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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~  References
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2012  |  Volume : 30  |  Issue : 3  |  Page : 302-307
 

Gene expression analysis of the SdeAB multidrug efflux pump in antibiotic-resistant clinical isolates of Serratia marcescens


1 Department of Pharmacology and Therapeutics, University 1, University of Manitoba, Winnipeg, MB, Canada
2 Director, University 1, University of Manitoba, Winnipeg, MB, Canada

Date of Submission06-Feb-2012
Date of Acceptance15-Apr-2012
Date of Web Publication8-Aug-2012

Correspondence Address:
S D Dalvi
Department of Pharmacology and Therapeutics, University 1, University of Manitoba, Winnipeg, MB
Canada
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Source of Support: Natural Sciences and Engineering Research Council of Canada (NSERC), Conflict of Interest: None


DOI: 10.4103/0255-0857.99491

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 ~ Abstract 

Purpose: Many isolates of Serratia marcescens, a well-known opportunistic pathogen, can be multidrug resistant. Fluoroquinolones are among the most important groups of antibiotics used for treatment of these organisms. However, fluoroquinolone resistance among S. marcescens isolates is fast increasing. Drug extrusion through efflux pumps like SdeAB/ HasF is one of the major mechanisms of resistance to fluoroquinolones. This study was carried out to analyze, through gene expression analysis of sdeB, the relative contribution of this mechanism toward fluoroquinolone resistance in clinical isolates of Serratia. Materials and Methods: Total RNA from 45 clinical isolates of S. marcescens was isolated. Quantitative real-time RT PCR was performed on the extracted RNA to study the gene expression of sdeB and was normalized to the sdeB expression in the standard strain of S. marcescens. Results: Of the 45 isolates analyzed, sdeB expression was found to be elevated in 20 isolates (44%). Of these 20 isolates, eight (40%) were fully resistant to at least one of the fluoroquinolones studied. Conversely, of the 20 isolates that over-expressed sdeB, 12 (60%) were fully sensitive to all fluoroquinolones tested. Conclusions: Drug efflux pumps are an important means of fluoroquinolone resistance among clinically important species ofSerratia. The expression of these pumps can be up-regulated in the presence of antibiotics and have the potential for changing the phenotype from sensitive to resistant, thus contributing to therapeutic failures.


Keywords: Antibiotic resistance, drug efflux pumps, fluoroquinolones, Serratia marcescens, SdeAB


How to cite this article:
Dalvi S D, Worobec E A. Gene expression analysis of the SdeAB multidrug efflux pump in antibiotic-resistant clinical isolates of Serratia marcescens. Indian J Med Microbiol 2012;30:302-7

How to cite this URL:
Dalvi S D, Worobec E A. Gene expression analysis of the SdeAB multidrug efflux pump in antibiotic-resistant clinical isolates of Serratia marcescens. Indian J Med Microbiol [serial online] 2012 [cited 2019 Sep 22];30:302-7. Available from: http://www.ijmm.org/text.asp?2012/30/3/302/99491



 ~ Introduction Top


The genus Serratia belongs to the family Enterobacteriaceae. The species within this genus are Gram negative, motile, facultative rods that grow well on most routine culture media. Among the Enterobacteriaceae, Serratia is the only genus that produces three enzymes: lipase, gelatinase and DNase. [1],[2] Serratia marcescens is the species most often implicated in human infections, followed by Serratia liquefaciens. [1],[2],[3],[4] S. marcescens is a nosocomial pathogen that commonly causes urinary tract infections, especially in catheterised patients, and respiratory tract infections. Less commonly, it may also cause wound infections, septicemia, meningitis, acute keratitis among contact lens wearers and serious infections in injection drug users. [1],[3],[4] Most of these infections are environmental in origin and spread in the hospital through contaminated hands of hospital personnel or through contaminated solutions like antiseptics. Treatment of infections due to Serratia spp. can be difficult not only because of their ability to possess various antibiotic resistance mechanisms, [5] but also due to the fact that in spite of displaying in vitro susceptibility to several antibiotics, they easily develop resistance to those drugs during therapy. [2] Notable among the resistance mechanisms is the presence of a constitutional Amp-C, which is inducible and chromosomally encoded, thus conferring resistance to most of the penicillins and cephalosporins. [3]

In Canada, Serratia spp. comprises about 3% of isolates from patients in critical care units, suffering from lower respiratory tract infections. Though this is a small percentage, Serratia spp. is usually resistant to multiple antibiotics, and thus causes a high degree of morbidity and mortality in critically ill patients.

