Efflux System Overexpression and Decreased OprD Contribute to the Carbapenem Resistance Among Extended-Spectrum Beta-Lactamase-Producing Pseudomonas aeruginosa Isolates from a Chinese University Hospital

Abstract

The aim of this study was to investigate, for the first time, the combinations of carbapenem resistance mechanisms in clinical isolates of extended-spectrum beta-lactamase (ESBL)-producing Pseudomonas aeruginosa in a Chinese hospital. Pulsed-field gel electrophoresis revealed the presence of eight clonal types among the 15 ESBL producers. Multilocus sequence typing of two isolates harboured bla_IMP-1 identified the clonal strain as ST325. All these genes were found either alone or simultaneously in the strains in the following five different arrangements:<bla_OXA-10>; <bla_OXA-10, bla_IMP-1>; <bla_PER-1, bla_OXA-10>; <bla_PER-1, bla_PSE-1>; <bla_OXA-10, bla_TEM-1>. Regarding mutation-driven resistance, all, but four of the isolates had a relevant decrease of oprD expression. In addition, 73.3% of the isolates overexpressed mexB, 40% mexD, and 33.3% mexY. A specific combination of overexpressed mexB or mexY and alteration in loop L7₁₀ of OprD were significantly associated with meropenem resistance. In conclusion, combination of several mutation-driven mechanisms leading to OprD inactivation and overexpression of efflux systems was the main carbapenem resistance mechanism among the ESBL-producing P. aeruginosa isolates, but acquisition of a transferable resistance determinant such as metallo-β-lactamase could be problematic in clinical settings in China.

Introduction

Coresistance, such as that found in a significant proportion of extended-spectrum beta-lactamase (ESBL)-producing Pseudomonas aeruginosa, precludes the empirical use of most of the antimicrobial families, particularly in the case of severe infections. The associated coresistance of P. aeruginosa isolates to different antimicrobials, such as cephalosporins and carbapenems, may play an important role in the spread and maintenance of these isolates. However, this associated coresistance also limits therapeutic options. Acquired carbapenem resistance in P. aeruginosa is often associated with acquired Ambler class B metallo-β-lactamase (MBL) production.¹⁶ In China, the prevalence of MBL-producing P. aeruginosa isolates was lower than in some developed countries.¹⁸ Regardless of the fact that efflux system upregulation, MBL production, and OprD alterations have been well described as multidrug resistance mechanisms in laboratory mutants,¹¹ however, there is still little information on the relevance of this mechanism in ESBL-producing isolates.

To gain a better understanding of the contribution of several mechanisms to carbapenem resistance in ESBL-producing P. aeruginosa isolates from China, this study examined the expression of oprD and several efflux pump genes as well as sequence variations of the oprD gene and the presence of MBL genes in ESBL-producing P. aeruginosa isolates.

Materials and Methods

Strains and susceptibility testing

A total of 83 nonduplicate P. aeruginosa isolates was collected from a Chinese tertiary hospital during the period from July 2010 to July 2011. Fifteen isolates P. aeruginosa previously characterized as ESBL producers were studied. Bacterial identification and antimicrobial susceptibility were performed using the MicroScan Walkaway 96SI automatized System (Dade Behring, Inc.), which were translated into clinical categories according to the interpretive criteria of the Clinical and Laboratory Standards Institute.¹ P. aeruginosa ATCC 27853 was used as a control throughout.

Phenotypic detection of ESBLs and MBLs

Combination disc tests were performed to seek ESBL producers, comparing the inhibition zones of discs containing ceftazidime (30 μg) alone and with clavulanate (30+10 μg) on Mueller-Hinton agar (Difco) with 400 mg/L phenylboronic acid added to inhibit interference by AmpC cephalosporinase. Inocula and conditions were as in disc testing and a zone expansion >5 mm in the presence of clavulanate was taken to imply ESBL production.⁹ Isolates were screened for MBL production by a combined disk test and double disk synergy test (DDST) as described previously.¹⁰

Detection of genes encoding acquired β-lactamases

Isolates were screened by PCR targeting bla_SHV, bla_TEM, bla_OXA, bla_PER, bla_CTX-M, bla_VEB, bla_KPC, and bla_GES alleles, as described previously.^6,7 In addition, bla_MBL alleles that encode enzymes of the IMP, VIM, GIM, SPM, and SIM groups were sought by PCR.^4,5 Sequence analyses were performed with the BLAST program at the National Center for Biotechnology Information server (http://ncbi.nlm.nih.gov/).

