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The synergistic activity of triclosan and cipro£oxacin on bio¢lms ofSalmonella Typhimurium Mina Tabak1,2, Keren Scher1, Michael L. Chikindas2 & Sima Yaron1 1Technion-Israel Institute of Technology, Faculty of Biotechnology and Food Engineering, Haifa, Israel; and 2Department of Food Science, Cook College,Rutgers, The State University of New Jersey, New Brunswick, NJ, USA Israel Institute of Technology, Faculty of Triclosan is a biocide whose wide use has raised a debate about the potential benefits Biotechnology and Food Engineering, Haifa32000, Israel. Tel.: 1972 4 829 2940; fax: vs. hazards of the incorporation of antimicrobials in consumer products. The purpose of the present study was to determine whether exposure of biofilms of Salmonella enterica serovar Typhimurium to triclosan influences the tolerance of thebacteria towards antibiotics such as ciprofloxacin and vice versa. A synergistic Received 15 April 2009; accepted 8 September antibiofilm activity was observed when the biofilms were treated with triclosan before or together with ciprofloxacin, and an additive activity was observed with planktonic Final version published online 16 October 2009.
cells. For example 500 mg mLÀ1 triclosan and 500 mg mLÀ1 ciprofloxacin reduced thenumber of viable cells in the biofilm by 1.6 and 0.5 log, respectively. However, the sequential treatment of 500 mg mLÀ1 triclosan followed by ciprofloxacin resulted in4.8 log reduction. Combination indexes (CI) for biofilms treated with triclosan followed by ciprofloxacin were 0.7, 0.32 and 0.25 for reduction of 90%, 99% and 99.9%, respectively, indicating a synergism. For planktonic cells, CIs were 1 Æ 0.1, Salmonella; triclosan; ciprofloxacin; biofilm; indicating an additive effect. Therefore, it was suggested that triclosan weakens the ability of biofilm-associated cells to survive exposure to ciprofloxacin in the biofilm,probably by improving the permeability or the activity of ciprofloxacin.
Triclosan is a bactericide frequently added to antiseptic products as diverse as toothpastes, deodorants, soaps and lotions (Russell, 2004). It is also used to reduce microbial loads from different surfaces such as food-processing plants Bacteria use multiple mechanisms to overcome the antimi- crobial activity of triclosan, including mutations in the enoyl reductase, alterations of the cell envelope, active efflux and expression of triclosan-degradative enzymes (Meade et al., 2001; Schweizer, 2001; Yazdankhah et al., 2006). In Escherichia coli and Salmonella enterica, overexpression of the AcrAB-TolC efflux pumps decreased the susceptibility to triclosan (McMurry et al., 1998b; Webber et al., 2008b). These efflux pumps are mem- brane proteins that transport a variety of toxic compounds out of the cell, and contribute to the development of a low-level resistance to various antibiotics, including tetracycline, chlor- amphenicol, cephalosporins, penicillins, nalidixic acid and fluoroquinolones (George & Levy, 1983; Cohen et al., 1989).
The fluoroquinolone ciprofloxacin is routinely used for the treatment of severe gastrointestinal infections in adults.
 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Other fluoroquinolones such as enrofloxacin, norfloxacine Under these conditions, MAE52 forms a biofilm at the and ofloxacine are widely used in farm animals. Resistance air–liquid surface (pellicle). This biofilm is stable and can to fluoroquinolones is commonly mapped to the genes be easily removed from the interface (Tabak et al., 2007). As encoding DNA gyrases, topoisomerases or multidrug efflux was shown before, each biofilm contains approximately pumps such as the AcrAB-TolC efflux pump (Yoshida et al., 108 CFU (Scher et al., 2005, 2007). For various analyses, 1990; Kaatz et al., 1993; Ferrero et al., 1994; Takahashi et al., biofilms were gently removed from the surface of the broth 1998). The global increase in the prevalence of S. enterica with sterile tweezers and washed with 10 mL saline. Enu- strains with a reduced susceptibility to fluoroquinolones meration of the cells in the biofilms before and after each constitutes a major concern because these pathogens have treatment was conducted by disruption of the biofilm with been associated with a significant burden of hospitalization glass beads, followed by conventional plate counting as and mortality (Helms et al., 2002, 2004; Molbak, 2005), and described previously (Scher et al., 2005).
