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Phage therapy has been successfully used as an experimental therapy in the treatment of multidrug-resistant strains of Staphylococcus aureus (MDRSA)-caused skin infections and is seen as the most promising alternative to antibiotics. However, in recent years a number of reports indicating that phages can interact with eukaryotic cells emerged. Therefore, there is a need to re-evaluate phage therapy in light of safety. It is important to analyze not only the cytotoxicity of phages alone but also the impact their lytic activity against bacteria may have on human cells. As progeny virions rupture the cell wall, lipoteichoic acids are released in high quantities. It has been shown that they act as inflammatory agents and their presence could lead to the worsening of the patient's condition and influence their recovery. In our work, we have tested if the treatment of normal human fibroblasts with staphylococcal phages will influence the metabolic state of the cell and the integrity of cell membranes. We have also analyzed the effectiveness of bacteriophages in reducing the number of MDRSA attached to human fibroblasts and the influence of the lytic activity of phages on cell viability. We observed that, out of three tested anti-Staphylococcal phages-vB_SauM-A, vB_SauM-C and vB_SauM-D-high concentrations (109 PFU/mL) of two, vB_SauM-A and vB_SauM-D, showed a negative impact on the viability of human fibroblasts. However, a dose of 107 PFU/mL had no effect on the metabolic activity or membrane integrity of the cells. We also observed that the addition of phages alleviated the negative effect of the MDRSA infection on fibroblasts' viability, as phages were able to effectively reduce the number of bacteria in the co-culture. We believe that these results will contribute to a better understanding of the influence of phage therapy on human cells and encourage even more studies on this topic.

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Due to the misuse of antibiotics, the multidrug-resistant Staphylococcus aureus (MDRSA) has caused serious infections and become more difficult to deal with. Here we propose to synthesise copper oxide nanoparticles (CuO-NPs) using a cell-free filter of Streptomyces rochei to enhance antibiotics activity against (MDRSA) and kill them. Characterisation of CuO-NPs using ultraviolet, dynamic light scattering, zeta potential, transmission electron microscopic (TEM), and X-ray diffraction, were investigated. The antibacterial action of the CuO-NPs was tested against standard strain and clinical isolates using the agar well diffusion method and the microdilution assay. The results showed the monodispersed spherical shape CuO-NPs with a mean diameter of 10.7 nm and were found to be active against (MDRSA). By a combination of CuO-NPs with different antibiotics, the highest synergistic effect was observed with cefoxitin, the minimum inhibitory concentration (MIC) was reduced to 6.5 for CuO-NPs, and 19.5 for cefoxitin. Time-kill assay showed the highest reduction in log10 colony-forming unit (CFU)/ml of initial inoculum of MRSA after 24 h. The HFB-4 cells cultured in the presence of CuO-NPs showed normal morphology with 100% viability at 8 µg/ml. TEM showed that combination (1/4 MIC cefoxitin +1/16 MIC CuO-NPs) highly damages bacterial cells' shape. The biosynthesis CuO-NPs showed antibacterial activity against S. aureus suggesting a promising alternative in clinical.

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Biofilms are complex bacterial structures composed of bacterial cells embedded in extracellular polymeric substances (EPS) consisting of polysaccharides, proteins and lipids. As a result, biofilms are difficult to eradicate using both mechanical methods, i.e., scraping, and chemical methods such as disinfectants or antibiotics. Bacteriophages are shown to be able to act as anti-biofilm agents, with the ability to penetrate through the matrix and reach the bacterial cells. However, they also seem to have their limitations. After several hours of treatment with phages, the biofilm tends to grow back and phage-resistant bacteria emerge. Therefore, it is now recommended to use a mixture of phages and other antibacterial agents in order to increase treatment efficiency. In our work we have paired staphylococcal phages with lactoferrin, a protein with proven anti-biofilm proprieties. By analyzing the biofilm biomass and metabolic activity, we have observed that the addition of lactoferrin to phage lysate accelerated the anti-biofilm effect of phages and also prevented biofilm re-growth. Therefore, this combination might have a potential use in biofilm eradication procedures in medical settings.

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BACKGROUND: Staphylococcus aureus has prevailed against the majority of antibiotics currently in clinical use, making it a significant global public health problem. As a safer alternative, bioactive compounds have been explored. Annona muricata has been shown to possess antimicrobial activity. However, there are few reports on the molecular activity of A. muricata bioactive compounds against S. aureus. Thus, this study was aimed at evaluating the antimicrobial activity of its crude extract as well as investigating the potential of its bioactive compounds against the Cap5O capsular polysaccharides (CPS) of S. aureus via molecular docking. METHODS: Collection of plant leaves, preparation of extracts, anti-nutrient analysis, phytochemical screening via crude method and gas chromatography-mass spectrophotometer (GC-MS), isolation and characterization of S. aureus and the antimicrobial activity test were all done using standard protocols. Molecular docking was done using the MCULE online tool with emphasis on docking scores, toxicity, and other properties. RESULTS: Crude screening of the extracts showed the presence of polyphenols, hydroxyanthraquinones, reducing compounds, flavonoids, saponins, glycosides, alkaloids, anthraquinones, phlobatannins and tannins in different concentrations. Anti-nutrient analysis showed the presence of allowable levels of evaluated anti-nutrients. GC-MS revealed a total of twenty-nine (29) bioactive compounds, out of which only 4 (13.80%) docked without toxicity and these were bicyclo[4.1.0]heptan-2-one 6-methyl, trichloromethane, carbonic acid 2-dimethylaminoethyl propyl ester, and 1-methyl-4-phenyl-5-thioxo-1,2,4-triazolidin-3-one on either the NAD-binding or C-terminal substrate binding domain of Cap5O. CONCLUSION: Results obtained show that Cap5O could be a potential drug target for multi-drug resistant S. aureus, however, further studies aimed at evaluating these bioactive compounds individually and in combination are highly needed.

