Unité BioAgresseurs, Santé et Environnement, Institut National de la Recherche Agronomique, 37380 Nouzilly, France
Received 9 December 2004; returned 27 January 2005; revised 4 February 2005; accepted 7 February 2005
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Abstract |
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Methods: acrB and tolC mutants of multidrug-resistant S. Typhimurium DT104 and DT204 strains were constructed by insertional inactivation of the acrB gene and deletion of the tolC gene. MICs of detergent and bile salts were determined for the wild-type strains, the acrB and the tolC mutant strains, in presence and in absence of the efflux pump inhibitor Phe-Arg ß-naphthylamide. The effect of sodium choleate on the in vitro growth of these strains was also evaluated. The LD50s of the strains were measured in a day-old chicken model, inoculated with several doses (1 x 103 to 1 x 108 cfu) by the oral route, for 7 days post-inoculation. The colonization levels were assessed at the sublethal dose 7 days post-inoculation by determining the number of cfu of Salmonella in the faeces, caecum, spleen and liver.
Results: The decrease in resistance levels to bile salts was 64- to 256-fold higher for the tolC mutants than for the acrB mutants relative to those of the parental strains. Addition of choleate in culture medium did not affect the growth of the wild-type strains or that of the acrB mutants, but inhibited completely the growth of the tolC mutants. The LD50s were 1.0 x 106 and 1.2 x 107 cfu for one wild-type S. Typhimurium DT104 strain and the acrB mutant, respectively, and were >1.0 x 108 for the tolC mutants or the S. Typhimurium DT204 strains. In contrast to the acrB mutants, the tolC mutants were unable to colonize the caecum, spleen and liver after 1 week of infection. Moreover, in most chicks, no intestinal excretion was detected for the tolC mutants. The colonization levels of the acrB mutants were not significantly different from those of the wild-type strains.
Conclusions: TolC but not AcrB appears to be essential for multidrug-resistant S. Typhimurium DT104 and DT204 colonization of chicks, which is in accordance with their respective roles in resistance to detergents and bile salts. Therefore, TolC could be a better target than AcrB for the development of efflux pump inhibitors.
Keywords: efflux systems , inhibitors , bile salts , detergents , infections
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Introduction |
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Emergence of resistance to fluoroquinolones in Salmonella spp. is a problem in both veterinary and human medicine since these drugs are often the treatment of choice in the cases of life-threatening salmonellosis due to multidrug-resistant strains. This is particularly the case for epidemic S. Typhimurium DT104 strains which over the last few years in addition to their multidrug resistance pattern to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines (ACSSuT), conferred by an antibiotic resistance gene cluster located within a chromosomal genomic island called Salmonella genomic island 1,2 have shown decreased susceptibility to fluoroquinolones.3,4 High-level fluoroquinolone resistance in Salmonella has rarely been documented. The first reported example was the emergence and probably clonal spread of S. Typhimurium variant Copenhagen of phage type DT204 (S. Typhimurium DT204) in the early 1990s in Europe.57
In Salmonella, quinolone resistance has been attributed to point mutations in the quinolone resistance-determining region (QRDR) of the target gene gyrA.3,8 Additionally, high-level fluoroquinolone-resistant isolates have shown mutations in the QRDR of gyrB and parC.5,7,9,10 We have recently reported participation of active efflux as an important mechanism of resistance to fluoroquinolones in S. Typhimurium, due to overproduction of the AcrAB-TolC efflux system.7,8,10,11 This efflux system has been previously identified in Escherichia coli conferring a multidrug resistance phenotype including clinically important antimicrobials.12,13 The AcrB efflux pump belongs to the resistance-nodulation-division (RND) family of transporters and the AcrAB-TolC tripartite efflux system has been shown to export a broad range of components across both the inner and outer membrane to the extracellular space.1216 Moreover, this and other homologous systems found in other Gram-negative bacteria have been shown to play an important role in detergent and bile salt resistance and in in vivo colonization.12,1722 Bile salts are detergent-like substances assisting fat digestion and absorption.18,19,21,23 Cholate and deoxycholate are primary constituents of the bile and they are conjugated with lipids.18,19,21 Bile is secreted into the duodenum in high concentrations,18,19,21 although a concentration of 2 mM is considered to be necessary for lipid digestion.18 Bile salts also have antimicrobial properties, degrading the lipid bilayer structure of bacteria.18,21,23 Thus, adaptation to high bile concentration is essential for enteric commensal or pathogenic bacteria to survive in the intestinal tract.