with clinical failures of therapy (Crump et al., 2003; Rupali MAE52 planktonic cells at the stationary phase were et al., 2004; Slinger et al., 2004; Molbak, 2005). Several prepared by collecting the broth under the biofilm after observations pointed to potential cross-resistance between 24 h of incubation at 37 1C. The stationary cultures of disinfectants and fluoroquinolones (Braoudaki & Hilton, MAE190, which do not form biofilms due to deletion of 2004a, b; Karatzas et al., 2007). For example, triclosan- the genes encoding for the production of cellulose (bcsA) resistant E. coli and S. enterica strains, achieved by exposure and curli (agfBA), were prepared by incubation at 37 1C to sublethal concentrations of triclosan, showed decreased under the same conditions as MAE52. Bacteria at the susceptibility to a range of antimicrobial agents, including logarithmic phase of growth were collected after 4 h of ciprofloxacin (Braoudaki & Hilton, 2004a; Randall et al., incubation. All planktonic cells were collected by centrifuga- 2004). However, the link between these substances has not tion (4500 g, 15 min) and resuspended in saline to a final been investigated thoroughly, especially not in biofilms.
In a previous study, we observed that S. enterica serovar Typhimurium in a biofilm is resistant to triclosan. There Effect of triclosan and ciprofloxacin on viability was only 1 log reduction in biofilms treated with 1000 mg mLÀ1 triclosan, a concentration that is in the rangeof the triclosan concentrations in commercial preparations To determine the effect of triclosan or ciprofloxacin on the (600–20 000 mg mLÀ1) (Tabak et al., 2007). Resistance of viability of biofilm-associated cells, each prewashed biofilm was Salmonella in the biofilm was attributed to low diffusion placed in 2 mL triclosan solution (0–1000 mg mLÀ1) at 25 1C or through the extracellular matrix and to an adaptive 37 1C, or in 2 mL ciprofloxacin solution (0–1000 mg mLÀ1) at response, which was obtained by changes in the expression 37 1C for 0, 30 and 60 min. At each time point, a biofilm was of genes such as acrAB and marA (encoding the AcrAB taken, washed twice with saline, disrupted with glass beads efflux pump and its activator MarA, respectively). Because as described (Scher et al., 2005) and plated onto LB agar the induced systems might provide further tolerance to triclosan and to other antimicrobials, we hypothesized that To study the effect of triclosan before ciprofloxacin treat- the exposure of biofilms to triclosan could influence the susceptibility to some antibiotics such as ciprofloxacin. The 0–500 mg mLÀ1 triclosan for 30 min at 37 1C. Then, the aim of the present study was to determine whether exposure biofilms were washed in saline three times and treated with of S. Typhimurium biofilm and planktonic cells to one ciprofloxacin (0–500 mg mLÀ1) for 1 h at 37 1C. To study the antimicrobial agent influences the tolerance to the second.
effect of ciprofloxacin before triclosan treatment, biofilmswere placed in solutions containing 0–500 mg mLÀ1 ciproflox-acin for 1 h at 37 1C. The biofilms were then washed in saline and treated with triclosan (0–500 mg mLÀ1) for 30 min. Tostudy the effect of ciprofloxacin together with triclosan, Bacterial strains and preparation of cells in biofilms were placed in solutions containing 0–500 mg mLÀ1 ciprofloxacin and 0–500 mg mLÀ1 triclosan for 1 h at 37 1C.
Salmonella enterica serovar Typhimurium ATCC 14028 and The treated biofilms were washed, disrupted with glass beads its mutants MAE52 and MAE190 were described previously and diluted for plate counting. Sequential and combined (Romling et al., 2000; Gerstel & Romling, 2001; Zogaj et al., treatments of triclosan and ciprofloxacin were also conducted 2001). For biofilm formation, overnight cultures of MAE52 with the planktonic cells at the logarithmic and stationary were diluted (1 : 30) in fresh Luria–Bertani (LB) broth phases as described above. The treated planktonic cells were without NaCl and incubated in 24-well microplates collected by centrifugation, washed three times and resus- (1.5 mL) for 24 h at 37 1C with gentle shaking (130 r.p.m.).
pended in saline, diluted 10-fold and plated.
