Stepwise Upregulation of the Pseudomonas aeruginosa Chromosomal Cephalosporinase Conferring High-Level β-Lactam Resistance Involves Three AmpD Homologues

Bacterial strains, plasmids, and antibiotic susceptibility testing.The wild-type Pseudomonas aeruginosa strain used in this work was the completely sequenced reference strain PAO1 (40). The PAO1 mutant derivatives constructed, the plasmids used or constructed, and the E. coli strains used in this work are described in Table 1. The MICs of the antipseudomonal β-lactams ceftazidime, cefepime, ticarcillin, piperacillin, piperacillin-tazobactam, aztreonam, imipenem, and meropenem were determined in Müller-Hinton agar plates with Etest strips (AB Biodisk, Solna, Sweden) by following the manufacturer's recommendations.

Cloning of ampD homologues ampDh2 and ampDh3.The P. aeruginosa ampD homologues ampDh2 (PA5485) and ampDh3 (PA0807) were detected by homology searches with the BLAST program at www.ncbi.nlm.nih.gov/BLAST . For cloning of ampDh2 and ampDh3, the PAO1 wild-type genes were PCR amplified with the primers described in Table 2. PCR products were ligated to plasmid pGEM-T to obtain pGTADh2 or pGTADh3, respectively, which were transformed into E. coli XL1-Blue made competent by CaCl₂. Transformants were selected in 50 μg/ml ampicillin MacConkey agar plates. The ampDh2 and ampDh3 genes obtained from three independent experiments were fully sequenced to ascertain the absence of mutations in the cloned fragments produced during PCR amplification. The BigDye Terminator Kit (PE-Applied Biosystems) was used to perform the sequencing reactions, which were analyzed with an ABI Prism 3100 DNA sequencer (PE-Applied Biosystems). Plasmid DNAs from pGTADh2 and pGTADh3 digested with BamHI were ligated to plasmid pUCP24 digested with the same enzyme to obtain plasmids pUCPADh2 and pUCPADh3, which were transformed into E. coli XL1-Blue. Transformants were selected in 20 μg/ml gentamicin MacConkey agar plates. In both cases, recombinant plasmids with DNA inserts with an orientation opposite to that of the lacZ promoter were selected.

Inactivation of P. aeruginosa ampD homologues.PAO1 ampD (PAΔD), ampDh2 (PAΔDh2), and ampDh3 (PAΔDh3) knockout mutants, as well as the three double mutants (ampD ampDh2 [PAΔDDh2], ampD ampDh3 [PAΔDDh3], and ampD2 ampDh3 [PAΔDh2Dh3]) and the triple mutant (ampD ampDh2 ampDh3 [PAΔDDh2Dh3]), were constructed by following the procedure previously described by Quénée et al. (36) for gene deletion and antibiotic marker recycling in P. aeruginosa. Upstream and downstream PCR products (Table 2) of ampD, ampDh2, or ampDh3 were digested with either BamHI or EcoRI and HindIII and cloned by three-way ligation into pEX100Tlink with the HindIII site deleted and opened by EcoRI and BamHI to obtain plasmids pEXTAD, pEXTADh2, and pEXTADh3, which were transformed into E. coli XL1-Blue. Transformants were selected in 50 μg/ml ampicillin MacConkey agar plates. The lox-flanked gentamicin resistance cassette (aacC1) obtained by HindIII restriction of plasmid pUCGmlox was cloned into the single site for this enzyme formed by ligation of the two flanking fragments, producing plasmids pEXTADGm, pEXTADh2Gm, and pEXTADh3Gm, which were transformed into E. coli XL1- Blue. Transformants were selected in 50 μg/ml ampicillin-20 μg/ml gentamicin MacConkey agar plates. These plasmids were then transformed into E. coli helper strain S17-1. The PAΔD, PAΔDh2, and PAΔDh3 PAO1 mutants were generated by, respectively, introducing pEXTADGm, pEXTADh2Gm, and pEXTADh3Gm from E. coli S17-1 by conjugation and selection for double recombinants with 5% sucrose-30 μg/ml gentamicin-1 μg/ml cefotaxime Luria-Bertani (LB) agar plates. Double recombinants were checked first by screening for ticarcillin (250 μg/ml) susceptibility and afterwards by PCR amplification. For removal of the gentamicin resistance cassettes, plasmid pCM157 was electroporated into the different mutants as previously described (38). Transformants were selected in 250 μg/ml tetracycline LB agar plates. One transformant for each mutant was grown overnight in 250 μg/ml tetracycline LB broth in order to allow expression of the cre recombinase. Plasmid pCM157 was then cured from the strains by three successive passages on LB broth. Selected colonies were then screened for tetracycline and gentamicin susceptibility. Finally, the knockout mutants were checked by PCR amplification and sequencing to ascertain that the corresponding genes were properly disrupted. The three double mutants and the triple mutant were then constructed from the single mutants sequentially by the same procedure.

