First report of a Tn 402-like class 1 integron carrying bla VIM-2 in Pseudomonas putida from Argentina

1 Departamento de Microbiología, Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2 Departamento de Química Biológica, Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 3 Servicio de Bacteriología, Hospital Intendente Carrasco, Secretaría de Salud Pública, Municipalidad de Rosario, 4 Servicio de Bacteriología, Hospital Provincial de Rosario, 2000 Rosario, Argentina

Pseudomonas putida colonizes several niches including soil, fresh water, and animal surfaces.Although rarely isolated from human infections, metallo-beta-lactamase (MBL)-containing P. putida clinical strains resistant to most beta-lactams) have recently been described and proposed to act as likely reservoirs of MBL genes [1,2].This, in addition to the lack of an effective clinical inhibitor of these metallo-enzymes, poses a serious challenge to antimicrobial therapy [3].bla VIM-2 represents the most widely distributed MBL gene worldwide, and is most generally present in class 1 integrons [4,5, Pasteran F et al. (2005) 45th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) Abstract C2 108/151].These elements may be contained within transposons, and the whole arrangement subjected in turn to rapid spread among different bacterial species by the aid of wide-range plasmids [5].It follows that the elucidation of the genetic platform(s) in which bla VIM-2 genes are contained enables us to rationally speculate on how they evolve and disseminate.We report here the identification for the first time of an unusual class 1 integron carrying bla VIM-2 and aacA4 aminoglycoside acetyl transferase gene cassettes embedded in a complete Tn402-like transposon, all elements carried in turn by a self-transferable plasmid present in a carbapenem-resistant P. putida clinical strain.
MBL activity in LD209 bacterial extracts was revealed by the EDTA-imipenem microbiological assay (EIM) [6].Identification of bla VIM , bla IMP , and bla SPM by PCR with specific primers (Table 1) followed by sequencing revealed only bla VIM-2 .Further characterization of the genomic context of bla VIM-2 using primers designed after the 5' and 3' conserved segments of class 1 integrons (5'-CS and 3'-CS, respectively; Table 1) systematically failed to produce amplification bands.Conversely, the use of 5'-CS (forward) and TniC-R (reverse) primers (Table 1) followed by sequencing analyses showed that bla VIM-2 is present in an unusual class 1 integron which lacks the 3'-CS region (Figure 1A).As shown in the figure, bla VIM-2 is located immediately adjacent to the 5'-CS region of the integron followed by an aacA4 gene encoding an AAC(6')-Ib type aminoglycoside acetyl transferase [7], preceding in turn the 3´ region of a tniC gene encoding the Tn5090 resolvase [5] (Figure 1A).It is worth noting that a class 1 integron almost identical to that reported here (In71, Figure 1) was detected in a chromosomal location of clonally related carbapenem-resistant P. aeruginosa strains responsible for an outbreak occurring in 2000 in Trieste, Italy [8].
All class 1 integrons have been proposed to have originated from multiple excision/rearrangements of a transposon derived from Tn402/Tn5090 originally containing a complete tni module [5,7,9].Characterization of the immediate genomic context of the unusual class 1 integron described here in P. putida by PCR using appropriate primer combinations (Figure 1B and Table 1) followed by sequencing and database searching analyses revealed the presence of a complete tni module displaying 99% identity at the nucleotide level (between 99-100% identity at the level of the corresponding encoded proteins) with the transposase (tniA), transposition auxiliary proteins (tniB, tniQ) and resolvase (tniC) genes of Klebsiella aerogenes Tn402 (Figures 1, C and D).As seen in Figure 1A, the genes composing this tni module are located downstream of the aacA4 gene cassette and transcribed in the opposite direction from the resistance genes.
The finding of a Tn402-like class 1 integron containing bla VIM-2 and aacA4 gene cassettes accompanied by a complete tni module constitutes to our knowledge the first report of such an arrangement in P. putida.A Tn402-like class 1 integron carrying aacA4 and bla VIM-2 gene cassettes as well as a complete tni module has been described recently in P. putida clinical strains isolated in the Balearic Islands, Spain [10].However, several differences exist between this element and the one described here, including an inverted order of bla VIM-2 and aacA4 genes, significant sequence divergence of the corresponding tni modules (only ~68% identical at the nucleotide level between the tni region shown in Figure 1 and the equivalent region deposited at GenBank GQ227991) [10], and the absence of a specific polymorphism (Ala108Thr) in the AacA4 enzyme present in LD209 which is characteristic of the equivalent enzymes produced by P. putida strains isolated in Spain.Also, an unusual class 1 integron carrying bla VIM-2 immediately downstream of the 5'CS has been recently reported in a P. putida clinical strain isolated in Portugal [11].In this case, however, the bla VIM-2 gene was followed by an unknown open reading frame rather which was in turn followed by the 3' region of tniC.
