Characterization of Tn6238 with a new allele of aac(6')-Ib-cr

Abstract

Here, we report that the genetic structure of Tn1331 remained conserved in Argentina from 1989 to 2013 (72 of 73 isolates), with the exception being the plasmid-borne Tn1331-like transposon Tn6238 containing a new aac(6')-Ib-cr allele recovered from a colistin-resistant Klebsiella pneumoniae clinical isolate. A bioinformatic analysis of aac(6')-Ib-like gene cassettes suggests that this new aac(6')-Ib-cr allele emerged through mutation or homologous recombination in the Tn1331 genetic platform. Tn6238 is a novel platform for the dissemination of aminoglycoside and fluoroquinolone resistance determinants.

FIG 1

Structure of Tn6238 (GenBank accession no. KJ511462). The transposon number was assigned by the Tn number registry Web page (http://www.ucl.ac.uk/eastman/research/departments/microbial-diseases/tn). In the central diagram, the horizontal bars represent inverted repeats, the arrows represent ORFs, the white bars represent attI1*, and the gray arrows and ovals represent the ORFs and attC sites of the gene cassettes, respectively. The top strands of the 11 different attC sites found in the aac(6′)-Ib-cr and aac(6′)-Ib gene cassettes are shown above the Tn6238 structure. The dots indicate identities and the dashes indicate insertions. The 1R, 2R, 1L, and 2L sites are marked with horizontal bars, and the extrahelical bases are marked with an asterisk. The most important mutations related to their potential impact on the attC functionality are underlined. The ORFs of the aac(6′)-Ib-cr alleles are shown below the Tn6238 structure. They are compared with the AAC(6′)-Ib protein encoded by aac(6′)-Ib1 [aac(6′)-Ib allele of the reference, GenBank accession no. U59183.1, bases 301 to 859]. The dots indicate identities. The numbers between dashes in the aac(6′)-Ib1 ORF indicate the number of bases not shown. The codons with mutations are underlined, and above them are shown the codified amino acids, numbered from the GTG start codon. The aac(6′)-Ib2 allele found in Tn1331 is shown (GenBank accession no. AF479774.1). The key mutations that make the protein capable of modifying quinolones are highlighted with gray boxes. All the aac(6′)-Ib-cr variants share the mutation GAT to TAT at position 490 of the aac(6′)-Ib1 ORF (D164Y). Position 259, also responsible for the ciprofloxacin-resistant phenotype, exhibits the mutation TGG to CGG [variant C, called here aac(6′)-Ib-cr1] or to AGG [variant A, called here aac(6′)-Ib-cr2], both generating W87R (13). ^aMost frequent are attC_{aac(6′)-Ib-cr1} and attC_{aac(6′)-Ib1}, which are found in all alleles of the aac(6′)-Ib-cr gene cassettes other than aac(6′)-Ib-cr3 (e.g., GenBank accession no. KJ511462, EF636461.1, and EF415651.2), and they are also found in the prevalent aac(6′)-Ib gene cassette in integrons, the Tn1331, Tn1331.2, and Tn1332 transposons, and all the AAC(6′)-Ib protein variants except 3 and 4 [e.g., GenBank accession no. U59183.1 used as reference of aac(6′)-Ib gene cassette] (14); ^battC_{aac(6′)-Ib-cr2} is linked to aac(6′)-Ib-cr2 (e.g., GenBank accession no. HM998988.1); ^cattC_{aac(6′)-Ib-cr3} is associated with aac(6′)-Ib-cr3, found only in GenBank accession no. HQ170516.1; ^dattC_{aac(6′)-Ib2} is found in Tn1331 of pJHCMW1 (GenBank accession no. AF479774.1); ^eattC_{aac(6′)-Ib3} is found in gene cassettes, with the constant region of the ORF identical to the aac(6′)-Ib of reference, but whose complete ORF encodes variants 4 and 39 of the AAC(6′)-Ib protein (GenBank accession no. AY370764.1 and X60321.1, respectively) (14); ^fattC_{aac(6′)-Ib4} and attC_{aac(6′)-Ib7} are found in gene cassettes in which the complete ORF encodes variants 8 and 14 of the AAC(6′)-Ib protein, respectively (GenBank accession no. DQ767903.1 and GQ293499.1) (14); ^gattC_{aac(6′)-Ib5} is found in the aac(6′)-Ib gene cassette in a Tn1331-like structure in which the complete ORF encodes variant 3 of the AAC(6′)-Ib protein (GenBank accession no. M23634.1) (14); ^hThis is the second attC_aac(6′)-Ib in terms of frequency (e.g., GenBank accession no. AF458080.1); ⁱattC_{aac(6′)-Ib6}, attC_{aac(6′)-Ib7}, attC_{aac(6′)-Ib8}, and attC_{aac(6′)-Ib9} are found in gene cassettes, with the constant region of the ORF identical to the aac(6′)-Ib of reference, in which the complete ORF encodes variants 13, 14, and 17 of AAC(6′)-Ib protein (e.g., GenBank accession no. AF458080.1, AJ313334.1, JF262177.1, and FO203354.1).

FIG 2

Evolutionary scheme of Tn6238. The horizontal bars represent inverted repeats, the arrows represent ORFs, the white bars represent attI1*, and the gray arrows and ovals represent the ORFs and attC sites of gene cassettes, respectively.