In vitro activity of ceftazidime/avibactam, cefiderocol, meropenem/vaborbactam and imipenem/relebactam against clinical strains of the Stenotrophomonas maltophilia complex.

BACKGROUND: Infections caused by Stenotrophomonas maltophilia and related species are increasing worldwide. Unfortunately, treatment options are limited, whereas the antimicrobial resistance is increasing. METHODS: We included clinical isolates identified as S. maltophilia by VITEK 2 Compact. Ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam, cefiderocol, quinolones, and tetracycline family members were evaluated by broth microdilution method and compared with first-line treatment drugs. Minimum inhibitory concentrations (MICs) were reported for all antibiotics. We sequenced the Whole Genome of cefiderocol resistant strains (CRSs) and annotated their genes associated with cefiderocol resistance (GACR). Presumptive phylogenetic identification employing the 16S marker was performed. RESULTS: One hundred and one clinical strains were evaluated, sulfamethoxazole and trimethoprim, levofloxacin and minocycline showed susceptibilities of 99.01%, 95.04% and 100% respectively. Ceftazidime was the antibiotic with the highest percentage of resistance in all samples (77.22%). Five strains were resistant to cefiderocol exhibiting MIC values ≥ 2 µg/mL (4.95%). The ß-lactamase inhibitors meropenem/vaborbactam and imipenem/relebactam, failed to inhibit S. maltophilia, preserving both MIC50 and MIC90 ≥64 µg/mL. Ceftazidime/avibactam restored the activity of ceftazidime decreasing the MIC range. Tigecycline had the lowest MIC range, MIC50 and MIC90. Phylogeny based on 16S rRNA allowed to identify to cefiderocol resistant strains as putative species clustered into Stenotrophomonas maltophilia complex (Smc). In these strains, we detected GARCs such as Mutiple Drug Resistance (MDR) efflux pumps, L1-type ß-lactamases, iron transporters and type-1 fimbriae. CONCLUSION: Antimicrobial resistance to first-line treatment is low. The in vitro activity of new ß-lactamase inhibitors against S. maltophilia is poor, but avibactam may be a potential option. Cefiderocol could be considered as a potential new option for multidrug resistant infections. Tetracyclines had the best in vitro activity of all antibiotics evaluated.

Plasmid-encoded gene duplications of extended-spectrum ß-lactamases in clinical bacterial isolates.

Introduction: The emergence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is an urgent and alarming One Health problem. This study aimed to investigate duplications of plasmid-encoded ESBL genes and their impact on antimicrobial resistance (AMR) phenotypes in clinical and screening isolates. Methods: Multi-drug-resistant bacteria from hospitalized patients were collected during routine clinical surveillance from January 2022 to June 2023, and their antimicrobial susceptibility patterns were determined. Genotypes were extracted from long-read whole-genome sequencing data. Furthermore, plasmids and other mobile genetic elements associated with ESBL genes were characterized, and the ESBL genes were correlated to ceftazidime minimal inhibitory concentration (MIC). Results: In total, we identified four cases of plasmid-encoded ESBL gene duplications that match four genetically similar plasmids during the 18-month surveillance period: five Escherichia coli and three Klebsiella pneumoniae isolates. As the ESBL genes were part of transposable elements, the surrounding sequence regions were duplicated as well. In-depth analysis revealed insertion sequence (IS)-mediated transposition mechanisms. Isolates with duplicated ESBL genes exhibited a higher MIC for ceftazidime in comparison to isolates with a single gene copy (3-256 vs. 1.5-32 mg/L, respectively). Conclusion: ESBL gene duplications led to an increased phenotypic resistance against ceftazidime. Our data suggest that ESBL gene duplications by an IS-mediated transposition are a relevant mechanism for how AMR develops in the clinical setting and is part of the microevolution of plasmids.

Zein nanoparticles containing ceftazidime and tobramycin: antibacterial activity against Gram-negative bacteria.