Quantification of gene expression by reverse-transcription-polymerase chain reaction

Total RNA was prepared using the TRIzol Max^™ method (TaKaRa). Real-time reverse-transcription-polymerase chain reaction was performed using the PrimeScript^™ RT Reagent Kit (TaKaRa). The primers of mexB, mexD, mexY, and oprD for PCR amplification of cDNA were according to Wang et al.²¹ Expression of the endogenous control gene rpsL was used to normalize data. Relative quantification was determined by the 2^−ΔΔCT method, where CT is the cycle threshold. Normalized expression of each gene of interest was calibrated against the corresponding mRNA expression by P. aeruginosa PAO1; results are given as the relative expression of mRNA compared with that of P. aeruginosa PAO1. In all cases, the mean values of mRNA expression obtained in three experiments were considered.

Sequencing of the oprD gene and outer membrane protein analysis

The presence of inactivating mutations in oprD was investigated through PCR amplification of the entire gene with specific primers followed by sequencing, as described before.¹² The nucleotide sequences were transcribed into the amino acid sequence using the Vector NTI Advanced 9.0.0 (InforMax; Invitrogen). Nucleotide and amino acid sequences were compared with those of the reference strain PAO1. Bacterial outer membrane proteins (OMPs) were examined using a previously reported method.⁸

Genotyping

Genomic DNA was digested overnight with 10 U of SpeI (TaKaRa) and was subjected to pulsed-field gel electrophoresis (PFGE) as described previously.¹⁴ Results were interpreted using BioNumerics 5.1 software according to the criteria of Tenover et al.¹⁹ PFGE patterns were compared with the Fingerprinting II software version 3.0 (Bio-Rad Laboratories) using the Dice coefficient, according to the manufacturer's instructions, and a dendrogram was constructed using the Unweighted Pair Group Method with arithmetic mean algorithm.

For MBL-positive isolates, multilocus sequence typing (MLST) was performed on several representatives of each pulsotype as described previously.²² Nucleotide sequences were determined for both strands and compared with existing sequences in the MLST database (http://pubmlst.org/paeruginosa/) for assignment of allelic numbers and STs.

Statistical analysis

Strains were considered to be MexAB-OprM hyperproducers and OprD hypoproducers if the relative expression levels were >2.5 and <0.4, respectively.²¹ However, strains were considered positive for mexD or mexY overexpression when the corresponding mRNA level was at least 10-fold higher compared with PAO1.

Results

Molecular typing and susceptibility to antibiotics

PFGE revealed a remarkable clonal diversity, with up to eight different patterns identified among the 15 isolates. The PFGE patterns are shown in Fig. 1. A dendrogram was constructed to show the degree of relatedness among the strains of eight clusters designated A to H. MLST experiments showed that two MBL-positive isolates shared an identical ST, ST235. All isolates showed resistance to ceftazidime and imipenem, while only seven (46.7%) were resistant to meropenem. Furthermore, 100% (15/15) of the strains met the criteria commonly used for the definition of multidrug resistance.¹⁶ Combination disc tests for ESBLs were applied to the 15 isolates, with 15 (100%) giving a positive result, while DDST tests gave positive MBL results for 2/15 (13.3%). Table 1 summarizes the antimicrobial susceptibility profiles, molecular typing, and gene expression data.

FIG. 1.

The unweighted pair group method with arithmetic mean dendrogram for percentage of similarity among PFGE profiles of SpeI-digested DNA from Pseudomonas aeruginosa isolates producing extended-spectrum beta-lactamase. A large strain cluster with >80% similarity is encircled. PFGE, pulsed-field gel electrophoresis; ER, emergence room; PICU, pediatric intensive care unit; NICU, neonatal intensive care unit; ND, neurology department; CD, cardiology department; SC, surgical ward; RU, respiratory unit.

Table 1.

