In vitro activity of human defensins HNP-1 and hBD-3 against multidrug-resistant ESKAPE Gram-negatives of clinical origin and selected peptidoglycan recycling-defective mutants.

The use of immune compounds as antimicrobial adjuvants is a classic idea recovering timeliness in the current antibiotic resistance scenario. However, the activity of certain antimicrobial peptides against ESKAPE Gram-negatives has not been sufficiently investigated. The objective of this study was to determine the activities of human defensins HNP-1 and hBD-3 alone or combined with permeabilizing/peptidoglycan-targeting agents against clinical ESKAPE Gram-negatives [Acinetobacter baumannii (AB), Enterobacter cloacae (EC), Klebsiella pneumoniae (KP), and acute/chronic Pseudomonas aeruginosa (PA)]. Lethal concentrations (LCs) of HNP-1 and hBD-3 were determined in four collections of multidrug resistant EC, AB, KP, and PA clinical strains (10-36 isolates depending on the collection). These defensins act through membrane permeabilization plus peptidoglycan building blockade, enabling that alterations in peptidoglycan recycling may increase their activity, which is why different recycling-defective mutants were also included. Combinations with physiological lysozyme and subinhibitory colistin for bactericidal activities determination, and with meropenem for minimum inhibitory concentrations (MICs), were also assessed. HNP-1 showed undetectable activity (LC > 32 mg/L for all strains). hBD-3 showed appreciable activities: LC ranges 2-16, 8-8, 8->32, and 8->32 mg/L for AB, EC, KP, and PA, being PA strains from cystic fibrosis significantly more resistant than acute origin ones. None of the peptidoglycan recycling-defective mutants showed greater susceptibility to HNP-1/hBD-3. Combination with colistin or lysozyme did not change their bactericidal power, and virtually neither did meropenem + hBD-3 compared to meropenem MICs. This is the first study comparatively analyzing the HNP-1/hBD-3 activities against the ESKAPE Gram-negatives, and demonstrates interesting bactericidal capacities of hBD-3 mostly against AB and EC. IMPORTANCE: In the current scenario of critical need for new antimicrobials against multidrug-resistant bacteria, all options must be considered, including classic ideas such as the use of purified immune compounds. However, information regarding the activity of certain human defensins against ESKAPE Gram-negatives was incomplete. This is the first study comparatively assessing the in vitro activity of two membrane-permeabilizing/peptidoglycan construction-blocking defensins (HNP-1 and hBD-3) against relevant clinical collections of ESKAPE Gram-negatives, alone or in combination with permeabilizers, additional peptidoglycan-targeting attacks, or the blockade of its recycling. Our data suggest that hBD-3 has a notable bactericidal activity against multidrug-resistant Acinetobacter baumannii and Enterobacter cloacae strains that should be considered as potential adjuvant option. Our results suggest for the first time an increased resistance of Pseudomonas aeruginosa strains from chronic infection compared to acute origin ones, and provide new clues about the predominant mode of action of hBD-3 against Gram-negatives (permeabilization rather than peptidoglycan-targeting).

Transferable AmpCs in Klebsiella pneumoniae: interplay with peptidoglycan recycling, mechanisms of hyperproduction, and virulence implications.

