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Antimicrobial Agents and Chemotherapy, June 2004, p . 2292-2294, Vol . 48, No . 6
Salmonella Gene rma (ramA) and Multiple-Drug-Resistant Salmonella enterica Serovar Typhimurium
Tahar van der Straaten,1 Riny Janssen,1 Dik J . Mevius,2 and Jaap T . van Dissel1*
Department of Infectious Diseases, Leiden University Medical Center, 2300 RC Leiden,1
Central Institute for Animal Disease Control, CIDC-Lelystad, Lelystad, The Netherlands2
Received 3 November 2003/
Returned for modification 23 December 2003/
Accepted 9 February 2004
MarA and its homologue, RamA, have been implicated in multidrug resistance (MDR) . RamA overexpression in Salmonella enterica serovar Typhimurium and Escherichia coli conferred MDR independently of marA . Inactivation of ramA did not affect the antibiotic susceptibilities of wild-type S . enterica serovar Typhimurium or 15 unrelated clinical MDR isolates . Thus, ramA overexpression is not a common MDR mechanism in Salmonella .
Multiple antibiotic resistance in Salmonella enterica serovar Typhimurium, an etiologic agent of food-borne enterocolitis in humans, is becoming a serious health problem . A multiple-drug-resistant (MDR) phenotype can likely develop in gram-negative microorganisms by many mechanisms (4); most of these have been elucidated in Escherichia coli . Among other mechanisms, an important route involves activation of the mar locus: MarA, the transcriptional activator of this locus, mediates drug resistance by causing decreased expression of the porin OmpF and overexpression of the multidrug efflux pump ArcB (1, 9) . Additional genetic mechanisms of MDR have been proposed . For instance, homologues of MarA, such as Rob and SoxS, have been shown to bind to the mar box; and constitutive soxS or rob mutants display MDR as well (3, 8) . George and coworkers (2) identified the ramA gene in MDR Klebsiella pneumoniae and suggested that the MDR phenotype of this strain was caused by constitutive overexpression of RamA . Because RamA displays close homology to MarA, SoxS, and Rob, the suggestion was made that RamA mediates MDR in Klebsiella via activation of the mar locus . Recently, a gene identical to ramA was also identified in S . enterica serovar Paratyphi B and was designated rma (11) . In this report, we describe a gene identical to ramA (rma) in S . enterica serovar Typhimurium that, when overexpressed on a plasmid in E . coli which lacks ramA, conferred an MDR phenotype to this bacterium and investigate whether this gene has a role in MDR in S . enterica serovar Typhimurium .
The strains and plasmids used in this study are listed in Table 1 . E . coli marA mutants were kindly provided by S . L . Levy (6) . The MDR S . enterica serovar Typhimurium strains were obtained from the surveillance collection of CIDC-Lelystad, Lelystad, The Netherlands, and are representatives of unrelated clinical MDR isolates obtained in The Netherlands over a 2-year period . The ramA gene was inactivated in these strains by transduction with a P22 lysate of the ramA::kanamycin Salmonella mutant (10) .
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TABLE 1 . Salmonella strains and plasmids used in this study
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To induce expression of RamA, the RamA-coding sequence was ligated into the isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible vector pTrcHisA (Invitrogen) by standard techniques . For constitutive overexpression, ramA was ligated into pBluescript (Stratagene) .
Disk diffusion assays were performed as follows . End-log-phase bacteria (optical density at 600 nm, 0.8) were diluted 1:10 in phosphate-buffered saline and plated on minimal M9 medium . For E . coli the plates were supplemented with thiamine (0.01%) and Casamino Acids (0.1%) . If required, ampicillin (50 µg/ml) or IPTG (0.1 mM) was added . Cotton disks containing antibiotics were placed in the centers of the plates . After overnight incubation at 37°C, the bacterium-free zone was determined as a measure of resistance . The disk diffusion assay was used to test the antibiotic susceptibilities of the bacterial mutant strains, for which the classical MIC broth microdilution method is not adequate (5) .
