ABC transporter inhibition by beauvericin partially overcomes drug resistance in Leishmania tropica.

Leishmaniasis is a neglected tropical disease infecting the world's poorest populations. Miltefosine (ML) remains the primary oral drug against the cutaneous form of leishmaniasis. The ATP-binding cassette (ABC) transporters are key players in the xenobiotic efflux, and their inhibition could enhance the therapeutic index. In this study, the ability of beauvericin (BEA) to overcome ABC transporter-mediated resistance of Leishmania tropica to ML was assessed. In addition, the transcription profile of genes involved in resistance acquisition to ML was inspected. Finally, we explored the efflux mechanism of the drug and inhibitor. The efficacy of ML against all developmental stages of L. tropica in the presence or absence of BEA was evaluated using an absolute quantification assay. The expression of resistance genes was evaluated, comparing susceptible and resistant strains. Finally, the mechanisms governing the interaction between the ABC transporter and its ligands were elucidated using molecular docking and dynamic simulation. Relative quantification showed that the expression of the ABCG sub-family is mostly modulated by ML. In this study, we used BEA to impede resistance of Leishmania tropica. The IC50 values, following BEA treatment, were significantly reduced from 30.83, 48.17, and 16.83 µM using ML to 8.14, 11.1, and 7.18 µM when using a combinatorial treatment (ML + BEA) against promastigotes, axenic amastigotes, and intracellular amastigotes, respectively. We also demonstrated a favorable BEA-binding enthalpy to L. tropica ABC transporter compared to ML. Our study revealed that BEA partially reverses the resistance development of L. tropica to ML by blocking the alternate ATP hydrolysis cycle.

Field evaluation of metal bioaccumulation in the gastropod Helix aspersa at agricultural and industrial sites in Lebanon.

Juvenile Helix aspersa Müller exposed in field microcosms were used to assess the spatial and temporal bioaccumulation of Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn at two agricultural and two industrial sites in Lebanon. The study was performed over a 12-week period where caged snails were sampled once every 3 weeks and assessed for metal bioaccumulation and partitioning between soft tissue and shells. Results showed that metal bioaccumulation by snails was site dependent, with Fe and Cd being the greatest and least accumulated metals, respectively. Significant differences between bioaccumulation in each of the matrices (soft tissue and shells) were also observed. Time-dependent bioaccumulation results showed an increasing accumulation trend at both agricultural sites, while a slight decline was observed at the end of the sampling campaign for the industrial sites. The study of the bioaccumulation factors (BAF) revealed that tested H. aspersa were macroconcentrators for Zn and Cd (BAF > 2) and deconcentrators for all other analyzed elements (BAF < 1). The high partitioning factor values obtained for Cu and Zn indicate an affinity of these two elements for the soft tissues of the snails. The results of this field study indicate that H. aspersa are well suited for active biomonitoring and could provide reliable information on metal pollution and bioavailability.

Molecular mechanism of fluconazole resistance and pathogenicity attributes of Lebanese Candida albicans hospital isolates.

Hospital infections caused by the opportunistic fungus Candida albicans are increasingly common and life threatening. The first line of defense consists of administering antifungal drugs such as azoles including fluconazole that prevent ergosterol biosynthesis. C. albicans is rapidly developing resistance towards antifungal drugs through various mechanisms including mutations in ERG11 which is a gene involved in the ergosterol biosynthesis pathway. These mutations prevent the binding of the drug and inactivate ergosterol synthesis. Alternatively, upregulation of cell membrane ergosterol content generates resistance by countering the effect of the drug. In this study we sequenced the ERG11 gene in 6 fluconazole sensitive and 8 fluconazole resistant C. albicans isolates recovered from clinical settings in Lebanon and quantified the ergosterol content of their plasma membranes to identify mechanisms linked to fluconazole resistance. A number of pathogenicity attributes were also analyzed to determine any correlation with fluconazole resistance. Our results revealed an increase in ergosterol content in the fluconazole resistant isolates. In addition, we identified both novel and previously reported amino acid substitutions in ERG11 as well as frameshift mutations that might contribute to resistance. The fluconazole resistant isolates did not exhibit an increased virulence potential in a mouse model of systemic infection and showed decreased in vitro potential to form biofilms. No discrepancy between drug resistant and sensitive isolates to cell surface disrupting agents was observed. This approach is the first of its kind to be carried out in Lebanon to identify possible mechanisms and phenotypes of drug resistant C. albicans isolates.

