Editor: Robert Burne
Effect of triclosan on Salmonella typhimurium at different growth stages and in biofilms
Article first published online: 6 DEC 2006
FEMS Microbiology Letters
Volume 267, Issue 2, pages 200–206, February 2007
How to Cite
Tabak, M., Scher, K., Hartog, E., Romling, U., Matthews, K. R., Chikindas, M. L. and Yaron, S. (2007), Effect of triclosan on Salmonella typhimurium at different growth stages and in biofilms. FEMS Microbiology Letters, 267: 200–206. doi: 10.1111/j.1574-6968.2006.00547.x
- Issue published online: 6 DEC 2006
- Article first published online: 6 DEC 2006
- Received 12 July 2006; revised 30 October 2006; accepted 7 November 2006.First published online 6 December 2006.
- antibiotic resistance;
- efflux pump
Triclosan is a potent biocide that is included in a diverse range of products. This research was aimed to investigate the susceptibility of planktonic and biofilm-associated Salmonella enterica serovar Typhimurium to triclosan, and to identify potential mechanisms of adaptation. The effect of triclosan was studied on planktonic Salmonella (log and stationary phases), on biofilm-associated cells, on bacteria derived from disrupted biofilms and on a biofilm-deficient mutant. An eight-log reduction of exponentially growing cells was observed with 1000 μg mL−1 triclosan within 10 min, a 3.6-log reduction in stationary cells and a 6.3-log reduction in stationary cells of a biofilm-deficient mutant (P<0.05). Biofilm-associated cells were tolerant (1-log reduction). However, biofilm-derived cells showed sensitivity to triclosan similar to stationary-phase cells. Triclosan induced the transcription of fabI and micF. Within biofilms, triclosan also up-regulated the transcription of acrAB, encoding for an efflux pump, marA, and the cellulose-synthesis-coding genes bcsA and bcsE. Thus, Salmonella within biofilms could experience reduced influx, increased efflux and enhanced exopolysaccharides production. Our results demonstrated that the tolerance of Salmonella towards triclosan in the biofilm was attributed to low diffusion through the extracellular matrix, while changes of gene expression might provide further resistance to triclosan and to other antimicrobials.
Triclosan is a bisphenol antimicrobial agent that has a broad range of activity (Russell, 2004). Triclosan is bacteriostatic at concentrations ranging between 0.025 and 100 μg mL−1, and bactericidal at higher levels (Suller & Russell, 1999, 2000; Escalada et al., 2005). It is used as a preservative, antiseptic and disinfectant in a diverse range of products. Owing to its extensive use and stability, triclosan and its derivatives can now be found in wastewater, deposited in environmental sediments and concentrated in aquatic biota (Adolfsson-Erici et al., 2002).
The inhibitory activity of triclosan results from blocking lipid synthesis through specific inhibition of the NADH-dependent enoyl-acyl carrier protein reductase FabI (McMurry et al., 1998b; Levy et al., 1999; Heath & Rock, 2000). At higher concentrations, triclosan is likely to damage the bacterial membrane (Villalain et al., 2001). Gram-negative bacteria use multiple mechanisms to develop resistance to triclosan, including mutations in the enoyl reductase, alteration of the cell envelope, active efflux and expression of triclosan-degradative enzymes (Schweizer, 2001a; Yazdankhah et al., 2006). The main physiological change resulting from adaptation to triclosan, as described so far in Escherichia coli and Salmonella, is the overexpression of efflux pumps, particularly the AcrAB efflux pump (Levy, 2002; Braoudaki & Hilton, 2004, 2005). As active AcrAB was also associated with increased resistance to many other structurally unrelated antimicrobials (McMurry et al., 1998a; Randall & Woodward, 2002; Randall et al., 2004), there might be a link between triclosan usage and antibiotic resistance (McMurry et al., 1998a; Levy, 2000, 2002).
Most data on the antimicrobial activity of triclosan were collected from studies using planktonic microorganisms rather than biofilm-associated microorganisms. Plastics incorporated with triclosan are now heavily marketed for use in the food processing industry, but very little is known about the effect of triclosan-containing plastics on the microbial loads of the foods, and none of these studies examined the effect of triclosan on biofilms (Yazdankhah et al., 2006). A number of research projects studied the antimicrobial activity of triclosan in environments that may contain biofilms, like on the floor (Moretro et al., 2006), in sink drains (McBain et al., 2003b) or within mouthrinse formulations (Arweiler et al., 2002). In these experiments, triclosan prevented the formation of biofilms, but its effect, if any, on viability in existing biofilm was equivocal or not studied.
