Biology subjects

El-Halfawy, O. M.

Publications and source records attributed to El-Halfawy, O. M..

7 recordsLinked to original sources

bioRxiv2026

Clinically-relevant altered antibiotic responses and mechanisms of β-lactam sensitization of MRSA in cystic fibrosis artificial sputum

Staphylococcus aureus is the most common bacterial pathogen affecting pediatric patients with Cystic fibrosis (CF), a genetic disorder that causes thick mucus buildup in the lungs, providing a scaffold for chronic infections. Antibiotic treatment is typically guided by standard in vitro antimicrobial susceptibility testing (AST) in Mueller-Hinton broth (MHB), which does not represent the infection site in CF lungs. Notably, discordances between AST predictions and antibiotic therapeutic outcomes were reported in up to 50% of CF cases. To address this gap, we conducted ASTs against methicillin-resistant S. aureus (MRSA) in CF sputum-mimetic media compared with MHB, demonstrating [≥]4-fold discordances across four of nine antibiotic classes. Most significantly, we observed unexpected {beta}-lactam sensitization of MRSA strains (up to 128-fold) in CF sputum-like media, crossing the CLSI clinical breakpoint, suggesting this shift may alter therapeutic outcomes. Genome-wide screens and follow-up assays revealed underlying cell envelope remodelling and alterations to cell envelope stress responses. On the other hand, mucin binding to daptomycin may have led to an apparent 8-fold increase in resistance to this antibiotic in one of the CF sputum-like media. Overall, our AST results in CF sputum-mimetic conditions provide insights into bacterial responses during CF infections. Importantly, they suggest {beta}-lactams may be effective in treating MRSA infections in CF patients, warranting further investigation in relevant in vivo systems.

microbiology↗

bioRxiv2025

Uncovering clinically-relevant altered antibiotic responses of Staphylococcus aureus under wound infection-mimetic conditions

Standard in vitro antimicrobial susceptibility testing (AST) using Mueller-Hinton broth (MHB) does not reflect infection-site conditions, and its results often do not correlate with therapeutic outcomes. Here, we compared the antibiotic susceptibility of methicillin-resistant Staphylococcus aureus (MRSA), a common chronic wound pathogen, in simulated wound fluid (SWF) resembling wound exudate versus MHB, revealing discordant AST results across six of nine tested antibiotic classes. The most significant were 128-fold increased resistance to tetracyclines and 256-fold sensitization to {beta}-lactams in SWF. Tetracycline resistance was mediated by MntC, an extracellular manganese-binding protein, whereas {beta}-lactam sensitization was driven by cell envelope remodelling in SWF. Galleria mellonella wound infection results matched the SWF susceptibility phenotypes, suggesting SWF better predicts in vivo wound infection therapeutic outcomes. These comprehensive phenotypic and mechanistic insights into MRSA antibiotic responses under wound-infection-mimetic conditions with direct in vivo validation identify a potential new antibiotic adjuvant target and may guide improved antibiotic therapy for MRSA wound infections.

microbiology↗

bioRxiv2025

Photochemical modification of two fluorene-based molecules yields structurally distinct DNA intercalators with potent anti-MRSA activity

Staphylococcus aureus is a leading cause of skin and soft tissue infections, endocarditis, and bloodstream infections worldwide. The emergence of methicillin-resistant S. aureus (MRSA) and growing resistance to last-resort antibiotics like vancomycin have created an urgent need for new antimicrobials with distinct mechanisms of action. In this study, we characterize DB10, a planar, fluorene-based compound identified in a high-throughput screen for MRSA inhibitors. Upon UVA exposure, DB10 undergoes photoconversion from a red-colored form (DB10-R) to a yellow-colored form (DB10-Y). In comparison with DB10-R, DB10-Y exhibits reduced hydrophobicity, lower cytotoxicity, and modestly improved minimum inhibitory concentrations (MICs) towards a number of Gram-positive bacteria. DB10-Y intercalates into DNA and induces double-stranded breaks, yet resistance emerged only at low levels after prolonged serial passaging. To optimize this scaffold, we screened a panel of fluorene analogs and identified the photoconverting analog DB33, which in its yellow form (DB33-Y) is non-toxic and retained DNA intercalation activity. DB33-Y was effective against intracellular S. aureus in macrophages and epithelial cells and significantly reduced bacterial burden and lesion size in a murine skin infection model. DB10-Y and DB33-Y both also suppressed expression of -toxin at sub-MIC concentrations, indicating an additional anti-virulence effect. Together, these findings highlight the therapeutic potential of fluorene-based DNA intercalators as a new class of antimicrobial and anti-virulence agents against MRSA.