Detailed characterization of an IncFII plasmid carrying blaOXA-48 from Lebanon.

BACKGROUND: The spread of carbapenem-resistant Enterobacteriaceae is an important challenge and an increasing healthcare problem. OXA-48 is a class D carbapenemase that is usually localized on a conjugative plasmid belonging to the IncL incompatibility group. METHODS: In this study, we used a combination of short- and long-read WGS approaches and molecular typing techniques to characterize the genetic environment of the smallest reported 27 029 bp IncFII plasmid carrying blaOXA-48 (pLAU-OXA48). RESULTS: The plasmid recovered from a clinical Escherichia coli isolate was positive for blaOXA-48, which was located within the Tn6237 composite transposon. Primers targeting junctions between the IncF fragment and Tn6237 for the rapid identification of pLAU-OXA48-like plasmids were designed. CONCLUSIONS: To our knowledge, this is the first report showing the complete sequence of an IncFII plasmid carrying blaOXA-48 within Tn6237 using hybrid assembly of long- and short-read sequencing.

Genotypic and phenotypic characterization of Candida albicans Lebanese hospital isolates resistant and sensitive to caspofungin.

The fungus Candida albicans is both a commensal and an opportunistic human pathogen, present as part of the normal human microflora causing serious mucosal, and systemic life threatening infections. The antifungal drug caspofungin of the echinocandin family is the latest generation of antifungal drugs to be developed. It functions by inhibiting glucan synthase thus weakening the fungal cell wall leading to death. Recently reports of resistance to caspofungin have been reported mainly through mutations in the FKS encoded subunits of glucan synthase at hot spot 1 (amino acids 641 to 649, FSTLSLRDP) and hot spot 2 (amino acids 1357 to 1364, DWIRRYTL). Our study aimed at sequencing both hot spots from 16 C. albicans Lebanese hospital isolates resistant and sensitive to caspofungin to determine whether mutations in these hot spots are present, and whether such mutations also impart resistance to our isolates. In addition, we wanted to determine any relationship between resistance and pathogenicity related attributes such as virulence, resistance to cell wall disrupting agents, biofilm formation, and cell wall chitin deposition. Five isolates were found to contain mutations with the mutations restricted to resistant strains. Within hot spot 1 substitution at positions S642, T643, L644, R647, and D648 were found, while within hot spot 2 substitutions at positions L1364, T1363, and R1360, W1358 and R1361 were identified with some of the mutations not previously documented. Strains that were resistant to caspofungin also showed increased resistance to Congo red but decreased biofilm formation and attenuated virulence in a mouse model of infection. Caspofungin sensitive strains showed decreased resistance to Congo red yet increased virulence and biofilm formation. Chitin content analysis showed that caspofungin resistant strains had elevated levels of chitin resulting in cell wall thickening that counters the effect of caspofungin, while sensitive strains showed decreased chitin content. Our results demonstrate an inverse correlation between resistance and virulence whereby resistance is due to thickening of the cell wall preventing the cell from gaining virulence attributes, while a more susceptible cell wall increases susceptibility to drugs but allows increased virulence.

Genomic Features of Vibrio parahaemolyticus from Lebanon and Comparison to Globally Diverse Strains by Whole-Genome Sequencing.