Chromosomal and transferable AmpC ß-lactamases represent top resistance mechanisms in different gram-negatives, but knowledge regarding the latter, mostly concerning regulation and virulence-related implications, is far from being complete. To fill this gap, we used Klebsiella pneumoniae (KP) and two different plasmid-encoded AmpCs [DHA-1 (AmpR regulator linked, inducible) and CMY-2 (constitutive)] as models to perform a study in which we show that blockade of peptidoglycan recycling through AmpG permease inactivation abolished DHA-1 inducibility but did not affect CMY-2 production and neither did it alter KP pathogenic behavior. Moreover, whereas regular production of both AmpC-type enzymes did not attenuate KP virulence, when blaDHA-1 was expressed in an ampG-defective mutant, Galleria mellonella killing was significantly (but not drastically) attenuated. Spontaneous DHA-1 hyperproducer mutants were readily obtained in vitro, showing slight or insignificant virulence attenuations together with high-level resistance to ß-lactams only mildly affected by basal production (e.g., ceftazidime, ceftolozane/tazobactam). By analyzing diverse DHA-1-harboring clinical KP strains, we demonstrate that the natural selection of these hyperproducers is not exceptional (>10% of the collection), whereas mutational inactivation of the typical AmpC hyperproduction-related gene mpl was the most frequent underlying mechanism. The potential silent dissemination of this kind of strains, for which an important fitness cost-related contention barrier does not seem to exist, is envisaged as a neglected threat for most ß-lactams effectiveness, including recently introduced combinations. Analyzing whether this phenomenon is applicable to other transferable ß-lactamases and species as well as determining the levels of conferred resistance poses an essential topic to be addressed.IMPORTANCEAlthough there is solid knowledge about the regulation of transferable and especially chromosomal AmpC ß-lactamases in Enterobacterales, there are still gaps to fill, mainly related to regulatory mechanisms and virulence interplays of the former. This work addresses them using Klebsiella pneumoniae as model, delving into a barely explored conception: the acquisition of a plasmid-encoded inducible AmpC-type enzyme whose production can be increased through selection of chromosomal mutations, entailing dramatically increased resistance compared to basal expression but minor associated virulence costs. Accordingly, we demonstrate that clinical K. pneumoniae DHA-1 hyperproducer strains are not exceptional. Through this study, we warn for the first time that this phenomenon may be a neglected new threat for ß-lactams effectiveness (including some recently introduced ones) silently spreading in the clinical context, not only in K. pneumoniae but potentially also in other pathogens. These facts must be carefully considered in order to design future resistance-preventive strategies.

In vitro dynamics and mechanisms of cefiderocol resistance development in wild-type, mutator and XDR Pseudomonas aeruginosa.

OBJECTIVES: To analyse the dynamics and mechanisms of stepwise resistance development to cefiderocol in Pseudomonas aeruginosa. METHODS: Cefiderocol resistance evolution was analysed in WT PAO1, PAOMS (mutS mutator derivate) and three XDR clinical isolates belonging to ST111, ST175 and ST235 clones. Strains were incubated in triplicate experiments for 24 h in iron-depleted CAMHB with 0.06-128 mg/L cefiderocol. Tubes from the highest antibiotic concentration showing growth were reinoculated into fresh medium containing concentrations up to 128 mg/L for 7 consecutive days. Two colonies per strain and experiment were characterized by determining the susceptibility profiles and WGS. RESULTS: Evolution of resistance was significantly enhanced in PAOMS, but was variable for the XDR strains, including levels similar to PAOMS (ST235), similar to PAO1 (ST175) or even below PAO1 (ST111). WGS revealed 2-5 mutations for PAO1 lineages and 35-58 for PAOMS. The number of mutations in the XDR clinical strains ranged from 2 to 4 except for one of the ST235 experiments in which a mutL lineage was selected, thus increasing the number of mutations. The most frequently mutated genes were piuC, fptA and pirR, related to iron uptake. Additionally, an L320P AmpC mutation was selected in multiple lineages and cloning confirmed its major impact on cefiderocol (but not ceftolozane/tazobactam or ceftazidime/avibactam) resistance. Mutations in CpxS and PBP3 were also documented. CONCLUSIONS: This work deciphers the potential resistance mechanisms that may emerge upon the introduction of cefiderocol into clinical practice, and highlights that the risk of resistance development might be strain-specific even for XDR high-risk clones.

Biochemical Insights into Imipenem Collateral Susceptibility Driven by ampC Mutations Conferring Ceftolozane/Tazobactam Resistance in Pseudomonas aeruginosa.

Several Pseudomonas aeruginosa AmpC mutants have emerged that exhibit enhanced activity against ceftazidime and ceftolozane, while also evading inhibition by avibactam. Interestingly, P. aeruginosa strains harboring these AmpC mutations fortuitously exhibit enhanced carbapenem susceptibility. This acquired susceptibility was investigated by comparing the degradation of imipenem by wild-type and cephalosporin-resistant AmpC. We show that cephalosporin-resistant AmpC enzymes lose their efficacy for hydrolyzing imipenem and suggest that this may be due to their increased flexibility and dynamics relative to the wild type.

