JAC Advance Access originally published online on September 13, 2007
Journal of Antimicrobial Chemotherapy 2007 60(5):947-955; doi:10.1093/jac/dkm314
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Prolonged treatment of Salmonella enterica serovar Typhimurium with commercial disinfectants selects for multiple antibiotic resistance, increased efflux and reduced invasiveness
1 Zoonotic Infections Group, Division of Veterinary Pathology, Infection and Immunity, School of Clinical Veterinary Science, University of Bristol, Langford House, Langford, Bristol BS40 5DU, UK 2 Antimicrobial Agents Research Group, Division of Immunity and Infection, The Medical School, The University of Birmingham, Birmingham B15 2TT, UK 3 Food Borne Zoonoses Unit, Health Protection Agency South West, School of Clinical Veterinary Science, University of Bristol, Langford House, Langford, Bristol BS40 5DU, UK 4 Department of Food and Environmental Safety, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK
* Correspondence address. Bacterial Stress Response Group, Department of Microbiology National University of Ireland Galway, Galway, Ireland. Tel: +353-91-495091; Fax: +353-91-494598; E-mail: kimon-andreas.karatzas{at}nuigalway.ie
Received 24 May 2007; returned 20 June 2007; revised 30 July 2007; accepted 31 July 2007
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Objectives: To study how disinfectants affect antimicrobial susceptibility and phenotype of Salmonella enterica serovar Typhimurium SL1344.
Methods: Wild-type strain SL1344 and its isogenic gyrA mutant were passaged daily for 7 days in subinhibitory concentrations, and separately for 16 days in gradually increasing concentrations of a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde (QACFG), an oxidizing compound blend (OXC), a phenolic tar acids-based disinfectant (TOP) and triclosan. The MICs of antimicrobials and antibiotics for populations and representative isolates and the proportion of cells resistant to the MICs for the wild-type were determined. Expression of acrB gene, growth at 37°C and invasiveness of populations in Caco-2 intestinal epithelial cells were assessed.
Results: QACFG and triclosan showed the highest selectivity for variants with reduced susceptibility to chloramphenicol, tetracycline, ampicillin, acriflavine and triclosan. Populations treated with the above biocides had reduced invasiveness in Caco-2 cells, and altered growth kinetics. Resistance to disinfectants was observed only after exposure to gradually increasing concentrations of triclosan, accompanied with a 2000-fold increase in its MIC. Growth in OXC and TOP did not affect the MICs of antibiotics, but resulted in the appearance of a proportion of cells resistant to the MIC of acriflavine and triclosan for the wild-type. Randomly selected stable variants from all populations, except the one treated with TOP, over-expressed acrB.
Conclusions: In vitro exposure to QACFG and triclosan selects for Salmonella Typhimurium cells with reduced susceptibility to several antibiotics. This is associated with overexpression of AcrAB efflux pump, but accompanied with reduced invasiveness.
Keywords: quaternary ammonium , triclosan , AcrAB
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Non-typhoidal Salmonella caused over 12 000 reported cases of enteritis in England and Wales in 2006,1 and over 65 000 across Europe in 2004,2 resulting in significant morbidity and mortality. While most cases require only simple bed rest and re-hydration, a proportion, particularly among the more vulnerable, may require antibiotic therapy. Clinically significant resistance to multiple antibiotics in Salmonella has been increasing and over 30% of isolates of Salmonella enterica serovar Typhimurium reported to the Enter-net surveillance network in 2004 were resistant to three or more antibiotics resulting in reduced therapeutic options.2 Recent regulations preventing the use of antibiotics as growth promoters in animals have been introduced in an attempt to reduce the emergence of antibiotic resistance in the food chain.3 This has necessitated an increase in the use of biosecurity measures that include the application of disinfectants to reduce microbial contamination of animal houses. In addition, antiseptics and disinfectants are used extensively by all industrial components of the food-chain.