Aims: This work describes the encapsulation of ceftazidime and tobramycin in zein nanoparticles (ZNPs) and the characterization of their antibacterial and antibiofilm activities against Gram-negative bacteria. Materials & methods: ZNPs were synthesized by nanoprecipitation. Cytotoxicity was assessed by MTT assay and antibacterial and antibiofilm assays were performed by broth microdilution and violet crystal techniques. Results: ZNPs containing ceftazidime (CAZ-ZNPs) and tobramycin (TOB-ZNPs) showed drug encapsulation and thermal stability. Encapsulation of the drugs reduced their cytotoxicity 9-25-fold. Antibacterial activity, inhibition and eradication of biofilm by CAZ-ZNPs and TOB-ZNPs were observed. There was potentiation when CAZ-ZNPs and TOB-ZNPs were combined. Conclusion: CAZ-ZNPs and TOB-ZNPs present ideal physical characteristics for in vivo studies of antibacterial and antibiofilm activities.

A nanotechnology product was developed to treat diseases caused by bacteria. This prototype showed the ideal characteristics and could be administered by ingestion through the mouth, aspiration through the nose or injection into the veins. The prototype did not harm or kill human cells. It killed the bacteria and prevented the formation of a type of protection against antibiotics that bacteria can produce, called a biofilm. Nanotechnology products are a promising alternative for the treatment of bacterial infections.

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.

In-depth analysis of the treatment effect and synergistic mechanism of TanReQing injection on clinical multi-drug resistant Pseudomonas aeruginosa.

Antibiotic resistance is a recognized and concerning public health issue. Gram-negative bacilli, such as Pseudomonas aeruginosa (P. aeruginosa), are notorious for their rapid development of drug resistance, leading to treatment failures. TanReQing injection (TRQ) was chosen to explore its pharmacological mechanisms against clinical multidrug-resistant P. aeruginosa (MDR-PA), given its antibacterial and anti-inflammatory properties. We revealed the expression of proteins and genes in P. aeruginosa after co-culture with TRQ. This study developed an assessment method to evaluate clinical resistance of P. aeruginosa using MALDI-TOF MS identification and Biotyper database searching techniques. Additionally, it combined MIC determination to investigate changes in MDR-PA treated by TRQ. TRQ effectively reduced the MICs of ceftazidime and cefoperazone and enhanced the confidence scores of MDR-PA as identified by mass spectrometry. Using this evaluation method, the fingerprints of standard P. aeruginosa and MDR-PA were compared, and the characteristic peptide sequence (Seq-PA No. 1) associated with flagellum was found. The phenotypic experiments were conducted to confirm the effect of TRQ on the motility and adhesion of P. aeruginosa. A combination of co-immunoprecipitation and proteome analysis was employed, and 16 proteins were significantly differentially expressed and identified as potential candidates for investigating the mechanism of inhibiting resistance in P. aeruginosa treated by TRQ. The candidates were verified by quantitative real-time PCR analysis, and TRQ may affect these core proteins (MexA, MexB, OprM, OprF, OTCase, IDH, and ASL) that influence resistance of P. aeruginosa. The combination of multiple methods helps elucidate the synergistic mechanism of TRQ in overcoming resistance of P. aeruginosa.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen closely associated with various life-threatening acute and chronic infections. The presence of antimicrobial resistance and multidrug resistance in P. aeruginosa infections significantly complicates antibiotic treatment. The expression of ß-lactamase, efflux systems such as MexAB-OprM, and outer membrane permeability are considered to have the greatest impact on the sensitivity of P. aeruginosa. The study used a method to assess the clinical resistance of P. aeruginosa using matrix-assisted laser desorption ionization time of flight mass spectrometry identification and Biotyper database search techniques. TanReQing injection (TRQ) effectively reduced the MICs of ceftazidime and cefoperazone in multidrug-resistant P. aeruginosa (MDR-PA) and improved the confidence scores for co-cultured MDR-PA. The study found a characteristic peptide sequence for distinguishing whether P. aeruginosa is resistant. Through co-immunoprecipitation and proteome analysis, we explored the mechanism of TRQ overcoming resistance of P. aeruginosa.

Reduce susceptibility to cefiderocol in gram negative bacteria in children: Is hope already lost before it's even arrived?

