The Safety of Aztreonam Versus Ceftazidime in Patients Labeled With Penicillin Allergy: A Cohort Study.

PURPOSE: Penicillin allergy is the most common drug allergy among hospitalized patients. Traditionally, aztreonam is recommended for patients labeled with penicillin allergy (PLWPA) in our institutional empirical antibiotic guidelines. Due to a global aztreonam shortage in December 2022, the antimicrobial stewardship unit recommended ceftazidime as a substitute. There is a paucity of real-world data on the safety profile of ceftazidime in PLWPA. Hence, we evaluated tolerability outcomes of ceftazidime use in PLWPA. METHODS: This retrospective cohort study compared PLWPA in Singapore General Hospital who received aztreonam (October 2022-December 2022) or ceftazidime (December 2022-February 2023). Patients were stratified according to their risk of allergic reaction (AR) based on history of penicillin allergy. The severity of AR was based on the Delphi study grading system. The primary outcome was development of AR after initiation of aztreonam or ceftazidime. The secondary tolerability outcomes include hepatotoxicity and neurotoxicity. FINDINGS: There were 168 patients in the study; 69 were men (41.1%) and the median age was 69 years (interquartile range: 59-76 years). Incidence of AR was statistically similar in both arms: 1 of 102 patients (0.98%) in the aztreonam arm vs 2 of 66 patients (3.03%) in the ceftazidime arm (P = 0.33). The patient in the aztreonam arm was deemed at medium risk of having an AR and developed localized rashes (grade 1). Both patients in the ceftazidime arm were deemed at high risk of AR and developed localized skin reaction (grade 1). Hepatotoxicity was observed in 1 patient prescribed aztreonam. No patients in the ceftazidime arm developed adverse events. IMPLICATIONS: Ceftazidime appears to be better tolerated and cheaper compared with aztreonam in PLWPA, and serves as an antimicrobial stewardship strategy to conserve broader-spectrum antibiotics use.

Confronting multidrug-resistant Klebsiella meningitis after mid-clival Cerebrospinal Fluid leak repair: a therapeutic odyssey.

A man in his 40s with type 2 diabetes mellitus had persistent right-sided watery nasal discharge for 6 months following cerebrospinal fluid (CSF) leak repair at another hospital, prompting his visit to us due to recurring symptoms. Imaging revealed a CSF leak from the mid-clivus for which revision endoscopic CSF leak repair was done. Regrettably, he developed postoperative meningitis caused by multidrug-resistant (MDR) Klebsiella pneumoniaeManaging this complex case was a challenging task due to the pathogen's resistance to conventional drugs and the scarcity of scientific evidence. We initiated a culture-guided combination regimen with ceftazidime, avibactam, aztreonam and tigecycline. This decision stemmed from meticulous literature review and observed antibiotic synergy while testing for this organism.After 4 weeks of vigilant treatment, the patient's symptoms improved significantly, and CSF cultures were sterile. We present our approach to effectively confront and manage a challenging instance of postoperative MDR bacterial meningitis.

Evaluation of a simple method for testing aztreonam and ceftazidime-avibactam synergy in New Delhi metallo-beta-lactamase producing Enterobacterales.

NDM-producing carbapenem-resistant bacterial infections became a challenge for clinicians. Combination therapy of aztreonam and ceftazidime-avibactam is a prudent choice for these infections. However, there is still no recommendation of a practically feasible method for testing aztreonam and ceftazidime-avibactam synergy. We proposed a simple method for testing aztreonam and ceftazidime-avibactam synergy and compared it with reference broth micro-dilution and other methods. Carbapenem-resistant Enterobacterales clinical isolates were screened for the presence of the NDM gene by the Carba R test. NDM harbouring isolates were tested for aztreonam and ceftazidime-avibactam synergy by broth microdilution (reference method), E strip-disc diffusion, double disc diffusion, and disc replacement methods. In the newly proposed method, the MHA medium was supplemented with ceftazidime-avibactam (corresponding to an aztreonam concentration of 4µg/ml). The MHA medium was then inoculated with the standard inoculum (0.5 McFarland) of the test organism. An AZT disc (30 µg) was placed on the supplemented MHA medium, and the medium was incubated overnight at 37°C. Aztreonam zone diameter on the supplemented MHA medium (in the presence of ceftazidime-avibactam) was compared with that from a standard disc diffusion plate (without ceftazidime-avibactam), performed in parallel. Interpretation of synergy was based on the restoration of aztreonam zone diameter (in the presence of ceftazidime-avibactam) crossing the CLSI susceptibility breakpoint, i.e., ≥ 21 mm. Of 37 carbapenem-resistant NDM-producing isolates, 35 (94.6%) were resistant to aztreonam and tested synergy positive by the proposed method. Its sensitivity and specificity were 97.14% and 100%, respectively. Cohen's kappa value showed substantial agreement of the reference method with the proposed method (κ = 0.78) but no other methods. The proposed method is simple, easily interpretable, and showed excellent sensitivity, specificity, and agreement with the reference method. Therefore, the new method is feasible and reliable for testing aztreonam synergy with avibactam in NDM-producing Enterobacterales.

