Journal of Antimicrobial Chemotherapy (2003) 52, 699702 DOI: 10.

1093/jac/dkg416 Advance Access publication 1 September 2003

Dissemination in distinct Brazilian regions of an epidemic carbapenem-resistant Pseudomonas aeruginosa producing SPM metallo--lactamase
Ana C. Gales1,2*, Liana C. Menezes1,2, Suzane Silbert2 and Hlio S. Sader2
1Laboratrio

Alerta; 2Laboratrio Especial de Microbiologia Clnica, Division of Infectious Diseases, Universidade Federal de So Paulo, Rua Leandro Duprt, 188, So Paulo, SP 04025010, Brazil
Received 3 April 2003; returned 28 May 2003, revised 14 July 2003; accepted 15 July 2003
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Background: In Brazil, carbapenem use has been limited by high carbapenem-resistance rates among Pseudomonas aeruginosa isolates. Objective: The main objective of this study was to evaluate the presence of an epidemic P. aeruginosa strain in unrelated Brazilian hospitals. We also aimed to search for the gene blaSPM, which encodes production of SPM, a novel metallo--lactamase (MBL). Methods: A reference broth microdilution method was used for antimicrobial susceptibility testing. The isolates were typed by ribotyping and pulsed-field gel electrophoresis (PFGE). A disc-approximation test using MBL inhibitors was employed to screen isolates for MBL production. PCR was used to search for the gene blaSPM. Results: A total of 43 clinical isolates of carbapenem-resistant P. aeruginosa were collected from 12 hospitals. Colistin retained greatest activity in vitro. A single ribogroup included 17 P. aeruginosa isolates (39.5%) collected from seven unrelated hospitals located in five Brazilian states. Sixteen of these isolates showed an identical PFGE pattern, and 15 produced an SPM-1-like MBL. The remaining 26 isolates were grouped into 25 diverse ribogroups; none were MBL producers. Conclusions: The emergence and dissemination of an epidemic clone has contributed to the high carbapenem resistance rates among P. aeruginosa isolates in Brazil. In addition, the production of SPM MBL has an important role in carbapenem resistance in this region. This is the first report of dissemination of an SPM-1-like-MBL-producing strain of P. aeruginosa among unrelated Brazilian hospitals.
Keywords: Gram-negative bacilli, carbapenem resistance, Brazil

Introduction
Pseudomonas aeruginosa is a leading cause of nosocomial infections, ranking first as a cause of nosocomial pneumonia in Brazilian hospitals.1 In the USA, P. aeruginosa ranked first among all nosocomial pathogens related to pneumonia in intensive care units reported to the National Nosocomial Infection Surveillance System.2 Infections caused by this microorganism are often difficult to treat because of its virulence and antimicrobial resistance. Usually, P. aeruginosa isolates demonstrate resistance to many -lactam agents following hyperproduction of an inducible AmpC -lactamase. In addition, efflux pumps and alterations in outer membrane permeability can play an important role in limiting the access of antimicrobial agents to intracellular targets.3 Thus, carbapenems generally remain one of the best therapeutic choices for treatment of serious P. aeruginosa

infections. However, carbapenem use has been threatened by the emergence of carbapenem-hydrolysing enzyme-producing isolates and the dissemination of multidrug-resistant clones.35 In the Hospital So Paulo, a 600 bed Brazilian teaching hospital, high carbapenem resistance rates have been reported among P. aeruginosa isolates, resulting from the presence of an epidemic clone designated clone SP.1,4 The occurrence of a multidrug-resistant P. aeruginosa strain belonging to a unique genotype was also reported in various hospitals in the state of Rio de Janeiro. In that study, 91% of P. aeruginosa isolates produced a non-characterized metallo-lactamase (MBL).6 Recently, a new MBL, designated SPM-1, has been identified from a carbapenem-resistant P. aeruginosa strain (481997A) responsible for causing a urinary tract infection in a paediatric patient hospitalized at the Hospital So Paulo complex.7 Strain 481997A belongs to clone SP. The main objective of this

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*Corresponding author. Tel: +55-11-5081-2819; Fax: +55-11-5571-5180; E-mail: galesac@aol.com

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PrinterMicrobial Characterization System (E.I. duPont de Nemours or Qualicon, Wilmington, DE, USA). Isolates with indistinguishable ribotype profiles were also typed by pulsed-field gel electrophoresis (PFGE) to confirm genetic similarity using the restriction endonuclease SpeI. Analysis of PFGE patterns was performed by visual inspection of photographs of ethidium bromide-stained gels. Isolates were classified as identical if they shared the same bands, and as unrelated if they differed by more than three bands. The SPM-1producing P. aeruginosa strain (481997A) was included as a control strain.

Metallo--lactamase phenotypic detection

A disc-approximation test, using 2-mercaptopropionic acid (2-MPA) and EDTA, was employed to detect MBL production.9 The positive control strains were an IMP-1-producing P. aeruginosa (PSA319) and an SPM-1-producing P. aeruginosa (481997A).

