Combination antimicrobial therapy: in vitro synergistic effect of anti-staphylococcal drug oxacillin with antimicrobial peptide nisin against Staphylococcus epidermidis clinical isolates and Staphylococcus aureus biofilms.

The ability of Staphylococcus epidermidis and S. aureus to form strong biofilm on plastic devices makes them the major pathogens associated with device-related infections (DRIs). Biofilm-embedded bacteria are more resistant to antibiotics, making biofilm infections very difficult to effectively treat. Here, we evaluate the in vitro activities of anti-staphylococcal drug oxacillin and antimicrobial peptide nisin, alone and in combination, against methicillin-resistant S. epidermidis (MRSE) clinical isolates and the methicillin-resistant S. aureus ATCC 43,300. The minimum inhibitory concentrations (MIC) and minimum biofilm eradication concentrations (MBEC) of oxacillin and nisin were determined using the microbroth dilution method. The anti-biofilm activities of oxacillin and nisin, alone or in combination, were evaluated. In addition, the effects of antimicrobial agents on the expression of icaA gene were examined by quantitative real-time PCR. MIC values for oxacillin and nisin ranged 4-8 µg/mL and 64-128 µg/mL, respectively. Oxacillin and nisin reduced biofilm biomass in all bacteria in a dose-dependent manner and this inhibitory effect was enhanced with combinatorial treatment. MBEC ranges for oxacillin and nisin were 2048-8192 µg/mL and 2048-4096 µg/mL, respectively. The addition of nisin significantly decreased the oxacillin MBECs from 8- to 32-fold in all bacteria. At the 1× MIC and 1/2× MIC, both oxacillin and nisin decreased significantly the expression of icaA gene in comparison with untreated control. When two antimicrobial agents were combined at 1/2× MIC concentration, the expression of icaA were significantly lower than when were used alone. Nisin/conventional oxacillin combination showed considerable anti-biofilm effects, including inhibition of biofilm formation, eradication of mature biofilm, and down-regulation of biofilm-related genes, proposing its applications for treating or preventing staphylococcal biofilm-associated infections, including device-related infections.

Partnering essential oils with antibiotics: proven therapies against bovine Staphylococcus aureus mastitis.

Introduction: There is an urgent need to develop therapeutic options for biofilm-producing Staphylococcus aureus (S. aureus). Therefore, the renewed interest in essential oils (EOs), especially carvacrol, linalool and eugenol, has attracted the attention of our research group. Methods: Multidrug resistance and multivirulence profiles in addition to biofilm production of S. aureus strains isolated from cows with mastitis were evaluated using both phenotypic and genotypic methods. The antimicrobial and antibiofilm activities of EOs were tested using both in vitro and molecular docking studies. Moreover, the interactions between commonly used antibiotics and the tested EOs were detected using the checkerboard method. Results: We found that all our isolates (n= 37) were biofilm methicillin resistant S. aureus (MRSA) producers and 40.5% were vancomycin resistant S. aureus (VRSA). Unfortunately, 73 and 43.2% of the recovered MRSA isolates showed multidrug resistant (MDR) and multivirulence patterns, respectively. The antimicrobial activities of the tested EOs matched with the phenotypic evaluation of the antibiofilm activities and molecular docking studies. Linalool showed the highest antimicrobial and antibiofilm activities, followed by carvacrol and eugenol EOs. Fortunately, synergistic interactions between the investigated EOs and methicillin or vancomycin were detected with fractional inhibitory concentration index (FICI) values ≤ 0.5. Moreover, the antimicrobial resistance patterns of 13 isolates changed to sensitive phenotypes after treatment with any of the investigated EOs. Treatment failure of bovine mastitis with resistant S. aureus can be avoided by combining the investigated EOs with available antimicrobial drugs. Conclusion: We hope that our findings can be translated into a formulation of new pharmaceutical dosage forms against biofilm-producing S. aureus pathogens.

