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.

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.

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.

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.

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.

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.

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.

In vitro activity of meropenem-vaborbactam plus aztreonam against metallo-ß-lactamase-producing Klebsiella pneumoniae.

We evaluated the in vitro activity of meropenem-vaborbactam plus aztreonam (MEV-ATM) against 140 metallo-ß-lactamase (MBL)-producing Klebsiella pneumoniae isolates. Among them, 25 isolates (17.9%) displayed minimum inhibitory concentrations (MIC) ≥ 8 µg/mL, while 112 (80.0%) had MIC ≤ 2 µg/mL. Genomic analysis and subsequent gene cloning experiments revealed OmpK36 134-135GD-insertion and increased carbapenemase gene (blaNDM-1 and blaOXA-48-like) copy numbers are the main factors responsible for MEV-ATM non-susceptibility. Notably, MEV-ATM is actively against aztreonam-avibactam-resistant mutants due to CMY-16 mutations.

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.

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).

Activity of aztreonam-avibactam against Enterobacterales resistant to recently approved beta-lactamase inhibitor combinations collected in Europe, Latin America, and the Asia-Pacific Region (2020-2022).

BACKGROUND: Aztreonam-avibactam is under clinical development for treatment of infections caused by carbapenem-resistant Enterobacterales (CRE), especially those resistant to recently approved ß-lactamase inhibitor combinations (BLICs). OBJECTIVES: To evaluate a large collection of CRE isolates, including those non-susceptible to ceftazidime-avibactam, meropenem-vaborbactam, and/or imipenem-relebactam. METHODS: Overall, 24 580 Enterobacterales isolates were consecutively collected (1/patient) in 2020-2022 from 64 medical centres located in Western Europe (W-EU), Eastern Europe (E-EU), Latin America (LATAM), and the Asia-Pacific region (APAC). Of those, 1016 (4.1%) were CRE. Isolates were susceptibility tested by broth microdilution. CRE isolates were screened for carbapenemase genes by whole genome sequencing. RESULTS: Aztreonam-avibactam inhibited 99.6% of CREs at ≤8 mg/L. Ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam were active against 64.6%, 57.4%, and 50.7% of CRE isolates, respectively; most of the non-susceptible isolates carried metallo-beta-lactamases. Aztreonam-avibactam was active against ≥98.9% of isolates non-susceptible to these BLICs. The activity of these BLICs varied by region, with highest susceptibility rates observed in W-EU (76.9% for ceftazidime-avibactam, 72.5% for meropenem-vaborbactam, 63.8% for imipenem-relebactam) and the lowest susceptibility rates identified in the APAC region (39.9% for ceftazidime-avibactam, 37.8% for meropenem-vaborbactam, and 27.5% for imipenem-relebactam). The most common carbapenemase types overall were KPC (44.6% of CREs), NDM (29.9%), and OXA-48-like (16.0%). KPC predominated in LATAM (64.1% of CREs in the region) and W-EU (61.1%). MBL occurrence was highest in APAC (59.5% of CREs in the region), followed by LATAM (34.0%), E-EU (28.9%), and W-EU (23.6%). CONCLUSIONS: Aztreonam-avibactam demonstrated potent activity against CRE isolates resistant to ceftazidime-avibactam, meropenem-vaborbactam, and/or imipenem-relebactam independent of the carbapenemase produced.

Effect of modification of penicillin-binding protein 3 on susceptibility to ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and cefiderocol of Escherichia coli strains producing broad-spectrum ß-lactamases.

The impact of penicillin-binding protein 3 (PBP3) modifications that may be identified in Escherichia coli was evaluated with respect to susceptibility to ß-lactam/ß-lactamase inhibitor combinations including ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and to cefiderocol. A large series of E. coli recombinant strains producing broad-spectrum ß-lactamases was evaluated. While imipenem-relebactam showed a similar activity regardless of the PBP3 background, susceptibility to other molecules tested was affected at various levels. This was particularly the case for ceftazidime-avibactam, aztreonam-avibactam, and cefepime-taniborbactam.

Next generation antibiotic combinations to combat pan-drug resistant Klebsiella pneumoniae.

