S

Omf

JD

a

ARA

KCOI

1

domoiamenbopsnMst

0h

International Journal of Antimicrobial Agents 40 (2012) 168– 172

Contents lists available at SciVerse ScienceDirect

International Journal of Antimicrobial Agents

j our na l ho me p age: ht tp : / /www.e lsev ier .com/ locate / i jant imicag

hort communication

prD mutations and inactivation, expression of efflux pumps and AmpC, andetallo-�-lactamases in carbapenem-resistant Pseudomonas aeruginosa isolates

rom South Korea

i-Young Lee, Kwan Soo Ko ∗

epartment of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea

r t i c l e i n f o

rticle history:eceived 5 February 2012ccepted 11 April 2012

eywords:arbapenemsprD

MP-6

a b s t r a c t

Among 213 Pseudomonas aeruginosa isolates collected from 10 South Korean hospitals, 57 isolates (26.8%)were carbapenem-resistant. All but three of the isolates had a relevant decrease of oprD expression. How-ever, decreased oprD expression was also detected in five of ten carbapenem-susceptible isolates. Outermembrane protein analysis confirmed porin loss in carbapenem-resistant P. aeruginosa isolates. Based onthe mutations of oprD gene sequences, carbapenem-resistant P. aeruginosa isolates could be classified intofive oprD mutational groups. However, there was no difference of OprD expression or carbapenem mini-mum inhibitory concentrations among the five mutational groups. Among the 57 carbapenem-resistantP. aeruginosa isolates, 41 (71.9%) overexpressed efflux systems or ampC. MexAB–OprM and AmpC overex-

pression (56.1% and 47.4%, respectively) was prevalent and was significantly associated with carbapenemresistance. However, no synergistic effect of efflux systems and AmpC on carbapenem resistance was evi-dent. In conclusion, combination of several mutation-driven mechanisms leading to OprD inactivationand overexpression of efflux systems was the main carbapenem resistance mechanism, but acquisitionof a transferable resistance determinant such as metallo-�-lactamase could be problematic in clinicalsettings in South Korea.

lsevie

© 2012 E

. Introduction

The increasing prevalence of multidrug-resistant (MDR) Pseu-omonas aeruginosa isolates is severely compromising the selectionf appropriate treatments and is causing high morbidity andortality [1]. Although carbapenems remain effective antibi-

tics for therapy of infections caused by MDR P. aeruginosasolates, development of high carbapenem resistance rates in P.eruginosa isolates has been reported worldwide [2]. The outerembrane protein (OMP) OprD regulates the entry of carbapen-

ms, and loss of OprD function is the major determinant ofon-metallo-�-lactamase (non-MBL)-mediated resistance to car-apenems [1]. Carbapenem resistance due to inactivation of OprDften occurs in conjunction with other mechanisms such as dere-ressed AmpC or MexAB–OprM [1]. As a member of the effluxystems, MexAB–OprM is linked to meropenem resistance butot to resistance to imipenem. MexCD–OprJ, MexEF–OprN and

exXY–OprM efflux systems may also be involved in reduced

usceptibility to meropenem [3]. The increasing prevalence ofransferable resistance determinants, particularly those encoding

∗ Corresponding author. Tel.: +82 31 299 6223; fax: +82 31 299 6229.E-mail address: ksko@skku.edu (K.S. Ko).

924-8579/$ – see front matter © 2012 Elsevier B.V. and the International Society of Chemttp://dx.doi.org/10.1016/j.ijantimicag.2012.04.004

r B.V. and the International Society of Chemotherapy. All rights reserved.

class B carbapenemases or MBLs such as IMP and VIM, is notewor-thy [4].

To gain a better understanding of the contribution of severalmechanisms to carbapenem resistance in P. aeruginosa isolatesfrom South Korea, this study examined the expression of oprD,ampC and several efflux pump genes as well as sequence variationsof the oprD gene and the presence of MBL genes in carbapenem-resistant P. aeruginosa isolates.

