Figure 1.
Comparative genome hybridization of four spontaneous ceftazidime-resistant mutants revealing mutations in gene PA3047.
Genomic DNA from mutants 1A1, 1A5, 1D4, and 1A7 was analyzed on a whole genome DNA tiling microarray and compared to the parental wildtype PAO1. Data points (stems) represent the log2 ratio of signal intensity of each mutant against the wildtype signal. Mutants 1A5 and 1A7 showed strong decreases in signal at three consecutive positions (*), indicating deletions. In mutant 1A1 and 1D4, a slight decrease in signal (+) pointed towards a small genetic change, e.g., a single point mutation.
Table 1.
Susceptibility to β-lactams and expression of ampC and creD genes in the studied mutants.
Table 2.
Results of the complementation experiments of the PAO1 ampD and dacB mutants with different plasmids.
Figure 2.
Schematic representation of the regulation of the P. aeruginosa chromosomal β-lactamase AmpC and peptidoglycan recycling under different conditions.
(A) Wild-type strain in the absence of β-lactams. During regular bacterial growth, the peptidoglycan degradation products, MurNac-peptides [N-acetylglucosaminyl-1,6-anhydro- N-acetylmuramyl-tri (or tetra) peptides], are generated in the periplasm through the activity of PBP4 and several other enzymes. These products are then internalized through the permease AmpG, and processed in the cytoplasm by the β-N-acetylglucosaminidase NagZ and the N-acetyl-anhydromuramyl-L-alanine amidase AmpD. P. aeruginosa has two additional AmpD proteins, AmpDh2 that it is apparently located in the outer membrane and AmpDh3 that is still of unknown location. The generated tripeptides are then incorporated to the murein biosynthesis pathway to yield the UDP-MurNac-pentapeptides that will be exported to the periplasm and incorporated to the peptidoglycan, to complete the recycling process. In the absence of β-lactam antibiotics, these UDP-MurNac-pentapeptides interact with AmpR, which functions as a negative regulator of ampC expression. (B) Growth of wild-type strain in the presence AmpC inducer β-lactams. During growth in the presence of certain β-lactams (AmpC inducers), such as cefoxitin or imipenem, AmpC is produced at high levels, conferring natural (intrinsic) resistance to the antibiotic, provided it is a good substrate for the enzyme (as occurs for cefoxitin but not for imipenem). The exact mechanism responsible for the induction of the expression of AmpC in the presence of these antibiotics is still not fully understood. One of the components of the induction process is apparently the saturation of AmpD, due to the enhanced generation of its substrate (MurNac-tripeptides). The accumulated MurNAc-tripeptides are thought to displace the UDP-MurNAc-pentapeptides from AmpR, converting it into an activator of ampC transcription. Our results, and other previous indirect evidences, suggest that the inhibition of PBP4 by these β-lactam antibiotics (known to be the most potent PBP4 inhibitors) plays a major role in the ampC induction process, and determines the activation of the CreBC (BlrAB) two-component regulator. The exact function of the signal transducer AmpE, located in the inner membrane, still needs to be elucidated, but apparently interacts with both AmpD and PBP4. (C) Growth of the AmpD and/or PBP4 mutants in the presence of AmpC non-inducer β-lactams (most antipseudomonal cephalosporins and penicillins, such as ceftazidime or piperacillin, respectively). The inactivation of PBP4 or AmpD produces a very similar constitutive ampC overexpression. Both mechanisms ultimately relay in the activation of AmpR, which changes its activity from negative to positive regulator of ampC expression. Independently of the mechanism, AmpC overexpression itself is shown to confer only moderate (low-level) acquired resistance to non-inducer β-lactams. Additionally, the inactivation of PBP4 specifically activates the CreBC (BlrAB) system, which drives, in conjunction with the AmpC overexpression, the high-level β-lactam resistance response.
Table 3.
Characterization of β-lactam resistant clinical isolates.