Longitudinal genomic surveillance of multidrug-resistant Escherichia coli carriage in critical care patients.

Colonization with multidrug-resistant Escherichia coli strains causes a substantial health burden in hospitalized patients. We performed a longitudinal genomics study to investigate the colonization of resistant E. coli strains in critically ill patients and to identify evolutionary changes and strain replacement events within patients. Patients were admitted to the intensive care unit and hematology wards at a major hospital in Lebanon. Perianal swabs were collected from participants on admission and during hospitalization, which were screened for extended-spectrum beta-lactamases and carbapenem-resistant Enterobacterales. We performed whole-genome sequencing and analysis on E. coli strains isolated from patients at multiple time points. The E. coli isolates were genetically diverse, with 11 sequence types (STs) identified among 22 isolates sequenced. Five patients were colonized by E. coli sequence type 131 (ST131)-encoding CTX-M-27, an emerging clone not previously observed in clinical samples from Lebanon. Among the eight patients whose resident E. coli strains were tracked over time, five harbored the same E. coli strain with relatively few mutations over the 5 to 10 days of hospitalization. The other three patients were colonized by different E. coli strains over time. Our study provides evidence of strain diversity within patients during their hospitalization. While strains varied in their antimicrobial resistance profiles, the number of resistance genes did not increase over time. We also show that ST131-encoding CTX-M-27, which appears to be emerging as a globally important multidrug-resistant E. coli strain, is also prevalent among critical care patients and deserves further monitoring.IMPORTANCEUnderstanding the evolution of bacteria over time in hospitalized patients is of utmost significance in the field of infectious diseases. While numerous studies have surveyed genetic diversity and resistance mechanisms in nosocomial infections, time series of within-patient dynamics are rare, and high-income countries are over-represented, leaving low- and middle-income countries understudied. Our study aims to bridge these research gaps by conducting a longitudinal survey of critically ill patients in Lebanon. This allowed us to track Escherichia coli evolution and strain replacements within individual patients over extended periods. Through whole-genome sequencing, we found extensive strain diversity, including the first evidence of the emerging E. coli sequence type 131 clone encoding the CTX-M-27 beta-lactamase in a clinical sample from Lebanon, as well as likely strain replacement events during hospitalization.

Whole Genome-Based Characterization of Multidrug Resistant Enterobacter and Klebsiella aerogenes Isolates from Lebanon.

Enterobacter spp. and Klebsiella aerogenes are rod-shaped Gram-negative opportunistic pathogens. This study aimed at the molecular and genomic characterization of multidrug resistant Enterobacter spp. and K. aerogenes isolates recovered from hospitalized patients in a tertiary care hospital in Lebanon. A total of 59 Enterobacter spp. clinical isolates consisting of 41 carbapenem-resistant and 18 susceptible by Etest were included in this study. Genotypic identification through whole-genome sequencing (WGS) was performed and confirmed in silico. Resistance and plasmid profiles were studied using ResFinder4.0 and Plasmid-Finder2.1. Multilocus sequence typing (MLST) was used to determine the isolates' clonality. Using the average nucleotide identity (ANI) we identified and confirmed that 47 (80%) isolates were E. hormaechei, 11 (18%) were Klebsiella aerogenes and 1 (2%) was an E. cloacae. Carbapenem-resistance was detected among 41 isolates all showing an MIC90 of ≥ 32 µg/mL for ertapenem, imipenem, and meropenem. blaNDM-1 (58.5%), blaACT-16 (54%), and blaOXA-1 (54%) were the most common detected ß-lactamases, while blaCTX-M-15 (68%) was the main detected extended-spectrum ß-lactamase (ESBL) encoding gene. Chromosomal ampC, carbapenemase encoding genes, and porin modifications were among the detected carbapenem resistance determinants. The carbapenemase encoding genes were linked to three well-defined plasmid Inc groups, IncFII/IncFIB, IncX3, and IncL. MLST typing revealed the diversity within the studied isolates, with ST114 being the most common among the studied E. hormaechei.: The spread of carbapenem-resistant isolates in clinical settings in Lebanon is a serious challenge. Screening and continuous monitoring through WGS analysis could effectively limit the dissemination of drug-resistant isolates in hospitalized patients. IMPORTANCE Drug resistance is an increasing global public health threat that involves most disease-causing organisms and antimicrobial drugs. Drug-resistant organisms spread in health care settings, and resistance to multiple drugs is common. Our study demonstrated the mechanisms leading to resistance against the last resort antimicrobial agents among members of the Enterobacteriaceae family. The spread of carbapenem-resistant bacteria in clinical settings is a serious challenge. Screening and continuous monitoring could effectively limit the dissemination of drug-resistant isolates in hospitalized patients.