Antibiotic resistance due to the presence of drug efflux pumps, like the recently characterised SdeAB, is fast emerging as a bacterial weapon against fluoroquinolones. [6],[7] The SdeAB/HasF efflux pump of Serratia marcescens is structurally and functionally similar to the AcrAB/TolC of  Escherichia More Details coli, belonging to the resistance-nodulation-cell division (RND) family of efflux pumps. [8] SdeB is a transmembrane energy-dependent pump that spans the inner cytoplasmic membrane. The HasF, also a transmembrane protein within the outer membrane, acts as a channel through which drugs are extruded. SdeA is the anchoring protein that binds both SdeB and HasF and is located in the periplasmic space. Thus, the complete SdeAB/HasF efflux pump, simply called SdeAB, functions as a heterotrimeric protein and its components are encoded by three separate genes.

The present study was conducted to determine the relative contribution of SdeAB to drug resistance, especially to fluoroquinolones, among the clinical isolates of S. marcescens.


 ~ Materials and Methods Top


Bacterial strains

Forty-five isolates of S. marcescens from the CANWARD 2007 collection were selected for this study. [9],[10] All the isolates were collected from the respiratory tracts of patients hospitalised in critical care units from hospitals across major Canadian cities in the year 2007. This study was conducted in the Department of Microbiology at the University of Manitoba, Winnipeg, MB, Canada. UOC-67 was used as the standard strain for all the experiments. Eight of the 45 isolates and UOC-67 were grown in the presence of increasing amounts of ciprofloxacin in Luria-Bertani (LB) broth (Difco, BD, Missisauga, ON, Canada). Four of the selected isolates (21, 24, 38 and 41) were fluoroquinolone sensitive and had low baseline expression levels of the sdeB. The other four (23, 32, 50 and 52) were fluoroquinolone sensitive but had elevated expression of sdeB. Isolates were first grown in LB broth containing 0.25 μg/ml ciprofloxacin (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) overnight at 37 o C with shaking followed by plating on tryptic soy agar without ciprofloxacin (Difco, BD). The resulting colonies were subcultured in LB broth supplemented with twofold increments of ciprofloxacin up to 32 μg/ml.

Antibiotic susceptibility testing

The susceptibility of all the isolates was tested against ciprofloxacin, levofloxacin, moxifloxacin, imipenem, piperacillin, cefoxitin, ceftriaxone and ceftazidime. The minimum inhibitory concentration (MIC) was determined using both the E-test strip (bioMerieux, Durham, NC, USA) and the traditional broth macrodilution methods. [11]

DNA extraction

Total DNA was extracted from all the isolates by the alcohol extraction procedure. [12] All reagents and biochemicals were obtained either from Sigma-Aldrich or Fisher Scientific Company (Ottawa, ON, Canada). Traditional end-point polymerase chain reaction (PCR) was performed to amplify the sdeB gene using the FastStart PCR Master (Roche Diagnostics, Laval, QC, Canada).

RNA extraction

All the isolates were grown overnight in LB broth and harvested in the late log phase. Total RNA was extracted from all the strains using the RNeasy Protect Bacteria Mini Kit, using the silica membrane technology (Qiagen Inc., Toronto, ON, Canada). To further purify the RNA of trace amounts of DNA, an additional step was performed using the RNase-free DNase set (Qiagen Inc.). Trace amounts of DNA co-extracted with the RNA were removed from the column by DNase, followed by subsequent removal of the DNase.

RNA analysis

For assessing the purity and integrity of the extracted RNA from all the isolates, electrophoresis was performed using the denaturing gel component of the NorthernMax Kit (Ambion, Life Technologies Inc., Burlington, ON, Canada).