Resistance to a disinfectant is thought unlikely to occur because most disinfectants are often complexes of antimicrobials4 that inactivate multiple cellular targets. A single mutation would be unlikely to confer resistance.4,5 However, resistance to a disinfectant such as triclosan which inactivates a specific cellular target, does occur as a result of specific mutations altering the target, in this case fabI.6,7 Resistance to antiseptics and disinfectants associated with plasmids and integrons carrying efflux-related transporters, such as qacE, and also antibiotic resistance determinants has been well-studied.8 Reduced susceptibility to specific agents including quaternary ammonium compounds, chlorhexidine, diamidines and acridines is also known to occur.4,9 This phenomenon has been implicated as a possible cause for the selection and persistence of bacterial strains with reduced susceptibility to a range of agents, including antibiotics4 and it has been speculated that these bacteria could pass along the food chain.10
Efflux, together with membrane impermeability, is an important non-specific defence mechanism that can confer multiple antibiotic resistance (MAR) in S. enterica and Escherichia coli and a better understanding of the genes mediating this phenomenon and the pressures favouring selection of the MAR phenotype is urgently required.11,12 Efflux pumps are membrane proteins that actively export a wide range of toxic substrates, including antibiotics, dyes and disinfectants thereby preventing accumulation of toxic agents and mediating resistance.13,14 Various studies in E. coli and S. enterica have demonstrated that efflux pumps play an important role in intrinsic resistance to unrelated antibiotics such as fluoroquinolones and tetracycline, or disinfectants including quaternary ammonium compounds and triclosan.10–12,14,15 They also seem to be important for virulence.16–18 The wide spectrum of substrates recognized by efflux systems has prompted concern that exposure of a bacterium to one substrate could confer resistance to others.19,20 Thus exposure to disinfectants could result in selection of efflux mutants with reduced susceptibility to unrelated antibiotics and biocides, not previously encountered by the bacterium.
In the present study, we investigated the hypothesis that disinfectants can select MAR strains of Salmonella Typhimurium. The aim was to examine the effects of sequential growth of Salmonella Typhimurium SL1344 and its isogenic gyrA mutant for 7 days in subinhibitory concentrations of different disinfectants on the antimicrobial resistance, invasiveness and growth. In addition, we assessed the expression of the acrB gene as a marker for increased efflux.
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Bacterial strains and growth conditions
Wild-type Salmonella Typhimurium SL134421 and its isogenic mutant (L696) carrying the substitution Asp-87 
Gly within GyrA were used throughout. Purified cultures of strains were stored in microbank tubes (Pro-lab Diagnostics, Neston, UK) at –80°C and were grown routinely in Luria Bertani (LB) broth and on LB agar (Oxoid, Basingstoke, Hampshire, UK) with or without disinfectants at 37°C with shaking at 160 rpm for the broth.
Dyes and antibiotics were purchased from Sigma-Aldrich (Poole, UK), except for ciprofloxacin, which was obtained from Fluka Chemie (Buchs, Switzerland) and triclosan which was obtained from Ciba Geigy (Basel, Switzerland). The disinfectants used were: an oxidizing compounds blend containing an inorganic peroxygen compound, inorganic salts, organic acid, anionic detergent, fragrance and dye (OXC); a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde (QACFG); a phenolic biocide composed of tar acids, organic acids and surfactants (TOP); and a dairy quaternary ammonium sterilizer containing non-ionic surfactant and excipients (DQACS).
