Hadi Hussein

Background/Objectives: Antimicrobial resistance (AMR) in Escherichia coli is a growing public health concern, particularly in regions affected by environmental contamination and poor wastewater management. Data on locally isolated E. coli-targeting phages in Lebanon remain limited. This study aimed to isolate, characterize, and evaluate two lytic bacteriophages against AMR E. coli. Methods: Two phages, EPIMAM01 (gb:PQ493298) and EPIMRB01 (gb:PQ657784), were isolated from untreated sewage in Beirut using E. coli ATCC 25922. Characterization included double-layer agar assays, one-step growth analysis, and stability testing across temperature and pH ranges. Bacteriolytic activity was assessed in planktonic cultures and preformed biofilms. Host range and efficiency of plating (EOP) were evaluated using clinical isolates. Whole-genome sequencing and comparative analyses were performed. Results: Both phages produced clear plaques and showed a latent period of ~40 min. EPIMAM01 had a higher estimated burst size (140 PFU/infected cell) than EPIMRB01 (75 PFU/infected cell). Both phages remained stable between 4–50 °C and within a pH range of 5–10 but showed marked loss of activity at temperatures ≥60 °C and pH ≤3 or ≥12. EPIMAM01 effectively inhibited planktonic growth of E. coli ATCC 25922, whereas EPIMRB01 showed stronger biofilm-disrupting activity against preformed E. coli biofilms. Both phages lysed several of the 17 tested clinical E. coli isolates. Comparative analyses of gene presence/absence patterns, bacterial defense systems, and adsorption phenotypes among the tested E. coli strains identified mlaA, ydcQ, and ompD-2 as candidate adsorption-associated genes and suggested CRISPR systems may reduce susceptibility. Genomic analysis classified both phages as T4-like phages lacking lysogeny, virulence, or AMR genes. Conclusions: EPIMAM01 and EPIMRB01 are lytic phages with complementary antimicrobial properties, supporting their potential for further development as AMR control agents.

Escherichia coli sequence type 648 (ST648), a lineage within the clinically important phylogroup F, has disseminated worldwide in humans and animals. In this study, we performed whole-genome sequencing and comparative genomic analysis for two New Delhi metallo-beta-lactamase (bla_NDM-5) carrying E. coli strains: ECsOL198, recovered from a wild Eurasian otter in Northern Lebanon, and ECOL247, isolated from a hospitalized leukemia patient. Both isolates belonged to phylogroup F and serotype O9:H4, and exhibited IncFIA, IncFIB, and IncFII plasmids. They shared a similar antimicrobial resistance profile, including a carbapenemase gene (bla_NDM-5), β-lactamase genes (bla_TEM-1, bla_CTX-M-15, and bla_OXA-1), and other genes that confer resistance to aminoglycosides (acc(3)-Ile, aadA2), sulfonamides (sul1), tetracyclines (tet(A)), and fluoroquinolones (mutations in gyrA and parC). Both isolates also carried common virulence-associated genes related to adhesion, iron acquisition, environmental persistence, and immune evasion. Whole-genome multilocus sequence typing (wgMLST) revealed that both isolates formed a distinct subclade closely related to a bloodstream-derived ST648 isolate from India, indicating limited relatedness to global clones. These findings highlight the transmission of nearly clonal multidrug-resistant E. coli ST648 in both clinical and non-clinical settings, raising concerns about the threat to public health.

Introduction: 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. Methods: 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. Results: K-mer-based identification revealed that the predominant species among CR-GNB isolates were Pseudomonas spp. and Escherichia coli (26.7% each), followed by Klebsiella pneumoniae (19.8%), Acinetobacter baumannii (17.6%), Proteus mirabilis (4.6%), Enterobacter cloacae (2.3%), Achromobacter spp. (1.5%), and Citrobacter freundii (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% bla_VIM, 5% bla_NDM-1, 27% bla_NDM-5, 65% bla_OXA-type, and 1% bla_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. Conclusions: 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.

The overuse of carbapenems has driven the rise of carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Enterobacterales (CRE), against which ceftazidime–avibactam (CAZ-AVI) offers an alternative treatment. This study phenotypically determined resistance profiles of P. aeruginosa (PA), Escherichia coli (EC), and Klebsiella pneumoniae (KP) clinical isolates to CAZ-AVI and investigated molecular resistance mechanisms genotypically. A total of 394 PA, 90 EC, and 84 KP isolates were collected from the American University of Beirut Medical Center. Antimicrobial susceptibility testing (AST) and whole-genome sequencing (WGS) were performed on 30 isolates per species. Results showed that 100% of KP, 63% of EC, and 100% of PA isolates were carbapenem-resistant. Among these, 73% of KP, 79% of EC, and 60% of PA were CAZ-AVI-resistant. WGS revealed diverse sequence types, plasmids, and antimicrobial resistance genes. Additionally, 100% of KP, 93% of EC, and 89% of PA isolates produced metallo-β-lactamases (MBLs). Mutations in ampD, ampR, and mexR were identified in CAZ-AVI-resistant, non-MBL-producing PA, whereas mutations in ompC, marR, and ampC were detected in CAZ-AVI-resistant, non-MBL-producing EC. While CAZ-AVI remains effective against most CRE and CRPA lacking MBLs, resistance to CAZ-AVI is multifactorial, commonly involving overexpression of efflux pumps and AmpC β-lactamases, loss of porin channels, and the presence of oxacillinases.

