Impact of chromosomally encoded resistance mechanisms and transferable ß-lactamases on the activity of cefiderocol and innovative ß-lactam/ß-lactamase inhibitor combinations against Pseudomonas aeruginosa.

OBJECTIVES: We aimed to compare the stability of the newly developed ß-lactams (cefiderocol) and ß-lactam/ß-lactamase inhibitor combinations (ceftazidime/avibactam, ceftolozane/tazobactam, aztreonam/avibactam, cefepime/taniborbactam, cefepime/zidebactam, imipenem/relebactam, meropenem/vaborbactam, meropenem/nacubactam and meropenem/xeruborbactam) against the most clinically relevant mechanisms of mutational and transferable ß-lactam resistance in Pseudomonas aeruginosa. METHODS: We screened a collection of 61 P. aeruginosa PAO1 derivatives. Eighteen isolates displayed the most relevant mechanisms of mutational resistance to ß-lactams. The other 43 constructs expressed transferable ß-lactamases from genes cloned in pUCP-24. MICs were determined by reference broth microdilution. RESULTS: Cefiderocol and imipenem/relebactam exhibited excellent in vitro activity against all of the mutational resistance mechanisms studied. Aztreonam/avibactam, cefepime/taniborbactam, cefepime/zidebactam, meropenem/vaborbactam, meropenem/nacubactam and meropenem/xeruborbactam proved to be more vulnerable to mutational events, especially to overexpression of efflux operons. The agents exhibiting the widest spectrum of activity against transferable ß-lactamases were aztreonam/avibactam and cefepime/zidebactam, followed by cefepime/taniborbactam, cefiderocol, meropenem/xeruborbactam and meropenem/nacubactam. However, some MBLs, particularly NDM enzymes, may affect their activity. Combined production of certain enzymes (e.g. NDM-1) with increased MexAB-OprM-mediated efflux and OprD deficiency results in resistance to almost all agents tested, including last options such as aztreonam/avibactam and cefiderocol. CONCLUSIONS: Cefiderocol and new ß-lactam/ß-lactamase inhibitor combinations show promising and complementary in vitro activity against mutational and transferable P. aeruginosa ß-lactam resistance. However, the combined effects of efflux pumps, OprD deficiency and efficient ß-lactamases could still result in the loss of all therapeutic options. Resistance surveillance, judicious use of new agents and continued drug development efforts are encouraged.

Neonatal SARS-CoV-2 immunoglobulin G antibodies at delivery and their impact on COVID-19.

OBJECTIVE: To assess neonatal SARS-CoV-2 anti-spike IgG antibody levels after maternal mRNA COVID-19 vaccination and/or infection during pregnancy and evaluate their protective effect. METHODS: Prospective observational study, conducted from January 2021 to December 2022. Infants were tested for anti-spike IgG antibodies at birth and then every 3 months until disappearance of titer. A follow-up was done for SARS-CoV-2 infection up to 12 months. RESULTS: In total, 147 newborns were enrolled with a median (IQR) gestational age of 39.60 weeks (38.3-40.4). Median (IQR) titers in UA/ml at 2 days were higher (P < .001) in newborns of vaccinated 7063.7 (2841.4-14,448.1), than of infected mothers 372.7 (158.00-884.90). Titers dropped significantly during the follow-up but 50% still had a detectable titer at 6 months. A high antibody titer at 2 days led to a longer persistence (HR 0.89, IC 95% 0.83-0.96, P = .004). In total, 36 infants were infected during the first months of life coinciding with the Omicron variant. Fifty percent had detectable antibodies during the infection period. Relationship between high IgG titers and month of infection was inverse (RHO - 0.52, P = .009). CONCLUSION: Though a high antibody titer at birth led to longer persistence, no protective effect against infection was found. As newborns are a high risk group for COVID-19, avoiding transmission during the first year of life is important.

Clinical Characteristics and Microorganisms Isolated in Community-Acquired Pneumonia in the COVID-19 Period.