Metabolomics unveil key pathways underlying the synergistic activities of aztreonam and avibactam against multidrug-resistant Escherichia coli.

PURPOSE: Aztreonam/avibactam is effective against serious infections caused by Gram-negative bacteria including Enterobacterales harboring metallo-ß-lactamases. While the utility of this combination has been established in vitro and in clinical trials, the purpose of this study is to enhance our understanding of the underlying mechanism responsible for their activities through metabolomic profiling of a multidrug-resistant Escherichia coli clinical isolate. METHODS: Metabolomic analyses of time-dependent changes in endogenous bacterial metabolites in a clinical isolate of a multidrug-resistant E. coli treated with aztreonam and avibactam were performed. E. coli metabolomes were compared at 15 min, 1 h and 24 h following treatments with either avibactam (4 mg/L), aztreonam (4 mg/L), or aztreonam (4 mg/L) + avibactam (4 mg/L). RESULTS: Drug treatment affected 326 metabolites with magnitude changes of at least 2-fold, most of which are involved primarily in peptidoglycan biosynthesis, nucleotide metabolism, and lipid metabolism. The feedstocks for peptidoglycan synthesis were depleted by aztreonam/avibactam combination; a significant downstream increase in nucleotide metabolites and a release of lipids were observed at the three timepoints. CONCLUSION: The findings indicate that the aztreonam/avibactam combination accelerates structural damage to the bacterial membrane structure and their actions were immediate and sustained compared to aztreonam or avibactam alone. By inhibiting the production of crucial cell wall precursors, the combination may have inflicted damages on bacterial DNA.

Comparison of testing methods assessing the in vitro efficacy of the combination of aztreonam with avibactam on multidrug-resistant Gram-negative bacilli.

BACKGROUND: Aztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches. METHODS: 96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher). RESULTS: According to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%). CONCLUSIONS: The ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.

Emergence of aztreonam/avibactam and tigecycline-resistant Pseudomonas putida group Co-producing blaIMP-1, blaAFM-4 and blaOXA-1041 with a novel sequence type ST268 in Southwestern China.

OBJECTIVES: The emergence of carbapenem-resistant Pseudomonas putida (CRPP) has raised public awareness. This study investigated two strains from the Pseudomonas putida group that were resistant to carbapenem, tigecycline, and aztreonam-avibactam (ATM-AVI), with a focus on their microbial and genomic characteristics. METHODS: We assessed the antibiotic resistance profile using broth dilution, disk diffusion, and E-test methods. Efflux pump phenotype testing and real-time quantitative PCR were employed to evaluate efflux pump activity in tigecycline resistance, while polymerase chain reaction was utilized to detect common carbapenem genes. Additionally, whole-genome sequencing was performed to analyze genomic characteristics. The transferability of blaIMP-1 and blaAFM-4 was assessed through a conjugation experiment. Furthermore, growth kinetics and biofilm formation were examined using growth curves and crystal violet staining. RESULTS: Both strains demonstrated resistance to carbapenem, tigecycline, and ATM-AVI. Notably, NMP can restore sensitivity to tigecycline. Subsequent analysis revealed that they co-produced blaIMP-1, blaAFM-4, tmexCD-toprJ, and blaOXA-1041, belonging to a novel sequence type ST268. Although they were closely related on the phylogenetic tree, they exhibited different levels of virulence. Genetic environment analysis indicated variations compared to prior studies, particularly regarding the blaIMP-1 and blaAFM-4 genes, which showed limited horizontal transferability. Moreover, it was observed that temperature exerted a specific influence on their biological factors. CONCLUSION: We initially identified two P. putida ST268 strains co-producing blaIMP-1, blaAFM-4, blaOXA-1041, and tmexCD-toprJ. The resistance to tigecycline and ATM-AVI can be attributed to the presence of multiple drug resistance determinants. These findings underscore the significance of P. putida as a reservoir for novel antibiotic resistance genes. Therefore, it is imperative to develop alternative antibiotic therapies and establish effective monitoring of bacterial resistance.

