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Antimicrob Agents Chemother. 2012 Apr; 56(4): 2084–2090.
PMCID: PMC3318359

Involvement of the AcrAB-TolC Efflux Pump in the Resistance, Fitness, and Virulence of Enterobacter cloacae


Multidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence in Enterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates of E. cloacae were used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). The acrA and tolC genes were deleted in strains EcDC64 and Jc194 to produce, respectively, EcΔacrA and EcΔtolC and JcΔacrA and JcΔtolC knockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance of E. cloacae to several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that the acrA and tolC genes both affect the fitness of E. cloacae, as fitness was clearly reduced in the acrA and tolC KO strains. The median CI values obtained in vitro and in vivo were, respectively, 0.42 and 0.3 for EcDC64/EcΔacrA, 0.24 and 0.38 for EcDC64/EcΔtolC, 0.15 and 0.11 for Jc194/JcΔacrA, and 0.38 and 0.39 for Jc194/JcΔtolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in both E. cloacae clinical strains when either the acrA or tolC gene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates of E. cloacae.


The AcrAB-TolC system is a tripartite complex that is widely distributed in Gram-negative bacteria. The three components of the system are AcrB, the inner membrane transporter protein, AcrA, the periplasmic adaptor protein, and TolC, the outer membrane channel. AcrA protein is believed to be involved in vivo in the assembly and maintenance of a stable complex that transmits conformational changes in AcrB and TolC (20, 35), thus leading to opening of the outer membrane channel (23). Although the components of the AcrAB-TolC pump work together as a tripartite system, the individual components may also play certain roles in other efflux systems (30). For example, in Escherichia coli, TolC has been shown to be required for the functioning of many efflux systems, including the resistance-nodulation-division (RND) pumps AcrD (15), (26), AcrEF (26), MdsAB (25), and MdtABC (24), as well as the major facilitator superfamily (MFS) systems EmrAB and EmrKY (27, 26), and also the ABC drug transporter MacAB (18). Similarly, AcrA has been shown to associate with AcrD (10, 11) and AcrF (10, 17) as well as with AcrB and TolC.

Multidrug efflux pumps, such as the RND superfamily member AcrAB-TolC, have been shown to be clinically relevant mechanisms of resistance to multiple antibiotics used to treat human infections. These pumps confer innate resistance to a wide range of toxic compounds such as antibiotics, dyes, detergents, and disinfectants in members of the Enterobacteriaceae (30, 31) and may be associated with multidrug resistance (MDR). Some studies suggest that overexpression of AcrAB is a marker of multidrug resistance (34). In regard to Enterobacter cloacae, our research group previously described the importance of this AcrAB-TolC efflux pump in conferring resistance to different antimicrobial agents, as well as to dyes and biocides (29).

In addition to an established role in antimicrobial resistance, some MDR efflux pumps not only confer resistance to drugs used in therapy but have also been shown to have a role in bacterial pathogenicity, in the colonization of a variety of host organisms, and in their survival therein (21, 30). The efflux pumps are responsible for expelling host-derived antimicrobial agents, such as bile salts, long-chain fatty acids, and antimicrobial peptides (21, 30). Thus, several studies have demonstrated (21) that inactivation of the efflux pump AcrAB-TolC by the lack of one of its structural components directly affects the virulence of the bacteria, indicating that this system is required for the bacteria to be pathogenic.

Although E. cloacae and Enterobacter aerogenes are the most common Enterobacter species that cause nosocomial infections, little is known about their pathogenic potential. These organisms cause a wide variety of infections, sepsis, infections of the respiratory tract and urinary tract, wound infections, and meningitis (although the latter is rare). They are frequently found in the gastrointestinal tract, which is usually the source of infection. Multiple-antibiotic-resistant strains have caused outbreaks in hospitals, usually involving settings where seriously ill patients are housed, such as intensive care units. Greater resistance to disinfectants and antimicrobial agents than in other members of the Enterobacteriaceae probably plays a role in the increasing prevalence of these organisms as nosocomial pathogens. However, the role that the AcrAB efflux pump plays in the pathogenesis of infections caused by E. cloacae has not been investigated (33).

