Beta-Lactam Resistance Mechanisms in Pseudomonas aeruginosa Strains Causing Bloodstream Infections: Comparative Results Between Brazilian and American Isolates

Abstract

This study evaluated the presence of distinct mechanisms of beta-lactam resistance in 122 Pseudomonas aeruginosa isolates, causing bloodstream infections at Hospital São Paulo (HSP, Brazil; 82 isolates) and Virginia Commonwealth University Medical Center (VCU, United States; 40 isolates). By Clinical Laboratory Standards Institute agar dilution, Brazilian P. aeruginosa isolates showed higher resistance rates to most antimicrobials tested than those collected from the United States, except for ciprofloxacin. Carbapenem hydrolysis was detected in seven P. aeruginosa from HSP, in which bla_SPM-1 (n=5), bla_IMP-1 (n=1), and bla_IMP-16 (n=1) were detected by polymerase chain reaction (PCR) followed by DNA sequencing. The production of GES-5 was observed in 1.25% of HSP isolates. No extended-spectrum beta-lactamase-encoding genes were detected in the VCU isolates. Expression of efflux systems genes (mexB, mexD, mexF, and mexY) was evaluated by quantitative reverse transcriptase–PCR. In HSP isolates MexXY-OprM (41.4%) efflux system was more frequently overexpressed, in contrast to what was observed in the VCU isolates, where both MexXY-OprM (25.0%) and MexAB-OprM (25.0%) were equally overexpressed. The oprD downregulation was similar among isolates collected from the HSP (92.7%) and VCU (95.0%). On the other hand, ampC overexpression was observed only among HSP isolates (31.7%). The distinct antimicrobial susceptibility profile and mechanisms of beta-lactam resistance found among P. aeruginosa isolated from teaching hospitals located in Brazil and the United States exemplify the importance of local epidemiology in determining antimicrobial resistance rates.

Introduction

P seudomonas aeruginosa is an important opportunistic pathogen associated with serious nosocomial infections. High attributable mortality rates, ranging from 20% to 38%, in hospital-acquired bloodstream infections caused by this microorganism have been reported.²⁵ P. aeruginosa is intrinsically resistant to many antimicrobial agents and is able to acquire multiple mechanisms of drug resistance during antimicrobial therapy. These facts reduce the antimicrobial options available for adequate treatment of P. aeruginosa bloodstream infections.²⁴

Acquired beta-lactamases, such as class A extended-spectrum beta-lactamases (ESBLs) and carbapenemases, class B metallo-beta-lactamases (MBLs), and class D oxacilinases (OXAs), have been extensively reported worldwide as determinants of beta-lactam resistance.^24,30 Additionally, overexpression of MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM efflux systems has been implicated in multidrug resistance phenotypes, including beta-lactams, among P. aeruginosa clinical isolates. Further, alteration in the OprD porin expression can also contribute to carbapenem resistance, especially imipenem.⁹

P. aeruginosa clinical isolates collected from Latin America have shown higher resistance rates than those reported worldwide, including those isolated in the United States.⁵ In the present study, we compared the presence of distinct mechanisms of beta-lactam resistance in P. aeruginosa isolates causing bloodstream infections recovered from Brazilian and American teaching hospitals.

Materials and Methods

Bacterial strains

A total of 122 P. aeruginosa isolates were recovered from patients with bloodstream infections from two teaching hospitals, Hospital São Paulo (HSP; 82 isolates), a tertiary hospital located in São Paulo, Brazil, and Virginia Commonwealth University Medical Center (VCU; 40 isolates), located in Richmond, VA. A single isolate per patient was studied. These isolates were consecutively collected between January 2000 and May 2002. Isolates from both institutions were properly stored at −70°C at Special Microbiology Laboratory of Federal University of São Paulo, and were subcultured two times on Columbia blood agar (Oxoid) for viability and purity checking.

Antimicrobial susceptibility testing

Antimicrobial susceptibility profiles of the P. aeruginosa isolates were determined by agar dilution method³ on Mueller-Hinton agar (Oxoid) for amikacin (Eurofarma), aztreonam (Bristol-Myers Squibb), cefepime (Bristol-Myers Squibb), ceftazidime (Strides Arcolab), ciprofloxacin (Fresenius Robi), gentamicin (Sigma), imipenem (Merck), meropenem (Astra Zeneca), and piperacillin/tazobactam (Novafarma). Results were interpreted according to the Clinical Laboratory Standards Institute's guidelines.⁴ P. aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as quality-control strains with results within acceptable ranges.

