Molecular Analysis of Metallo-β-Lactamase Gene bla _SPM-1 -Surrounding Sequences from Disseminated Pseudomonas aeruginosa Isolates in Recife, Brazil

Abstract

The spread of clonally related carbapenem-resistant Pseudomonas aeruginosa producing the metallo-␤-lactamase SPM-1 was found in Recife, Brazil. Upstream of bla SPM-1 , a novel common region (CR4) was identified, comprising an open reading frame, orf495, whose product shares significant identity with putative recombinases, such as Orf513. CR4 may be responsible for mobilization and expression of bla SPM-1 .During the last decade, several acquired carbapenem-hydrolyzing enzymes have been identified in Pseudomonas aeruginosa, beginning with IMP-1 and derivatives, which are widespread in Japan (24, 25) and China (4) and are emerging in Europe and Canada (8,13). Then, VIM-1 and VIM-2 enzymes were characterized, first in Italy and France (11,22), respectively, but these enzymes are also highly prevalent in Korea and Greece (7,14,29). Recently a third member of acquired Ambler class B ␤-lactamases, SPM-1, has been identified from a P. aeruginosa isolate in Sao Paulo, Brazil (28).The aim of this study was to analyze carbapenem-resistant P. aeruginosa isolates recovered from different hospitals in Recife, Brazil, from November 2002 to January 2003. Nineteen P. aeruginosa strains resistant to ceftazidime and imipenem were isolated at the Marcelo Magalhaes clinical laboratory in Recife. Ten isolates were from patients of neurological wards and intensive care units of the Hospital Portugues, Recife, whereas nine strains were from patients hospitalized in similar units from several hospitals in Recife. Eleven imipenem-resistant isolates were identified as metallo-␤-lactamase producers by using the double disk-synergy test with EDTA (30), and these were retained for further genetical analysis. They were resistant to all ␤-lactams except aztreonam. Isoelectric focusing analysis performed as described previously (20) showed that the 11 isolates expressed 3 ␤-lactamases, with pI values of 6.5, 7.5, and 8.7, the last likely corresponding to the naturally occurring AmpC enzyme of P. aeruginosa. Under standard PCR conditions (20), a series of primers was used for detection of genes encoding Ambler class B ␤-lactamases of the VIM, IMP, or SPM type as described previously (21). Positive results were obtained using the bla SPM-1 -specific primers (SPM-1A, 5Ј-CTGCTTGGATTCATGGGCGC-3Ј; SPM-1B, 5Ј-CCTTT TCCGCGACCTTGATC-3Ј). The 11 isolates had the same bla SPM-1 gene previously identified in P. aeruginosa (28).A genotypic comparison of the bla SPM-1 -positive P. aeruginosa isolates performed by pulsed-field gel electrophoresis, using restriction enzyme SpeI, revealed that these isolates were clonally related (data not shown) (18, 27). Electroporation of putative plasmid DNA extracts from bla SPM-1 -positive P. aeruginosa isolates performed as described previously (19) failed. Using pulsed-field gel electrophoresis and I-CeuI digestion followed by hybridization with PCR-generated probes as previously reported (9), a strong signal (located at ca. 400 kb) was obtained with the bla SPM-1 -specific probe for SPM-1-producing P. aeruginosa that ...

Carbapenem-resistant Pseudomonas aeruginosa: clonal spread in southern Brazil and in the state of Goiás

“…[1][2][3]7 Toleman et al (2002) characterized the SPM-1 gene, and Poirel et al (2004) subsequently characterized a common region called CR4 as the genetic element responsible for expression and mobilization of this gene. The latter, however, failed to transfer the putative plasmid DNA by electroporation, 3,8 suggesting that the SPM-1 gene is different from the IMP and VIM genes in terms of its ability to be transmitted among microorganisms. Our data corroborate the fi nding of SPM-1 only in clonal isolates of P. aeruginosa, suggesting that bla SPM-1 , which is the predominant gene in Brazil, is not as mobile as the bla VIM or bla IMP genes.…”

“…Clonal dissemination of carbapenem-resistant P. aeruginosa was also reported in subsequent studies that included samples from other parts of the country. [2][3][4] Clinical isolates of carbapenem-resistant P. aeruginosa were fi rst detected in June 2003 at the teaching hospital of the University of Santa Catarina (UFSC), Florianópolis, Santa Catarina, Brazil. The increasing frequency strains with this resistance profi le have been isolated prompted us to perform this work, which aiming at evaluating the possible clonal relationship between 29 carbapenem-resistant P. aeruginosa isolates from inpatients in this hospital using macrorestriction analysis by pulsed fi eld gel electrophoresis -PFGE [SpeI (10U), CHEF -DRIII (Bio-rad Laboratories, USA)].…”

Caracterização molecular da resistência aos carbapenêmicos em enterobactérias isoladas em hospitais brasileiros

“…aeruginosa, uma vez que essa enzima tem sido detectada apenas nesta espécie, provocando alta mortalidade entre os pacientes hospitalizados no Brasil (Poirel et al, 2004b, Zavascki, et al, 2005. Análises genéticas das regiões próximas do gene bla SPM-1 revelaram que esta enzima não é parte de um integron, porém foi localizada em um plasmídio, associada a regiões comuns que contém um tipo de estrutura transponível, com uma recombinase em potencial e seqüências promotoras (Poirel, et al, 2004b).…”

“…Análises genéticas das regiões próximas do gene bla SPM-1 revelaram que esta enzima não é parte de um integron, porém foi localizada em um plasmídio, associada a regiões comuns que contém um tipo de estrutura transponível, com uma recombinase em potencial e seqüências promotoras (Poirel, et al, 2004b).…”

Alternativas terapêuticas para o tratamento de infecções por Pseudomonas aeruginosa multirresistentes endêmicas no Brasil.

“…Até o presente período, SPM-1 só foi encontrada em P. aeruginosa e sua disseminação epidêmica já foi evidenciada em vários estados brasileiros (Gales et al, 2003a;Poirel et al, 2004;Zavascki et al, 2005). Salabi et al (2010) publicou o primeiro caso de P. aeruginosa produtora de SPM-1 na Europa, de um paciente suíço que recebeu o primeiro atendimento em um hospital em Recife.…”