microbiology↗

bioRxiv2024

Discovery of broad-spectrum bacterial polyamine detoxification inhibitors as potential antivirulence agents and antibiotic adjuvants

The alarming rise in antimicrobial resistance reinforces an urgent need for new antimicrobial strategies. Host- or bacteria-derived chemicals, such as polyamines, present at infection sites often influence microbial virulence and antibiotic response. Polyamines are cationic small molecules overproduced by the host during infection, modulating immune responses. Polyamine detoxification by several pathogens correlates with increased virulence. We sought to uncover inhibitors of polyamine detoxification through a high-throughput whole-cell screen against the community-acquired methicillin-resistant Staphylococcus aureus (MRSA) USA300, identifying the polyamine analog OES2-0017 and the catechol derivative isoproterenol (OES1-1087), which synergized with polyamines in the low-micromolar range. Chemical genomics and enzymatic assays combined with computational studies revealed that both compounds prevented polyamine detoxification by spermine/spermidine acetyltransferase SSAT (SpeG) and another previously uncharacterized S. aureus SSAT (denoted PaiASa herein). OES2-0017 directly inhibited the enzymes, whereas OES1-1087 reduced intracellular levels of acetyl-CoA required for SSAT activity. OES2-0017 perturbed the bacterial membrane at higher concentrations, likely causing the observed growth-inhibitory effects, whereas OES1-1087 increased membrane fluidity, likely increasing susceptibility to spermine-mediated membrane perturbation. The inhibitors showed broad-spectrum activity against various Gram-positive and Gram-negative bacteria, as well as Candida albicans. OES2-0017 abolished the spermine-mediated protection of MRSA USA300 from antibiotics, including vancomycin, phenocopying the {Delta}speG mutant and suggesting its potential utility as an antibiotic adjuvant. OES2-0017 eradicated SpeG-expressing Salmonella Typhimurium inside murine macrophages, whereas OES1-1087 reduced the S. Typhimurium burden in the liver and spleen in a murine gastrointestinal infection model, demonstrating their potential as antivirulence agents. Neither inhibitor exhibited cytotoxic activity against eukaryotic cells at their respective antimicrobial ranges. Small-scale structure-activity relationship experimental and computational analyses identified OES2-0017 analogs with improved specificity for the bacterial enzyme compared to the human SAT1 and no toxicity. This study provides novel antimicrobial compounds with broad-spectrum activity and a novel mode of action for multidrug-resistant priority pathogens.

microbiology↗

bioRxiv2024

The antimicrobial potential of traditional remedies of Indigenous Peoples from Canada against MRSA planktonic and biofilm bacteria in wound-infection mimetic conditions

Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of wound infections, often progressing into serious invasive bloodstream infections. MRSA disproportionately affects Indigenous peoples in Canada with higher rates of skin and wound infections, an example of persistent gaps in health outcomes between Indigenous and non-Indigenous peoples precipitated by the legacy of colonialism. Conversely, Indigenous peoples have long used natural remedies for infections and other diseases; however, their knowledge was rarely considered for modern medicine. The stagnant antibiotic discovery pipeline and alarming rise of resistance to current antibiotics prompted us to turn to Indigenous medicine as an untapped source of antimicrobials. As such, we collected and prepared 85 extracts of medicinal plants of value to Indigenous Peoples spanning the Canadian Prairies. We explored the antimicrobial potential of these extracts against MRSA under wound infection-mimetic conditions compared to culture media typically used to study bacterial antibiotic responses and biofilms but not adequately representative of infection sites. We identified extracts with MRSA growth inhibitory [e.g., bergamot, dock, gaillardia, and dandelion extracts] and biofilm prevention and eradication [e.g., gumweed extracts] activities. Extracts, including those of chokecherry, hoary puccoon, and Northern bedstraw, were only active under wound infection-mimetic conditions, highlighting the relevance of antibiotic discovery under host-relevant conditions. Testing growth inhibitory extracts against an S. aureus cross-resistance platform suggested that they act through mechanisms likely distinct from known antibiotic classes. Together, through an interdisciplinary partnership leveraging Western approaches and traditional Indigenous knowledge, we identified plant extracts with promising antimicrobial potential for drug-resistant MRSA wound infections.

microbiology↗

bioRxiv2024

Polyamine-mediated sensitization of Klebsiella pneumoniae to macrolides through a dual mode of action

Chemicals bacteria encounter at the infection site could shape their stress and antibiotic responses; such effects are typically undetected in standard lab conditions. Polyamines are small molecules typically overproduced by the host during infection and have been shown to alter bacterial stress responses. We sought to determine the effect of polyamines on the antibiotic response of Klebsiella pneumoniae, a Gram-negative priority pathogen. Interestingly, putrescine and other natural polyamines sensitized K. pneumoniae to azithromycin, a macrolide protein translation inhibitor typically used for Gram-positive bacteria. This synergy was further potentiated in the physiological buffer, bicarbonate. Chemical genomic screens suggested a dual mechanism whereby putrescine acts at the membrane and ribosome levels. Putrescine permeabilized the outer membrane of K. pneumoniae (NPN and {beta}-lactamase assays) and the inner membrane (Escherichia coli {beta}-galactosidase assays). Chemically and genetically perturbing membranes led to a loss of putrescine-azithromycin synergy. Putrescine also inhibited protein synthesis in an E. coli-derived cell-free protein expression assay simultaneously monitoring transcription and translation. Profiling the putrescine-azithromycin synergy against a combinatorial array of antibiotics targeting various ribosomal sites suggested that putrescine acts as tetracyclines targeting the 30S ribosomal acceptor site. Next, exploiting the natural polyamine-azithromycin synergy, we screened a polyamine analog library for azithromycin adjuvants, discovering four azithromycin synergists with activity starting from the low micromolar range and mechanisms similar to putrescine. This work sheds light on the bacterial antibiotic responses under conditions more reflective of those at the infection site and provides a new strategy to extend the macrolide spectrum to drug-resistant K. pneumoniae.

microbiology↗

bioRxiv2019

Uncovering the hidden antibiotic potential of Cannabis

The spread of antimicrobial resistance continues to be a priority health concern worldwide, necessitating exploration of alternative therapies. Cannabis sativa has long been known to contain antibacterial cannabinoids, but their potential to address antibiotic resistance has only been superficially investigated. Here, we show that cannabinoids exhibit antibacterial activity against MRSA, inhibit its ability to form biofilms and eradicate pre-formed biofilms and stationary phase cells persistent to antibiotics. We show that the mechanism of action of cannabigerol is through targeting the cytoplasmic membrane of Gram-positive bacteria and demonstrate in vivo efficacy of cannabigerol in a murine systemic infection model caused by MRSA. We also show that cannabinoids are effective against Gram-negative organisms whose outer membrane is permeabilized, where cannabigerol acts on the inner membrane. Finally, we demonstrate that cannabinoids work in combination with polymyxin B against multi-drug resistant Gram-negative pathogens, revealing the broad-spectrum therapeutic potential for cannabinoids.

microbiology↗