Genomic Characterization of Mutli-Drug Resistant Pseudomonas aeruginosa Clinical Isolates: Evaluation and Determination of Ceftolozane/Tazobactam Activity and Resistance Mechanisms.

Resistance to ceftolozane/tazobactam (C/T) in Pseudomonas aeruginosa is a health concern. In this study, we conducted a whole-genome-based molecular characterization to correlate resistance patterns and ß-lactamases with C/T resistance among multi-drug resistant P. aeruginosa clinical isolates. Resistance profiles for 25 P. aeruginosa clinical isolates were examined using disk diffusion assay. Minimal inhibitory concentrations (MIC) for C/T were determined by broth microdilution. Whole-genome sequencing was used to check for antimicrobial resistance determinants and reveal their genetic context. The clonal relatedness was evaluated using MLST, PFGE, and serotyping. All the isolates were resistant to C/T. At least two ß-lactamases were detected in each with the blaOXA-4, blaOXA-10, blaOXA-50, and blaOXA-395 being the most common. blaIMP-15, blaNDM-1, or blaVIM-2, metallo-ß-lactamases, were associated with C/T MIC >256 µg/mL. Eight AmpC variants were identified, and PDC-3 was the most common. We also determined the clonal relatedness of the isolates and showed that they grouped into 11 sequence types (STs) some corresponding to widespread clonal complexes (ST111, ST233, and ST357). C/T resistance was likely driven by the acquired OXA ß-lactamases such as OXA-10, and OXA-50, ESBLs GES-1, GES-15, and VEB-1, and metallo- ß-lactamases IMP-15, NDM-1, and VIM-2. Collectively, our results revealed C/T resistance determinants and patterns in multi-drug resistant P. aeruginosa clinical isolates. Surveillance programs should be implemented and maintained to better track and define resistance mechanisms and how they accumulate and interact.

Replacement of the Alpha variant of SARS-CoV-2 by the Delta variant in Lebanon between April and June 2021.

The COVID-19 pandemic continues to expand globally, with case numbers rising in many areas of the world, including the Eastern Mediterranean Region. Lebanon experienced its largest wave of COVID-19 infections from January to April 2021. Limited genomic surveillance was undertaken, with just 26 SARS-CoV-2 genomes available for this period, nine of which were from travellers from Lebanon detected by other countries. Additional genome sequencing is thus needed to allow surveillance of variants in circulation. In total, 905 SARS-CoV-2 genomes were sequenced using the ARTIC protocol. The genomes were derived from SARS-CoV-2-positive samples, selected retrospectively from the sentinel COVID-19 surveillance network, to capture diversity of location, sampling time, sex, nationality and age. Although 16 PANGO lineages were circulating in Lebanon in January 2021, by February there were just four, with the Alpha variant accounting for 97 % of samples. In the following 2 months, all samples contained the Alpha variant. However, this had changed dramatically by June and July 2021, when all samples belonged to the Delta variant. This study documents a ten-fold increase in the number of SARS-CoV-2 genomes available from Lebanon. The Alpha variant, first detected in the UK, rapidly swept through Lebanon, causing the country's largest wave to date, which peaked in January 2021. The Alpha variant was introduced to Lebanon multiple times despite travel restrictions, but the source of these introductions remains uncertain. The Delta variant was detected in Gambia in travellers from Lebanon in mid-May, suggesting community transmission in Lebanon several weeks before this variant was detected in the country. Prospective sequencing in June/July 2021 showed that the Delta variant had completely replaced the Alpha variant in under 6 weeks.

SARS-CoV-2 genomic epidemiology: data and sequencing infrastructure.