The MICs for the clinical Salmonella isolates were determined by the broth microdilution method, according to the NCCLS guidelines (7) . An E-test was performed by standard procedures for determination of tetracycline resistance .
Overexpression of RamA confers MDR in S . enterica serovar Typhimurium.
Given the homology between RamA and MarA and the findings for Klebsiella and S . enterica serovar Paratyphi B, we investigated the ability of RamA to confer resistance to various unrelated antibiotics in S . enterica serovar Typhimurium by means of disk diffusion assays . We induced expression of RamA in wild-type Salmonella with the IPTG-inducible ramA plasmid and expressed RamA in E . coli with pBl-ramA . Both microorganisms displayed an MDR phenotype after overexpression of RamA (Table 2), which is in accordance with the published results of George et al . (2) on the expression of RamA in E . coli . Of note, the latter bacterium lacks ramA, and we found that ramA is highly confined to S . enterica serovars (10) and is not present in the genomes of many other members of the family Enterobacteriaceae, with the notable exceptions of K . pneumoniae and Enterobacter cloacae .
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TABLE 2 . Antibiotic susceptibilities of E . coli and S . enterica serovar Typhimurium strains
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The MDR phenotype mediated by RamA is independent of MarA.
Yassien et al . (11) showed that RamA (Rma) of S . enterica serovar Paratyphi B is a DNA binding protein that binds to the mar box . MarA is a transcriptional activator for marRAB and binds to the mar box located within marO . Homologues of MarA, such as SoxS, Rob, and RamA, have been shown to bind to the mar box and also to upregulate expression of the mar locus (3,8,11) . Thus, on the basis of experiments with E . coli, Yassien et al . (11) hypothesized that RamA can substitute for MarA and directly activate MarA-controlled genes, leading to an MDR phenotype . An alternative explanation for their data would be that the MDR phenotype conferred by overexpression of RamA is MarA dependent . To investigate this issue we expressed RamA on an IPTG-inducible multicopy plasmid in a marA-negative E . coli mutant and its parental strain . As assessed by disk diffusion assays, in both wild-type E . coli and the marA-negative mutant, RamA significantly (P < 0.025) increased the levels of resistance to multiple unrelated antibiotics and conferred an MDR phenotype (Table 2) . This result demonstrates that in E . coli RamA can mediate an MDR phenotype independently of a functionally intact marA, likely by direct activation of MarA-controlled genes .
The antibiotic susceptibility of wild-type Salmonella is not affected by inactivation of ramA.
Next, we assayed the resistance of ramA null mutants of S . enterica serovar Typhimurium to multiple unrelated antibiotics . These strains were obtained by gene replacement with suicide vector pGP704, which contains ramA inactivated by a kanamycin cassette (10) . Compared with the wild-type parental Salmonella strain, the null mutants did not display increased susceptibilities to tetracycline, chloramphenicol, ciprofloxacin, or nalidixic acid (Table 2) . The identical susceptibilities of the Salmonella strains to, for instance, ciprofloxacin were confirmed by E-test on Iso-Sensitest agar plates, with the MICs for all strains being 0.032 to 0.064 mg/liter .
The MDR phenotype of clinical isolates of Salmonella is not affected by inactivation of ramA.
Further evidence that a functionally intact ramA is dispensable for the expression of an MDR phenotype was obtained in experiments with 15 clinical S . enterica serovar Typhimurium isolates (including strain 12 DT104), all of which displayed an MDR phenotype, as defined by resistance to at least three unrelated antibiotics . These strains were obtained from the Dutch national surveillance collection of CIDC-Lelystad and are representative of unrelated clinical MDR isolates obtained in The Netherlands over a 2-year period . In these strains the ramA gene was inactivated by transduction with a P22 lysate of the ramA::kanamycin Salmonella mutant . The MDR phenotype was not reversed to a non-MDR, susceptible phenotype in any of these strains (Table 3), as determined by assays for MICs . In more than 270 assays for MICs, only 2 indicated a change in the MIC of more than 2 dilution steps by the broth microdilution method, according to the NCCLS guidelines (7) . The MICs of doxycycline, tetracycline, and florfenicol for six MDR strains showed slight decreases; however, according to the NCCLS guidelines, the interpretation of the final MICs still indicated a resistant phenotype .