Disinfectant exposure experiments
Two types of experiments were performed: type 1, where cultures were sequentially grown in LB broth containing no agent or 0.2% OXC, 0.025% TOP, 0.006% QACFG, 0.06 mg/L triclosan for up to 7 days. Each day, an inoculum of 1% (v/v) from the overnight culture was used to prepare the culture of the following day in LB supplemented with the same concentration of the corresponding disinfectant. In type 2 experiments, cultures were transferred daily for up to 16 days to gradually increasing concentrations of disinfectants. Concentrations were increased daily by 1 mg/L for triclosan and 25% of the MIC for the other disinfectants. On day 6, for sequential growth in constant disinfectant concentrations and day 15, for sequential growth in gradually increasing concentrations, one millilitre samples from cultures were harvested and stored in microbank tubes at –80°C for subsequent antibiotic susceptibility testing. Before exposure to antibiotics and antimicrobials, cultures were removed from storage and grown overnight in fresh LB with the corresponding subinhibitory concentrations of the disinfectants. Ten-fold serial dilutions of these cultures were prepared in sterile saline solution (Oxoid, Basingstoke, UK) and transferred in triplicate onto LB agar plates (Oxoid, Basingstoke, UK), with or without different antimicrobials at the MIC of the disinfectant or antibiotic for SL1344, or L696. These were: 0.06 mg/L of triclosan, 400 mg/L of acriflavine, 2 mg/L of ampicillin, 8 mg/L of chloramphenicol, 0.003 mg/L ciprofloxacin, 2 mg/L of kanamycin and 2 mg/L of tetracycline. Agar plates were incubated at 37°C for 24 h and numbers of cfu determined.
The minimal inhibitory concentrations of a range of antibiotics, dyes and disinfectants were determined using the agar doubling dilution method according to the recommendations of the BSAC.21
Gentamicin protection assay (invasiveness)
The gentamicin protection assay was performed for the wild-type strain and all populations derived from it, as described previously by Elsinghorst.22 In brief, Caco-2 human colon adenocarcinoma cells (ECACC number 86010202) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 2 mM glutamine, 1% non-essential amino acids and 10% FBS. Penicillin/streptomycin (Invitrogen Life Technologies, Paisley, UK) was used at a concentration of 100 U/mL in DMEM until 3 days before cells were used for infection studies. Culture medium was changed every 2–3 days. Just before experiments, Caco-2 cells were washed three times with sterile PBS and subsequently 2 mL antibiotic-free DMEM was added to each well. The OD600 of stationary phase bacterial cultures of the populations and the wild-type was measured and all cultures were adjusted to similar OD600 value. We previously confirmed that there was a good correlation between OD600 measurements and cell numbers for all strains, assessed by comparing cfu and OD values. Fifty microlitres of the adjusted bacterial suspension was added to the well, resulting in 
106 cfu per well and yielding an estimated ratio of bacteria to cells (multiplicity of infection) of 2.5:1. Cells were incubated for 1 h at 37°C and subsequently washed twice with PBS and suspended in PBS containing 100 mg/L of gentamicin. After 2 h at 37°C, Caco-2 cells exposed to gentamicin were rinsed twice with sterile PBS and lysed with 2 mL of 1% (v/v) Triton X-100 in PBS. Following incubation for 5 min at 37°C cell lysates were serially diluted and plated on COLBA to quantify the number of intracellular bacteria. The t-test for two samples assuming equal variances was used to determine statistical differences between the means in invaded cells of each population and of the wild-type. P values <0.05 were considered statistically significant.
Growth characteristics of all populations were assessed at 37°C with shaking (160 rpm). Five microlitres from an overnight culture of each population was inoculated into 200 µL of fresh LB broth. Samples were placed in a microtitre plate and bacterial growth was assessed by measuring the OD600 of the samples in a Bioscreen C microplate reader (Labsystems, Helsinki, Finland). Growth curves were constructed in triplicate.
Isolation of variants from populations
In order to investigate the composition and the possible mechanisms conferring any antimicrobial resistance observed in the populations, individual isolates TOP R1 and R2, QACFG R1 and R2, OXC R1 and R2 and TRIC R1 and R2 were obtained from each of the corresponding wild-type-derived populations TOP 7d, QACFG 7d, OXC 7d and 16TRIC 16d, respectively. Individual isolates from populations QACFG 7d and 16TRIC 16d were obtained by streaking a sample from each of the parent cultures on LB agar plates containing no antimicrobials, as these populations contained mainly variants with reduced susceptibility to antibiotics and antimicrobials. However, in order to obtain such variants from populations OXC 7d and TOP 7d, a selective pressure had to be applied, as the majority of their cells demonstrated a wild-type phenotype regarding antimicrobial resistance (Figure 1). A sample from the OXC 7d population was streaked on an agar plate containing 400 mg/L of acriflavine, and 10 randomly selected colonies were screened for decreased antibiotic susceptibility. The same procedure was followed for TOP 7d, and two colonies were isolated on an agar plate containing 6 mg/L of chloramphenicol. All isolates were identified as Salmonella spp. after growth on XLD agar plates, Gram staining, multiple-locus variable-number tandem-repeats (VNTR) analysis and molecular subtyping by PFGE, as described below.
