It has long been recognized that the modification of penicillin-binding proteins (PBPs) to reduce their affinity for β-lactams is an important mechanism (target modification) by which Gram-positive cocci acquire antibiotic resistance. Among Gram-negative rods (GNR), however, this mechanism has been considered unusual, and restricted to clinically irrelevant laboratory mutants for most species. Using as a model Pseudomonas aeruginosa, high up on the list of pathogens causing life-threatening infections in hospitalized patients worldwide, we show that PBPs may also play a major role in β-lactam resistance in GNR, but through a totally distinct mechanism. Through a detailed genetic investigation, including whole-genome analysis approaches, we demonstrate that high-level (clinical) β-lactam resistance in vitro, in vivo, and in the clinical setting is driven by the inactivation of the dacB-encoded nonessential PBP4, which behaves as a trap target for β-lactams. The inactivation of this PBP is shown to determine a highly efficient and complex β-lactam resistance response, triggering overproduction of the chromosomal β-lactamase AmpC and the specific activation of the CreBC (BlrAB) two-component regulator, which in turn plays a major role in resistance. These findings are a major step forward in our understanding of β-lactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious diseases.
Decades after their discovery, β-lactams remain key components of our antimicrobial armamentarium for the treatment of infectious diseases. Nevertheless, resistance to these antibiotics is increasing alarmingly. There are two major bacterial strategies to develop resistance to β-lactam antibiotics: the production of enzymes that inactivate them (β-lactamases), or the modification of their targets in the cell wall (the essential penicillin-binding proteins, PBPs). Using the pathogen Pseudomonas aeruginosa as a model microorganism, we show that high-level (clinical) β-lactam resistance in vitro and in vivo frequently occurs through a previously unrecognized, totally distinct resistance pathway, driven by the mutational inactivation of a nonessential PBP (PBP4) that behaves as a trap target for β-lactams. We show that mutation of this PBP determines a highly efficient and complex β-lactam resistance response, triggering overproduction of the chromosomal β-lactamase AmpC and the specific activation of a two-component regulator, which in turn plays a key role in resistance. These findings are a major step forward in our understanding of β-lactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious diseases.
Citation: Moya B, Dötsch A, Juan C, Blázquez J, Zamorano L, Haussler S, et al. (2009) β-Lactam Resistance Response Triggered by Inactivation of a Nonessential Penicillin-Binding Protein. PLoS Pathog 5(3): e1000353. https://doi.org/10.1371/journal.ppat.1000353
Editor: Frederick M. Ausubel, Massachusetts General Hospital, United States of America
Received: November 25, 2008; Accepted: February 26, 2009; Published: March 27, 2009
Copyright: © 2009 Moya et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Ministerio de Educacion y Ciencia of Spain (SAF2006-08154), the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III through the Spanish Network for the Research in Infectious Diseases (REIPI C03/14 and RD06/0008), and the Govern de les Illes Balears (PROGECIB-9A). AD is a recipient of a predoctoral stipend provided by the DFG-sponsored European Graduate School program “Pseudomonas: Pathogenicity and Biotechnology”. Financial support from the Helmholtz Gemeinschaft is also gratefully acknowledged. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Decades after their discovery, β-lactams remain key components of our antimicrobial armamentarium for the treatment of infectious diseases. Bacterial resistance to them is generally driven either by the production of enzymes that inactivate them (β-lactamases), or by the modification of their targets in the cell wall (penicillin-binding Proteins, PBPs), sometimes in conjunction with mechanisms leading to diminished permeability or active efflux .
While the acquisition of modified PBPs showing low affinity for β-lactams is well known to be a major resistance mechanism in Gram-positive cocci, such as penicillin-resistant Streptococcus pneumoniae or the much-feared methicillin-resistant Staphylococcus aureus, this mechanism has not been thought to be important for most species of Gram-negative rods (GNR) . The production of intrinsic or horizontally acquired β-lactamases is undoubtedly the predominant resistance mechanism in the latter organisms . Among GNRs, the most widely distributed β-lactamases are chromosomally-encoded AmpC variants, produced by most Enterobacteriaceae and Pseudomonas aeruginosa, high up the list of pathogens causing life-threatening infections in hospitalized patients world-wide .