Recent studies have shown the great interest of inhibiting efflux mechanisms to combat bacterial multidrug resistance.24 The efflux pump inhibitor Phe-Arg-ß-naphthylamide (PAßN), in association with fluoroquinolones, was shown to be effective against fluoroquinolone-resistant E. coli and Pseudomonas aeruginosa.24,25 We have previously shown that this efflux pump inhibitor was also highly effective in association with several fluoroquinolones, in particular enrofloxacin, against high-level fluoroquinolone-resistant S. Typhimurium DT204.7 The same decrease in resistance levels (16- to 32-fold) was indeed observed for mutants where acrB or tolC was inactivated.7,10
The aim of this study was to assess, in multidrug-resistant S. Typhimurium DT104 and DT204 strains, the importance of the AcrAB-TolC efflux system in detergent and bile salt resistance and in the colonization of the intestinal tract and other organs in a day-old Salmonella susceptible chick model.
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Materials and methods |
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S. Typhimurium DT104 strains used were isolated from cattle in France (multidrug- and quinolone-resistant strain BN10055) and in Belgium (multidrug-resistant and quinolone-susceptible strain 1948SA96). Multidrug- and high-level fluoroquinolone-resistant S. Typhimurium DT204 strain 102SA00 used in this study was isolated in Belgium in 2000 from animal feed imported from China (Table 1). acrB and tolC mutants of these three field strains were as previously described.7,10,26 Briefly, inactivation of the acrB gene was done by insertion of a kanamycin resistance cassette,7,17 and deletion of the tolC gene and its replacement with a kanamycin resistance gene was performed using the method of Datsenko & Wanner.27 The constructions were first realized in a susceptible S. Typhimurium DT104 strain, then mutations were introduced into the three multidrug-resistant S. Typhimurium strains from this study by P22 phage transduction.7,10,26
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Minimum inhibitory concentration (MIC) determinations
MICs of sodium deoxycholate (DOC) (SigmaAldrich, Steinheim, Germany), sodium choleate (CHOL) (SigmaAldrich), and sodium dodecyl sulphate (SDS) (Interchim Uptima, France), were determined by the doubling dilution method on Luria-Bertani (LB) medium in microtitre plates with inocula of 105 cfu per well. The plates were incubated for 18 h at 37°C. The lowest concentration of compound that completely inhibited growth was determined as the MIC. The inhibition of growth was considered when the difference in the absorbance value (between time 0 and time 18 h) at 600 nm was 0.1. MICs were also determined in the presence of the efflux pump inhibitor PAßN (SigmaAldrich) at a concentration of 80 mg/L. Experiments were repeated twice.
In vitro growth assays
Growth assays were performed in the presence and absence of bile salts for both the wild-type strains and their acrB and tolC mutants. CHOL is a crude bile extract that contains the sodium salts of cholic, glycocholic, deoxycholic and taurocholic acids, and represents the bile salt components in the animal intestine. BHI broth with and without 1 g/L (about 2 mM) of CHOL were inoculated at 1% with overnight cultures and incubated using the same culture conditions described above. During the incubation, samples were taken at different time points (every 30 min from 1.5 to 4.5 h). The absorbance values were measured at 600 nm and the enumeration of cfu was done as described above.
Infection model
The experiments comply with the current French laws on animal experimentation.
Poultry line B13 is a histocompatible inbred White Leghorn (line GB1), developed and produced by the Unité Expérimentale de Pathologie Aviaire et Parasitologie, INRA. This poultry line was chosen for its susceptibility to Salmonella infections.
Newly hatched chicks were distributed six to each isolator, allowing us to control the environmental conditions. Chicks were reared in wire-floored isolators with free access to feed and water and they were observed each day for a period of 8 days. At 1-day-old, the chicks were individually infected by gavage with 0.1 mL of a pure bacterial suspension calibrated at 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107 or 1 x 108 cfu of S. Typhimurium DT104 or DT204 strains. Six chicks per dose and per bacterial strain were inoculated. Assays were repeated twice for the S. Typhimurium DT204 strains.
The number of cfu in suspensions was verified as described above. Prior to inoculation, the chicks were screened for the presence of Salmonella in faeces by Muller Kauffmann (Diagnostic Pasteur, Marnes-la-Coquette, France) enrichment and plating out on Salmonella-Shigella agar medium (Bio-Rad, Marnes-la-Coquette, France). The results confirmed that all chicks were Salmonella-free before challenge.