In the present research, we studied the effect of triclosan on the viability of the foodborne pathogen Salmonella enterica serovar Typhimurium (S. typhimurium) within the biofilm in comparison with planktonic cells in different growth phases, and identified mechanisms of adaptive tolerance to the antimicrobial.
Materials and methods
Salmonella typhimurium ATCC 14028-1 s Nalr (wt) and its mutants MAE52 and MAE190 were described previously (Zogaj et al., 2001). While the wt forms biofilms at 28°C, MAE52 also forms biofilms at 37°C. The MAE190 mutant does not form a biofilm due to deletion of the genes encoding for the production of cellulose and curli (Romling et al., 2000; Gerstel & Romling, 2001).
Overnight cultures of MAE52 were diluted (1:30) in fresh Luria–Bertani (LB) broth without NaCl and incubated in 24-well micro-plates (1.5 mL) for 24 h at 37°C with gentle shaking (130 r.p.m.). Under these conditions, MAE52 forms a biofilm at the air–liquid surface. Biofilms were gently removed from the surface of the broth with sterile tweezers and washed with 10 mL saline. Enumeration of cells in the biofilm was conducted after disruption of the biofilm with glass beads as was described previously. As was shown before, each biofilm contained c. 108 CFU (Scher et al., 2005).
Preparation of cells in different phases of growth
The following cultures were used: MAE52 within biofilms (biofilm-associated cells), MAE52 originating from disrupted biofilms (biofilm-derived cells) and planktonic bacteria at the log phase of growth and at the stationary phase (wt, MAE52 and MAE190).
To prepare the MAE52 biofilm-associated cells and biofilm-derived cells, biofilms were formed as described above, and used in their entirety (biofilm-associated cells) or agitated with glass beads for 1 min in saline, followed by centrifugation (32 g, 5 min) to remove unbroken small particles of the biofilm matrix, and a second centrifugation step (4500 g, 15 min) to pellet the cells (biofilm-derived cells).
MAE52 planktonic cells at the stationary phase were prepared by collecting the broth under the biofilm after 24 h of incubation at 37°C. The stationary cultures of wt and MAE190 strains (which do not form biofilms at 37°C) were prepared by incubation under the same conditions as MAE52.
Log-phase cultures were obtained by growing all strains for 3.5 h to OD600 nm 0.4–0.5. All planktonic cells were then collected by centrifugation (4500 g, 15 min) and resuspended in saline to a final concentration of c. 108 CFU mL−1.
Determination of minimal inhibitory concentration (MIC) and minimal bacteriocidal concentration (MBC) for planktonic cells
A stock solution of 5000 μg mL−1 triclosan (Irgasan, Fluka BioChemika) was prepared in 50% ethanol (McBain et al., 2003a; Aiello et al., 2004). Dilutions of predetermined concentration were made either in medium or in saline before each experiment.
Determination of the MIC was conducted in broth according to the recommendation of the National Committee for Clinical Laboratory Standards (NCCLS, 2003) using triclosan concentrations of 0.06–2 μg mL−1 and a final inoculum of 108 CFU mL−1. The final concentration of ethanol did not exceed 0.02% and had no growth-inhibitory properties.
MBC, i.e. the concentration required to obtain at least a 3-log reduction (Sherris, 1990), was determined for 108 CFU mL−1 in saline, LB or Mueller–Hinton broth (MHB) supplemented with 2–1000 μg mL−1 triclosan. Following incubation for 24 h at 37°C, cultures were harvested (4500 g for 15 min), diluted 10-fold in saline and plated on LB agar. Colonies were counted after 24 h of incubation at 37°C.
Effect of triclosan on viability
To determine the effect of triclosan on the viability of biofilm-associated cells, each prewashed biofilm was placed in 2 mL triclosan solution (1000 μg mL−1) for 5, 10, 30 and 60 min at room temperature. Then, the biofilms were washed twice with 10 mL saline, disrupted with glass beads as previously described (Scher et al., 2005) and plated onto LB agar.
Triclosan (final concentration of 1000 μg mL−1) was also added to all the planktonic cells described above. Aliquots were taken at 5, 10, 30 and 60 min, collected by centrifugation, washed and resuspended in saline, diluted 10-fold and plated. Controls were treated with saline or with saline and ethanol. All experiments were conducted three to five times in duplicate.