Although AmpC is produced at very low basal levels in wild-type strains, its expression is highly inducible in the presence of certain β-lactams (β-lactamase inducers) such as cefoxitin or imipenem . In fact, the efficacy of the widely-used broad spectrum penicillins (such as piperacillin) and cephalosporins (such as ceftazidime) relies on the fact that they are very weak AmpC inducers, even though they are efficiently hydrolyzed by this enzyme . Unfortunately, mutants showing constitutive high level AmpC production (AmpC derepressed mutants) are frequently selected during treatment with these β-lactams, leading to the failure of antimicrobial therapy ,. In some natural strains of Enterobacteriaceae and P. aeruginosa –, the inactivation of AmpD (cytosolic N-acetyl-anhydromuramyl-L-alanine amidase involved in peptidoglycan recycling –), and point mutations in AmpR (LysR-type transcriptional regulator required for ampC induction –) have been found to lead to AmpC overexpression, and thus to β-lactam resistance.
In this paper we show that, in contrast to the current expectations, the mutations triggering β-lactam resistance in P. aeruginosa, whether in vitro, in vivo, or in the clinical setting, frequently arise within a PBP gene. Inactivation of the E. coli dacB ortholog, encoding the nonessential low molecular mass PBP4 ,, is demonstrated to be the principal route to one-step high level (clinical) β-lactam resistance, by triggering the overexpression of ampC and the specific activation of the CreBC two component regulator , which is also found to play a major role in resistance.
Mutation of P. aeruginosa PBP4 is the main driver of one-step high-level β-lactam resistance in vitro and in vivo
The mechanisms by which β-lactam resistance arises were studied in a previously described  collection of 36 independent ceftazidime resistant mutants. These mutants were obtained in vitro (one-step spontaneous mutants) or in vivo (after 3 days of treatment with humanized ceftazidime regimen in mouse model of lung infection), at two ceftazidime concentrations (4 and 16 µg/ml), and from the wild-type strain PAO1 (normal mutation rate supply) or its mutS deficient hypermutable derivative PAOΔmutS (high mutation rate supply). In the previous study, all the mutants were shown to be highly resistant to all tested penicillins, cephalosporins, and monobactams; overexpression of the chromosomal cephalosporinase AmpC (18 to 236-fold higher expression relative to wild-type) was found to be the instrument of β-lactam resistance in all cases.
In this work, in an attempt to find out the genetic mechanisms leading to AmpC hyperproduction, we sequenced and quantified the expression of all genes so far known to be involved in ampC regulation and overexpression (ampD, ampE, ampDh2, ampDh3 and ampR) in the 36 mutants. We also performed complementation experiments with plasmids harboring the wild-type ampD gene (pUCPAD) or the complete ampDE operon (pUCPADE). A complete report of the obtained results is provided in Table S1. In contrast to present models, almost none of the mutants (32 of 36) showed mutations in any of the loci examined. The only exceptions were 4 mutants obtained in vivo from PAOΔmutS (high mutation rate supply) at the low ceftazidime concentration (4 µg/ml), each showing a different mutation in ampD. A modified expression of any of the studied genes was neither observed in the 32 mutants. As expected, only the four ampD mutants showed positive complementation with pUCPAD, but, intriguingly, all the 36 mutants showed positive complementation with pUCPADE (Table S1). Furthermore, positive complementation required the simultaneous presence of both ampD and ampE, since plasmids harboring ampDE operons with a non functional ampD and a wild-type ampE also failed to complement the resistance phenotype.
These findings suggested that, contrary to current understanding, mutations in ampD or ampR are not at all the most common, in vitro or in vivo, leading to AmpC overexpression and high level (clinical) β-lactam resistance in P. aeruginosa. Furthermore, the results strongly suggest that one-step high-level ceftazidime resistance in P. aeruginosa mainly occurs through single mutations in a gene/genes previously unknown to be involved in β-lactam resistance or AmpC regulation. That a single mutation has to be responsible for the resistance phenotype is shown by the ceftazidime resistance mutation rates published previously . At two different ceftazidime concentrations (4 and 16 µg/ml), spontaneous resistant mutants were obtained with a rate of 10−8 mutations per cell division for wild-type PAO1 and of 10−5−10−6 for PAOΔmutS. These mutation rates should rule out the involvement of more than one mutation in the resistance phenotype.