Screening for carbapenemase producers

Carbapenemase activity in crude extracts was investigated by ultraviolet spectrophotometric assays. Briefly, a full 10-μl loop of the tested organism was inoculated into 500 μl of phosphate buffer 100 mM (pH 7.0) and disrupted by sonication. The cells were removed by centrifugation and the supernatants were used for further experiments. Protein quantification in the crude extracts was performed using Bradford strain. Hydrolytic activity of crude extracts was determined against 10 μM imipenem and 10 μM meropenem in 100 mM phosphate buffer (pH 7.0). Measurements were carried out at 297-nm wavelength. Positive control included P. aeruginosa 48-1997A, producer of the MBL SPM-1,²⁷ and negative control included P. aeruginosa ATCC 27853. Carbapenem hydrolysis inhibition was performed by incubating the crude extract with 25 mM ethylenediaminetetraacetic acid (EDTA) for 15 minutes before the assay with imipenem and meropenem.

Detection of β-lactamase genes by polymerase chain reaction and DNA sequencing

The presence of MBL-encoding genes (bla_SPM, bla_VIM, bla_IMP, bla_GIM, and bla_SIM),¹¹ ESBL (bla_KPC, bla_SHV, bla_CTX, bla_PER, and bla_GES),^{12,14,15,17,20} and OXA-type-carbapenemase (bla_OXA-23, bla_OXA-24, bla_OXA-51, bla_OXA-48, and bla_OXA-58)^19,28 were investigated by polymerase chain reaction (PCR) using previously described primers and cycling conditions. For amplicons sequencing, PCR products were purified using PCR purification columns (Qiagen). Sequencing reactions were performed using specific primers and an automated ABI Prisma 3100 sequencer (Applied Biosystems). The nucleotide and deduced protein sequences were analyzed with software available over the Internet at the National Center for Biotechnology Information Web site (www.ncbi.nlm.nih.gov).

Quantitative reverse transcriptase–PCR

Transcriptional levels of mexB, mexD, mexF, mexY, ampC, and oprD were determined by Mastercycler Realplex (Eppendorf). Briefly, total RNA was extracted using the RNeasy Mini Kit, following the manufacturer's recommendations (Qiagen). Five micrograms of total RNA was submitted to cDNA synthesis using High-Capacity cDNA Archive Kit (Applied Biosystems). Quantitative reverse transcriptase–PCR was performed by Platinum SYBR Green Supermix (Invitrogen), using specific primers for mexB, mexD, mexF, mexY, ampC, and oprD.²⁹ Amplification was carried out in triplicate from cDNA preparations. The ribosomal protein rpsL-encoding gene was used as a reference gene for normalizing the transcriptional levels of target genes. Transcription data were analyzed with Q-Gene software.¹³ The MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM efflux systems were considered overexpressed when the transcriptional levels of mexB and mexY were at least 2- and 4-fold, and mex D and mexF were at least 100-fold higher than those of the wild-type reference strain PA01.⁶ Reduced oprD expression and overexpression of ampC were considered relevant when their transcriptional levels were ≤70% and ≥10-fold, respectively, compared with that of the PA01 reference strain.^21,23

Statistical analysis

Data were analyzed by Epi-Info program 6.0 (Centers for Disease Control and Prevention, Atlanta, GA) using the Chi-square or Fisher exact test when applicable. A p-value ≤0.05 with a confidence interval of 95% was considered significant.

Results

Susceptibility testing (minimal inhibitory concentration)

The in vitro activity of each selected antimicrobial agent tested against P. aeruginosa is summarized in Table 1. Cephalosporins exhibited limited in vitro activity against HSP isolates, with cefepime (58.5%) showing higher susceptibility rate than that of ceftazidime (48.8%). Imipenem and meropenem showed identical in vitro activity (minimal inhibitory concentration (MIC)₅₀, 4 μg/ml) and susceptibility rates (51.2%) against these isolates. In general, VCU isolates exhibited higher susceptibility rates, except for amikacin and ciprofloxacin. Despite exhibiting the same in vitro potency (MIC₅₀ 4 μg/ml) against P. aeruginosa isolates, discrepant susceptibility rates for ciprofloxacin were observed for the VCU (5%) and HSP (46.3%) isolates. All of the VCU isolates were resistant to at least one antimicrobial tested. In contrast, ∼22.0% of the all HSP isolates (n=18) were susceptible to all tested antimicrobials.

