Ahmed Osama El-Gendy

Purpose: Multidrug-resistant bacterial infections are a substantial global health challenge. Targeting bacterial virulence factors that control the progression, severity, and pathogenicity of bacterial infections could prevent and combat infection by intractable, potentially life-threatening pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Laser-based antibacterial photodynamic therapy (aPDT) has emerged as a promising alternative for infection management. Methods: This study assessed the attenuation of adhesion and invasion of MRSA bacteria in two human epithelial cell lines, melanoma cells (A375) and breast ductal carcinoma (T47D), after exposure to different sublethal femtosecond laser doses. The INSPIRE HF100 laser system (Spectra Physics), pumped by a mode-locked femtosecond Ti: sapphire laser MAI TAI HP (Spectra Physics), was used to provide the femtosecond laser pulses at a wavelength of 400 nm for different durations; 15, 30, and 45 min., as well as different average powers; 50, 100, and 150 mW. Results: Our results showed that, an average power of 50 mW for 15 min. significantly reduced MRSA adhesion (by ~ 84–96%) and invasion (by ~ 84–98%), even though exposure durations of 30 and 45-min. resulted in reduced viability ( p < 0.0001 by ANOVA and Tukey test). Conclusion: All proposed femtosecond laser doses effectively impaired MRSA’s ability to adhere to and invade epithelial cells. Graphical This diagram illustrates the experimental workflow for identifying the optimal femtosecond laser parameters to attenuate MRSA’s adhesion to and invasion of different epithelial cell lines. Following exposure to femtosecond laser irradiation, MRSA was co-cultured with epithelial cells for various infection durations. Adhesion and Invasion assays were done, and the adherent and invasive MRSA were quantified by colony-forming unit (CFU) counting, allowing for recommending an optimal femtosecond laser treatment for mitigating bacterial adhesion and invasion.

Ocular melanoma, a malignant tumor that arises from the uveal tract, presents significant therapeutic challenges due to its intricate anatomy and limited accessibility. Recent advances propose a novel targeted treatment approach by combining femtosecond laser technology with Zinc Oxide Nanoparticles (ZnO-NPs) synthesized through laser ablation. This study investigates the combined effects of femtosecond laser irradiation and ZnO-NPs on A375 melanoma cells. ZnO-NPs were synthesized by ablating high-purity zinc in distilled water using a 532 nm Nd:YAG laser. The nanoparticles were characterized using UV-Vis spectroscopy, TEM, EDX, and concentration analysis, revealing optimal size distribution and surface properties suitable for biological applications. A375 ocular melanoma cells were treated with ZnO-NPs, followed by femtosecond laser irradiation at wavelengths of 420 and 700 nm. Cytotoxicity was assessed using MTT assays, which showed that the combination of ZnO-NPs and 420 nm laser irradiation significantly reduced cell viability compared to either treatment alone. This innovative approach underscores the potential of combining nanotechnology with femtosecond laser technology to achieve precise and effective cancer cell targeting. Further in vivo studies are required to evaluate the clinical applicability of this promising strategy.

Background: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CRhvKp) is increasingly recognized as the predominant nosocomial pathogen in healthcare environments. Objective: This study aims to investigate the prevalence, antibiotic resistance patterns, and molecular characteristics of CRhvKp in Egypt. Methods: Out of 150 non-duplicate clinical isolates collected from a hospital in Egypt, 63 were identified as K. pneumoniae via biochemical tests and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS). The CRKp isolates were identified using the VITEK^®2 system and KB disc diffusion. Subsequently, to define CRhvKp using PCR, a specific conjugation of rmpA and/or rmpA2 with iucA, iroB, or peg-344 was employed. Virulence and carbapenemase genes for CRKp isolates were screened via PCR, and the results of the carbapenemase genes were validated through Sanger sequencing. Results: Among the 63 K. pneumoniae isolates, 30 were identified as CRKp, and all exhibited multidrug resistance (MDR). CRKp was classified as 66.7% for CRhvKp and 33.3% for CR-non-hvKp. The presence of various virulence genes in CRhvKp and CR-non-hvKp samples was observed as follows: fimH (75% and 60%), mrkD (100% and 90%), entB (80% and 90%), irp2 (85% and 90%), wabG (90% and 60%), and uge (40% and 60%). The existence of carbapenemase genes in CRhvKp and CR-non-hvKp samples was observed as follows: bla _NDM (41.3% and 25.0%), bla _KPC (10% and 5%), and bla _OXA−48 (20.0% and 0.0%). Conclusion: Our study identified a significant percentage of MDR CRhvKp and CR-non-hvKp isolates harboring different carbapenemases, which sparked substantial concern. Herein, we highlight the concerning rapid increase of CRhvKp in Egypt. Hence, continued countrywide are surveillance is crucial to clarify the prevalence rate of these CRhvKp in Egypt.