Gastroenteritis is a disease that can be caused by virulent strains of Vibrio parahaemolyticus in humans upon the consumption of contaminated seafood. In summer 2017, a sudden increase in the number of patients suffering from gastroenteritis due to a V. parahaemolyticus infection was observed at the Middle East Institute of Health University Hospital in Lebanon. The aim of this study was to analyze the isolates recovered from stool specimens, and to compare them using different phenotypic assays, genomic profiling techniques, and whole-genome sequencing, to achieve a better understanding of the current V. parahaemolyticus strains available in Lebanon. Virulence potential was analyzed based on the detection of the hemolysins: thermostable direct hemolysin (tdh), thermostable direct hemolysin-related hemolysin (trh), and thermolabile hemolysin (tlh). Resistance was determined by testing antibiotic susceptibility and performing PCR assays for ß-lactamases and quinolone resistance determinants. Genetic relatedness was verified by multilocus sequence typing, pulsed-field gel electrophoresis, and whole genome-based single nucleotide polymorphism analysis. All of the isolates had the tdh+, trh-, group-specific PCR+ genotype, which is a characteristic of the O3:K6 pandemic clone. The isolates were resistant to ampicillin (100%), ceftazidime (86%), ticarcillin (14%), and amikacin (14%), belonged to the sequence type ST3, and had very similar phylogenetic fingerprints. The isolates undertaken in this study exhibited almost identical resistance, virulence, and phylogenetic patterns, confirming an outbreak linked to the spread of the pandemic O3:K6 serotype in the country.

First insights on the genetic diversity of MDR Mycobacterium tuberculosis in Lebanon.

BACKGROUND: Lebanon hosts a heterogeneous population coming from underdeveloped and developing countries, resulting in increasing incidences of tuberculosis over the past years. The genetic heterogeneity and lineages associated with tuberculosis, along with their resistance determinants have not been studied at the genomic level previously in the region. METHODS: Isolates were recovered from the American University of Beirut Medical Center (AUBMC). Antimicrobial susceptibility profiles were determined using the MGIT automated system for the first-line drugs at AUBMC, while second-line drug susceptibility was tested at Mayo Clinic Laboratories. Whole Genome Sequencing (WGS) was performed to classify mycobacterial lineages and highlight single nucleotide mutations causing resistance to both 1st line and 2nd line antimicrobials. wgSNP analysis provided insights on the phylogeny of the isolates along with spoligotyping and core genomic SNVs, IS6110 insertion sites, and variable number tandem repeats (VNTR). RESULTS: The analyzed isolates carry distinct resistance determinants to isoniazid, rifampicin, ethambutol, quinolones, and streptomycin. The isolates belonged to different lineages including the Euro/American lineage (Lineage 4) (53.8%), M. bovis (15.4%) and Delhi/Central Asia (Lineage 1) (15.4%), Beijing/East Asia (Lineage 2) (7.7%), and East Africa/Indian Ocean lineage (Lineage 3) (7.7%) showing great phylogenetic differences at the genomic level. CONCLUSIONS: The population diversity in Lebanon holds an equally diverse and uncharacterized population of drug resistant mycobacteria. To achieve the WHO "END-TB" milestones of 2025 and 2035, Lebanon must decrease TB incidences by 95% in the next decade. This can only be done through WGS-based patient centered diagnosis with higher throughput and genomic resolution to improve treatment outcomes and to monitor transmission patterns.

Characterization of blaNDM-19-producing IncX3 plasmid isolated from carbapenem-resistant Escherichia coli and Klebsiellapneumoniae.

The increase in the prevalence of carbapenem-producing Enterobacterales (CPE) is a major threat, with the New Delhi metallo-ß-lactamase (NDM) enzyme-producing CPEs being one of the major causative agents of healthcare settings infections. In this study, we characterized an IncX3 plasmid harboring blaNDM-19 in Lebanon, recovered from three Escherichia coli belonging to ST167 and one Klebsiella pneumoniae belonging to ST16 isolated from a clinical setting. Plasmid analysis using PBRT, Plasmid Finder, and PlasmidSPAdes showed that all four isolates carried a conjugative 47-kb plasmid having blaNDM-19, and was designated as pLAU-NDM19. We constructed a sequence-based maximum likelihood phylogenetic tree and compared pLAU-NDM19 to other representative IncX3 plasmids carrying NDM-variants and showed that it was closely linked to NDM-19 positive IncX3 plasmid from K. pneumoniae reported in China. Our findings also revealed the route mediating resistance transmission, the IncX3 dissemination among Enterobacterales, and the NDM-19 genetic environment. We showed that mobile elements contributed to the variability of IncX3 genomic environment and highlighted that clonal dissemination in healthcare settings facilitated the spread of resistance determinants. Antimicrobial stewardship programs implemented in hospitals should be coupled with genomic surveillance to better understand the mechanisms mediating the mobilization of resistance determinants among nosocomial pathogens and their subsequent clonal dissemination.

