However, the phenotypic analysis revealed that the C-NS and C-S isolates with high MICs of cefotaxime and ceftazidime (>16 μg mL−1) produced putative ESBLs (augmented AZD4547 price zones around cefotaxime and ceftazidime disks from the side of that with amoxicillin plus clavulanate in the DDST) or AmpC-like β-lactamases (zones around cefotaxime and ceftazidime disks augmented upon the presence of cloxacillin). The single C-S isolate P3/C154247 with lower cefotaxime and ceftazidime MICs was suggestive of the inducible AmpC expression (blunted zones around cefotaxime and ceftazidime disks from the side of amoxicillin with clavulanate). The results of the IEF and bioassay analyses are shown in Table
3. For the isolates with the
ESBL phenotype, Pembrolizumab clinical trial β-lactamases hydrolyzing cefotaxime and ceftazidime in the bioassay had a pI of 8.2 (Table 3). By PCR and sequencing, this enzyme was identified to be SHV-5. In crude extracts of the putative AmpC producers, the IEF and bioassay revealed the presumptive AmpC enzymes to have a pI of 7.9. The multiplex PCR, followed by amplification and sequencing of the entire gene, identified this AmpC as DHA-1. Both SHV-5 and DHA-1 were found in the isolate P4/C160267. Additionally, all of the isolates produced β-lactamases with pIs of 7.6 and 7.4, identified by PCR and sequencing to be SHV-1 and OXA-1, respectively. The blaDHA-1 gene was identified within a complex class 1 integron almost identical to that in the K. pneumoniae RBDHA strain from the Parisian region (Verdet et al., 2006). The Morganella morganii chromosomal fragment with the blaDHA-1 and ampR genes was separated from the ISCR1 element by a large insertion containing parts of operons sap and psp. The integronic gene cassette array differed from that in RBDHA only by a single mutation in the first cassette, converting it from the aminoglycoside acetyltransferase gene aac(6′)-Ib to the aminoglycoside and quinolone acetyltransferase gene aac(6′)-Ib-cr (Strahilevitz et al., 2009). The cassette was followed by blaOXA-1,
catB3, and arr3 (Table http://www.selleck.co.jp/products/Neratinib(HKI-272).html 3). Mapping of the 3′ part of the integron indicated the same arrangement of the region located between the gene sul1 and the IS6100 element (Verdet et al., 2006). The aac(6′)-Ib-cr and blaOXA-1 genes were also identified in all of the SHV-5-producing isolates, but their genetic context was not elucidated. Three PFGE types were discerned among the isolates, with type A grouping both DHA-1 (subtype A1) and SHV-5 (subtype A2) producers, type B grouping only DHA-1 producers, and type C with the single isolate P4/C160267 coexpressing the two enzymes (Table 3). The pulsotypes of the C-S isolates were identical to the ones of the C-NS isolates obtained from the same patients. By MLST, all of the isolates were assigned to the K. pneumoniae clone ST11 clone. The results of the porin analysis are presented in Tables 3 and 4, and, partially, in Fig. 1.