In an attempt to detect the mutations in the gene(s) yet unknown to be involved in β-lactam resistance, we followed a whole-genome analysis approach. Four of the PAO1 ceftazidime resistant in vitro mutants were analyzed by comparative hybridization on a recently described microarray for the discovery of single nucleotide polymorphisms (SNPs) in P. aeruginosa  using the parental PAO1 strain as reference. As shown in Figure 1, major decreases in hybridization ratios (indicating deletions of 50–100 base pairs) were detected for two of the mutants in the gene PA3047, the E. coli dacB ortholog, encoding the nonessential low molecular mass PBP4 ,. PCR and sequencing confirmed the presence of the deletions in this gene [(nts 1149–1231 for one of the mutants (1A5) and nts 1069–1138 for the other (1D7)] (Figure 1, Table S1). Furthermore, the two remaining mutants (1A1 and 1D4) also revealed a less pronounced decrease of hybridization ratio at a single position in gene PA3047 (Figure 1); PCR and sequencing identified as well the mutations, a G to A change in nt 819 leading to a premature stop codon (W273X) for 1A1 and a A to C change in nt 235 leading to a missense mutation (T79P) for 1D4 (Table S1).
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.
The function and structure of PBP4 (DacB) has been characterized mainly in E. coli. The protein is a nonessential low molecular mass class C PBP with DD-carboxypeptidase and DD-endopeptidase activity, that is thought to play an auxiliary role in morphology maintenance, peptidoglycan maturation and recycling, and cell separation during division ,,. The crystal structure of E. coli PBP4 has been recently determined  and found to be organized in three domains. Domain I has the characteristic SXXK, SXN, and KTG motifs of PBPs and β-lactamases, and contains the other two extra domains embedded within it. PBP4 from P. aeruginosa shows a 27% identity with that of E. coli, and contains all conserved motifs. The alignment of E. coli and P. aeruginosa PBP4 sequences is included in Figure S1.
Following the discovery of mutations within the dacB ortholog, we sequenced this gene in the rest of the collection of the 36 ceftazidime resistant mutants, and the complete list of the mutations detected is provided as Table S1. All but 2 of the 32 mutants not having mutations in ampD had mutations in dacB. A total of 28 different mutations were detected, and included deletions/insertions (9), nonsense mutations (7), and missense mutations (12). Many of the missense mutations occurred in sequences encoding highly conserved motifs, including the catalytic serine, at position 72 in P. aeruginosa (Figure S1, Table S1).
In order to further confirm the role of dacB mutations in β-lactam resistance, we constructed the dacB knockout mutant of PAO1 (PAΔdacB). As shown in Table 1, the inactivation of dacB in PAO1 yielded an almost identical phenotype to that documented in the ceftazidime-resistant dacB spontaneous mutants, with high level β-lactam resistance and ampC overexpression. Therefore, it is for the first time demonstrated that the inactivation of a particular PBP (which are supposed to be antibiotic targets) produces high-level β-lactam resistance.
Mutation of P. aeruginosa PBP4 determines an AmpR-dependent overexpression of the β-lactamase AmpC
In order to understand the role of PBP4 mutation in β-lactam resistance and upregulation of AmpC expression, we constructed the ampC, ampR (transcriptional regulator of AmpC), ampD (negative regulator of AmpC) and ampE (second component of the bicistronic ampDE operon, encodes an inner membrane-bound sensory transducer that modulates AmpD activity ), knockout mutants of strain 1A1 (DacB W273X in vitro spontaneous mutant of PAO1) and of strain PAO1 as control. As shown in Table 1, the inactivation of ampC completely restored ceftazidime susceptibility in 1A1, showing that the overexpression of the β-lactamase is essential for the resistance phenotype. Furthermore, the inactivation of ampR restored ceftazidime susceptibility and basal ampC expression levels, thus demonstrating that the effect of PBP4 mutation requires a functional AmpR. Therefore, considering that PBP4 has been shown to be involved in peptidoglycan recycling  it seems reasonable to believe that ampC overexpression driven by dacB inactivation, as occurs in the classical ampD mutation pathway, should be consequence of the qualitative or quantitative modification of muropeptides, that are the effector molecules for AmpC induction through their interaction with AmpR . Our results are also consistent with previous observations in the E. coli model, in which the strongest AmpC inducers (such as imipenem) were shown to be potent PBP4 inhibitors, suggesting a role of this PBP in the induction process .