 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved The effect of triclosan and ciprofloxacin in the biofilm cell counts was obtained in the biofilm after 1 h of incuba-tion at 37 1C with the highest concentration used To assess possible synergistic activity, the combination indexes (1000 mg mLÀ1). The MIC of triclosan for planktonic cells (CI) were calculated according to the equation: CI = (D)T/ was 0.5 mg mLÀ1. In biofilms, there was only a 1 log reduc- (Dx)T1(D)C/(Dx)C, where (D)T and (D)C are doses of triclo- tion even with 1000 mg mLÀ1 triclosan at 25 1C as was san and ciprofloxacin in combination, and (Dx)T and (Dx)C described (Tabak et al., 2007), but exposure to 500 and are doses of triclosan and ciprofloxacin that produce x% effect 1000 mg mLÀ1 triclosan at 37 1C resulted in 1.6 and 3 log when used alone. CI o 1, = 1, and 4 1 indicate synergism, reduction, respectively. Because treatments with triclosan at additive effect and antagonism, respectively. The simplest 37 1C were found to be more effective in killing the bacteria, definition for additive effect indicates that the effect of the we continued the experiments at 37 1C. When biofilms were two compounds together is greater than the effect of treated with triclosan followed by ciprofloxacin at concen- each alone, and synergism occurs when the combined effect trations of 50–500 mg mLÀ1 triclosan and 250–500 mg mLÀ1 exceeds that predicted by the sum of the individual actions of ciprofloxacin, a 4–5 log reduction was observed. This reduc- the compounds (Chou, 2006). For each set of experiments, we tion in viability was much larger than the sum of calculated the lowest concentrations that resulted in 90%, 99% both compounds treated alone, indicating a possible synergy and 99.9% reduction. Ciprofloxacin alone at a concentra- (Fig. 1). For example, treatment with 500 mg mLÀ1 triclosan tion as high as 1000 mg mLÀ1 resulted in approximately 80% or ciprofloxacin reduced the CFU by 1.6 and 0.5 log, reduction in the biofilm; thus, (Dx)C was displayed in all respectively. However, the sequential treatment with the calculations as 1000 mg mLÀ1, an assumption that does not same concentration of triclosan followed by ciproflo- affect the conclusions about synergy when the calculated xacin resulted in a 4.8 log reduction of the viable cell count.
CI o 1, because the real (Dx)C would reduce CI even more.
Even lower concentrations (50 mg mLÀ1 triclosan followed by Each experiment was conducted at least three times in 250 mg mLÀ1 ciprofloxacin) resulted in a 3 log reduction, duplicate. Data were analyzed with MICROSOFT EXCEL version 7, which is significantly higher than the effect of triclosan alone and statistically processed using the one-way ANOVA method, and ciprofloxacin alone, each compound at 500 mg mLÀ1 followed by the Tukey–Kramer test. P-values o 0.05 were (P o 0.05). The calculated CI indexes were CI = 0.7 for 90% reduction, CI = 0.32 for 99% and CI = 0.25 for 99.9%,indicating a synergism. This synergy was also observed when the biofilms were treated with triclosan at 25 1C followed bya treatment with ciprofloxacin at 37 1C (data not shown).
Susceptibility of S. Typhimurium in biofilm tosequential treatment with triclosan followed by Susceptibility of S. Typhimurium in biofilms to combined treatment of triclosan andciprofloxacin The minimal inhibitory concentration (MIC) of ciproflox-acin for planktonic S. Typhimurium (wt) used in this study To determine whether triclosan and ciprofloxacin have syner- was 0.125 mg mLÀ1, but a reduction of o 0.7 log of viable gistic activity when they are used together, the experiments Fig. 1. The effect of sequential treatments ofbiofilms of Salmonella Typhimurium withtriclosan (TR), followed by ciprofloxacin onbacterial viability. Biofilms were treated withtriclosan (50–500 mg mLÀ1) for 30 min at 37 1Cfollowed by ciprofloxacin treatment(1–500 mg mLÀ1) for 1 h at 37 1C, and culturablecells were counted. Data are the mean of CFUcounts of three experiments; each experimentwas conducted in duplicate, and bars indicate SD.
ÃSignificant difference (P o 0.05) in thereduction of CFU counts of biofilms treated withtriclosan and ciprofloxacin compared with thereduction in biofilms treated with eachcompound alone at the same concentrations.
 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Fig. 2. The effect of treatments of biofilms ofSalmonella Typhimurium with triclosan (TR)combined with ciprofloxacin on bacterial viability.