Quantification of β-lactamase activity.β-Lactamase specific activity (nanomoles of nitrocefin hydrolyzed per minute per milligram of protein) was determined spectrophotometrically on crude sonic extracts from strain PAO1 and the seven above-described ampD mutants as previously described (18). To determine the β-lactamase specific activity after induction, before the preparation of the crude sonic extracts, the strains were grown in the presence of 50 μg/ml cefoxitin for 3 h. Alternatively, when specifically indicated, induction experiments were performed by incubation in the presence of ceftazidime (0.5 or 20 μg/ml). In all cases, the mean β-lactamase activity values obtained in three independent experiments were considered. The phenotypic determination of AmpC inducibility was performed by assessing the presence of antagonism between imipenem and ceftazidime disks in Müller-Hinton agar plates.

Quantification of the expression of ampC, ampD, ampE, ampDh2, and ampDh3.The levels of expression of ampC, ampD, ampE, ampDh2, and ampDh3 were determined by real-time PCR in strain PAO1 and the seven ampD mutants with and without cefoxitin induction. Total RNA from logarithmic-phase-grown cultures (with and without 50 μg/ml cefoxitin) was obtained with the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and adjusted to a final concentration of 50 ng/μl. A 500-ng sample of purified RNA was then used for one-step reverse transcription and real-time PCR amplification with the QuantiTect SYBR Green RT-PCR Kit (QIAGEN, Hilden, Germany) in a SmartCycler II (Cepheid, Sunnyvale, CA). The primers listed in Table 2 and previously described RpsL-1 and RpsL-2 (34) were used for amplification of ampC, ampD, ampE, ampDh2, ampDh3, and rpsL (used as a reference to calculate the relative amount of mRNA). In all cases, the mean values of relative mRNA expression obtained in three independent duplicate experiments were considered.

Complementation assays.For complementation experiments, plasmids pUCPADh2 and pUCPADh3; previously described plasmids pUCPAD and pUCPADE, harboring the PAO1 wild-type ampD gene and the complete ampDE operon, respectively (18); and plasmid pUCP24 (as a control) were electroporated into the different ampD mutants or PAO1. Transformants were selected in 50 μg/ml gentamicin LB agar plates. Ceftazidime MICs and β-lactamase activity were determined to evaluate the complementation of the AmpC hyperproduction phenotypes.

ampDh2 and ampDh3 are functional ampD homologues.Figure 1 shows a ClustalW multiple-sequence alignment of the predicted amino acid sequences of AmpDh2 and AmpDh3 compared to the AmpD proteins from P. aeruginosa and several Enterobacteriaceae. AmpDh2 and AmpDh3 were 27 and 25% identical to P. aeruginosa AmpD and 26 and 26% identical to E. cloacae AmpD, respectively. The percentage of identity between AmpDh2 and AmpDh3 was 40%. PCR and sequencing of ampDh2 and ampDh3 from 10 different clonal types of P. aeruginosa clinical strains confirmed that both genes are highly conserved in this species. Furthermore, both AmpDh2 and AmpDh3 contain all of the residues previously found to be essential for C. freundii AmpD catalytic activity, including His34, His154, Lys162, and Asp164 (9). ampDh2 is located in the chromosome of PAO1, between the alginate production regulatory gene kinB (PA5484) and PA5486, coding for a protein of unknown function, whereas ampDh3 is located between PA0806 and PA0808, both encoding theoretical proteins of unknown function.