Table 1.

Comparison of the In Vitro Activity of Selected Antimicrobial Agents Tested Against Pseudomonas aeruginosa Isolates Causing Bloodstream Infection at Hospital São Paulo and Virginia Commonwealth University Hospitals

	Hospital São Paulo (n=82)				Virginia Commonwealth University (n=40)
	MIC (μg/ml)		% by category ^a		MIC (μg/ml)		% by category ^a
Antimicrobial agents	MIC₅₀	MIC₉₀	S	R	MIC₅₀	MIC₉₀	S	R
Aztreonam	16	>32	37.8	43.9	8	32	60.0	17.5
Piperacillin/tazobactam	32	256	59.8	40.2	8	256	80.0	20.0
Ceftazidime	16	>32	48.8	40.2	8	>32	65.0	20.0
Cefepime	8	32	58.5	20.7	4	32	77.5	15.0
Ciprofloxacin	4	>4	46.3	52.4	4	>4	5.0	75.0
Meropenem	4	32	51.2	37.8	1	8	82.5	10.0
Imipenem	4	32	51.2	28.1	1	16	80.0	15.0
Gentamicin	8	>16	47.6	42.7	<2	16	67.5	27.5
Amikacin	<8	>64	56.1	28.0	16	>64	62.5	15.0

Breakpoint criteria as published by the CLSI (2010).

MIC, minimal inhibitory concentration; S, susceptible; R, resistant.

Carbapenem hydrolysis and β-lactamase detection

Only seven (8.5%) P. aeruginosa isolates collected from HSP showed carbapenem hydrolysis activity (p=0.05). These isolates had their carbapenemase activity inhibited by EDTA, and the presence of the MBL-encoding genes bla_SPM-1 (n=5), bla_IMP-1 (n=1), and bla_IMP-16 (n=1) was confirmed by multiplex PCR and DNA sequencing, respectively. The gene bla_GES-5, a class A carbapenemase, was detected in a single Brazilian isolate. OXA-encoding genes were not identified in any isolates.

Gene expression

Gene expression analysis showed that overexpression of efflux pumps was more frequently detected among P. aeruginosa clinical isolates from HSP (70.7%) than those from MCV (60.0%) (p=0.2). The most frequently overexpressed system was MexXY-OprM (41.4%; from 4.13- to 958-fold compared with PA01) in Brazilian isolates, while the MexAB-OprM (25.0%; from 3.27- to 89.31-fold compared with PA01) and MexXY-OprM (25.0%; from 7.41- to >10,000-fold compared with PA01) were equally detected among American isolates. Overexpression of MexEF-OprN was not observed among Brazilian isolates, but was detected in 7.5% (n=3) of the VCU isolates (108 to>10,000-fold than PA01). Overall, 31.7% of P. aeruginosa isolates from HSP overexpressed the ampC gene (13- to >10,000-fold compared with PA01) (p≤0.05). Decreased expression of the oprD gene was identified in P. aeruginosa isolates collected from both hospitals (92.7% to HSP and 95.0% to VCU isolates, respectively; p=0.4) (Fig. 1).

FIG. 1.

Frequency of mexB, mexD, mexF, mexY, and ampC gene overexpression, and decreased oprD gene expression among Pseudomonas aeruginosa isolates causing bloodstream infection at Hospital São Paulo (HSP) and Virginia Commonwealth University (VCU).