Additionally, we show that the AmpDE pathway of AmpC repression is functional in the PBP4 mutants, since the inactivation of ampD dramatically increased further ampC expression and ceftazidime resistance in 1A1 (Table 1). Furthermore, while the inactivation of ampE in PAO1 (or in its ampD mutant) did not produce significant effects, it also determined a marked increase of ampC expression and ceftazidime resistance in the dacB mutant.These results suggest that both genes of the ampDE operon play a major role in the dacB mutant background. This conclusion is further supported by the positive complementation of the PBP4 mutants with the complete ampDE operon expressed from a multicopy plasmid (Table 2). Moreover, the expression of dacB from a multicopy plasmid (pUCPdB) also complemented both, the dacB and the ampD mutants (Table 2). Therefore, these results show that PBP4 and AmpDE are parallel synergic ampC regulatory pathways (a defect in one of them can be complemented by increasing the amount of the other), both ultimately relying on a functional AmpR.
While both pathways have a very similar effect on ampC expression, PBP4 mutation confers high level (clinical) β-lactam resistance (i.e. resistant according to current breakpoints), while ampD inactivation confers only moderate resistance (i.e. still susceptible according to current resistance breakpoints) (Table 1). In fact, the resistance level conferred by PBP4 mutation is more similar to that conferred by the simultaneous inactivation of the three ampD genes of P. aeruginosa (ampD plus the two additional homologous genes, ampDh2 and ampDh3 ), that produces a much higher increase in ampC expression (Table 1). Nevertheless, this mechanism of high-level resistance is not found among clinical strains ,, because it requires the acquisition of several mutations and because it causes a marked reduction of fitness and virulence . Here we show that in vivo (murine systemic infection model) fitness is not affected in the PAO1 dacB mutant, as shown by the competition index (CI) of 0.92, in sharp contrast to the previously documented CIs of less than 0.01 for the double and triple ampD mutants . Therefore, in contrast to the ampD inactivation pathway, PBP4 mutation in P. aeruginosa is a very efficient one-step trigger of high level β-lactam resistance mechanism of potentially enormous clinical relevance.
Mutation of P. aeruginosa PBP4 specifically triggers the activation of the CreBC two-component regulator, which in turn plays a major role in resistance
In order to explore further the effects of PBP4 mutation, we performed a whole-genome analysis of gene expression in two selected mutant strains (1A1 and 2A2) compared to wild-type PAO1, using the Affymetrix GeneChip P. aeruginosa genome array. In addition to ampC (and co-transcribed PA4111), which obviously was upregulated, only one further gene showed a significantly (>2-fold change) modified expression. This gene, creD, was upregulated in both mutants analyzed (not shown). creD encodes an inner membrane protein of yet unknown function that is regulated by the CreBC two-component regulator. The CreBC system has been deeply studied in E. coli, and it is shown to be a global regulator involved in metabolic control . Interestingly, the homolog of this system in Aeromonas Spp. (designated BlrAB) has been shown to play a role in the regulation of β-lactamase expression in these species –. As first approach, we analyzed whether creD overexpression was a signature feature of the PBP4 mutants. Indeed, real time RT-PCR experiments confirmed the overexpression of this gene in the two selected mutants (Table 1) as well as in the complete collection of in vivo and in vitro PBP4 mutants, with creD mRNA levels ranging from 25 to 60-fold higher than those of wild-type PAO1 (not shown). Moreover, the inactivation of dacB in wild-type PAO1 produced a similar creD overexpression (Table 1). The effect on creD expression seemed to be specific for the PBP4 mutants, rather than a direct consequence of AmpC overexpression, since this gene was not upregulated in the ampD mutants (Table 1). Interestingly, creD expression in wild-type PAO1 was found to be highly inducible by β-lactamase inducers (cefoxitin) (Table 1). Furthermore, creD inducibility was significantly reduced in the ampD mutants and the reduction in expression was dependent on a functional AmpR, since its inactivation restored the inducibility (Table 1). Overall, these results suggested a link between the CreBC regulator, PBP4 mutations, and the components of the regulatory system of ampC expression.
