show Abstracthide AbstractMultidrug resistance in clinically relevant pathogens continues to escalate, driven by factors such as the overuse of antimicrobials and limited access to medical services, which foster the widespread dissemination of multidrug-resistant bacterial strains. Globally, approximately 5 to 12% of newborns necessitate specialized care, with a staggering 20% of these neonates contracting infections upon admission and up to 15% developing infections during their clinical care. Gram-negative bacteria play a significant role in causing these infections, with a notable percentage demonstrating multidrug resistance (MDR). To assess the prevalence of antibiotic resistance in hospitalized neonates in the Neonatal Intensive Care Unit (NICU) at the Tertiary Level or those brought to the Emergency Room (ER) of the Hospital Regional Materno Infantil de Monterrey (HRMIM) in Nuevo Leon, Mexico, a prospective audit was conducted from July 2021 to July 2022. Bacterial isolates collected from neonates were characterized to determine their antibiotic susceptibility and resistance profiles at phenotypic and genotypic level. This analysis aimed to establish correlations and measure risk factors between Gram-negative infections with MDR organisms and concurrent illnesses, as well as treatments offered in the special care nursery. These treatments encompassed medical interventions like the use of antibiotics, non-steroidal anti-inflammatory drugs, steroids, and others. As part of this study, 83 Gram-negative isolates were newly sequenced using the Illumina platform, enabling a comprehensive molecular characterization of Gram-negative MDR bacterial isolates. This molecular analysis provides valuable insights into the nature and extent of multidrug resistance in these clinically significant pathogens.