In this study, both the AcrA and the TolC components of the AcrAB-TolC efflux pump were deleted in two clinical isolates of Enterobacter cloacae in order to investigate the effect of the lack of both structural components on the resistance, fitness, and virulence of this microorganism in an experimental mouse model of infection.


Strains, culture media, and plasmids.

Laboratory strains and plasmids used in the study are listed in Table 1. The clinical isolates used were two strains of Enterobacter cloacae, EcDC64 and Jc194. Both strains were isolated from a patient admitted to the Complexo Hospitalario Universitario A Coruña (A Coruña, Spain). The EcDC64 strain showed an MDR phenotype overexpressing the AcrAB-TolC efflux pump, while the Jc194 strain showed basal efflux pump expression and a more sensitive antibiotic resistance profile than strain EcDC64 (29). Escherichia coli strain TG1 was used for cloning procedures. All strains used in the study were maintained at −80°C in 15% (vol/vol) glycerol (for cryoprotection) until use. The strains were grown on MacConkey agar plates (Becton, Dickinson and Company, NJ), in Luria-Bertani (LB) broth, or on LB agar in the presence of 50 μg of ampicillin/ml, 25 μg of kanamycin/ml, 30 μg of chloramphenicol/ml, 4 μg of gentamicin/ml, or 30 μg of tetracycline/ml (all from Sigma-Aldrich, St. Louis, MO) when required.

Table 1
Bacterial strains and plasmids used in this study

Construction of knockout strains.

Disruption of the acrA and tolC genes in strains EcDC64 and Jc194 was performed by the method described by Datsenko and Wanner (9), with some modifications. The Red helper pKOBEG (kindly donated by J. M. Ghigo, Institut Pasteur, France) (Table 1) is a low-copy-number plasmid that contains a gene for chloramphenicol resistance, a temperature-sensitive origin of replication, and the Red system, which comprises an exonuclease and the β and γ functions of phage λ. The pKOBEG plasmid (Table 1) was introduced into strains EcDC64 and Jc194 by heat shock, and transformants were selected on LB agar with chloramphenicol, after incubation for 24 h at 30°C. One transformant carrying the Red helper plasmid was made electrocompetent. A selectable resistance gene was amplified by PCR from genomic DNA by use of primers, including 5′ extensions with homology for the acrA or tolC loci listed in Table 2. The PCR product was used to disrupt the acrA and tolC genes of strains EcDC64 and Jc194 by electroporation. Electroporation (25 μF, 200 Ω, and 2.5 kV) of the electrocompetent strains was carried out according to the manufacturer's instructions (Bio-Rad Laboratories, Madrid, Spain), with 50 μl of cells and 1 μg of the purified and dialyzed PCR products (0.025-μm nitrocellulose membranes; Millipore, Billerica, MA). Shocked cells were added to 1 ml of LB broth, incubated overnight at 30°C, spread on LB agar containing kanamycin and tetracycline, and incubated at 30°C for 24 h. The mutant strains were then grown on LB agar containing kanamycin and tetracycline at 44°C for 24 h and incubated overnight on LB agar containing kanamycin, tetracycline, and chloramphenicol, at 30°C, to test for loss of the helper plasmid.

Table 2
Oligonucleotides used in this study

Cloning procedures.

Recombinant plasmids were first constructed and then introduced into the acrA and tolC knockout strains for complementation studies. For construction of pAPG-1 (Table 1), specific primers were designed to amplify the acrA gene from E. cloacae strain EcDC64. The amplified DNA was purified, digested with EcoRV and HindIII, and then ligated with the T4 DNA ligase (Promega Corporation, Madison, WI) into a similarly digested pACYC184 expression vector under the control of the CTX-M-14 gene promoter (29). The accuracy of the construct was checked by restriction analysis.