In vitro and in vivo activity of ceftazidime/avibactam and aztreonam alone or in combination against mcr-9, serine- and metallo-ß-lactamases-co-producing carbapenem-resistant Enterobacter cloacae complex.

PURPOSE: Enterobacteriaceae carrying mcr-9, in particularly those also co-containing metallo-ß-lactamase (MBL) and TEM type ß-lactamase, present potential transmission risks and lack adequate clinical response methods, thereby posing a major threat to global public health. The aim of this study was to assess the antimicrobial efficacy of a combined ceftazidime/avibactam (CZA) and aztreonam (ATM) regimen against carbapenem-resistant Enterobacter cloacae complex (CRECC) co-producing mcr-9, MBL and TEM. METHODS: The in vitro antibacterial activity of CZA plus ATM was evaluated using a time-kill curve assay. Furthermore, the in vivo interaction between CZA plus ATM was confirmed using a Galleria mellonella (G. mellonella) infection model. RESULTS: All eight clinical strains of CRECC, co-carrying mcr-9, MBL and TEM, exhibited high resistance to CZA and ATM. In vitro time-kill curve analysis demonstrated that the combination therapy of CZA + ATM exerted significant bactericidal activity against mcr-9, MBL and TEM-co-producing Enterobacter cloacae complex (ECC) isolates with a 100% synergy rate observed in our study. Furthermore, in vivo survival assay using Galleria mellonella larvae infected with CRECC strains co-harboring mcr-9, MBL and TEM revealed that the CZA + ATM combination significantly improved the survival rate compared to the drug-treatment alone and untreated control groups. CONCLUSION: To our knowledge, this study represents the first report on the in vitro and in vivo antibacterial activity of CZA plus ATM against CRECC isolates co-harboring mcr-9, MBL and TEM. Our findings suggest that the combination regimen of CZA + ATM provides a valuable reference for clinicians to address the increasingly complex antibiotic resistance situation observed in clinical microorganisms.

Effect of ceftazidime-avibactam combined with different antimicrobials against carbapenem-resistant Klebsiella pneumoniae.

This study aimed to assess the in vitro efficacy of ceftazidime-avibactam (CZA) in combination with various antimicrobial agents against carbapenem-resistant Klebsiella pneumoniae (CRKP). We selected 59 clinical CRKP isolates containing distinct drug resistance mechanisms. The minimum inhibitory concentrations (MICs) of meropenem (MEM), colistin (COL), eravacycline (ERA), amikacin (AK), fosfomycin (FOS), and aztreonam (ATM), both individually and in combination with CZA, were tested using the checkerboard method. The interactions of antimicrobial agent combinations were assessed by fractional inhibitory concentration index (FICI) and susceptible breakpoint index (SBPI). The time-kill curve assay was employed to dynamically evaluate the effects of these drugs alone and in combination format. In the checkerboard assay, the combination of CZA+MEM showed the highest level of synergistic effect against both KPC-producing and carbapenemase-non-producing isolates, with synergy rates of 91.3% and 100%, respectively. Following closely was the combination of FOS+CZA . For metallo-beta-lactamases (MBLs) producing strains, ATM+CZA displayed complete synergy, while the combination of MEM+CZA showed a synergy rate of only 57.14% for NDM-producing strains and 91.67% for IMP-producing strains. In the time-kill assay, MEM+CZA also demonstrated significant synergistic effects against the two KPC-2-producing isolates (Y070 and L70), the two carbapenemase-non-producing isolates (Y083 and L093), and the NDM-1-producing strain L13, with reductions in log10 CFU/mL exceeding 10 compared to the control. Against the IMP-producing strain Y047, ATM+CZA exhibited the highest synergistic effect, resulting in a log10 CFU/mL reduction of 10.43 compared to the control. The combination of CZA and MEM exhibited good synergistic effects against KPC-producing and non-enzyme-producing strains, followed by the FOS+CZA combination. Among MBL-producing strains, ATM+CZA demonstrated the most pronounced synergistic effect. However, the combinations of CZA with ERA, AK, and COL show irrelevant effects against the tested clinical isolates. IMPORTANCE: Our study confirmed the efficacy of the combination CZA+MEM against KPC-producing and non-carbapenemase-producing strains. For metalloenzyme-producing strains, CZA+ATM demonstrated the most significant synergy. Additionally, CZA exhibited a notable synergy effect when combined with FOS. These combination therapies present promising new options for the treatment of CRKP infection.