Role of Enzymatic Activity in the Biological Cost Associated with the Production of AmpC ß-Lactamases in Pseudomonas aeruginosa.

In the current scenario of growing antibiotic resistance, understanding the interplay between resistance mechanisms and biological costs is crucial for designing therapeutic strategies. In this regard, intrinsic AmpC ß-lactamase hyperproduction is probably the most important resistance mechanism of Pseudomonas aeruginosa, proven to entail important biological burdens that attenuate virulence mostly under peptidoglycan recycling alterations. P. aeruginosa can acquire resistance to new ß-lactam-ß-lactamase inhibitor combinations (ceftazidime-avibactam and ceftolozane-tazobactam) through mutations affecting ampC and its regulatory genes, but the impact of these mutations on the associated biological cost and the role that ß-lactamase activity plays per se in contributing to the above-mentioned virulence attenuation are unknown. The same questions remain unsolved for plasmid-encoded AmpC-type ß-lactamases such as FOX enzymes, some of which also provide resistance to new ß-lactam-ß-lactamase inhibitor combinations. Here, we assessed from different perspectives the effects of changes in the active center and, thus, in the hydrolytic spectrum resistance to inhibitors of AmpC-type ß-lactamases on the fitness and virulence of P. aeruginosa, using site-directed mutagenesis; the previously described AmpC variants T96I, G183D, and ΔG229-E247; and, finally, blaFOX-4 versus blaFOX-8. Our results indicate the essential role of AmpC activity per se in causing the reported full virulence attenuation (in terms of growth, motility, cytotoxicity, and Galleria mellonella larvae killing), although the biological cost of the above-mentioned AmpC-type variants was similar to that of the wild-type enzymes. This suggests that there is not an important biological burden that may limit the selection/spread of these variants, which could progressively compromise the future effectiveness of the above-mentioned drug combinations. IMPORTANCE The growing antibiotic resistance of the top nosocomial pathogen Pseudomonas aeruginosa pushes research to explore new therapeutic strategies, for which the resistance-versus-virulence balance is a promising source of targets. While resistance often entails significant biological costs, little is known about the bases of the virulence attenuations associated with a resistance mechanism as extraordinarily relevant as ß-lactamase production. We demonstrate that besides potential energy and cell wall alterations, the enzymatic activity of the P. aeruginosa cephalosporinase AmpC is essential for causing the full attenuation associated with its hyperproduction by affecting different features related to pathogenesis, a fact exploitable from the antivirulence perspective. Less encouraging, we also show that the production of different chromosomal/plasmid-encoded AmpC derivatives conferring resistance to some of the newest antibiotic combinations causes no significantly increased biological burdens, which suggests a free way for the selection/spread of these types of variants, potentially compromising the future effectiveness of these antipseudomonal therapies.

Activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam against ceftolozane/tazobactam- and ceftazidime/avibactam-resistant Pseudomonas aeruginosa.

OBJECTIVES: To evaluate the activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam against a clinical and laboratory collection of ceftolozane/tazobactam- and ceftazidime/avibactam-resistant Pseudomonas aeruginosa ß-lactamase mutants. METHODS: The activity of cefiderocol, imipenem/relebactam, cefepime/taniborbactam, cefepime/zidebactam and comparators was evaluated against a collection of 30 molecularly characterized ceftolozane/tazobactam- and/or ceftazidime/avibactam-resistant P. aeruginosa isolates from patients previously treated with cephalosporins. To evaluate how the different ß-lactamases in the clinical isolates affected the resistance to these agents, a copy of each blaPDC, blaOXA-2 and blaOXA-10 ancestral and mutant allele from the clinical isolates was cloned in pUCp24 and expressed in dual blaPDC-oprD (for blaPDC-like genes) or single oprD (for blaOXA-2-like and blaOXA-10-like genes) PAO1 knockout mutants. MICs were determined using reference methodologies. RESULTS: For all isolates, MICs were higher than 4 and/or 8 mg/L for ceftolozane/tazobactam and ceftazidime/avibactam, respectively. Cefiderocol was the most active agent, showing activity against all isolates, except one clinical isolate that carried an R504C substitution in PBP3 (MIC = 16 mg/L). Imipenem/relebactam was highly active against all isolates, except two clinical isolates that carried the VIM-20 carbapenemase. Cefepime/zidebactam and cefepime/taniborbactam displayed activity against most of the isolates, but resistance was observed in some strains with PBP3 amino acid substitutions or that overexpressed mexAB-oprM or mexXY efflux pumps. Evaluation of transformants revealed that OXA-2 and OXA-10 extended-spectrum variants cause a 2-fold increase in the MIC of cefiderocol relative to parental enzymes. CONCLUSIONS: Cefiderocol, imipenem/relebactam, cefepime/taniborbactam and cefepime/zidebactam show promising and complementary in vitro activity against ceftolozane/tazobactam- and ceftazidime/avibactam-resistant P. aeruginosa. These agents may represent potential therapeutic options for ceftolozane/tazobactam- and ceftazidime/avibactam-resistant P. aeruginosa infections.