Contribution of Different Resistance Mechanisms to Carbapenems Among the Clinical Isolates of Pseudomonas aeruginosa Showing Different Extended-Spectrum Beta-Lactamase Phenotypes

				Relative gene expression					Mutations detected
Strain no.	Clinical isolates antibiotic resistance	ESBL genotype	MBL genotype	MexB	MexD	MexY	OprD	OprD alteration/mutations	mexR mutations	nalC mutations
PA1	AZT, CAZ, CTX, FEP, IMP, PIP, TCC, TOB	OXA-10	ND	1.3	11.39	0.02	1.18	D43N, T103S, K115T, F170L	None	Gly₇₁Glu
PA2	AZT, CAZ, CTX, FEP, IMP, PIP, TIC/CLAV, TOB	OXA-10	ND	9.58	13.27	1.93	2.33	D43N, T103S, K115T, P157R, F170L	Val₁₂₆Glu	Gly₇₁GluSer₂₀₉Arg
PA3	AZT, CIP, CAZ, FEP, IMP, LEV, MEM, PIP, TCC, TOB	OXA-10	ND	10.93	5.46	1.64	0.24	D43N, T103S, K115T, Y120H, T144K, A145S, F170L	None	Gly₇₁GluSer₂₀₉Arg
PA4	AMK, AZT, CAZ, CIP, CTX, FEP, IMP, LEV, PIP, TCC, TOB	PER-1, OXA-10	ND	11.75	15.03	1.28	0.19	D43N, S57E, S59R, T103S, T115K, V127L, E171 delete, P186G, V189T, E202Q, I210A	Val₁₂₆Glu	Gly₇₁GluSer₂₀₉Arg
PA5	AZT, CIP, CAZ, FEP, IMP, MEM, PIP, TCC, TOB	PER-1, OXA-10	ND	18.25	18.51	1.55	0.039	F170L, Q185E, P186G, V189T, E230K, Y283H, R310E, N312G, A315G	None	Gly₇₁GluSer₂₀₉Arg
PA6	AMK, AZT, CAZ, CIP, CTX, FEP, LEV, PIP, TCC, TOB	PER-1, OXA-10	ND	2.35	17.27	2.1	0.153	S57E, S59R, V127L, K137E, E185Q, P186G, V189T, I210A, E230K, S240T	None	Gly₇₁GluSer₂₀₉Arg
PA7	AMK, AZT, CAZ, CIP, CTX, FEP, IMP, LEV, PIP, TCC, TOB	TEM-1, OXA-10	ND	0.93	0.67	40.5	0.0188	S57E, S59R, V127L, E185Q, P186G, V189T, E202Q, I210A, E230K, S240T, T276A, A281G, R310E, N312G, A315G, L374M, 372(VDSSSS-YAGL)383, S403A	Val₁₂₆Glu	Gly₇₁GluAla₁₈₆Thr
PA8	AZT, CAZ, CIP, CTX, FEP, IMP, LEV, PIP, TCC, TOB, TZP	OXA-10	ND	12.55	3.78	0.08	0.0006	S57E, S59R, V127L, E185Q, P186G, V189T, E202Q, I210A, E230K, S240T, T276A, A281G, R310E, N312G, A315G, L374M, 372(VDSSSS-YAGL)383, S403A	None	Gly₇₁GluSer₂₀₉Arg
PA9	AMK, AZT, CAZ, CIP, CTX, FEP, IMP, LEV, MEM, PIP, TCC, TOB, TZP	PSE-1, OXA-10	ND	10.41	1.04	12.03	0.1183	D43N, S57E, S59R, F148L, I210A, S240T, N262T, R310E, 372(VDSSSS-YAGL)383	None	Gly₇₁GluSer₂₀₉Arg
PA10	AZT, CAZ, CIP, CTX, FEP, IMP, LEV, MEM, PIP, TCC, TOB, TZP	OXA-10	ND	10.56	1.66	0.29	0.0007	D43N, S57E, S59R, 206S delete, I210A, S240T, N262T, R310E, 372(VDSSSS-YAGL)383	None	Gly₇₁GluSer₂₀₉Arg
PA11	AMK, AZT, CAZ, CIP, CTX, FEP, IMP, LEV, MEM, PIP, TCC, TOB, TZP	OXA-10	IMP-1	12.26	19.03	27.28	0.0115	D43N, S57E, S59R, S121R, I210A, E230K, S240T, N262T, 372(VDSSSS-YAGL)383	None	None
PA12	AZT, CRO, CAZ, CIP, FEP, IMP, LEV, PIP, TCC, TOB	PSE-1, PER-1	ND	5.9	0.91	4.8	0.514	D43N, S57E, S59R, L136V, I210A, E230K, S240T, N262T, 372(VDSSSS-YAGL)383	Val₁₂₆Glu	None
PA13	AMK,AZT, CIP, CAZ, FEP, IMP, LEV,MEM, PIP, TCC, TZP, TOB	PSE-1, PER-1	ND	11.88	1.38	160.9	0.0017	D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A281G, Y283N, K296Q, Q301E, R310E, A315G, 372(VDSSSS-YAGL)383	None	Gly₇₁GluSer₂₀₉Arg
PA14	AZT, CAZ, CIP, CTX, FEP, IMP, LEV, PIP, TCC, TOB	OXA-10	ND	1.51	1.32	13.12	2.64	D43N, T103S, K115T, F170L	Val₁₂₆Glu	None
PA15	AMK, AZT, CAZ, CIP, CTX, FEP, IMP, LEV, MEM, PIP, TCC, TOB, TZP	OXA-10	IMP-1	9.13	8.69	1.16	0.025	S57E, S59R, V127L, E185Q, P186G, V189T, I210A, E230K, S240T, N262T, T276A, A281G, K296Q, Q301E, R310E, A315G, M347L, 372(VDSSSS-YAGL)383	None	Gly₇₁GluSer₂₀₉Arg

