In vitro evolution of cefepime/zidebactam (WCK 5222) resistance in Pseudomonas aeruginosa: dynamics, mechanisms, fitness trade-off and impact on in vivo efficacy.

OBJECTIVES: To study the dynamics, mechanisms and fitness cost of resistance selection to cefepime, zidebactam and cefepime/zidebactam in Pseudomonas aeruginosa. METHODS: WT P. aeruginosa PAO1 and its ΔmutS derivative (PAOMS) were exposed to stepwise increasing concentrations of cefepime, zidebactam and cefepime/zidebactam. Selected mutants were characterized for change in susceptibility profiles, acquired mutations, fitness, virulence and in vivo susceptibility to cefepime/zidebactam. Mutations were identified through WGS. In vitro fitness was assessed by measuring growth in minimal medium and human serum-supplemented Mueller-Hinton broth. Virulence was determined in Caenorhabditis elegans and neutropenic mice lung infection models. In vivo susceptibility to a human-simulated regimen (HSR) of cefepime/zidebactam was studied in neutropenic mice lung infection. RESULTS: Resistance development was lower for the cefepime/zidebactam combination than for the individual components and high-level resistance was only achieved for PAOMS. Cefepime resistance development was associated with mutations leading to the hyperexpression of AmpC or MexXY-OprM, combined with PBP3 mutations and/or large chromosomal deletions involving galU. Zidebactam resistance was mainly associated with mutations in PBP2. On the other hand, resistance to cefepime/zidebactam required multiple mutations in genes encoding MexAB-OprM and its regulators, as well as PBP2 and PBP3. Cumulatively, these mutations inflicted significant fitness cost and cefepime/zidebactam-resistant mutants (MIC = 16-64 mg/L) remained susceptible in vivo to the HSR. CONCLUSIONS: Development of cefepime/zidebactam resistance in P. aeruginosa required multiple simultaneous mutations that were associated with a significant impairment of fitness and virulence.

Molecular Analysis of the Contribution of Alkaline Protease A and Elastase B to the Virulence of Pseudomonas aeruginosa Bloodstream Infections.

Pseudomonas aeruginosa is a major cause of nosocomial bloodstream infections. This microorganism secretes two major proteases, alkaline protease A (AprA) and elastase B (LasB). Despite several in vitro studies having demonstrated that both purified proteases cleave a number of components of the immune system, their contribution to P. aeruginosa bloodstream infections in vivo remains poorly investigated. In this study, we used a set of isogenic mutants deficient in AprA, LasB or both to demonstrate that these exoproteases are sufficient to cleave the complement component C3, either soluble or deposited on the bacteria. Nonetheless, exoprotease-deficient mutants were as virulent as the wild-type strain in a murine model of systemic infection, in Caenorhabditis elegans and in Galleria mellonella. Consistently, the effect of the exoproteases on the opsonization of P. aeruginosa by C3 became evident four hours after the initial interaction of the complement with the microorganism and was not crucial to survival in blood. These results indicate that exoproteases AprA and LasB, although conferring the capacity to cleave C3, are not essential for the virulence of P. aeruginosa bloodstream infections.

In vitro dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in Pseudomonas aeruginosa.

OBJECTIVES: We analysed the dynamics and mechanisms of resistance development to imipenem alone or combined with relebactam in Pseudomonas aeruginosa WT (PAO1) and mutator (PAOMS; ΔmutS) strains. METHODS: PAO1 or PAOMS strains were incubated for 24 h in Mueller-Hinton Broth with 0.125-64 mg/L of imipenem ± relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64 mg/L of imipenem ± relebactam for 7 days. Two colonies per strain, replicate experiment and antibiotic from early (Day 1) and late (Day 7) cultures were characterized by determining the susceptibility profiles, WGS and determination of the expression of ampC and efflux-pump-coding genes. Virulence was studied in a Caenorhabditis elegans infection model. RESULTS: Relebactam reduced imipenem resistance development for both strains, although resistance emerged much faster for PAOMS. WGS indicated that imipenem resistance was associated with mutations in the porin OprD and regulators of ampC, while the mutations in imipenem/relebactam-resistant mutants were located in oprD and regulatoras of MexAB-OprM. High-level imipenem/relebactam resistance was only documented in the PAOMS strain and was associated with an additional specific (T680A) mutation located in the catalytic pocket of ponA (PBP1a) and with reduced virulence in the C. elegans model. CONCLUSIONS: Imipenem/relebactam could be a useful alternative for the treatment of MDR P. aeruginosa infections, potentially reducing resistance development during treatment. Moreover, this work deciphers the potential resistance mechanisms that may emerge upon the introduction of this novel combination into clinical practice.