In addition, the association of resistance determinants and susceptibility testing profile among the P. aeruginosa isolates were analyzed (Table 2). Brazilian isolates presented association of different determinants of beta-lactam resistance, including acquired mechanisms such as MBL-encoding genes. Among 26 HSP isolates that overexpressed ampC, 15.4% carried MBL-encoding genes and 61.5% overexpressed at least one efflux system, respectively. Among HSP isolates that possessed MBL-encoding genes, 71.4% also showed overexpression of MexXY-OprM. Curiously, no ampC overexpression was observed in the VCU isolates. Decrease in oprD gene expression was the main associated mechanism of beta-lactam resistance among P. aeruginosa isolates, independent of geographic location. Additionally, 37.8% of Brazilian and 67.5% of American isolates showed only the decreased oprD gene. AmpC derepression together with decreased OprD was observed in 30.5% of HSP isolates. GES-5 was associated only with decreased OprD in a Brazilian isolate. Beta-lactamase resistance mechanisms could not be found in two (2.4%) Brazilian isolates evaluated, and these were susceptible to most of the antimicrobials tested.

Table 2.

Mechanisms of Resistance to Selected Antimicrobial Agents Detected Among Pseudomonas aeruginosa Isolates Causing Bloodstream Infection at Hospital São Paulo and Medical College of Virginia Hospitals

			Antimicrobial tested/% of resistant isolates
Determinant of antimicrobial resistance ^a	No. of isolates	No. of isolates with OprD reduced	AZT	PTZ	CAZ	FEP	CIP	MER	IMI	GEN	AMI
Brazilian isolates (n=82)
XY++; AmpC	8	8	R	87.5	87.5	87.5	R	87.5	87.5	R	87.5
XY++	7	7	71.4	71.4	71.4	71.4	85.7	57	57	85.6	57.2
AmpC	6	6	S/R	33.3	S/R	33.4	33.3	33.3	33.4	33.3	33.3
ABM++; XY++	6	3	S/R	33.3	S/R	16.7	S/R	S/R	66.7	33.3	33.3
ABM++; XY++; AmpC	5	4	80	60	60	80	80	60	80	80	60
ABM++	4	4	75	S	S/R	S	25	S/R	25	S	S
ABM++; AmpC	2	2	S	S	S	S	S	S	S/R	S/R	S
SPM-1	2	2	R	R	R	R	R	R	R	R	R
ABM++; XY++; AmpC; SPM-1	1	1	R	R	R	R	R	R	R	R	R
ABM++; XY++; AmpC; IMP-16	1	1	R	R	R	R	R	R	R	S	S
ABM++; CDJ++; XY++, AmpC	1	1	S	S	S	S	S	S	S	R	S
ABM++; CDJ++; XY++	1	1	R	S	S	S	S	S	S	R	R
CDJ++; XY++	1	1	R	R	R	R	R	R	R	R	R
XY++; AmpC; SPM-1	1	1	R	R	R	R	R	R	R	R	R
XY++; SPM-1	1	1	S	R	R	R	R	R	R	R	R
XY++; AmpC; IMP-1	1	1	R	S	S	R	R	R	R	S	S
GES-5	1	1	R	R	R	R	R	R	R	R	R
oprD−	31	31	51.6	22.6	35.5	19.4	32.3	19.4	13	32.3	29
No mechanisms of resistance identified	2	0	S	S	S	S	S/R	S	S	S/R	S/R
American isolates (n=40)
ABM++	4	4	75	25	75	S/R	75	S	S	75	S
ABM++; EFN++; XY++	2	2	S	S	S	S	S/R	S	S	S	S/R
ABM++; XY++	3	2	33.3	66.7	33.3	33.3	66.7	33.3	33.3	S	33.3
ABM++; CDJ++; EFN++; XY++	1	1	R	S	S	S	R	S	S	S	S
XY++	3	2	33.3	33.3	66.7	33.3	66.7	66.7	66.7	S	66.7
oprD−	27	27	37	14.8	29.6	18.5	R	3.7	11.1	37	37

The abbreviations ABM+, CDJ+, EFN+, XY+, and AmpC+ designated MexAB, MexCD, MexEF, MexXY, and AmpC overexpression, respectively.

AMI, amikacin; AZT, aztreonam; CAZ, ceftazidime; CIP, ciprofloxacin; FEP, cefepime; GEN, gentamicin; IMI, imipenem; MER, meropenem; PTZ, piperacillin/tazobactam; OprD−, reduced expression of OprD porin; S, 100% of isolates evaluated were susceptible; R, 100% of isolates evaluated were resistant; S/R, 50% of isolates were susceptible and 50% were resistant.

