Identification and characterization of a novel aac(6')-Iag associated with the blaIMP-1-integron in a multidrug-resistant Pseudomonas aeruginosa

Abstract

In a continuing study from Dec 2006 to Apr 2008, we characterized nine multi-drug resistant Pseudomonas aeruginosa strains isolated from four patients in a ward at the Hiroshima University Hospital, Japan. Pulsed-field gel electrophoresis of SpeI-digested genomic DNAs from the isolates suggested the clonal expansion of a single strain; however, only one strain, NK0009, was found to produce metallo-β-lactamase. PCR and subsequent sequencing analysis indicated NK0009 possessed a novel class 1 integron, designated as In124, that carries an array of four gene cassettes: a novel aminoglycoside (AG) resistance gene, aac(6')-Iag, blaIMP-1, a truncated form of blaIMP-1, and a truncated form of aac(6')-Iag. The aac(6')-Iag encoded a 167-amino-acid protein that shows 40% identity with AAC(6')-Iz. Recombinant AAC(6')-Iag protein showed aminoglycoside 6'-N-acetyltransferase activity using thin-layer chromatography (TLC) and MS spectrometric analysis. Escherichia coli carrying aac(6')-Iag showed resistance to amikacin, arbekacin, dibekacin, isepamicin, kanamycin, sisomicin, and tobramycin; but not to gentamicin. A conjugation experiment and subsequent Southern hybridization with the gene probes for blaIMP-1 and aac(6')-Ig strongly suggested In124 is on a conjugal plasmid. Transconjugants acquired resistance to gentamicin and were resistant to virtually all AGs, suggesting that the In124 conjugal plasmid also possesses a gene conferring resistance to gentamicin.

Figure 1. DNA restriction fragment polymorphism (a) and Southern blot hybridization analyses (b) of clinically isolated MDRP (NK0001–NK0009), transconjugant of NK0009 (TC), and PAO1Rp (recipient).

(a) Genomic DNAs of P. aeruginosa strains were digested with SpeI and analyzed using PFGE as described in the Materials and Methods. P. aeruginosa 060123 was used as a positive control as a bla _IMP-1 carrier . (b) Southern blot hybridization using a bla _IMP-1 probe of the genomic DNAs.

Figure 2. Schematic representation of a restriction map (a) and PCR scanning analysis (b) of the class 1 integron In124.

The novel ORF, aac(6′)-Iag is shown as a black filled arrow and the truncated aac(6′)-Iag is shown as a gray filled arrow. Orf2 located downstream of bla _IMP-1 is a truncated form of bla _IMP-1 with a 5′-terminal 8-bp deletion and a 5′-terminal 62-bp addition. The attIl and 59-base elements are shown as a white circle and black circles, respectively. Enzyme restriction sites: B, BamHI; Bg, BglI; E, EcoRV; H, HindIII; P, PstI; Sa, SalI; Sm, SmaI; and Sp, SpeI. PCR scanning analysis with nine primer sets confirmed the gene cassette arrays in In124.

Figure 3. Phylogenetic tree of aminoglycoside 6′-N-acetyltransferase-I compared to AAC(6′)-Iag.

A neighbor-joining tree was constructed using the 121 aac(6′)-I gene products. Each AAC(6′)-I protein is indicated by a lowercase character followed by the EMBL/GenBank/DDBJ accession number. The scale bar represents the amino acid substitutions per site. AAC(6′)-Iag is indicated by an arrowhead.

Figure 4. Alignment of 59-base elements (a) and nucleotide sequences of the 367-bp fragment (position 869 to 1,235) (b) of In124.

(a) Seven-base-pair putative core site in the left-hand (LH) and right-hand (RH) consensus sequences are designated 1L, 2L, 2R, 1R, respectively. The sequences of the seven-base-pair putative core site are boxed and the asterisk indicates the extra base in 2L. (b) The −10 and −35 sequences of the putative integrase promoter are underlined with dotted lines and are also located on the complementary strand (P promoter). Analysis of the adjacent region preceding aac(6′)-Iag, bla _IMP-1, Δbla _IMP-1 and Δaac(6′)-Iag found two potential −35 and −10 promoters: P1 promoter (TGGACA…N17…TAAGCT) and P2 promoter (TTGTTA…N14…TACAGT) are present between 899 to 927 and 1,018 to 1,043, respectively. Finally, the putative promoter of aac(6′)-Iag is thought of as the P1. The proposed ribosome-binding site of aac(6′)-Iag is double underlined and is located 3-bp upstream of the start codon of aac(6′)-Iag. The 7-bp core site is wavy lined.

Figure 5. Thin-layer chromatogram of AGs incubated with AAC(6′)-Iag in the presence (+) or absence (−) of acetyl coenzyme A.

KAN, kanamycin; AMK, amikacin; GEN, gentamicin; TOB, tobramycin; DBK, dibekacin; ABK, arbekacin; SISO, sisomicin; ISP, isepamicin.

Figure 6. Structure of AGs.

Upper figure denotes basic chemical structure of three rings (A, B and C). R1–8 represents substituents listed in table below.