ESBL, extended-spectrum beta-lactamase; MBL, metallo-β-lactamase; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; AZT, aztreonam; PIP, piperacillin; TZP, piperacillin-tazobactam; IMP, imipenem; MEM, meropenem; CIP, ciprofloxacin; LEV, levofloxacin; TCC, ticarcillin-clavulanic acid; TOB, tobramycin; AMK, amikacin; ND, not determined.

Characterization of acquired β-lactamases and prevalence of carbapenemases

Genes encoding ESBLs were detected in all the isolates exhibiting an ESBL phenotype. The bla_OXA-10-like genes were found in 13 isolates (86.7%), variously accompanying the genes for PER, PSE,TEM, and/or IMP beta-lactamases. bla_PER-1 was found in five isolates, bla_PSE-1 was detected in three isolates, and bla_TEM-1 was detected in one isolate. bla_SHV and bla_CTX-M alleles were not detected. Five isolates harbored both bla_OXA-10-like and bla_PER-1. Two carried bla_OXA-10-like with bla_IMP-1. Two had bla_PER-1 and bla_PSE-1 genes. And one isolate contained bla_OXA-10-like and bla_TEM-1 (Table 1). bla_IMP-1 was found in two isolates giving a positive MBL test, whereas bla_VIM, bla_GIM, bla_SIM, bla_SPM, bla_KPC, and bla_NDM alleles were not detected.

Porin OprD studies

In this study, 73.3% (11/15) of the ESBL-producing isolates showed decreased transcription of oprD to variable degrees; in 53.3% (8/15) of the isolates, the reduction was significant (<0.4-fold compared with P. aeruginosa PAO1) and in three isolates (20%), oprD transcription was zero or very low (<0.01 compared with P. aeruginosa PAO1). Reduced expression of OprD was also assessed by OMP analysis (data not shown). These data therefore confirm that OprD inactivation is a nearly universal signature of imipenem resistance.

Correlation of these oprD nucleotide sequences, the mRNA levels of oprD, and the molecular characterization are shown in Table 1. Amino acid changes were found in loops 1 and 2 of OprD (amino acid D43N, T103S, K115T, and F170L), but the isolate had normal amounts of oprD mRNA. These amino acid changes were frequent in the cluster A strains tested in this study, while these changes could not reduce the expression of OprD (data not shown). It is suggested that these amino acid changes are not correlated with the transcription levels of oprD. The OprD types of six strains resistant to meropenem showed a stretch of 12 amino acid residues located in loop L15 (formerly loop L7) replaced by a sequence of 10 amino acid residues (372-VDSSSSYAGL-384). These data show that decreased expression of the oprD gene contributes to resistance to imipenem, but that the contribution to meropenem resistance is somewhat limited. To confirm this conclusion, more P. aeruginosa isolates need to be studied further.

