Antimicrobial^a susceptibility profiles of recipient strain <i>E. coli</i> and <i>P. aeruginosa</i>.

a<p>PIPC, piperacillin; CAZ, ceftazidime; IPM, imipenem; MEM, meropenem; LVX, levofloxacin; CIP, ciprofloxacin; AMK, amikacin; ABK, arbekacin; DBK, dibekacin; GAN, gentamicin; ISP, isepamicin; KAN, kanamycin; SISO, sisomicin; TOB, tobramycin.</p>b<p>Structure of plasmid KK1 is shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070557#pone-0070557-g002" target="_blank">Fig. 2a</a>.</p

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

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

PCR primers used in this study.

<p>PCR primers used in this study.</p

Schematic representation of a restriction map (a) and PCR scanning analysis (b) of the class 1 integron <i>In</i>124.

<p>The novel ORF, <i>aac(6′)-Iag</i> is shown as a black filled arrow and the truncated <i>aac(6′)-Iag</i> is shown as a gray filled arrow. <i>Orf2</i> located downstream of <i>bla</i>_IMP-1 is a truncated form of <i>bla</i>_IMP-1 with a 5′-terminal 8-bp deletion and a 5′-terminal 62-bp addition. The <i>att</i>Il 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 <i>In</i>124.</p

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

<p>(a) Genomic DNAs of <i>P. aeruginosa</i> strains were digested with <i>Spe</i>I and analyzed using PFGE as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070557#s2" target="_blank">Materials and Methods</a>. <i>P. aeruginosa</i> 060123 was used as a positive control as a <i>bla</i>_IMP-1 carrier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070557#pone.0070557-Kouda1" target="_blank">[2]</a>. (b) Southern blot hybridization using a <i>bla</i>_IMP-1 probe of the genomic DNAs.</p

Structure of AGs.

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

Alignment of 59-base elements (a) and nucleotide sequences of the 367-bp fragment (position 869 to 1,235) (b) of <i>In</i>124.

<p>(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 <i>aac(6′)-Iag</i>, <i>bla</i>_IMP-1, <i>Δbla</i>_IMP-1 and <i>Δaac(6′)-Iag</i> found two potential −35 and −10 promoters: P1 promoter (<u>TGGACA</u>…N17…<u>TAAGCT</u>) and P2 promoter (<u>TTGTTA</u>…N14…<u>TACAGT</u>) are present between 899 to 927 and 1,018 to 1,043, respectively. Finally, the putative promoter of <i>aac(6′)-Iag</i> is thought of as the P1. The proposed ribosome-binding site of <i>aac(6′)-Iag</i> is double underlined and is located 3-bp upstream of the start codon of <i>aac(6′)-Iag</i>. The 7-bp core site is wavy lined.</p

Fragment ions of protonated molecules in ESI-MS/MS analysis of aminoglycosides.

<p>P^*: Product ions produced by the ESI were selected to follow ESI-MS/MS analysis as precursor ions.</p><p>n.d.: not detected due to low signal.</p><p>-: not determined due to out of measurement ranges.</p

Kinetic parameters for aminoglycosides.

<p>Each value is mean ± SD of three experiments.</p