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

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 β-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-β-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 β-lactams except aztreonam. Isoelectric focusing analysis performed as described previously (20) showed that the 11 isolates expressed 3 β-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 β-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, 5′-CTGCTTGGATTCATGGGCGC-3′; SPM-1B, 5′-CCTTTTCCGCGACCTTGATC-3′). 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 did not correspond to one of the bands that hybridized with the 16S ribosomal DNA-specific probe (data not shown). Thus, the bla_SPM-1 gene was likely plasmid located. In addition, Southern hybridization experiments with BamHI-restricted DNA of SPM-1-producing P. aeruginosa isolates gave a unique signal (data not shown), indicating that the bla_SPM-1 gene was present as a single copy in the P. aeruginosa genome.

To characterize the genetic structures surrounding the bla_SPM-1 gene in P. aeruginosa, cloning experiments were performed using whole-cell DNA of a bla_SPM-1-positive P. aeruginosa isolate and pBK-CMV as a cloning vector, as described previously (17). Selection of recombinant clones was performed using trypticase soy agar plates containing 100 μg of amoxicillin and 30 μg of kanamycin per ml, and two phenotypes were obtained.

A recombinant clone containing plasmid pMM-1 expressed a β-lactamase with a pI value of 7.5 and possessed the bla_SPM-1 gene, which conferred resistance to aminopenicillins, ceftazidime, and cefoxitin and reduced suceptibility to imipenem. Another recombinant clone, containing plasmid pMM-2, expressed a restricted-spectrum oxacillinase with a pI value of 6.5, not inhibited by clavulanic acid. Sequencing revealed a novel class D β-lactamase gene, bla_OXA-56, that was part of a bla_OXA-10-like gene cassette structure. OXA-56 had three amino acid substitutions compared to OXA-35 (2) and OXA-7 (23).

Sequencing of the 3,312-bp insert of pMM-1 revealed an open reading frame upstream of bla_SPM-1 encoding 495 amino acids (Fig. 1). The product of orf495 had 81% amino acid identity with Orf2, identified in the multidrug resistance region of an SXT-type conjugative transposon from Vibrio cholerae (10) and considered a recombinase of the so-called CR2 element (16), 58% identity with OrfA, identified in an Escherichia coli strain (5) and part of the so-called CR3 (16), and 54% identity with Orf513, identified upstream of several antibiotic resistance genes, such as bla_CMY-9, bla_CTX-M-2, bla_CTX-M-9, catA2, dfrA10, and part of the so-called CR1 (1, 3, 6, 15). The definition of these conserved regions (CRs) included right-hand ends exhibiting a degree of identity between the CRs (16). Analysis of the nucleotide sequences located between orf495 and bla_SPM-1 revealed a similar 33-bp right-hand boundary, sharing 31, 24, and 24 bp with those of CR3, CR2, and CR1, respectively (Fig. 1). Thus, orf495 may be part of a novel transposable structure called CR4. The putative recombinases of the CRs shared an overall important degree of identity (Fig. 2), whereas their functionality remain to be determined. Partridge and Hall (16) showed that CR1 is able to move by itself by homologous recombination events and that the right-hand end of this structure may be active by incorporating adjacent sequences. Upstream of orf495, the sequence encoding the C-terminal extremity of a putative GroEL chaperonin protein (called GroEL1) was present, with its product sharing 89 and 87% amino acid identity with GroEL proteins from Desulfitobacterium hafniense and Stenotrophomonas maltophilia, respectively, with two hairpin structures likely playing a role in transcription ending (Fig. 1).

Downstream of bla_SPM-1, sequence encoding an N-terminally truncated part of a similar protein (called GroEL2) was present (Fig. 1). PCR experiments using primers designed from the sequence reported by Toleman et al. (28) in combination with SPM-1A followed by sequencing revealed perfect identity with sequences identified in the bla_SPM-1-positive P. aeruginosa strain 48-1997A, starting with a 16-bp-long hairpin structure located just after the putative recombination crossover point (Fig. 1). The C-terminal extremity of the GroEL2-encoding sequence present downstream of bla_SPM-1 was very similar (94% nucleotide identity) to that of GroEL1 (Fig. 1). In addition, the noncoding sequences located downstream of both groEL genes were identical. Surprisingly, the 5′ end of orf495 was identified in the 3′-end extremity of the groEL2 gene in our sequence, as well as in that reported by Toleman et al. (28). Thus, at least part of orf495 is also present on both sides of bla_SPM-1 (Fig. 1).

These data indicated a possible duplication of part of the target genetic structures subsequent to a mobilization process that could result, as suggested (16), from a one-ended mobilization event enhancing the spread of bla_SPM-1.

The genetic structures involved in this mobilization process may also enhance expression of the adjacent antibiotic resistance genes, as demonstrated for insertion sequence elements (12). Indeed, analysis of the 220 bp separating orf495 from bla_SPM-1 revealed putative promoter sequences made of a −35 motif (TTGAAT) provided by the CR4 boundary and a putative original −10 motif (TACAAT) of bla_SPM-1, constituting a hybrid promoter (Fig. 1).

Finally, the origin of this novel element, CR4, has to be determined, since orf495 is not present at least in the P. aeruginosa PAO1 genome (26). Although bla_SPM-1 has a GC content of 48%, that of orf495 is 70%, consistent with a Pseudomonaceae origin.

This study describes a novel genetic element associated with a metallo-β-lactamase gene that may constitute a tool for its dissemination.