ISCR2, Another Vehicle for bla_VEB Gene Acquisition

Abstract

The expanded-spectrum β-lactamase (ESBL) gene bla_VEB-1, identified worldwide in Enterobacteriaceae and Pseudomonas aeruginosa, is associated with either class 1 integrons or repeated elements. We report here the first association of bla_VEB-1a with the insertion sequence ISCR2 in six Acinetobacter species isolates recovered from Argentina. That genetic structure was likely at the origin of the mobilization of this ESBL gene.

Among the different Ambler class A expanded-spectrum β-lactamase (ESBL) genes, the bla_VEB-1 gene is considered to be an emerging one since its presence has been detected in many gram-negative organisms from different parts of the world during the last decade (14, 25). It has been identified in Enterobacteriaceae and Pseudomonas aeruginosa in many countries, such as France, Spain, Algeria, Turkey, Canada, Korea, and Thailand (1, 2, 8, 23, 29). In addition, P. aeruginosa isolates producing the VEB-1a variant (differing from VEB-1 by just a single amino acid located in the leader peptide of the premature protein) have been found in Kuwait and India (3, 24). VEB-1-producing Acinetobacter baumannii strains have been identified in France, having disseminated on a nationwide scale (13, 20).

The bla_VEB-1 gene is often part of a gene cassette located in class 1 integrons. In A. baumannii strain AYE from France, the corresponding class 1 integron was itself part of an 86-kb resistance island, the largest identified so far (7). However, in some cases, the bla_VEB-1 and bla_VEB-1a genes have not been identified in the form of gene cassettes, being associated with peculiar genetic structures called repeated elements (Re) in P. aeruginosa and Enterobacteriaceae (3, 12, 22).

From 2002 to 2004, several Acinetobacter isolates producing the ESBL VEB-1a were recovered from different cities in Argentina (16). Six VEB-1a-positive isolates were retained: four A. baumannii isolates, one Acinetobacter johnsonii isolate, and one Acinetobacter genomospecies 10 isolate (17). Five of them were shown previously to produce the narrow-spectrum β-lactamase SCO-1 (17). All six isolates are resistant to all β-lactams except carbapenems, with the exception of Acinetobacter genomospecies 10, which coproduces the carbapenem-hydrolyzing oxacillinase OXA-58 and is resistant to imipenem (17). Preliminary experiments performed by PCR mapping using corresponding primers (3) showed that the bla_VEB-1a gene is neither part of a class 1 integron nor associated with Re elements in those isolates. Accordingly, we have examined the genetic context of the bla_VEB-1a gene in those six isolates in order to predict its acquisition mechanism.

First, cloning experiments were performed using DNA from a whole-cell preparation of one of the isolates, A. johnsonii isolate 7037, and pBK-CMV as a cloning vector as described previously (17). BamHI-restricted DNA fragments of A. johnsonii 7037 were ligated into a BamHI-digested plasmid and transferred into Escherichia coli DH10B by electroporation as described previously (18). Selection of recombinant clones was performed using Trypticase soy agar plates containing 100 μg of amoxicillin (amoxicilline) and 30 μg of kanamycin per ml. Recombinant plasmid p7037B1, expressing an ESBL phenotype, was retained for further analysis. Sequencing of the 3,630-bp insert in p7037B1 identified the bla_VEB-1a gene (Fig. 1). Upstream of bla_VEB-1a, sequences corresponding to insertion sequence ISCR2 (formerly designated the CR2 element) were identified. In fact, the ISCR2 right-end extremity defined as oriIS was located 223 bp upstream of bla_VEB-1a, those 223 bp including the GTTAGCG sequence usually defining the core site of the bla_VEB-1a gene cassette when bla_VEB-1a is present in class 1 integrons. Downstream of bla_VEB-1a, a truncated copy of ISCR2 (ΔISCR2) was present, the corresponding tnpA transposase gene being truncated at its 5′ extremity, resulting in a protein lacking 119 of its 497 amino acids at the N-terminal extremity. ISCR2 belongs to the ISCR family, which currently includes 20 members, all presenting some similarities to IS91- like insertion sequences (http://www.cardiff.ac.uk/medic/aboutus/departments/medicalmicrobiology/genetics/iscr/iscr_elements.html). The ISCR2 transposase shares less than 57% amino acid identity with all other ISCR transposases. ISCRs are peculiar since they do not possess inverted repeats, do not generate target site duplications upon transposition, and transpose through a process called rolling-circle transposition (28, 29).

FIG. 1.

Nucleotide sequence of a 3,630-bp BamHI fragment of recombinant plasmid p7037B1 containing the bla_VEB-1a gene. The deduced amino acid sequence is designated in single-letter code below the nucleotide sequence. The transcription orientations of the ORFs are indicated by horizontal arrows (orf is the unknown gene, and xre is the gene encoding a putative transcriptional regulator). The right-end boundary of ISCR2, defined as oriIS, is shaded. The putative promoter sequences of the bla_VEB-1a gene, consisting of the −35 and −10 regions, are boxed. The ΔtnpA truncated transposase gene of ISCR2 is indicated. The stars indicate stop codons. The vertical arrows indicate the boundaries of the bla_VEB-1a gene cassette as found in class 1 integrons (with the core site sequence in bold) and also the location where ΔISCR2 is identified. The BamHI sites of the cloned fragment are underlined.