Biofilms were placed in solutions containing0–500 mg mLÀ1 ciprofloxacin and 0–500 mg mLÀ1triclosan for 1 h at 37 1C and culturable cells werecounted. Data are the mean of CFU counts ofthree experiments; each experiment wasconducted in duplicate, and bars indicate SD.
ÃSignificant difference (P o 0.05) in thereduction of CFU counts of biofilms treated withtriclosan and ciprofloxacin compared with thereduction in biofilms treated with eachcompound alone at the same concentrations.
Fig. 3. The effect on bacterial viability of se-quential treatments of biofilms of SalmonellaTyphimurium with ciprofloxacin, followed bytriclosan (TR). Biofilms were treated withciprofloxacin (125–500 mg mLÀ1) for 1 h at 37 1Cfollowed by triclosan treatment (50–500 mg mLÀ1)for 30 min at 37 1C andculturable cells were counted. Data are the meanof CFU counts of three experiments; eachexperiment was conducted in duplicate, and barsindicate SD. ÃSignificant difference (P o 0.05) inthe reduction of CFU counts of biofilms treatedwith triclosan and ciprofloxacin compared withthe reduction in biofilms treated with eachcompound alone at the same concentrations.
described above were repeated with both agents at the same Susceptibility of S. Typhimurium to sequential time. A very low effect on the viability of biofilm-associated cells ( o 1.5 log reduction) was observed when the cells were treated with triclosan and ciprofloxacin simultaneously, atciprofloxacin concentrations of 0–16 mg mLÀ1 (Fig. 2). How- A sequential treatment with ciprofloxacin followed by ever, a significant antibiofilm activity was observed at higher triclosan was also more effective against biofilms than the sum of each treatment alone at high concentrations of 500 mg mLÀ1 triclosan reduced 1.6 log and 250 mg mLÀ1 cipro- ciprofloxacin ( 4 125 mg mLÀ1) (Fig. 3). Yet the decrease floxacin reduced 0.25 log, the combined treatment resulted in was smaller compared with treatments with triclosan before a 5.0 log reduction (P o 0.05). The calculated CI indexes were ciprofloxacin or with both compounds together, and the CI = 1.0 for 90% reduction, CI = 0.32 for 99% and CI = 0.45 effect was less significant statistically. For example, for 99.9%, indicating a synergism at higher concentrations 500 mg mLÀ1 triclosan and 250–500 mg mLÀ1 ciprofloxacin and additive effect at the lower concentrations.
reduced 4.8 log when the biofilms were first treated with  2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved The effect of triclosan and ciprofloxacin in the biofilm Fig. 4. Effect of treatment of SalmonellaTyphimurium with 500 mg mLÀ1 triclosan (TR) and500 mg mLÀ1ciprofloxacin (Cip) and combinedtreatments for 1 h on the viability of planktonic(gray) and biofilm-associated (black) cells. Dataare the average of three experiments; eachexperiment was conducted in duplicate.
ÃSignificant difference (P o 0.05) betweenplanktonic and biofilm cells.
triclosan, 5.0 log in the mixed treatment and 3.8 log when influences the activity of ciprofloxacin in the biofilm.
they were first treated with ciprofloxacin (P o 0.05). The However, we did not study the evolutionary process of calculated CI indexes were CI = 1.0 for 90% reduction, emergence and selection of resistant mutants, as had CI = 0.35 for 99% and CI = 0.6 for 99.9%, indicating a been shown and discussed in the past (McMurry et al., synergism at the higher concentrations.
1998b; Mazzariol et al., 2000; Chuanchuen et al., 2001;Tkachenko et al., 2007), but focused on mechanisms of Susceptibility of planktonic cells to treatment adaptation in the biofilms. To the best of our knowledge, the effect of a sequential or combined exposure of Salmonellain biofilms to biocides and antibiotics had not been investi- When planktonic cells were treated with triclosan and ciprofloxacin, an additive effect was observed (CIs were We investigated the dual effect of triclosan and ciproflox- 1 Æ 0.1). In planktonic cells, the order of treatments did not acin (used together or sequentially) against S. Typhimurium have any effect on cell viability, and very similar results were biofilms. Initially, we determined the susceptibility of bio- obtained with all strains (wt, MAE52 and MAE190). A film-associated Salmonella to ciprofloxacin, and confirmed summary of the comparison between planktonic cells at the that biofilm-associated Salmonella are less susceptible to stationary phase of growth and biofilm-associated cells is ciprofloxacin as compared with planktonic cells. Previous shown in Fig. 4. Interestingly, although each compound had articles also showed that different biofilm-associated cells low activity against biofilm-associated cells as compared resist exposure to ciprofloxacin (Desai et al., 1998; Anderl with planktonic cells, the combined treatment reached a et al., 2000; Aiassa et al., 2006; Rodriguez-Martinez et al., similar reduction in biofilm and planktonic cells.