Real-time polymerase chain reaction

One-step reverse transcriptase (RT) PCR was performed on the RNA extracted from all isolates using the EXPRESS One-Step SYBR GreenER Universal Kit (Invitrogen, Life Technologies Inc., Burlington, ON, Canada). A 123 bp region of the sdeB gene was amplified using forward primer 5′-ATCCAGTGGACCGATCTGAG-3′ and reverse primer 5′-CAGCGTCCAGCTTTCATACA-3′. A 190 bp fragment of the housekeeping gene rplU[13] was amplified using forward primer 5′-GCTTGGAAAAGCTGGACATC-3′ and reverse primer 5′-TACGGTGGTGTTTACGACGA-3′. Relative basal expression of the sdeB gene was studied using the UOC-67 as the calibrator strain, which has an antibiotic-sensitive phenotype. [14] The primers for sdeB were designed by the OligoPerfect Designer software by Invitrogen. Both the sdeB and the rplU primers were synthesised by Invitrogen. All real-time RT PCR experiments were performed on the ABI 7500 Fast Real-Time Thermal Cycler (Applied Biosystems, Burlington, ON, Canada) at the Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, MB.

The one-step RT-PCR reaction was set up for each of the 45 isolates as follows: EXPRESS SYBR GreenER qPCR Supermix Universal: 40 μl; Primer 1 (F): 1.6 μl; Primer 2 (R): 1.6 μl; ROX: 0.16 μl; Superscript RT: 2 μl; template RNA: 30 μl; distilled water: 4.64 μl (to make up a total reaction volume of 80 μl). The reaction was set up in triplicate where 20 μl of the above mixture was loaded into each of three wells of a 96-well reaction plate (MicroAmp Fast optical 96-well reaction plate with barcode, 0.1 ml, Applied Biosystems). Appropriate no-RT and no-template controls were run with every plate.

The entire gene expression analysis was repeated using fresh RNA extracted from each of the 45 isolates. The final data are the average of two separate readings. To calculate the change in gene expression, the cycle threshold (Ct) for the housekeeping gene (rplU) was subtracted from the cycle threshold (Ct) of the gene in question (sdeB). The resulting value, the DCt, was similarly calculated for the calibrator strain (UOC-67). Subtracting the DCt of the calibrator strain from the DCt of each of the experimental strains resulted in the DDCt value. The formula (2−DDCt ) was then applied to calculate the fold change in gene expression relative to the calibrator strain. [15]

Statistical analysis

To determine the significance and validity of the results, a chi-square test was performed. A P value of <0.05 was considered significant.


 ~ Results Top


Antibiotic susceptibility testing of the isolates was performed using by both E-test strip and the broth macrodilution methods. [11] Both methods showed excellent correlation, with the E-test strip method being the more convenient. Results are found in [Table 1].
Table 1: Antibiotic susceptibility and sdeB expression of S. marcescens CANWARD 2007 isolates

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Relative gene expression of sdeB as determined by RT-PCR for all strains is found in [Table 1]. Fivefold or higher overexpression was considered to be significant based on the chi-square test (P < 0.05). The MIC breakpoints for resistance to fluoroquinolones were as follows: ciprofloxacin ≥4 μg/ml; levofloxacin ≥8 μg/ml; moxifloxacin ≥2 μg/ml.

Of the 45 isolates analysed, sdeB expression was found to be significantly elevated (more than fivefold as compared to that of calibrator strain UOC-67) in 20 isolates (44%). Of these 20 isolates, 8 (40%) were fully resistant to at least one of the fluoroquinolones studied. Conversely, of the 20 isolates that overexpressed sdeB, 12 (60%) were fully or moderately sensitive to all fluoroquinolones tested.

Fluoroquinolone susceptible isolates 21, 24, 38, and 41, expressing sdeB at levels equal to or below UOC-67, [Table 1] along with UOC-67 were grown in increasing concentrations of ciprofloxacin. Resultant sdeB expression, as detected by RT-PCR, drastically increased, as found in [Table 2]. Conversely, four fluoroquinolone susceptible isolates found to originally overexpress sdeB (23, 32, 50, and 52) did not significantly alter the expression of sdeB when grown in the presence of increasing amounts of ciprofloxacin. Results are found in [Table 2].
Table 2: Ciprofl oxacin-induced expression of sdeB in select S. marcescens CANWARD 2007 isolates

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 ~ Discussion Top


A wide variety of methods exist naturally among bacteria for inactivating antimicrobial agents, leading to clinically significant resistance to antibiotics. The most common among these include production of enzymes that inactivate antibiotics (e.g. ß-lactamases), alteration of the target of antimicrobial action (e.g. gyrA mutations), and the presence of efflux pumps that actively extrude antibiotics from within the bacterial cell. [5],[8],[16] Fluoroquinolones are among the most widely used antibiotics for both in-patient and out-patient therapy. Because of widespread use, resistance to this class of antibiotics has become rampant.