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Stability of phenotype and typing of individual isolates
All individual isolates were assessed for phenotypic stability. They were subcultured for 10 consecutive days using 0.3% (v/v) inocula in fresh LB medium in the absence of any disinfectant at 37°C. On day 10, cultures were tested for their antibiotic and disinfectant resistance as described earlier.
The individual isolates were typed by multiple-locus VNTR analysis using the method of Lindstedt et al.,23 and molecular subtyping by PFGE. PFGE was performed following XbaI digestion of genomic DNA according to the standard 1 day protocol of the Centers for Disease Control and Prevention.24
RT–PCR analysis of expression of acrB
The expression of acrB in all representative isolates that demonstrated the MAR phenotype was investigated using comparative RT–PCR.25 Three separate cultures for each isolate were used to obtain RNA. Mid-exponential phase cultures grown in LB broth were harvested and pellets re-suspended in one-fifth volume of ice-cold 95% ethanol and 5% phenol. Cultures were harvested during mid-exponential growth. Suspensions were kept on ice for 30 min before cells were re-harvested by centrifugation at 1500 g for 10 min at 4°C. Pellets were resuspended in 100 µL of TE buffer containing 50 mg/mL lysozyme and incubated at room temperature for 5 min. RNA was purified using the Promega SV total RNA purification kit (Promega, Southampton, UK) according to the manufacturer's recommendations. The quality and quantity of RNA extracted was determined by agarose gel electrophoresis and by analysis using a NanoDrop apparatus (NanoDrop Technologies, Wilmington, USA). cDNA was synthesized from 2 µg of total RNA for each sample in triplicate, using the SuperScript III system (Invitrogen Life Technologies, Paisley, UK) with random primers according to the manufacturer's instructions. RT–PCR reactions and DHPLC analysis for 16S rRNA and acrB (Transgenomic Ltd, Crewe, UK) were performed as previously described.25 Data were normalized to the expression of 16S rRNA to minimize cell density-dependent errors, by calculating the mean 16S expression from all RNA preparations. Subsequently, the ‘area under the curve’ for acrB from the same biological repeat were normalized as described previously.25 The mean and standard deviation of the nine repeats for each data set were calculated, followed by two-tailed, two-sample, equal variance Student's t-tests to compare gene expression relative to SL1344.
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Disinfectant exposure experiments
In general, sequential growth of SL1344 and L696 in the presence of QACFG for type 1 experiments and triclosan for type 2 resulted in the appearance of a proportion of cells in each population with reduced susceptibility to antibiotics and antimicrobials. Results for SL1344 and L696 were similar throughout. In addition, exposure to all disinfectants had no effect on the susceptibility of the final populations to kanamycin (Table 1). Except for triclosan, reduced susceptibility to increased concentrations of disinfectants and biocides was not achieved.
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In type 1 experiments, sequential growth in a subinhibitory concentration of OXC resulted in 0.03 and 0.02% of the cells of the final population being resistant to the MICs of the control parent strain for triclosan (Figure 1a) and acriflavine (Figure 1b), respectively. In type 1 experiments with TOP, the final population had 7 and 100% of its cells resistant to the MICs of the control parent strain for triclosan (Figure 1a) and acriflavine (Figure 1b), respectively. Extended growth in QACFG and in gradually increasing concentrations of triclosan (maximum: 16 mg/L) resulted in 100% of the cells of these populations being resistant to the MICs of the control parent strain for triclosan, acriflavine, tetracycline, chloramphenicol and ampicillin (Figure 1a–e).
