We evaluated 14 304 Pseudomonas aeruginosa isolates from 56 US medical centers (2015-2024). Ceftazidime-avibactam, ceftolozane-tazobactam, and imipenem-relebactam were active against 97.0%-97.9% of isolates overall. Piperacillin-tazobactam and ceftazidime activities decreased slightly during the study period, whereas the activities of meropenem, levofloxacin, and tobramycin improved since 2017.

Acinetobacter baumannii is a formidable nosocomial pathogen responsible for severe hospital-acquired infections. This study aimed to investigate the relationship between genotypic and phenotypic resistance profiles in 36 non-duplicate invasive A. baumannii isolates collected at a tertiary care hospital in Bulgaria over a six-year period (2019-2024). Identification was performed by MALDI-TOF MS and PCR-based detection of blaOXA-51-like and gyrB. Antimicrobial susceptibility testing to ten agents was performed by gradient diffusion and broth microdilution, and selected resistance genes were detected by conventional PCR. Whole-genome sequencing of 17 representative isolates was conducted to confirm the resistance genotype-phenotype associations and perform MLST. Carbapenem resistance was detected in 34/36 isolates (94.4%), mediated by blaOXA-23-like (29/34) or blaOXA-72 (5/34). The armA gene associated with high-level resistance to all three aminoglycosides was identified exclusively in blaOXA-23-like-positive isolates (18/29). In armA-negative isolates, gentamicin and tobramycin MICs decreased markedly, whereas amikacin MICs remained elevated. In addition, minocycline MICs ≥ 8 mg/L were observed in the presence of armA. Seven distinct genotypic-phenotypic profiles were defined based on these gene-MIC associations. Resistome and phylogenetic analyses confirmed these relationships, with MLST assigning the isolates to established clonal lineages ST2 and ST636 (International Clone II). These findings support the use of targeted genes, such as armA, combined with phenotypic testing to guide empirical therapy and strengthen local surveillance of high-risk A. baumannii clones in hospital settings.

Background: The dissemination of extensively drug-resistant Pseudomonas aeruginosa strains across the human-food interface represents a critical threat to global public health, complicating empirical therapeutic choices and challenging traditional nosocomial restriction paradigms. Objective: This study investigated the prevalence, phenotypic resistome landscapes, and underlying integron-mediated mobilization mechanics of P. aeruginosa across the One Health continuum using an optimized real-time quantitative PCR (qPCR) platform. Methods: A total of 116 samples comprising human clinical specimens (n = 61) and retail food matrices (n = 55) were screened. Phenotypic resistance was profiled against 16 antipseudomonal agents, with polymyxin and colistin resistance validated via reference broth microdilution. A novel real-time SYBR Green qPCR assay backed by derivative melting curve analysis was developed to track Class 1 (intI1) and Class 2 (intI2) integrons. Results:Pseudomonas aeruginosa was recovered from 36.2% (n = 42/116) of total samples, exhibiting a homogeneous distribution across clinical (32.8%) and retail food matrices (40.0%, p = 0.445). Antibiogram characterization revealed a critical resistance burden: 100% of isolates fell into extreme epidemiological tiers, with 71.4% (n = 30) exhibiting an extensively drug-resistant (XDR) phenotype and 28.6% (n = 12) reaching absolute pandrug-resistant (PDR) status. Absolute phenotypic resistance (100%) was recorded across both cohorts for meropenem, ceftazidime, aztreonam, tobramycin, polymyxins, and novel combination therapeutics (ceftazidime-avibactam and ceftolozane-tazobactam). Pairwise tracking demonstrated an exceptionally strong positive correlation in resistance distribution patterns between human and food isolates (Spearman's rho = 0.948, p < 0.001). Analytical validation of the qPCR assay demonstrated tight, reproducible specific derivative melting peaks (Tm) at 83.2 °C for intI1 and 83.8 °C for intI2. Genotypic screening revealed universal conservation of plasmid-borne intI1 (100%), whereas plasmidic intI2 was variably distributed (61.9%). Crucially, intI2 carriage served as a definitive marker for the transition to complete pandrug resistance, being detected in 100% (n = 12/12) of PDR isolates compared to 46.7% (n = 14/30) of XDR strains (p = 0.001), supported by a strong positive correlation with the PDR phenotype (rho = 0.496, p = 0.0008). Conclusions: These findings reveal an overlapping, extreme resistome across the food-clinical interface and identify intI2 as a genetic tipping point for absolute pandrug resistance. The validated real-time assay provides a highly sensitive, proactive molecular surveillance framework capable of mapping high-risk mobile genetic platforms before they manifest as catastrophic empirical treatment failures.