Activity of Imipenem-Relebactam against a Large Collection of Pseudomonas aeruginosa Clinical Isolates and Isogenic ß-Lactam-Resistant Mutants.

Imipenem and imipenem-relebactam MICs were determined for 1,445 Pseudomonas aeruginosa clinical isolates and a large panel of isogenic mutants showing the most relevant mutation-driven ß-lactam resistance mechanisms. Imipenem-relebactam showed the highest susceptibility rate (97.3%), followed by colistin and ceftolozane-tazobactam (both 94.6%). Imipenem-relebactam MICs remained ≤2 µg/ml in all 16 isogenic PAO1 mutants and in 8 pairs of extensively drug-resistant clinical strains that had developed resistance to ceftolozane-tazobactam and ceftazidime-avibactam due to mutations in OXA-10 or AmpC.

Pathogenic characteristics of Pseudomonas aeruginosa bacteraemia isolates in a high-endemicity setting for ST175 and ST235 high-risk clones.

Multidrug-resistant (MDR) Pseudomonas aeruginosa represents a major clinical concern. The interplay between antimicrobial resistance and virulence of P. aeruginosa was investigated in in vitro and in vivo studies. Thirty-eight well-characterized (21 MDR and 17 non-MDR) P. aeruginosa strains from patients with bacteraemia were analysed. Resistance phenotype, carbapenemase production, clonal relatedness, type III secretion system genotype, O-antigen serotype, cytotoxicity (ability to lyse cells) on A549 cells, and virulence (lethality in nematodes) in a Caenorhabditis elegans model were investigated. MDR strains showed lower cytotoxicity (35.4 ± 21.30% vs. 45.0 ± 18.78 %; P = 0.044) and virulence (66.7% vs. 100%; P = 0.011) than non-MDR strains. However, the pathogenicity of MDR high-risk clones varied broadly, with ST235 and ST175 clones being the most and least cytotoxic (51.8 ± 10.59% vs. 11.0 ± 1.25%; P < 0.0001) and virulent ([100% vs. 73.1; P = 0.075] and [0% vs. 93.9%; P < 0.0001], respectively). The pathogenicity of the ST235 clone was similar to that of non-MDR strains, and its ability to lyse cells and high virulence were related with the exoU-positive genotype. Furthermore, the O11 serotype was more frequent among the ST235 clone and exoU-positive genotype strains and was also essential for the pathogenicity of P. aeruginosa. Our data suggest that the pathogenicity of MDR high-risk clones is the result not only of the resistance phenotype but also of the virulence genotype. These findings have implications for the clinical management of patients and infection control programmes.

Association between Pseudomonas aeruginosa O-antigen serotypes, resistance profiles and high-risk clones: results from a Spanish nationwide survey.

