TolC but not AcrB is essential for multidrug-resistant Salmonella enterica serotype Typhimurium colonization of chicks

Sylvie Baucheron, Christian Mouline, Karine Praud, Elisabeth Chaslus-Dancla and Axel Cloeckaert*

Unité BioAgresseurs, Santé et Environnement, Institut National de la Recherche Agronomique, 37380 Nouzilly, France


* Corresponding author. Tel: +33-2-47-42-77-50; Fax: +33-2-47-42-77-74; Email: cloeckae{at}tours.inra.fr

Received 9 December 2004; returned 27 January 2005; revised 4 February 2005; accepted 7 February 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To study the role of the multidrug efflux system AcrAB-TolC in resistance of multidrug-resistant Salmonella enterica serotype Typhimurium (S. Typhimurium) phage type DT104 and DT204 strains to detergents and bile salts. To evaluate the importance of the inner membrane transporter AcrB and the outer membrane component TolC of this efflux system in the colonization of two multidrug-resistant S. Typhimurium DT104 and one DT204 strain in chicks.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Salmonella enterica serotype Typhimurium (S. Typhimurium) is an important gastrointestinal pathogen that causes diseases ranging from mild gastro-enteritis accompanied by local inflammation and diarrhoea, to a life-threatening systemic infection.1 Non-typhoidal Salmonella has emerged as a common cause of illness in industrial countries, and much of this increase is accounted for by two Salmonella enterica serotypes, Enteritidis and Typhimurium.1 Their entry into the human food chain results most often from disease-free carriage in the intestine of poultry.1

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and culture conditions

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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Table 1. Characteristics of the S. enterica serotype Typhimurium strains studied

 
Bacteria were grown overnight at 37°C in brain heart infusion (BHI) broth (Difco, Becton Dickinson, MD, USA) under aerobic conditions. For in vivo experiments, overnight culture concentrations were adjusted to calculated doses (1 x 101 to 1 x 108 cfu) in saline by measurement of absorbance at 600 nm. The correspondence between absorbance values and cfu was previously established for each strain (data not shown). Enumerations of cfu in suspensions were determined by plating 10-fold serial dilutions on BHI agar.

Minimum inhibitory concentration (MIC) determinations

MICs of sodium deoxycholate (DOC) (Sigma–Aldrich, Steinheim, Germany), sodium choleate (CHOL) (Sigma–Aldrich), 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 (Sigma–Aldrich) 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) (Sigma–Aldrich) 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Role of AcrB and TolC in resistance to detergents and bile salts

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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Figure 1. Cumulated deaths of chicks 7 days post-inoculation by the oral route with S. Typhimurium DT104 strains 1948SA96 and BN10055, and S. Typhimurium DT204 strain 102SA00 and their derived acrB and tolC mutants. Numbers indicate the total number and values in parentheses indicate the total percentage of dead chicks.

 
TolC but not AcrB is essential for colonization

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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Table 2. Means and standard deviations of number of cfu in the faeces, caecal contents, livers and spleens of chicks at 7 days after challenge with the indicated doses of the S. enterica serotype Typhimurium strains

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The multidrug-resistant S. Typhimurium strains studied were shown to colonize efficiently the intestinal tract, spleen and liver of the chicks. The most interesting findings of this study were that the tolC mutants shown to be susceptible to detergents and bile salts failed to colonize the intestinal tract, spleen and liver of the chicks, whereas the acrB mutants that were still significantly resistant to detergents and bile salts did not show any difference to the respective wild-type strains in the ability to colonize these organs. In addition, TolC appeared to play a significant role in virulence since at least for one of the S. Typhimurium DT104 strains, deletion of tolC resulted in a significant increase in the LD50, as was previously demonstrated for S. Enteritidis in mice.22 The role of TolC homologues in colonization was also previously demonstrated for other Gram-negative bacteria such as Vibrio cholerae in a mouse infection model.19 Lin et al. demonstrated that CmeABC, an efflux system homologous to AcrAB-TolC, is essential for the colonization of chickens by Campylobacter jejuni.18 In contrast to our results, cmeB (acrB counterpart) as well as cmeC (tolC counterpart) mutants failed to colonize the intestinal tract of the chickens. Besides, TolC has been shown to be involved in secretion of virulence factors.15,19,22

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


    Acknowledgements
 
We thank A. Brée for helpful advice in the experimental assays, C. Limouzin and his team from the Unité expérimentale de Pathologie Aviaire et Parasitologie for assistance in animal husbandry, and S. Payot for helpful discussions in the methodological choices.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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