1 Laboratório Alerta; 2 Laboratório Especial de Microbiologia Clínica, Division of Infectious Diseases, Universidade Federal de São Paulo, Rua Leandro Duprét, 188, São Paulo, SP 04025010, Brazil
Received 3 April 2003; returned 28 May 2003, revised 14 July 2003; accepted 15 July 2003
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Abstract |
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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-ß-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
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Introduction |
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In the Hospital São 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- ß-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 São Paulo complex.7 Strain 481997A belongs to clone SP. The main objective of this 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.
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Material and methods |
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The Brazilian hospitals were asked to submit only carbapenem-resistant 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 Laboratório Especial de Microbiologia Clínica (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.
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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.
Molecular methods
Confirmed carbapenem-resistant isolates were genotyped by ribotyping, following the instructions of the manufacturer of the RiboPrinterMicrobial 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-1-producing P. aeruginosa strain (481997A) was included as a control strain.
Metallo-ß-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 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 40°C for 1 min and extension at 68°C for 1 min.9 PCR products were visualized by electrophoresis on 0.8% agarose gels stained with 1% ethidium bromide.
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Results |
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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.
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Discussion |
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The existence of common PFGE types among carbapenem-resistant 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.
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Acknowledgements |
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This study was financed by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, No. 2001/033497).
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Footnotes |
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References |
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