Introduction

Multidrug efflux pumps cause serious problems in cancer chemotherapy and bacterial infection treatments. In bacteria, drug resistance is often associated with multidrug efflux pumps that decrease intracellular drug accumulation.1, 2 These efflux pumps are classified into following five families on the basis of sequence similarity: (1) major facilitator; (2) resistance-nodulation cell division (RND); (3) small multidrug resistance; (4) multidrug and toxic compound extrusion; and (5) ATP-binding cassette.3 The pumps belonging to the RND family are particularly effective in producing resistance because they form a tripartite complex with the periplasmic proteins belonging to the membrane fusion protein (MFP) family and the outer membrane channels, facilitating direct efflux of drugs to the external medium. RND pumps often possess wide substrate specificity.1

Salmonella enterica is a pathogen that causes a variety of diseases in humans ranging from gastroenteritis to bacteremia and typhoid fever.4 In the 1990s, the prevalence of multidrug-resistant S. enterica increased dramatically in the UK,5, 6 USA7, 8 and Canada.9 In addition, many countries have documented outbreaks associated with drug-resistant Salmonella in poultry, beef and pork.10, 11, 12, 13, 14 Emerging resistance to antibiotics in Salmonella has been found in pathogens isolated from humans and animals and is thus a potentially serious public health problem.15, 16

Determination of bacterial genome sequences enables us to trace putative drug resistance genes in bacteria.17, 18 A recent study showed that S. enterica serovar Typhimurium has five putative RND transporter systems (see Figure 1): AcrAB, AcrD, AcrEF, MdtABC and MdsABC.19 In S. enterica, the function of all RND transporter systems requires TolC (outer membrane channel), except for MdsABC, which requires either MdsC or TolC for drug resistance.20 Four of these system genes, acrAB, acrEF, mdtABC and mdsABC also code for an MFP (acrA, acrE, mdtA and mdsA, respectively) in the same operon (Figure 1). However, an MFP gene is not located near acrD, and the MFP, with which AcrD functions, remains to be studied in detail (Figure 1). Previously, we reported that the overproduction of AcrD conferred multidrug resistance to the ΔacrB mutant against novobiocin, sodium dodecyl sulfate (SDS) and sodium deoxycholate;19 however, its role in resistance against β-lactam antibiotics and requirement of MFP have not been elucidated.

Figure 1
figure 1

Resistance-nodulation cell division (RND) transporter system genes encoded in the S. enterica serovar Typhimurium genome. Chromosomal positions of genes encoding RND drug transporters, outer membrane proteins and membrane fusion proteins (MFPs) are indicated by ‘kb’ (kilobase pair) in the S. enterica serovar Typhimurium strain LT2 genome.29 Figure was modified from Nishino et al. (2006).19 Arrows correspond to the lengths and directions of the genes.

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In this study, we describe that AcrD contributes to Salmonella resistance against β-lactam antibiotics including oxacillin, cloxacillin, nafcillin, carbenicillin, sulbenicillin and aztreonam. In addition to the role of AcrA as MFP in the AcrAB multidrug efflux pump, we report that AcrA is required for the multidrug resistance conferred by the AcrD efflux pump (Figure 2). The results suggest multiple roles of AcrA as an MFP in S. enterica.

Figure 2
figure 2

Model for AcrA usage as an membrane fusion protein (MFP) by the AcrAB–TolC and AcrD efflux systems in S. enterica. In S. enterica, AcrA functions with AcrB and AcrD efflux pumps, resulting in an increase in multidrug resistance.

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Materials and methods

Bacterial strains, plasmids and growth conditions

The bacterial strains and plasmids used in this study are listed in Table 1. The S. enterica serovar Typhimurium strains were derived from the wild-type strain ATCC 14028s (ref. 21). These strains were grown at 37 °C in Luria–Bertani (LB) broth.22

Table 1 S. enterica strains and plasmids used in this study
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Construction of gene deletion mutants

To construct the ΔacrB mutant NKS1202, the kanamycin resistance marker of the ΔacrB::KmR strain EG16565 (ref. 19) was eliminated using the pCP20 plasmid as described previously.23 To construct the ΔacrABΔacrD mutant NKS1245, P22 phages isolated from the ΔacrAB::KmR strain NKS147 and the ΔacrD::CmR strain EG16567 (ref. 19) were transduced to the wild-type strain ATCC 14028s, and kanamycin and chloramphenicol resistance markers were eliminated using the pCP20 plasmid. To construct the ΔacrAB strain NKS234 or the ΔtolC strain NKS233, gene disruption was performed as described by Datsenko and Wanner.23 The following oligonucleotide primers were used for the construction of the mutants:

acrA-P2

(5′-ACCATTGACCAATTTGAAATCGGACACTCGAGGTTTACATCATATGAATATCCTCCTTAG-3′);

acrB-P1

(5′-AAAAAAGGCCGCTTGCGCGGCCTTATCAACAGTGAGCAAAGTGTAGGCTGGAGCTGCTTC-3′);

tolC-P2

(5′-TACAAATTGATCAGCGCTAAATACTGCTTCACAACAAGGACATATGAATATCCTCCTTAG-3′);

and tolC-P1

(5′-GGGCACAGGTCTGATAAGCGCAGCGCCAGCGAATAACTTAGTGTAGGCTGGAGCTGCTTC-3′).