Background: Genomic surveillance of SARS-CoV-2 is critical in monitoring viral lineages. Available data reveal a significant gap between low- and middle-income countries and the rest of the world. Methods: The SARS-CoV-2 sequencing costs using the Oxford Nanopore MinION device and hardware prices for data computation in Lebanon were estimated and compared with those in developed countries. SARS-CoV-2 genomes deposited on the Global Initiative on Sharing All Influenza Data per 1000 COVID-19 cases were determined per country. Results: Sequencing costs in Lebanon were significantly higher compared with those in developed countries. Low- and middle-income countries showed limited sequencing capabilities linked to the lack of support, high prices, long delivery delays and limited availability of trained personnel. Conclusion: The authors recommend the mobilization of funds to develop whole-genome sequencing-based surveillance platforms and the implementation of genomic epidemiology to better identify and track outbreaks, leading to appropriate and mindful interventions.

Lebanon and other low- and middle-income countries have limited sequencing capabilities. Sequencing costs using MinION in Lebanon were higher than the approximate sequencing costs in developed countries. The challenges faced by low- and middle-income countries include lack of support, few established sequencing facilities, high prices, long delivery delays and the limited availability of trained personnel. There is a need to focus on the development of whole-genome sequencing-based surveillance platforms and the implementation of genomic epidemiology to improve sequencing efforts in many resource-limited settings and to contain and prevent future pandemic-level outbreaks.

In silico evidence of beauvericin antiviral activity against SARS-CoV-2.

BACKGROUND: Scientists are still battling severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus 2019 (COVID-19) pandemic so human lives can be saved worldwide. Secondary fungal metabolites are of intense interest due to their broad range of pharmaceutical properties. Beauvericin (BEA) is a secondary metabolite produced by the fungus Beauveria bassiana. Although promising anti-viral activity has previously been reported for BEA, studies investigating its therapeutic potential are limited. METHODS: The objective of this study was to assess the potential usage of BEA as an anti-viral molecule via protein-protein docking approaches using MolSoft. RESULTS: In-silico results revealed relatively favorable binding energies for BEA to different viral proteins implicated in the vital life stages of this virus. Of particular interest is the capability of BEA to dock to both the main coronavirus protease (Pockets A and B) and spike proteins. These results were validated by molecular dynamic simulation (Gromacs). Several parameters, such as root-mean-square deviation/fluctuation, the radius of gyration, H-bonding, and free binding energy were analyzed. Computational analyses revealed that interaction of BEA with the main protease pockets in addition to the spike glycoprotein remained stable. CONCLUSION: Altogether, our results suggest that BEA might be considered as a potential competitive and allosteric agonist inhibitor with therapeutic options for treating COVID-19 pending in vitro and in vivo validation.

Tracking the international spread of SARS-CoV-2 lineages B.1.1.7 and B.1.351/501Y-V2 with grinch.

Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501Y-V2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected.

Genomic characterization and phylogenetic analysis of the first SARS-CoV-2 variants introduced in Lebanon.

BACKGROUND: In December 2019, the COVID-19 pandemic initially erupted from a cluster of pneumonia cases of unknown origin in the city of Wuhan, China. Presently, it has almost reached 94 million cases worldwide. Lebanon on the brink of economic collapse and its healthcare system thrown into turmoil, has previously managed to cope with the initial SARS-CoV-2 wave. In this study, we sequenced 11 viral genomes from positive cases isolated between 2 February 2020 and 15 March 2020. METHODS: Sequencing data was quality controlled, consensus sequences generated, and a maximum-likelihood tree was generated with IQTREE v2. Genetic lineages were assigned with Pangolin v1.1.14 and single nucleotide variants (SNVs) were called from read files and manually curated from consensus sequence alignment through JalView v2.11 and the genomic mutational interference with molecular diagnostic tools was assessed with the CoV-GLUE pipeline. Phylogenetic analysis of whole genome sequences confirmed a multiple introduction scenario due to international travel. RESULTS: Three major lineages were identified to be circulating in Lebanon in the studied period. The B.1 (20A clade) was the most prominent, followed by the B.4 lineage (19A clade) and the B.1.1 lineage (20B clade). SNV analysis showed 15 novel mutations from which only one was observed in the spike region.

Molecular characterization of a carbapenem-resistant Enterobacter hormaechei ssp. xiangfangensis co-harbouring blaNDM-1 and a chromosomally encoded phage-linked blaCTX-M-15 genes.