OBJECTIVES: To evaluate the correlation of O-antigen serotypes with resistance profiles and high-risk clones in a Spanish nationwide survey. METHODS: Up to 30 consecutive healthcare-associated Pseudomonas aeruginosa isolates were collected during October 2017 from each of 51 hospitals (covering all Spanish regions) with a total of 1445 isolates studied. MICs of 13 antipseudomonal agents and MDR/XDR profiles had been previously determined, as well as whole-genome sequences of 185 representative XDR isolates. O-antigen serotypes (O1-O16) were determined by agglutination using serotype-specific antisera (BioRad). The Pseudomonas aeruginosa serotyper (PAst) program was used for in silico serotyping. RESULTS: The most frequent serotypes were O6 (17.8%), O1 (15.4%) and O11 (13.3%). In contrast, the most frequent serotype among XDR isolates (17.3%) was O4 (34.1%), distantly followed by O11 (15.9%). Within serotypes, XDR phenotypes were more frequent for O12 (60.0%) and O4 (57.3%). The most frequent clone among the XDR isolates was ST175 (40.9%), followed by CC235 (10.7%), ST308 (5.2%) and CC111 (3.6%). Up to 81.6% of XDR ST175 isolates typed O4, whereas 18.4% were non-typeable. O4 genotype was detected in all sequenced (n=55) ST175 isolates. On the other hand, CC235 and ST308 were associated with O11, whereas CC111 was linked to serotype O12. CONCLUSIONS: O4 serotype is linked to the MDR/XDR profile of widespread ST175 (typically only susceptible to colistin, amikacin and the novel combinations ceftolozane/tazobactam and ceftazidime/avibactam) and therefore, after local validation, its detection in the microbiology laboratory might be useful for guiding semi-empirical antipseudomonal therapies and infection control measures in Spanish hospitals.

In Vivo Validation of Peptidoglycan Recycling as a Target to Disable AmpC-Mediated Resistance and Reduce Virulence Enhancing the Cell-Wall-Targeting Immunity.

BACKGROUND: Searching for new strategies to defeat Pseudomonas aeruginosa is of paramount importance. Previous works in vitro showed that peptidoglycan recycling blockade disables AmpC-dependent resistance and enhances susceptibility against cell-wall-targeting immunity. Our objective was to validate these findings in murine models.This study shows for the first time in different murine models of infection that blocking the peptidoglycan recycling in Pseudomonas aeruginosa causes an important virulence impairment and disables AmpC-mediated resistance, being hence validated as a promising therapeutic target. METHODS: Wildtype PAO1, recycling-defective AmpG and NagZ mutants, an AmpC hyperproducer dacB mutant, and their combinations were used to cause systemic/respiratory infections in mice. Their survival, bacterial burden, inflammation level, and effectiveness of ceftazidime or subtherapeutic colistin to treat the infections were assessed. RESULTS: Inactivation of AmpG or NagZ significantly attenuated the virulence in terms of mice mortality, bacterial load, and inflammation. When inactivating these genes in the dacB-defective background, the ß-lactam resistance phenotype was abolished, disabling the emergence of ceftazidime-resistant mutants, and restoring ceftazidime for treatment. Subtherapeutic colistin was shown to efficiently clear the infection caused by the recycling-defective strains, likely due to the combined effect with the mice cell-wall- targeting immunity. CONCLUSIONS: This study brings us one step closer to new therapies intended to disable P. aeruginosa AmpC-mediated resistance and dampen its virulence, and strongly support the interest in developing efficient AmpG and/or NagZ inhibitors.

Spanish nationwide survey on Pseudomonas aeruginosa antimicrobial resistance mechanisms and epidemiology.

OBJECTIVES: To undertake a Spanish nationwide survey on Pseudomonas aeruginosa molecular epidemiology and antimicrobial resistance. METHODS: Up to 30 consecutive healthcare-associated P. aeruginosa isolates collected in 2017 from each of 51 hospitals were studied. MICs of 13 antipseudomonal agents were determined by broth microdilution. Horizontally acquired ß-lactamases were detected by phenotypic methods and PCR. Clonal epidemiology was evaluated through PFGE and MLST; at least one XDR isolate from each clone and hospital (n = 185) was sequenced. RESULTS: The most active antipseudomonals against the 1445 isolates studied were colistin and ceftolozane/tazobactam (both 94.6% susceptible, MIC50/90 = 1/2 mg/L) followed by ceftazidime/avibactam (94.2% susceptible, MIC50/90 = 2/8 mg/L). Up to 252 (17.3%) of the isolates were XDR. Carbapenemases/ESBLs were detected in 3.1% of the isolates, including VIM, IMP, GES, PER and OXA enzymes. The most frequent clone among the XDR isolates was ST175 (40.9%), followed by CC235 (10.7%), ST308 (5.2%) and CC111 (4.0%). Carbapenemase production varied geographically and involved diverse clones, including 16.5% of ST175 XDR isolates. Additionally, 56% of the sequenced XDR isolates showed horizontally acquired aminoglycoside-modifying enzymes, which correlated with tobramycin resistance. Two XDR isolates produced QnrVC1, but fluoroquinolone resistance was mostly caused by QRDR mutations. Beyond frequent mutations (>60%) in OprD and AmpC regulators, four isolates showed AmpC mutations associated with resistance to ceftolozane/tazobactam and ceftazidime/avibactam. CONCLUSIONS: ST175 is the most frequent XDR high-risk clone in Spanish hospitals, but this nationwide survey also indicates a complex scenario in which major differences in local epidemiology, including carbapenemase production, need to be acknowledged in order to guide antimicrobial therapy.