BACKGROUND: In last years the diffusion of carbapenem resistance in Gram-negative bacteria (CR-GNB) is increasing worldwide, mainly due to the expression of carbapenemases. Cefiderocol has molecular characteristics that ideally confers activity against all CR-GNB, but resistant strains have already been identified. We describe cefiderocol susceptibility profile among multi-drug resistant Gram-negative isolated from pediatric patients. METHODS: Prospective, single pediatric center study, 1st January 2020-15th June 2023. All GNB carbapenemases producers or phenotypically carbapenem-resistant isolated in the study period were tested for cefiderocol susceptibility. Clinical and microbiological data were collected. A descriptive analysis was performed, comparing the groups of cefiderocol-resistant vs. cefiderocol-susceptible Enterobacterales and non-fermenting Gram-negative bacteria (NF-GNB). RESULTS: Forty-seven GNB were tested for cefiderocol susceptibility; 38% were cefiderocol-resistant: 16/30 (52%) among Enterobacterales and 2/17 (12%) among NF-GNB. None of the patients were previously exposed to cefiderocol. Looking at Enterobacterales, resistance to ceftazidime/avibactam was higher among cefiderocol-resistant vs. cefiderocol-susceptible strains (62% vs 36%, respectively), as MBL expression (67% vs. 36%, respectively). Too few NF-GNB were cefiderocol-resistance to draw any conclusion. No difference in ICU admission and mortality was identified comparing cefiderocol-resistant vs. susceptible strains. Patients colonized/infected by cefiderocol-resistant strains had been previously hospitalized more frequently. CONCLUSION: In our cohort cefiderocol resistance was mostly registered among Enterobacterales, and especially among MBL producers' strains (that were alongside resistant to ceftazidime/avibactam). This could be explained by the known possible cross resistance mechanism among ceftazidime/avibactam and cefiderocol. Also, correlation of cefiderocol-resistance with previous hospitalization could be associated with horizontal resistance transmission. Looking at our data, we believe that cefiderocol should be use cautiously, especially empirically and in monotherapy, due to the high resistance rate.

Genomic epidemiology and ceftazidime-avibactam high-level resistance mechanisms of Pseudomonas aeruginosa in China from 2010 to 2022.

Ceftazidime-avibactam (CZA) resistance is a huge threat in the clinic; however, the underlying mechanism responsible for high-level CZA resistance in Pseudomonas aeruginosa (PA) isolates remains unknown. In this study, a total of 5,763 P. aeruginosa isolates were collected from 2010 to 2022 to investigate the ceftazidime-avibactam (CZA) high-level resistance mechanisms of Pseudomonas aeruginosa (PA) isolates in China. Fifty-six PER-producing isolates were identified, including 50 isolates carrying blaPER-1 in PA, and 6 isolates carrying blaPER-4. Of these, 82.1% (46/56) were classified as DTR-PA isolates, and 76.79% (43/56) were resistant to CZA. Importantly, blaPER-1 and blaPER-4 overexpression led to 16-fold and >1024-fold increases in the MICs of CZA, respectively. WGS revealed that the blaPER-1 gene was located in two different transferable IncP-2-type plasmids and chromosomes, whereas blaPER-4 was found only on chromosomes and was carried by a class 1 integron embedded in a Tn6485-like transposon. Overexpression of efflux pumps may be associated with high-level CZA resistance in blaPER-1-positive strains. Kinetic parameter analysis revealed that PER-4 exhibited a similar kcat/Km with ceftazidime and a high (∼3359-fold) IC50 value with avibactam compared to PER-1. Our study found that overexpression of PER-1 combined with enhanced efflux pump expression and the low affinity of PER-4 for avibactam contributes to high-level resistance to CZA. Additionally, the Tn6485-like transposon plays a significant role in disseminating blaPER. Urgent active surveillance is required to prevent the further spread of high-level CZA resistance in DTR-PA isolates.

The pharmacokinetics/pharmacodynamics of ceftazidime/avibactam for central nervous system infections caused by carbapenem-resistant Gram-negatives: a prospective study.