Comparative analysis of in vitro dynamics and mechanisms of ceftolozane/tazobactam and imipenem/relebactam resistance development in Pseudomonas aeruginosa XDR high-risk clones.

OBJECTIVES: To analyse the dynamics and mechanisms of stepwise resistance development to ceftolozane/tazobactam and imipenem/relebactam in XDR Pseudomonas aeruginosa clinical strains. METHODS: XDR clinical isolates belonging to ST111 (main resistance mechanisms: oprD-, dacB-, CARB-2), ST175 (oprD-, ampR-G154R) and ST235 (oprD-, OXA-2) high-risk clones were incubated for 24 h in Müeller-Hinton Broth with 0.125-64 mg/L of ceftolozane + tazobactam 4 mg/L or imipenem + relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated into fresh medium containing concentrations up to 64 mg/L for 7 consecutive days. Two colonies per strain from each of the triplicate experiments were characterized by determining the susceptibility profiles, whole genome sequencing (WGS), and in vitro fitness through competitive growth assays. RESULTS: Resistance development occurred more slowly and reached a lower level for imipenem/relebactam than for ceftolozane/tazobactam in all tested XDR strains. Moreover, resistance development to imipenem/relebactam remained low even for ST175 isolates that had developed ceftolozane/tazobactam resistance during therapy. Lineages evolved in the presence of ceftolozane/tazobactam showed high-level resistance, imipenem/relebactam hypersusceptibility and low fitness cost, whereas lineages evolved in the presence of imipenem/relebactam showed moderate (borderline) resistance, no cross-resistance to ceftolozane/tazobactam and high fitness cost. WGS evidenced that ceftolozane/tazobactam resistance was mainly caused by mutations in the catalytic centres of intrinsic (AmpC) or acquired (OXA) ß-lactamases, whereas lineages evolved in imipenem/relebactam frequently showed structural mutations in MexB or in ParS, along with some strain-specific mutations. CONCLUSIONS: Imipenem/relebactam could be a useful alternative for the treatment of XDR P. aeruginosa infections, potentially reducing resistance development during therapy.

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.

A Genomic Snapshot of the SARS-CoV-2 Pandemic in the Balearic Islands.

Objective: To analyze the SARS-CoV-2 genomic epidemiology in the Balearic Islands, a unique setting in which the course of the pandemic has been influenced by a complex interplay between insularity, severe social restrictions and tourism travels. Methods: Since the onset of the pandemic, more than 2,700 SARS-CoV-2 positive respiratory samples have been randomly selected and sequenced in the Balearic Islands. Genetic diversity of circulating variants was assessed by lineage assignment of consensus whole genome sequences with PANGOLIN and investigation of additional spike mutations. Results: Consensus sequences were assigned to 46 different PANGO lineages and 75% of genomes were classified within a VOC, VUI, or VUM variant according to the WHO definitions. Highest genetic diversity was documented in the island of Majorca (42 different lineages detected). Globally, lineages B.1.1.7 and B.1.617.2/AY.X were identified as the 2 major lineages circulating in the Balearic Islands during the pandemic, distantly followed by lineages B.1.177/B.1.177.X. However, in Ibiza/Formentera lineage distribution was slightly different and lineage B.1.221 was the third most prevalent. Temporal distribution analysis showed that B.1 and B.1.5 lineages dominated the first epidemic wave, lineage B.1.177 dominated the second and third, and lineage B.1.617.2 the fourth. Of note, lineage B.1.1.7 became the most prevalent circulating lineage during first half of 2021; however, it was not associated with an increased in COVID-19 cases likely due to severe social restrictions and limited travels. Additional spike mutations were rarely documented with the exception of mutation S:Q613H which has been detected in several genomes (n = 25) since July 2021. Conclusion: Virus evolution, mainly driven by the acquisition and selection of spike substitutions conferring biological advantages, social restrictions, and size population are apparently key factors for explaining the epidemic patterns registered in the Balearic Islands.