2. Materials and methods

2.1. Bacterial isolates

All non-duplicate P. aeruginosa isolates from patients withbacteraemia or urinary tract infection (UTI) were collected fromNovember 2006 to August 2007 from 10 South Korean hospitals. Asa result, a total of 213 isolates (bacteraemia, n = 101; UTI, n = 112)were included in this study. Species identification was performedwith a VITEK-2 system (bioMérieux, Hazelwood, MO). Bacteraemiawas diagnosed if there were two or more of the following conditions

of systemic inflammatory response syndrome: temperature >38 ◦Cor <36 ◦C; heart rate >90 beats/min; respiratory rate >20 beats/minor partial pressure of carbon dioxide (PaCO2) of <32 mmHg; andwhite blood cell count of >12 000 cells/�L or <4000 cells/�L and

otherapy. All rights reserved.

of An

>naim

2

bLtmctIpwwa

2a

fstnrOgC

2

se

2

msaToMgEloptpmits

2

A

J.-Y. Lee, K.S. Ko / International Journal

10% bands. A bacterial isolate with a density ≥105 CFU/mL in uri-ary culture or ≥108 CFU/mL in suprapubic puncture was identifieds a causative pathogen of UTI. Carbapenem-resistant P. aeruginosasolates were selected on the basis of resistance to imipenem and/or

eropenem.

.2. Antimicrobial susceptibility testing

In vitro antimicrobial susceptibility testing was performedy the broth microdilution method according to Clinical andaboratory Standards Institute (CLSI) guidelines [5]. Thir-een antimicrobial agents were tested, including imipenem,

eropenem, piperacillin/tazobactam, ampicillin/sulbactam,efepime, ceftriaxone, ceftazidime, cefoperazone/sulbactam,etracycline, ciprofloxacin, amikacin, polymyxin B and colistin.nterpretation of susceptibility was according to CLSI break-oints. Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853ere employed as quality control strains. Multidrug resistanceas defined as acquired non-susceptibility to three or more

ntimicrobial categories [6].

.3. Sequencing of the oprD gene and outer membrane proteinnalysis

Inactivating mutations in the oprD gene were investigatedor 57 carbapenem-resistant and 10 representative carbapenem-usceptible P. aeruginosa isolates. Sequences were compared withhat of the reference strain P. aeruginosa PAO1 (GenBank accessiono. CAA78448). Bacterial OMPs were examined using a previouslyeported method [7]. Following sonication and ultracentrifugation,MPs were separated by sodium dodecyl sulphate–polyacrylamideel electrophoresis (SDS-PAGE) and gels were stained withoomassie blue.

.4. Identification of metallo-ˇ-lactamases

All of the carbapenem-resistant P. aeruginosa isolates werecreened by PCR for blaIMP, blaVIM, blaSPM, blaGIM and blaSIM. Pres-nce of MBL genes was confirmed by sequencing of PCR products.

.5. Quantitative reverse transcription PCR (qRT-PCR)

mRNA transcription levels of oprD, ampC, mexB, mexD, mexF andexY were determined by qRT-PCR as described previously with

ome modifications [8]. Reverse transcription was performed inccordance with the protocol for the use of Omniscript® Reverseranscriptase (QIAGEN GmbH, Hilden, Germany). Quantification ofprD transcripts was performed using SYBR® Green PCR Masterix (Applied Biosystems, Foster City, CA). Relative quantities of

ene expression were calculated using the standard curve method.xpression of the 30S ribosomal gene rpsL was assessed in paral-el to normalise the transcriptional levels of target genes. ReducedprD expression was considered relevant when it was ≤30% com-ared with that of P. aeruginosa PAO1 [3]. Isolates were consideredo be AmpC, MexCD–OprJ, MexEF–OprN and MexXY–OprM hyper-roducers when the levels of expression of ampC, mexD, mexF andexY genes were ≥10-fold higher than that of the strain PAO1, and

solates were considered to be MexAB–OprM hyperproducers whenhe level of mexB expression was ≥3-fold higher than that of thetrain PAO1, according to previously defined criteria [9].

.6. Statistical analysis

Pearson’s �2 test was used to compare categorical variables.ll data were analysed using the SPSS statistical software package

timicrobial Agents 40 (2012) 168– 172 169

v.11.5 (SPSS Inc., Chicago, IL). Differences were considered statisti-cally significant at a P-value of <0.05 for all tests.