OBJECTIVES: To describe the pharmacokinetics/pharmacodynamics (PK/PD) of ceftazidime/avibactam in critically ill patients with CNS infections. METHODS: A prospective study of critically ill patients with CNS infections who were treated with ceftazidime/avibactam and the steady-state concentration (Css) of ceftazidime/avibactam in serum and/or CSF was conducted between August 2020 and May 2023. The relationship between PK/PD goal achievement, microbial eradication and the clinical efficacy of ceftazidime/avibactam was evaluated. RESULTS: Seven patients were finally included. The ceftazidime/avibactam target attainment in plasma was optimal for three, quasi-optimal for one and suboptimal for three. In three patients with CSF drug concentrations measured, ceftazidime/avibactam target attainment in CSF was 100% (3/3), which was optimal. The AUCCSF/serum values were 0.59, 0.44 and 0.35 for ceftazidime and 0.57, 0.53 and 0.51 for avibactam. Of the seven patients, 100% (7/7) were treated effectively, 71.4% (5/7) achieved microbiological eradication, 85.7% (6/7) survived and 14.3% (1/7) did not survive. CONCLUSIONS: The limited clinical data suggest that ceftazidime/avibactam is effective in the treatment of CNS infections caused by MDR Gram-negative bacilli (MDR-GNB), can achieve the ideal drug concentration of CSF, and has good blood-brain barrier penetration.

High ceftazidime-avibactam resistance among carbapenem-resistant Enterobacter species: Data from the Antimicrobial Testing Leadership and Surveillance (ATLAS) programme, 2014-2021.

OBJECTIVES: Trends in the susceptibility to ceftazidime-avibactam (CZA) and tigecycline (TGC) among Enterobacter species from different geographic areas are unknown.This study aimed to analyse the trends in CZA and TGC susceptibility changes across different continents from 2014 to 2021 utilizing Antimicrobial Testing Leadership and Surveillance (ATLAS) data. METHODS: A total of 23 669 isolates of Enterobacter species were collected over an 8-y period. RESULTS: The overall non-susceptibility rate of Enterobacter isolates to both CZA and TGC was 3.2%. India (16.5%), Guatemala (15.4%), and the Philippines (13.1%) exhibited the highest resistance to CZA. The increase in CZA resistance rates was particularly evident in Asia, with an increase from 4.0% to 8.3%, and in Latin America, from 1.5% to 5%. The non-susceptibility rate for TGC mildly increased in Africa/Middle East but decreased in other continents during the study period. The overall rate of carbapenem resistance increased from 2.9% in 2014-2017 to 4.3% in 2018-2021. Among carbapenem-resistant Enterobacter isolates, the CZA resistance rate was highest in Asia (87.4%), followed by Europe (69.2%) and Africa/Middle East (60.8%). Among the 380 Enterobacter isolates resistant to CZA and carbapenem, the most common genotype of carbapenemase genes was blaNDM (59.2%), followed by blaVIM (24.2%), blaOXA (4.2%), blaIMP (1.1%), and blaKPC (1.1%). The susceptibility of carbapenem-resistant Enterobacter to TGC remained high, with an overall susceptibility rate of 90%. CONCLUSIONS: The heterogeneous distribution of CZA resistance rates among different geographical regions highlights the divergent therapeutic options for drug-resistant Enterobacter species.

Global trends in nonsusceptibility rates of Escherichia coli isolates to meropenem and ceftazidime/avibactam: Data from the Antimicrobial Testing Leadership and Surveillance (ATLAS) programme, 2014-21.

OBJECTIVE: To understand the global changes in the nonsusceptibility rates of Escherichia coli to meropenem and ceftazidime-avibactam (CZA), we conducted a study using the Antimicrobial Testing Leadership and Surveillance database. METHODS: A total of 49 394 E. coli isolates were collected during the 8-year study period. RESULT: The countries with the highest nonsusceptible rates for meropenem were India (16.6%), followed by Pakistan (6.7%), Ukraine (5.4%), Qatar (5.3%), and Guatemala (3.2%). For CZA, the nonsusceptible rate was highest in India (15.6%), followed by Qatar (4.0%), Guatemala (3.9%), China (2.6%), and Thailand (2.5%). During the study period, the nonsusceptible rates of meropenem and CZA in E. coli increased in Asia, Latin America, and Africa/Middle East. Isolates from the medical ICU (odds ratio [OR], 4.62) and surgical ICU (OR, 3.98) were associated with a higher risk of CZA nonsusceptible rates. Compared to intestinal specimens, respiratory and genitourinary specimens had the highest OR (2.32 and 2.17) associated with CZA resistance. Further analysis of carbapenemase distribution showed an increase in the percentage of blaNDM-positive isolates and a decrease in blaKPC-positive isolates worldwide, especially in Latin America. Additionally, we observed a gradual decline in the prevalence of blaOXA-positive E. coli without concomitant carriage of metallo-ß-lactamase genes in the worldwide surveillance. CONCLUSIONS: Further surveillance is necessary to determine whether blaNDM -positive E. coli (i.e., CZA-resistant isolates) is increasing and leading to more superbugs spreading worldwide.

