Novel insights about class 2 integrons from experimental and genomic epidemiology

Abstract

In order to contribute to the knowledge of the architecture and epidemiology of class 2 integrons, we performed a class 2 integron molecular survey in which we analyzed 726 isolates in two bacterial populations from environmental and nonepidemiologically related clinical samples, respectively, collected from 1982 to 2007. We recovered the intI2 gene from 130 of 726 isolates, most of which were clinical isolates, and only 1 (a psychrophilic Pseudomonas sp.) was from a water sample. Unlike the widespread distribution of class 1 integrons within Gram-negative bacilli, only Acinetobacter baumannii and Enterobacter cloacae harbored class 2 integrons at a high frequency in our collection. Class 2 integrons with six novel cassette arrays were documented. Characterization of the transposition module of Tn7, the genetic platform in which class 2 integrons have always been reported, showed tns modules with a mosaic genetic structure. A bioinformatic analysis performed with the tns genes present in sequence databases, the finding of intI2 not associated with tns genes, and the genetic examination of novel tns-like genes found in three isolates indicated the possibility of the independent evolution of the two components related to horizontal gene transfer, the class 2 integrons and the Tn7 transposons.

FIG. 1.

Schematic representation of class 2 integrons. (a) Variable region of the class 2 integrons described in the GenBank database with the corresponding accession number and/or the given name. The thin vertical closed bar represents the attI2 site, and the ovals represent the attC sites of the gene cassettes. Both recently described alleles of a functional intI2 gene correspond to the sequences with GenBank accession numbers DQ533990 and EU780012. The rest of the intI2 genes exhibited a stop codon of 179 amino acids. The graphic is not to scale. (b) Description of the PCR cartography of class 2 integrons used in this study, showing as an example the organization of the class 2 integron Tn7::In2-8. The genetic localization of each primer is indicated by an arrow. The numbers in parentheses correspond to the expected sizes of the products amplified by PCR. In order to search for hybrid genetic structures between the class 1 and 2 integrons described previously (34), the sequences were also investigated for both of the 3′ ends usually found for class 1 integrons (the 3′ conserved segment and tniC). The primers are indicated by arrows. Dashed lines represent the expected amplification product. The thin vertical open bar represents the attI2 site, and the ovals represent the attC sites of the gene cassettes. ND, not positive for amplification; there is not a previous report of this positive amplification for this pair of primers.

FIG. 2.

Schematic representation of arrays of class 2 integrons found among the bacterial population (n = 126) analyzed in the present survey. Integrons In2-0, In2-2, In2-3, In2-5, Tn7::IS1, and In2-6::IS26 were described for the first time in this study. The thin vertical closed bar represents the attI2 site, and the ovals represent the attC sites of the gene cassettes. All intI2 genes from these arrays were sequenced, and they revealed the usual internal stop codon.

FIG. 3.

Bioinformatic analysis of the Tn7 and Tn7-like elements showing the genetic architecture found in diverse bacterial species. The Tn7 genes are represented in different colors. The different patterns of filling indicate the percent amino acid identity of the proteins codified by the Tn7-like elements found in the chromosomes of the listed species compared to the Tn7 sequence with GenBank accession number NC_002525. Vertical lines indicate less than 30% amino acid identity, diagonal lines denote less than 50% identity, and horizontal lines indicate less than 70% identity. The open reading frames are arbitrarily named orf simply to indicate the genes for hypothetical proteins. Integrases, represented by Int, not related to the integron integrase gene are indicated in reddish orange. The transposases of the IS3/IS911 family are represented as Tnp and are orange. Genes related to the integron integrase were not found in the flanking sequences (boundaries of 10 kb each), except for the class 2 integron from Shigella sonnei Ss046. A search for genes related to the glmS gene in the 10-kb flanking sequences was also performed. Percent amino acid identities and similarities were calculated by use of the same criteria used by the BLAST subroutine (NCBI database). The figure is not to scale; small arrows are used to indicate significant deletions in some of the Tns proteins.