Analysis of colonization experiments
Seven days post-inoculation, the LD50 was calculated by the method of Reed & Muench.28 Faeces samples were taken from the six chicks surviving at the sublethal dose. The chicks were then sacrificed by carbon dioxide inhalation. Livers, spleens and the whole caecal contents were removed, weighed and homogenized in the appropriate volume of saline to obtain a 10-fold dilution. The bacterial loads of the samples were quantified by plating serial 10-fold dilutions of the homogenates onto agar plates containing BHI supplemented with kanamycin (50 mg/L) (SigmaAldrich) or Rambach agar (Merck, Darmstadt, Germany) for the mutants and the wild-type strains, respectively (detection limit: 102 cfu/g). PCRs were performed to confirm that colonies recovered on BHI agar supplemented with kanamycin corresponded to the acrB or tolC mutants.
Statistical analysis
The number of cfu per sample was transformed to log10 to normalize the distribution of individual counts required for statistical analysis. Means and standard deviations of log10-transformed cfu were calculated and compared. The significance of the difference between the mutants and the wild-type strains was tested using Dunnett's multiple comparison t-test. Statistical significance was defined as a P value of < 0.01.
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Results |
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We have previously described the importance of the AcrB and TolC components of the AcrAB-TolC efflux system in the multidrug resistance of two S. Typhimurium strains, i.e. high-level fluoroquinolone-resistant DT204 strain 102SA00 and quinolone-resistant DT104 strain BN10055.7,26 To complete the substrate spectrum of the AcrAB-TolC efflux system and to determine the role of its components, we tested the susceptibility of these strains and of the multidrug-resistant but quinolone-susceptible S. Typhimurium DT104 strain 1948SA96 and their tolC and acrB mutants to detergents and bile salts. For all wild-type strains (BN10055, 1948SA96 and 102SA00), high MIC values of the detergent SDS and of the bile salts DOC and CHOL were obtained (Table 1). In the presence of the efflux pump inhibitor PAßN, the MIC value of SDS decreased 2048- to 4096-fold and the MIC values of DOC and CHOL decreased 256-fold. Thus, these results indicated efflux as an important mechanism of resistance to detergents and bile salts in multidrug-resistant S. Typhimurium DT104 and DT204.
The role of active efflux by AcrAB-TolC was further confirmed by inactivation of the acrB and tolC genes in these strains. For the acrB mutants, MIC values of SDS and DOC decreased 64- to 128-fold and 2- to 8-fold, respectively, but the MIC values of CHOL did not decrease significantly (Table 1). In the case of the tolC mutants, MIC values of SDS, DOC and CHOL decreased 1024- to 2048-fold, 512-fold and 256-fold, respectively. Thus, the decrease in resistance levels to SDS and bile salts was much higher for the tolC mutants than for the acrB mutants.
Choleate inhibits the growth of tolC mutants but not that of acrB mutants
We compared the in vitro growth of the tolC and the acrB mutants with that of the wild-type strains in the absence and presence of CHOL in broth medium. The wild-type strains and the acrB mutants displayed similar growth curves whereas the tolC mutants did not grow at all in the presence of CHOL (data not shown). In the absence of CHOL, the growth curves of the wild-type strain and the tolC and acrB mutant strains were similar (data not shown). Thus, these results are in accordance with the bile salt MIC values and confirm that TolC is much more important in resistance to bile salts than AcrB.
Role of TolC in virulence
Figure 1 shows the results of cumulated deaths 1 week after challenge of the chicks, which allowed us to calculate the LD50. The infection of the chicks by the oral route with S. Typhimurium DT104 strain 1948SA96 gave LD50 values of 1.0 x 106 and 1.2 x 107 cfu for the wild-type and the acrB mutant, respectively, and was >1.0 x 108 for the tolC mutant because the number of dead chicks was too low. The LD50 values of the S. Typhimurium DT104 strain BN10055 and its acrB or tolC mutant were also > 1.0 x 108 (Figure 1). The infections with the S. Typhimurium DT204 strains caused no deaths at all (Figure 1). S. Typhimurium DT104 strain 1948SA96 was thus the most virulent among the three wild-type strains tested and at least for this strain it appeared that the TolC channel played an important role in virulence in this chicken model.
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Seven days after challenge, chickens were taken for analysis of colonization at the inoculation doses 1 x 105 and 1 x 106 cfu, which corresponded to the sublethal doses of the S. Typhimurium DT104 and S. Typhimurium DT204 strains, respectively. The inability of the tolC mutants to grow in the presence of bile salts would suggest that TolC is required for S. Typhimurium intestinal colonization of the chicks. As shown in Table 2, the three wild-type strains as well as their acrB mutants were recovered at high levels, on average 6, 8, 4 and 4 log10 cfu/g of faeces, caecal contents, livers and spleens, respectively. The differences in colonization levels between the wild-type strains and the acrB mutants were statistically not significant. However, the three tolC mutants failed to colonize organs and were not recovered in the faeces or from caecal contents or in some cases the level of colonization was 10 000-fold lower than that of the wild-type strains (Table 2). The differences in the viable counts in faeces, caecal content, liver and spleen, between the tolC mutants and the wild-type strains or their acrB mutants were thus always statistically highly significant (P < 0.01). These results indicated that TolC but not AcrB is essential for the in vivo colonization of multidrug-resistant S. Typhimurium in the intestinal tract and in the spleen and liver of chicks.