RNA isolation, cDNA synthesis and qRT PCR
Stationary MAE52 cells and biofilms were treated with 500 μg mL−1 triclosan for 10 and 30 min as described above. Controls were biofilms and stationary cells treated with 5% ethanol. Treated cells were washed with 10 mL saline, and following disruption of the biofilms with glass beads, all samples were harvested by centrifugation, resuspended in 2 mL saline and were added to 4 mL of RNAprotect Bacteria Reagent (QIAGEN, Valencia, CA). Total RNA was extracted with the RNeasy Mini Kit (QIAGEN). The DNA remaining in the sample was removed with RNase-Free DNase (QIAGEN). cDNA was synthesized from the RNA template (250 ng) using the Reverse-iT 1st-strand synthesis Kit (ABgene) and was diluted 1:5 in water to obtain the working solution.
Real-time PCR for the quantification of micF, acrA, marA, fabI, bcsA, bcsE and agfB mRNAs was performed using 4 μL working solution of cDNA, 100 to 250 nM concentrations of each primer (Table 1) and 10 μL ABsolute™ QPCR SYBR Green Mix (ABgene) in 20 μL total volume. A three-step protocol was used in Rotor-Gene 3000 (Corbett Research): (i) denaturization (15 min at 95°C); (ii) an amplification and extension program repeated 40 times (1 s at 95°C, 15 s at appropriate annealing temperature and 20 s at 72°C); and (iii) a melting curve program of heating from 72 to 99°C, at a heating rate of 1°C per 5 s.
|* 16S rRNA gene F/R||CGGGGAGGAAGGTGTTGTG/GAGCCCGGGGATTTCACATC|
The concentrations of the experimental transcripts were calculated from the linear regression of a standard curve and standardized by referring the 16S rRNA gene calculated concentrations. For each culture, the concentration of the sample that was treated without triclosan was set as 1, and the other samples were calculated proportionally. Analysis was performed twice in triplicates, and the average numbers are shown. No amplification product was obtained when the total RNA was used as a template in PCR reactions using the same primers.
Data were analyzed with microsoft excel version 7, and statistically processed using the One-Way anova method, followed by the Tukey–Kramer test in case of anova showing significance. P values<0.05 were regarded as significant.
Susceptibility to triclosan depends on the growth phase
The MIC for wt, MAE52 and MAE190 in the log phase was 0.5 μg mL−1. For the same three strains, the MBC was 500 μg mL−1, which is 1000-fold higher than the MIC, and was not dependent on the media (saline, LB or MHB).
We compared the capability of triclosan (1000 μg mL−1 i.e. 2 × MBC) to kill log-phase cells, stationary cells, biofilm-associated cells and biofilm-derived cells (Fig. 1). Exposure of the log-phase cells (all three strains) to 1000 μg mL−1 triclosan resulted in at least 8-log reduction of viable cell counts. A significant difference (P<0.05) was observed between the mutant incapable of biofilm formation (6.3-log reduction) and the other two strains (3.6-log reduction) at the stationary phase (Fig. 1). Only a 1-log reduction was observed with regard to the CFU of the cells within the biofilm. However, the biofilm-derived cells, which were treated with triclosan after disruption of the biofilm, had susceptibility to triclosan similar to the stationary cells (3.9-log reduction, Fig. 1). In all cultures, the majority of the cell death was achieved within the first 10 min.
Triclosan up-regulated transcription of specific genes in the biofilm
Quantification of the transcription of micF, acrA, marA, fabI, bcsA, bcsE and agfB in biofilm and stationary MAE52 cells before- and after-exposure to a subinhibitory concentration (500 μg mL−1) of triclosan was achieved by qRT PCR. 16S rRNA gene was used to standardize the results as its amount was similar in biofilm and stationary cells and did not change after exposure to triclosan (Fig. 2).
Transcription of fabI in stationary cells was fourfold higher than in biofilm-associated cells (P<0.05) (data not shown). Triclosan up-regulated transcription of fabI in biofilm-associated cells and stationary cells by 10-fold after 30 min (Fig. 2). Triclosan also induced micF transcription in both cultures, but just by twofold. Transcription of marA and acrA was 17-fold lower in biofilm-associated cells (P<0.05) (data not shown). Triclosan did not affect significantly the transcription of marA and acrA in the stationary cells, but induced the transcription of these genes in the biofilm-associated cells (P<0.05; Fig. 2).