Antimicrobial resistance has emerged as one of the leading public health threats of the twenty-first century. Gram-negative pathogens have been a major contributor to the declining efficacy of antibiotics through both acquired resistance and tolerance. In this study, a pan-drug resistant (PDR), NDM-1 and CTX-M-15 co-producing isolate of K. pneumoniae, CDC Nevada, (Kp Nevada) was exposed to the clinical combination of aztreonam + ceftazidime/avibactam (ATM/CAZ/AVI) to overcome metallo-ß-lactamases. Unexpectedly, the ß-lactam combination resulted in long filamentous cell formation induced by PBP3 inhibition over 168 h in the hollow fiber infection model experiments with eventual reversion of the total population upon drug removal. However, the addition of imipenem to the two drug ß-lactam combination was highly synergistic with suppression of all drug resistant subpopulations over 5 days. Scanning electron microscopy and fluorescence microscopy for all imipenem combinations in time kill studies suggested a role for imipenem in suppression of long filamentous persisters, via the formation of metabolically active spheroplasts. To complement the imaging studies, salient transcriptomic changes were quantified using RT-PCR and novel cassette assay evaluated ß-lactam permeability. This showed significant upregulation of both spheroplast protein Y (SPY), a periplasmic chaperone protein that has been shown to be related to spheroplast formation, and penicillin binding proteins (PBP1, PBP2, PBP3) for all combinations involving imipenem. However, with aztreonam alone, pbp1, pbp3 and spy remained unchanged while pbp2 levels were downregulated by > 25%. Imipenem displayed 207-fold higher permeability as compared with aztreonam (mean permeability coefficient of 17,200 nm/s). Although the clinical combination of aztreonam/avibactam and ceftazidime has been proposed as an important treatment of MBL Gram-negatives, we report the first occurrence of long filamentous persister formation. To our knowledge, this is the first study that defines novel ß-lactam combinations involving imipenem via maximal suppression of filamentous persisters to combat PDR CDC Nevada K. pneumoniae.

Activity of aztreonam/avibactam and ceftazidime/avibactam against Enterobacterales with carbapenemase-independent carbapenem resistance.

Enterobacterales with carbapenemase-independent resistance to carbapenems are sometimes selected during therapy and, on rare occasions, cause outbreaks. Most have extended-spectrum or AmpC ß-lactamases, together with changes to permeability or penicillin-binding proteins (PBPs). Newer ß-lactam-ß-lactamase inhibitor combinations may present useful options for infections due to these organisms. Accordingly, Clinical and Laboratory Standards Institute/European Committee on Antimicrobial Susceptibility Testing broth-microdilution was used to measure the minimum inhibitory concentrations (MICs) of ceftazidime/avibactam and aztreonam/avibactam for 51 carbapenemase-negative Enterobacterales with resistance or reduced susceptibility to carbapenems: genomic sequencing of the least-susceptible organisms was also undertaken. MICs of the two avibactam combinations cross-correlated closely, but with fewer MICs (2/51 vs. 10/51) exceeding 8+4 mg/L in the case of ceftazidime/avibactam. Raised MICs for Escherichia coli were associated with PBP3 inserts together with CMY-42 ß-lactamase; correlates among Enterobacter cloacae complex isolates remain elusive, with AmpC and PBP3 sequences found to be species specific. In the case of Klebsiella spp., no MICs exceeding 2 mg/L were seen for either combination. It appears that these avibactam combinations have potential against Enterobacterales with carbapenemase-independent carbapenem resistance or reduced susceptibility, with ceftazidime/avibactam being more reliably active than aztreonam/avibactam.

Clone dissemination of IncX3 plasmid carrying blaNDM-1 in ST76 carbapenem resistance Klebsiella pneumoniae and bactericidal efficiency of aztreonam combined with avibactam in vitro and in vivo.

OBJECTIVES: The rapid spread of the New Delhi Metal-ß-lactamase-1 (NDM-1) gene in Klebsiella pneumoniae poses a substantial challenge to pediatric therapeutic care. Here, we aimed to characterise the IncX3-type plasmid carrying the blaNDM-1 gene in ST76 carbapenem resistance K. pneumoniae (CRKP) strains and assess the in vitro and in vivo bactericidal efficacy of Aztreonam (ATM) combined with Avibactam (AVI) (ATM+AVI) against CRKP. METHODS: The broth microdilution method and PCR were used to detect antimicrobial susceptibility and antibiotic resistance genes. Genetic relatedness was determined using Pulsed-Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST). The plasmid conjugation assay was used to verify the transmissibility of drug-resistant plasmids. Whole-Genome Sequencing (WGS) was employed to elucidate the genomic attributes of the genes. The Fractional Inhibitory Concentration (FIC) was calculated based on the checkerboard titration assay to determine the antimicrobial effect of ATM+AVI. The time-kill curve assay and a mouse anti-infection model were used to investigate the in vitro and in vivo bactericidal efficiency of ATM+AVI. RESULTS: Seven blaNDM-1-producing strains were found to be highly resistant to carbapenems, and they all belonged to the same sequence type (ST76) and were classified into the same PFGE clusters with an 89.1% similarity. The conjugation assay showed that the blaNDM-1-carrying plasmid was successfully transferred to Escherichia coli 600, resulting in transconjugants with carbapenem antibiotic resistance. A 54-kb IncX3 plasmid (pNDM-XZA88) carried the blaNDM-1 gene located on a Tn125 transposon-like element structure, demonstrating the transferability of resistance genes. Genome comparative analysis revealed that pNDM-XZA88 was highly similar to pCQ17 × 3 and pRor-30818cz and had relatively conserved backbones and variable accessory regions compared to the other four plasmids (pC39-334 kb, pNDM-1-DY1928, pNDM-K725, and pNDM-Z244). The checkerboard titration and time-kill curve assays revealed that the ATM+AVI combination therapy exerted significant bactericidal efficacy against the blaNDM-1-producing strains in vitro. The ATM+AVI combination also significantly reduced the bacterial burden in a mouse infection model constructed using the blaNDM-1-producing K. pneumoniae. CONCLUSION: This study demonstrated the clone dissemination of blaNDM-1-harboring IncX3 plasmids among the ST76 K. pneumoniae isolated from pediatric patients. Therefore, more attention should be paid to preventing this high-risk clone from harming pediatric patients. Moreover, we deduced that the ATM+AVI combination therapy is an effective strategy for treating blaNDM-1-producing K. pneumoniae.