Molecular mechanisms driving the in vivo development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR Pseudomonas aeruginosa infections.

BACKGROUND: The development of resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of Pseudomonas aeruginosa infections is concerning. OBJECTIVES: Characterization of the mechanisms leading to the development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR P. aeruginosa infections. METHODS: Four paired ceftolozane/tazobactam- and ceftazidime/avibactam-susceptible/resistant isolates were evaluated. MICs were determined by broth microdilution. STs, resistance mechanisms and genetic context of ß-lactamases were determined by genotypic methods, including WGS. The OXA-10 variants were cloned in PAO1 to assess their impact on resistance. Models for the OXA-10 derivatives were constructed to evaluate the structural impact of the amino acid changes. RESULTS: The same XDR ST253 P. aeruginosa clone was detected in all four cases evaluated. All initial isolates showed OprD deficiency, produced an OXA-10 enzyme and were susceptible to ceftazidime, ceftolozane/tazobactam, ceftazidime/avibactam and colistin. During treatment, the isolates developed resistance to all cephalosporins. Comparative genomic analysis revealed that the evolved resistant isolates had acquired mutations in the OXA-10 enzyme: OXA-14 (Gly157Asp), OXA-794 (Trp154Cys), OXA-795 (ΔPhe153-Trp154) and OXA-824 (Asn143Lys). PAO1 transformants producing the evolved OXA-10 derivatives showed enhanced ceftolozane/tazobactam and ceftazidime/avibactam resistance but decreased meropenem MICs in a PAO1 background. Imipenem/relebactam retained activity against all strains. Homology models revealed important changes in regions adjacent to the active site of the OXA-10 enzyme. The blaOXA-10 gene was plasmid borne and acquired due to transposition of Tn6746 in the pHUPM plasmid scaffold. CONCLUSIONS: Modification of OXA-10 is a mechanism involved in the in vivo acquisition of resistance to cephalosporin/ß-lactamase inhibitor combinations in P. aeruginosa.

Whole-genome sequence-guided PCR for the rapid identification of the Pseudomonas aeruginosa ST175 high-risk clone directly from clinical samples.

OBJECTIVES: Pseudomonas aeruginosa frequently show MDR/XDR profiles, which are associated with worldwide-disseminated high-risk clones (HRCs). We developed a PCR assay for the detection in clinical samples of ST175, an HRC that is widespread in European countries. METHODS: The whole-genome sequence was obtained for one ST175 isolate using a PacBio RSII sequencer. Reads from multiple isolates belonging to ST175 and the PAO1 reference strain were mapped against the ST175 genome to identify potentially specific regions. Once curated, using the BLAST database to search for the presence of those regions in any other organism, we designed a specific PCR for the detection of ST175. RESULTS: Assembly of the ST175 PacBio-sequenced genome resulted in three contigs with a total length of 7 087 985 bases, encoding 6566 coding sequences. Specific regions for ST175 genomes were detected and a PCR targeting a 318 bp fragment located within a 3177 bp ORF coding for a putative reverse transcriptase was designed. The PCR test was first evaluated in silico against 229 XDR P. aeruginosa genomes (73 ST175) from two multicentre studies, yielding 100% sensitivity and specificity. Then, the PCR was evaluated in vitro in 25 isolates (12 ST175) and in 120 clinical samples (30 urine samples, 30 blood cultures, 30 sputum samples and 30 rectal swabs) of which 10% contained ST175, yielding again 100% sensitivity and specificity. CONCLUSIONS: The PCR assay developed, showing high sensitivity and specificity for the detection of the ST175 HRC directly from clinical samples, could become a useful tool for guiding infection control and treatment strategies in areas with a high prevalence of this clone.