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Discussion |
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Our results of colonization obtained in the chicks are in accordance with the susceptibility testing results. The tolC mutants were much more susceptible to detergents and bile salts than the acrB mutant strains. Moreover, only the tolC mutant strains were inhibited in their growth in the presence of choleate. Interestingly, the efflux pump inhibitor PAßN affected the susceptibilities of the wild-type strains to the several compounds to a similar extent as that obtained by deletion of tolC. This efflux pump inhibitor has been previously shown to target RND multidrug transporters such as AcrB or MexB24 and thus possibly it could act on other transporters involved in export of detergents and bile salts. These results contrast to the susceptibility patterns to antibiotic families such as phenicols, quinolones and tetracycline which were not significantly different between the acrB and tolC mutant strains (Table 1).7,10 Thus, in contrast to export of these antibiotic families, it appears that, in Salmonella, TolC probably also functions with other transporters involved in the intrinsic resistance to detergents and bile salts as shown previously for E. coli.12,20
Several recent studies suggest that the use of an efflux pump inhibitor combined with an antimicrobial could reduce the acquired resistance to fluoroquinolones and would decrease the frequency of emergence of strains clinically resistant to fluoroquinolones.24 In our previous studies, the efflux pump inhibitor PAßN was shown to be effective in decreasing the resistance level to several fluoroquinolones and other unrelated drugs in multidrug-resistant S. Typhimurium DT104 and DT204 strains.7,26 The S. Typhimurium DT104 strains studied showed a low-level of resistance to fluoroquinolones (MIC up to 4 mg/L) and carried only one mutation in the target gene gyrA or none at all. In this case PAßN decreased the level of resistance to a fluoroquinolone-susceptible phenotype. For S. Typhimurium DT204 strains highly resistant to fluoroquinolones (MIC of 32 or 64 mg/L depending on the fluoroquinolone), despite the presence of multiple target gene mutations in gyrA, gyrB, and parC, PAßN was also able to decrease the fluoroquinolone resistance level up to 32-fold although not to a susceptible phenotype but for example to an intermediate-resistant phenotype for enrofloxacin. In this study, we show that the use of the efflux pump inhibitor PAßN also resulted in a significant decrease in the resistance levels to bile salts and detergents. Moreover, the results of this study also indicated that TolC is more importantly involved than AcrB in resistance to bile salts and detergents and suggest that transporters other than AcrB interacting with TolC could be involved in resistance to bile salts and detergents. Therefore the TolC channel constitutes an interesting key target in vivo, rather than the transporter components spanning the inner membrane because of its major role in resistance to a broad spectrum of toxic compounds including antibiotics and detergents. Thus, TolC could be a useful target for the development of efflux system inhibitors against multidrug-resistant pathogenic bacteria.29
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Acknowledgements |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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2
.
Boyd, D., Peters, G. A., Cloeckaert, A. et al. (2001). Complete nucleotide sequence of a 43-kilobase genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104 and its identification in phage type DT120 and serovar Agona. Journal of Bacteriology 183, 572532.
3
.
Threlfall, E. J. (2000). Epidemic Salmonella Typhimurium DT104: a truly international multiresistant clone. Journal of Antimicrobial Chemotherapy 46, 710.
4
.
Molbak, K., Baggesen, D. L., Aarestrup, F. M. et al. (1999). An outbreak of multidrug-resistant, quinolone-resistant Salmonella enterica serotype Typhimurium DT104. New England Journal of Medicine 341, 14205.
5
.
Guerra, B., Malorny, B., Schroeter, A. et al. (2003). Multiple resistance mechanisms in fluoroquinolone-resistant salmonella isolates from Germany. Antimicrobial Agents and Chemotherapy 47, 2059.
