In vitro activity of cefiderocol in Pseudomonas aeruginosa isolates from people with cystic fibrosis recovered during three multicentre studies in Spain.

OBJECTIVES: Despite the introduction of cystic fibrosis transmembrane conductance regulator (CFTR) modulators, Pseudomonas aeruginosa is still a major pathogen in people with cystic fibrosis (pwCF). We determine the activity of cefiderocol and comparators in a collection of 154 P. aeruginosa isolates recovered from pwCF during three multicentre studies performed in 17 Spanish hospitals in 2013, 2017 and 2021. METHODS: ISO broth microdilution was performed and MICs were interpreted with CLSI and EUCAST criteria. Mutation frequency and WGS were also performed. RESULTS: Overall, 21.4% were MDR, 20.8% XDR and 1.3% pandrug-resistant (PDR). Up to 17% of the isolates showed a hypermutator phenotype. Cefiderocol demonstrated excellent activity; only 13 isolates (8.4%) were cefiderocol resistant by EUCAST (none using CLSI). A high proportion of the isolates resistant to ceftolozane/tazobactam (71.4%), meropenem/vaborbactam (70.0%), imipenem/relebactam (68.0%) and ceftazidime/avibactam (55.6%) were susceptible to cefiderocol. Nine out of 13 cefiderocol-resistant isolates were hypermutators (P < 0.001). Eighty-three STs were detected, with ST98 being the most frequent. Only one isolate belonging to the ST175 high-risk clone carried blaVIM-2. Exclusive mutations affecting genes involved in membrane permeability, AmpC overexpression (L320P-AmpC) and efflux pump up-regulation were found in cefiderocol-resistant isolates (MIC = 4-8 mg/L). Cefiderocol resistance could also be associated with mutations in genes related to iron uptake (tonB-dependent receptors and pyochelin/pyoverdine biosynthesis). CONCLUSIONS: Our results position cefiderocol as a therapeutic option in pwCF infected with P. aeruginosa resistant to most recent ß-lactam/ß-lactamase inhibitor combinations.

Evaluation of CHROMagar™ B. cepacia agar for the detection of Burkholderia cepacia complex species from sputum samples of patients with cystic fibrosis.

INTRODUCTION: Burkholderia cepacia complex (BCC) are non-fermenting Gram-negative bacteria that can chronically colonize the lungs of people with cystic fibrosis (pwCF), causing a severe and progressive respiratory failure, post-transplant complications and epidemic outbreaks. Therefore, rapid and accurate identification of these bacteria is relevant for pwCF, in order to facilitate early eradication and prevent chronic colonization. However, BCCs are often quite difficult to detect on culture media as they have a slow growth rate and can be hidden by other fast-growing microorganisms, including Pseudomonas aeruginosa and filamentous fungi. MATERIAL AND METHODS: We evaluated the sensitivity of CHROMagar™ B. cepacia agar using 11 isolates from a well-characterized BCC collection, using BCA agar (Oxoid, UK) as a gold standard. We also studied 180 clinical sputum samples to calculate positive (PPV) and negative (NPV) predictive values. Furthermore, we used three of the well-characterized BCC isolates to determine the limit of detection (LOD). RESULTS: Eleven isolates grew on CHROMagar™ B. cepacia at 37ºC after 48 h. The NPV and PPV of CHROMagar™ B. cepacia were 100% and 87.5%, respectively. The LOD of CHROMagar™ B. cepacia was around 1 × 103 CFU/ml, requiring a ten-fold dilution lower bacterial load than BCA for BCC detection. CONCLUSION: CHROMagar™ B. cepacia agar proved to have a very good sensitivity and specificity for the detection of clinical BCCs. Moreover, the chromogenic nature of the medium allowed us to clearly differentiate BCC from other Gram-negative species, filamentous fungi and yeasts, thereby facilitating the identification of contaminants.

Occurrence of multi-carbapenemase-producing Enterobacterales in a tertiary hospital in Madrid (Spain): A new epidemiologic scenario.

INTRODUCTION: Multi-carbapenemase-producing Enterobacterales (M-CPE) are increasingly described. We characterized the M-CPE isolates prospectively recovered in our hospital (Madrid, Spain) over two years (2021-2022). METHODS: We collected 796 carbapenem resistant Enterobacterales (CRE) from clinical and surveillance samples. Carbapenemase production was confirmed with phenotypic (immunochromatographic, disk diffusion) and molecular (PCR, WGS) techniques. Antimicrobial susceptibility was evaluated by a standard broth microdilution method. Clinical and demographic data were collected. RESULTS: Overall, 23 M-CPE (10 Klebsiella pneumoniae, 6 Citrobacter freundii complex, 3 Escherichia coli, 2 Klebsiella oxytoca, and 2 Enterobacter hormaechei) isolates were recovered from 17 patients (3% with CPE, 0.26-0.28 cases per 1000 admissions). OXA-48 + KPC-3 (7/23) and KPC-3 + VIM-1 (5/23) were the most frequent carbapenemase combinations. All patients had prior antibiotics exposure, including carbapenems (8/17). High resistance rates to ceftazidime/avibactam (14/23), imipenem/relebactam (16/23) and meropenem/vaborbactam (7/23) were found. Ceftazidime/avibactam + aztreonam combination was synergistic in all metallo-ß-lactamase producers. Clonal and non-clonal related isolates were found, particularly in K. pneumoniae (5 ST29, 3 ST147, 3 ST307) and C. freundii (3 ST8, 2 ST125, 1 ST563). NDM-1 + OXA-48 was introduced with the ST147-K. pneumoniae high-risk clone linked to the transfer of a Ukrainian patient. We identified four possible nosocomial clonal transmission events between patients of the same clone with the same combination of carbapenemases (KPC-3 + VIM-1-ST29-K. pneumoniae, NDM-1 + OXA-48-ST147-K. pneumoniae and KPC-2 + VIM-1-ST145-K. oxytoca). Carbapenemase-encoding genes were located on different plasmids, except for VIM-1 + KPC-2-ST145-K. oxytoca. Cross-species transmission and a possible acquisition overtime was found, particularly between K. pneumoniae and E. coli producing OXA-48 + KPC-3. CONCLUSION: M-CPE is an emerging threat in our hospital. Co-production of different carbapenemases, including metallo-ß-lactamases, limits therapeutic options and depicts the need to reinforce infection control measures.