Outcomes of 23 patients diagnosed with New Delhi metallo-beta-lactamase (NDM)-producing Klebsiella pneumoniae infection treated with ceftazidime/avibactam and aztreonam at a single center in Poland.

PURPOSE: Amongst all etiologic hospital-acquired infection factors, K. pneumoniae strains producing New Delhi metallo-ß-lactamase (KP-NDM) belong to pathogens with the most effective antibiotic resistance mechanisms. Clinical guidelines recommend using ceftazidime/avibactam with aztreonam (CZA + AT) as the preferred option for NDM-producing Enterobacterales. However, the number of observations on such treatment regimen is limited. This retrospective study reports the clinical and microbiological outcomes of 23 patients with KP-NDM hospital-acquired infection treated with CZA + AT at a single center in Poland. METHODS: The isolates were derived from the urine, lungs, blood, peritoneal cavity, wounds, and peritonsillar abscess. In microbiological analysis, mass spectrometry for pathogen identification, polymerase chain reaction, or an immunochromatographic assay for detection of carbapenemase, as well as VITEK-2 system, broth microdilution, and microdilution in agar method for antimicrobial susceptibility tests were used, depending of the pathogens' nature. CZA was administered intravenously (IV) at 2.5 g every eight hours in patients with normal kidney function, and aztreonam was administered at 2 g every eight hours IV. Such dosage was modified when renal function was reduced. RESULTS: KP-NDM was eradicated in all cases. Four patients (17.4%) died: three of them had a neoplastic disease, and one - a COVID-19 infection. CONCLUSION: The combination of CZA + AT is a safe and effective therapy for infections caused by KP-NDM, both at the clinical and microbiological levels. The synergistic action of all compounds resulted in a good agreement between the clinical efficacy of CZA + AT and the results of in vitro susceptibility testing.

In vitro activity of antibiotics potentially effective against difficult-to-treat strains of Gram-negative rods: retrospective study.

Bacterial resistance surveillance is one of the main outputs of microbiological laboratories and its results are important part of antimicrobial stewardship (AMS). In this study, the susceptibility of specific bacteria to selected antimicrobial agents was tested. The susceptibility of 90 unique isolates of pathogens of critical priority obtained from clinically valid samples of ICU patients in 2017-2021 was tested. 50% of these fulfilled difficult-to-treat resistance (DTR) criteria and 50% were susceptible to all antibiotics included in the definition. 10 Enterobacterales strains met DTR criteria, and 2 (20%) were resistant to colistin (COL), 2 (20%) to cefiderocol (FCR), 7 (70%) to imipenem/cilastatin/relebactam (I/R), 3 (30%) to ceftazidime/avibactam (CAT) and 5 (50%) to fosfomycin (FOS). For Enterobacterales we also tested aztreonam/avibactam (AZA) for which there are no breakpoints yet. The highest MIC of AZA observed was 1 mg/l, MIC range in the susceptible cohort was 0.032-0.064 mg/l and in the DTR cohort (incl. class B beta-lactamase producers) it was 0.064-1 mg/l. Two (13.3%) isolates of Pseudomonas aeruginosa (15 DTR strains) were resistant to COL, 1 (6.7%) to FCR, 13 (86.7%) to I/R, 5 (33.3%) to CAT, and 5 (33.3%) to ceftolozane/tazobactam. All isolates of Acinetobacter baumannii with DTR were susceptible to COL and FCR, and at the same time resistant to I/R and ampicillin/sulbactam. New antimicrobial agents are not 100% effective against DTR. Therefore, it is necessary to perform susceptibility testing of these antibiotics, use the data for surveillance (including local surveillance) and conform to AMS standards.