Introduction: Community-acquired pneumonia is a leading cause of mortality and hospital admissions. The aetiology remains unknown in 30-65% of the cases. Molecular tests are available for multiple pathogen detection and are under research to improve the causal diagnosis. Methods: We carried out a prospective study to describe the clinical characteristics and aetiology of community-acquired pneumonia during the COVID-19 pandemic and to assess the diagnostic effectivity of the microbiological tests, including a molecular test of respiratory pathogens (FilmArray™ bioMérieux). Results: From the 1st of February 2021 until the 31st of March 2022, 225 patients were included. Failure in microorganism identification occurred in approximately 70% of patients. Streptococcus pneumoniae was the most common isolate. There were 5 cases of viral pneumonia. The tested FilmArray exhibited a low positivity rate of 7% and mainly aided in the diagnosis of viral coinfections. Conclusions: Despite our extensive diagnostic protocol, there is still a low rate of microorganism identification. We have observed a reduction in influenza and other viral pneumoniae during the COVID-19 pandemic. Having a high NEWS2 score on arrival at the emergency department, an active oncohematological disease or chronic neurological conditions and a positive microbiological test result were related to worse outcomes. Further research is needed to determine the role of molecular tests in the microbiological diagnosis of pneumonia.

Elevated complement C3 and increased CD8 and type 1 helper lymphocyte T populations in patients with post-COVID-19 condition.

BACKGROUND: Biological markers associated to post-COVID-19 condition (PCC) have not been clearly identified. METHODS: Eighty-two patients attending our post-COVID-19 outpatient clinic were recruited and classified as fully recovered (40.2%) or presenting with PCC (59.8%). Clinical and radiological data, laboratory markers, cytokines, and lymphocyte populations were analyzed. RESULTS: Median number of days after hospitalization was 78.5 [p25-p75: 60-93] days. PCC was significantly more frequent in women, in patients with a previously critical COVID-19, and in those with two or more comorbidities. No differences were found in lymphocyte counts, ferritin, C-reactive protein, D-dimer or sCD25, IL-1ß, IL-1Ra, IL-6, CXCL8, IL-17A, IL-18, IL-22, IFN-γ, TNF-α, and IL-10 cytokines levels. PCC patients showed significantly higher levels of complement factor C3 than fully recovered patients: median C3 128 mg/dL [p25-p75:107-135] vs 111 mg/dL [p25-p75: 100-125] (p =.005), respectively. In the flow cytometry assessment of peripheral blood lymphocyte subpopulations, PCC patients showed significantly increased CD8 populations compared to fully recovered patients: median CD8: 529 [p25-p75: 384-683] vs 370/mm3 [p25-p75:280-523], p =.007. When type 1, 2, 17/22, and 17.1 helper and follicular T lymphocyte subpopulations were analyzed, the frequency of Th1 was significantly higher in PCC patients compared to fully recovered patients (30% vs 38.5%, p =.028). CONCLUSION: Patients with a post-COVID-19 condition showed significantly increased immunological parameters of inflammation (complement factor C3 and CD8 and Th1 T lymphocyte populations) compared to fully recovered patients. These parameters could be used as biological markers of this condition.

Selection of AmpC ß-Lactamase Variants and Metallo-ß-Lactamases Leading to Ceftolozane/Tazobactam and Ceftazidime/Avibactam Resistance during Treatment of MDR/XDR Pseudomonas aeruginosa Infections.