The incidence and mortality rates associated with melanoma are increasing. Due to their high proliferation rate, ability to self-renew, and resistance mechanisms, cancer cells often withstand conventional therapies such as radiation and chemotherapy. Therefore, further research is required to develop novel melanoma therapies with fewer adverse effects, but effective therapeutic impacts. This study aims to investigate how femtosecond laser treatment affects melanoma cells using the A375 cell line as an in vitro model. A375 melanoma cells were plated at a concentration of 10⁴ cells per well in 96-well plates and incubated overnight; then, they were subjected to femtosecond laser irradiation for durations of 3, 5, or 10 min, maintaining a steady power of 100 mW. The laser operated across different wavelengths in the ultraviolet, visible, and infrared ranges. Cell viability was evaluated 24 h after irradiation using the MTT assay. The results showed the significant inhibition of melanoma cell growth with various femtosecond laser parameters, particularly at 380 and 400 nm. At 380 nm, the cell viability was reduced by approximately 90%, and at 400 nm by 73%, after 10 min of exposure. Additional reductions were observed at 420 nm (47%) and 440 nm (18%), while no significant effects were found at 700–780 nm. The most effective exposure time was 10 min. Femtosecond laser radiation exerts a noteworthy anticancer effect on A375 cells, particularly at specific wavelengths and exposure durations, underscoring the potential of femtosecond laser therapy for treating melanoma. Exploring the underlying mechanisms of these effects and evaluating the clinical potential of this treatment modality requires further research.

Background: Klebsiella is one of the most challenging superbugs having a high tendency to acquire rapid resistance to many antibiotics, even the ones recognized as the last resort. In several hospitals and environmental niches, Klebsiella is continuously exposed to residual amounts of antibiotics at sub-inhibitory concentrations forming an environmental stress motivating them to adapt and evolve antimicrobial resistance. In the present study, meropenem (MEM) resistance was induced experimentally in a MEM-sensitive strain of K. quasipneumoniae ATCC 700603 through sequential sub-culturing in presence of sub-inhibitory concentrations of MEM over a period of 20 days. To uncover the possible mechanisms standing behind the evolution of antimicrobial resistance upon successive exposure to stress of MEM rather than horizontal gene transfer (HGT) of antibiotic resistance genes. Results: Fully adapted cells of the 20th generation (G20) showed MEM-resistance with elevated minimum inhibitory concentration (MIC) by 256-fold compared to the parent cells (G0). The main mechanism of resistance was the production of carbapenemases, which was assured by different tests including nitrocefin, modified-Hodge test (MHT), and modified carbapenem inactivation method (mCIM). The degradation of MEM reached 65.93% by the produced carbapenemases of G20 as determined by the HPLC analysis. Transcriptomics analysis of the class D carbapenemase encoding gene, bla _OXA-2 , revealed that it was significantly over-expressed by a 3.12-fold (p < 0.05) in G20 compared to G0. Conclusion: The evolved MEM resistance aroused mainly from MEM degradation by carbapenemases, neither increased efflux nor decreased influx of MEM. The rational use of antibiotics is essential to reduce bacterial exposure to the environmental basal levels of antibiotics and decreasing the evolution of antimicrobial resistance. Graphical

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most alarming antibiotic-resistant pathogens causing nosocomial and community-acquired infections. Actinomycetes, particularly Streptomycetes, have historically been a major source of natural products including anti-MRSA agents. Hence, we designed this study to isolate new anti-MRSA natural products from Streptomycetes aided by liquid chromatography-electrospray ionization-high resolution mass spectrometry (LC-ESI-HRMS)-based metabolomics data analysis and dereplication study to select the best fractions for isolation work. Following this approach, two new natural products, namely, (4-propylphenyl) methane-diazonium (1) and (4E)-6-oxooctadec-4-enoic acid (2) and three known compounds, 8-methyl-5-oxononanoic acid (4), 15-methylhexadecanoic acid (5), and (Z)-14-methylpentadecenoic acid (6) were isolated from two soil Streptomyces sp. Additionally, (9E)-11-hydroxyoctadec-9-enoic acid (3) was recovered for the first time from Streptomycetes. The minimum inhibitory concentration (MIC) of compounds 1, 5, and 6 against MRSA were 128, 64, and 64 µg/mL respectively. The compounds were slightly more active against B. subtilis with MIC values of 64, 64, and 32 µg/mL, respectively. Our research adds to the growing knowledge about the importance of combining sophisticated analytical methods in the chemical profiling of the fractions with the outcomes of the bioassays throughout the subsequent fractionation processes to help isolate new secondary metabolites from natural sources. Graphical abstract

