This study was conducted to investigate decreased susceptibility (minimum inhibitory concentrations [MICs] 0.25-4 mg/L) and resistance (MICs > 4 mg/L) to aztreonam-avibactam (ATM-AVI). Contemporary non-replicate clinical isolates of carbapenemase-producing Escherichia coli (CP-EC) (n=90) and ESBL-producing E. coli (EP-EC) (n=12) were used. CP-EC belonged to 25 distinct sequence types (STs) and all EP-EC belonged to ST405. All strains were isolated from 2019 to 2022 at the Karolinska University Laboratory, Stockholm, Sweden. ATM-AVI MICs were determined using broth microdilution. The EUCAST epidemiological cut-off value of 0.125 mg/L was used to define the wild type (WT). Whole-genome sequences (Illumina) were analysed for detecting resistance determinants among WT vs. non-WT isolates. Among 102 isolates, 40 (39%) and 62 (61%) were WT and non-WT, respectively. Among non-WT isolates, resistance was noted for 20 and decreased susceptibility for 42. Resistance was observed among 14/47 New Delhi metallo-β-lactamase (NDM)-producers, 5/43 OXA-48 group producers, and 1/12 EP-EC. Decreased susceptibility was observed among 29/47 NDM, 13/43 OXA-48 group, and 3/12 EP-EC. Resistant isolates predominantly belonged to ST405, followed by STs 410, 361, 167, 617, and 1284. Penicillin-binding protein 3 (PBP3) inserts (YRIK/YRIN) were observed in 20/20 and CMY-42 in 5/20 resistant isolates. Several mutations in the ftsI (encoding PBP3) and regulatory genes of outer membrane proteins (OmpC and OmpF) and efflux pumps (AcrAB-TolC) were detected. A ≥ 2-fold reduction in MICs was observed among 20/20 vs. 7/20 isolates tested in the presence of the membrane permeabiliser, polymyxin B nanopeptide (PMBN) and efflux inhibitor, phenylalanine arginine β-naphthylamide (PAβN), respectively. In conclusion, resistance to ATM-AVI is a result of interplay of various determinants, including target alterations, deactivating enzymes, and decreased permeability.

Carbapenem-resistant Enterobacterales represent a major health threat and have few approved therapeutic options. Enterobacterales isolates were collected from hospitalized inpatients from 49 sites in six European countries (1 January-31 December 2020) and underwent susceptibility testing to cefiderocol and β-lactam/β-lactamase inhibitor combinations. Meropenem-resistant (MIC >8 mg/L) and cefiderocol-susceptible isolates were analyzed by PCR, and cefiderocol-‍resistant isolates by whole-genome sequencing, to identify resistance mechanisms. Overall, 1,909 isolates (including 970 Klebsiella spp., 382 Escherichia coli, and 244 Enterobacter spp.) were collected, commonly from bloodstream infections (43.6%). Cefiderocol susceptibility was higher than approved β-lactam/β-lactamase inhibitor combinations and largely comparable to cefepime-taniborbactam and aztreonam-avibactam against all Enterobacterales (98.1% vs 78.1%-‍97.4% and 98.7%-99.1%, respectively) and Enterobacterales resistant to meropenem (n = 148, including 125 Klebsiella spp.; 87.8% vs 0%-71.6% and 93.2%-98.6%, respectively), β-lactam/β-lactamase inhibitor combinations (66.7%-‍92.1% vs 0%-‍88.1% and 66.7%-97.9%, respectively), and to both meropenem and β-‌lactam/β-lactamase inhibitor combinations (61.9%-65.9% vs 0%-‍20.5% and 76.2%-97.7%, respectively). Susceptibilities to approved and developmental β-lactam/β-lactamase inhibitor combinations against cefiderocol-resistant Enterobacterales (n = 37) were 10.8%-‍56.8% and 78.4%-94.6%, respectively. Most meropenem-resistant Enterobacterales harbored Klebsiella pneumoniae carbapenemase (110/148) genes, although metallo-β-lactamase (35/148) and oxacillinase (OXA) carbapenemase (6/148) genes were less common; cefiderocol susceptibility was retained in β-lactamase producers, other than NDM, AmpC, and non-carbapenemase OXA producers. Most cefiderocol-resistant Enterobacterales had multiple resistance mechanisms, including ≥1 iron uptake-related mutation (37/37), carbapenemase gene (33/37), and ftsI mutation (24/37). The susceptibility to cefiderocol was higher than approved β-lac‌tam/β-lactamase inhibitor combinations against European Enterobacterales, including meropenem- and β-lactam/β-lactamase inhibitor combination-resistant isolates.
OBJECTIVE: This study collected a notably large number of Enterobacterales isolates from Europe, including meropenem- and β-lactam/β-lactamase inhibitor combination-resistant isolates against which the in vitro activities of cefiderocol and developmental β-lactam/β-lactamase inhibitor combinations were directly compared for the first time. The MIC breakpoint for high-dose meropenem was used to define meropenem resistance, so isolates that would remain meropenem resistant with doses clinically available to patients were included in the data. Susceptibility to cefiderocol, as a single active compound, was high against Enterobacterales and was higher than or comparable to available β-lactam/β-lactamase inhibitor combinations. These results provide insights into the treatment options for infections due to Enterobacterales with resistant phenotypes. Early susceptibility testing of cefiderocol in parallel with β-lactam/β-lactamase inhibitor combinations will allow patients to receive the most appropriate treatment option(s) available in a timely manner. This is particularly important when options are more limited, such as against metallo-β-lactamase-producing Enterobacterales.