3. Results

3.1. Bacterial isolates and antimicrobial susceptibility

Among 213 P. aeruginosa isolates, 49 (23.0%) and 40 (18.8%) iso-lates were resistant to imipenem and meropenem, respectively.Eight meropenem-resistant P. aeruginosa isolates displayed inter-mediate resistance to imipenem, and seven and ten imipenem-resistant isolates were intermediately resistant and susceptibleto meropenem, respectively. Thus, a total of 57 isolates (26.8%)resistant to imipenem and/or meropenem were determined tobe resistant to carbapenems. Among these isolates, 42 isolateswere from patients with UTIs and 15 isolates were from patientswith bacteraemia. The carbapenem resistance rate among iso-lates from UTIs was significantly higher than that among isolatesfrom bacteraemia (37.5% vs. 14.9%; P < 0.001). The 57 carbapenem-resistant isolates were from seven hospitals in different regions.No carbapenem-resistant P. aeruginosa isolates were found in theother three hospitals. Forty-six (80.7%) of the 57 carbapenem-resistant isolates showed multidrug resistance, and 12 isolateswere resistant to all antibiotics tested except polymyxins. Among156 carbapenem-intermediate or -susceptible isolates, 26 isolates(16.7%) were MDR, which was significantly lower than that amongcarbapenem-resistant isolates (P < 0.001).

The antimicrobial resistance of carbapenem-resistant and-susceptible P. aeruginosa isolates was compared (Table 1).Carbapenem-resistant P. aeruginosa isolates were significantlymore resistant to all �-lactam and non-�-lactam antibiotics, excepttetracycline, than the carbapenem-susceptible isolates. Sixteencolistin-non-susceptible P. aeruginosa isolates, all of which weresusceptible to carbapenems, were found.

3.2. Analysis of oprD expression and mutations in the oprD gene

All carbapenem-resistant P. aeruginosa isolates except three hada relevant decrease in oprD expression (≤30%) compared with thatof PAO1 (Fig. 1A). Three isolates that were outside the limit of rel-evant decrease also showed decreased expression ≤50% comparedwith that of PAO1. Decrease of oprD expression that appeared to berelevant was also detected in five of ten carbapenem-susceptible P.aeruginosa isolates studied.

Based on the mutations of oprD gene sequences, carbapenem-resistant P. aeruginosa isolates could be categorised into five oprDmutational groups (I–V). In Group I, five carbapenem-resistant iso-lates having no apparent inactivating mutations were included.However, they lacked OprD expression as judged by their OMPprofile as well as by qRT-PCR (Fig. 1). Group II, which showedpoint mutations leading to premature stop codons, included sevencarbapenem-resistant P. aeruginosa isolates. All isolates belong-ing to Group II except one (P10) showed significantly decreasedoprD expression in qRT-PCR analysis (Fig. 1A). However, isolateP10 lacked the 48 kDa OprD-compatible protein (Fig. 1B). Themajority of the carbapenem-resistant P. aeruginosa isolates (n = 35;61.4%) belonged to Group III, which displayed frameshift muta-tions by insertion or deletion of 1 bp. Eight carbapenem-resistantP. aeruginosa isolates showed deletions or insertions of more than1 bp, which resulted in frameshift as in Group III, and were desig-nated Group IV. Among these isolates of Group IV, seven isolates

showed 2–76 bp deletions in oprD genes, and one isolate (P59)showed an insertion of 5 bp at position 129. Group V includedtwo carbapenem-resistant P. aeruginosa isolates in which oprDgenes were disrupted by insertion sequences, ISPpu21 and ISPal1

170 J.-Y. Lee, K.S. Ko / International Journal of Antimicrobial Agents 40 (2012) 168– 172

Table 1Antimicrobial resistance of Pseudomonas aeruginosa isolates.

Antimicrobial agent No. (%) of resistant isolates P-value

Carbapenem-resistant isolates (n = 57) Carbapenem-susceptible isolates (n = 156)

Imipenem 49 (86.0) 0 –Meropenem 40 (70.2) 0 –Piperacillin/tazobactam 38 (66.7) 38 (24.4) <0.001Ampicillin/sulbactam N/D N/D –Cefoperazone/sulbactam N/D N/D –Cefepime 40 (70.2) 31 (19.9) <0.001Ceftriaxone 54 (94.7) 92 (59.0) <0.001Ceftazidime 36 (63.2) 34 (21.8) <0.001Tetracycline 50 (87.7) 130 (83.3) 0.664Ciprofloxacin 34 (59.6) 30 (19.2) <0.001Amikacin 27 (47.4) 21 (13.5) <0.001Polymyxin B 0 0 –Colistin 0 16 (10.3)a 0.007