The complete creB, creC and creD genes, as well as their promoter regions, were fully sequenced in 1A1, 2A2 and five additional randomly selected mutants from the collection. The absence of mutations supported further the notion that the mutations in PBP4 are solely responsible for the complete β-lactam resistance response. The single mutation hypothesis is definitively confirmed by the fact that direct dacB inactivation produces the same phenotype (i.e. the same MICs and ampC and creD expression levels) observed in the spontaneous dacB mutants.
To gain insights into the role of the CreBC system in β-lactam resistance, we constructed creBC and creD knockout mutants of the PBP4 mutants 1A1 and 2A2, as well as of PAO1 and its single and triple ampD mutants as controls. Interestingly, the inactivation of creBC in the PBP4 mutants (1A1 and 2A2) not only decreased creD expression back to wild-type levels, but also drastically decreased β-lactam MICs, leaving them well within the susceptible (treatable) range according to current breakpoints (Table 1). Furthermore, the effect was specific to the PBP4 mutants, since β-lactam susceptibility was not affected by creBC inactivation in wild-type PAO1 or in its ampD mutants (Table 1). Overall, these results strongly suggest that PBP4 mutations specifically trigger the activation of the CreBC two-component regulator, leading to creD upregulation and β-lactam resistance. Nevertheless, CreD seems not to be the only CreBC-dependent driver of resistance, since the direct inactivation of creD in the PBP4 mutants decreased resistance slightly, but did not give the drastic reduction seen on CreBC inactivation (Table 1). The extra resistance of the PBP4 mutants compared to the ampD mutant (despite showing similar levels of ampC expression) is therefore apparently driven by the specific activation of the CreBC system in the PBP4 mutants. Indeed, the resistance level of 1A1 after CreBC inactivation was more similar to that of the ampD mutant (Table 1). Further evidence showing that the CreBC system is a key component in one-step high level β-lactam resistance development was provided by mutation rates experiments. While high level ceftazidime resistant mutants were readily selected from PAO1 wild-type strain [mutation rate to ceftazidime (at breakpoint concentration, 16 µg/ml) resistance of 3×10−8 mutants per cell division], mutation rates were below the detection limit (<1×10−11) for its CreBC knockout mutant (PAΔCreBC), consistently with the interpretation that a functional CreBC system is required for one-step high level (clinical) β-lactam resistance development in P. aeruginosa.
Nevertheless, in contrast to the previous experiences with the BlrAB system in Aeromonas Spp., CreBC mediated resistance was not directly driven by an effect on ampC expression, since 1A1ΔCreBC (despite showing a drastic reduction of resistance) had similar (still overexpressed) ampC levels than the parent 1A1 (Table 1). Moreover, β-lactamase activity was also similar (data not shown), showing that apparently the effect is neither produced by posttranscriptional modification of AmpC. Therefore, although we demonstrate that mutation of PBP4 specifically activates the CreBC two-component regulator, and that this event plays a major role in β-lactam resistance, the underlying mechanism is still uncertain. Despite only creD showed a modified expression greater 2-fold in the transcriptome analysis, even small modifications of expression of genes involved in outer membrane permeability, antibiotic efflux or general metabolism could significantly enhance the effect of AmpC overexpression and thus β-lactam resistance. In any case, our findings indicate that the nomenclature for this two-component system in P. aeruginosa should be changed to follow that used for Aeromonas Spp. (Blr, standing for β-lactam resistance) and not that used in E. coli (Cre, standing for carbon-source responsive) , –.