Discussion

The multidrug resistance phenotype, frequently observed in P. aeruginosa clinical isolates, can be attributed to the association of different mechanisms of resistance such as overexpression of efflux pumps, reduced permeability of outer membrane, and the presence of beta-lactamase enzymes. Efflux systems pump out many classes of antimicrobial agents used to treat infections caused by this pathogen, reducing their concentration inside the cells.⁹ However, there is little evidence of the impact that efflux system overexpression can cause in the clinical setting.⁷ Moreover, currently, there are no widely accepted criteria that correlate the transcriptional levels of efflux system-encoding genes with the corresponding increase in the MIC values.²⁹

Wild-type P. aeruginosa express basal levels of AmpC enzymes. However, the expression level of this beta-lactamase is inducible upon beta-lactam exposure, especially carbapenems and cephamycins. Beta-lactams are extensively employed to treat bacterial infections in HSP. We believe that AmpC overexpression among Brazilian isolates observed in our study might be related to antimicrobial consumption, especially broad-spectrum cephalosporins that were able to derepress this gene. This observation is corroborated by the high prevalence of ESBL-producing Enterobacteriaceae isolated from several Brazilian hospitals.¹ The percentage of isolates that showed a decrease of oprD was similar in both groups of isolates studied. Despite some studies describing that OprD inactivation confers reduced susceptibility to carbapenems,² our results suggested that a decrease of oprD may not be an important resistance determinant to these antimicrobials, since 84.2% and 86.8% of Brazilian and U.S. isolates that showed oprD downregulation were susceptible to carbapenems. However, it is important to note that we analyzed only oprD gene transcription and a more detail study about the expression and structure of OprD protein should be done for a better comprehension about the relationship between the OprD expression and the antimicrobial resistance phenotype in these set of clinical isolates.

Since fluoroquinolones are substrates for all efflux systems evaluated in this study, higher resistance rates to ciprofloxacin among VCU isolates might be attributed to the interplay of the efflux pump systems and mutations in the quinolone resistance-determining regions of gyrA and parC genes and topoisomerase IV, but further studies are necessary to confirm this hypothesis.^10,16 Although meropenem resistance was less frequent among VCU isolates than HSP isolates, the MexAB-OprM efflux system was more prevalent among VCU isolates. Tanimoto and colleagues²⁶ reported that pre-exposure to ciprofloxacin increased the chance of selecting meropenem-resistant P. aeruginosa mutants due to MexAB-OprM efflux system overexpression.

In this study, we have observed distinct antimicrobial susceptibility profiles among P. aeruginosa isolates originating from Brazil and United States. This finding could be attributed to the presence of different mechanisms of resistance encountered in the respective collections. All resistance mechanisms evaluated in this study (OprD downregulation, efflux system overexpression, and beta-lactamase production) were observed among Brazilian isolates, which is in accordance to what was previously reported by Xavier and colleagues.²⁹ On the other hand, the efflux system overexpression and OprD downregulation were the only associated mechanisms of resistance observed in the VCU isolates. Our results are in agreement with those reported by some studies conducted in Europe,² which described the presence of multiple resistance mechanisms simultaneously in P. aeruginosa isolates.²³

Our findings agree with multinational studies that denote that resistance rates may vary significantly among different countries.^5,18 The local resistance rates are driven by many factors such as patient-related disease, consumption of antibiotics, and implementation of and/or adherence to infection control polices. Appropriate empirical antimicrobial treatment is crucial for patient outcome, as demonstrated by previous studies.²² Kollef and colleagues⁸ have reported that mortality rates were lower in those patients who had received adequate initial therapy than in those who had received inadequate therapy (31.3% and 56.8%, respectively). The presence of multiple resistance mechanisms in a single isolate, as observed in this study, together with other factors discussed above, further restricts the therapeutic options available for empirical treatment and the chance of clinical success in infections caused by P. aeruginosa.

Footnotes

Acknowledgments

We would like to thank Renata C. Picão and Cecilia G. Carvalhaes for the critical reading of this article. We are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for providing a researcher grant to A.C.G. (process # 307816/2009-5). This work was financially supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, process # 06/55937-3) and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) that conceded a grant to L.C.C.F.

Disclosure Statement

None declared.

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