Molecular Epidemiology and Virulence Gene Analysis of Methicillin-Resistant Staphylococcus aureus Associated with Skin and Soft Tissue Infections in the Shaoxing Region.

Objective: This study aimed to investigate the prevalence, molecular types, and virulence genes of methicillin-resistant Staphylococcus aureus (MRSA) causing skin and soft tissue infections (SSTIs) in the Shaoxing region. Methods: MRSA strains were collected from patients with SSTIs in Shaoxing People's Hospital from January 2019 to December 2019. We conducted SCCmec typing, Staphylococcus protein A (SPA) typing, multilocus sequence typing (MLST), and virulence gene analysis using whole-genome sequencing on all MRSA strains. Results: The detection rate of community-acquired MRSA (CA-MRSA) isolated from SSTI patients in our hospital was 33.3% (6/18). The primary SCCmec types of CA-MRSA strains were IV and V, with IVg(2B) and V(5C2&5) accounting for 16.7% each. Hospital-acquired MRSA (HA-MRSA) strains primarily exhibited SCCmec types IVa(2B) (25.0%), followed by II(2A) (16.7%), V(5C2) (16.7%), and V(5C2&5) (8.3%). SPA typing indicated that CA-MRSA strains causing SSTIs were predominantly t437 (14.3%), t034 (14.3%), t309 (14.3%), t4549 (14.3%), and t7637 (14.3%). The primary SPA type of HA-MRSA strains was t311 (16.7%). MLST typing revealed that the main sequence types (STs) of CA-MRSA strains causing SSTIs were ST22 (33.3%), followed by ST398, ST59, ST88, and ST630, each accounting for 16.7%. The principal STs of HA-MRSA strains were ST398 (16.7%), ST59 (16.7%), ST88 (16.7%), and ST5 (16.7%), followed by ST22, ST630, ST6, and ST188, each at 8.3%. The primary clones of CA-MRSA strains causing SSTIs were ST59-t437-IVg(2B) (16.7%) and ST630-t4549-V(5C2&5) (16.7%), while the primary clones of HA-MRSA strains were ST59-t437-IVa(2B), ST630-t4549-V(5C2&5), ST6-t304-IVa(2B), ST5-t311-II(2A), ST59-t172-IVa(2B), ST398-t571-V(5C2), ST398-t034-V(5C2), and ST5-t311-II(2A), each accounting for 8.3%. The detection rate of the lukSF-PV virulence gene was higher in CA-MRSA strains (50.0%) than in HA-MRSA strains (16.7%). Conclusions: The isolation rate of CA-MRSA strains causing SSTIs was high in Shaoxing People's Hospital, with ST59-t437-IVg(2B) and ST630-t4549-V(5C2&5) being the predominant clones. MRSA strains exhibited multiple virulence genes, with the lukSF-PV gene having a higher detection rate in CA-MRSA strains, signifying its importance as a virulence factor in CA-MRSA.

Targeting and arginine-driven synergizing photodynamic therapy with nutritional immunotherapy nanosystems for combating MRSA biofilms.

The resistance and immune escape of methicillin-resistant Staphylococcus aureus (MRSA) biofilms cause recalcitrant infections. Here, we design a targeting and synergizing cascade PDT with nutritional immunotherapy nanosystems (Arg-PCN@Gel) containing PCN-224 as PDT platform for providing reactive oxygen species (ROS), incorporating arginine (Arg) as nitric oxide (NO) donor to cascade with ROS to produce more lethal ONOO- and promote immune response, and coating with gelatin as targeting agent and persistent Arg provider. The nanosystems adhered to the autolysin of MRSA and inhibited Arg metabolism by down-regulating icdA and icaA. It suppressed polysaccharide intercellular adhesin and extracellular DNA synthesis to prevent biofilm formation. The NO broke mature biofilms and helped ROS and ONOO- penetrate into biofilms to inactivate internal MRSA. Arg-PCN@Gel drove Arg to enhance immunity via inducible NO synthase/NO axis and arginase/polyamine axis and achieve efficient target treatment in MRSA biofilm infections. The targeting and cascading PDT synergized with nutritional immunotherapy provide an effective promising strategy for biofilm-associated infections.