We next analyzed whether the above Tn402 derivative was plasmid-borne.LD209 cells were subjected to alkaline lysis and the obtained DNA was transformed into electrocompetent Escherichia coli (E.coli) DH5 cells [12] followed by a selection of resistant colonies in LB agar containing 16 g/ml ampicillin.Analysis of plasmid content by restriction enzyme digestion (EcoRI and HindIII) and agarose gel electrophoresis indicated the same plasmid in all cases, which was designated as pLD20.MBL production by E. coli harbouring pLD20 was confirmed by a microbiological assay [6].Also, a three-fold increment in the MIC values for both imipenem and meropenem was found as compared to non-transformed cells.Concerning expression of the aacA4 gene present in the integron described here, E. coli MIC values for gentamicin and amikacin increased three-and one-fold, respectively, as compared to non-transformed bacteria.Similar  [14].The sequences representing the 5´ CS and the antimicrobial gene cassettes of the integron as well as the Tn402 tni module are indicated below the figure.The IRi, IRt, the attI1 site, and the 59-bp elements are slightly enlarged for clarity, as well as the duplicated GTTTT sequences at the site of insertion in the plasmid.B. PCR-overlapping assay used for the identification and characterization of the different gene and other components of the integron/transposon element described here.The different primers employed and their corresponding target sites are indicated by small arrows (see Table 1 for sequence details).DNA sequences of IRi, IRt, and corresponding outer boundaries were obtained by primer walking starting with primers attI1-R and TniA-F (5'-CGTAAGGCCACGGTATTGCGC-3') as indicated in the lower part of the figure.C. Percent identities at the nucleotide sequence level between the indicated regions and the corresponding GenBank [8,14] sequences.D. Percent identities at the amino acid sequence level between the polypeptides encoded in the indicated regions and the corresponding GenBank sequences.For further details see GenBank accession number GQ857074.
results were observed by other authors using E. coli cells transformed with plasmids directing production of AAC(6`)-Ib 7/8 type enzymes [13].Agar mating studies [12] confirmed the ability of pLD20 to undergo conjugative mobilization from P. putida LD209 (donor) to rifampicin-resistant E. coli DH5 .Moreover, agar mating analyses [12] indicated that pLD20 can mobilize from E. coli DH5 (donor) to P. aeruginosa PAO1 or E. coli MC4100 (recipients).Thus pLD20 constitutes a self-mobilisable plasmid capable of spreading the bla VIM-2 containing the Tn402-like class 1 integron described in this work among a wide range of bacterial species.
The plasmid location of the above Tn402-like derivative allowed us to accurately characterize the transposon outer boundaries.Sequencing analyses (Figure 1) revealed the presence of the 25 bp IRi and IRt inverted repeats and all associated 19 bp adjoining repeats characteristic of the Tn402 transposon family [14].Moreover, the two GTTTT direct repeats bracketing the IRs (Figure 1) strongly indicate small target duplications associated with a recent transposition event.In toto, all the above features indicate that P. putida LD209 carries a selftransferable, wide-range plasmid containing a functional Tn402 transposon carrying a class 1 integron bearing bla VIM-2 and aacA4 gene cassettes.
It remains to be tested whether bla VIM-2 genes captured by highly mobile genetic platforms such as those identified here in a likely reservoir of antimicrobial resistance genes such as P. putida [1,10] could account for the high levels of incidence of this MBL in P. aeruginosa both in our geographic region [4, Pasteran F et al. (2005) 45th ICAAC Abstract C2 108/151] and other areas worldwide [2,4,5,8,[10][11][12].

Figure 1 .
Figure 1.Schematic representation of the P. putida plasmid-borne Tn402 derivative described in this work.

Table 1 .
PCR primers used in this study.
Y: C, T; M: A, C; H: A, T, C a Primer TniC-R is identical to TniCF of ref.5.