There are two important mechanisms of fluoroquinolone resistance. The first involves a mutation in the binding site for fluoroquinolones, which is DNA gyrase (in Gram-negative organisms) and/or topoisomerase IV (in Gram-positive organisms). The other mechanism of resistance is active efflux of fluoroquinolones through transmembrane pumps like SdeAB and SdeXY. [5],[7],[17]

It has been previously shown that fluoroquinolones are the major class of antibiotics extruded by the SdeAB pump. [6],[15] In the present study, of the 20 isolates that overexpressed sdeB, 40% were completely resistant to fluoroquinolones, whereas 77% were resistant to cefoxitin and only 3% were resistant to ceftriaxone and ceftazidime, confirming that fluoroquinolones are major substrates for this pump. The reason for high frequency of resistance to cefoxitin is the presence of a wide variety of ß-lactamases, such as AmpC, that are intrinsically present in Serratia spp. [2],[3],[4]

Of the 45 isolates studied, 10 were fully resistant to at least one of the fluoroquinolones. Of these, eight overexpressed the sdeB gene. Conversely, of the 25 isolates with wild-type levels of sdeB expression, 23 strains (92%) were fully susceptible to fluoroquinolones, thus linking resistance and overexpression of sdeB.

Of the 35 fluoroquinolone-sensitive isolates, 13 overexpressed the sdeB gene. This could possibly be due to mutations in sdeR, the transcriptional regulator of sdeB, leading to constitutive overexpression of the drug efflux pump, or in hasF, the gene encoding the transmembrane channel, resulting in a non-functional outer membrane component. [6],[18],[19],[20]

One limitation of this study was that we measured the expression of sdeB, but expression does not always correlate with function. If the overexpressed efflux pump components are mutated, they become nonfunctional, and hence there will not be an equivalent increase in resistance. Also, we assessed the expression of only one of the three components of the efflux pump. Overexpression of sdeB with normal expression of sdeA and hasF will result in the strain being phenotypically sensitive to the antimicrobials studied. This might be a possible explanation why the overexpression of the sdeB component does not always result in resistance to fluoroquinolones. Further gene expression studies on the sdeA and hasF components are required to confirm this.

When four of the sensitive but sdeB overexpressing isolates were exposed to increasing concentrations of ciprofloxacin, efflux pump expression remained at almost the same level even though their phenotype changed from sensitive to resistant. This could be due to the emergence of resistant clones that possessed a different mechanism of resistance to ciprofloxacin, such as mutation in gyrA.

The passaging experiments also suggest that the sdeB is inducible, being up-regulated in the presence of elevated amounts of antibiotics. Such strains have an obvious survival advantage over those with normal levels of efflux pump expression, especially in environments exerting a selection pressure, including hospital-intensive care units with high usage of antibiotics. This may have tremendous implications in infectious disease diagnostics and therapeutics. Monotherapy with fluoroquinolones against such strains could potentially lead to serious consequences as it would select for resistance. Sensitive quantitative PCR techniques could be developed to analyse the expression of efflux pumps that recognize this subset of otherwise fluoroquinolone-susceptible strains to alert the physician in a timely manner and prevent development of resistance.

Further studies need to be carried out on more fluoroquinolone-resistant isolates from several species of clinically significant bacteria to find out the relative contribution of SdeAB or its homologues to fluoroquinolone resistance.

 
 ~ References Top

1.Abbott SL. Klebsiella, Enterobacter, Citrobacter, Serratia, Plesiomonas, and other Enterobacteriaceae, In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, editors. Manual of Clinical Microbiology, 9 th ed. Washington, D.C.: ASM Press; 2007. p. 698-715.  Back to cited text no. 1
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3.Donnenberg MS. Enterobacteriaceae, In: Mandell GL, Bennett JE, Dolin R, editors. Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases, 6 th ed. Philadelphia, Pa: Elsevier Churchill Livingstone; 2005. p. 2567-86.  Back to cited text no. 3
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4.Gruber WC, Fisher RG, Boyce TG. Serratia, In: Feigin RD, Cherry JD, Demmler GJ, Kaplan SL, editors. Textbook of Pediatric Infectious Diseases, 5 th ed. Philadelphia, Pa: Saunders; 2004. p. 1469-73.  Back to cited text no. 4
    
