Molecular Epidemiological Surveillance of Carbapenem-Resistant Gram-Negative Bacteria in Southern Lebanon

<b>Introduction:</b> Carbapenem-resistant Gram-negative bacteria (CR-GNB) are rapidly spreading pathogens that increase morbidity and mortality in hospital settings and significantly restrict available treatment options worldwide. The lack of molecular epidemiological data and the limited use of next-generation sequencing (NGS) in South Lebanon have hindered comprehensive surveillance efforts. This study represents the first molecular characterization of CR-GNB in this region. <b>Methods:</b> A total of 477 clinical Gram-negative bacterial isolates were collected from intensive care unit (ICU) patients admitted to various hospitals in South Lebanon in 2023. Of these, 131 CR-GNB were subjected to whole-genome sequencing using the Illumina MiSeq platform. K-mer-based species identification, multilocus sequence typing (MLST), antimicrobial resistance (AMR) gene profiling, and plasmid analysis were performed using multiple bioinformatic tools. Phylogenetic analysis was conducted using SaffronTree. <b>Results:</b> K-mer-based identification revealed that the predominant species among CR-GNB isolates were <i>Pseudomonas</i> spp. and <i>Escherichia coli</i> (26.7% each), followed by <i>Klebsiella pneumoniae</i> (19.8%), <i>Acinetobacter baumannii</i> (17.6%), <i>Proteus mirabilis</i> (4.6%), <i>Enterobacter cloacae</i> (2.3%), <i>Achromobacter</i> spp. (1.5%), and <i>Citrobacter freundii</i> (0.8%). Based on antimicrobial susceptibility testing, isolates were classified as follows: 0.8% as pan drug-resistant (PDR), 40.5% as extensively drug-resistant (XDR), and 52.7% as multidrug-resistant (MDR) and 6.1% as antimicrobial-resistant (AMR). All isolates harbored AMR genes, with the following distribution: 2% <i>bla</i>_VIM, 5% <i>bla</i>_NDM-1, 27% <i>bla</i>_NDM-5, 65% <i>bla</i>_OXA-type, and 1% <i>bla</i>_DIM-1. Plasmid-associated AMR genes were detected in 58% of isolates; among these, 96% carried Inc-family plasmids, 57% Col plasmids, and 11% replication-associated elements (rep). Phylogenetic analysis demonstrated that certain isolates exhibited both hospital-specific and shared genetic profiles, indicating widespread dissemination across multiple healthcare facilities, as well as evidence of local emergence and ongoing transmission. <b>Conclusions:</b> The high prevalence of CR-GNB harboring resistance genes and plasmids underscores the urgent need for NGS-based genomic surveillance in South Lebanon. Implementing such strategies is essential for tracking resistance genes, identifying clonal outbreaks, and guiding effective infection control interventions to mitigate the spread of CR-GNB.