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Molecular mechanisms of beta-lactam resistance mediated by AmpC hyperproduction in Pseudomonas aeruginosa clinical strains.

ABSTRACT: The molecular mechanisms of beta-lactam resistance mediated by AmpC hyperproduction in natural strains of Pseudomonas aeruginosa were investigated in a collection of 10 isogenic, ceftazidime-susceptible and -resistant pairs of isolates, each sequentially recovered from a different intensive care unit patient treated with beta-lactams. All 10 ceftazidime-resistant mutants hyper-produced AmpC (beta-lactamase activities were 12- to 657-fold higher than those of the parent strains), but none of them harbored mutations in ampR or the ampC-ampR intergenic region. On the other hand, six of them harbored inactivating mutations in ampD: four contained frameshift mutations, one had a C-->T mutation, creating a premature stop codon, and finally, one had a large deletion, including the complete ampDE region. Complementation studies revealed that only three of the six ampD mutants could be fully trans-complemented with either ampD- or ampDE-harboring plasmids, whereas one of them could be trans-complemented only with ampDE and two of them (including the mutant with the deletion of the ampDE region and one with an ampD frameshift mutation leading to an ampDE-fused open reading frame) could not be fully trans-complemented with any of the plasmids. Finally, one of the four mutants with no mutations in ampD could be trans-complemented, but only with ampDE. Although the inactivation of AmpD is found to be the most frequent mechanism of AmpC hyperproduction in clinical strains, our findings suggest that for certain types of mutations, AmpE plays an indirect role in resistance and that there are other unknown genes involved in AmpC hyperproduction, with at least one of them apparently located close to the ampDE operon.

SUBMITTER: Juan C

PROVIDER: S-EPMC1280133 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Molecular mechanisms of beta-lactam resistance mediated by AmpC hyperproduction in Pseudomonas aeruginosa clinical strains.

Juan Carlos C Maciá María D MD Gutiérrez Olivia O Vidal Carmen C Pérez José L JL Oliver Antonio A

Antimicrobial agents and chemotherapy 20051101 11

The molecular mechanisms of beta-lactam resistance mediated by AmpC hyperproduction in natural strains of Pseudomonas aeruginosa were investigated in a collection of 10 isogenic, ceftazidime-susceptible and -resistant pairs of isolates, each sequentially recovered from a different intensive care unit patient treated with beta-lactams. All 10 ceftazidime-resistant mutants hyper-produced AmpC (beta-lactamase activities were 12- to 657-fold higher than those of the parent strains), but none of them ...[more]

PMID: 16251318

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Project description:This study aimed to characterize the role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins (LMM PBPs), namely, PBP4 (DacB), PBP5 (DacC), and PBP7 (PbpG), in peptidoglycan composition, ?-lactam resistance, and ampC regulation. For this purpose, we constructed all single and multiple mutants of dacB, dacC, pbpG, and ampC from the wild-type P. aeruginosa PAO1 strain. Peptidoglycan composition was determined by high-performance liquid chromatography (HPLC), ampC expression by reverse transcription-PCR (RT-PCR), PBP patterns by a Bocillin FL-binding test, and antimicrobial susceptibility by MIC testing for a panel of ?-lactams. Microscopy and growth rate analyses revealed no apparent major morphological changes for any of the mutants compared to the wild-type PAO1 strain. Of the single mutants, only dacC mutation led to significantly increased pentapeptide levels, showing that PBP5 is the major dd-carboxypeptidase in P. aeruginosa. Moreover, our results indicate that PBP4 and PBP7 play a significant role as dd-carboxypeptidase only if PBP5 is absent, and their dd-endopeptidase activity is also inferred. As expected, the inactivation of PBP4 led to a significant increase in ampC expression (around 50-fold), but, remarkably, the sequential inactivation of the three LMM PBPs produced a much greater increase (1,000-fold), which correlated with peptidoglycan pentapeptide levels. Finally, the ?-lactam susceptibility profiles of the LMM PBP mutants correlated well with the ampC expression data. However, the inactivation of ampC in these mutants also evidenced a role of LMM PBPs, especially PBP5, in intrinsic ?-lactam resistance. In summary, in addition to assessing the effect of P. aeruginosa LMM PBPs on peptidoglycan structure for the first time, we obtained results that represent a step forward in understanding the impact of these PBPs on ?-lactam resistance, apparently driven by the interplay between their roles in AmpC induction, ?-lactam trapping, and dd-carboxypeptidase/?-lactamase activity.

Project description:AmpC is a group I, class C -lactamase present in most Enterobacteriaceae and in Pseudomonas aeruginosa and other nonfermenting gram-negative bacilli. The β-lactam class of antibiotics is one of the most important structural classes of antibacterial compounds and act by inhibiting the bacterial D ,D - transpeptidases that are responsible for the final step of peptidoglycan cross-linking. Our main aim in the study is to screen possible inhibitors against AmpC / β - lactamase (an enzyme responsible for antimicrobial activity in Pseudomonas aeruginosa), through virtual screening of 1364 NCI (National Cancer Institute) diversity set II compounds. Homology Model of AmpC / β - lactamase was constructed using MODELLER and the Model was validated using PROCHECK and Verify 3D programs to obtain a stable structure, which was further used for virtual screening of NCI (National Cancer Institute) diversity set II compounds through molecular Docking studies using Autodock. The amino acid sequence of the β - lactamase was also subjected to ScanProsite web server to find any pattern present in the sequence. After the prediction of 3-dimensional model of AmpC/ β-lactamase, the possible Active sites ofβ - lactamase were determined using LIGSITE(csc) and CastP web servers simultaneously. The Docked complexes were validated and Enumerated based on the Autodock Scoring function to pick out the best inhibitor based on Autodock energy score. Thus from the entire 1364 NCI diversity set II compounds which were Docked, the best four docking solutions were selected (ZINC12670903, ZINC17465965, ZINC11681166 and ZINC13099024). Further the Complexes were analyzed through LIGPLOT for their interaction for the 4 best docked NCI diversity set II compounds. Thus from the Complex scoring and binding ability it is deciphered that these NCI diversity set II compounds could be promising inhibitors for Pseudomonas aeruginosa using AmpC /β - lactamase as Drug target yet pharmacological studies have to confirm it.

Dataset Information

Molecular mechanisms of beta-lactam resistance mediated by AmpC hyperproduction in Pseudomonas aeruginosa clinical strains.

Publications

Molecular mechanisms of beta-lactam resistance mediated by AmpC hyperproduction in Pseudomonas aeruginosa clinical strains.

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