Transcription of MexAB-OprM, MexCD-OprJ, and MexXY-OprM

Among the efflux systems, mexB overexpression (73.3%) was the most prevalent mechanism, followed by mexD (40%) and mexY (33.3%). Regarding meropenem-resistant isolates, a highly significant link to mexY and mexB overexpression was documented for the first time: isolates with meropenem minimum inhibitory concentration (MICs) of 1∼8 μg/ml produced mexY mRNA levels of 0.02–4.8-fold that of PAO1, while four isolates with meropenem MICs of 32–64 μg/ml produced mexY mRNA levels of 12.03–160.9-fold, one of these P. aeruginosa isolates possessed bla_IMP-1 genes. Additionally, eight isolates with meropenem MICs of 8–64 μg/ml were more likely to have increased expression of mexB than were the seven isolates with meropenem MICs of 1–4 μg/ml (p<0.05).

Mutations in at least two genes (mexR and nalC) reportedly lead to increased expression of MexAB-OprM.²⁰ We found numerous mutations in these genes in all clinical isolates (Table 1). Three isolates had a Val126Glu change in MexR; however, mexB expression in these isolates was similar to that in isolates lacking the mutation. Thus, there was no evidence of a correlation of hyperexpression of the MexAB-OprM and MexR mutation. NalC Gly71Glu and Se209Arg were found in nine strains, but these varied widely in resistance, including seven- up to 10-fold change in mexB expression. Ser209Arg in NalC is known to have an impact on efflux activity,¹² whereas Gly71Glu is considered insignificant. The significance of this combination for mexB expression merits further investigation.

The relationship of carbapenem resistance with efflux system overexpression and OprD reduction was investigated between the ESBL-producers and the non-ESBL isolates (Table 2). While 8 (53.3%) of the 15 ESBL-producers overexpressed one or more genes of the efflux systems and reduced oprD, overexpression of at least one gene and reduced oprD was found only in 4 (16%) non-ESBL isolates. Overexpression of the mexY gene was observed in five ESBL-producers (33.3%), but no non-ESBL isolates overexpressed the mexY gene. Interestingly, the same link to mexB overexpression was observed (Table 2).

Table 2.

Relationship of Carbapenem Resistance with Efflux Systems Overexpression and oprD Reduction Between the Extended-Spectrum Beta-Lactamase Producers and the Non-Extended-Spectrum Beta-Lactamase Isolates

	No. (%) of carbapenem-resistant isolates
Resistance mechanism	ESBL-producing isolates (n=15)	Non-ESBL-producing isolates (n=25)	p-Value
mexB overexpression	11 (73.3)	5 (20)	0.002
mexD overexpression	6 (40)	3 (12)	0.050
mexY overexpression	5 (33.3)	—	0.005
oprD reduction	11 (73.3)	15 (60)	0.502
Any efflux pumps overexpression + oprD reduction	8 (53.3)	4 (16)	0.030

Discussion

This study aimed to investigate the mechanisms underlying carbapenem resistance in a collection of ESBL-producing P. aeruginosa isolates. Although subject to several limitations, discussed below, this study does reveal something important, namely, that most ESBL-producing P. aeruginosa isolates from one hospital in China exhibiting coresistance to carbapenem is due to upregulation of efflux or reduced OprD.

The ESBLs reported for P. aeruginosa are SHV, TEM, PER, VEB, BEL, GES, OXA, and, more recently, CTX-M types.^17,23 In this study, OXA types were predominant, followed by PER and PSE types. The dominance of OXA types among the ESBLs of P. aeruginosa disagrees with data reported previously.¹⁵ It is striking that OXA-10 like was found in four different beta-lactamase combinations (alone, with PER-1, TEM-1, or PSE-1 enzymes), implying to some extent that it is well established. The coexistence of bla_OXA-10-like with bla_IMP-1 in P. aeruginosa was observed, and is presently reported for the first time. Nevertheless, the absence of the genes for CTX-M and SHV ESBLs was confirmed by PCR, supporting the ESBL screening tests and refuting this concern.

An important issue to consider for understanding resistance dynamics is the interconnections between the different resistance mechanisms. Previous studies have indicated that mutational inactivation of oprD is known to be the main mechanism of imipenem resistance in the absence of acquired carbapenemases.¹³ In the present study, 13.3% of the ESBL-producing P. aeruginosa isolates were found to be MBL producers. However, decreased expression of oprD was found in 73.3% of the isolates regardless of the presence of other factors. In 4 (11.3%) of such OprD-reduced isolates, expression levels of oprD could be reduced by more than 1,000 times compared with the control strain. Although the percentage of isolates showing oprD reduction was higher in the ESBL-producers (73.3%) than in the non-ESBL-producers (60%), the difference was not statistically significant. The discrepancy may be due to the small number of ESBL-producers evaluated in the present study.