Evolution of ceftazidime-avibactam and cefiderocol resistance in ST131-H30R1-Escherichia coli isolates with KPC-3 mutants and application of FTIR biotyping.

Ceftazidime-avibactam and cefiderocol represent two of the few alternatives for infections by KPC-producing Enterobacterales. We reported the emergence of resistance to both ceftazidime-avibactam and cefiderocol in a KPC-producing ST131-Escherichia coli (KPC-ST131-Ec) clinical isolate. Antimicrobial susceptibility testing, Fourier-transform infrared (FTIR) spectroscopy, whole-genome sequencing, and cloning experiments were performed. A KPC-49-Ec isolate resistant to ceftazidime-avibactam (MICCZA > 16/4 mg/L) and susceptible to cefiderocol (MICFDC: 2 mg/L) was recovered in a blood sample from an oncologic patient hospitalized in the medical ICU (June 2019) during ceftazidime-avibactam treatment. After 44 days, a KPC-31-Ec resistant to both ceftazidime-avibactam and cefiderocol (MICCZA > 16/4 mg/L, MICFDC: 8 mg/L) was found in a rectal sample during a second cycle of ceftazidime-avibactam treatment. Both KPC-49 (R163S) and KPC-31 (D179Y) were detected in the epidemic ST131-H30R1-Ec high-risk clone and showed a phenotype resembling that of ESBL producers. FTIR spectroscopy managed to differentiate cefiderocol-susceptible and resistant ST131-Ec isolates, and these from others belonging to different clones. After cloning and transformation experiments, KPC-49 and KPC-31 were responsible for ceftazidime-avibactam resistance (MICCZA > 16/4 mg/L) and decreased carbapenem MICs (MICMER ≤ 0.12 mg/L, MICIMI ≤ 1 mg/L). KPC-31 was also shown to be associated with increased MICs of cefiderocol (twofold and threefold dilutions over KPC-3 and KPC-49, respectively). However, mutations in proteins participating in outer membrane stability and integrity, such as TolR, could have a more relevant role in cefiderocol resistance. The effects of ceftazidime-avibactam and cefiderocol co-resistance in clinical isolates of Enterobacterales producing KPC mutants make their identification challenging for clinical laboratories.IMPORTANCEThroughout four admissions in our hospital of a single patient, different KPC-3 variants (KPC-3, KPC-49, and KPC-31) were found in surveillance and clinical ST131-Escherichia coli isolates, after prolonged therapies with meropenem and ceftazidime-avibactam. Different patterns of resistance to cefiderocol and ceftazidime-avibactam emerged, accompanied by restored carbapenem susceptibility. The inability to detect these variants with some phenotypic methods, especially KPC-31 by immunochromatography, and the expression of a phenotype similar to that of ESBL producers, posed challenge to identify these variants in the clinical microbiology laboratory. Molecular methods and whole-genome sequencing are necessary and new techniques able to cluster or differentiate related isolates could also be helpful; this is the case of Fourier-transform infrared spectroscopy, which managed in our study to discriminate isolates by cefiderocol susceptibility within ST131, and those from the non-ST131 ones.

Cefiderocol resistance genomics in sequential chronic Pseudomonas aeruginosa isolates from cystic fibrosis patients.

OBJECTIVE: To evaluate the activity of cefiderocol against sequential P. aeruginosa isolates from chronically-infected cystic fibrosis patients as well as to investigate the potential mechanisms involved in resistance through whole genome sequencing. METHODS: Three sequential P. aeruginosa isolates from each of 50 chronically-colonized cystic fibrosis patients were studied. MICs for novel and classical antipseudomonal agents were determined by broth microdilution and whole genome sequences (n = 150) were obtained to investigate the presence of mutations within a set of chromosomal genes involved in P. aeruginosa antibiotic resistance (n = 40) and iron uptake (n = 120). RESULTS: Cefiderocol showed the lowest MIC50/90 values and its susceptibility rate was comparable to other novel antipseudomonal agents. Clinical resistance was documented in 9 isolates from 6 patients. Resistance genes associated with a statistically significant increase in cefiderocol MICs included ampC, pmrAB, galU, fusA1 and those coding the penicillin-binding proteins PBP2 and PBP3. Likewise, mutations within several genes participating in different iron-uptake systems were found to be significantly associated with resistance, including genes participating in the pyochelin and pyoverdin biosynthesis and several tonB-dependent receptors. Mutator and small colony variants isolates were also associated with increased cefiderocol MICs. DISCUSSION: Cefiderocol resistance is modulated by a complex mutational resistome, potentially conferring cross-resistance to novel beta-lactam beta-lactamase combinations, as well as an extended list of mutated iron-uptake genes. Monitoring the acquisition of mutations in all these genes will be helpful to guide treatments and mitigate the emergence and spread of resistance to this novel antibiotic.