5.Hooper DC. Emerging mechanisms of fluoroquinolone resistance. Emerg Infect Dis 2001;7:337-41.  Back to cited text no. 5
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6.Begic S, Worobec EA. The role of the Serratia marcescens SdeAB multidrug efflux pump and TolC homologue in fluoroquinolone resistance studied via gene-knockout mutagenesis. Microbiology 2008;154:454-61.  Back to cited text no. 6
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7.Fujimaki K, Fujii T, Aoyama H, Sato K, Inoue Y, Inoue M, et al. Quinolone resistance in clinical isolates of Serratia marcescens. Antimicrob Agents Chemother 1989;33:785-7.  Back to cited text no. 7
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9.Zhanel GG, Karlowsky JA, DeCorby M, Nichol KA, Wierzbowski A, Baudry PJ, et al. Prevalence of antimicrobial-resistant pathogens in Canadian hospitals: Results of the Canadian Ward Surveillance Study (CANWARD 2007). Can J Infect Dis Med Microbiol 2009;20 (Suppl A):9A-19A.  Back to cited text no. 9
    
10.Zhanel GG, DeCorby M, Nichol KA, Wierzbowski A, Baudry PJ, Tailor F et al. Antimicrobial susceptibility of 6685 organisms isolated from Canadian hospitals: CANWARD 2007. Can J Infect Dis Med Microbiol 2009;20 (Suppl A):20A-30A.  Back to cited text no. 10
    
11.Clinical and Laboratory Standards Institute / NCCLS. Methods for Dilution Antimicrobial Susceptibility tests for Bacteria That Grow Aerobically. Approved standard M7-A6. Wayne, Pa: Clinical and Laboratory Standards Institute; 2006.  Back to cited text no. 11
    
12.Ausubel FM, Bent R, Kingston RE, Moore DD, Seidman JG, Smith JA, et al. Current protocols in molecular biology. New York, N.Y: John Wiley and Sons; 1989.  Back to cited text no. 12
    
13.Shanks RMQ, Stella NA, Kalivoda EJ, Doe MR, O'Dee DM, Lathrop KL, et al. A Serratia marcescens OxyR homolog mediates surface attachment and biofilm formation. J Bacteriol 2007;189:7262-72.  Back to cited text no. 13
    
14.Szabo D, Silveira F, Hujer AM, Bonomo RA, Hujer KM, Marsh JW, et al. Outer membrane protein changes and efflux pump expression together may confer resistance to ertapenem in Enterobacter cloacae. Antimicrob Agents Chemother 2006;50:2833-5.  Back to cited text no. 14
    
15.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 -∆∆Ct method. Methods 2001;25:402-8.  Back to cited text no. 15
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16.Minato Y, Shahcheraghi F, Ogawa W, Kuroda T, Tsuchiya T. Functional gene cloning and characterization of the SsmE multidrug efflux pump from Serratia marcescens. Biol Pharm Bull 2008;31:516-9.  Back to cited text no. 16
    
17.Chen J, Kuroda T, Huda MN, Mizushima T, Tsuchiya T. An RND-type multidrug efflux pump SdeXY from Serratia marcescens. J Antimicrob Chemother 2003;52:176-9.  Back to cited text no. 17
    
18.Bornet C, Chollet R, Mallea M, Chevalier J, Davin-Regli A, Pages J, et al. Imipenem and expression of multidrug efflux pump in Enterobacter aerogenes. Biochem Biophys Res Commun 2003;301:985-90.  Back to cited text no. 18
    
19.Kumar A, Worobec EA. HasF, a TolC-homolog of Serratia marcescens, is involved in energy-dependent efflux. Can J Microbiol 2005;51:497-500.  Back to cited text no. 19
    
20.Kumar A, Worobec EA. Cloning, sequencing, and characterization of the SdeAB multidrug efflux pump of Serratia marcescens. Antimicrob Agents Chemother 2005;49:1495-501.  Back to cited text no. 20
    



 
 
    Tables

  [Table 1], [Table 2]

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