Characterization of AmpC Beta-lactamase mutations of extensively drug-resistant Pseudomonas aeruginosa isolates that develop resistance to ceftolozane/tazobactam during therapy / Caracterización de las mutaciones en la betalactamasa AmpC de aislados de Pseudomonas aeruginosa XDR que desarrollaron resistencia a ceftolozano/tazobactam durante el tratamiento

INTRODUCTION: We characterized AmpC β-lactamase mutations that resulted in ceftolozane/tazobactam resistance in extensively drug-resistant (XDR) Pseudomonas aeruginosa isolates recovered from patients treated with this agent from June 2016 to December 2018. METHODS: Five pairs of ceftolozane/tazobactam susceptible/resistant P. aeruginosa XDR isolates were included among a total of 49 patients treated. Clonal relationship among isolates was first evaluated by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) was further performed. AmpC mutations were investigated by PCR amplification of the blaPDC gene followed by sequencing. RESULTS: The ST175 high-risk clone was detected in four of the pairs of isolates and the ST1182 in the remaining one. All resistant isolates showed a mutation in AmpC: T96I in two of the isolates, and E247K, G183V, and a deletion of 19 amino acids (G229-E247) in the other three. The G183V mutation had not been described before. The five isolates resistant to ceftolozane/tazobactam showed cross-resistance to ceftazidime/avibactam and lower MICs of imipenem and piperacillin/tazobactam than the susceptible isolates. CONCLUSIONS: Ceftolozane/tazobactam resistance was associated in all of the cases with AmpC mutations, including a novel mutation (G183V) not previously described. There is a vital need for surveillance and characterization of emerging ceftolozane/tazobactam resistance, in order to preserve this valuable antipseudomonal agent

INTRODUCCIÓN: Se han caracterizado las mutaciones en la betalactamasa AmpC que han producido resistencia a ceftolozano/tazobactam en aislados de Pseudomonas aeruginosa extremadamente resistente (XDR) en pacientes tratados con este agente desde junio de 2016 hasta diciembre de 2018. MÉTODOS: Se incluyeron 5 pares de aislados (sensibles/resistentes a ceftolozano/tazobactam) de P. aeruginosa XDR entre un total de 49 pacientes tratados. Se estudió la relación clonal mediante electroforesis en campo pulsado y MLST. Las mutaciones en AmpC se caracterizaron mediante amplificación por PCR del gen blaPDC y posterior secuenciación. RESULTADOS: Se detectó el clon de alto riesgo ST175 en 4 pares de aislados y el ST1182 en el restante. Todos los aislados resistentes mostraron una mutación en AmpC: T96I en 2 aislados, E247K, G183V y una deleción de 19 aminoácidos (G229-E247) en los otros 3. La mutación G183V no había sido descrita antes. Los 5 aislados resistentes a ceftolozano/tazobactam mostraron resistencia cruzada a ceftazidima/avibactam y CMI inferiores de imipenem y piperacilina/tazobactam que los aislados sensibles. CONCLUSIONES: La resistencia a ceftolozano/tazobactam se asoció con mutaciones en AmpC en todos los casos, incluida una nueva mutación G183V no descrita con anterioridad. La vigilancia y caracterización de la resistencia emergente a ceftolozano/tazobactam es de gran importancia para preservar este nuevo agente antipseudomónico

Molecular and biochemical insights into the in vivo evolution of AmpC-mediated resistance to ceftolozane/tazobactam during treatment of an MDR Pseudomonas aeruginosa infection.