Rapid characterization of carbapenem-resistant Enterobacterales by multiplex lateral flow assay and detection of ceftazidime-avibactam-aztreonam synergy.

PURPOSE: The choice of antibiotics for treatment of Carbapenem-Resistant Enterobacterales (CRE) is increasing becoming limited due to co-expression of Metallo-beta-lactamases (MBL) along with other carbapenemases in these isolates. The study aimed to investigate the occurrence of CRE and to determine the in-vitro synergy and clinical outcomes of Ceftazidime-Avibactam and Aztreonam combination in CRE infections in adult Intensive Care Units (ICUs). METHODS: 79 CRE isolates recovered from adult ICUs during January to March 2023 were tested by O.K.N.V.I. RESIST-5, a lateral flow multiplex assay for rapid detection of OXA-48-like, NDM, IMP, VIM, and KPC carbapenemases. Ceftazidime-Avibactam MIC was determined by microbroth dilution method and in vitro synergy between Ceftazidime-Avibactam and Aztreonam was assessed by Modified E-test/disc diffusion method for these isolates. RESULTS: The study revealed 7.5 % occurrence of CRE in our hospital, with high occurrence of NDM (n = 42, 53.1 %) and OXA-48-like (n = 63, 79.7 %) carbapenemase. Production of more than one type of carbapenemases was found in 44 isolates. A total of 57 isolates (72 %) had Ceftazidime-Avibactam resistance and 44 of them displayed Ceftazidime-Avibactam and Aztreonam in-vitro synergy. Successful clinical outcome was observed in two patients who received Ceftazidime-Avibactam and Aztreonam combination therapy for 7 days or more. CONCLUSIONS: Despite the preponderance of Ceftazidime-Avibactam resistant CRE expressing NDM and OXA-48-like carbapenemase in our hospital, 77.2 % of them displayed in-vitro synergy of Ceftazidime-Avibactam with Aztreonam. It emphasizes the potential therapeutic utility of this combination in CRE strains showing coproduction of MBL and serine carbapenemases. Greater therapeutic potential of Ceftazidime-Avibactam and Aztreonam combination was observed with extended duration of therapy. However, further clinical evidence is needed to establish the efficacy of this combination and consider other factors that influence treatment outcomes.

Ceftazidime-avibactam and aztreonam combination for Carbapenem-resistant Enterobacterales bloodstream infections with presumed Metallo-ß-lactamase production: a systematic review and meta-analysis.

INTRODUCTION: Carbapenem-resistant Enterobacterales (CRE) due to Metallo-ß-lactamase (MBL) production are treated with either polymyxins or the novel combination of ceftazidime-avibactam and aztreonam (AA). This study aims to evaluate the 30-day mortality of AA in patients with BSI caused by MBL-CRE infections. METHODOLOGY: In this systematic review and meta-analysis, all articles up to June 2023 were screened using search terms like 'CRE', 'MBL', 'AA' and 'polymyxins'. The risk ratio for AA vs polymyxins was pooled using a random-effect model, and the results were represented by a point estimate with a 95% confidence interval. RESULTS: After removing the duplicates, the titles and abstracts of 455 articles were screened, followed by a full-text screening of 50 articles. A total of 24 articles were included for systematic review, and four comparative studies were included in the meta-analysis. All four studies had a moderate or serious risk of bias. The pooled risk ratio for 30-day mortality for AA vs. polymyxins was 0.51 (95%CI: 0.34-0.76), p < 0.001. There was no significant heterogeneity. CONCLUSION: The meta-analysis from studies with a high risk of bias shows that AA is associated with lesser 30-day mortality when compared to polymyxins in patients with MBL-producing CRE BSI. Registration with PROSPERO- CRD42023433608.