Figure 2 summarizes the current knowledge on P. aeruginosa ampC regulation, peptidoglycan recycling, and the described similarities and differences of the β-lactam resistance response driven by AmpD inactivation or PBP4 mutation.
(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.
Role of PBP4 mutation in the clinical setting
To find out whether PBP4 mutations and the linked CreBCD mediated resistance documented for in vitro and in vivo mutants occurred also in natural human infections, we investigated a previously described collection of clinical strains . This collection included 10 isogenic pairs of ceftazidime susceptible and resistant clinical isolates obtained from 10 Intensive Care Unit patients. All patients had severe P. aeruginosa infections that were treated with β-lactams, experiencing therapy failure due to resistance development. In all cases, the subsequent ceftazidime resistant isolate showed AmpC hyperproduction, but only in four of them could the resistance phenotype be attributed to known mechanisms (ampD mutations) . As shown in Table 3, all 6 remaining ceftazidime isolates contained mutations in dacB (not present in the preceding isogenic susceptible isolate). Interestingly, two of the isolates (despite them being genetically distinct) have sustained the same PBP4 mutation (T428P); this same mutation was found in one of the PAO1 in vivo mutants (Table S1) and involves a conserved residue close to the KTG motif . Furthermore, consistent with the findings for in vitro and in vivo mutants, all six natural PBP4 mutants overexpressed creD (2.8–38 fold higher expression than their respective wild-type isolates) (Table 3). The inactivation of creBC significantly reduced ceftazidime resistance in all but one of the natural PBP4 mutants (Table 3). On the other hand, the expression of creD was not modified in the four clinical strains containing only mutations in ampD (not shown).
Using P. aeruginosa as a model organism, we have shown that the most prevalent mutations causing immediate onset of high level β-lactam resistance are found in the dacB gene, encoding the nonessential PBP4. This is the first demonstration of the acquisition of β-lactam resistance through such a mechanism. All the previous examples by which PBP-mediated resistance develops involve modified enzymes showing low affinity for β-lactams (target modification) . Even though inactivation of the classical AmpC negative regulator AmpD upregulates AmpC, only mutations in dacB confer high level (clinical) β-lactam resistance. The inactivation of PBP4 is found to trigger an AmpR-dependent overproduction of the chromosomal β-lactamase AmpC, and the specific activation of the CreBC (BlrAB) two-component regulator, which in turn plays a major role in the β-lactam resistance response. This interplay between mutation of supposed antibiotic targets, production of antibiotic inactivating enzymes, and global regulators, is an unexpected layer of complexity of β-lactam resistance biology, which provides new perspectives on potential antibiotic targets for the treatment of infectious diseases. Since all the components of the described resistance mechanism (dacB, ampC, ampR, and creBCD) are found in the genomes of many GNR (E. coli might be an example of exception since ampR is not present in this species ), the results presented here are expected to have broad implications for the development of new antimicrobial compounds. Particularly, the CreBCD system is envisaged as an attractive candidate target to develop molecules capable of reducing the development of resistance when used together with β-lactam antibiotics.
Materials and Methods
Bacterial strains and plasmids
A complete list of laboratory strains and plasmids used or constructed in this study is provided as Table S2. A previously described  collection of 36 independent ceftazidime resistant mutants was used. These mutants were obtained either in vitro (one-step spontaneous mutants) or in vivo (after 3 days of treatment with humanized ceftazidime regimen in mouse model of lung infection), at two ceftazidime concentrations (4 and 16 µg/ml), and from the wild-type strain PAO1 (normal mutation rate supply) or its mutS deficient derivative PAOΔmutS (high mutation rate supply). Additionally, a previously reported  collection of 10 isogenic pairs of ceftazidime susceptible and resistant clinical isolates obtained from 10 Intensive Care Unit patients suffering from severe P. aeruginosa infections was used.