BACKGROUND: Pseudomonas aeruginosa may develop resistance to novel cephalosporin/ß-lactamase inhibitor combinations during therapy through the acquisition of structural mutations in AmpC. OBJECTIVES: To describe the molecular and biochemical mechanisms involved in the development of resistance to ceftolozane/tazobactam in vivo through the selection and overproduction of a novel AmpC variant, designated PDC-315. METHODS: Paired susceptible/resistant isolates obtained before and during ceftolozane/tazobactam treatment were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. Characterization of the novel PDC-315 variant was performed through genotypic and biochemical studies. The effects at the molecular level of the Asp245Asn change were analysed by molecular dynamics simulations using Amber. RESULTS: WGS identified mutations leading to modification (Asp245Asn) and overproduction of AmpC. Susceptibility testing revealed that PAOΔC producing PDC-315 displayed increased MICs of ceftolozane/tazobactam, decreased MICs of piperacillin/tazobactam and imipenem and similar susceptibility to ceftazidime/avibactam compared with WT PDCs. The catalytic efficiency of PDC-315 for ceftolozane was 10-fold higher in relation to the WT PDCs, but 3.5- and 5-fold lower for piperacillin and imipenem. IC50 values indicated strong inhibition of PDC-315 by avibactam, but resistance to cloxacillin inhibition. Analysis at the atomic level explained that the particular behaviour of PDC-315 is linked to conformational changes in the H10 helix that favour the approximation of key catalytic residues to the active site. CONCLUSIONS: We deciphered the precise mechanisms that led to the in vivo emergence of resistance to ceftolozane/tazobactam in P. aeruginosa through the selection of the novel PDC-315 enzyme. The characterization of this new variant expands our knowledge about AmpC-mediated resistance to cephalosporin/ß-lactamase inhibitors in P. aeruginosa.

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.

Nosocomial outbreak linked to a flexible gastrointestinal endoscope contaminated with an amikacin-resistant ST17 clone of Pseudomonas aeruginosa.

Endoscope contamination is infrequent but can be the source of nosocomial infections and outbreaks. In August 2016, an unexpected increase in the incidence of amikacin-resistant P. aeruginosa isolates (AK-Pae) was observed at a tertiary care center in the south of Spain. An epidemiological and microbiological investigation (August-October 2016) was performed to explain this finding. Isolates from clinical and environmental samples (2 endoscopes used for retrograde cholangiopancreatography; ERCP) were identified by MALDI-TOF. Antimicrobial susceptibility testing was performed using the MicroScan system. Whole-Genome-Sequencing (Miseq, Illumina) was performed to determine the resistome and virulome. Clonal relatedness among isolates was assessed by SpeI-PFGE and MLST. A Caenorhabditis elegans killing assay was performed for virulence testing. Biofilm formation was performed using a colorimetric assay. Four of the 5 patients infected and/or colonized with AK-Pae in August 2016 had undergone ERCP ≤5 days before sample collection. Two endoscopes were contaminated with AK-Pae. Isolates from one endoscope showed an identical PFGE pattern to 9 isolates (cluster I) and differed (1-2 bands) to 5 isolates (cluster II). Isolates from these clusters belonged to the ST17 clone. This S17 clone was characterized by its low virulence in the C. elegans killing assay, and its biofilm-forming ability, slightly superior to that of high-risk clones of P. aeruginosa ST175 and ST235. This outbreak was caused by an endoscope used for ERCP contaminated with an invasive, moderately virulent, biofilm-forming AK-Pae ST17 clone, suggesting the possible emergence of a new high-risk lineage of this clone.

Characterization of AmpC ß-lactamase mutations of extensively drug-resistant Pseudomonas aeruginosa isolates that develop resistance to ceftolozane/tazobactam during therapy.