N

FPgrt

/D, not determined (Breakpoints are not available from the Clinical and Laboratory Stana Colistin-non susceptible P. aeruginosa isolates [minimum inhibitory concentration (M

ig. 1. (A) oprD expression of 10 carbapenem-susceptible and 57 carbapenem-resistanCR). mRNA transcription levels of oprD genes are represented by values relative to thatroups of oprD gene are indicated below the isolate numbers. The dashed line indicateepresentative isolates determined by sodium dodecyl sulphate–polyacrylamide gel electhe left. Mutational groups of the oprD gene are indicated above the isolate numbers. M, m

dards Institute).IC) ≥ 4 mg/L].

t P. aeruginosa isolates evaluated by quantitative reverse transcription PCR (qRT- of P. aeruginosa PAO1, a reference strain susceptible to carbapenems. Mutationals 30% level compared with that of PAO1. (B) Outer membrane protein profiles ofrophoresis (SDS-PAGE). The banding position of OprD is indicated by the arrow on

olecular size marker.

J.-Y. Lee, K.S. Ko / International Journal of Antimicrobial Agents 40 (2012) 168– 172 171

Table 2Relationship of carbapenem resistance with efflux systems and AmpC.

Overexpressed gene No. (%) of resistant isolates P-value

Carbapenem-resistantisolates (n = 57)

Representativecarbapenem-susceptibleisolates (n = 10)

mexB 32 (56.1) 2 (20) 0.045mexD 11 (19.3) – 0.195mexF 2 (3.5) – 1.000mexY 9 (15.8) 1 (10) 1.000Any efflux pumps 38 (66.7) 3 (30) 0.038

(pmIbt

sidor

3

a(ollwsitmasoao

m>abcsoatPns

3

rpi

ampC 27 (47.4)

Any efflux pumps + ampC 24 (42.1)

Any genes 41 (71.9)

GenBank accession nos. FJ905320 and AF173167, respectively), atositions 13 and 148, respectively. As ten imipenem-resistant buteropenem-susceptible P. aeruginosa isolates belonged to Groups

I (three isolates), III (six isolates) and IV (one isolate), the differenceetween imipenem and meropenem susceptibility did not appearo be due to the specific pattern of oprD mutations.

oprD genes of 10 carbapenem-susceptible isolates were alsoequenced. Although seven carbapenem-susceptible P. aeruginosasolates showed 3 bp deletions at positions 1115 and 1147, sucheletions were irrelevant to premature stop of protein processingr frameshift mutation and were also found in 23 carbapenem-esistant P. aeruginosa isolates.

.3. Overexpression of efflux systems and ampC

The relationship of carbapenem resistance with efflux systemnd ampC overexpression was investigated (Table 2). Whilst 4171.9%) of the 57 carbapenem-resistant isolates overexpressed oner more genes of the efflux systems or ampC, overexpression of ateast one gene was found only in 3 carbapenem-susceptible iso-ates. Among the efflux systems, overexpression of the mexB gene

as most prevalent (56.1%). Although mexD and mexY overexpres-ion was found in 11 and 9 carbapenem-resistant P. aeruginosasolates, respectively, their prevalence was not significantly higherhan in carbapenem-susceptible isolates. Overexpression of theexF gene was found only in two carbapenem-resistant P.

eruginosa isolates. All isolates with mexD and mexF overexpressionimultaneously hyperexpressed the mexB gene. Overexpressionf the ampC gene was observed in 27 carbapenem-resistant P.eruginosa isolates (47.4%), but no carbapenem-susceptible isolatesverexpressed the ampC gene (Table 2).

Although P. aeruginosa isolates with both imipenem anderopenem minimum inhibitory concentrations (MICs) of

64 mg/L showed overexpression both of efflux systems and thempC gene, the imipenem MICs of isolates with overexpressionoth of efflux systems and ampC gene did not appear to be signifi-antly higher than for isolates with overexpression of only effluxystems or the ampC gene, or even than those with overexpressionf neither efflux systems nor the ampC gene. Although ten P.eruginosa isolates with overexpression of both efflux systems andhe ampC gene showed meropenem MICs > 64 mg/L, nine of these. aeruginosa isolates possessed blaIMP-6 genes. Thus, there waso evidence of a synergistic effect of co-hyperexpression of effluxystems and AmpC.