Background/Objectives: Antimicrobial resistance among ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens has become a major global health threat, significantly reducing the effectiveness of conventional antibiotics. Plant-derived compounds have emerged as promising antibiotic adjuvants capable of restoring antibiotic activity through multiple resistance-modulating mechanisms. This review aims to critically evaluate the current evidence regarding phytochemicals that enhance antibiotic efficacy against drug-resistant ESKAPE pathogens. Methods: Relevant studies investigating plant-derived compounds with antibiotic adjuvant activity against ESKAPE pathogens were identified and critically analyzed. Particular attention was given to mechanisms of action, synergistic interactions with conventional antibiotics, structure-activity relationships, and translational evidence from in vivo studies. Results: Numerous phytochemicals have demonstrated the ability to potentiate antibiotic activity through efflux pump inhibition, biofilm disruption, membrane permeabilization, and quorum sensing interference. For instance, the flavonoid quercetin inhibits the NorA efflux pump in Staphylococcus aureus, restoring ciprofloxacin susceptibility, while the sulfur-containing compound ajoene from garlic disrupts quorum sensing in Pseudomonas aeruginosa, sensitizing biofilms to tobramycin. Although several compounds exhibit promising synergistic effects in vitro, significant barriers remain regarding bioavailability, standardization, pharmacokinetics, and clinical translation. Conclusions: Plant-derived antibiotic adjuvants represent a promising strategy to combat multidrug-resistant ESKAPE pathogens. However, greater emphasis on translational research, standardized methodologies, and clinically relevant experimental models is required to facilitate their development toward therapeutic applications.

Background/Objectives: Antimicrobial resistance (AMR) poses a major global health challenge, particularly in opportunistic pathogens such as Pseudomonas aeruginosa. This study aimed to identify metabolic adaptations associated with antibiotic resistance by integrating transcriptomic data from drug-resistant clinical isolates with a genome-scale metabolic model (GEM) of P. aeruginosa under four antibiotic treatments: ceftazidime (CAZ), ciprofloxacin (CIP), meropenem (MEM), and tobramycin (TOB). Methods: Transcriptomic data from 414 clinical isolates were integrated with the iPau21 genome-scale metabolic model (GEM) of P. aeruginosa. Differential gene expression analysis was performed using DESeq2, and differentially expressed genes (DEGs) were identified using a false discovery rate (FDR)-adjusted p-value < 0.05 and a fold-change threshold of ≥2 or ≤0.5. Reporter metabolites (RMs) were identified using the Reporter Metabolite algorithm with an FDR-adjusted p-value < 0.05. Pathway enrichment analysis was performed to characterize condition-specific metabolic alterations, and pathway significance was determined using the Benjamini-Hochberg procedure with an adjusted p-value < 0.05. Results: The analysis revealed predominantly antibiotic-specific transcriptional responses, with limited overlap in DEGs across treatment conditions. Reporter metabolite and pathway enrichment analyses identified distinct metabolic adaptations associated with biofilm formation, virulence, and stress response pathways. Several metabolites, including propionic acid, acetic acid, L-inositol, glutamine, glutarate, fumarate, and melatonin, were computationally prioritized candidate metabolites for future metabolite-based adjuvant strategies aimed at enhancing antibiotic efficacy. Conclusions: This systems biology approach provides a comprehensive framework for identifying metabolic vulnerabilities associated with AMR in P. aeruginosa. The identified metabolites represent candidate antibiotic adjuvant molecules generated through computational prioritization and should be regarded as hypotheses for future experimental validation rather than validated therapeutic interventions. These findings provide a foundation for future studies exploring metabolism-based strategies to improve antibiotic efficacy and combat antimicrobial resistance.