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TABLE 3 . MICs for MDR S . enterica serovar Typhimurium strains and their ramA knock-out mutants
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In conclusion, overexpression of RamA in E . coli and S . enterica serovar Typhimurium confers an MDR phenotype in a MarA-independent manner that is likely mediated by direct activation of mar-regulated genes, although formal proof for this is not yet available . However, inactivation of ramA does not lead to enhanced antibiotic susceptibility and does not reverse the antibiotic resistance phenotypes of 15 unrelated clinical MDR S . enterica serovar Typhimurium isolates . Thus, the findings for Salmonella rule against a common role of this gene in the MDR phenotypes of clinical Salmonella isolates .
* Corresponding author . Mailing address: Department of Infectious Diseases, Leiden University Medical Center, P.O . Box 9600, 2300 RC Leiden, The Netherlands . Phone: 31-71-5262613 . Fax: 31-71-5266758 . E-mail: j.t.van_dissel{at}lumc.nl .
- Alekshun, M . N., and S . B . Levy. 1997 . Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon . Antimicrob . Agents Chemother . 41:2067-2075.
- George, A . M., R . M . Hall, and H . W . Stokes. 1995 . Multidrug resistance in Klebsiella pneumoniae: a novel gene, ramA, confers a multidrug resistance phenotype in Escherichia coli . Microbiology 141:1909-1920.
- Jair, K . W., X . Yu, K . Skarstad, B . Thony, N . Fujita, A . Ishihama, and R . E . Wolf, Jr. 1996 . Transcriptional activation of promoters of the superoxide and multiple antibiotic resistance regulons by Rob, a binding protein of the Escherichia coli origin of chromosomal replication . J . Bacteriol . 178:2507-2513.
- Livermore, D . M. 2003 . Bacterial resistance: origins, epidemiology, and impact . Clin . Infect . Dis . 36:S11-S23.
- Maloy S . R., V . J . Stewart, and R . K . Taylor (ed.). 1996 . Genetic analysis of pathogenic bacteria . Cold Spring Harbor course . Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
- Maneewannakul, K., and S . B . Levy. 1996 . Identification for mar mutants among quinolone-resistant clinical isolates of Escherichia coli . Antimicrob . Agents Chemother . 40:1695-1698.
- National Committee for Clinical Laboratory Standards. 2000 . Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed . Approved standard . NCCLS document M7-A5 . National Committee for Clinical Laboratory Standards, Wayne, Pa.
- Oethinger, M., I . Podglajen, W . V . Kern, and S . B . Levy. 1998 . Overexpression of the marA or soxS regulatory gene in clinical topoisomerase mutants of Escherichia coli . Antimicrob . Agents Chemother . 42:2089-2094.
- Sulavik, M . C., M . Dazer, and P . F . Miller. 1997 . The Salmonella typhimurium mar locus: molecular and genetic analyses and assessment of its role in virulence . J . Bacteriol . 179:1857-1866.
- van der Straaten, T., L . Zulianello, A . van Diepen, D . L . Granger, R . Janssen, and J . T . van Dissel. 2004 . Salmonella enterica serovar Typhimurium RamA, intracellular oxidative stress response, and bacterial virulence . Infect . Immun . 72:996-1003.
- Yassien, M . A., H . E . Ewis, C . D . Lu, and A . T . Abdelal. 2002 . Molecular cloning and characterization of the Salmonella enterica serovar Paratyphi B rma gene, which confers multiple drug resistance in Escherichia coli . Antimicrob . Agents Chemother . 46:360-366.
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