For construction of pAPG-2 (Table 1), the tolC gene was amplified from strain EcDC64 with specific oligonucleotides (Table 2). The amplified fragment of 1,485 bp was purified and digested with BamHI/HindIII and ligated into the pUCP24 expression vector (Table 1), previously digested with the same enzymes, under the control of the placZ promoter.

Antimicrobial susceptibility testing.

MICs of the following antibiotics were determined by Etest (bioMérieux, Durham, NC), following the manufacturer's instructions: oxacillin, erythromycin, clindamycin, linezolid, ciprofloxacin, chloramphenicol, tetracycline, and tigecycline.

In vitro competition experiments.

In vitro competition experiments were performed with each of the isogenic pairs EcDC64/EcΔacrA, EcDC64/EcΔtolC, Jc194/JcΔacrA and Jc194/JcΔtolC. Exponentially growing cells of the corresponding mutant and wild-type strains in LB broth were mixed in a 1:1 ratio and diluted in 0.9% saline solution. Approximately 103 cells from each of the mixtures were inoculated into eight flasks containing 10 ml of LB broth and grown at 37°C and 180 rpm for 16 to 18 h, corresponding to approximately 20 generations. Serial 10-fold dilutions were plated in duplicate on LB agar alone and on LB agar containing 50 μg/ml of kanamycin or 30 μg/ml of tetracycline (depending on the knockout mutant strain), in order to determine the total CFU and the CFU of the mutant, respectively, after overnight incubation at 37°C. The competition index (CI) was defined as the mutant/wild-type ratio. The CI values were calculated for each of the eight independent competition experiments, and the median values were recorded. To assess the growth rates under noncompetitive conditions, growth curves were constructed for all strains. The cells were cultured for 24 h in LB broth at 37°C and 180 rpm. The OD600 was measured at 30-min intervals during the exponential phase and thereafter every hour. Three independent experiments were performed for each of the mutants. The growth rate (μ) was calculated on the basis of the exponential segment of the growth curve, and defined as ln 2g−1, where g is the time taken for an exponentially growing culture to double in size (1).

In vivo competition experiments and virulence assays.

In vivo fitness was assessed by competition experiments in the mouse model of systemic infection. For this purpose, 1:1 mixtures of each of the mutant–wild-type pairs containing a total of approximately 2 × 106 exponentially growing cells were injected intraperitoneally into eight 18- to 25-g C57 mice (General Animal Experimentation and Production Service, University of Seville, Spain). Mice were sacrificed 24 h after injection, and their spleens were aseptically extracted and homogenized in 1 ml of 0.9% saline solution (in a Retsch MM200 mixer mill) to enable the number of CFU of each strain to be determined. The CI values were determined as described for in vitro competition experiments.

To assess the effect on virulence, approximately 3 × 107 exponentially growing cells of EcDC64, Jc194, EcΔacrA, EcΔtolC, JcΔacrA, and JcΔtolC were injected intraperitoneally into groups of six immunosuppressed C57 mice, weighing 18 to 22 g (General Animal Experimentation and Production Service, University of Seville, Spain), previously treated with cyclophosphamide (100 mg/kg) for 3 days. The mice were processed as described above to determine the number of CFU of each strain.


Role of AcrAB-TolC efflux pump in antibiotic resistance in E. cloacae.