Construction of knockout mutants
P. aeruginosa single or multiple knockout mutants in ampD, ampE, ampR, ampC, creBC, creD, or dacB were constructed using the Cre-lox system for gene deletion and antibiotic resistance marker recycling . Upstream and downstream PCR products (primers used provided as Table S3) of each gene were digested with either BamHI or EcoRI and HindIII and cloned by a three way ligation into pEX100Tlink deleted for the HindIII site and opened by EcoRI and BamHI. The resulting plasmids were transformed into E. coli XL1Blue strain and transformants were selected in 30 µg/ml ampicillin LB agar plates. The lox flanked gentamicin resistance cassette (aac1) obtained by HindIII restriction of plasmid pUCGmlox was cloned into the single site for this enzyme formed by the ligation of the two flanking fragments. The resulting plasmids were again transformed into E. coli XL1Blue strain and transformants were selected in 30 µg/ml ampicillin-5 µg/ml gentamicin LB agar plates. Plasmids were then transformed into the E. coli S17-1 helper strain. Knockout mutants were generated by conjugation followed by selection of double recombinants using 5% sucrose-1 µg/ml cefotaxime-30 µg/ml gentamicin LB agar plates. Double recombinants were checked first screening for carbellicin (200 µg/ml) susceptibility and afterwards by PCR amplification and sequencing. For the recycling of the gentamicin resistance cassettes, plasmid pCM157 was electroporated into the different mutants. 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 the expression of the cre recombinase. Plasmid pCM157 was then cured from the strains by successive passages on LB broth. Selected colonies were then screened for tetracycline (250 µg/ml) and gentamicin (30 µg/ml) susceptibility and checked by PCR amplification and DNA sequencing.
PCR, sequencing, and quantification of gene expression
In order to explore the β-lactam resistance mechanisms in the above described collection of bacterial strains, ampD, ampE, ampR, ampDh2, ampDh3, creB, creC, creD and dacB genes were amplified by PCR, using primers described in Table S3, and fully sequenced. All mutations detected were checked by sequencing a fresh independent PCR product. Sequencing reactions were performed with the BigDye Terminator Kit (PE Applied Biosystems, Foster City, Calif.) and sequences were analyzed on an ABI prism 3100 DNA sequencer (PE Applied Biosystems). Resulting sequences were then compared (www.ncbi.nih.gov/BLAST) with those of the wild-type PAO1 strain ,.
The levels of expression of ampC, ampD, ampE, ampDh2, ampDh3 and creD were determined by real time RT-PCR with and without cefoxitin induction. Total RNA from logarithmic-phase-grown cultures (grown with and without 50 µg/ml cefoxitin) was obtained with the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and treated with 2 U of TURBO DNase (Ambion) for 30 min at 37°C to remove contaminating DNA. The reaction was stopped by the addition of 5 µl of DNase inactivation reagent and the samples were 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 using the QuantiTect SYBR Green RT-PCR Kit (QIAGEN, Hilden, Germany) in a SmartCycler II (Cepheid, Sunnyvale, CA). The primers listed in Table S3 were used for amplification of ampC, ampD, ampE, ampDh2, ampDh3, creD, and rpsL (used as reference to normalize the relative amount of mRNA). In all cases, the mean values of relative mRNA expression obtained in at least three independent duplicate experiments were considered.
Susceptibility testing, quantification of β-lactamase activity, complementation studies, estimation of mutation rates, and fitness experiments
Minimal inhibitory concentrations (MICs) for ceftazidime, cefepime, ticarcillin, piperacillin, piperacillin/tazobactam, aztreonam, imipenem, meropenem ciprofloxacin, tobramycin, tetracycline, chloramphenicol and colistin were determined in Müller-Hinton (MH) agar plates using E-test strips (AB Biodisk, Sweden) following the manufacturers recommendations. β-lactamase specific activity (nanomoles of nitrocefin hydrolyzed per minute per milligram of protein) was determined spectrophotometrically on crude sonic extracts as previously described . 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. In all cases, the mean β-lactamase activity values obtained in three independent experiments were considered. Complementation experiments were performed following previously described protocols . Briefly, plasmids pUCPAD (harboring the wild-type ampD gene), pUCPADE (harboring the complete wild-type ampDE operon) or pUCP24 (cloning vector) were electroporated into the different β-lactam resistant strains or PAO1 (as control). Additionally, plasmids pUCPADE2A1, pUCPADE1C5 or pUCPADE2C2 containing a non functional ampD and a wild-type ampE were electroporated in selected mutants. Finally, complementation experiments using the cloned wild-type dacB gene (pUCPdB) were also performed in selected strains. Transformants were selected in 50 µg/ml gentamicin LB agar plates. Complementation was considered positive when the MICs of ceftazidime for the transformants were at least 3 two-fold dilutions lower than those of the parent strains. The rates of mutation to high level (16 µg/ml) ceftazidime resistance were estimated following previously described protocols (19). To determine the effect on bacterial fitness of particular resistance mutations, in vitro (LB growth) and in vivo (murine model of systemic infection) competition experiments were performed following previously described procedures . Median Competition Indexes (CIs), defined as the mutant/wild-type ratio, were calculated from at least 8 independent experiments.