2007). The resistance to ciprofloxacin was not attributed tothe low diffusion of ciprofloxacin through the biofilm matrix, because ciprofloxacin readily penetrated biofilms In 2005, the Non-Prescription Drug Advisory Committee of formed by different bacteria such as Klebsiella pneumoniae, the US Food and Drug Administration (FDA) discussed the E. coli or Pseudomonas aeruginosa (Anderl et al., 2000; potential benefits and risks associated with antiseptic pro- Rodriguez-Martinez et al., 2007). Hence, it was suggested ducts marked as ‘antimicrobial’. Much of the debate regard- that ciprofloxacin failed to kill bacteria in the biofilm ing consumer antiseptic products was focused on the use of because the bacteria do not grow or grow slowly (Anderl products that contain triclosan, and the conclusions of the FDA meeting resulted in a call for further research regarding In line with our previous studies, demonstrating that, in a the benefits and risks of consumer antiseptic products used biofilm, triclosan induces the transcription of the acrAB and in the community setting (Aiello et al., 2007). In a recent marA (Tabak et al., 2007), we expected that triclosan would study, we showed that the ‘in use’ concentrations of triclosan have increased the tolerance of biofilm-associated cells to are probably not efficient in killing Salmonella embedded in ciprofloxacin, because overexpression of MarA or AcrAB biofilms (Tabak et al., 2007). In the present research, we leads to a low level of resistance to antibiotics such as went further and tried to determine whether triclosan quinolones (Hachler et al., 1991; Miller et al., 1994; Okusu  2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved et al., 1996; Alekshun & Levy, 1997; Randall et al., 2004). In It was shown that triclosan resistance is multifactorial and contrast to our expectations, the results pointed towards that there are at least three distinct mechanisms of triclosan synergistic activity between triclosan and ciprofloxacin.
resistance in Salmonella (Webber et al., 2008a, b). Our Exposure of biofilms to triclosan before ciprofloxacin re- findings show that mechanisms of resistance in biofilms sulted in a significant reduction in viable counts. This and particularly mechanisms of cross-resistance cannot be reduction was dependent on the concentrations of both predicted from data about mechanisms of resistance in triclosan and ciprofloxacin. A similar, but less notable, planktonic cells, and that observations about resistance due phenomenon was observed when the biofilms were treated to the selection of specific mutants is not always correlated with triclosan and ciprofloxacin together, because in this with an adaptive response in the biofilm. Thus, we conclude case an additive effect was observed at the lower concentra- that knowledge about the consequences of the usage of tions and synergism at the higher concentrations. The lowest biocides alone or combined with other antimicrobials is still effect was observed when biofilms were treated with cipro- incomplete, particularly in microbial biofilms.
floxacin before triclosan. Furthermore, the biofilm did notgive the embedded cells any advantage over planktonic cells insurviving the combined treatment, because both the biofilm and the planktonic cells were equally susceptible to thistreatment. Similarly, Aaron et al. (2002) showed that while This work was supported by the Technion Research and biofilm-grown P. aeruginosa were tolerant to the treatment Development and Lando/Ben-David Funds.
with ciprofloxacin or to the combined treatment with tobra-mycin and piperacillin–tazobactam, the same isolates weresensitive to a combined treatment of tobramycin, piperacil- lin–tazobactam and ciprofloxacin. Two possible mechanismsmay explain the synergistic activity in the biofilm: (1) bacteria Aaron SD, Ferris W, Ramotar K, Vandemheen K, Chan F & use the same cellular mechanisms to adapt to ciprofloxacin Saginur R (2002) Single and combination antibiotic and triclosan, such as the AcrAB efflux pumps (McMurry susceptibilities of planktonic, adherent, and biofilm-grownPseudomonas aeruginosa isolates cultured from sputa of adults et al., 1998b; Webber et al., 2008b), and the double attack is with cystic fibrosis. J Clin Microbiol 40: 4172–4179.
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