Difficult-to-treat (DTR) Pseudomonas aeruginosa harboring Verona-Integron metallo-ß-lactamase (blaVIM): infection management and molecular analysis.

Pseudomonas aeruginosa harboring Verona Integron-encoded metallo-ß-lactamase enzymes (VIM-CRPA) have been associated with infection outbreaks in several parts of the world. In the US, however, VIM-CRPA remain rare. Starting in December 2018, we identified a cluster of cases in our institution. Herein, we present our epidemiological investigation and strategies to control/manage these challenging infections. This study was conducted in a large academic healthcare system in Miami, FL, between December 2018 and January 2022. Patients were prospectively identified via rapid molecular diagnostics when cultures revealed carbapenem-resistant P. aeruginosa. Alerts were received in real time by the antimicrobial stewardship program and infection prevention teams. Upon alert recognition, a series of interventions were performed as a coordinated effort. A retrospective chart review was conducted to collect patient demographics, antimicrobial therapy, and clinical outcomes. Thirty-nine VIM-CRPA isolates led to infection in 21 patients. The majority were male (76.2%); the median age was 52 years. The majority were mechanically ventilated (n = 15/21; 71.4%); 47.6% (n = 10/21) received renal replacement therapy at the time of index culture. Respiratory (n = 20/39; 51.3%) or bloodstream (n = 13/39; 33.3%) were the most common sources. Most infections (n = 23/37; 62.2%) were treated with an aztreonam-avibactam regimen. Six patients (28.6%) expired within 30 days of index VIM-CRPA infection. Fourteen isolates were selected for whole genome sequencing. Most of them belonged to ST111 (12/14), and they all carried blaVIM-2 chromosomally. This report describes the clinical experience treating serious VIM-CRPA infections with either aztreonam-ceftazidime/avibactam or cefiderocol in combination with other agents. The importance of implementing infection prevention strategies to curb VIM-CRPA outbreaks is also demonstrated.

Performance of disk diffusion method for aztreonam in combination with avibactam against Enterobacteriales.

OBJECTIVES: To evaluate the performance of an in-house developed disk diffusion method for aztreonam in combination with avibactam against Enterobacteriales. METHODS: The in vitro antibacterial activity of aztreonam with avibactam against 204 carbapenemase-producing Enterobacteriales was determined by a disk diffusion method, with a broth microdilution method as a reference. RESULTS: The optimal S/R breakpoints for disk diffusion tests of 30/20 and 10/4 µg disks, calculated by the dBETs software using the model-based approaches, were ≥22/≤21 and ≥12/≤11 mm, respectively. On the basis of the estimated breakpoints, the CAs for disk diffusion tests of 30/20 and 10/4 µg aztreonam/avibactam disks were both 98.0%, with 0.5% major error and 37.5% very major error. CONCLUSIONS: The home-made disk diffusion method is an economical and practical method for clinical microbiology laboratories to determine the antibacterial susceptibility of aztreonam with avibactam against Enterobacteriales.

Iron efflux by IetA enhances ß-lactam aztreonam resistance and is linked to oxidative stress through cellular respiration in Riemerella anatipestifer.

BACKGROUND: Riemerella anatipestifer encodes an iron acquisition system, but whether it encodes the iron efflux pump and its role in antibiotic resistance are largely unknown. OBJECTIVES: To screen and identify an iron efflux gene in R. anatipestifer and determine whether and how the iron efflux gene is involved in antibiotic resistance. METHODS: In this study, gene knockout, streptonigrin susceptibility assay and inductively coupled plasma mass spectrometry were used to screen for the iron efflux gene ietA. The MIC measurements, scanning electron microscopy and reactive oxygen species (ROS) detection were used to verify the role of IetA in aztreonam resistance and its mechanism. Mortality and colonization assay were used to investigate the role of IetA in virulence. RESULTS: The deletion mutant ΔietA showed heightened susceptibility to streptonigrin, and prominent intracellular iron accumulation was observed in ΔfurΔietA under excess iron conditions. Additionally, ΔietA exhibited increased sensitivity to H2O2-produced oxidative stress. Under aerobic conditions with abundant iron, ΔietA displayed increased susceptibility to the ß-lactam antibiotic aztreonam due to heightened ROS production. However, the killing efficacy of aztreonam was diminished in both WT and ΔietA under anaerobic or iron restriction conditions. Further experiments demonstrated that the efficiency of aztreonam against ΔietA was dependent on respiratory complexes Ⅰ and Ⅱ. Finally, in a duckling model, ΔietA had reduced virulence compared with the WT. CONCLUSION: Iron efflux is critical to alleviate oxidative stress damage and ß-lactam aztreonam killing in R. anatipestifer, which is linked by cellular respiration.