The chloramphenicol resistance gene, cat, or the kanamycin resistance gene, aph, flanked by Flp recognition sites, was amplified by PCR using the aforementioned primers. The resulting PCR products were used to transform the recipient ATCC 14028s strain harboring the pKD46 plasmid expressing Red recombinase. The chromosomal structure of the mutated loci was verified by PCR, as described previously.23 cat and aph were eliminated using the pCP20 plasmid, as described previously.23

Plasmid construction

acrA was amplified from ATCC 14028s genomic DNA using the primers 5′-CGCAGTACTATGTCGGTGAATTTACAGGCG-3′ and 5′-CGCGGATCCGTCTTAACGGCTCCTGTTTAA-3′, which introduced ScaI and BamHI sites (underlined in the primer sequences above), respectively. The PCR fragment contained a region from 141 bp upstream to 22 bp downstream of acrA. This fragment was digested with ScaI and BamHI and cloned into the corresponding sites of pACYC177, where an ampicillin resistance gene was located. The resulting plasmid, pacrA, carried acrA instead of the ampicillin marker (Table 1). acrD was amplified using the primers 5′-CGCGGATCCCATTATCTCCTTTATTTCTCC-3′ and 5′-CGCGCATGCTTATTTCGGGCGCGGCTTCAG-3′, which introduced BamHI and SphI sites (underlined in the primer sequences above), respectively. The PCR fragment contained a region from 100 bp upstream to the stop codon of acrD. This fragment was digested with BamHI and SphI and cloned into the corresponding sites of pHSG398 to produce pacrD (Table 1). To obtain pACYC177 without the ampicillin marker, pACYC177 was digested at two sites with Psp1406I (AclI) and self-ligated. The resulting vector was named pACYC177KmR.

Determination of MICs of toxic compounds

The antibacterial activities of various agents were determined on LB agar (1% tryptone, 0.5% yeast extract, 0.5% NaCl) plates containing oxacillin, cloxacillin, nafcillin, carbenicillin, sulbenicillin, aztreonam, SDS, or novobiocin (Sigma, St Louis, MO, USA) at various concentrations. Agar plates were made by the twofold agar dilution technique, as described previously.24 To determine the MICs, bacteria were grown in LB broth at 37 °C overnight, diluted into the same medium, and then tested at a final inoculum size of 105 CFU per μl by using a multipoint inoculator (Sakuma Seisakusyo, Tokyo, Japan) after incubation at 37 °C for 20 h. The MIC was the lowest concentration of a compound that inhibited cell growth.

Results

Effect of acrD overexpression on the multidrug resistance of the ΔacrB strain

To investigate the role of the AcrD drug efflux pump in multidrug resistance, acrD in S. enterica serovar Typhimurium ATCC14028s was cloned into the vector pHSG398, and the constructed plasmid was transformed into the ΔacrB strain. Compared with the wild-type strain, the ΔacrB strain showed increased susceptibility to all eight antimicrobial agents and chemical compounds tested (Table 2). The transformant (ΔacrB/pacrD) showed increased resistance to SDS and novobiocin as we previously reported.19, 20 Because we had previously cloned acrD into the pUC118 vector, which had the β-lactamase gene as a marker, the effect of acrD overexpression on the β-lactam resistance of S. enterica was unknown.19, 20 In this study, acrD was cloned into the pHSG398 vector that had a chloramphenicol resistance marker. In addition to SDS and novobiocin, the pacrD plasmid conferred resistance to β-lactam antibiotics such as oxacillin, cloxacillin, nafcillin, carbenicillin, sulbenicillin and aztreonam. These data indicate that the AcrD efflux system confers multidrug resistance to S. enterica against SDS, novobiocin and β-lactam antibiotics.

Table 2 Susceptibility of S. enterica strains to toxic compounds
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Effect of acrD overexpression on the drug susceptibility of the ΔacrAB or ΔtolC strains

We previously reported that only the AcrAB–TolC efflux system was expressed under laboratory conditions, whereas other efflux systems required additional cues for detectable expression.19 Thus, to determine the components required for the function of the AcrD efflux system, we focused on the outer membrane channel, TolC and the MFP, AcrA. We constructed the ΔtolC and ΔacrAB mutants of S. enterica, and the plasmid carrying acrD was transformed into these mutants (Table 1). In contrast to the results of the ΔacrB mutant, overexpression of acrD did not confer multidrug resistance to ΔtolC and ΔacrAB mutants (Table 2). These data suggest that the multidrug resistance conferred by AcrD overproduction is dependent on the presence of TolC and AcrA proteins.