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FIG. 1.

ClustalW multiple-sequence alignment of P. aeruginosa AmpDh2 and AmpDh3 and the previously described AmpD proteins from P. aeruginosa, E. coli, E. cloacae, and C. freundii. Conserved residues previously found to be essential for C. freundii AmpD catalytic activity (9) are in bold. Dashes indicate gaps. Asterisks, colons, and periods indicate identical, conserved, and semiconserved residues, respectively.

As previously noted (23), inactivation of ampD in strain PAO1 (PAΔD) led to a partially derepressed phenotype characterized by increased AmpC production but retaining further inducibility. This phenotype was associated with an eightfold increase in the ceftazidime MICs. As shown in Table 3, the plasmids harboring ampDh2 (pUCPADh2) and ampDh3 (pUCPADh3) completely transcomplemented the AmpC hyperproduction phenotype of PAΔD as readily as those harboring the regular ampD gene (pUCPAD) and the complete ampDE operon (pUCPADE), demonstrating that they both have functional N-acetyl-anhydromuramyl-l-alanine amidase activity.

Role of ampD homologues in ampC regulation and β-lactam resistance.Table 4 shows the levels of ampC expression under basal and cefoxitin-induced conditions and the susceptibilities to the antipseudomonal β-lactams of strains PAO1 and the seven ampD single, double, and triple mutants. ampD inactivation in strain PAO1 (PAΔD) led to a 60-fold increase in ampC expression, reaching 150-fold under cefoxitin induction, and was associated with a significant increase, albeit not surpassing CLSI nonsusceptibility breakpoints, of the MICs of all of the antipseudomonal penicillins and cephalosporins. The resistance increase was highest for piperacillin, piperacillin-tazobactam, and ceftazidime and lowest for ticarcillin and cefepime. As for the highly β-lactamase hydrolysis-resistant carbapenems, the MICs of imipenem were not significantly affected but those of meropenem were considerably raised, albeit they remained far from the nonsusceptibility breakpoints. On the other hand, the inactivation of neither ampDh2 nor ampDh3 (PAΔDh2 and PAΔDh3) significantly modified ampC expression or the antipseudomonal β-lactam MICs. Furthermore, the ampDh2-ampDh3 double inactivation (PAΔDh2Dh3) only slightly increased ampC basal and induced expression (twofold compared to PAO1) but did not produce any increase in β-lactam resistance, rather the opposite; MICs were 1 dilution lower than those of some of the antibiotics for PAO1 (Table 4).

This apparent lack of effect of the ampD homologues on the regulation of the chromosomal cephalosporinase and β-lactam resistance drastically changed when the ampD ampDh2 (PAΔDDh2) and ampD ampDh3 (PAΔDDh3) double mutants were analyzed. Regarding ampC expression, PAΔDDh2 caused only a modest increase in the inducibility of β-lactamase compared to PAΔD, whereas PAΔDDh3 caused a dramatic increase in both the basal and induced ampC levels (Table 4). The effect of PAΔDDh2 on β-lactam resistance was variable; it greatly increased the cefepime, aztreonam, and ticarcillin MICs, modestly increased those of ceftazidime, and modestly reduced those of piperacillin (with and without tazobactam) and meropenem compared to PAΔD. On the other hand, PAΔDDh3 dramatically increased the MICs (surpassing the clinical resistance breakpoints) of all of the antipseudomonal penicillins and cephalosporins and further increased the MICs of the carbapenem β-lactam meropenem. Finally, the triple mutant PAΔDDh2Dh3 caused a dramatic increase in the basal ampC expression level (1,000-fold compared to PAO1, 17-fold compared to PAΔD), which was not further cefoxitin inducible, showing that, finally, complete derepression was reached. Initially surprising, the antipseudomonal β-lactam MICs were not significantly raised for the completely derepressed triple mutant compared to the double mutant PAΔDDh3. The explanation for this apparently odd finding was found to be actually simple: subinhibitory concentrations of the, in theory, weak AmpC inducer β-lactams such as ceftazidime induced PAΔDDh3 AmpC production to values 1,000-fold higher than that of PAO1 (reaching the level of complete derepression) just as well as the potent inducer cefoxitin, as shown in Table 5.

