Klebsiella pneumoniae strain KG525, which showed high-level resistance to broad-spectrum cephalosporins, was isolated from the neonatal intensive care unit (NICU) of a Japanese hospital in March 2002. The ceftazidime resistance of strain KG525 was transferable to Escherichia coli CSH-2 by conjugation. Cloning and sequence analysis revealed that production of a novel extended-spectrum class A -lactamase (pI 7.0), designated GES-3, which had two amino acid substitutions of M62T and E104K on the basis of the sequence of GES-1, was responsible for resistance in strain KG525 and its transconjugant. The bla GES-3 gene was located as the first gene cassette in a class 1 integron that also contained an aacA1-orfG fused gene cassette and one unique cassette that has not been described in other class 1 integrons and ended with a truncated 3 conserved segment by insertion of IS26. Another five ceftazidime-resistant K. pneumoniae strains, strains KG914, KG1116, KG545, KG502, and KG827, which were isolated from different neonates during a 1-year period in the same NICU where strain KG525 had been isolated, were also positive for GES-type -lactamase genes by PCR. Pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-PCR analyses displayed genetic relatedness among the six K. pneumoniae strains. Southern hybridization analysis with a GES-type -lactamase gene-specific probe showed that the locations of bla GES were multiple and diverse among the six strains. These findings suggest that within the NICU setting genetically related K. pneumoniae strains carrying the bla GES gene were ambushed with genetic rearrangements that caused the multiplication and translocation of the bla GES gene.
The nosocomial spread of six genetically related Klebsiella pneumoniae strains producing GES-type -lactamases was found in a neonatal intensive care unit, and we previously reported that one of the six strains, strain KG525, produced a new -lactamase, GES-3. In the present study, the molecular mechanism of cephamycin resistance observed in strain KG502, one of the six strains described above, was investigated. This strain was found to produce a variant of GES-3, namely, GES-4, which was responsible for resistance to both cephamycins (cefoxitin MIC, >128 g/ml) and -lactamase inhibitors (50% inhibitory concentration of clavulanic acid, 15.2 ؎ 1.7 M). The GES-4 enzyme had a single G170S substitution in the ⍀-loop region compared with the GES-3 sequence. This single amino acid substitution was closely involved with the augmented hydrolysis of cephamycins and carbapenems and the decreased affinities of -lactamase inhibitors to GES-4. A cloning experiment and sequencing analysis revealed that strain KG502 possesses duplicate bla GES-4 genes mediated by two distinct class 1 integrons with similar gene cassette configurations. Moreover, the genetic environments of the bla GES-4 genes found in strain KG502 were almost identical to that of bla GES-3 in strain KG525. From these findings, these two phenotypically different strains were suggested to belong to a clonal lineage. The bla GES-4 gene found in strain KG502 might well emerge from a point mutation in the bla GES-3 gene harbored by its ancestor strains, such as strain KG525, under heavy antibiotic stress in order to acquire extended properties of resistance to cephamycins and carbapenems.
A series of [5]carbohelicene derivatives substituted by electron-withdrawing maleimide and electron-donating methoxy such as maleimide-substituted [5]carbohelicene (HeliIm), and methoxy-substituted HeliIm (MeO-HeliIm) were newly designed and synthesized to examine the electrochemical properties, excited-state dynamic and circularly polarized luminescence (CPL). First, electrochemical measurements and DFT calculations of [5]carbohelicene derivatives were performed by comparing with the structural isomers: picene derivatives. Introduction of an electron-withdrawing maleimide group onto a [5]carbohelicene core contributes to the stabilized LUMO state in HeliIm as compared to that of [5]carbohelicene (Heli), whereas the energy level of HOMO state in MeO-HeliIm increases by introducing electron-donating methoxy (MeO) groups onto a HeliIm skeleton. The HOMO-LUMO gap of MeO-HeliIm is smaller than those of HeliIm and Heli, which is similar to the steady-state spectroscopic measurements. The absolute fluorescence quantum yield (Φ FL ) of HeliIm (0.37) largely increased as compared to [5]carbohelicene, Heli (0.04), whereas Φ FL of MeO-HeliIm (0.22) was slightly smaller than that of HeliIm. Theses photophysical processes including intersystem crossing are successfully explained by the kinetic discussions. Since [5]carbohelicene derivatives show the chirality, measurements of circular dichroism (CD) and circularly polarized luminescence (CPL) were successfully performed. In particular, HeliIm and MeO-HeliIm have provide excellent circularly polarized luminescence (CPL) and the values of the anisotropy factor g lum were estimated to be 2.4 × 10 −3 and 2.3 × 10 −3 , relatively. This is the first observation of CPL in [5]carbohelicene derivatives.
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