We showed that cells within biofilms were protected from the effects of exposure to triclosan. To elucidate whether triclosan up-regulates bcsA, bcsE and agfB, genes involved in biofilm formation, we compared their transcription levels before and after exposure to triclosan. Increased transcription of bcsA and bcsE was observed after exposure of biofilm-associated cells to triclosan, while the transcription in stationary cells was not affected. Induction was noticeably higher after 30 min (P<0.05; Fig. 2). A slight but not statistically significant reduction in the agfB transcription was observed only in the triclosan-exposed biofilms (Fig. 2).
This is the first study that compared the effect of triclosan on the viability of planktonic and biofilm-associated cells, and showed a significant difference between the susceptibility of log-phase Salmonella cells, stationary cells and cells within biofilms. Triclosan was efficient in controlling exponentially growing cells, and the MIC (0.5 μg mL−1) was in the scale reported for other clinical and environmental bacteria (Randall et al., 2001, 2004; Aiello et al., 2004; Braoudaki & Hilton, 2005). Stationary and biofilm cells, the most widely represented states of microorganisms living in the environment, survived exposure to 1000 μg mL−1 triclosan (2000 × MIC), a concentration that was lethal for log-phase cells. Our observation that cells in a biofilm were resistant to triclosan supports the study of Junker & Hay (2004). It can also explain why plastic containing 1500 μg mL−1 triclosan did not effectively reduce bacterial populations on meat surfaces, whereas the same bacteria were sensitive to triclosan in plate assays (Cutter, 1999). The considerable difference between susceptibility of log-phase and stationery cells was also observed in E. coli (Escalada et al., 2005), but was unlike the previous observations in Enterococcus hirae and Staphylococcus aureus (Suller & Russell, 2000; Escalada et al., 2005), supporting the hypothesis that the inhibitory activity of triclosan against E. coli and Salmonella may result from multiple mechanisms and not from just targeted action against the bacterial cytoplasmic membrane (McMurry et al., 1998b; Levy et al., 1999; Heath & Rock, 2000).
The triclosan sensitivity of the biofilm-derived cells was identical to that of stationary cells, suggesting that the matrix has a significant role in the resistance of biofilm-associated cells probably via reduction of diffusion. The low activity of triclosan attributed to low diffusion was also reported for biofilm-associated oral Streptococcus mutans (Steinberg et al., 2006). Furthermore, curli or cellulose, the main components of the biofilm matrix, might also have a protective effect on planktonic Salmonella via diffusion into the cells from the broth, as the mutant MAE190, which has deletions in the genes coding for curli and cellulose synthesis, was considerably more sensitive to triclosan compared with the other strains. This was not seen when we treated the same Salmonella mutant with chlorine (Scher et al., 2005).
The lower triclosan susceptibility of stationary and biofilm-associated cells compared with log-phase cells suggests that the cells probably adapted to triclosan by an alteration of gene expression or by changes in the permeability of triclosan through the envelope, and not by mutations. The effect of subinhibitory concentrations of triclosan on expression of genes has not been studied before. Based on previous reports about triclosan activity and about strains that developed resistance to triclosan (McMurry et al., 1998a; Heath et al., 1999; Levy et al., 1999; Braoudaki & Hilton, 2004, 2005), we chose to focus on specific genes that might be activated by triclosan during the adaptation process. We hypothesized that as FabI is the triclosan target, Salmonella cells would increase the FabI synthesis after exposure to triclosan, in order to eliminate the inhibition of fatty acid synthesis. Indeed, fabI transcription was induced in response to triclosan by stationary and biofilm-associated cells. This is an observation that, to the best of our knowledge, has not been described previously.
The transcription of marA and acrA was investigated, as mutants with decreased susceptibility to triclosan showed higher expression of broad-spectrum efflux pumps like AcrAB (McMurry et al., 1998a; Schweizer, 1998, 2001b; Braoudaki & Hilton, 2005). Other potential changes in the outer membrane that might affect the permeability like alteration in LPS were not observed during exposure of Salmonella to triclosan (Braoudaki & Hilton, 2005). We found that triclosan increased the transcription of acrAB and its activator marA, adding triclosan to the antimicrobial agents that induce marA like tetracycline (Seoane & Levy, 1995). However, this induction was just in biofilms. The marA gene encodes for MarA, a global regulator that controls the expression of more than 60 genes, many of which have a role in bacterial stress response including acrAB (Barbosa & Levy, 2000). If we assume that a higher transcription of the genes acrAB and marA is directly correlated with a higher expression of these proteins, and given that the overexpression of MarA, AcrAB and micF has also been linked to the increased resistance to many different antibiotics, organic solvents and disinfectants (Alekshun & Levy, 1997, 1999; Yaron et al., 2003), we propose that within the biofilm, there is a relationship between the use of triclosan and antibiotic resistance.