Cystic fibrosis (CF) is a polymicrobial infection characterized by interactions among various bacterial species that affect one another’s cohabitation. The investigation of interspecies interactions in dual infections is essential to understand their reaction in the environment better and assist in the development of treatment regimens and innovative disease control approaches. Our hypothesis posits that co-infection interactions promote the adaptation of Staphylococcus aureus and Pseudomonas aeruginosa, potentially leading to synergistic action. To explore this, we examined dual-species interactions in co-isolated pairs of these organisms from Egyptian CF patients using laboratory media and artificial sputum media (ASM). Based on demographic data, 82 collected bacterial isolates from single, dual, and triple cultures were identified from 50 enrolled patients. In the interaction of the pairs in mimic media, P. aeruginosa exo-products significantly enhanced the biofilm formation and growth of S. aureus. Conversely, S. aureus did not inhibit P. aeruginosa biofilm formation. Furthermore, the biofilm mode of dual-organism growth provides protection in the CF context, as bacterial biofilms can withstand much higher antimicrobial levels compared to planktonically grown bacteria. Additionally, key biofilm genes regulated by quorum sensing were differentially expressed in both species in an isolate-dependent manner, highlighting their significant role in coexistence dual-species biofilm coexistence. In conclusion, our study illuminates the competitive and cooperative interactions between these two pathogens, which impact their coexistence and encourage biofilm production. This, in turn, accelerates disease progression and compromises patient health.

Background: Methicillin-Resistant Staphylococcus aureus (MRSA) is a global concern owing to the increasing prevalence of multidrug-resistant (MDR) strains. Vancomycin has been the primary treatment for MRSA; however, Vancomycin-resistant strains are being increasingly reported worldwide. Therefore, comparative studies are essential to support antimicrobial stewardship and improving clinical management. Ultimately, the findings from this study are expected to inform treatment strategies and guide public health interventions effectively. Material and methods: This study investigated the prevalence, antimicrobial resistance, and virulence characteristics of Vancomycin-sensitive S. aureus (VSSA) and Vancomycin-resistant S. aureus (VRSA) within MRSA strains. By employing a combination of phenotypic methods, such as antimicrobial susceptibility testing, and genotypic techniques, including molecular typing and identification of virulence genes, we obtained comprehensive insights into VRSA and VSSA profiles. Results: Of 250 clinical samples, 62 (24.8%) were S. aureus and 27 (43.5%) were identified as MRSA. All MRSA isolates exhibited MDR patterns. Most MRSA strains were VSSA (20/27, 74.1%), while 7 (25.9%) were VRSA. The VRSA isolates showed more antimicrobial resistance than VSSA isolates; however, the VRSA isolates had less virulence than VSSA isolates. Linezolid was the most effective treatment, with a 3.7% resistance rate. A higher percentage of biofilm-producing MRSA (96.3%) was confirmed by both phenotypic and genotypic methods. All isolates, except one VRSA, showed multi-virulence patterns (harbored more than 3 virulence genes). High diversity and low clonality (D-value = 0.99) were found in both VSSA and VRSA. Based on our correlation findings, the emergence of vancomycin resistance could modify the association between antimicrobial resistance and virulence, potentially affecting the pathogenic profile of these strains. The study also revealed complex interactions among host factors (including age and gender), sample origin, antimicrobial resistance, biofilm production, and virulence genes. Conclusion: This study highlights the alarming spread of MRSA and VRSA, which show significant resistance and virulence.

Enterococcus faecalis is among the most resistant bacteria found in infected root canals. The demand for cutting-edge disinfection methods has rekindled research on photoinactivation with visible light. This study investigated the bactericidal activity of femtosecond laser irradiation against vancomycin-resistant Enterococcus faecalis V583 (VRE). The effect of parameters such as wavelength and energy density on the viability and growth kinetics of VRE was studied to design an optimized laser-based antimicrobial photoinactivation approach without any prior addition of exogenous photosensitizers. The most effective wavelengths were 430 nm and 435 nm at a fluence of 1000 J/cm², causing a nearly 2-log reduction (98.6% and 98.3% inhibition, respectively) in viable bacterial counts. The colony-forming units and growth rate of the laser-treated cultures were progressively decreased as energy density or light dose increased at 445 nm but reached a limit at 1250 J/cm². At a higher fluence of 2000 J/cm², the efficacy was reduced due to a photobleaching phenomenon. Our results highlight the importance of optimizing laser exposure parameters, such as wavelength and fluence, in bacterial photoinactivation experiments. To our knowledge, this is the first study to report an optimized wavelength for the inactivation of VRE using visible femtosecond laser light.