The downstream 1,170-bp fragment separating the bla_VEB-1a gene from the ΔISCR2 element contained two open reading frames (ORFs), in addition to the entire 59-bp sequence defining the bla_VEB-1a gene cassette when bla_VEB-1a is integron borne. One ORF corresponded to a 92-amino-acid putative protein of unknown function, and the second corresponded to a 103-amino-acid protein showing homology to helix-turn-helix XRE family transcriptional regulators (Fig. 1). The latter protein shared 84% amino acid identity with a protein identified by analysis of the genome of Psychroflexus torquis ATCC 700755 (GenBank accession number ZP_01253739).

We subsequently investigated the five other isolates of our collection for the ISCR2-bla_VEB-1a association. PCR assays performed using bla_VEB-1-specific primers (27) in combination with ISCR2-specific primer ISCR2A (5′-AAGAATTTCTCCAATGCGGG-3′) or ISCR2B (5′-GCGGCTCCTTTTCCGACAAC-3′) showed that in all isolates the bla_VEB-1a gene was bracketed by the ISCR2 elements, as found in isolate 7037. Attempts to identify plasmids by using the Kieser technique (10) permitted visualization of several plasmids in all the bla_VEB-1a-positive isolates. However, subsequent Southern hybridization performed with a probe specific for bla_VEB-1a indicated that this ESBL gene was very likely chromosomally located in all the isolates tested since a single hybridization signal corresponding only to the chromosomal band was obtained (data not shown). This result is in accordance with the results of electrotransformation assays, performed as described previously (17), that did not allow the transfer of any β-lactam resistance marker into an A. baumannii recipient strain.

The finding of two copies of the ISCR2 element (including one truncated copy) at the extremities of bla_VEB-1a strongly suggests that ISCR2 was at the origin of the gene's mobilization. According to the hypotheses raised by Toleman et al. (28), it is very likely that an intact ISCR2 copy originally mobilized bla_VEB-1a by a rolling-circle transposition process and that a secondary process of homologous recombination between two ISCR2 copies led to the observed structure. To confirm the hypothesis that such recombination events may occur, we performed a PCR assay using bla_VEB-1a-specific outward primers VEB-inv1 (5′-CAGTTTGAGCATTTGAATACAC-3′) and VEB-inv2 (5′-AGCGTATTTGTTGCAGAGTCC-3′). Using DNA samples from all VEB-1a-positive isolates as templates, a ca. 2,900-bp amplicon from each isolate was obtained. Sequencing identified a structure encompassing the bla_VEB-1a gene and the corresponding downstream sequences, together with the downstream ΔISCR2 truncated element. However, the sequence of the upstream, intact ISCR2 copy was not included in that amplicon. Therefore, homologous recombination may have been at the source of the mobilization of the bla_VEB-1a gene in its present genetic context. Further studies will be conducted to explore the possible role of the ISCR2 transposase in that recombination process, with regard to the fact that the ISCR transposases were initially thought to be recombinases (15).

The genetic structures involved in the mobilization process for resistance genes often play an additional role in the genes’ expression by providing promoter sequences, as demonstrated previously for many IS elements and class 1 integrons (11, 21) and in particular for ISCR1 and ISCR4 (19, 27). In the ISCR2-bla_VEB-1a configuration in evidence here, no obvious feature indicates a possible role of ISCR2 in bla_VEB-1a expression. Indeed, the distance separating the oriIS extremity of ISCR2 from the bla_VEB-1a start codon is quite long and no putative promoter is found in silico inside the ISCR2 sequence. In contrast, a precise analysis of the sequences separating ISCR2 from the bla_VEB-1a gene revealed putative promoter sequences made of a −35 motif (TTGTAA) and a −10 motif (TGGTTT) separated by the optimal 17-bp distance (Fig. 1), thus suggesting that bla_VEB-1a gene expression is driven by its original promoter in those Acinetobacter isolates.

This study demonstrates further that bla_VEB-like gene acquisition may be linked to a variety of genetic elements (Fig. 2). This is an uncommon observation since the widespread ESBL genes of the bla_TEM, bla_SHV, and bla_CTX-M types are usually associated with very conserved genetic structures (28). In addition, this finding is the first evidence of ISCR2-mediated acquisition of a β-lactamase gene, while ISCR2 has been identified previously in association with the sul2 sulfonamide resistance gene (26), the dfr18 trimethoprim resistance gene (4, 9), and the floR florfenicol resistance gene (5, 6). Interestingly, in the isolates described herein, a fragment larger than the known bla_VEB-1 gene cassette had been mobilized by ISCR2, likely directly from the bacterial progenitor.

FIG. 2.

Comparison of the different genetic structures in which the bla_VEB-1-like genes have been identified. The diagram in panel A corresponds to the present description of an ISCR2-associated bla_VEB-1a gene, that in panel B shows the bla_VEB-1a gene in the form of a gene cassette inside a class 1 integron as reported previously (21), and the diagram in panel C depicts the bla_VEB-1a gene bracketed by the Re1 and Re2 elements as described elsewhere (3, 12). The core and inverse core sites bracketing the bla_VEB-1a gene and defining its corresponding cassette are indicated by black circles.

Finally, the origin of this ISCR2 element remains be determined to better understand where and how the capture of bla_VEB-1a from its natural reservoir has occurred. Although the bla_VEB-1a gene has a GC content of 31.3%, that of ISCR2 is 59.5%, clearly not consistent with the elements’ having the same origin. ISCR2 has been so far identified in diverse bacterial species, including A. baumannii (1).

Nucleotide sequence accession number.

The sequences determined in this study have been assigned GenBank accession number FJ808975.