Synergistic effects of inhaled aztreonam plus tobramycin on hypermutable cystic fibrosis Pseudomonas aeruginosa isolates in a dynamic biofilm model evaluated by mechanism-based modelling and whole genome sequencing.

OBJECTIVE: Hypermutable Pseudomonas aeruginosa strains are highly prevalent in chronic lung infections of patients with cystic fibrosis (CF). Acute exacerbations of these infections have limited treatment options. This study aimed to investigate inhaled aztreonam and tobramycin against clinical hypermutable P. aeruginosa strains using the CDC dynamic in vitro biofilm reactor (CBR), mechanism-based mathematical modelling (MBM) and genomic studies. METHODS: Two CF multidrug-resistant strains were investigated in a 168 h CBR (n = 2 biological replicates). Regimens were inhaled aztreonam (75 mg 8-hourly) and tobramycin (300 mg 12-hourly) in monotherapies and combination. The simulated pharmacokinetic profiles of aztreonam and tobramycin (t1/2 = 3 h) were based on published lung fluid concentrations in patients with CF. Total viable and resistant counts were determined for planktonic and biofilm bacteria. MBM of total and resistant bacterial counts and whole genome sequencing were completed. RESULTS: Both isolates showed reproducible bacterial regrowth and resistance amplification for the monotherapies by 168 h. The combination performed synergistically, with minimal resistant subpopulations compared to the respective monotherapies at 168 h. Mechanistic synergy appropriately described the antibacterial effects of the combination regimen in the MBM. Genomic analysis of colonies recovered from monotherapy regimens indicated noncanonical resistance mechanisms were likely responsible for treatment failure. CONCLUSION: The combination of aztreonam and tobramycin was required to suppress the regrowth and resistance of planktonic and biofilm bacteria in all biological replicates of both hypermutable multidrug-resistant P. aeruginosa CF isolates. The developed MBM could be utilised for future investigations of this promising inhaled combination.

Emergence of ceftazidime-avibactam resistance in blaKPC-33-harbouring ST11 Klebsiella pneumoniae in a paediatric patient.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses immense threats to the health of infected patients worldwide, especially children. This study reports the infection caused by CRKP in a paediatric intensive care unit (PICU) child and its drug-resistant mutation during the treatment. Twelve Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains were isolated from the child. Broth microdilution method, plasmid transformation assay, and whole genome sequencing (WGS) were performed to investigate the antimicrobial susceptibility, resistance mechanisms, and genetic structural features of CRKPs. The results showed that 12 strains were highly resistant to most available antimicrobial agents. Among them, K. pneumoniae FD11 and K. pneumoniae FD12 were resistant to ceftazidime-avibactam (CZA, MIC >64 mg/L) and restored the carbapenem susceptibility (Imipenem, MIC =0.25 mg/L; Meropenem, MIC =2 mg/L). The patient improved after treatment with CZA in combination with aztreonam. Plasmid transformation assay demonstrated that the blaKPC-33-positive transformant increased MICs of CZA by at least 33-fold and 8-fold compared with the recipient Escherichia coli DH5α and blaKPC-2-positive transformants. WGS analysis revealed that all strains belonged to the ST11-KL64 type and showed highly homologous (3-26 single nucleotide polymorphisms [SNPs]). A single base mutation (G532T) of blaKPC-2 resulted in a tyrosine to aspartic acid substitution at Ambler amino acid position 179 (D179Y), which conferred CZA resistance in K. pneumoniae. This is the first report of a drug-resistant mutation evolving into blaKPC-33 during the treatment of blaKPC-2-positive CRKP in paediatric-infected patients. It advises clinicians that routine sequential antimicrobial susceptibility testing and KPC genotyping are critical during CZA therapy in children infected with CRKP.