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The rise of antibiotic resistance (ABR) and the drying up of the pipeline for the development of new antibiotics demands an urgent search for new antibiotic leads. While the majority of clinically available antibiotics were discovered from terrestrial Streptomyces, related species from marine sediments as a source of antibiotics remain underexplored. Here, we utilized culture-dependent isolation of thirty-five marine sediment-derived actinobacterial isolates followed by a screening of their antibacterial activity against multidrug-resistant S. aureus ATCC BAA-44. Our results revealed that the crude extract of Streptomyces griseorubens strain DSD069 isolated from marine sediments collected in Romblon, Philippines displays the highest antibacterial activity, with 96.4% growth inhibition. The S. aureus ATCC BAA-44 cells treated with crude extract of Streptomyces griseorubens strain DSD069 showed cell membrane damage as demonstrated by (a) leakage and loss of vital cell constituents, including DNA and proteins, (b) irregular shrinkage of cells, and (c) increase membrane permeability. The antibiotic compounds were identified as Bisanhydroaklavinone and 1-Hydroxybisanhydroaklavinone with MIC value of 6.25 µg/mL and 50.00 µg/mL, respectively. Bisanhydroaklavinone and 1-Hydroxybisanhydroaklavinone are shunt metabolites in the biosynthesis of anticancer anthracycline derivatives namely doxorubicin, daunorubicin, and cinerubins. It is rare, however, that shunt metabolites are accumulated during fermentation of marine sediment-derived Streptomyces strain without genetic modification. Thus, our study provides evidence that natural bacterial strain can produce Bisanhydroaklavinone and 1-Hydroxybisanhydroaklavinone as antibiotic leads to combat ABR.

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Staphylococcus aureus and Mycobacterium tuberculosis are major causative agents responsible for serious nosocomial and community-acquired infections impacting healthcare systems globally. Over several decades, these pathogens have developed resistance to multiple antibiotics significantly affecting morbidity and mortality. Thus, these recalcitrant pathogens are amongst the most formidable microbial pathogens for which international healthcare agencies have mandated active identification and development of new antibacterial agents for chemotherapeutic intervention. In our present work, a series of new quinazolin-4(3H)-one derivatives were designed, synthesized and evaluated for their antibacterial activity against ESKAP pathogens and pathogenic mycobacteria. The experiments revealed that 4'c, 4'e, 4'f and 4'h displayed selective and potent inhibitory activity against Staphylococcus aureus with MIC values ranging from 0.03-0.25 µg/mL. Furthermore, compounds 4'c and 4'e were found to be benign to Vero cells (CC50 = >5 µg/mL) and displayed promising selectivity index (SI) > 167 and > 83.4 respectively. Additionally, 4'c and 4'e demonstrated equipotent MIC against multiple drug-resistant strains of S. aureus including VRSA, concentration dependent bactericidal activity against S. aureus and synergized with FDA approved drugs. Moreover, compound 4'c exhibited more potent activity in reducing the biofilm and exhibited a PAE of ∼2 h at 10X MIC which is comparable to levofloxacin and vancomycin. In vivo efficacy of 4'c in murine neutropenic thigh infection model revealed that 4'c caused a similar reduction in cfu as vancomycin. Gratifyingly, compounds 4d, 4e, 9a, 9b, 14a, 4'e and 4'f also exhibited anti-mycobacterial activity with MIC values in the range of 2-16 µg/mL. In addition, the compounds were found to be less toxic to Vero cells (CC50 = 12.5->100 µg/mL), thus displaying a favourable selectivity index. The interesting results obtained here suggest the potential utilization of these new quinazolin-4(3H)-one derivatives as promising antibacterial agents for treating MDR-Staphylococcal and mycobacterial infections.

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Methicillin-resistant Staphylococcus aureus (MRSA) has been detected in retail meats, although large-scale studies are scarce. We conducted a one-year survey in 2010-2011 within the framework of the National Antimicrobial Resistance Monitoring System. Among 3520 retail meats collected from eight U.S. states, 982 (27.9%) contained S. aureus and 66 (1.9%) were positive for MRSA. Approximately 10.4% (107/1032) of S. aureus isolates, including 37.2% (29/78) of MRSA, were multidrug-resistant (MDRSA). Turkey had the highest MRSA prevalence (3.5%), followed by pork (1.9%), beef (1.7%), and chicken (0.3%). Whole-genome sequencing was performed for all 66 non-redundant MRSA. Among five multilocus sequence types identified, ST8 (72.7%) and ST5 (22.7%) were most common and livestock-associated MRSA ST398 was assigned to one pork isolate. Eleven spa types were represented, predominately t008 (43.9%) and t2031 (22.7%). All four types of meats harbored t008, whereas t2031 was recovered from turkey only. The majority of MRSA (84.8%) possessed SCCmec IV and 62.1% harbored Panton-Valentine leukocidin. Pulsed-field gel electrophoresis showed that all ST8 MRSA belonged to the predominant human epidemic clone USA300, and others included USA100 and USA200. We conclude that a diverse MRSA population was present in U.S. retail meats, albeit at low prevalence.

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