Detection of the blaSPM gene

After boiling the bacterial cells, PCR was performed on total DNA using SPM primers (forward: 5-CCTACAATCTAACGGCGACC-3, reverse: 5-TCGCCGTGTCCAGGTATAAC-3). The cycling parameters were 95C for 5 min, followed by 30 cycles of denaturation at 95C for 1 min, annealing at 40C for 1 min and extension at 68C for 1 min.9 PCR products were visualized by electrophoresis on 0.8% agarose gels stained with 1% ethidium bromide.
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Figure 1. Location of the Brazilian hospital-based laboratories that provided carbapenem-resistant P. aeruginosa isolates. The cities marked with an asterisk symbol submitted genotype SP isolates.

Results
study was to evaluate the presence of clone SP in other Brazilian hospitals. We also aimed to search for the gene blaSPM among carbapenem-resistant P. aeruginosa isolates. The 43 P. aeruginosa isolates examined were all confirmed to be resistant to the carbapenems imipenem and meropenem, according to the NCCLS breakpoints.8 All P. aeruginosa isolates were inhibited by colistin 0.5 mg/L. Colistin showed the highest susceptibility rate (100.0%), followed by piperacillin/tazobactam (39.5%), aztreonam (30.2%) and gentamicin (27.9%).8 The fluoroquinolones, gatifloxacin (MIC90 > 8 mg/L; 11.6% susceptible) and ciprofloxacin (MIC90 > 4 mg/L; 11.6% susceptible), showed poor activity against the P. aeruginosa isolates tested. A single ribogroup (723) was found among 17 P. aeruginosa isolates (39.5%) collected from seven unrelated medical centres located in five Brazilian states (Table 1). Sixteen of these isolates showed an identical PFGE profile, designated pattern A, which was the same as that displayed by the clone SP. P. aeruginosa strains belonging to clone SP usually exhibit high levels of resistance to carbapenems, broad-spectrum cephalosporins, fluoroquinolones and aminoglycosides. Among the 16 isolates showing the genotype SP, 15 exhibited an MBL phenotype according to screening tests and were positive by PCR for blaSPM. The remaining 26 isolates were represented by 25 diverse ribogroups; none showed an MBL phenotype in screening tests, and none was positive for blaSPM.

Material and methods

Bacterial strains
The Brazilian hospitals were asked to submit only carbapenemresistant P. aeruginosa isolates to the coordinating laboratory. A total of 43 clinical isolates of carbapenem-resistant P. aeruginosa were collected from 12 hospital-based laboratories (see Figure 1) between March and August 2002. One isolate per patient was evaluated. Upon receipt at the Laboratrio Especial de Microbiologia Clnica (LEMC), UNIFESP/EPM, the isolates were subcultured onto blood agar to ensure their viability and purity. Confirmation of species identification was performed with API NF (bioMerieux Vitek, St Louis, MO, USA), or conventional methods as required.

Susceptibility testing
At the coordinating laboratory, antimicrobial susceptibility testing was performed using a reference broth microdilution method recommended by the NCCLS.8 Quality control was conducted using Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, P. aeruginosa ATCC 27853 and Enterococcus faecalis ATCC 29212.

Discussion
The presence of epidemic carbapenem-resistant P. aeruginosa strains has been reported in many intensive care units around the world.10 This is the first paper to report the spread of an epidemic carbapenemresistant P. aeruginosa strain among Brazilian hospitals located in dfferent states. MBLs are emerging worldwide as an important mechanism of carbapenem resistance among non-fermentative Gram-negative isolates.3,5 In this study, the remaining 15 of 16

Molecular methods
Confirmed carbapenem-resistant isolates were genotyped by ribotyping, following the instructions of the manufacturer of the Ribo-

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Carbapenem-resistant P. aeruginosa
Table 1. Characterization of the carbapenem-resistant P. aeruginosa isolates that belonged to the ribogroup 723 Medical centre 3 3 5 6 8 8 8 8 8 9 9 9 15 15 20 20 20 MBL screening test positive positive positive positive positive positive positive positive positive positive positive positive negative negative positive positive positive PFGE pattern A A A A A A A A A A A A B A A A A

Bank number 2477 2479 2528 2807 2430 2382 2431 2432 2434 2613 2608 2756 2821 2703 2818 2819 2820

City Salvador Salvador Londrina Braslia Santo Andr Santo Andr Santo Andr Santo Andr Santo Andr Fortaleza Fortaleza Fortaleza Maring Maring Curitiba Curitiba Curitiba

State Bahia Bahia Paran DF So Paulo So Paulo So Paulo So Paulo So Paulo Cear Cear Cear Paran Paran Paran Paran Paran

Body site LRT urine urine LRT skin stool urine urine urine CVL urine urine skin urine LRTb urine urine

blaSPM gene positive positive positive positive positive positive positive positive positive positive positive positive negative negative positive positive positive

Ribogroup 723 723 723 723 723 723 723 723 723 723 723 723 723 723 723 723 723

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DF, Distrito Federal; CVL, central venous line; LRT, lower respiratory tract.