Model for stepwise upregulation of ampC and high-level β-lactam resistance.From the above-described results, it can be deduced that upregulation to full derepression of the expression of P. aeruginosa ampC is achieved by a three-step escalating mechanism rendering four relevant expression states (shown in Fig. 2) and resistance phenotypes (pictures shown in Fig. 3): basal-level inducible expression (PAO1 wild type), moderate-level hyperinducible expression with increased antipseudomonal β-lactam resistance (PAΔD), high-level hyperinducible expression with high-level β-lactam resistance (PAΔDDh3), and very high-level derepressed expression not further increasing the already high-level β-lactam resistance (PAΔDDh2Dh3).

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FIG. 2.

Representation of the three-step escalating mechanism rendering four relevant expression states: basal-level inducible expression (PAO1), moderate-level hyperinducible expression (PAΔD), high-level hyperinducible expression (PAΔDDh3), and very high-level derepressed expression (PAΔDDh2Dh3).

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FIG. 3.

Assessment of AmpC inducibility and antipseudomonal β-lactam resistance phenotypes with imipenem (IPM) and ceftazidime (CAZ) disks. PAO1, highly ceftazidime susceptible, AmpC-inducible phenotype; PAΔD, moderately ceftazidime resistant, AmpC-inducible phenotype; PAΔDDh3, highly ceftazidime resistant, apparently derepressed phenotype (produced by ceftazidime induction of AmpC to the maximum production level); PAΔDDh2Dh3, highly ceftazidime resistant, AmpC-derepressed phenotype.

Interestingly, as shown in Table 6, any of the three ampD homologues, when produced from the high-copy-number pUCP24 derivatives (pUCPAD, pUCPADh2, and pUCPADh3), returned the very high-level derepressed expression and high-level β-lactam resistance of PAΔDDh2Dh3 to wild-type PAO1 levels, showing that the multiple-step upregulation model responds to quantitative rather than qualitative differences among the three ampD homologues involved.

Expression of ampD homologues is not regulated by AmpC inducers.The levels of expression of ampD, ampE, ampDh2, and ampDh3 genes from strain PAO1 were quantified under basal and cefoxitin-induced conditions to find out if the expression of any of them was modified (up- or downregulated) by incubation in the presence of AmpC inducers, but no significant differences in the levels of mRNA were detected (data not shown). Similarly, the inactivation of any of the ampD homologues did not modify the expression of the other ampD genes (i.e., the expression of ampDh2 or ampDh3 was not modified in PAΔD compared to PAO1, and the same for the other combinations), showing that the expression of the different ampD homologues apparently is not interregulated. Finally, ampD inactivation did not cause an increase in its own transcription, which would be consistent with the constitutive expression of this gene. In contrast, the transcription of ampDh2 or ampDh3 was slightly increased when the respective gene was inactivated: a two- to fivefold increase in ampDh2 or ampDh3 mRNA was observed for all of the single, double, and triple ampDh2 (PAΔDh2, PAΔDDh2, PAΔDh2Dh3, and PAΔDDh2Dh3)- or ampDh3 (PAΔDh3, PAΔDDh3, PAΔDh2Dh3, and PAΔDDh2Dh3)-inactivated mutants, respectively, compared to wild-type PAO1. These results suggest that the expression of ampDh2 and ampDh3 is inducible.