INTRODUCTION: We characterized AmpC ß-lactamase mutations that resulted in ceftolozane/tazobactam resistance in extensively drug-resistant (XDR) Pseudomonas aeruginosa isolates recovered from patients treated with this agent from June 2016 to December 2018. METHODS: Five pairs of ceftolozane/tazobactam susceptible/resistant P. aeruginosa XDR isolates were included among a total of 49 patients treated. Clonal relationship among isolates was first evaluated by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) was further performed. AmpC mutations were investigated by PCR amplification of the blaPDC gene followed by sequencing. RESULTS: The ST175 high-risk clone was detected in four of the pairs of isolates and the ST1182 in the remaining one. All resistant isolates showed a mutation in AmpC: T96I in two of the isolates, and E247K, G183V, and a deletion of 19 amino acids (G229-E247) in the other three. The G183V mutation had not been described before. The five isolates resistant to ceftolozane/tazobactam showed cross-resistance to ceftazidime/avibactam and lower MICs of imipenem and piperacillin/tazobactam than the susceptible isolates. CONCLUSIONS: Ceftolozane/tazobactam resistance was associated in all of the cases with AmpC mutations, including a novel mutation (G183V) not previously described. There is a vital need for surveillance and characterization of emerging ceftolozane/tazobactam resistance, in order to preserve this valuable antipseudomonal agent.

Publisher Correction: Profiling the susceptibility of Pseudomonas aeruginosa strains from acute and chronic infections to cell-wall-targeting immune proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Effective inhibition of PBPs by cefepime and zidebactam in the presence of VIM-1 drives potent bactericidal activity against MBL-expressing Pseudomonas aeruginosa.

OBJECTIVES: The combination of cefepime and the novel ß-lactam enhancer zidebactam (WCK 5222) is under development for the treatment of difficult-to-treat Gram-negative infections. Against MBL-producing pathogens, cefepime and zidebactam induce cell elongation and spheroplast formation, indicating PBP3 and PBP2 dysfunction, respectively, having a potent bactericidal effect as a combination. The objective of the present study was to determine the mechanistic basis of the bactericidal effect of cefepime/zidebactam on MBL-expressing pathogens. METHODS: Pseudomonal PBP-binding affinities of cefepime, zidebactam and imipenem were assessed at different timepoints and also in the presence of purified VIM-1 using a Bocillin FL competition assay. The antibacterial activity of cefepime/zidebactam against three VIM-expressing Pseudomonas aeruginosa isolates was assessed by time-kill and neutropenic mouse lung/thigh infection studies. RESULTS: Amidst cefepime-hydrolysing concentrations of VIM-1, substantial cefepime binding to target PBPs was observed. High-affinity binding of zidebactam to PBP2 remained unaltered in the presence of VIM-1; however, MBL addition significantly affected imipenem PBP2 binding. Furthermore, the rate of cefepime binding to the primary target PBP3 was found to be higher compared with the imipenem PBP2 binding rate. Finally, complementary PBP inhibition by cefepime/zidebactam resulted in enhanced bactericidal activity in time-kill and neutropenic mouse lung/thigh infection studies against VIM-6-, VIM-10- and VIM-11-expressing P. aeruginosa, thus revealing the mechanistic basis of ß-lactam enhancer action. CONCLUSIONS: For the first time ever (to the best of our knowledge), this study demonstrates that in the presence of VIM-1 MBL, ß-lactamase-labile cefepime and ß-lactamase-stable zidebactam produce effective inhibition of respective target PBPs. For cefepime, this seems to be a result of a faster rate of PBP binding, which helps it overcome ß-lactamase-mediated hydrolysis.

Predicting antimicrobial resistance in Pseudomonas aeruginosa with machine learning-enabled molecular diagnostics.

Limited therapy options due to antibiotic resistance underscore the need for optimization of current diagnostics. In some bacterial species, antimicrobial resistance can be unambiguously predicted based on their genome sequence. In this study, we sequenced the genomes and transcriptomes of 414 drug-resistant clinical Pseudomonas aeruginosa isolates. By training machine learning classifiers on information about the presence or absence of genes, their sequence variation, and expression profiles, we generated predictive models and identified biomarkers of resistance to four commonly administered antimicrobial drugs. Using these data types alone or in combination resulted in high (0.8-0.9) or very high (> 0.9) sensitivity and predictive values. For all drugs except for ciprofloxacin, gene expression information improved diagnostic performance. Our results pave the way for the development of a molecular resistance profiling tool that reliably predicts antimicrobial susceptibility based on genomic and transcriptomic markers. The implementation of a molecular susceptibility test system in routine microbiology diagnostics holds promise to provide earlier and more detailed information on antibiotic resistance profiles of bacterial pathogens and thus could change how physicians treat bacterial infections.

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.