Application and Limitations of 16S rRNA Gene Sequencing for Identifying WHO Priority Pathogenic Gram-Negative Bacilli

Paula Araujo de Souza,1,2 Juliana Nunes Ramos,1,* Luiza Vasconcellos,1,* Luciana Veloso Costa,1 Stephen James Forsythe,3 Marcelo Luiz Lima Brandão1,* 1Department of Experimental and Preclinical Development, Institute of Technology in Immunobiologicals, Oswaldo Cruz Foundation, Rio de Janeiro, Rio de Janeiro, Brazil; 2Department of Microbiology, National Institute of Quality Control in Health, Oswaldo Cruz Foundation, Rio de Janeiro, Rio de Janeiro, Brazil; 3Foodmicrobe.com Ltd., Adams Hill, Nottingham, UK*These authors contributed equally to this workCorrespondence: Marcelo Luiz Lima Brandão, Department of Experimental and Preclinical Development, Institute of Technology in Immunobiologicals, Oswaldo Cruz Foundation, Av. Brasil, 4365. Manguinhos, Rio de Janeiro, RJ, CEP 21040-900, Brazil, Email [email protected]: Antimicrobial resistance (AMR) poses one of the greatest global health challenges, particularly in healthcare-associated infections caused by multidrug-resistant Gram-negative bacilli. Rapid and reliable identification of these pathogens is critical to guide therapy, improve patient outcomes, and support infection control measures. This review explores the application of 16S ribosomal RNA (rRNA) gene sequencing for the identification of pathogenic Gram-negative bacilli included in the World Health Organization (WHO) antimicrobial resistance priority list. The 16S rRNA gene, with its conserved and hypervariable regions, provides a robust molecular marker widely used in bacterial taxonomy and clinical diagnostics. The analysis covers conventional Sanger sequencing, next-generation sequencing (NGS), and third-generation approaches, outlining their advantages, limitations, and clinical applicability. Results indicate that while 16S rRNA sequencing is a valuable tool for genus-level identification, comparative analysis reveals its resolution is often insufficient for distinguishing closely related species such as Escherichia coli and Shigella spp. or for taxa with low interspecies variability. In these cases, complementary strategies – such as multilocus sequence analysis, whole genome sequencing, or advanced mass spectrometry-based methods – are required to achieve accurate identification. Furthermore, the reliability of 16S-based identification depends heavily on the quality of reference databases, as demonstrated by in silico analysis of type strains, and adherence to interpretative guidelines. In conclusion, 16S rRNA sequencing remains a cornerstone of molecular diagnostics and epidemiological surveillance of multidrug-resistant Gram-negative pathogens, but its integration with additional molecular and proteomic tools is essential to overcome its limitations and strengthen infection management strategies. Keywords: infectious diseases, antimicrobial resistance, multidrug resistance, gram-negative bacilli, one health, molecular characterization