Rapid pathogen identification, resistance detection, and susceptibility profiling improve antimicrobial prescribing and associated outcomes, but fragmented workflows lead to inefficiencies and are costly. We evaluated a research-use-only (RUO) approach using ASTar remnant bacterial suspension from routine antimicrobial susceptibility testing (AST) for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) pathogen identification and lateral flow assay (LFA)-based detection of targeted resistance mechanisms. Gram-negative bacterial strains from reference and curated resistance collections (CDC1, ARLG2, and ATCC3) ( n = 129) were contrived into blood culture bottles and processed in the ASTar System using the ASTar BC G- Kit (Q-linea AB, Sweden). Under RUO conditions, remnant bacterial suspensions were collected ~1–2 h after ASTar run initiation and analyzed using NG Test CTX-M Multi, NG Test CARBA-5, NG Test Acineto-5 RUO, and MALDI-TOF MS. Mean (±SD) remnant suspension volume was 2,722 μL (±300 µL). All samples yielded high-confidence MALDI-TOF MS scores (>2.0), with five initially scoring <2.0 and resolving on repeat testing. LFA results showed full agreement with reference isolates for blaCTX-M positive/negative (32/32) and with 60 or 61 target carbapenemase-positive/negative isolates. Testing of a subset of samples to mimic reflex workflows with ASTar phenotypic results did not affect LFA performance ( n = 35; 23 Enterobacterales, 3 Pseudomonas aeruginosa , and 9 Acinetobacter baumannii ). Cost savings can be realized versus commercial multiplex PCR. This integrated approach of ~6 h rapid phenotypic AST with same-run identification and resistance detection (1–2 h from instrument start) or reflex testing upon availability of ASTar results may support earlier susceptibility results and offer cost savings to current workflows. IMPORTANCE: Rapid, integrated diagnostic workflows are recommended and essential for improving patient outcomes in bloodstream infections, where delays in pathogen identification and resistance detection can compromise timely antimicrobial therapy. Current workflows often rely on multiple platforms, increasing turnaround time and laboratory burden. Additionally, the clinical impact of rapid diagnostic results is dependent on timely interpretation and implementation within antimicrobial stewardship frameworks, which may involve complex decision algorithms. This study demonstrates that remnant bacterial suspensions generated during routine antimicrobial susceptibility testing (AST) using the ASTar System can be recovered and utilized for rapid matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and lateral flow assay-based resistance detection. By enabling pathogen identification and/or resistance detection within ~1–2 h of AST initiation or resistance detection via reflex testing, this approach reduces duplicate sample testing and may enable earlier access to rapid microbiological results.

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