Biomonitoring of PAHs and PCBs in industrial, suburban, and rural areas using snails as sentinel organisms.

There is a worldwide concern about the presence of persistent organic pollutants (POPs) in the environment because of their toxicity, bioaccumulation, and resistance to degradation. Various conventional monitoring techniques have been used to assess their presence in diverse environmental compartments. Most currently available methods, however, have limitations with regards to long-term monitoring. In the present work, juvenile Cornu aspersum (O. F. Müller, 1774) snails were tested in field microcosms as biomonitors for two major classes of organic pollutants, namely, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). The study assessed their deployment in one suburban, one rural, and two industrial sites over an 18-week period and monitored for temporal variations of 16 PAHs and 22 PCBs. Sampling was conducted once every 3 weeks. Targeted pollutants were extracted from the caged snails using the QuEChERS extraction procedure and subsequently analyzed using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). The results showed that the bioaccumulation of specific pollutants was site dependent; significantly higher levels of PCBs were observed at the industrial sites as compared to the suburban and rural ones. PAHs were bioaccumulated by the snails via ingestion of air and soil whereas PCBs were mainly bioaccumulated via soil contact and ingestion. The findings of this study indicate that C. aspersum is a reliable model organism for the biomonitoring of organic pollutants in air and soil compartments and can be used as part of an integrated environmental assessment.

Phenotypic and molecular characterization of multi-drug resistant Klebsiella spp. isolates recovered from clinical settings.

Klebsiella pneumoniae is a Gram-negative bacterium that colonizes the gastrointestinal tract and nasopharynx with many being linked to nosocomial infections. Extended-spectrum ß-lactamases (ESBL)-producing and carbapenem-resistant K. pneumoniae is recognized by the World Health Organization (WHO) as a critical public health concern. In this study, whole-genome sequencing (WGS) - based analysis was performed to understand the molecular epidemiology of multi-drug resistant Klebsiella spp. clinical isolates. Genome comparison, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and whole-genome-SNP-based phylogenetic analysis (wg-SNP) were used for in-depth molecular characterization. in silico typing was used to determine the resistance genes, virulence factors, Inc. groups, and capsular types. All except one isolate were non-susceptible to meropenem and 89% were non-susceptible to ertapenem and imipenem. blaNDM, blaOXA-48, and blaKPC were the detected carbapenemases with blaNDM-1 found in half of the sequenced genomes. Resistance to colistin was detected in one isolate and was linked to several genetic alterations in crrB, pmrB, and pmrC genes. The most common plasmid type was IncFIB followed by IncR, and the Type 3 fimbriae, encoded by the mrkABCDF operon, was conserved among all isolates. The most common sequence- (ST) and K-type detected were ST147 and K64. The prevelance and the genomic relatedness of ST147 isolates, as shown by the data from SNP-based phylogenetic analysis, PFGE, and genomic clustering, may be an outbreak marker. However, this can only be validated through a more comprehensive study encompassing a wider sampling scheme and over an extended timeframe.

Tracking SARS-CoV-2 variants during the 2023 flu season and beyond in Lebanon.

BACKGROUND: Early SARS-CoV-2 variant detection relies on testing and genomic surveillance. The Omicron variant (B.1.1.529) has quickly become the dominant type among the previous circulating variants worldwide. Several subvariants have emerged exhibiting greater infectivity and immune evasion. In this study we aimed at studying the prevalence of the Omicron subvariants during the flu season and beyond in Lebanon through genomic screening and at determining the overall standing and trajectory of the pandemic in the country. METHODS: A total of 155 SARS-CoV-2 RNA samples were sequenced, using Nanopore sequencing technology. RESULTS: Nanopore sequencing of 155 genomes revealed their distribution over 39 Omicron variants. XBB.1.5 (23.29 %) was the most common, followed by XBB.1.9.1 (10.96 %) and XBB.1.42 (7.5 %). The first batch collected between September and November 2022, included the BA.2.75.2, BA.5.2, BA.5.2.20, BA.5.2.25 and BQ.1.1.5 lineages. Between December 2022 and January 2023, those lineages were replaced by BA.2.75.5, BN.1, BN.1.4, BQ.1, BQ.1.1, BQ.1.1.23, CH.1.1, CM.4 and XBK. Starting February 2023, we observed a gradual emergence and dominance of the recombinant XBB and its sub-lineages (XBB.1, XBB.1.5, XBB.1.5.2, XBB.1.5.3, XBB.1.9, XBB.1.9.1, XBB.1.9.2, XBB.1.16, XBB.1.22 and XBB.1.42). CONCLUSIONS: The timely detection and characterization of SARS-CoV-2 variants is important to reduce transmission through established disease control measures and to avoid introductions into animal populations that could lead to serious public health implications.