OBJECTIVE: 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.
CONCLUSIONS: 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.

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.

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The combination of ceftazidime-avibactam (CAZ-AVI) and aztreonam (ATM) is used to treat MBL-producing Enterobacterales-related infections. The new combination aztreonam-avibactam (AZA) is currently in development. We compared results obtained with the new MIC test strip (MTS) AZA (Liofilchem) with broth microdilution method (BMD) on 41 MBL-producing Enterobacterales from 41 clinical samples. The MTS AZA was also compared to combination testing method using CAZ-AVI and ATM strips. Compared to BMD, categorical agreement (CA) was 100%. Compared with combination testing method, CA was 97.6%. The MTS AZA can be used to determine MICs levels of AZA or CAZ-AVI/ATM combinations.

OBJECTIVE: Metallo-β-lactamase (MBL)-producing Enterobacterales are a major challenge worldwide due to limited treatment options. Aztreonam-avibactam (ATM-AVI), which is under clinical development, has shown activity against MBL-positive isolates. This study evaluated the prevalence of MBL producers and the nature of enzymes among a global collection of clinical isolates of Enterobacterales from the Antimicrobial Testing Leadership and Surveillance program (ATLAS) surveillance program (2016-2020), and the antimicrobial activity of ATM-AVI and comparators against this collection.
METHODS: Non-duplicate clinical isolates of Enterobacterales (N = 106 686) collected across 63 countries were analysed. Antimicrobial susceptibility was performed using broth microdilution. Minimum inhibitory concentrations (MICs) were interpreted using Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing breakpoints. Provisional pharmacokinetic/pharmacodynamic breakpoint of ≤8 mg/L was considered for ATM-AVI. β-lactamase genes were characterized by polymerase chain reaction and sequencing. The Cochran Armitage Trend test was used to determine significant trends in percentage of isolates over time.
RESULTS: Overall, MBL-positive isolates were 1.6% of total Enterobacterales isolates globally, with a significant increasing trend observed over time, globally and across regions (P < 0.05). New Delhi MBL (NDM) was the most common MBL (83.3%). ATM-AVI demonstrated potent activity against MBL-positive isolates (MIC ≤8 mg/L: 99.4% isolates inhibited; MIC90, 1 mg/L). Consistent activity was also noted across different regions. Potent activity was demonstrated against different NDM variants and MBL-positive isolates co-carrying other carbapenemases (98.1% and 99.7% isolates inhibited at ≤8 mg/L, respectively). About 0.6% MBL-positive isolates (10/1707) had MICs >8 mg/L for ATM-AVI.
CONCLUSIONS: ATM-AVI demonstrated potent activity against MBL-positive isolates, including NDM variants and MBL-positive isolates co-carrying other carbapenemases, and may represent a good option for treating infections caused by MBL-positive Enterobacterales.