Thus, triclosan and antibiotics not only share multidrug efflux systems as a common mechanism of resistance, but triclosan also induces expression of these efflux pumps. However, the up-regulation of acrAB and marA by triclosan in the biofilm will probably mediate resistance only to low concentrations of antibiotics, because it was shown that expression of AcrAB only protected E. coli in biofilms against low concentrations of ciprofloxacin (Maira-Litran et al., 2000).
The extracellular matrix produced by Salmonella in the biofilm is composed of proteinaceous components and exopolysaccharides. The main exopolysaccharide is cellulose, which is produced and secreted by enzymes encoded in the bcsABZC and bcsEFG operons (Zogaj et al., 2001; Solano et al., 2002). In Salmonella, the bcs genes are constitutively transcribed (Zogaj et al., 2001). Enhanced cellulose biosynthesis via transcriptional up-regulation of the structural genes bcsA and bcsE by triclosan in biofilm-associated cells might contribute to extended biofilm formation. It should be noted, however, that triclosan did not induce the bcs genes in planktonic cells, but only in cells that were already within the biofilm matrix. Induction of biofilm formation by aminoglycosides antibiotics has been observed in Pseudomonas aeruginosa and E. coli (Hoffman et al., 2005), but to the best of our knowledge has not been observed for biocides.
We demonstrated that the concentrations used in consumer products (usually ranging from 600 to 20 000 μg mL−1) (Suller & Russell, 1999) should be effective for the prevention of growth, but might not be effective in killing Salmonella, particularly not within biofilms. This underscores the need for a revision of the issue of the antimicrobial efficacy of triclosan against microorganisms in biofilms when triclosan is incorporated into products such as soaps, dishwashing liquids, food storage containers and other kitchen utensils and medical devices. Two mechanisms contributed to the enhanced resistance of the biofilm-associated cells: the presence of the exopolysaccharide matrix that probably reduced diffusion, and specific changes in transcription of genes. These changes exhibit a strong potential to enhance the resistance of biofilm-associated cells to low concentrations of other antimicrobials, an issue that should be further investigated in the future.
This work was supported by grant 2001083 from the Binational Science Foundation (BSF).
- 2002) Triclosan, a commonly used bactericide found in human milk and in the aquatic environment in Sweden. Chemosphere 46: 1485–1489. , , & (
- 2004) Relationship between triclosan and susceptibilities of bacteria isolated from hands in the community. Antimicrob Agents Chemother 48: 2973–2979. , , , & (
- 1997) Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob Agents Chemother 41: 2067–2075. & (
- 1999) The mar regulon: multiple resistance to antibiotics and other toxic chemicals. Trends Microbiol 7: 410–413. & (
- 2002) Effect of an amine-fluoride-triclosan mouthrinse on plaque regrowth and biofilm vitality. J Clin Periodontol 29: 358–363. , , & (
- 2000) Differential expression of over 60 chromosomal genes in Escherichia coli by constitutive expression of MarA. J Bacteriol 182: 3467–3474. & (
- 2004) Adaptive resistance to biocides in Salmonella enterica and Escherichia coli O157 and cross-resistance to antimicrobial agents. J Clin Microbiol 42: 73–78. & (
- 2005) Mechanisms of resistance in Salmonella enterica adapted to erythromycin, benzalkonium chloride and triclosan. Int J Antimicrob Agents 25: 31–37. & (
- 1999) The effectiveness of triclosan-incorporated plastic against bacteria on beef surfaces. J Food Prot 62: 474–479. (
- 2005) Triclosan-bacteria interactions: single or multiple target sites? Lett Appl Microbiol 41: 476–481. , , & (
- 2004) Establishment of a real-time PCR-based approach for accurate quantification of bacterial RNA targets in water, using Salmonella as a model organism. Appl Environ Microbiol 70: 3618–3623. , , , , & (
- 2001) Oxygen tension and nutrient starvation are major signals that regulate agfD promoter activity and expression of the multicellular morphotype in Salmonella typhimurium. Environ Microbiol 3: 638–648. & (