In parallel, the MICs for all populations were determined (Table 1). Growth of cultures for 7 days in LB without antimicrobials did not have any effect on the MICs with one exception, that of triclosan which increased 4-fold. For all populations, the MICs of all disinfectants, except triclosan, remained stable, indicating that growth with or without disinfectants for seven daily subcultures did not have any effect. MICs of OXC, TOP, QACFG and DQACS were 0.2%, 0.4%, 0.03% and 0.2%, respectively. Sequential growth in OXC increased the MIC of ampicillin by 4-fold. Sequential growth in TOP did not significantly affect the MICs of any agent. However, sequential growth in QACFG increased the MICs of tetracycline, ampicillin and triclosan by 4-, 8- and 16-fold, respectively. Extended growth in 0.06 mg/L triclosan did not result in reduced susceptibility to antimicrobials (data not shown). However, the population grown in gradually increasing concentrations of triclosan had an increased MIC of ampicillin and triclosan by 8- and 1000–2000-fold, respectively (Table 1).
Gentamicin protection assay (invasiveness)
All populations demonstrated reduced ability to invade Caco-2 human colon adenocarcinoma epithelial cells compared with the wild-type (Figure 2). However, only for populations QACFG 7d and 16TRIC 16d was this difference statistically significant (P < 0.05) compared with the wild-type. QACFG 7d and 16TRIC 16d demonstrated numbers of invaded cells by 1.3 and 2.2 logarithmic cycles lower than those of the wild-type. Similar results were obtained with L696 (data not shown).
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Analysis of growth kinetics
As the S. enterica populations were a mix of different strains, growth curves did not show the classical growth pattern of monocultures and did not allow us to calculate values like µmax. However, all OD600 values for 16TRIC 16d were significantly lower than those of the wild-type (P < 0.05) during exponential phase denoting impaired growth. This population also showed an increased lag phase but its cell density at stationary phase was the same as wild-type (Figure 3). QACFG 7d showed increased cell density at stationary phase compared with the wild-type, but OXC 7d and TOP 7d showed growth patterns similar to the wild-type.
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Isolation of variants from the populations
Ten isolates were obtained from the OXC 7d population by growth on LB containing 400 mg/L acriflavine. Nine, including isolate OXC R2, did not show any reduced susceptibility to antibiotics. The MIC of triclosan was unchanged or increased by 2-fold. One isolate (OXC R1) demonstrated a 4-fold increase in the MICs of all antibiotics except that of kanamycin which remained unchanged (Table 2).
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From two randomly selected isolates obtained from the QACFG 7d population, only one, QACFG R2, showed reduced susceptibility to multiple antibiotics (Table 2). For this isolate, the MICs of ciprofloxacin, chloramphenicol and ampicillin were increased by 4-fold, and that of tetracycline by 8-fold. The MIC of kanamycin remained unchanged. However, the MICs of TOP and DQACS were reduced by 4- and 2-fold, respectively. QACFG R1 showed similar resistance to the wild-type with the exception of 4-fold increases in the MICs of ampicillin and triclosan.
TOP R2 showed a 4- and 8-fold increase in the MICs of triclosan and tetracycline, respectively (Table 2). This strain also had a 2-fold reduction in its susceptibility to DQACS. TOP R1 had similar phenotype to the wild-type regarding antimicrobial resistance.
TRIC R1 and R2 had the same susceptibilities as 16TRIC 16d population from which they were isolated.
Stability of phenotype and typing of individual isolates
All isolates had a stable phenotype following growth in fresh LB for 10 days in the absence of disinfectants. VNTR (Table 3) and PFGE (data not shown) analysis results for all variants were similar to those of the wild-type.
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acrB expression of representative isolates from the populations
The mRNA levels for the acrB transcript were determined for variants OXC R1, QACFG R2, TOP R2, TRIC R1 and TRIC R2 and compared with those of the parent strain SL1344. TOP R2 demonstrated levels of expression for acrB similar to those of the wild-type (P > 0.05), while QACFG R2, OXC R1, TRIC R1 and TRIC R2 demonstrated a 3- to 4-fold higher expression (P < 0.05) as shown in Figure 4.