Effect of acrA overexpression on the drug susceptibility of S. enterica

In the drug susceptibility tests, we observed that the ΔacrAB strain was slightly (twofold change) susceptible to oxacillin, cloxacillin, nafcillin, carbenicillin and sulbenicillin compared with the ΔacrB strain (Table 2). This phenotype was complemented with the plasmid carrying acrA (Table 2). The ΔacrAB strain harboring the pacrA plasmid behaved like the ΔacrB strain. This result suggests the possibility that AcrA may be required for other drug resistance factors in addition to AcrB. The aforementioned results obtained using the plasmid carrying acrD and the ΔacrAB strain suggests that AcrD may require AcrA for its function. To determine whether the function of chromosomally encoded AcrD causes the difference in MICs between the ΔacrB and ΔacrAB strains, the plasmid carrying acrA was transformed into the ΔacrAB and ΔacrABΔacrD strains (Table 1). The drug susceptibility of the ΔacrABΔacrD strain was similar to that of the ΔacrAB strain (Table 2). In contrast to the case of the ΔacrAB mutant, the plasmid carrying acrA did not increase the drug resistance of the ΔacrABΔacrD mutant. These data suggest that increased susceptibility of the ΔacrAB strain compared with the ΔacrB strain is due to loss of function of the chromosomally expressed AcrD efflux pump along with the deletion of acrA.

Effect of the co-expression of acrD and acrA on the drug susceptibility of S. enterica

The aforementioned results suggest the possibility that the AcrD efflux pump requires AcrA for its function as an MFP. To verify whether AcrA functions as the MFP of the AcrD efflux system, pacrD and pacrA were co-transformed into the ΔacrAB strain (Table 1). Interestingly, co-expression of acrA and acrD significantly conferred drug resistance to the ΔacrAB strain against all eight compounds tested, whereas overexpression of acrD alone did not confer any resistance to this strain. In addition, co-expression of acrA and acrD conferred multidrug resistance to the ΔacrABΔacrD strain. These results indicate that AcrA (an MFP) is required for the function of the AcrD efflux pump and contributes to AcrD-mediated multidrug resistance (Figure 2).

Discussion

In this study, we identified that the AcrD efflux system confers resistance to various compounds including β-lactam antibiotics and requires AcrA as an MFP in S. enterica. In a previous study, we determined that the overexpression of acrD conferred resistance to the ΔacrB strain of S. enterica against SDS and novobiocin. However, the effect of acrD on Salmonella susceptibility to β-lactam antibiotics had not been investigated because acrD was cloned into the pUC118 vector, which had the β-lactamase gene as a marker.19 Here, we demonstrated that the plasmid carrying acrD conferred resistance not only to SDS and novobiocin, but also to oxacillin, cloxacillin, nafcillin, carbenicillin, sulbenicillin and aztreonam in the ΔacrB mutant. Although this plasmid did not increase the multidrug resistance of the ΔacrAB mutant, the co-expression of acrD with acrA increased the multidrug resistance of this mutant. Furthermore, in contrast to the result that the ΔacrAB mutant complemented with pacrA behaved like ΔacrB mutant, the plasmid carrying acrA did not increase the drug resistance of the ΔacrABΔacrD mutant. These data suggest that chromosomally expressed AcrD, despite the low expression level,19 induces intrinsic multidrug resistance in S. enterica. In Escherichia coli, it was suggested that the function of the AcrD system depends on AcrA, although a co-expression study of acrD with acrA was not previously performed.25, 26 In this study, on the basis of the results of co-expression of acrD with acrA, we revealed that AcrD of S. enterica requires AcrA for its function. In addition to the role of AcrA in the AcrAB–TolC system, our study showed that it was required for the function of the AcrD efflux system in S. enterica (Figure 2).

One possibility for AcrD using AcrA as an MFP when it is coded in a different operon is that AcrD has a compensatory role when AcrB function is disrupted. In other words, AcrD may form a complex with AcrA and TolC when mutations occur in AcrB and compensate for the lost function of AcrAB–TolC multidrug efflux system. Another possibility is that AcrA contributes to different biological functions by forming complexes with two different RND pumps, AcrB and AcrD. As described previously,1 AcrB is an important factor for bacterial multidrug resistance. It was recently reported that AcrD contributes to resistance against metals such as copper or zinc in addition to its role in multidrug resistance.27, 28 Because AcrA is required for the functioning of AcrD, AcrA may be involved in AcrD-mediated metal resistance. Such a functional network of multidrug efflux pumps may contribute to bacterial adaptation to various environmental conditions.

Further investigation of the cooperation of multidrug efflux systems is required to elucidate their biological significance in natural environments. Such investigations may provide further insights into the role of these systems in cell physiology.