Increasing antimicrobial resistance among multidrug-resistant (MDR), extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Enterobacterales (CPE), in particular metallo-β-lactamase (MBL)-positive strains, has led to limited treatment options in these isolates. This study evaluated the activity of aztreonam-avibactam (ATM-AVI) and comparator antimicrobials against Enterobacterales isolates and key resistance phenotypes stratified by wards, infection sources and geographic regions as part of the ATLAS program between 2016 and 2020. Minimum inhibitory concentrations (MICs) were determined per Clinical and Laboratory Standards Institute (CLSI) guidelines. The susceptibility of antimicrobials were interpreted using CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. A tentative pharmacokinetic/pharmacodynamic breakpoint of 8 µg/mL was considered for ATM-AVI activity. ATM-AVI inhibited ≥99.2% of Enterobacterales isolates across wards and ≥99.7% isolates across infection sources globally and in all regions at ≤8 µg/mL. For resistance phenotypes, ATM-AVI demonstrated sustained activity across wards and infection sources by inhibiting ≥98.5% and ≥99.1% of multidrug-resistant (MDR) isolates, ≥98.6% and ≥99.1% of ESBL-positive isolates, ≥96.8% and ≥90.9% of carbapenem-resistant (CR) isolates, and ≥96.8% and ≥97.4% of MBL-positive isolates, respectively, at ≤8 µg/mL globally and across regions. Overall, our study demonstrated that ATM-AVI represents an important therapeutic option for infections caused by Enterobacterales, including key resistance phenotypes across different wards and infection sources.

OBJECTIVE: To our knowledge, this is the first study to report the in vitro activity of two novel antimicrobial drugs, including imipenem-relebactam (IMR) and aztreonam-avibactam (AZA), toward carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKP) strains. Our in vitro activity study revealed that only few antibacterial agents (including several novel agents) exhibit high antimicrobial activity toward carbapenem-resistant Klebsiella pneumoniae (CRKP) and CR-hvKP isolates. IMR and AZA may be promising therapeutic agents for the treatment of infections caused by CRKP and CR-hvKP isolates.

Aztreonam-avibactam (AZA), a newly developed β-lactam/β-lactamase inhibitor combination, is a treatment option for infections due to carbapenem-resistant Enterobacterales (CRE), including metallo-ß-lactamase producers, regardless of additional production of broad-spectrum serine-ß-lactamases. However, AZA-resistance has already been reported in Enterobacterales and its early detection could be a valuable tool for faster and more accurate clinical decision-making. We therefore developed a rapid culture-based test for the identification of AZA resistance among multidrug-resistant Enterobacterales. The Rapid Aztreonam/Avibactam NP test is based on resazurin reduction when bacterial growth occurs in the presence of AZA at 8/4 µg/mL (protocol 1) or 12/4 µg/mL (protocol 2). Given the absence of guidelines on AZA susceptibility testing, two tentative breakpoints were indeed used to categorize AZA-susceptible isolates: ≤4 µg/mL in protocol 1 and ≤ 8 µg/mL in protocol 2. Bacterial growth was visually detectable by a blue-to-purple or blue-to-pink color change of the medium. A total of 78 enterobacterial isolates (among which 34 AZA-resistant and 13 AZA-resistant according to protocols 1 and 2, respectively) were used to evaluate the test performance using protocol 1 or protocol 2. The sensitivity and specificity of the test were found to be 100% and 97.7%, respectively, following protocol 1 and 100% and 100%, respectively, following protocol 2, in comparison with broth microdilution. All results were obtained within 4.5 hours corresponding to a time saving of ca. 14 hours compared with currently available methods for AZA susceptibility testing. The Rapid Aztreonam/Avibactam NP test is rapid, highly sensitive, specific, easily interpretable, and easy to implement in routine.