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. 2025 Jun 3:15:1597700.
doi: 10.3389/fcimb.2025.1597700. eCollection 2025.

Detection of β-lactam resistance genes in Gram-negative bacteria from positive blood cultures using a microchip-based molecular assay

Vittorio Ivagnes #  1 , Flavio De Maio #  2 , Ilaria Baccani  3 , Alberto Antonelli  3 , Giulia Menchinelli  2 , Roberto Rosato  1 , Giordana Cafaro  2 , Giulia Santarelli  1 , Federico Falletta  1 , Tiziana D'Inzeo  1   2 , Maurizio Sanguinetti  1   2 , Teresa Spanu  2 , Giulia De Angelis  1   2 , Gian Maria Rossolini  3   4 , Brunella Posteraro  1   5
Affiliations

Detection of β-lactam resistance genes in Gram-negative bacteria from positive blood cultures using a microchip-based molecular assay

Vittorio Ivagnes et al. Front Cell Infect Microbiol. .

Abstract

Background: Accurate detection of β-lactam resistance genes in bloodstream infections is critical for guiding antimicrobial therapy. This study evaluates the Alifax Gram-negative resistance (GNR) microchip assay for detecting β-lactam resistance genes directly from positive blood cultures (PBCs) for Gram-negative (GN) bacteria, including Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii.

Methods: Simulated (n=146) and clinical (n=106) GN-PBC samples were tested for bla KPC, bla VIM, bla NDM, bla IMP, bla OXA-23-like, bla OXA-48-like, bla SHV-ESBL, bla CTX-M-1/9 group, and bla CMY-2-like genes using the GNR microchip assay. Whole-genome sequencing (WGS) served as the reference assay for simulated samples and, selectively, for clinical samples. The bioMérieux BioFire Blood Culture Identification 2 (BCID2) panel assay was used as a comparator for clinical samples.

Results: The GNR microchip assay correctly identified 203 (99.5%) of 204 β-lactam resistance genes in simulated samples. One sample tested false negative for a bla SHV-ESBL gene but true positive for a bla KPC gene. In clinical samples, GNR results were concordant with BCID2 for 113 (100%) of 113 genes included in both assays. Additionally, the GNR assay detected bla CMY-2 -like (n=6), bla OXA-23-like (n=5), and bla SHV-ESBL (n=2), which are not targeted by BCID2, all confirmed by WGS. In two β-lactam-resistant P. aeruginosa samples but negative by the GNR assay, WGS confirmed the absence of acquired β-lactam resistance genes, suggesting alternative resistance mechanisms.

Conclusion: The GNR microchip assay demonstrated high concordance and broader β-lactam resistance gene coverage compared to BCID2, supporting its potential role in routine diagnostics. Further validation in larger, prospective studies is warranted.

Keywords: GNR microchip assay; Gram-negative bacteria; antimicrobial resistance; molecular detection; positive blood cultures; β-lactamase.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Study design overview. Two sets of positive blood cultures for Gram-negative (GN) bacterial organisms were analyzed to detect β-lactam resistance genes using the GNR microchip assay. In the first set, detection results were compared with whole-genome sequencing (WGS) analysis. In the second set, detection results were compared with the BioFire Blood Culture Identification 2 (BCID2) panel assay and, in specific cases, with WGS analysis.
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