Infections caused by ceftolozane-tazobactam and ceftazidime-avibactam-resistant P. aeruginosa infections are an emerging concern. We aimed to analyze the underlying ceftolozane-tazobactam and ceftazidime-avibactam resistance mechanisms in all multidrug-resistant or extensively drug-resistant (MDR/XDR) P. aeruginosa isolates recovered during 1 year (2020) from patients with a documented P. aeruginosa infection. Fifteen isolates showing ceftolozane-tazobactam and ceftazidime-avibactam resistance were evaluated. Clinical conditions, previous positive cultures, and ß-lactams received in the previous month were reviewed for each patient. MICs were determined by broth microdilution. Multilocus sequence types (MLSTs) and resistance mechanisms were determined using short- and long-read whole-genome sequencing (WGS). The impact of Pseudomonas-derived cephalosporinases (PDCs) on ß-lactam resistance was demonstrated by cloning into an ampC-deficient PAO1 derivative (PAOΔC) and construction of 3D models. Genetic support of acquired ß-lactamases was determined in silico from high-quality hybrid assemblies. In most cases, the isolates were recovered after treatment with ceftolozane-tazobactam or ceftazidime-avibactam. Seven isolates from different sequence types (STs) owed their ß-lactam resistance to chromosomal mutations and all displayed specific substitutions in PDC: Phe121Leu and Gly222Ser, Pro154Leu, Ala201Thr, Gly214Arg, ΔGly203-Glu219, and Glu219Lys. In the other eight isolates, the ST175 clone was overrepresented (6 isolates) and associated with IMP-28 and IMP-13, whereas two ST1284 isolates produced VIM-2. The cloned PDCs conferred enhanced cephalosporin resistance. The 3D PDC models revealed rearrangements affecting residues involved in cephalosporin hydrolysis. Carbapenemases were chromosomal (VIM-2) or plasmid-borne (IMP-28, IMP-13) and associated with class-1 integrons located in Tn402-like transposition modules. Our findings highlighted that cephalosporin/ß-lactamase inhibitors are potential selectors of MDR/XDR P. aeruginosa strains producing PDC variants or metallo-ß-lactamases. Judicious use of these agents is encouraged.

In vivo translational assessment of the GES genotype on the killing profile of ceftazidime, ceftazidime/avibactam and meropenem against Pseudomonas aeruginosa.

OBJECTIVES: To evaluate the in vivo killing profile of human-simulated exposures of ceftazidime, ceftazidime/avibactam and meropenem against GES-harbouring Pseudomonas aeruginosa in the murine thigh infection model. METHODS: Five P. aeruginosa isolates [three isogenic (GES-1, GES-5 and GES-15) and two clinical (GES-5 and GES-15)] were evaluated. MICs were determined using broth microdilution. Human-simulated regimens (HSRs) of ceftazidime 2 g IV q8h as a 2 h infusion, ceftazidime/avibactam 2.5 g IV q8h as a 2 h infusion and meropenem 2 g IV q8h as a 3 h infusion were administered. Change in bacterial burden relative to baseline was assessed. RESULTS: Modal MICs ranged from 8 to >64 mg/L for ceftazidime, from 1 to 16 mg/L for ceftazidime/avibactam and from 1 to >64 mg/L for meropenem. In vivo, for the isogenic strains, avibactam augmented ceftazidime activity against the GES-1- and GES-15-harbouring isolates. Both ceftazidime and ceftazidime/avibactam resulted in significant kill against the GES-5 isogenic isolate. The meropenem HSR produced >1 log10 kill against each isogenic isolate (MICs of 1-4 mg/L). Against the GES-5 clinical isolate, ceftazidime and ceftazidime/avibactam resulted in >1 log10 kill compared with bacterial growth with the meropenem HSR. In the clinical isolate harbouring GES-15, the elevated MICs of ceftazidime and ceftazidime/avibactam reduced the effectiveness of both compounds, while the observed reduction in meropenem MIC translated into in vivo efficacy of the HSR regimen, predictive of clinical efficacy. CONCLUSIONS: In GES-harbouring P. aeruginosa, quantitative reductions in bacterial density observed with the translational murine model suggest that the phenotypic profile of ceftazidime, ceftazidime/avibactam and meropenem is predictive of clinical efficacy when using the evaluated dosing regimens.

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.

Spread of SARS-CoV-2 in hospital areas.