The susceptibilities to a number of antibiotics, of both wild-type E. cloacae strains described above and their derived isogenic efflux pump knockouts, EcΔacrA, EcΔtolC, JcΔacrA, and JcΔtolC, were determined (Table 3). The MICs of most of the antibiotics tested in wild-type strains were high. This is because the antibiotics tested are good substrates of the efflux pumps belonging to the RND family, such as AcrAB-TolC, which are expressed by Gram-negative bacteria and provide intrinsic resistance to some of the antibiotics studied. However, for E. cloacae strain Jc194, the MICs of the antibiotics tested were lower, which is consistent with lower expression of AcrAB-TolC (29). The MICs of all antibiotics were lower for the acrA knockout strains than for the wild-type isolates (Table 3). In regard to the various antibiotics, the MICs of erythromycin, clindamycin, and linezolid were greatly reduced in both strains, as were those of ciprofloxacin, chloramphenicol, and tigecycline for EcΔacrA, while there were modest decreases in the MICs of these antibiotics in the case of JcΔacrA. However, the loss of the acrA gene had a greater effect on the MIC of oxacillin in strain JC194. Although both wild-type strains are resistant to erythromycin, clindamycin, linezolid, and oxacillin, these antibiotics were selected for susceptibility testing since they are good substrates of AcrAB-TolC, which allowed us to clarify the role of the distinct components of the efflux pump in both strains. Antibiotic resistance was fully restored when the AcrA protein was overexpressed in acrA knockout strains (Table 3). The role of AcrA in the JcΔacrA tetracycline MIC was not studied because this strain has a tetracycline marker in its genome.

Table 3
Antibiotic susceptibility profiles (MICs) for the bacterial strains indicated

Analysis of EcΔtolC and JcΔtolC derivative isolates revealed a similar antibiotic susceptibility profile to that of their parental strains. Oxacillin and tetracycline MICs were unaffected, as were the MICs of tigecycline and ciprofloxacin, which remained almost unchanged. There were slight decreases in the MICs of chloramphenicol and erythromycin, while susceptibility to clindamycin and linezolid was significantly increased and was higher in strain Jc194. The antibiotic susceptibility was lower in tolC mutants when the TolC protein was overexpressed, although the MIC of some antibiotics was not fully restored. Overall, deletion of acrA as well as tolC was associated with antimicrobial resistance in E. cloacae.

Role of the AcrAB-TolC efflux pump in the fitness of E. cloacae.

To investigate the involvement of the AcrAB-TolC efflux pump on the fitness of E. cloacae, acrA and tolC knockouts were constructed in strains EcDC64 and Jc194 by replacing part of the genes with a resistance cassette, as described in Materials and Methods. To clarify whether the inactivation of the efflux pump affects the growth rate, growth curves were constructed for all strains studied. None of the EcDC64 knockout strains showed significant modification of the growth rate (0.263 h−1 for EcDC64, 0.260 h−1 for EcΔacrA, and 0.265 h−1 for EcΔtolC) relative to the wild-type strain. Moreover, JcΔacrA showed a clearly lower growth rate (0.199 h−1) in the first phase of growth (during the first 2 h) than the wild-type strain (0.256 h−1) and JcΔtolC (0.259 h−1). From 2 h onwards, growth of strain Jc194 equates to the wild-type and JcΔtolC strains (Fig. 1). After 12 h of growth in culture, the optical density of Jc194 and those of its KO derivatives were almost the same.

Fig 1
Growth curves for wild-type E. cloacae clinical isolates and their isogenic derivates in LB medium. Data are mean values of three measurements. For simplification, error bars have been omitted, although in all cases the standard deviation was <10%. ...