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.

Olive oil nanoemulsion containing curcumin: antimicrobial agent against multidrug-resistant bacteria.

The present work aimed to develop, characterize, and evaluate the antibacterial and antibiofilm activity of two nanoemulsions (NEs) containing 500 µg/mL of curcumin from Curcuma longa (CUR). These NEs, produced with heating, contain olive oil (5%) and the surfactants tween 80 (5%) and span 80 (2.5%), water q.s. 100 mL, and were stable for 120 days. NE-2-CUR presented Ø of 165.40 ± 2.56 nm, PDI of 0.254, ζ of - 33.20 ± 1.35 mV, pH of 6.49, and Entrapment Drug Efficiency (EE) of 99%. The NE-4-CUR showed a Ø of 105.70 ± 4.13 nm, PDI of 0.459, ζ of - 32.10 ± 1.45 mV, pH of 6.40 and EE of 99.29%. Structural characterization was performed using DRX and FTIR, thermal characterization using DSC and TG, and morphological characterization using SEM, suggesting that there is no significant change in the CUR present in the NEs and that they remain stable. The MIC was performed by the broth microdilution method for nine gram-positive and gram-negative bacteria, as well as Klebsiella pneumoniae clinical isolates resistant to antibiotics and biofilm and efflux pump producers. The NEs mostly showed a bacteriostatic profile. The MIC varied between 125 and 250 µg/mL. The most sensitive bacteria were Staphylococcus aureus and Enterococcus faecalis, for which NE-2-CUR showed a MIC of 125 µg/mL. The NEs and ceftazidime (CAZ) interaction was also evaluated against the K. pneumoniae resistant clinical isolates using the Checkerboard method. NE-2-CUR and NE-4-CUR showed a synergistic or additive profile; there was a reduction in CAZ MICs between 256 times (K26-A2) and 2 times (K29-A2). Furthermore, the NEs inhibited these isolates biofilms formation. The NEs showed a MBIC ranging from 15.625 to 250 µg/mL. Thus, the NEs showed physicochemical characteristics suitable for future clinical trials, enhancing the CAZ antibacterial and antibiofilm activity, thus becoming a promising strategy for the treatment of bacterial infections caused by multidrug-resistant K. pneumoniae. KEY POINTS: • The NEs showed physicochemical characteristics suitable for future clinical trials. • The NEs showed a synergistic/additive profile, when associated with ceftazidime. • The NEs inhibited biofilm formation of clinical isolates.

Assessing the in vivo impact of novel ß-lactamase inhibitors on the efficacy of their partner ß-lactams against serine carbapenemase-producing Pseudomonas aeruginosa using human-simulated exposures.

OBJECTIVES: To evaluate the efficacy of human-simulated regimens (HSRs) of ceftazidime, ceftazidime/avibactam, imipenem, imipenem/relebactam, meropenem and meropenem/vaborbactam in a murine thigh infection model against serine carbapenemase-producing Pseudomonas aeruginosa. METHODS: Nine P. aeruginosa clinical isolates harbouring GES-5 (n = 1), GES-20 (n = 1), GES-5/20 (n = 1), GES-19, GES-20 (n = 3) and KPC (n = 3) were evaluated. Six mice were administered HSRs of ceftazidime 2 g q8h (2 h infusion), ceftazidime/avibactam 2.5 g q8h (2 h infusion), meropenem 2 g q8h (3 h infusion), imipenem 0.5 g q6h (0.5 h infusion), imipenem/relebactam 1.25 g q6h (0.5 h infusion) and meropenem/vaborbactam 4 g q8h (3 h infusion). Change in bacterial burden relative to baseline and the percent of isolates meeting the 1 log10 kill endpoint were assessed. RESULTS: The addition of avibactam to ceftazidime increased the percentage of isolates meeting 1 log10 kill from 33% to 100% of GES- or KPC-harbouring isolates. Imipenem/relebactam HSR produced ≥1 log10 of kill against 83% and 100% of GES- and KPC-harbouring isolates, respectively, while imipenem alone failed to reach 1 log10 kill for any isolates. Vaborbactam resulted in variable restoration of meropenem activity as 1 log10 kill was achieved in only 33% and 66% of GES- and KPC-harbouring isolates, respectively, compared with no isolates for meropenem alone. CONCLUSIONS: Ceftazidime/avibactam and imipenem/relebactam were active against 100% and 89% of KPC- or GES-harbouring isolates tested in vivo. The activity of meropenem/vaborbactam was variable, suggesting this may be an inferior treatment option in this setting. Further studies to evaluate clinical outcomes in GES- and KPC-producing P. aeruginosa are warranted given their increasing prevalence worldwide.