Clinical and genomic characteristics of IMP-producing Enterobacter cloacae complex and Klebsiella pneumoniae.

Carbapenemase-producing Enterobacterales (CPEs) are one of the top priority antimicrobial-resistant pathogens. Among CPEs, those producing acquired metallo-ß-lactamases (MBLs) are considered particularly problematic as few agents are active against them. Imipenemase (IMP) is the most frequently encountered acquired MBL in Japan, but comprehensive assessment of clinical and microbiological features of IMP-producing Enterobacterales infection remains scarce. Here, we retrospectively evaluated 62 patients who were hospitalized at a university hospital in Japan and had IMP-producing Enterobacterales from a clinical culture. The isolates were either Enterobacter cloacae complex or Klebsiella pneumoniae, and most of them were isolated from sputum. The majority of K. pneumoniae, but not E. cloacae complex isolates, were susceptible to aztreonam. Sequence type (ST) 78 and ST517 were prevalent for E. cloacae complex and K. pneumoniae, respectively, and all isolates carried blaIMP-1. Twenty-four of the patients were deemed infected with IMP-producing Enterobacterales. Among the infected patients, therapy varied and largely consisted of conventional ß-lactam agents, fluoroquinolones, or combinations. Three (13%), five (21%), and nine (38%) of them died by days 14, 30, and 90, respectively. While incremental mortality over 90 days was observed in association with underlying comorbidities, active conventional treatment options were available for most patients with IMP-producing Enterobacterales infections, distinguishing them from more multidrug-resistant CPE infections associated with globally common MBLs, such as New Delhi metallo-ß-lactamase (NDM) and Verona integron-encoded metallo-ß-lactamase (VIM).

Determination of aztreonam/ceftazidime-avibactam synergism and proposal of a new methodology for the evaluation of susceptibility in vitro.

We proposed a new methodology, the microelution ATM/CZA (mATM/CZA), based on the antibiotic disc elution and the use of resazurin, for rapid (<4h) determination of in vitro susceptibility to aztreonam combined with ceftazidime-avibactam among Enterobacterales. The mATM/CZA presented excellent accuracy with 1.9 %, 98.1 % and 100 % of major error, specificity and sensitivity, respectively. Furthermore, we assessed synergism between aztreonam and ceftazidime-avibactam in Enterobacterales and Pseudomonas aeruginosa, which was observed in 37/55 Enterobacterales and 31/56 P. aeruginosa. As reference methodologies (checkerboard, time-kill curve) are not compatible with the routine of the clinical microbiology laboratories, mATM/CZA is an important alternative to evaluate susceptibility of the combination in a scenario where its clinical use is increasingly important.

Comparative genomic analysis of Pseudomonas aeruginosa strains susceptible and resistant to carbapenems and aztreonam isolated from patients with healthcare-associated infections in a Mexican hospital.

Pseudomonas aeruginosa (PA) is an important opportunistic pathogen that causes different infections on immunocompromised patients. Within PA accessory genome, differences in virulence, antibiotic resistance and biofilm formation have been described between strains, leading to the emergence of multidrug-resistant strains. The genome sequences of 17 strains isolated from patients with healthcare-associated infections in a Mexican hospital were genomically and phylogenetically analyzed and antibiotic resistance genes, virulence genes, and biofilm formation genes were detected. Fifteen of the 17 strains were resistant to at least two of the carbapenems meropenem, imipenem, and the monobactam aztreonam. The antibiotic resistance (mexA, mexB, and oprM) and the biofilm formation (pslA and pslD) genes were detected in all strains. Differences were found between strains in accessory genome size. The strains had different sequence types, and seven strains had sequence types associated with global high risk epidemic PA clones. All strains were represented in two groups among PA global strains. In the 17 strains, horizontally acquired resistance genes to aminoglycosides and beta-lactams were found, mainly, and between 230 and 240 genes that encode virulence factors. The strains under study were variable in terms of their accessory genome, antibiotic resistance, and virulence genes. With these characteristics, we provide information about the genomic diversity of clinically relevant PA strains.

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.