion and MIC testing. This revertant strain was originally produced from the industrial isolate, having lost tolerance to triclosan after repeated subculture in the absence of the biocide (Lear et al., 2002). Benzalkonium chloride tolerance and chloramphenicol resistance were evidently retained in the absence of selective pressure from triclosan. This would suggest that the reduced susceptibilities may be intrinsic features of the industrial and revertant strains, and not ‘‘linked’’ to
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triclosan tolerance as such. Furthermore, the elevated benzalkonium chloride MICs were not reflected in terms of sensitivity to the lethal effect of the biocide, except where the very low concentration of 5 mg ml 1 was used. In addition, a concentration of 20 mg ml 1 produced a44 log reduction in concentration within 5 min and therefore the tolerance of these isolates to benzalkonium chloride, in terms of MIC only, is unlikely to be of significance in practice. Differences between effective concentrations reported with suspension tests or in agar for a specific biocide have been discussed previously (Lear et al., 2002). In disk diffusion testing, the industrial para-chloro-metaxylenol-tolerant strain of P. stutzeri showed a ‘‘resistant’’ response to chloramphenicol, compared with ‘‘sensitive’’ for the standard strain. Although a higher chloramphenicol MIC was also observed later, both MICs were categorised as ‘‘resistant’’ under the guidelines used. It must be borne in mind that different methodologies and resistance thresholds may produce such differences in results, but again the MIC result suggests that chloramphenicol resistance is an intrinsic feature of both strains and is unrelated to triclosan tolerance. Hence, the only possible association observed, although not linked to triclosan tolerance, was that between benzalkonium chloride and chloramphenicol in the A. johnsonii strains. Such crossresistance has previously been documented in P. aeruginosa (Loughlin et al., 2002) and E. coli (Langsrud et al., 2004). Resistance to chloramphenicol has been associated with a loss of outer membrane porin proteins in Gram-negative bacteria (Toro et al., 1990), and the resulting lack of permeability or uptake could confer some degree of resistance to biocides. Drug expulsion by efflux has also been linked to chloramphenicol and benzalkonium chloride cross-resistance in E. coli (Langsrud et al., 2004). In summary, the triclosan and para-chloro-meta-xylenoltolerant industrial strains of bacteria investigated in this study did not demonstrate relevant tolerance in practice to other biocides or cross-resistance to the antibiotics tested. Where some decrease in susceptibility was demonstrated it appeared to be innate to the microorganisms, relatively insignificant and/or not ‘‘linked’’ with previously observed biocide tolerance per se. Given the debate over such issues (Schweizer, 1999; Russell, 2000; Fraise, 2002), these are interesting and significant results. Instances of linked biocide and antibiotic resistance (Sanchez et al., 2005), and the lack of it (Reynaldo et al., 2004), continue to appear in the literature. However, there is currently no evidence that the phenomenon has caused a real problem in practice (Gilbert and McBain, 2003). Nonetheless, it is clear that continued investigation of biocide and antibiotic resistance is essential in order to monitor this phenomenon (Fraise, 2002). Acknowledgements The authors would like to thank Reckitt Benckiser Healthcare, Hull, UK for their professional and financial
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