Deciphering ß-lactamase-independent ß-lactam resistance evolution trajectories in Pseudomonas aeruginosa.

Background: While resistance related to the expression of ß-lactamases, such as AmpC from Pseudomonas aeruginosa, has been deeply studied, this work addresses the gap in the knowledge of other potential bacterial strategies to overcome the activity of ß-lactams when ß-lactamases are not expressed. Methods: We analysed ß-lactam resistance evolution trajectories in a WT strain and in isogenic mutants either lacking AmpC (AmpC mutant) or unable to express it (AmpG mutant), exposed to increasing concentrations of ceftazidime for 7 days in quintuplicate experiments. Characterization of evolved lineages included susceptibility profiles, whole-genome sequences, resistance mechanisms, fitness (competitive growth assays) and virulence (Caenorhabditis elegans model). Results: Development of resistance was faster for the WT strain but, after 7 days, all strains reached clinical ceftazidime resistance levels. The main resistance mechanism in the WT strain was ampC overexpression, due to mutations in dacB and ampD or mpl. In contrast, ampC overexpression did not evolve in any of the AmpG lineages. Moreover, sequencing of the ΔAmpC and ΔAmpG evolved lineages revealed alternative resistance mutations (not seen in WT lineages) that included, in all cases, large (50-600 kb) deletions of specific chromosomal regions together with mutations leading to ß-lactam target [ftsI (PBP3)] modification and/or the overexpression or structural modification of the efflux pump MexAB-OprM. Finally, evolved lineages from the AmpC and, especially, AmpG mutants showed a reduced fitness and virulence. Conclusions: In addition to providing new insights into ß-lactam resistance mechanisms and evolution, our findings should be helpful for guiding future strategies to combat P. aeruginosa infections.

Interplay among Resistance Profiles, High-Risk Clones, and Virulence in the Caenorhabditis elegans Pseudomonas aeruginosa Infection Model.

The increasing prevalence of nosocomial infections produced by multidrug-resistant (MDR) or extensively drug-resistant (XDR) Pseudomonas aeruginosa is frequently linked to widespread international strains designated high-risk clones. In this work, we attempted to decipher the interplay between resistance profiles, high-risk clones, and virulence, testing a large (n = 140) collection of well-characterized P. aeruginosa isolates from different sources (bloodstream infections, nosocomial outbreaks, cystic fibrosis, and the environment) in a Caenorhabditis elegans infection model. Consistent with previous data, we documented a clear inverse correlation between antimicrobial resistance and virulence in the C. elegans model. Indeed, the lowest virulence was linked to XDR profiles, which were typically linked to defined high-risk clones. However, virulence varied broadly depending on the involved high-risk clone; it was high for sequence type 111 (ST111) and ST235 but very low for ST175. The highest virulence of ST235 could be attributed to its exoU+ type III secretion system (TTSS) genotype, which was found to be linked with higher virulence in our C. elegans model. Other markers, such as motility or pigment production, were not essential for virulence in the C. elegans model but seemed to be related with the higher values of the statistical normalized data. In contrast to ST235, the ST175 high-risk clone, which is widespread in Spain and France, seems to be associated with a particularly low virulence in the C. elegans model. Moreover, the previously described G154R AmpR mutation, prevalent in ST175, was found to contribute to the reduced virulence, although it was not the only factor involved. Altogether, our results provide a major step forward for understanding the interplay between P. aeruginosa resistance profiles, high-risk clones, and virulence.