We performed a systematic sampling and analysis of airborne SARS-CoV-2 RNA in different hospital areas to assess viral spread. Systematic air filtration was performed in rooms with COVID-19 infected patients, in corridors adjacent to these rooms, to rooms of intensive care units, and to rooms with infected and uninfected patients, and in open spaces. RNA was extracted from the filters and real-time reverse transcription polymerase chain reaction was performed using the LightMix Modular SARS-CoV-2 E-gene. The highest occurrence of RNA was found in the rooms with COVID-19 patients (mean 2600 c/m3) and the adjacent corridor (mean 4000 c/m3) which was statistically significant more exposed (p < 0.01). This difference was related to the ventilation systems. As is commonly found in many hospitals, each of the rooms had an individual air inlet and outlet, while in the corridors these devices were located at the distance of every four rooms. There was a significant transfer of viruses from the COVID-19 patients' rooms to the corridors. The airborne SARS-CoV-2 RNA in the corridors of ICUs with COVID-19 patients or care rooms of uninfected patients were ten times lower, averages 190 c/m3 and 180 c/m3, respectively, without presenting significant differences. In all COVID-19 ICU rooms, patients were intubated and connected to respirators that filtered all exhaled air and prevented virus release, resulting in significantly lower viral concentrations in adjacent corridors. The results show that the greatest risk of nosocomial infection may also occur in hospital areas not directly exposed to the exhaled breath of infected patients. Hospitals should evaluate the ventilation systems of all units to minimize possible contagion and, most importantly, direct monitoring of SARS-CoV-2 in the air should be carried out to prevent unexpected viral exposures.

In Vivo Evolution of GES ß-Lactamases Driven by Ceftazidime/Avibactam Treatment of Pseudomonas aeruginosa Infections.

The mechanisms underlying an in vivo switch in the resistance phenotype of P. aeruginosa after ceftazidime-avibactam treatment was investigated. The initial isolate (a blood culture) was resistant to meropenem but remained susceptible to antipseudomonal cephalosporins and combinations with ß-lactamase inhibitors. One week after ceftazidime-avibactam therapy, a subsequent isolate (a rectal swab) recovered from the same patient showed the opposite phenotype. Whole-genome sequence analysis revealed a single SNP difference between both (ST235) isolates, leading to a P162S change in blaGES-5, creating blaGES-15. Thus, blaGES-1, blaGES-5, and blaGES-15 were cloned and expressed in the wild-type strain PAO1. Susceptibility profiles confirmed the P162S substitution reverted the carbapenemase phenotype determined by the G170S change of GES-5 back into the ESBL phenotype of GES-1.

Predictive Immunological, Virological, and Routine Laboratory Markers for Critical COVID-19 on Admission.

BACKGROUND: Early identification of COVID-19 patients at risk of critical illness is a challenging endeavor for clinicians. We aimed to establish immunological, virological, and routine laboratory markers, which, in combination with clinical information, may allow identifying such patients. METHODS: Blood tests to measure neutrophil/lymphocyte ratio (NLR) and levels of ferritin, CRP, D-dimer, complement components (C3 and C4), cytokines, and lymphocyte subsets, as well as SARS-Cov-2 RT-PCR tests, were performed in COVID-19-confirmed cases within 48 hours of admission. RT-PCR cycle threshold (Ct) values from oropharyngeal or nasopharyngeal swabs were determined on the day of admission. Symptom severity was categorized as mild (grade 1), severe (grade 2), or critical (grade 3). RESULTS: Of 120 patients who were included, 49 had mild, 32 severe, and 39 critical COVID-19. Levels of ferritin >370 ng/mL (OR 16.4, 95% CI 5.3-50.8), D-dimer >440 ng/mL (OR 5.45, 95% CI 2.36-12.61), CRP >7.65 mg/dL (OR 11.54, 95% CI 4.3-30.8), NLR >3.77 (OR 13.4, 95% CI 4.3-41.1), IL-6 >142.5 pg/mL (OR 8.76, 95% CI 3.56-21.54), IL-10 >10.8 pg/mL (OR 16.45, 95% CI 5.32-50.81), sIL-2rα (sCD25) >804.5 pg/mL (OR 14.06, 95% CI 4.56-43.28), IL-1Ra >88.4 pg/mL (OR 4.54, 95% CI 2.03-10.17), and IL-18 >144 pg/mL (OR 17.85, 95% CI 6.54-48.78) were associated with critical COVID-19 in the univariate age-adjusted analysis. This association was confirmed in the multivariate age-adjusted analysis only for ferritin, CRP, NLR, IL-10, sIL-2rα, and IL-18. T, B, and NK cells were significantly decreased in critical patients. SARS-CoV-2 was not detected in blood except in 3 patients who had indeterminate results. RT-PCR Ct values from oropharyngeal or nasopharyngeal swabs on admission were not related to symptom severity. CONCLUSION: Ferritin, D-dimer, CRP, NLR, cytokine (IL-18 and IL-10), and cytokine receptor (IL-6, IL1-Ra, and sCD25) test results combined with clinical data can contribute to the early identification of critical COVID-19 patients.