Filter-feeding bivalves can accumulate fecal bacteria and antimicrobial resistance determinants, yet routine regulatory monitoring relies mainly on quantitative Escherichia coli criteria. This study provides a whole-genome characterization of an ExPEC-like E. coli ST536 (O21) isolate recovered from commercially depurated retail mussels originating from a Class B harvesting area in Portugal. The isolate was phenotypically identified by MicroScan WalkAway Plus and subjected to antimicrobial susceptibility testing and Oxford Nanopore long-read sequencing, followed by genome assembly, annotation, antimicrobial resistance and virulence screening, plasmid and prophage detection, and phylogenomic analysis. Phenotypically, only resistance to tobramycin was detected using the MicroScan WalkAway system. Genomic analysis revealed a complex mobile element-rich architecture, including multiple resistance-associated determinants, 116 virulence-associated loci, prophage regions, and a mobilizable IncY plasmid carrying CTX-like class A β-lactamase-associated hits detected by ABRicate. Despite its ST536 assignment and ExPEC-associated traits, phylogenomic analysis placed the isolate within a phylogroup A/commensal-like background rather than among classical B2 ExPEC lineages, suggesting acquisition of pathoadaptive modules by an atypical environmental lineage. These findings highlight the potential utility of WGS as a complementary tool and suggest that depurated bivalves may serve as sentinels, warranting further investigation.

The rise of multidrug-resistant microorganisms highlights the need for rapid antimicrobial susceptibility testing (AST) to guide early and effective therapy. This study evaluated a novel MALDI-TOF MS-based assay (MBT FAST, Bruker, Germany), which employs the direct-on-target microdroplet growth assay (DOT-MGA) for phenotypic AST. A total of 243 Enterobacteriaceae isolates representing diverse species and resistance patterns were tested against a broad panel of antibiotics. Each isolate was incubated in 6-µL microdroplets of cation-adjusted Mueller-Hinton broth, with and without antibiotics at two-fold serial dilutions, at 35°C for 6 h inside a humidity-controlled chamber (MBT FAST Shuttle, Bruker). Broth was removed using a prototype device (MBT FAST Stamp, Bruker), and MALDI-TOF MS measurements were performed with the MALDI Biotyper Sirius system (Bruker). Results were compared with broth microdilution as the standard for determining minimum inhibitory concentrations (MIC). Valid growth control was achieved for 100% of isolates. Essential agreement (EA), defined as MICs within ±1 two-fold dilution step of the standard method, was as follows: amoxicillin/clavulanic acid 96.3%, amikacin 97.5%, aztreonam 95.5%, ceftazidime/avibactam 95.5%, ceftazidime 94.6%, cefepime 92.2%, ciprofloxacin 91.8%, colistin 92.2%, ceftriaxone 95.5%, cefotaxime 94.2%, gentamicin 95.5%, imipenem 91.8%, levofloxacin 97.9%, meropenem 87.2%, meropenem/vaborbactam 94.2%, piperacillin/tazobactam 90.9%, trimethoprim/sulfamethoxazole 90.9%, temocillin 93.3%, and tobramycin 97.5%. The overall EA across all antimicrobials tested reached 96.0%. The research-use-only MBT FAST assay provides reliable AST within 6 h, substantially reducing turnaround time. Building on technology widely implemented in diagnostic laboratories, this approach holds promise for integration into microbiology practice.