- 2000) A triclosan-resistant bacterial enzyme. Nature 406: 145–146. & (
- 1999) Mechanism of triclosan inhibition of bacterial fatty acid synthesis. J Biol Chem 274: 11110–11114. , , , , & (
- 2005) Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 436: 1171–1175. , , , , & (
- 2004) Effects of triclosan incorporation into ABS plastic on biofilm communities. J Antimicrob Chemother 53: 989–996. & (
- 1999) Molecular basis of triclosan activity. Nature 398: 383–384. , , , , , , & (
- 2000) Antibiotic and antiseptic resistance: impact on public health. Pediatr Infect Dis J 19: S120–S122. (
- 2002) Active efflux, a common mechanism for biocide and antibiotic resistance. Symp Ser Soc Appl Microbiol S65–S71. (
- 2000) An evaluation of the potential of the multiple antibiotic resistance operon (mar) and the multidrug efflux pump acrAB to moderate resistance towards ciprofloxacin in Escherichia coli biofilms. J Antimicrob Chemother 45: 789–795. , & (
- 2003a) Effects of triclosan-containing rinse on the dynamics and antimicrobial susceptibility of in vitro plaque ecosystems. Antimicrob Agents Chemother 47: 3531–3538. , , , , & (
- 2003b) Exposure of sink drain microcosms to triclosan: population dynamics and antimicrobial susceptibility. Appl Environ Microbiol 69: 5433–5442. , , , , , & (
- 1998a) Overexpression of marA, soxS, or acrAB produces resistance to triclosan in laboratory and clinical strains of Escherichia coli. FEMS Microbiol Lett 166: 305–309. , & (
- 1998b) Triclosan targets lipid synthesis. Nature 394: 531–532. , & (
- 2006) Evaluation of the antibacterial effect of a triclosan-containing floor used in the food industry. J Food Prot 69: 627–633. , , & (
- NCCLS (2003) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 6th ed. Approved standard M7-A6. National Committee for Clinical Laboratory Standards, Wayne, Pa.
- 2002) The multiple antibiotic resistance (mar) locus and its significance. Res Vet Sci 72: 87–93. & (
- 2001) Association between cyclohexane resistance in Salmonella of different serovars and increased resistance to multiple antibiotics, disinfectants and dyes. J Med Microbiol 50: 919–924. , , & (
- 2004) Effect of triclosan or a phenolic farm disinfectant on the selection of antibiotic-resistant Salmonella enterica. J Antimicrob Chemother 54: 621–627. , , & (
- 2000) AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways. Mol Microbiol 36: 10–23. , , , & (
- 2004) Whither triclosan? J Antimicrob Chemother 53: 693–695. (
- 2005) Effect of heat, acidification, and chlorination on Salmonella enterica serovar Typhimurium cells in a biofilm formed at the air-liquid interface. Appl Environ Microbiol 71: 1163–1168. , & (
- 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 mutations for the study of efflux systems. Antimicrob Agents Chemother 42: 394–398. (
- 2001a) Antibiotic and biocide and its link to antibiotics. J Hosp Infect 43: 1–7. (
- 2001b) Triclosan: a widely used biocide and its link to antibiotics. FEMS Microbiol Lett 202: 1–7. (
- 1995) Characterization of MarR, the repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli. J Bacteriol 177: 3414–3419. & (
- 1990) Medical Microbiology: An Introduction to Infectious Diseases, 2nd edn. Prentice Hall International, Englewood Cliffs, NJ. (
- 2002) Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose. Mol Microbiol 43: 793–808. , , , , , & (
- 2006) Sustained-release delivery systems of triclosan for treatment of Streptococcus mutans biofilm. J Biomed Mater Res B Appl Biomater 77: 282–286. , & (
- 1999) Antibiotic and biocide resistance in methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococcus. J Hosp Infect 43: 281–291. & (
- 2000) Triclosan and antibiotic resistance in Staphylococcus aureus. J Antimicrob Chemother 46: 11–18. & (
- 2001) Membranotropic effects of the antibacterial agent Triclosan. Arch Biochem Biophys 390: 128–136. , , , & (
- 2003) Characterization of an Escherichia coli O157:H7 marR mutant. Int J Food Microbiol 85: 281–291. , & (
- 2006) Triclosan and antimicrobial resistance in bacteria: an overview. Microb Drug Resist 12: 83–90. , , , , , , & (
- 2001) The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix. Mol Microbiol 39: 1452–1463. , , , & (