The overuse of carbapenems has driven the rise of carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Enterobacterales (CRE), against which ceftazidime-avibactam (CAZ-AVI) offers an alternative treatment. This study phenotypically determined resistance profiles of P. aeruginosa (PA), Escherichia coli (EC), and Klebsiella pneumoniae (KP) clinical isolates to CAZ-AVI and investigated molecular resistance mechanisms genotypically. A total of 394 PA, 90 EC, and 84 KP isolates were collected from the American University of Beirut Medical Center. Antimicrobial susceptibility testing (AST) and whole-genome sequencing (WGS) were performed on 30 isolates per species. Results showed 100% of KP, 63% of EC, and 100% of PA isolates were carbapenem-resistant. Among these, 73% of KP, 79% of EC, and 60% of PA were CAZ-AVI-resistant. WGS revealed diverse sequence types, plasmids, and antimicrobial resistance genes. Additionally, 100% of KP, 93% of EC, and 89% of PA isolates produced metallo-β-lactamases (MBLs). Mutations in ampD, ampR, and mexR were identified in CAZ-AVI-resistant, non-MBL-producing PA, whereas mutations in ompC, marR, and ampC were detected in CAZ-AVI-resistant, non-MBL-producing EC. While CAZ-AVI remains effective against most CRE and CRPA lacking MBLs, resistance to CAZ-AVI is multifactorial, commonly involving overexpression of efflux pumps and AmpC β-lactamases, loss of porin channels, and the presence of oxacillinases.

Cholera is a life-threatening gastrointestinal infection caused by a toxigenic bacterium, Vibrio cholerae. After a lull of almost 30 years, a first case of cholera was detected in Lebanon in October 2022. The outbreak lasted three months, with 8007 suspected cases (671 laboratory-confirmed) and 23 deaths. In this study, we use phenotypic methods and microbial genomics to study 34 clinical and environmental Vibrio cholerae isolates collected throughout this outbreak. All isolates are identified as V. cholerae O1, serotype Ogawa strains from wave 3 of the seventh pandemic El Tor (7PET) lineage. Phylogenomic analysis unexpectedly reveals the presence of two different strains of the seventh pandemic El Tor (7PET) lineage. The dominant strain has a narrow antibiotic resistance profile and is phylogenetically related to South Asian V. cholerae isolates and derived African isolates from the AFR15 sublineage. The second strain is geographically restricted and extensively drug-resistant. It belongs to the AFR13 sublineage and clusters with V. cholerae isolates collected in Yemen. In conclusion, the 2022-2023 Lebanese cholera outbreak is caused by the simultaneous introduction of two different 7PET strains. Genomic surveillance with cross-border collaboration is therefore crucial for the identification of new introductions and routes of circulation of cholera, improving our understanding of cholera epidemiology.

(1) Background: Infections with pan-drug-resistant (PDR) bacteria, such as A. baumannii, are becoming increasingly common, especially in healthcare facilities. In this study, we selected 15 colistin-resistant clinical A. baumannii isolates from a hospital in Beirut, Lebanon, to test combination therapies and determine their sequence types (STs) and the mechanism of colistin resistance using whole-genome sequencing (WGS). (2) Methods: Antimicrobial susceptibility testing via broth microdilution against 12 antimicrobials from different classes and growth rate assays were performed. A checkerboard assay was conducted on PDR isolates using six different antimicrobials, each in combination with colistin. Genomic DNA was extracted from all isolates and subjected to WGS. (3) Results: All isolates were resistant to all tested antimicrobials with the one exception that was susceptible to gentamicin. Combining colistin with either meropenem, ceftolozane–tazobactam, or teicoplanin showed synergistic activity. Sequencing data revealed that 67% of the isolates belonged to Pasteur ST2 and 33% to ST187. Furthermore, these isolates harbored a number of resistance genes, including bla_OXA-23. Mutations in the pmrC gene were behind colistin resistance. (4) Conclusions: With the rise in antimicrobial resistance and the absence of novel antimicrobial production, alternative treatments must be found. The combination therapy results from this study suggest treatment options for PDR ST2 A. baumannii-infected patients.

Epstein-Barr virus is a pathogen that causes persistent infection with potential consistent viral DNA shedding. The enhancement of production of proinflammatory cytokines by viral DNA itself may contribute to autoimmune disease development or exacerbation. In this project, we identified that endosomal Toll-like receptors are involved in triggering proinflammatory mediators in response to viral DNA. Pathways and receptors involved may serve as future therapeutic targets for autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus.