Article Figures & Data
Figures
- Fig. 1.
Comparative structures of the class 1 integrons In58 and In59 that contain the blaVIM-2 gene cassette from P. aeruginosa RON-1 and RON-2 clinical isolates, respectively. The intI1 integrase gene, which encodes the integrase, is contained in the 5′-CS, and the 3′-CS found downstream of the integrated gene cassette includes the sulfonamide resistance genesul1 and the disinfectant resistance determinantqacEΔ1. Inserted genes are indicated by boxes, and the arrows indicate their transcriptional orientation. The 59-be's are represented by black circles and the attI1 recombination sites by white circles.
- Fig. 2.
Nucleotide sequence of a 5,061-bp BamHI fragment of pNOR-2003 containing the VIM-2 coding sequence and part of integron In59. The start codons of the ORFs are indicated by horizontal arrows, and the deduced amino acid sequences are reported below the nucleotide sequence. Stop codons for each ORF are indicated by asterisks. Dashes in the nucleotide sequence indicate where the reported sequence was identical to published sequences. The −35 and −10 sequences of promoters Pc and putativeqacE/qacEΔ1 are indicated. The conserved core and inverse core sites located at each cassette boundary are boxed, and the composite 59-be's are italicized. The cassette boundaries are indicated by vertical arrows as well as the putative fusion points of the 5′ end of part of the qacE cassette to theaacA29 gene cassettes. The attI1 site is underlined with a dashed line.
- Fig. 3.
Comparisons of the deduced amino acid sequences of AAC(6′)-29a and AAC(6′)-29b proteins with those of the most closely related aminoglycoside acetyltransferases. Amino acid differences between AAC(6′)-29a and AAC(6′)-29b appear in grey. Identical amino acids in at least 17 sequences are indicated by asterisks; conserved amino acid substitutions are indicated by dots according to the following exchange groups: A, G, P, S, and T; H, K, and R; F, W, and Y; D, E, N, and Q; and I, L, M, and V. Boxed motifs at the carboxy terminal end of the proteins are conserved in most of the enzymes and are absent in AAC(6′)-29 proteins.
Tables
- Table 1.
Bacterial strains and plasmids
Strain or plasmid Relevant genotype or phenotypea Source or reference Strains E. coliDH10B araD139Δ(ara leu)7697 deoR endA1 galK1 galU nupG recA1 rpsL F′-mcrA Δ(mrr-hsdRMS-mrcBC) φ80lacZΔM15 ΔlacX74 Gibco BRL, Paris, France E. coli JM109 endA1 gyrA96 hsdR17Δ(lac proA) relA recA1 supE44 thi F′ (lacIqlacZΔM15proAB+ traD36) 15 In vitro-obtained rifampin-resistant E. coliJM109 Rifampinr 15 In vitro-obtained rifampin-resistant P. aeruginosa PU21 ilv/leu, streptomycinr, rifampinr 15 P. aeruginosa COL-1 blaVIM-2 expressing clinical isolate 16 P. aeruginosaRON-1 Carbapenem-hydrolyzing clinical isolate This study P. aeruginosa RON-2 Carbapenem-hydrolyzing clinical isolate This study Plasmids pBK-CMV Neomycinr, kanamycinr Stratagene Inc., Ozyme, Amsterdam, The Netherlands pPCRScript-Cam (SK+) Chloramphenicolr Stratagene Inc. pNOR-2002 5,648-bp BamHI-fragment from P. aeruginosa RON-1 DNA in pBK-CMV This study pNOR-2003 5,061-bp BamHI-fragment from P. aeruginosa RON-2 DNA in pBK-CMV This study pLO-1 1,541-bp PCR fragment containing aacA29a from pNOR-2003 in pPCRScript-Cam This study pLO-2 1,655-bp PCR fragment containing aacA29b from pNOR-2003 in pPCRScript-Cam This study ↵a A superscript, “r” indicates resistance.
- Table 2.
MICs of β-lactams for VIM-2-possessing P. aeruginosa clinical strains, E. coli DH10B harboring recombinant plasmids pNOR-2002 and pNOR-2003, and reference strainE. coli DH10B
β-Lactam (s)a MIC (μg/ml) P. aeruginosa RON-1 P. aeruginosaRON-2 P. aeruginosa COL-1b E. coli DH10B (pNOR-2002 or pNOR-2003)c E. coli DH10B Amoxicillin >512 >512 >512 >512 4 Amoxicillin + CLA >512 >512 >512 >512 4 Ticarcillin 512 >512 >512 >512 4 Ticarcillin + CLA >512 >512 >512 >512 4 Piperacillin 64 32 64 4 1 Piperacillin + TZB 128 64 16 4 1 Cephalothin >512 >512 >512 128 2 Cefoxitin >512 >512 >512 64 1 Ceftazidime 64 128 256 16 0.5 Cefotaxime 256 256 >512 8 0.06 Cefepime 64 32 64 0.25 0.03 Cefsulodin 512 512 512 512 0.25 Aztreonam 2 2 0.25 0.12 0.12 Imipenem 32 64 128 2 0.12 Meropenem 4 8 128 1 0.06 ↵a CLA, clavulanic acid at a fixed concentration of 2 μg/ml; TZB, tazobactam at a fixed concentration of 4 μg/ml.
↵b MICs of β-lactams for P. aeruginosa COL-1 have been reported previously (16).
↵c Recombinant plasmids pNOR-2002 and pNOR-2003 possess the blaVIM-2 gene from P. aeruginosa RON-1 and RON-2, respectively.
- Table 3.
MICs of various aminoglycosides for P. aeruginosa RON-2, E. coli JM109 harboring recombinant plasmids pLO-1 and pLO-2 containing aacA29a andaacA29b genes, respectively, and reference strain E. coli JM109
Strain MIC (μg/ml)a AMK DIB GEN ISE NET TOB KAN SIS P. aeruginosaRON-2 256 256 4 256 8 256 >256 16 E. coli JM109 (pLO-1) 2 2 <0.12 1 <0.12 8 32 0.25 E. coli JM109 (pLO-2) 4 4 <0.12 1 <0.12 4 16 0.25 E. coli JM109 (pNOR-2003) 32 32 <0.12 16 0.25 16 >256 1 E. coliJM109 <0.12 0.25 <0.12 <0.12 <0.12 <0.12 0.5 <0.12 ↵a AMK, amikacin; DIB, dibekacin; GEN, gentamicin; ISE, isepamicin; NET, netilmicin; TOB, tobramycin; KAN, kanamycin; SIS, sisomicin.