Computational Applications: Beauvericin from a Mycotoxin into a Humanized Drug.

Drug discovery was initially attributed to coincidence or experimental research. Historically, the traditional approaches were complex, lengthy, and expensive, entailing costly random screening of synthesized compounds or natural products coupled with in vivo validation largely depending on the availability of appropriate animal models. Currently, in silico modeling has become a vital tool for drug discovery and repurposing. Molecular docking and dynamic simulations are being used to find the best match between a ligand and a molecule, an approach that could help predict the biomolecular interactions between the drug and the target host. Beauvericin (BEA) is an emerging mycotoxin produced by the entomopathogenic fungus Beauveria bassiana, being originally studied for its potential use as a pesticide. BEA is now considered a molecule of interest for its possible use in diverse biotechnological applications in the pharmaceutical industry and medicine. In this manuscript, we provide an overview of the repurposing of BEA as a potential therapeutic agent for multiple diseases. Furthermore, considerable emphasis is given to the fundamental role of in silico techniques to (i) further investigate the activity spectrum of BEA, a secondary metabolite, and (ii) elucidate its mode of action.

NEK8-Associated Nephropathies: Do Autosomal Dominant Forms Exist?

INTRODUCTION: Nephronophthisis (NPHP) is a group of autosomal recessive renal diseases characterized by a reduced ability of the kidneys to concentrate solutes, chronic tubulointerstitial nephritis, and cystic kidney disease. It represents the most common genetic cause of childhood renal failure. To date, around 20 different genes, encoding primary cilia proteins, have been linked to NPHP. These contribute to one-third of cases with NPHP while the majority of patients remain molecularly undiagnosed. MATERIALS AND METHODS: Whole exome sequencing (WES) was carried out on a 2-year-old Lebanese boy with infantile NPHP characterized by multicystic kidney dysplasia, kidney insufficiency, and enlarged kidneys in addition to chronic anemia. The candidate variant, detected by WES, was then tested in the patient and his parents by Sanger sequencing. Copy number variation (CNV) analysis was subsequently performed in the proband. RESULTS: Our studies enabled the detection of a heterozygous de novo variant in NEK8 (NM_178170: p.Arg45Trp) in the proband. CNV analysis excluded the presence of big deletions or insertions in this gene. CONCLUSION: Here we report a de novo heterozygous variant in the NEK8 gene in infantile NPHP. This variant was previously detected at a de novo state in a patient presenting with the same clinical features as the proband. This suggests that autosomal dominant forms of NEK8-linked nephropathies may exist. Reporting further patients with NEK8 mutations is essential to confirm these findings and assess whether dominant forms of the disease are restricted to a specific mutational spot or are linked to variants scattered throughout the NEK8 gene.

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.

Whole-genome sequence analysis of carbapenem-resistant Enterobacteriaceae recovered from hospitalized patients.

