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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 β-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.

The aim was to develop and validate a Pseudomonas aeruginosa genotypic resistance score, based on analysis of the whole genome sequence resistome, to predict antimicrobial susceptibility phenotypes.A scoring system based on the analysis of mutation-driven resistance in 40 chromosomal genes and horizontally acquired resistance (Resfinder) was developed for ceftazidime, ceftolozane/tazobactam, meropenem, ciprofloxacin and tobramycin. Resistance genes/mutations were scored from 0 (no effect) to 1 (EUCAST clinical resistance). One hundred wild-type strains obtained from 51 different hospitals during a 2017 multicentre study were fully sequenced and analysed in order to define a catalogue of natural polymorphisms in the 40 chromosomal resistance genes. The capacity of genotypic score to predict the susceptibility phenotype was tested in 204 isolates randomly selected from the 51 hospitals (four from each hospital).The analysis of the 100 wild-type isolates yielded a catalogue of 455 natural polymorphisms in the 40 genes involved in mutational resistance. However, resistance mutations and high-risk clones (such as ST235) were also documented among a few wild-type isolates. Overall, the capacity of the genotypic score (0.5) for predicting phenotypic susceptibility (S + I in the case of meropenem) was very high (95-100%). In contrast, the capacity of the genotypic score to predict resistance (≥1) was far more variable depending on the agent. Prediction of meropenem clinical resistance was particularly low (18/39, 46.1%), whereas it classified clinical ceftolozane/tazobactam resistance in 100% (7/7) of cases.Although a margin for improvement was evidenced in this proof of concept study, an overall good correlation between the genotypic resistance score and the susceptibility profile was documented. Further refining of the scoring system, automatization and testing of large international cohorts should follow.

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.

[Objectives]: We assessed the association between the lethality of Pseudomonas aeruginosa in a Caenorhabditis elegans model and outcomes of P. aeruginosa bloodstream infections. [Methods]: A total of 593 P. aeruginosa bloodstream isolates recovered from a prospective Spanish multicentre study were analysed. Clinical variables, susceptibility profiles and Type III Secretion System (TTSS) genotypes (exoU/exoS genes) were available from previous studies. A C. elegans virulence score (CEVS) was used, classifying the isolates into high (CEVS 4–5), intermediate (CEVS 3) and low (CEVS 1–2) virulence. The main outcome analysed was 30-day mortality. [Results]: Up to 75% (446/593) of the isolates showed a high-virulence phenotype, and 17% (101/593) a low-virulence one. No association between virulence phenotype and the main outcome variable (30-day mortality) was found (29/101 (28.7%) versus 127/446 (28.5%), p 1). However, an inverse association between C. elegans virulence and multidrug-resistant and extensively drug-resistant profiles was documented (OR 0.655 (95% CI 0.571–0.751) and OR 0.523 (95% CI 0.436–0.627), p, This work was supported by Planes Nacionales de I+D+i 2008–2011/2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016) and grants PI12/00734, PI15/00088 and PI18/00076, co-financed by the European Development Regional Fund ‘A way to achieve Europe’ and operative programme Intelligent Growth 2014–2020. Sánchez-Diener I is the recipient of a P-FIS fellowship from Instituto de Salud Carlos III, Ministerio de Economía y Competitividad.

Background While resistance related to the expression of β-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 β-lactams when β-lactamases are not expressed. Methods We analysed β-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 β-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 β-lactam resistance mechanisms and evolution, our findings should be helpful for guiding future strategies to combat P. aeruginosa infections.

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, β-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 β-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 β-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 β-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 β-lactam resistance, apparently driven by the interplay between their roles in AmpC induction, β-lactam trapping, and DD-carboxypeptidase/β-lactamase activity., This work was supported by the Ministerio de Economía y Competitividad of Spain and the Instituto de Salud Carlos III through the Spanish Network for the Research in Infectious Diseases (grants RD06/0008 and RD12/0015) and grants PS09/00033 and PI12/00103. The work at CBMSO was supported by grants BFU2009-09200 from the Ministerio de Economía y Competitividad of Spain and 223431 DIVINOCELL from the European Union. We acknowledge the predoctoral grant JAE/Predoc from the Consejo Superior de Investigaciones Cientificas to A.R. The study is also supported by the Direcció General d’Universitats, Recerca i Transferència del Coneixement del Govern de les Illes Balears, Spain.

The increasing prevalence of nosocomial infections produced by multidrugresistant (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 highrisk 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., This work was supported by the Planes Nacionales de IDi 2008-2011/2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0006, RD12/0015/004, RD16/0016/ 0004, RD16/0016/008, and RD16/0016/0011) and grants PI15/00088 and PI12/00734. This work was cofinanced by grants from the European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth (2014-2020). I.S.-D. is the recipient of a P-FIS fellowship from Instituto de Salud Carlos III, Ministerio de Economía y Competitividad.

Objectives To evaluate the interconnection between peptidoglycan (PG) recycling, fosfomycin susceptibility and synergy between fosfomycin and β-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 β-lactams and fosfomycin and therefore further development of these molecules is encouraged.

ObjectivesWe 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.MethodsPAO1 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.ResultsRelebactam 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.ConclusionsImipenem/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.