Adding Insult to Injury: Mechanistic Basis for How AmpC Mutations Allow Pseudomonas aeruginosa To Accelerate Cephalosporin Hydrolysis and Evade Avibactam.

Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide and notorious for its broad-spectrum resistance to antibiotics. A key mechanism that provides extensive resistance to ß-lactam antibiotics is the inducible expression of AmpC ß-lactamase. Recently, a number of clinical isolates expressing mutated forms of AmpC have been found to be clinically resistant to the antipseudomonal ß-lactam-ß-lactamase inhibitor (BLI) combinations ceftolozane-tazobactam and ceftazidime-avibactam. Here, we compare the enzymatic activity of wild-type (WT) AmpC from PAO1 to those of four of these reported AmpC mutants, bearing mutations E247K (a change of E to K at position 247), G183D, T96I, and ΔG229-E247 (a deletion from position 229 to 247), to gain detailed insights into how these mutations allow the circumvention of these clinically vital antibiotic-inhibitor combinations. We found that these mutations exert a 2-fold effect on the catalytic cycle of AmpC. First, they reduce the stability of the enzyme, thereby increasing its flexibility. This appears to increase the rate of deacylation of the enzyme-bound ß-lactam, resulting in greater catalytic efficiencies toward ceftolozane and ceftazidime. Second, these mutations reduce the affinity of avibactam for AmpC by increasing the apparent activation barrier of the enzyme acylation step. This does not influence the catalytic turnover of ceftolozane and ceftazidime significantly, as deacylation is the rate-limiting step for the breakdown of these antibiotic substrates. It is remarkable that these mutations enhance the catalytic efficiency of AmpC toward ceftolozane and ceftazidime while simultaneously reducing susceptibility to inhibition by avibactam. Knowledge gained from the molecular analysis of these and other AmpC resistance mutants will, we believe, aid in the design of ß-lactams and BLIs with reduced susceptibility to mutational resistance.

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.

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.

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.

Challenging Antimicrobial Susceptibility and Evolution of Resistance (OXA-681) during Treatment of a Long-Term Nosocomial Infection Caused by a Pseudomonas aeruginosa ST175 Clone.

Selection of extended-spectrum mutations in narrow-spectrum oxacillinases (e.g., OXA-2 and OXA-10) is an emerging mechanism for development of in vivo resistance to ceftolozane-tazobactam and ceftazidime-avibactam in Pseudomonas aeruginosa Detection of these challenging enzymes therefore seems essential to prevent clinical failure, but the complex phenotypic plasticity exhibited by this species may often lead to their underestimation. The underlying resistance mechanisms of two sequence type 175 (ST175) P. aeruginosa isolates showing multidrug-resistant phenotypes and recovered at early and late stages of a long-term nosocomial infection were evaluated. Whole-genome sequencing (WGS) was used to investigate resistance genomics, whereas molecular and biochemical methods were used for characterization of a novel extended-spectrum OXA-2 variant selected during therapy. The metallo-ß-lactamase blaVIM-20 and the narrow-spectrum oxacillinase blaOXA-2 were present in both isolates, although they differed by an inactivating mutation in the mexB subunit, present only in the early isolate, and in a mutation in the blaOXA-2 ß-lactamase, present only in the final isolate. The new OXA-2 variant, designated OXA-681, conferred elevated MICs of the novel cephalosporin-ß-lactamase inhibitor combinations in a PAO1 background. Compared to OXA-2, kinetic parameters of the OXA-681 enzyme revealed a substantial increase in the hydrolysis of cephalosporins, including ceftolozane. We describe the emergence of the novel variant OXA-681 during treatment of a nosocomial infection caused by a Pseudomonas aeruginosa ST175 high-risk clone. The ability of OXA-681 to confer cross-resistance to ceftolozane-tazobactam and ceftazidime-avibactam together with the complex antimicrobial resistance profiles exhibited by the clinical strains harboring this new enzyme argue for maintaining active surveillance on emerging broad-spectrum resistance in P. aeruginosa.

