Differential  -lactam resistance response driven by ampD or dacB (PBP4) inactivation in genetically diverse Pseudomonas aeruginosa strains

Sir, The hyperproduction of the chromosomal β-lactamase AmpC is the most frequent and relevant mechanism of resistance to penicillins and cephalosporins in Pseudomonas aeruginosa.1,2 The aim of this study was to explore whether the available information on the P. aeruginosa PAO1 background,3 showing that dacB (encoding PBP4) inactivation leads to AmpC hyperproduction and the specific activation of the CreBC (BlrAB) system, determining high-level β-lactam resistance, in contrast to classical ampD inactivation,4 is a general principle in P. aeruginosa. For this purpose, ampD and dacB knockout mutants of strains PAO1, PA14 and eight genetically unrelated ceftazidime-susceptible clinical isolates were constructed following well-established procedures based on the cre-lox system for gene deletion and antibiotic resistance marker recycling in P. aeruginosa.3,4 All clinical isolates were obtained from blood cultures of patients admitted to Son Dureta Hospital Intensive Care Unit (ICU) during 2008. Isolates were selected using the following criteria: (i) wild-type ceftazidime susceptibility (defined as MICs <4 mg/L); and (ii) each belonging to a different PFGE clonal type. The MICs (Etest) of ceftazidime, cefepime, piperacillin/tazobactam, imipenem and meropenem, and the ampC and creD expression levels, determined by real-time reverse transcription PCR (RT–PCR) as previously described,3 for wild-type strains and their respective ampD or dacB mutants are shown in Table 1. The inactivation of ampD or dacB significantly increased ampC expression, but a remarkable variability was observed between strains, ranging from 1.6- to 294-fold (median 67-fold) for ampD and from 5.6- to 298-fold (median 106-fold) for dacB. The lowest increases were observed for PA14, while PAO1 showed increases close to the median values (Table 1). Despite diversity, no significant differences were observed in the quantitative effect on ampC expression produced by ampD or dacB inactivation. On the other hand, dacB inactivation produced significantly higher MICs of antipseudomonal penicillins and cephalosporins than ampD inactivation (Table 1). As an example, while ampD inactivation produced a median ceftazidime MIC increase of 2 log2, dacB inactivation produced a 3.5 log2 increase (P = 0.001, paired t-test). In agreement with previous data for PAO1, increased MICs correlated with the activation of the CreBC (BlrAB) system; whereas ampD inactivation did not significantly modify the expression of creD (blrD), a gene known to be regulated by this system, dacB inactivation resulted in creD overexpression in all strains (median increase 7.5-fold). Effects of ampD or dacB inactivation on the 10 P. aeruginosa strains with regard to β-lactam MICs and ampC and creD (blrD) expression aCAZ, ceftazidime; FEP, cefepime; TZP, piperacillin/tazobactam; IPM, imipenem; MEM, meropenem. bRelative mRNA level with respect to wild-type PAO1. cAmpC polymorphisms with respect to the wild-type PAO1 sequence. Effects of ampD or dacB inactivation on the 10 P. aeruginosa strains with regard to β-lactam MICs and ampC and creD (blrD) expression aCAZ, ceftazidime; FEP, cefepime; TZP, piperacillin/tazobactam; IPM, imipenem; MEM, meropenem. bRelative mRNA level with respect to wild-type PAO1. cAmpC polymorphisms with respect to the wild-type PAO1 sequence. The presence of frequent additional resistance mechanisms that could modify β-lactam susceptibility was explored in the clinical strains. The expression of genes coding for MexAB-OprM (mexB) and MexXY-OprM (mexY) efflux pumps was determined by real-time RT–PCR, following previously described protocols,5 and ampC and oprD genes (the latter only for imipenem-resistant isolates) were fully sequenced in the studied strains from two independent PCR products. None of them overexpressed mexB, whereas three (AAR, JCC and FRM) overexpressed mexY (19- to 41-fold higher expression than PAO1), probably accounting for the observed higher basal cefepime MICs, compared with those of ceftazidime, in these strains. Additionally, one of the clinical strains (GGF) was carbapenem resistant, due to a G to A mutation in nucleotide 1017 of oprD, leading to the origination of a premature stop codon (W339X). The polymorphisms detected in AmpC sequences of the clinical isolates and strain PA14, compared with PAO1, are also shown in Table 1. Previously reported polymorphisms T21A, G27D, A55T, T105A and L176R were detected in several isolates.6 The T105A substitution has been previously detected in a high proportion of imipenem non-susceptible strains, and has been shown to enhance hydrolysis of cefepime and imipenem.6 For this reason, AmpC enzymes containing this polymorphism have been designated extended-spectrum AmpC (ESAC). We nevertheless did not observe any effect of this polymorphism on cefepime or imipenem MICs, either for wild-type strains or for their respective ampD or dacB AmpC-hyperproducing mutants (Table 1). In conclusion, dacB inactivation leads to increased β-lactam MICs and ampC and creD (blrD) overexpression in diverse P. aeruginosa strains, but with a significant strain-specific variability with regard to the quantitative effect. This work was supported by the Ministerio de Ciencia e Innovación of Spain and Instituto de Salud Carlos III, through the Spanish Network for Research in Infectious Diseases (REIPI C03/14 and RD06/0008), and grants SAF2006-08154 and PS09/00033. None to declare.