.4. Prevalence of metallo-ˇ-lactamases

MBL genes were identified in 15 (26.3%) of the 57 carbapenem-esistant P. aeruginosa isolates. The overall prevalence of MBLroduction among 213 P. aeruginosa isolates was 7.0%. Thirteen

solates were positive for the blaIMP-like gene and two isolates

– 0.004– 0.011

3 (30) 0.025

were positive for the blaVIM-like gene. All of the blaIMP-like andblaVIM-like genes identified were blaIMP-6 and blaVIM-2, respectively.No blaSPM-like, blaGIM-like or blaSIM-like genes were identified in anyisolates.

4. Discussion

Mutational inactivation of oprD is the main mechanism of car-bapenem resistance in the absence of acquired carbapenemases.Sequence analysis of the oprD gene also revealed various routesof inactivation, including a single nucleotide change resulting inpremature stop codon (Group II), 1 bp insertion or deletion result-ing in frameshift (Group III), insertion or deletion of two or morenucleotides (Group IV) and disruption of protein by insertionsequences (Group V). Some P. aeruginosa isolates (Group I) werealso found to be resistant to carbapenems despite no apparentinactivating mutations in the oprD gene. These isolates showedrepression of oprD expression in qRT-PCR analysis as well as OprDloss in OMP analysis. Amino acid changes in the oprD gene mayaffect expression, although it has previously been suggested thatamino acid changes are not correlated with transcription levels ofoprD [10]. Otherwise, other unknown mechanisms regulating theexpression of porins may exist.

As expected, oprD genes of carbapenem-susceptible isolatesshowed no mutational change contributing to a loss of OprD func-tion. However, one-half of the carbapenem-susceptible isolatesshowed decreased expression of oprD. Although OprD loss of thesefive carbapenem-susceptible isolates with oprD repression was notinvestigated by the OMP profiling method, this finding may showthat carbapenem resistance in P. aeruginosa isolates requires addi-tional mechanisms, such as overexpression of efflux systems orAmpC [11].

Seventeen carbapenem-resistant P. aeruginosa isolates wereresistant to imipenem (MICs of 16–32 mg/L) but not to meropenem(MICs of 4–8 mg/L), although their oprD genes showed mutationalchanges leading to their inactivation. One of these isolates (P42)displayed porin loss in OMP analysis. OprD inactivation is knownto confer reduced susceptibility to imipenem, but further resistancemechanisms such as overexpression of efflux systems or ampCwould be required to lead to meropenem resistance [12,13]. Thesedata show that decreased expression of the oprD gene contributesto resistance to imipenem but that the contribution to meropenemresistance is somewhat limited.

Of the carbapenem-resistant P. aeruginosa isolates, 71.9% over-expressed at least one gene among the four major efflux systemsand ampC. Overexpression of MexAB–OprM was the most preva-lent, followed by AmpC. However, the association of MexCD–OprJ,

MexEF–OprN and MexXY–OprM with carbapenem resistance wasnot statistically significant. All isolates with overexpression ofMexCD–OprJ and MexEF–OprN also overexpressed MexAB–OprM.Thus, the contribution of MexCD–OprJ and MexEF–OprN may not

1 of An

bdohoisMlpts

obSitSont

A

oF

B

R

[

[

[

[

[ter species and Pseudomonas aeruginosa. Clin Infect Dis 2006;43(Suppl. 2):

72 J.-Y. Lee, K.S. Ko / International Journal

e critical in P. aeruginosa isolates from South Korea. A recent reportocumented a statistically significant link between mexB and mexYverexpression [9]. According to the findings of the current study,owever, only three isolates co-overexpressed both genes. As twof these three isolates showed relatively low imipenem MICs, its considered that there is no synergistic effect of the two effluxystems co-regulated by the same exit portal, MexAB–OprM andexXY–OprM. Instead, overexpression of MexXY–OprM has been

inked to fluoroquinolone and aminoglycoside resistance [14,15];resently, nine of ten isolates that overexpressed mexY were resis-ant to both ciprofloxacin and amikacin. A synergistic effect of effluxystems and AmpC on carbapenem resistance was not observed.