Analysis of whole-genome gene expression
Three independent replicates of PAO1 and of two selected ceftazidime resistant mutants (1A1 and 2A2) were grown in 10 ml of LB broth in a 50-ml baffled flask vigorously shaken at 37°C to an optical density at 600 nm (OD600) of 1. The cells were collected by centrifugation (8,000 g for 5 min at 4°C) and total RNA was isolated using the RNeasy minikit (QIAGEN) following the manufacturer's instructions. RNA was dissolved in water and treated with 2 U of TURBO DNase (Ambion) for 30 min at 37°C to remove contaminating DNA. The reaction was stopped by the addition of 5 µl of DNase inactivation reagent. Ten micrograms of total RNA were checked by running on an agarose gel prior to cDNA synthesis. cDNA synthesis, fragmentation, labeling and hybridization were performed according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol. Expression analysis was performed as described previously . Only transcripts showing higher than two-fold increases or decreases were considered as differentially expressed. In all cases the PPDE (posterior probability for differential expression) was between 0.999 and 1.
Whole-genome analysis to detect the mutations involved in ceftazidime resistance
In order to detect the presence of mutations in genes yet unknown to be involved in β-lactam resistance, a whole-genome analysis approach was followed. For this purpose, four ceftazidime resistant mutants were analyzed and compared with wild-type PAO1 using a recently described microarray for the discovery of single nucleotide polymorphisms (SNPs) in P. aeruginosa . Cultures were grown in brain-heart infusion (BHI) medium for 12 h at 37°C in shaking glass flasks at 180 rpm and genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen). Cell lysates were treated with RNase I (Qiagen) to prevent accidental carryover of RNA to the microarray. Genomic DNA was partially digested with DNase I (Amersham Biosciences, Piscataway, NJ) to a fragment size of ∼50–250 bp, confirmed by gel electrophoresis, and fragments were labeled at the 3′-ends with biotin-ddUTP (Roche Diagnostics, Indianapolis, IN) using Terminal deoxynucleotidyl transferase (Roche). Samples were hybridized to an identical lot of PATA1 arrays  for 16 hours at 50°C. After hybridization the microarrays were washed, stained with SA-PE and read using an Affymetrix GeneChip fluidic station and scanner according to Affymetrix standard protocols (Affymetrix, Santa Clara, CA). Analysis of microarray data was performed using the Affymetrix GCOS 1.4 to generate the raw data files (cel data). The raw data files were further analyzed using ‘Tiling Analysis Software’ (TAS) version 1.1 by Affymetrix.
Characterization of in vitro and in vivo ceftazidime-resistant mutants. The sequences corresponding to the signal peptides are shown in italics. SXXK, SXN, and KTG motifs are shown within boxes and the delimitations of the three domains are indicated with arrows. Asterisks, colons, and periods indicate identical, conserved, and semiconserved residues, respectively.
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Strains and plasmids used or constructed in this study
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We are grateful to Professor Jeremy Tame of Yokohama City University for highly constructive comments and suggestions. We also thank J. L. Pérez, Chief of the Microbiology Department of H. Son Dureta, for continuous support to this project, and C. Vidal and C. Santos for inestimable collaboration in the sequencing experiments.
Conceived and designed the experiments: AO. Performed the experiments: BM AD CJ LZ. Analyzed the data: BM AD JB SH AO. Contributed reagents/materials/analysis tools: AD JB SH. Wrote the paper: BM JB SH AO.
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