P. aeruginosa isolates belonging to clone SP were SPM producers. The production of SPM-1 was associated with broad-spectrum -lactam resistance, including carbapenem resistance. As with other MBLs, aztreonam is not a good substrate for SPM-1.7 However, additional mechanisms of -lactam resistance could be present in the P. aeruginosa isolates evaluated in the current study since most of the isolates, even those non-producers of the SPM enzyme, were highly resistant to aztreonam and most of the antimicrobial agents tested. The accumulation of resistance determinants imposes an immense limitation on the therapeutic choices available for treatment of such infections. The existence of common PFGE types among carbapenemresistant P. aeruginosa isolates from distinct geographic locations is troublesome. The interhospital spread of the P. aeruginosa clone SP among hospitals situated nearby could be explained by the transfer of infected patients and/or sharing of common healthcare staff. However, this explanation is unlikely for hospitals distant from each other. To have a better understanding of the role of clone SP in the dissemination of carbapenem resistance among P. aeruginosa isolates in Brazilian hospitals, it is important to evaluate a large number of P. aeruginosa strains collected from hospitals located in other Brazilian regions. In contrast to what it is observed with S. aureus, where a single oxacillin-resistant clone is present throughout Brazil, our results indicate that carbapenem resistance among P. aeruginosa isolates has risen because of both the emergence of resistant strains under antimicrobial selective pressure and the dissemination of epidemic clones.

Andrade), Bahia; Hospital de Clnicas da Universidade Federal do Paran, (M.L. DallaCosta), Hospital de Clnicas da Universidade Estadual de Londrina (E.G. Cavassim), Hospital de Clnicas da Universidade Estadual de Maring (J.M.G. Mikcha), Paran; Hospital de Base de Braslia (J. Ribeiro), Braslia, DF; Hospital e Maternidade Brasil (R. Pimenta, E.B. Siqueira), Hospital do Servidor Pblico Estadual (E. Kusano), Hospital Geral de Carapicuiba Sanatorinhos (L. Alamo), Instituto de Cardiologia Dante Pazzanese (C.E. Ferreira), So Paulo; Hospital Felcio Rocho (C. Starling, J.A. Ferreira), Minas Gerais; Laboratrio Louis Pasteur (T. Bandeira), and Hospital Messejana (T. Bandeira), Cear. This study was financed by the Fundao de Amparo Pesquisa do Estado de So Paulo (FAPESP, No. 2001/033497).

References
1. Sader, H. S., Gales, A. C., Pfaller, M. A. et al. (2001). Pathogen frequency and resistance patterns in Brazilian hospitals: summary of results from three years of the SENTRY Antimicrobial Surveillance Program. Brazilian Journal of Infectious Disease 5, 20014. 2. Richards, M. J., Edwards, J. R., Culver, D. H. et al. (1999). Nosocomial infections in medical intensive care units in the United States. National Nosocomial Infections Surveillance System. Critical Care Medicine 27, 88792. 3. Livermore, D. M. (2002). Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: our worst nightmare? Clinical Infectious Diseases 34, 63440. 4. Gales, A. C., Jones, R. N., Turnidge, J. et al. (2001). Characterization of Pseudomonas aeruginosa isolates: occurrence rates, antimicrobial susceptibility patterns, and molecular typing in the global SENTRY Antimicrobial Surveillance Program, 19971999. Clinical Infectious Diseases 32, Suppl. 2, 14655. 5. Nordmann, P. & Poirel, L. (2002). Emerging carbapenemases in Gram-negative aerobes. Clinical Microbiology and Infection 8, 32131. 6. Pellegrino, F. L., Teixeira, L. M., Carvalho Md, M. G. et al. (2002). Occurrence of a multidrug-resistant Pseudomonas aeruginosa clone in

Acknowledgements
We are very grateful to Timothy Walsh and Mark Toleman from the Department of Pathology & Microbiology, University of Bristol (Bristol, UK), for providing the SPM primers, and to the Brazilian clinical microbiology laboratories for submitting the P. aeruginosa isolates. The participant institutions were: Hospital Aliana (M.G.

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different hospitals in Rio de Janeiro, Brazil. Journal of Clinical Microbiology 40, 24204. 7. Toleman, M. A., Simm, A. M., Murphy, T. A. et al. (2002). Molecular characterization of SPM-1, a novel metallo-beta-lactamase isolated in Latin America: report from the SENTRY antimicrobial surveillance programme. Journal of Antimicrobial Chemotherapy 50, 6739. 8. National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Susceptibility TestingTwelfth Informational Supplement M100-S12. NCCLS, Wayne, PA, USA. 9. Arakawa, Y., Shibata, N., Shibayama, K. et al. (2000). Convenient test for screening metallo-beta-lactamase-producing gram-negative bacteria by using thiol compounds. Journal of Clinical Microbiology 38, 403. 10. Bertrand, X., Thouverez, M., Talon, D. et al. (2001). Endemicity, molecular diversity and colonisation routes of Pseudomonas aeruginosa in intensive care units. Intensive Care Medicine 27, 12638.

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