Mechanisms leading to in vivo ceftolozane/tazobactam resistance development during the treatment of infections caused by MDR Pseudomonas aeruginosa.

Objectives: Characterization of the mechanisms driving ceftolozane/tazobactam resistance development in 5 of 47 (10.6%) patients treated for MDR Pseudomonas aeruginosa infections in a Spanish hospital. Methods: Five pairs of ceftolozane/tazobactam-susceptible/resistant P. aeruginosa isolates were studied. MICs were determined by broth microdilution, clonal relatedness was assessed by MLST and resistance mechanisms were investigated by phenotypic and genotypic methods, including WGS. ampC variants were cloned to assess their impact on resistance. Results: In all five cases, the same clone was detected for the susceptible/resistant pairs; the widespread ST175 high-risk clone in four of the cases and ST179 in the remaining case. Genomic analysis of the four initial ST175 isolates revealed the characteristic OprD mutation (Q142X) responsible for carbapenem resistance and the AmpR mutation (G154R) responsible for AmpC overexpression and ß-lactam resistance. The final isolates had developed ceftolozane/tazobactam and ceftazidime/avibactam resistance, and each additionally showed a mutation in AmpC: E247K in one of the isolates, T96I in two isolates and a deletion of 19 amino acids (G229-E247) in the remaining isolate. The cloned AmpC variants showed greatly increased ceftolozane/tazobactam and ceftazidime/avibactam MICs compared with WT AmpC, but, in contrast, yielded lower MICs of imipenem, cefepime and particularly piperacillin/tazobactam. On the other hand, ceftolozane/tazobactam resistance development in ST179 was shown to be driven by the emergence of the extended-spectrum OXA ß-lactamase OXA-14, through the selection of an N146S mutation from OXA-10. Conclusions: Modification of intrinsic (AmpC) and horizontally acquired ß-lactamases appears to be the main mechanism leading to ceftolozane/tazobactam resistance in MDR P. aeruginosa.

Ceftolozane/tazobactam for the treatment of multidrug resistant Pseudomonas aeruginosa: experience from the Balearic Islands.

A prospective, descriptive observational study of consecutive patients treated with ceftolozane/tazobactam in the reference hospital of the Balearic Islands (Spain), between May 2016 and September 2017, was performed. Demographic, clinical, and microbiological variables were recorded. The later included resistance profile, molecular typing, and whole genome sequencing of isolates showing resistance development. Fifty-eight patients were treated with ceftolozane/tazobactam. Thirty-five (60.3%) showed respiratory tract infections, 21 (36.2%) received monotherapy, and 37 (63.8%) combined therapy for ≥ 72 h, mainly with colistin (45.9%). In 46.6% of the patients, a dose of 1/0.5 g/8 h was used, whereas 2/1 g/8 h was used in 41.4%. In 56 of the cases (96.6%), the initial Pseudomonas aeruginosa isolates recovered showed a multidrug resistant (MDR) phenotype, and 50 of them (86.2%) additionally met the extensively drug resistant (XDR) criteria and were only susceptible colistin and/or aminoglycosides (mostly amikacin). The epidemic high-risk clone ST175 was detected in 50% of the patients. Clinical cure was documented in 37 patients (63.8%) and resistance development in 8 (13.8%). Clinical failure was associated with disease severity (SOFA), ventilator-dependent respiratory failure, XDR profile, high-risk clone ST175, negative control culture, and resistance development. In 6 of the 8 cases, resistance development was caused by structural mutations in AmpC, including some mutations described for the first time in vivo, whereas in the other 2, by mutations in OXA-10 leading to the extended spectrum OXA-14. Although further clinical experience is still needed, our results suggest that ceftolozane/tazobactam is an attractive option for the treatment of MDR/XDR P. aeruginosa infections.

