The emergence of carbapenem-hydrolysing metallo-b-lactamases (MBLs) is a serious threat to the clinical utility of carbapenems. This study identified plasmid-and integron-borne bla IMP-1 and bla in clinical isolates of Serratia marcescens. The bla IMP-1 and bla IMP-10 gene cassettes were carried by a class 1 integron and followed by the aac(69)-IIc gene cassette. The bla IMP-1 and bla IMP-10 gene cassettes were preceded by a weak P ant promoter, TGGACA(N) 17 TAAGCT, and an inactive P2 promoter, TTGTTA(N) 14 TACAGT. These genes were easily transferred to Escherichia coli by conjugation and transformation, indicating that they are located on transferable plasmids. Due to the acquisition of bla IMP-1 , the susceptibility of E. coli transconjugants to imipenem, meropenem, panipenem and biapenem decreased by 32-, 256-, 64-and 128-fold, respectively. In comparison, after gaining bla IMP-10 , the susceptibility of E. coli transconjugants to the four carbapenems decreased by 64-, 2048-, 256-and 64-fold, respectively. Strains harbouring bla IMP-10 showed higher-level resistance to imipenem, meropenem and panipenem than the strains harbouring bla IMP-1 , although the nucleotide sequences of the class 1 integrons carrying bla IMP-10 and bla IMP-1 were identical except for a single point mutation. INTRODUCTIONCarbapenems are relatively stable agents that act against extended-spectrum b-lactamases produced by Gram-negative rods, and are one of the first-choice antibiotics for the treatment of Serratia marcescens infections (Gilbert et al., 2005). However, the occurrence and spread of genes encoding metallo-b-lactamases (MBLs) including IMP, VIM, SPM and GIM have been reported in a variety of clinical isolates of Enterobacteriaceae from 28 countries (Walsh et al., 2005). The clinical significance of MBLs is further highlighted by their ability to hydrolyse almost all b-lactams except for monobactams, and by the fact that to date there is no clinically available inhibitor (Bush, 1998;Livermore & Woodford, 2000;Nordmann & Poirel, 2002).The bla IMP-1 gene, which encodes IMP-1 MBL, was the first MBL determinant reported, initially in a clinical isolate of S. marcescens from Japan in 1994 (Ito et al., 1995; Osano et al., 1994). To date, the nucleotide sequences of 24 IMP variants have been determined (Lahey Clinic, 2008). bla IMP-10 is a point mutation derivative of bla IMP-1 and has been identified in clinical isolates of Pseudomonas aeruginosa and Alcaligenes xylosoxidans (Iyobe et al., 2002;Zhao et al., 2008). To our knowledge, the bla IMP-10 gene has not been identified among Enterobacteriaceae strains.MBL genes are usually found as gene cassettes in integrons, mostly in class 1 integrons (Shibata et al., 2003). An integron is a mobile DNA element that can capture and carry genes, particularly antibiotic-resistance genes, by site-specific recombination (Bennett, 1999;Stokes & Hall, 1989). Five classes of integron are known to play a role in the dissemination of antibiotic resistance, and class 1 integrons are the most ext...
Background: The emergence of carbapenem-hydrolyzing metallo-β-lactamases (MBLs) is a serious threat to clinical medication of carbapenems. We evaluated the contributions of IMP-10 MBL, the outer membrane barrier and the MexAB-OprM efflux system to high-level carbapenem resistance in Pseudomonas aeruginosa clinical isolates. Methods: MBL-producing strains were screened by ceftazidime and mercaptoacetic acid double-disk synergy testing. Genes were determined by PCR analysis and DNA sequencing. IMP-10 MBL activity was assayed using nitrocefin as a substrate. Results: The blaIMP-10+ strains showed high-level resistance to carbapenems, with minimum inhibitory concentrations of 512 to >4,096 μg/ml. The minimum inhibitory concentrations were decreased by the inhibitors of MBLs, sodium mercaptoacetate (SMA) and ethylenediaminetetraacetic acid (EDTA), 16 and 64 fold, respectively. However, against the activity of the prepared IMP-10 MBL, the 50% inhibitory concentrations of SMA and EDTA were 5.5 and 211.6 μM, respectively, indicating that SMA was much more effective than EDTA in blocking the enzyme activity. The contradictory results may be explained by the additional effect of EDTA as an outer membrane permeabilizer because the susceptibility of the blaIMP– strains to carbapenems was increased 2 fold by EDTA, but not by SMA. In the presence of carbonyl cyanide m-chlorophenylhydrazone, a proton motive force inhibitor, the susceptibility of the P. aeruginosa isolates to carbapenems was increased 1–4 fold. Conclusion: The acquired IMP-10 MBL is a crucial factor in the high-level resistance to carbapenems in P. aeruginosa clinical isolates, while the outer membrane barrier and the MexAB-OprM efflux system play a basic and minor role, respectively.
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