In vitro and in vivo competition experiments were performed for each knockout strain versus the wild-type strain, as described in Materials and Methods. The CI values obtained for each of the in vitro competition experiments are shown in Fig. 2. In all cases, the lack of a structural component of the efflux pump AcrAB-TolC had a fitness cost for both strains. When the efflux pump was inactivated, disruption of the acrA gene in the multidrug-resistant EcDC64 strain lead to a slight reduction in fitness (median CI for the EcΔacrA/EcDC64 competition, 0.42) compared with the more susceptible Jc194 strain, in which the inactivation of AcrA resulted in a marked decrease in fitness, as shown by the median CI of 0.15 for the JcΔacrA/Jc194 competition experiments. On the other hand, the lack of the TolC component had a similar effect on the fitness of both strains. The median CI value for EcΔtolC/EcDC64 competition was 0.24, and that for JcΔtolC/Jc194 competition was 0.32. The same trend was observed when the same isogenic pairs of isolates were analyzed by in vivo competition experiments performed in a mouse model of systemic infection. The results for these experiments are presented in Fig. 2. The lack of AcrA was significant and caused a decrease in the competitiveness of EcΔacrA (median CI for the EcΔacrA/EcDC64 competition, 0.3) and JcΔacrA (median CI for the JcΔacrA/Jc194 competition, 0.11). The CI values were slightly lower than those observed in the in vitro competition experiments. The fitness costs caused by inactivation of TolC (the median CI value for EcΔtolC/EcDC64 competition was 0.38, and for JcΔtolC/Jc194 competition it was 0.39) were almost identical in both strains. Compared with data from in vitro competition experiments, the role of TolC in the fitness of the Jc194 strain was almost identical. However, EcΔtolC was fairly much more competitive in vivo than in vitro. Together, these results indicate that AcrAB-TolC efflux pump is involved in the maintenance of biological competitiveness of E. cloacae in both environments, in vitro and in vivo, in a mouse model.

Fig 2
(A) Results of in vitro competition experiments. The experiments were performed in flasks of LB broth in which bacteria were grown at 37°C and 180 rpm for 16 to 18 h, corresponding to approximately 20 generations, as described in Materials and ...

Virulence in a mouse model of systemic infection.

The ability of the knockout strains to cause systemic infection in a mouse model was tested. To assess the effect on virulence of the inactivation of the acrA and tolC genes, 6 mouse groups were injected intraperitoneally with each of the different strains studied, EcDC64, EcΔacrA, EcΔtolC, Jc194, JcΔacrA, and JcΔtolC. The bacterial loads in spleen homogenates were determined at 24 h postinfection, as described above. As shown in Table 4, spleens from mice infected with acrA knockout strains presented significantly lower (P < 0.0001) bacterial loads than those infected with the wild-type strains. The same trend was observed when spleens from mice infected with EcΔtolC and JcΔtolC were analyzed. The ability of JcΔtolC to cause infection was slightly lower (P = 0. 0242) than that of strain Jc194. In the case of mice infected with EcΔtolC, the bacterial load in the spleen decreased markedly (P = 0.0003) in comparison with the decrease observed in the wild-type strain.

Table 4
Effect of AcrAB-TolC deficiency on E. cloacae systemic infection in vivo


The role of the AcrAB-TolC efflux pump in antimicrobial resistance has previously been described for some species of Enterobacteriaceae (30, 31). Enterobacter cloacae encodes an AcrAB-TolC system homologous to the system described in Escherichia coli (29). So far, most clinical studies on this type of system have focused on the involvement of these pumps in antibiotic resistance. However, few studies have examined the contributions of the pumps to bacterial fitness and virulence. In this study, the role of the AcrAB-TolC efflux pump in antibiotic resistance was analyzed by constructing mutant strains with acrA and tolC knockout genes in MDR EcDC64 and Jc194 clinical isolates. Inactivation of acrA led to an increase in the susceptibility to a range of antibiotics, relative to the wild-type strain, as previously reported (29). The MIC values conferred by JcΔacrA to antibiotics were generally similar to those of strain EcΔacrA, but in some cases an increase in susceptibility, relative to strain EcΔacrA, was observed, such as with oxacillin, erythromycin, and linezolid (Table 3). However, the MICs of ciprofloxacin, chloramphenicol, and tigecycline decreased more sharply when the acrA gene was inactivated in EcDC64, which may be related to higher levels of acrAB expression. In this study, only certain antibiotics were tested, as we have previously reported the susceptibility profile shown by AcrAB-TolC in E. cloacae (29).