In Vivo Emergence of Resistance to Novel Cephalosporin-ß-Lactamase Inhibitor Combinations through the Duplication of Amino Acid D149 from OXA-2 ß-Lactamase (OXA-539) in Sequence Type 235 Pseudomonas aeruginosa.

Resistance development to novel cephalosporin-ß-lactamase inhibitor combinations during ceftazidime treatment of a surgical infection by Pseudomonas aeruginosa was investigated. Both initial (97C2) and final (98G1) isolates belonged to the high-risk clone sequence type (ST) 235 and were resistant to carbapenems (oprD), fluoroquinolones (GyrA-T83I, ParC-S87L), and aminoglycosides (aacA7/aacA8/aadA6). 98G1 also showed resistance to ceftazidime, ceftazidime-avibactam, and ceftolozane-tazobactam. Sequencing identified blaOXA-2 in 97C2, but 98G1 contained a 3-bp insertion leading to the duplication of the key residue D149 (designated OXA-539). Evaluation of PAO1 transformants producing cloned OXA-2 or OXA-539 confirmed that D149 duplication was the cause of resistance. Active surveillance of the emergence of resistance to these new valuable agents is warranted.

Activity of cefiderocol and innovative ß-lactam/ß-lactamase inhibitor combinations against isogenic strains of Escherichia coli expressing single and double ß-lactamases under high and low permeability conditions.

OBJECTIVES: To analyse the impact of the most clinically relevant ß-lactamases and their interplay with low outer membrane permeability on the activity of cefiderocol, ceftazidime/avibactam, aztreonam/avibactam, cefepime/enmetazobactam, cefepime/taniborbactam, cefepime/zidebactam, imipenem/relebactam, meropenem/vaborbactam, meropenem/xeruborbactam and meropenem/nacubactam against recombinant Escherichia coli strains. METHODS: We constructed 82 E. coli laboratory transformants expressing the main ß-lactamases circulating in Enterobacterales (70 expressing single ß-lactamase and 12 producing double carbapenemase) under high (E. coli TG1) and low (E. coli HB4) permeability conditions. Antimicrobial susceptibility testing was determined by reference broth microdilution. RESULTS: Aztreonam/avibactam, cefepime/zidebactam, cefiderocol, meropenem/xeruborbactam and meropenem/nacubactam were active against all E. coli TG1 transformants. Imipenem/relebactam, meropenem/vaborbactam, cefepime/taniborbactam and cefepime/enmetazobactam were also highly active, but unstable against most of MBL-producing transformants. Combination of ß-lactamases with porin deficiency (E. coli HB4) did not significantly affect the activity of aztreonam/avibactam, cefepime/zidebactam, cefiderocol or meropenem/nacubactam, but limited the effectiveness of the rest of carbapenem- and cefepime-based combinations. Double-carbapenemase production resulted in the loss of activity of most of the compounds tested, an effect particularly evident for those E. coli HB4 transformants in which MBLs were present. CONCLUSIONS: Our findings highlight the promising activity that cefiderocol and new ß-lactam/ß-lactamase inhibitors have against recombinant E. coli strains expressing widespread ß-lactamases, including when these are combined with low permeability or other enzymes. Aztreonam/avibactam, cefiderocol, cefepime/zidebactam and meropenem/nacubactam will help to mitigate to some extent the urgency of new compounds able to resist MBL action, although NDM enzymes represent a growing challenge against which drug development efforts are still needed.