This study demonstrates that decreased oprD expression andverexpression of MexAB–OprM and AmpC are the main car-apenem resistance mechanisms of P. aeruginosa isolates fromouth Korea. Combination of several mechanisms leading to OprDnactivation as well as overexpression of efflux systems may con-ribute to most carbapenem resistance in P. aeruginosa isolates fromouth Korea. However, we could not confirm a synergistic effectn carbapenem resistance. Along with these resistance determi-ants, acquired MBLs such as IMP-6 are also becoming a seriousherapeutic threat in South Korea.

cknowledgments

Pseudomonas aeruginosa isolates used in this study werebtained from the Asian Bacterial Bank (ABB) of the Asia Pacificoundation for Infectious Diseases (APFID) (Seoul, South Korea).

Funding: This research was partially supported by the Samsungiomedical Research Institute (Suwon, South Korea).

Competing interests: None declared.Ethical approval: Not required.

eferences

[1] Poole K. Pseudomonas aeruginosa: resistance to the max. Front Microbiol2011;2:65.

[

timicrobial Agents 40 (2012) 168– 172

[2] Davies TA, Marie Queenan A, Morrow BJ, Shang W, Amsler K, He W, et al.Longitudinal survey of carbapenem resistance and resistance mechanisms inEnterobacteriaceae and non-fermenters from the USA in 2007–09. J AntimicrobChemother 2011;66:2298–307.

[3] Rodríguez-Martínez JM, Poirel L, Nordmann P. Molecular epidemiology andmechanisms of carbapenem resistance in Pseudomonas aeruginosa. AntimicrobAgents Chemother 2009;53:4783–8.

[4] Cornaglia G, Giamarellou H, Rossolini GM. Metallo-�-lactamases: a last frontierfor �-lactams? Lancet Infect Dis 2011;11:381–93.

[5] Clinical and Laboratory Standards Institute. Performance standards forantimicrobial susceptibility testing: twenty-first informational supplement.Document M100-S21. Wayne, PA: CLSI; 2011.

[6] Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG,et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistantbacteria: an international expert proposal for interim standard definitions foracquired resistance. Clin Microbiol Infect 2012;18:268–81.

[7] Quale J, Bratu S, Landman D, Heddurshetti R. Molecular epidemiology andmechanisms of carbapenem resistance in Acinetobacter baumannii endemic inNew York City. Clin Infect Dis 2003;37:214–20.

[8] Dumas JL, van Delden C, Perron K, Köhler T. Analysis of antibiotic resistance geneexpression in Pseudomonas aeruginosa by quantitative real-time-PCR. FEMSMicrobiol Lett 2006;254:217–25.

[9] Cabot G, Ocampo-Sosa AA, Tubau F, Macia MD, Rodríguez C, Moya B, et al.Overexpression of AmpC and efflux pumps in Pseudomonas aeruginosa iso-lates from bloodstream infections: prevalence and impact on resistancein a Spanish multicenter study. Antimicrob Agents Chemother 2011;55:1906–11.

10] Wang J, Zhou JY, Qu TT, Shen P, Wei ZQ, Yu YS, et al. Molecular epidemiologyand mechanisms of carbapenem resistance in Pseudomonas aeruginosa isolatesfrom Chinese hospitals. Int J Antimicrob Agents 2010;35:486–91.

11] Riera E, Cabot G, Mulet X, García-Castillo M, del Campo R, Juan C, et al. Pseu-domonas aeruginosa carbapenem resistance mechanisms in Spain: impact onthe activity of imipenem, meropenem, and doripenem. J Antimicrob Chemother2011;66:2022–7.

12] Gutiérrez O, Juan C, Cercenado E, Navarro F, Bouza E, Coll P, et al. Molecu-lar epidemiology and mechanisms of carbapenem resistance in Pseudomonasaeruginosa isolates from Spanish hospitals. Antimicrob Agents Chemother2007;51:4329–35.

13] Quale J, Bratu S, Gupta J, Landman D. Interplay of efflux system, ampC, andoprD expression in carbapenem resistance of Pseudomonas aeruginosa clinicalisolates. Antimicrob Agents Chemother 2006;50:1633–41.

14] Bonomo RA, Szabo D. Mechanisms of multidrug resistance in Acinetobac-

S49–56.15] Fraud S, Poole K. Oxidative stress induction of the MexXY multidrug efflux

genes and promotion of aminoglycoside resistance development in Pseu-domonas aeruginosa. Antimicrob Agents Chemother 2011;55: 1068–74.