6 . Heisig, P., Kratz, B., Halle, E. et al. (1995). Identification of DNA gyrase A mutations in ciprofloxacin-resistant isolates of Salmonella typhimurium from men and cattle in Germany. Microbial Drug Resistance 1, 2118.[ISI][Medline]
7 . Baucheron, S., Imberechts, H., Chaslus-Dancla, E. et al. (2002). The AcrB multidrug transporter plays a major role in high-level fluoroquinolone resistance in Salmonella enterica serovar Typhimurium phage type DT204. Microbial Drug Resistance 8, 2819.[CrossRef][ISI][Medline]
8 . Cloeckaert, A. & Chaslus-Dancla, E. (2001). Mechanisms of quinolone resistance in Salmonella. Veterinary Research 32, 291300.[CrossRef][ISI][Medline]
9 . Hansen, H. & Heisig, P. (2003). Topoisomerase IV mutations in quinolone-resistant Salmonellae selected in vitro. Microbial Drug Resistance 9, 2532.[ISI][Medline]
10
.
Baucheron, S., Chaslus-Dancla, E. & Cloeckaert, A. (2004). Role of TolC and parC mutation in high-level fluoroquinolone resistance in Salmonella enterica serovar Typhimurium DT204. Journal of Antimicrobial Chemotherapy 53, 6579.
11
.
Giraud, E., Cloeckaert, A., Kerboeuf, D. et al. (2000). Evidence for active efflux as the primary mechanism of resistance to ciprofloxacin in Salmonella enterica serovar Typhimurium. Antimicrobial Agents and Chemotherapy 44, 12238.
12
.
Fralick, J. A. (1996). Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli. Journal of Bacteriology 178, 58035.
13 . Poole, K. (2004). Efflux-mediated multiresistance in Gram-negative bacteria. Clinical Microbiology and Infection 10, 1226.
14 . Murakami, S., Nakashima, R., Yamashita, E. et al. (2002). Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419, 58793.[CrossRef][ISI][Medline]
15 . Koronakis, V., Sharff, A., Koronakis, E. et al. (2000). Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405, 9149.[CrossRef][ISI][Medline]
16
.
Zgurskaya, H. I. & Nikaido, H. (1999). Bypassing the periplasm: reconstitution of the AcrAB multidrug efflux pump of Escherichia coli. Proceedings of the National Academy of Sciences USA 96, 71905.
17 . Lacroix, F. J., Cloeckaert, A., Grepinet, O. et al. (1996). Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection. FEMS Microbiology Letters 135, 1617.[CrossRef][ISI][Medline]
18
.
Lin, J., Sahin, O., Michel, L. O. et al. (2003). Critical role of multidrug efflux pump CmeABC in bile resistance and in vivo colonization of Campylobacter jejuni. Infection and Immunity 71, 42509.
19
.
Bina, J. E. & Mekalanos, J. J. (2001). Vibrio cholerae tolC is required for bile resistance and colonization. Infection and Immunity 69, 46815.
20
.
Sulavik, M. C., Houseweart, C., Cramer, C. et al. (2001). Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes. Antimicrobial Agents and Chemotherapy 45, 112636.
21
.
Thanassi, D. G., Cheng, L. W. & Nikaido, H. (1997). Active efflux of bile salts by Escherichia coli. Journal of Bacteriology 179, 25128.
22 . Stone, B. J. & Miller, V. L. (1995). Salmonella enteritidis has a homologue of tolC that is required for virulence in BALB/c mice. Molecular Microbiology 17, 70112.[CrossRef][ISI][Medline]
23 . Gunn, J. S. (2000). Mechanisms of bacterial resistance and response to bile. Microbes and Infection 2, 90713.[CrossRef][ISI][Medline]
24
.
Lomovskaya, O., Warren, M. S., Lee, A. et al. (2001). Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. Antimicrobial Agents and Chemotherapy 45, 10516.
25
.
Mazzariol, A., Tokue, Y., Kanegawa, T. M. et al. (2000). High-level fluoroquinolone-resistant clinical isolates of Escherichia coli overproduce multidrug efflux protein AcrA. Antimicrobial Agents and Chemotherapy 44, 34413.
26
.
Baucheron, S., Tyler, S., Boyd, D. et al. (2004). AcrAB-TolC directs efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium DT104. Antimicrobial Agents and Chemotherapy 48, 372935.
27
.
Datsenko, K. A. & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences USA 97, 66405.
28 . Davis, B. D., Dulbecco, R., Eisen, H. N., et al. (1973). Computation of 50% endpoints (LD50 or ID50) by method of Reed and Muench. In Microbiology, pp. 6634. Harper International edn, Hagerstown, MD, USA.
29 . Higgins, M. K., Eswaran, J., Edwards, P. et al. (2004). Structure of the ligand-blocked periplasmic entrance of the bacterial multidrug efflux protein TolC. Journal of Molecular Biology 342, 697702.[CrossRef][ISI][Medline]