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In the present study, the ability of several commonly used disinfectants to select for populations and isolates of Salmonella Typhimurium with reduced susceptibility to antibiotics or biocides was assessed. Stable variants were isolated, indicating that such exposures were responsible for the reduced susceptibility observed and that this phenomenon was not due to transient stress responses of wild-type cells. The gyrA mutation did not affect any of the phenotypic characteristics, apart from reduced susceptibility to fluoroquinolones.
Although sequential growth in subinhibitory concentrations of triclosan did not result in any phenotypic alterations in the population, it was the only disinfectant where its MIC was increased by sequential growth in gradually increasing concentrations of the compound in the growth medium. This resulted in a population that was highly triclosan resistant (1000–2000-fold increase in the MIC). This is probably because, unlike most disinfectants, triclosan inactivates a specific cellular target, FabI which is involved in lipid synthesis.6,26–28 Population 16TRIC 16d comprised only variants with reduced susceptibility to multiple antibiotics, acriflavine and triclosan. This phenomenon was associated with overexpression of acrB, as found in two randomly selected isolates (TRIC R1 and R2). Overexpression of efflux pumps including AcrAB can confer resistance to triclosan and acriflavine in E. coli and Salmonella Typhimurium, as well as low-level resistance to several antibiotics including ß-lactams (ampicillin), tetracycline and chloramphenicol.26,29 The ease by which triclosan-resistant variants can occur in a pure culture, even in the absence of selective pressure is demonstrated by the 4-fold increase in the MIC of this compound following sequential growth in LB alone for 7 days.
QACFG selected for populations with a similar phenotype to 16TRIC 16d and reduced susceptibility to antimicrobials which was also linked to overexpression of acrAB as demonstrated in the randomly selected QACFG R2 variant. This confirms the important role of AcrAB in tolerance of Salmonella Typhimurium to quaternary ammonium compounds as previously shown in E. coli.8 The 8-fold increase in the MIC of triclosan for QACFG 7d might be linked with a similar action of AcrAB against both disinfectants, although such increase was not observed with individual isolates derived from this population. Interestingly, QACFG R2 was highly sensitive to TOP (4-fold decrease in MIC) and its MIC of DQACS, which was the other quaternary ammonium disinfectant, was reduced by 2-fold, although it was isolated after exposure to a quaternary ammonium-based disinfectant. It is possible that the surfactants present in both TOP and DQACS, but not in QACFG were highly active against this variant. These data indicate that rotational use of different disinfectants in premises might prevent the selection of antibiotic resistant strains. Despite the above results with QACFG, it is important to note that the concentration used (MIC) was around 166 times lower than the recommended in-use concentration of 1%, suggesting that adhering to the manufacturer's recommendations for usage of the disinfectant should prevent the development of strains with reduced susceptibility to antibiotics and antimicrobials.
OXC and TOP had a lower potential for selection of S. enterica cells with reduced susceptibility to antibiotics and antimicrobials compared with triclosan and QACFG. Exposure to OXC and TOP had no major effects on the MICs of the antibiotics. However, both disinfectants resulted in the appearance of cells resistant to the antibiotics at the MICs of the control parent strain, and more markedly to those of acriflavine and triclosan. This was more pronounced with TOP than with OXC and all cells of TOP 7d were resistant to the MIC of acriflavine for the control parent strain. The randomly selected isolate TOP R2 also had an increased MIC of tetracycline and chloramphenicol. Although this was not seen in the TOP 7d population, there was a small proportion of cells resistant to the MICs of these antibiotics for the control parent strain, probably associated with the presence of variants like TOP R2. The expression of acrB in TOP R2 was similar to that of the wild-type, suggesting that alternate mechanisms like overexpression of other efflux pumps or reduced permeability might be responsible for the above phenomena. TOP R2 was also more susceptible to DQACS compared with the wild-type, again supporting the rotational use of different disinfectants.
