Novel resistance to imipenem associated with an altered PBP-4 in a Pseudomonas aeruginosa clinical isolate

Mechanisms of resistance to pefloxacin were investigated in four isogenic Pseudomonas aeruginosa strains: S (parent isolate; MIC, 2 ,ug/ml), PT1 and PT2 (posttherapy isolates obtained in animals; MICs, 32 and 128 ,ug/ml, respectively), and PT2-r (posttherapy isolate obtained after six in vitro subpassages of PT2; MIC, 32 ,ug/ml). [2-3H]adenine incorporation (indirect evidence of DNA gyrase activity) in EDTA-permeabilized cells was less affected by pefloxacin in PT2 and PT2-r (50% inhibitory concentration, 0.27 and 0.26 ,ug/ml, respectively) than it was in S and PT1 (50% inhibitory concentration, 0.04 and 0.05 ,ug/ml, respectively). Reduced [14C]pefloxacin labeling of intact cells in strains PT1 and PT2 correlated with more susceptibility to EDTA and the presence of more calcium (P < 0.05) and phosphorus in the outer membrane fractions. Outer membrane protein analysis showed reduced expression of protein D2 (47 kDa) in strains PT1 and PT2. Other proteins were apparently similar in all strains. The addition of calcium chloride (2 mM) to the sodium dodecyl sulfate-solubilized samples of outer membrane proteins, before heating and Western blotting, probed with monoclonal antibody anti-OmpF showed electrophoretic mobility changes of OmpF in strains PT1 and PT2 which were not seen in strain S. Calcium-induced changes were reversed with ethyleneglycoltetraacetate.Decreased [14C]pefloxacin labeling was further correlated with an altered lipopolysaccharide pattern and increased 3-deoxy-D-mannooctulosonic acid concentration (P < 0.01). These findings suggested that resistance to pefloxacin is associated with altered DNA gyrase in strain PT2-r, with altered permeability in PT1, and with both mechanisms in PT2. The decreased expression of protein D2 and the higher calcium and lipopolysaccharide contents of the outer membrane could be responsible for the permeability deficiency in P. aeruginosa.The activities of fluoroquinolones against Pseudomonas aeruginosa have been fully documented (45), but bacteria can develop resistance to these agents (12,18,19,36,38). This resistance results from reduced permeability of the outer membrane (OM), altered DNA gyrase, or a combination of the two processes (7,8,18,36,38). However, mechanisms of resistance at the molecular level are not yet entirely elucidated (45).In a pseudomonal peritonitis model, we showed previously that resistance emerges rapidly when mice are treated with pefloxacin or ciprofloxacin (26). In mice infected with a quinolone-susceptible P. aeruginosa isolate, posttherapy variants (strain PT1) emerged after a single dose of pefloxacin, showing an 8-to 16-fold decrease in susceptibility to quinolones. Subinoculation of a PT1 strain, followed by therapeutic exposure to ciprofloxacin, produced a highly resistant posttherapy variant (strain PT2) for which the MIC was increased 64-fold (26).We report here results of further studies on the mechanism of resistance of these strains. As expected, we observed involvements of DNA gyrase and permeability, but we were surprised by ...

Section: Discussionmentioning

confidence: 99%

Resistance to pefloxacin in Pseudomonas aeruginosa

Michéa-Hamzehpour

Lucain

Péchère

1991

“…Expression of PBPs in P. aeruginosa, A. baumannii, P. mirabilis, and Rhodococcus equi is decreased, resulting in carbapenem resistance (57, 68, 71, 150, 154). In addition, amino (6,17,31,56,100,109,134,150,158,165,184,185,218).…”