According to the findings of the current study, up to 100% of the isolates overexpressed at least one of the mechanisms, with mexB (73.3%), mexD (40%), or mexY (33.3%) being the genes more frequently overexpressed. It should be noted, however, that the first large outbreak of a P. aeruginosa strain hyperproducing MexXY-OprM was recently reported in a Chinese hospital. External loop 7 is especially important for the uptake of meropenem.² In our study, 13 isolates with overexpression of mexX or mexB showed meropenem MIC 2∼64 mg/L, whereas eight out of them showed meropenem MIC 8–64 mg/L had concomitant alterations in loop L7₁₀ of OprD and increase of mexB or mexY mRNA. Our results confirm and extend the data from a previous study showing that meropenem appeared to be significantly affected by overexpression of mexB or mexY and alteration in loop L7₁₀ of OprD. Future studies, including more such isolates may be able to provide more definite information. Molecular typing by PFGE showed a wide diversity of patterns. All isolates from blood presented a high coefficient of similarity and closely related PFGE patterns, suggesting a close genetic relationship. The spread of bla_IMP-1-positive isolates observed in the study was caused by a member of the ST258 clone using MLST analysis, suggesting that clonal expansion played a predominant role in the dissemination of these isolates.

In summary, this study demonstrates that combination of several mechanisms leading to OprD inactivation as well as overexpression of efflux systems may contribute to most carbapenem resistance in ESBL-producing P. aeruginosa isolates in a Chinese hospital, whereas MBL production, although increasing, is still infrequent.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

[CLSI] Clinical, Laboratory Standards Institute. 2011. Performance Standards for Antimicrobial Susceptibility Testing, 31No. 1, 20th Informational Supplement M100-S20CLSI: Wayne, PA.

Elena

, Cabot

, Mulet

, García-Castillo

, del Campo

, Juan

, Cantón

, Oliver

2011. Pseudomonas aeruginosa carbapenem resistance mechanisms in Spain: impact on the activity of imipenem, meropenem and doripenem. J. Antimicrob. Chemother., 66:2022–2027.

Glupczynski

, Bogaerts

, Deplano

, Berhin

, Huang

T.D.

, Van Eldere

, Rodriguez-Villalobos

2010. Detection and characterization of class A extended-spectrum-betalactamase-producing Pseudomonas aeruginosa isolates in Belgian hospitals. J. Antimicrob. Chemother., 65:866–871.

Gutiérrez

, Juan

, Cercenado

, Navarro

, Bouza

, Coll

, Pérez

J.L.

, Oliver

2007. Molecular epidemiology and mechanisms of carbapenem resistance in Pseudomonas aeruginosa isolates from Spanish hospitals. Antimicrob. Agents Chemother., 51:4329–4335.

Lee

J.Y.

, Ko

K.S.

2012. OprD mutations and inactivation, expression of efflux pumps and AmpC, and metallo-β-lactamases in carbapenem-resistant Pseudomonas aeruginosa isolates from South Korea. Int. J. Antimicrob. Agents, 40:168–172.

Lin

S.P.

, Liu

M.F.

, Lin

C.F.

, Shi

Z.Y.

2012. Phenotypic detection and polymerase chain reaction screening of extended-spectrum β-lactamases produced by Pseudomonas aeruginosa isolates. J. Microbiol. Immunol. Infect., 45:200–207.

Liu

, Li

X.Y.

, Wan

L.G.

, Jiang

W.Y.

, Li

F.Q.

, Yang

J.H.

2012. Molecular characterization of the bla(KPC-2) gene in clinical isolates of carbapenem-resistant Klebsiella pneumoniae from the pediatric wards of a Chinese hospital. Can. J. Microbiol., 58:1167–1173.

Ocampo-Sosa

A.A.

, Cabot

, Rodríguez

, Roman

, Tubau

, Macia

M.D.

, Moya

, Zamorano

, Suárez

, Peña

, Domínguez

M.A.

, Moncalián

, Oliver

, Martínez-Martínez

Spanish Network for Research in Infectious Diseases (REIPI). 2012. Alterations of OprD in carbapenem-intermediate and -susceptible strains of Pseudomonas aeruginosa isolated from patients with bacteremia in a Spanish multicenter study. Antimicrob. Agents Chemother., 56:1703–1713.

Picão

R.C.

, Poirel

, Gales

A.C.

