Identification of genotypes of plasmid-encoded AmpC beta-lactamases from clinical isolates and characterization of mutations in their promoter and attenuator regions.

We investigated the occurrence of AmpC beta-lactamases among Escherichia coli and Klebsiella pneumoniae isolates and determined the genotype of plasmid-mediated AmpC beta-lactamases at a medical center. The AmpC beta-lactamase promoter and attenuator were amplified from chromosomal DNA of high AmpC-producing E. coli isolates and sequenced. Antibiotic screening and 3D extract tests showed the presence of AmpC beta-lactamase in 3.56% of K. pneumoniae and 1.88% of E. coli isolates. Ten isolates (six K. pneumoniae and four E. coli) were positive for extended spectrum beta-lactamase (ESBL) as indicated by the double disc diffusion method. DHA-1 plasmid-encoded AmpC beta-lactamase was present in 10 K. pneumoniae isolates and four E.coli isolates. E. coli chromosomal AmpC beta-lactamase carried polymorphisms in the -42, -32, and -18 bases of the promoter and in the +26 and +27 bases of the attenuator, which may play a role in antibiotic resistance. The observed mutations may have clinical implications for the management of antibiotic-resistant infections.

DHA-1 plasmid-mediated AmpC ß-lactamase expression and regulation of Klebsiella pnuemoniae isolates.

The present study aimed to investigate the regulatory mechanism of the AmpC enzyme by analyzing the construction and function of AmpCR, AmpE and AmpG genes in the Dhahran (DHA)­1 plasmid of Klebsiella pneumoniae (K. pneumoniae). The production of AmpC and extended­spectrum ß­lactamase (ESBL) were determined following the cefoxitin (FOX) inducing test for AmpC, preliminary screening and confirmation tests for ESBL in 10 DHA­1 plasmid AmpC enzymes of K. pneumoniae strains. AmpCR, AmpD, AmpE and AmpG sequences were analyzed by polymerase chain reaction. The pACYC184­X plasmid analysis system was established and examined by regulating the pAmpC enzyme expression. The electrophoretic bands of AmpCR, AmpD, AmpE and AmpG were expressed. Numerous mutations in AmpC + AmpR (AmpCR) and in the intergenic region cistron of AmpC­AmpR, AmpD, AmpE and AmpG were observed. The homology of AmpC and AmpR, in relation to the Morganella morganii strain, was 99%, which was determined by comparing the gene sequences of Kp1 with those of Kp17 AmpCR. The specific combination of AmpR and labeled probe demonstrated a band retarded phenomenon and established a spatial model of AmpR. All the enzyme production strains demonstrated Val93→Ala in AmpG; six transmembrane domains were found in AmpE in all strains, with the exception of Kp1 and Kp4, which had only three transmembrane segments that were caused by mutation. The DHA­1 plasmid AmpC enzymes encoded by plasmid are similar to the inducible chromosomal AmpC enzymes, which are also regulated by AmpD, AmpE, AmpR and AmpG.