Genome analysis of secondary metabolite­biosynthetic gene clusters of Photorhabdus akhurstii subsp. akhurstii and its antibacterial activity against antibiotic-resistant bacteria.

Xenorhabdus and Photorhabdus can produce a variety of secondary metabolites with broad spectrum bioactivity against microorganisms. We investigated the antibacterial activity of Xenorhabdus and Photorhabdus against 15 antibiotic-resistant bacteria strains. Photorhabdus extracts had strong inhibitory the growth of Methicillin-resistant Staphylococcus aureus (MRSA) by disk diffusion. The P. akhurstii s subsp. akhurstii (bNN168.5_TH) extract showed lower minimum inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The interaction between either P. akhurstii subsp. akhurstii (bNN141.3_TH) or P. akhurstii subsp. akhurstii (bNN168.5_TH) or P. hainanensis (bNN163.3_TH) extract in combination with oxacillin determined by checkerboard assay exhibited partially synergistic interaction with fractional inhibitory concentration index (FICI) of 0.53. Time-killing assay for P. akhurstii subsp. akhurstii (bNN168.5_TH) extract against S. aureus strain PB36 significantly decreased cell viability from 105 CFU/ml to 103 CFU/ml within 30 min (P < 0.001, t-test). Transmission electron microscopic investigation elucidated that the bNN168.5_TH extract caused treated S. aureus strain PB36 (MRSA) cell membrane damage. The biosynthetic gene clusters of the bNN168.5_TH contained non-ribosomal peptide synthetase cluster (NRPS), hybrid NRPS-type l polyketide synthase (PKS) and siderophore, which identified potentially interesting bioactive products: xenematide, luminmide, xenortide A-D, luminmycin A, putrebactin/avaroferrin and rhizomide A-C. This study demonstrates that bNN168.5_TH showed antibacterial activity by disrupting bacterial cytoplasmic membrane and the draft genome provided insights into the classes of bioactive products. This also provides a potential approach in developing a novel antibacterial agent.

Methicillin Resistant Staphylococci Isolated from Goats and Their Farm Environments in Saudi Arabia Genotypically Linked to Known Human Clinical Isolates: a Pilot Study.

We conducted a pilot whole genome sequencing (WGS) study to characterize the genotypes of nine methicillin resistant staphylococci (MRS) isolates recovered from goats and their farm environments in Eastern Province, Saudi Arabia, between November 2019 to August 2020. Seven out of nine isolates were methicillin resistant Staphylococcus aureus (MRSA), and two were methicillin resistant Staphylococcus epidermidis (MRSE). All MRSA isolates possessed genotypes previously identified to infect humans, including isolates harboring ST6-SCCmec IV-t304 (n = 4), ST5-SCCmec VI- t688 (n = 2) and ST5-SCCmec V-t311 (n = 1). 2 MRSA isolates possessed plasmids that were genetically similar to those identified in S. aureus isolates recovered from humans and poultry. In contrast, plasmids found in three MRSA isolates and one MRSE isolate were genetically similar to those recovered from humans. All MRSA isolates harbored the host innate modulate genes sak and scn previously associated with human infections. The genotypes of MRSE isolates were determined as ST35, a well-known zoonotic sequence type and ST153, which has been associated with humans. However, the MRSE isolates were untypeable due to extra ccr complexes identified in their SCCmec elements. Moreover, we identified in ST153 isolate SCCmec element also harbored the Arginine Catabolic Mobile Element (ACME) IV. All MRS isolates were phenotypically resistant to trimethoprim-sulfamethoxazole, an antibiotic for the decolonization of MRS. Three isolates carried antibiotic resistance genes in their SCCmec elements that were not previously described, including those encoding fusidic acid resistance (fusC) and trimethoprim resistance (dfrC) incorporated in the MRSA SCCmec VI. IMPORTANCE Our findings demonstrate a possible cross-transmission of methicillin resistant staphylococci between goats and their local environments and between goats and humans. Due to ever increasing resistance to multiple antibiotics, the burden of MRS has a significant impact on livestock farming, public health, and the economy worldwide. This study highlights that implementing a holistic approach to whole genome sequencing surveillance in livestock and farm environments would aid our understanding of the transmission of methicillin resistant staphylococci and, most importantly, allow us to implement appropriate infection control and hygiene practices.