Synergistic activity of fosfomycin, ß-lactams and peptidoglycan recycling inhibition against Pseudomonas aeruginosa.

OBJECTIVES: To evaluate the interconnection between peptidoglycan (PG) recycling, fosfomycin susceptibility and synergy between fosfomycin and ß-lactams in Pseudomonas aeruginosa METHODS: Fosfomycin MICs were determined by broth microdilution and Etest for a panel of 47 PAO1 mutants defective in several components of PG recycling and/or AmpC induction pathways. PAO1 fosfomycin MICs were also determined in the presence of a 5 mM concentration of the NagZ inhibitor PUGNAc. Population analysis of fosfomycin susceptibility and characterization of the resistant mutants that emerged was also performed for selected strains. Finally, fosfomycin, imipenem and fosfomycin + imipenem killing curves were assessed. RESULTS: Mutants defective in AmpG, NagZ or all three AmpD amidases showed a marked increase in fosfomycin susceptibility (at least two 2-fold dilutions with respect to WT PAO1). Moreover, PAO1 fosfomycin MICs were consistently reduced from 48 to 24 mg/L in the presence of a 5 mM concentration of PUGNAc. Fosfomycin hypersusceptibility of the ampG, nagZ and triple ampD mutants was also clearly confirmed in the performed population analysis, although the emergence of resistant mutants, through GlpT mutations, was not avoided. Synergy between fosfomycin and imipenem was evidenced for the WT strain, the AmpC-hyperproducing strain (triple AmpD mutant) and the NagZ and AmpG mutants in killing curves. Moreover, regrowth of resistant mutants was not evidenced for the combination. CONCLUSIONS: PG recycling inhibitors are envisaged as useful adjuvants in the treatment of P. aeruginosa infections with ß-lactams and fosfomycin and therefore further development of these molecules is encouraged.

Role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins in AmpC expression, ß-lactam resistance, and peptidoglycan structure.

This study aimed to characterize the role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins (LMM PBPs), namely, PBP4 (DacB), PBP5 (DacC), and PBP7 (PbpG), in peptidoglycan composition, ß-lactam resistance, and ampC regulation. For this purpose, we constructed all single and multiple mutants of dacB, dacC, pbpG, and ampC from the wild-type P. aeruginosa PAO1 strain. Peptidoglycan composition was determined by high-performance liquid chromatography (HPLC), ampC expression by reverse transcription-PCR (RT-PCR), PBP patterns by a Bocillin FL-binding test, and antimicrobial susceptibility by MIC testing for a panel of ß-lactams. Microscopy and growth rate analyses revealed no apparent major morphological changes for any of the mutants compared to the wild-type PAO1 strain. Of the single mutants, only dacC mutation led to significantly increased pentapeptide levels, showing that PBP5 is the major dd-carboxypeptidase in P. aeruginosa. Moreover, our results indicate that PBP4 and PBP7 play a significant role as dd-carboxypeptidase only if PBP5 is absent, and their dd-endopeptidase activity is also inferred. As expected, the inactivation of PBP4 led to a significant increase in ampC expression (around 50-fold), but, remarkably, the sequential inactivation of the three LMM PBPs produced a much greater increase (1,000-fold), which correlated with peptidoglycan pentapeptide levels. Finally, the ß-lactam susceptibility profiles of the LMM PBP mutants correlated well with the ampC expression data. However, the inactivation of ampC in these mutants also evidenced a role of LMM PBPs, especially PBP5, in intrinsic ß-lactam resistance. In summary, in addition to assessing the effect of P. aeruginosa LMM PBPs on peptidoglycan structure for the first time, we obtained results that represent a step forward in understanding the impact of these PBPs on ß-lactam resistance, apparently driven by the interplay between their roles in AmpC induction, ß-lactam trapping, and dd-carboxypeptidase/ß-lactamase activity.