Enterobacter cloacae complex (ECC) members are rapidly emerging as successful nosocomial pathogens, especially, with the emergence of carbapenem-resistant clones. In this study, we performed a comprehensive molecular characterization of a carbapenem-resistant E. hormaechei ssp. xiangfangensis LAU_ENC1. hsp60 and average nucleotide identity (ANI) were used for its identification. The repertoire of resistance genes and phage content were analyzed. Plasmid sequences were extracted and compared to closest references. The isolate LAU_ENC1 was identified as an E. hormaechei ssp. xiangfangensis and belonged to ST-114A sub-cluster. blaNDM-1, blaCTX-M-15, blaOXA-1, and blaACT-16 genes were detected as ß-lactam resistance determinants. A chromosomal hybrid intact phage, Enterobacter phage LAU1, with blaCTX-M-15 integrated in its direct vicinity within an ISEcp1 - blaCTX-M-15 - wbuC - ∆Tn2 rare cassette was detected. blaNDM-1 was integrated within a novel IncFII conjugative plasmid, pLAU_ENC1, through an IS3000- ΔISAba125-blaNDM-1-bleMBL-//-Tn5403 cassette. To our knowledge, this is the first report of a multi-drug resistant (MDR) E. hormaechei ssp. xiangfangensis carrying a blaCTX-M-15 integrated within the proximity of a provirus chromosomal region. Treatment options for MDR ECC members are becoming scarce, thus warranting an increased monitoring of the dissemination of these pathogens in clinical settings.

COVID-19 Pandemic in Lebanon: One Year Later, What Have We Learnt?

Lebanon is witnessing an unprecedented crisis with the rapid spread of coronavirus disease 2019 (COVID-19), financial meltdown, economic collapse, and the Beirut Port explosion. The first wave began in February 2020, following which the country experienced several episodes and peaks while alternating between lockdowns and phased liftings. One year of the pandemic revealed that effective mitigation could not be separated from the collapse of the ongoing economic, political, and health sectors. Scaling up vaccination, preparedness, and response capacities is essential to control community transmission. The World Health Organization (WHO), National Council for Scientific Research-Lebanon (CNRS-L), nongovernmental organizations (NGOs), and humanitarian responses proved to be the safety net for the country during the current pandemic.

Interleukin-37: A Link Between COVID-19, Diabetes, and the Black Fungus.

The COVID-19 pandemic involved millions of people and diabetes was identified as an associated comorbidity. Initiation of systemic corticosteroids in patients suffering from severe COVID-19 was associated with lower mortality. A surge of invasive fungal infections of the maxillofacial region, namely mucormycosis, was linked to a deadly infection known as black fungus. Black fungus, diabetes, corticosteroids, and coronavirus disease 2019 (COVID-19) all have a dysregulated immune response in common, which partly could also be attributed to interleukin 37 (IL-37). IL-37, a new cytokine of the IL-1 family, known for broadly reducing innate inflammation as well as acquired immune responses. The use of corticosteroids in diabetic COVID-19 patients, crowded hospitals, and lack of medical oxygen should be carefully considered to reduce COVID-associated secondary infections.

SARS-CoV-2 and ORF3a: Nonsynonymous Mutations, Functional Domains, and Viral Pathogenesis.

The effect of the rapid accumulation of nonsynonymous mutations on the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not yet known. The 3a protein is unique to SARS-CoV and is essential for disease pathogenesis. Our study aimed at determining the nonsynonymous mutations in the 3a protein in SARS-CoV-2 and determining and characterizing the protein's structure and spatial orientation in comparison to those of 3a in SARS-CoV. A total of 51 different nonsynonymous amino acid substitutions were detected in the 3a proteins among 2,782 SARS-CoV-2 strains. We observed microclonality within the ORF3a gene tree defined by nonsynonymous mutations separating the isolates into distinct subpopulations. We detected and identified six functional domains (I to VI) in the SARS-CoV-2 3a protein. The functional domains were linked to virulence, infectivity, ion channel formation, and virus release. Our study showed the importance of conserved functional domains across the species barrier and revealed the possible role of the 3a protein in the viral life cycle. Observations reported in this study merit experimental confirmation.IMPORTANCE At the surge of the coronavirus disease 2019 (COVID-19) pandemic, we detected and identified six functional domains (I to VI) in the SARS-CoV-2 3a protein. Our analysis showed that the functional domains were linked to virulence, infectivity, ion channel formation, and virus release in SARS-CoV-2 3a. Our study also revealed the functional importance of conserved domains across the species barrier. Observations reported in this study merit experimental confirmation.