Molecular mechanisms of ceftazidime resistance in Pseudomonas aeruginosa isolates from canine and human infections

• Published in: Veterinární medicína Vol. 55; no. 4; pp. 172 - 182
• Main Authors: Du, S.-J., Kuo, H.-C., Cheng, C.-H., Fei, A.C.Y., Wei, H.-W., Chang, S.-K.
• Format: Journal Article
• Language: English
• Published: Prague Czech Academy of Agricultural Sciences (CAAS) 01.01.2010; Czech Academy of Agricultural Sciences
• Subjects: ampc; ampr; Antiinfectives and antibacterials; Antimicrobial agents; Ceftazidime; Efflux; extended spectrum β; Gene expression; Genes; Infections; lactamases; Molecular modelling; Multidrug resistance; Mutation; oprm efflux system; Otitis; Point mutation; Pseudomonas aeruginosa; β Lactamase
• ISSN: 0375-8427; 1805-9392
• DOI: 10.17221/64/2010-VETMED

Sixty-six clinical P. aeruginosa isolates, 17 obtained from canine otitis specimens and 49 received from human patients with bloodstream infections, were collected between February 2007 and January 2008. The minimal inhibitory concentrations (MICs) of the antimicrobial agents of these isolates were determined. Multidrug resistance was common, with 23 (34.8%) isolates found to be ceftazidime resistant. To explore the mechanisms of ceftazidime resistance, PCR analyses were performed to detect drug-resistance genes. The prevalence rate of Ambler class A, B, and D β-lactamase genes were obtained, with blaTEM-1 100%, blaPSE-1 100%, blaOXA-2 96.2%, blaSHV-18 91.3%, blaOXA-17 78.3%, blaVIM-3 26.1%, blaOXA-10 21.7% and blaSHV-1 8.7%. An efflux inhibition assay with the PAβN compound was conducted. The ceftazidime resistance isolates were also tested by RT-qPCR to determine the mRNA expression levels of the oprM and ampC genes. Five (21.7%) of the ceftazidime resistance isolates appeared to overactivate the OprM efflux system. The ampD, ampE, and ampR genes and the ampC-ampR intergenic region were subsequently amplified and sequenced. Five (21.7%) of the ceftazidime resistance isolates from humans and canines had a point mutation in AmpR (Asp135-Asn, n = 3; Als194-Ser, n = 2), which induces AmpC overproduction from 10- to 80-fold. This study first reported ceftazidime resistance in P. aeruginosa from canine otitis specimens, which are closely related to ESBLs (50%), including the overproduction of AmpC (25%) and the OprM efflux system (25%). The ESBLs (100%) played an important role in all ceftazidime resistance isolates from humans, and either AmpC (21.1%) or OprM (21.1%) might be overexpressed within the same isolate. A human patient isolate (H307B) showed simultaneous expression of ESBLs, the OprM efflux system, and AmpC overproduction.