, Nordmann

2009. Further identification of CTX-M-2 extended-spectrum beta-lactamase in Pseudomonas aeruginosa. Antimicrob. Agents Chemother, 53:2225–2226.

10.

Picão

R.C.

, Andrade

S.S.

, Nicoletti

A.G.

, Campana

E.H.

, Moraes

G.C.

, Mendes

R.E.

, Gales

A.C.

2008. Metallo-beta-lactamase detection: comparative evaluation of double-disk synergy versus combined disk tests for IMP-, GIM-, SIM-, SPM-, or VIM-producing isolates. J. Clin. Microbiol., 46:2028–2037.

11.

Polotto

, Casella

, de Lucca Oliveira

M.G.

, Rúbio

F.G.

, Nogueira

M.L.

, de Almeida

M.T.

, Nogueira

M.C.

2012. Detection of P. aeruginosa harboring bla CTX-M-2, bla GES-1 and bla GES-5, bla IMP-1 and bla SPM-1 causing infections in Brazilian tertiary-care hospital. BMC Infect. Dis., 12:176.

12.

Quale

, Bratu

, Gupta

, Landman

2006. Interplay of efflux system, ampC, and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates. Antimicrob. Agents Chemother., 50:1633–1641.

13.

Riera

, Cabot

, Mulet

, García-Castillo

, del Campo

, Juan

, Cantón

, Oliver

2011. Pseudomonas aeruginosa carbapenem resistance mechanisms in Spain: impact on the activity of imipenem, meropenem and doripenem. J. Antimicrob. Chemother., 66:2022–2027.

14.

Rodríguez-Martínez

J.M.

, Poirel

, Nordmann

2009. Molecular epidemiology and mechanisms of carbapenem resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother., 53:4783–4788.

15.

Rodriguez-Martinez

J.-M.

, Poirel

, Nordmann

2009. Extended spectrum cephalosporinases in Pseudomonas aeruginosa. Antimicrob. Agents Chemother., 53:1766–1771.

16.

Shu

J.C.

, Chia

J.H.

, Siu

L.K.

, Kuo

A.J.

, Huang

S.H.

, Su

L.H.

, Wu

T.L.

2012. Interplay between mutational and horizontally acquired resistance mechanisms and its association with carbapenem resistance amongst extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) Int. J. Antimicrob. Agents, 39:217–222.

17.

Strateva

, Yordanov

2009. Pseudomonas aeruginosa - a phenomenon of bacterial resistance. J. Med. Microbiol., 58,Pt 9:1133–1148.

18.

T.T.

, Zhang

J.L.

, Wang

, Tao

, Yu

Y.S.

, Chen

Y.G.

, Zhou

J.Y.

, Li

L.J.

2009. Evaluation of phenotypic tests for detection of metallo-β-lactamase-producing Pseudomonas aeruginosa strains in China. J. Clin. Microbiol., 47:1136–1142.

19.

Tenover

F.C.

, Arbeit

R.D.

, Goering

R.V.

, Mickelsen

P.A.

, Murray

B.E.

, Persing

D.H.

, Sweaminathan

1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol., 33:2233–2239.

20.

Tomás

, Doumith

, Warner

, Turton

J.F.

, Beceiro

, Bou

, Livermore

D.M.

, Woodford

2010. Efflux pumps, OprD porin, AmpC beta-lactamase, and multiresistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob. Agents Chemother., 54:2219–2224.

21.

Wang

, Zhou

J.Y.

, Qu

T.T.

, Shen

, Wei

Z.Q.

, Yu

Y.S.

, Li

L.J.

2010. Molecular epidemiology and mechanisms of carbapenem resistance in Pseudomonas aeruginosa isolates from Chinese hospitals. Int. J. Antimicrob. Agents, 35:486–491.

22.

Yoo

J.S.

, Yang

J.W.

, Kim

H.M.

, Byeon

, Kim

H.S.

, Yoo

J.I.

, Chung

G.T.

, Lee

Y.S.

2012. Dissemination of genetically related IMP-6-producing multidrug-resistant Pseudomonas aeruginosa ST235 in South Korea. Int. J. Antimicrob. Agents, 39:300–304.

23.

Zhao

W.H.

, Hu

Z.Q.

2010. Beta-lactamases identified in clinical isolates of Pseudomonas aeruginosa. Crit. Rev. Microbiol., 36:245–258.