mentioning

confidence: 99%

Carbapenems: Past, Present, and Future

Papp-Wallace

Endimiani

Taracila

et al. 2011

1,146

962

In this review, we summarize the current "state of the art" of carbapenem antibiotics and their role in our antimicrobial armamentarium. Among the ␤-lactams currently available, carbapenems are unique because they are relatively resistant to hydrolysis by most ␤-lactamases, in some cases act as "slow substrates" or inhibitors of ␤-lactamases, and still target penicillin binding proteins. This "value-added feature" of inhibiting ␤-lactamases serves as a major rationale for expansion of this class of ␤-lactams. We describe the initial discovery and development of the carbapenem family of ␤-lactams. Of the early carbapenems evaluated, thienamycin demonstrated the greatest antimicrobial activity and became the parent compound for all subsequent carbapenems. To date, more than 80 compounds with mostly improved antimicrobial properties, compared to those of thienamycin, are described in the literature. We also highlight important features of the carbapenems that are presently in clinical use: imipenem-cilastatin, meropenem, ertapenem, doripenem, panipenem-betamipron, and biapenem. In closing, we emphasize some major challenges and urge the medicinal chemist to continue development of these versatile and potent compounds, as they have served us well for more than 3 decades.Carbapenems play a critically important role in our antibiotic armamentarium. Of the many hundreds of different ␤-lactams, carbapenems possess the broadest spectrum of activity and greatest potency against Gram-positive and Gram-negative bacteria. As a result, they are often used as "last-line agents" or "antibiotics of last resort" when patients with infections become gravely ill or are suspected of harboring resistant bacteria (23,(174)(175)(176)229). Unfortunately, the recent emergence of multidrug-resistant (MDR) pathogens seriously threatens this class of lifesaving drugs (189). Several recent studies clearly show that resistance to carbapenems is increasing throughout the world (35,64,73,123,151,155,173,200). Despite this menacing trend, our understanding of how to best use these agents is undergoing a renaissance, especially concerning their role with regard to ␤-lactamase inhibition. In this context, we view the number, type, and diversity of carbapenems as compelling reasons to explore these compounds for new insights into drug development.

“…Similar to resistance to other ,B-lactams, the mechanisms of the carbapenem resistance may result from altered penicillin-binding proteins, as described for Acinetobacter baumanii, Listeria monocytogenes, and Pseudomonas aeruginosa (3,12,30), from a decrease in the uptake of carbapenems, as has been described for P. aeruginosa (5,32,39), or from a high level of biosynthesis of P-lactamases with a low level of activity against carbapenems that are normally present combined with a decrease in outer membrane permeability, as described for overproduction of Ambler class C cephalosporinase in Enterobacter cloacae (18). However, the main mechanism of the carbapenem resistance is linked to the presence of a P-lactamase with strong hydrolytic activities against these 3-lactams.…”

mentioning

confidence: 99%

Biochemical properties of a carbapenem-hydrolyzing beta-lactamase from Enterobacter cloacae and cloning of the gene into Escherichia coli

Nordmann

Naas

et al. 1993

148

121

A clinical isolate of Enterobacter cloacae, strain NOR-1, exhibited resistance to imipenem and remained susceptible to extended-spectrum cephalosporins. Clavulanic acid partially restored the susceptibility of the strain to imipenem. Two 13-lactamases with isoelectric points (pI) of 6.9 and >9.2 were detected in strain E.cloacae NOR-1; the higher pl corresponded to AmpC cephalosporinase. Plasmid DNA was not detected in E. cloacae NOR-1 and imipenem resistance could not be transferred into Escherichia coli JM109. The carbapenem-hydrolyzing 13-lactamase gene was cloned into plasmid pACYC184. One recombinant plasmid, pPTN1, harbored a 5.3-kb Sau3A fragment from E. cloacae NOR-1 expressing the carbapenem-hydrolyzing 13-lactamase. This enzyme (pl 6.9) hydrolyzed ampicillin, cephalothin, and imipenem more rapidly than it did meropenem and aztreonam, but it hydrolyzed extended-spectrum cephalosporins only weakly and did not hydrolyze cefoxitin. Hydrolytic activity was partially inhibited by clavulanic acid, sulbactam, and tazobactam, was nonsusceptible to chelating agents such as EDTA and 1,10-o-phenanthroline, and was independent of the presence of ZnCl2. Its relative molecular mass was 30,000 Da. Induction experiments concluded that the carbapenem-hydrolyzing ,3-lactamase biosynthesis was inducible by cefoxitin and imipenem. Subcloning experiments with HindIII partial digests of pPTN1 resulted in a recombinant plasmid, designated pPTN2, which contained a 1.3-kb insert from pPTN1 and which conferred resistance to 13-lactam antibiotics.Hybridization studies performed with a 1.2-kb Hindlll fragment from pPTN2 failed to determine any homology with ampC of E. cloacae, with other known 13-lactamase genes commonly found in members of the family Enterobacteriaceae (blaTEM-l and blasHv.3 derivatives), and with previously described carbapenemase genes such as those from Xanthomonas maltophilia, Bacilus cereus, Bacteroidesfragilis (cfA), and Aeromonas hydrophila (cphA). This work describing the biochemical properties of a novel chromosome-encoded 13-lactamase from E. cloacae indicates that this enzyme differs from all the previously described carbapenemases. This is the first reported cloning of a Enterobacteriaceae.