Inactivation of the ampD Gene in Pseudomonas aeruginosa Leads to Moderate-Basal-Level and Hyperinducible AmpC β-Lactamase Expression

Abstract

It has been shown in enterobacteria that mutations in ampD provoke hyperproduction of chromosomal ␤-lactamase, which confers to these organisms high levels of resistance to ␤-lactam antibiotics. In this study, we investigated whether this genetic locus was implicated in the altered AmpC ␤-lactamase expression of selected clinical isolates and laboratory mutants of Pseudomonas aeruginosa. The sequences of the ampD genes and promoter regions from these strains were determined and compared to that of wild-type ampD from P. aeruginosa PAO1. Although we identified numerous nucleotide substitutions, they resulted in few amino acid changes. The phenotypes produced by these mutations were ascertained by complementation analysis. The data revealed that the ampD genes of the P. aeruginosa mutants transcomplemented Escherichia coli ampD mutants to the same levels of ␤-lactam resistance and ␤-lactamase expression as wild-type ampD. Furthermore, complementation of the P. aeruginosa mutants with wild-type ampD did not restore the inducibility of ␤-lactamase to wild-type levels. This shows that the amino acid substitutions identified in AmpD do not cause the altered phenotype of AmpC ␤-lactamase expression in the P. aeruginosa mutants. The effects of AmpD inactivation in P. aeruginosa PAO1 were further investigated by gene replacement. This resulted in moderatebasal-level and hyperinducible expression of ␤-lactamase accompanied by high levels of ␤-lactam resistance. This differs from the stably derepressed phenotype reported in AmpD-defective enterobacteria and suggests that further change at another unknown genetic locus may be causing total derepressed AmpC production. This genetic locus could also be altered in the P. aeruginosa mutants studied in this work.

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

“…A novel amino acid substitution in position 194 is thought to have caused the overexpression of ampC. Bratu et al (2007) have demonstrated that increased ampC expression is associated with major deletions that affect ampD, and Langaee et al (2000) have shown that mutations within the structural gene of ampD can lead to ampC overexpression. These studies also found that increased β-lactam MICs in organisms with inducible ampC and ampE genes can modulate ampC repression in hyperproducing strains in the absence of inducers.…”

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…The role of many orthologous proteins including AmpR, AmpC, AmpD and AmpG have been demonstrated in P. aeruginosa and appear to have similar roles to their Enterobacteriaceae counterparts [151,200,211]. In addition, new players such as ampP, ampDh2 and ampDh3 have been identified in P. aeruginosa [139,151].…”

“…The absence of ampD leads to the accumulation of anhMurNAc-tripeptide in the cytosol and constitutive overproduction of b-lactamase even in the absence of induction, resulting in a high resistance to b-lactams [163]. The most common mechanism for constitutive ampC overexpression in clinical strains leading to b-lactam resistance in Enterobacteriaceae as well as P. aeruginosa is to mutate ampD [200][201][202][203] (Table 3). P. aeruginosa has three different AmpD homologues, AmpD (PA4522), AmpDh2 (PA5485) and AmpDh3 (PA0807) that are responsible for de-repression of ampC expression in a step-wise manner [139,204].…”

Gene Mutations Responsible for Overexpression of AmpC β-Lactamase in Some Clinical Isolates ofEnterobacter cloacae

“…In AmpD-defective bacterial strains, unhydrolyzed anhydromuropeptides accumulate in the cytoplasm (7,13), and this causes activation of AmpR, which results in the semiconstitutive or constitutive expression of ampC. The role of AmpD in the induction of AmpC in gram-negative bacilli has been studied in detail (15,18,20).…”