OBJECTIVES: Carbapenems are among the few effective antibiotics against multidrug-resistant Enterobacteriaceae. This study aimed at characterizing the plasmid content and resistome of clinical carbapenem-resistant Enterobacteriaceae (CRE) recovered from 2016 to 2019 from hospitalized patients in Lebanon. METHODS: Plasmid typing and whole-genome sequencing were used to study the genomic characteristics of 65 clinical CREs including 27 Escherichia coli, 24 Klebsiella pneumoniae, one Klebsiella quasipneumoniae, three Morganella morganii, three Citrobacter freundii, five Enterobacter hormaechei, and two Serratia marcescens. RESULTS: blaOXA-48 (33.8%; n = 22) and blaOXA-48-like genes were among the detected resistance determinants, with two isolates co-harbouring blaNDM-5. Various blaNDM variants, blaNDM-1 (16.9%; n = 11), blaNDM-5 (9.2%; n = 6), blaNDM-7 (9.2%; n = 6), and blaNDM-19 (4.6%; n = 3), different ESBLs, and AmpC ß-lactamases were detected. Carbapenem resistance determinants were linked to a variety of incompatibility groups with IncFIB(K) (43.1%; n = 28) being the most prevalent, followed by IncFIA (40.0%), IncL (35.4%), IncX3 (32.3%), IncI1 (32.3%), and IncFIIK (29.2%). CONCLUSIONS: We analysed the clonality and resistance determinants of 65 multidrug-resistant (MDR) Enterobacteriaceae recovered in the period from 2016 to 2019 from a large tertiary hospital in Lebanon. NDM variants, OXA-48, and OXA-181 were the most prevalent detected carbapenemases and were mostly linked to the dissemination of IncL, IncX3, and IncF. This study reinforces the need to track the spread and dominance of clinically relevant carbapenemase-encoding plasmids in healthcare settings.

Carbapenem resistance determinants and their transmissibility among clinically isolated Enterobacterales in Lebanon.

BACKGROUND: The occurrence of carbapenem-resistant bacterial infections has increased significantly over the years with Gram-negative bacteria exhibiting the broadest resistance range. In this study we aimed to investigate the genomic characteristics of clinical carbapenem-resistant Enterobacterales (CRE). METHODS: Seventeen representative multi-drug resistant (MDR) isolates from a hospital setting showing high level of resistance to carbapenems (ertapenem, meropenem and imipenem) were chosen for further characterization through whole-genome sequencing. Resistance mechanisms and transferability of plasmids carrying carbapenemase-encoding genes were also determined in silico and through conjugative mating assays. RESULTS: We detected 18 different ß-lactamases, including four carbapenemases (blaNDM-1, blaNDM-5, blaNDM-7, blaOXA-48) on plasmids with different Inc groups. The combined results from PBRT and in silico replicon typing revealed 20 different replicons linked to plasmids ranging in size between 80 and 200 kb. The most prevalent Inc groups were IncFIB(K) and IncM. OXA-48, detected on 76-kb IncM1 conjugable plasmid, was the most common carbapenemase. We also detected other conjugative plasmids with different carbapenemases confirming the role of horizontal gene transfer in the dissemination of antimicrobial resistance genes. CONCLUSION: Our findings verified the continuing spread of carbapenemases in Enterobacterales and revealed the types of mobile elements circulating in a hospital setting and contributing to the spread of resistance determinants. The occurrence and transmission of plasmids carrying carbapenemase-encoding genes call for strengthening active surveillance and prevention efforts to control antimicrobial resistance dissemination in healthcare settings.

Absolute quantification of gene expression in drug discovery using RT-qPCR: Case of a drug used in the treatment of leishmaniasis.

Leishmaniasis is a neglected disease and a public health concern. Chemotherapeutic agents available for the treatment of parasitic infections, including leishmaniasis, have several limitations. For that, we designed a highly sensitive assay using RT-aqPCR to evaluate the efficacy of antileishmanial drugs using SYBR Green to quantify the expression of marker genes. A matrix of reactions using different annealing temperatures and primer concentrations was tested to obtain optimum assay performance. The standard curves designed for quantification of parasites and macrophages showed linearity over a 9-log DNA concentration range. The amount of input target sequence was determined by plotting the Ct value of drug-exposed cells on the standard curves. We then tested the efficacy of miltefosine against Leishmania tropica. The RT-aqPCR assay was more sensitive, reproducible, and time-efficient than the conventional microscopic counting method. Most of the anti-parasitic drugs available have significant drawbacks, and there is an urgent need to develop new alternatives. Our assay expedites preclinical testing efficacy of candidate anti-parasitic compounds.

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.

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.