Disruption of tolC also resulted in a moderate increase in antimicrobial susceptibility, although it did not affect the MIC of some antibiotics such as oxacillin and tetracycline, which revealed different functionalities between AcrA and TolC. The greatest increase in susceptibility was observed with the disruption of acrA. These data suggest the important role for the periplasmic adaptor AcrA in efflux efficiency. Studies in Salmonella spp. (3) have shown that there are differences in susceptibility patterns depending on which gene (acrA, acrB, or tolC) is inactivated and that the effect is substrate specific. In the present study, the most susceptible mutant to the antibiotic tested was the acrA mutant. The differences in the resistance pattern by inactivating acrA or tolC may be partly explained by the reported promiscuity or redundancy of both proteins with other efflux pumps. Indeed, and according to our data, the function of AcrA is more restrictive and less redundant than that of the TolC protein. The possibility that another porin may assume the role of TolC in the efflux pump cannot be disregarded, in accordance with the data reported here.

The involvement of RND efflux pump proteins in pathogenicity has been studied in different bacteria. Species of Salmonella, Klebsiella, Pseudomonas, Neisseria and others have shown that the lack of an AcrAB-TolC component has an effect on the maintenance of biological competitiveness and the ability to cause infection (26, 13, 14, 16, 19, 22, 25, 28, 36).

The goal of the present study was to determine how the lack of a functional AcrAB-TolC efflux system affects the fitness and virulence of E. cloacae, a microorganism that is increasing in importance because of the high level of resistance to antibiotics that it displays (12). It is widely assumed that in vitro resistance to antibiotic substrates of efflux pumps is predictive of fitness advantages in the presence of such molecules. However, during infection or colonization, bacteria do not always need to deal with an antimicrobial agent. For this reason, it can be interesting to analyze the fitness cost of a specific gene without the presence of a selective stimulus, such as a specific antimicrobial molecule. It appears that both acrA and tolC genes are key to the fitness of E. cloacae isolates, although some minor differences between both clinical isolates of AcrA and TolC were observed.

In regard to antibiotic resistance, deletion of the acrA gene was associated with a greater decrease in antibiotic MICs than those caused by deletion of the tolC gene (Table 3), thus emphasizing the importance of the AcrA protein in conferring resistance to several antimicrobial agents. With respect to competition experiments, abrogation of either AcrA or TolC was associated with a reduction in the CI both in vitro and in vivo, although the CI value of the JcΔacrA strain was much lower than that of its isogenic wild-type pair. Overall, the data suggest that separate deletion of each component may have different physiological and biological impacts in bacterial cells, even though both AcrA and TolC proteins are components of the same efflux pump mechanism.

Consistent with these data, virulence assays with mouse in vivo experiments revealed lower bacterial loads in the spleens of mice infected with knockout strains than in those infected with wild-type strains. The in vivo attenuation observed in acrA mutants was more dramatic than that observed in tolC knockout strains.

In summary, we described here the use of specific knockouts to test the effects of the AcrAB-TolC efflux system on antibiotic resistance, fitness, and virulence in E. cloacae. We demonstrated that the components of this efflux pump not only are involved in resistance to a range of antibiotics but also play an important role in the maintenance of biological competitiveness and are required for the full virulence of E. cloacae.

The possibility that deletion of these genes may be important for attenuation of E. cloacae isolates as a putative vaccine strategy to combat bacterial infections deserves further attention.


This work was supported by an Agustí Pumarola grant (SCMIMC and SEIMC), by the Fondo de Investigaciones Sanitarias (PI081368, PS09/00687) and SERGAS (PS07/90), and by grants from the Xunta de Galicia (07CSA050916PR) to G.B.

A.P. and M.M. are in receipt of scholarships from REIPI (Spanish Network for Research in Infectious Diseases). A.F. is in receipt of a Rio Hortega research support contract from Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación. M.P.C. was supported by a doctoral grant (SFRH/BD/6474/2009). M.P. and S.R.-F. are in receipt of research contracts from the Xunta de Galicia (Programa Isidro Parga Pondal and Instituto de Salud Carlos III, respectively).

We thank COST Action BM0701 members for their helpful collaboration and advice.


Published ahead of print 30 January 2012


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