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.

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.

Structural insights into the molecular mechanism of high-level ceftazidime-avibactam resistance conferred by CMY-185.

ß-Lactamases can accumulate stepwise mutations that increase their resistance profiles to the latest ß-lactam agents. CMY-185 is a CMY-2-like ß-lactamase and was identified in an Escherichia coli clinical strain isolated from a patient who underwent treatment with ceftazidime-avibactam. CMY-185, possessing four amino acid substitutions of A114E, Q120K, V211S, and N346Y relative to CMY-2, confers high-level ceftazidime-avibactam resistance, and accumulation of the substitutions incrementally enhances the level of resistance to this agent. However, the functional role of each substitution and their interplay in enabling ceftazidime-avibactam resistance remains unknown. Through biochemical and structural analysis, we present the molecular basis for the enhanced ceftazidime hydrolysis and impaired avibactam inhibition conferred by CMY-185. The substituted Y346 residue is a major driver of the functional evolution as it rejects primary avibactam binding due to the steric hindrance and augments oxyimino-cephalosporin hydrolysis through a drastic structural change, rotating the side chain of Y346 and then disrupting the H-10 helix structure. The other substituted residues E114 and K120 incrementally contribute to rejection of avibactam inhibition, while S211 stimulates the turnover rate of the oxyimino-cephalosporin hydrolysis. These findings indicate that the N346Y substitution is capable of simultaneously expanding the spectrum of activity against some of the latest ß-lactam agents with altered bulky side chains and rejecting the binding of ß-lactamase inhibitors. However, substitution of additional residues may be required for CMY enzymes to achieve enhanced affinity or turnover rate of the ß-lactam agents leading to clinically relevant levels of resistance.IMPORTANCECeftazidime-avibactam has a broad spectrum of activity against multidrug-resistant Gram-negative bacteria including carbapenem-resistant Enterobacterales including strains with or without production of serine carbapenemases. After its launch, emergence of ceftazidime-avibactam-resistant strains that produce mutated ß-lactamases capable of efficiently hydrolyzing ceftazidime or impairing avibactam inhibition are increasingly reported. Furthermore, cross-resistance towards cefiderocol, the latest cephalosporin in clinical use, has been observed in some instances. Here, we clearly demonstrate the functional role of the substituted residues in CMY-185, a four amino-acid variant of CMY-2 identified in a patient treated with ceftazidime-avibactam, for high-level resistance to this agent and low-level resistance to cefiderocol. These findings provide structural insights into how ß-lactamases may incrementally alter their structures to escape multiple advanced ß-lactam agents.

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.

Rapid Detection of Ceftazidime/Avibactam Susceptibility/Resistance in Enterobacterales by Rapid CAZ/AVI NP Test.

We developed a novel culture-based test, the Rapid CAZ/AVI NP test, for rapid identification of ceftazidime/avibactam susceptibility/resistance in Enterobacterales. This test is based on glucose metabolization upon bacterial growth in the presence of a defined concentration of ceftazidime/avibactam (128/53 µg/mL). Bacterial growth is visually detectable by a red to yellow color change of red phenol, a pH indicator. A total of 101 well characterized enterobacterial isolates were used to evaluate the test performance. This test showed positive percent agreement of 100% and negative percent agreement of 98.5% with overall percent agreement of 99%, by comparison with the MIC gradient strip test (Etest) taken as the reference standard method. The Rapid CAZ/AVI NP test had only 1.5% major errors and 0% extremely major errors. This test is rapid (result within 2 hours 45 minutes), reliable, affordable, easily interpretable, and easy to implement in clinical microbiology laboratories without requiring any specific equipment.