Under the experimental conditions used in this study, OXC showed the lowest potential for selection of reduced susceptibility to antibiotics and antimicrobials resulting in lower than 0.1% proportion of cells resistant to the MIC of the wild-type for all antimicrobials. Since OXC 7d probably comprised mainly wild-type-like cells, isolate OXC R1 was not a representative isolate of the population as it was obtained by plating on acriflavine. Like most isolates described in this study, OXC R1 demonstrated reduced susceptibility to chloramphenicol and ampicillin probably associated with increased efflux mediated by AcrAB. Together with QACFG R2 they were the only isolates with increased MIC of ciprofloxacin, which could also be attributed to the latter mechanism, as described previously.30,31
Populations QACFG 7d and 16TRIC 16d that demonstrated the lowest reduced susceptibility to multiple antibiotics, also showed statistically significant reduced invasiveness in Caco-2 intestinal epithelial cells. The other S. enterica populations also showed lower invasiveness, but the differences were not statistically significant. These results were also confirmed with the populations obtained in the gyrA mutant background. To our knowledge, this is the first report demonstrating that triclosan and quaternary ammonium compounds select for strains with impaired invasiveness. Invasiveness of cells in QACFG 7d might have been even lower if an overnight growth in fresh LB without antimicrobials was not included. During this step, wild-type cells might have increased their proportion in the population raising its overall invasiveness. However, we had to include this step to alleviate any stress responses of S. enterica cells due to the presence of disinfectants in the medium that could possibly affect invasiveness. Presence of wild-type cells in the populations obtained with constant concentrations of disinfectants is certain since the concentrations used were subinhibitory. These findings might explain the discrepancies between in vitro studies clearly linking the use of certain disinfectants with antibiotic resistance, and epidemiological data that do not point to this direction.5,32 Triclosan is known to down-regulate virulence factors in E. coli, but this is a transient stress response and not a permanent phenotypic change.33 Whether the mechanisms of antimicrobial resistance are involved in impairment of invasiveness warrants further investigation. Previous work in Pseudomonas aeruginosa has suggested that overexpression of the Mex efflux pumps resulted in reduced expression of virulence determinants.34 Population 16TRIC 16d had impaired growth during exponential phase but its final population density was similar to that of the wild-type. In contrast, QACFG 7d reached a higher cell density than the wild-type. It has previously been shown that cells of E. coli overexpressing AcrAB grow to lower cell densities at stationary phase but no adverse effects on virulence have been reported.35
Our work demonstrates that subinhibitory concentrations of disinfectants, which might occur during poor disinfection and cleaning procedures, can lead to selection of strains with reduced susceptibility, or even resistance to antibiotics. These effects were more pronounced with a quaternary ammonium-based biocide and with the use of high concentrations of triclosan. However, the dissemination of these strains might be low due to impaired virulence. In further work, we studied the phenotype (e.g. virulence) of several isolates obtained from these populations and analysed their proteomes, identifying alterations in the expression of several proteins. Further research on this is important, to reveal the genetic basis of disinfectant tolerance, its contribution to antibiotic resistance and the possible role of antimicrobial resistance mechanisms in virulence.
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This study was funded by the Department for Environment, Food and Rural Affairs UK (project OD2010).
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None to declare.
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Acknowledgements |
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We thank Nick Coldham and Luke Randall for helpful discussions and advice.
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2 Enter-net, International Surveillance Network for Enteric Infections-Salmonella, VTEC 0157 and Campylobacter. Annual report-Surveillance of Enteric Pathogens in Europe and Beyond (2004) http://www.hpa.org.uk/hpa/inter/enter-net/Enter-net%20annual%20report%202004.pdf (26 March 2007, date last accessed).
3 European Commission Press Releases (22/01/2005). Ban on Antibiotics as Growth Promoters in Animal Feed Enters into Effect. Ref. IP/05/1687. http://europa.eu/rapid/pressReleasesAction.do?reference=IP/05/1687&format=HTML&aged=0&language=EN&guiLanguage=en (26 March 2007, date last accessed).
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