Traditional Chinese Medicine Tanreqing Targets Both Cell Division and Virulence in Staphylococcus aureus.

Staphylococcus aureus has been recognized as an important human pathogen and poses a serious health threat worldwide. With the advent of antibiotic resistance, such as the increased number of methicillin-resistant Staphylococcus aureus (MRSA), there is an urgent need to develop new therapeutical agents. In this study, Chinese traditional medicine Tanreqing (TRQ) has been used as an alternative treating agent against MRSA and we aim to unravel the mode of action of TRQ underlying MRSA inhibition. TRQ treatment affected numerous gene expression as revealed by RNA-seq analysis. Meanwhile, TRQ targeted cell division to inhibit cell growth as shown by illumination microscopy. Besides, we confirmed that TRQ downregulates the expression of virulence factors such as hemolysin and autolysin. Finally, we used a murine model to demonstrate that TRQ efficiently reduces bacterial virulence. Altogether, we have proved TRQ formula to be an effective agent against S. aureus infections.

Multi-targeted metallo-ciprofloxacin derivatives rationally designed and developed to overcome antimicrobial resistance.

Antimicrobial resistance is a major global threat to human health due to the rise, spread and persistence of multi-drug-resistant bacteria or 'superbugs'. There is an urgent need to develop novel chemotherapeutics to overcome this overarching challenge. The authors derivatized a clinically used fluoroquinolone antibiotic ciprofloxacin (Cip), and complexed it to a copper phenanthrene framework. This resulted in the development of two novel metallo-antibiotics of general formula [Cu(N,N)(CipHA)]NO3 where N,N represents a phenanthrene ligand and CipHA represents a hydroxamic acid of Cip derivative. Comprehensive studies, including a detailed proteomic study in which Staphylococcus aureus cells were exposed to the complexes, were undertaken to gain an insight into their mode of action. These new complexes possess potent antibacterial activity against S. aureus and methicillin-resistant S. aureus. In addition, they were found to be well tolerated in vivo in Galleria mellonella larvae, which has both functional and structural similarities to the innate immune system of mammals. These findings suggest that proteins involved in virulence, pathogenesis, and the synthesis of nucleotides and DNA repair mechanisms are most affected. In addition, both complexes affected similar cell pathways when compared with clinically used Cip, including cationic antimicrobial peptide resistance. The Cu-DPPZ-CipHA (DPPZ = dipyrido[3,2-a:2',3'-c]phenazine) analogue also induces cell leakage, which leads to an altered proteome indicative of reduced virulence and increased stress.

Copper Resistance Promotes Fitness of Methicillin-Resistant Staphylococcus aureus during Urinary Tract Infection.

Urinary tract infection (UTI) is one of the most common infectious conditions affecting people in the United States and around the world. Our knowledge of the host-pathogen interaction during UTI caused by Gram-positive bacterial uropathogens is limited compared to that for Gram-negative pathogens. Here, we investigated whether copper and the primary copper-containing protein, ceruloplasmin, are mobilized to urine during naturally occurring UTI caused by Gram-positive uropathogens in patients. Next, we probed the role of copper resistance in the fitness of methicillin-resistant Staphylococcus aureus (MRSA) during experimental UTI in a murine model. Our findings demonstrate that urinary copper and ceruloplasmin content are elevated during UTI caused by Enterococcus faecalis, S. aureus, S. epidermidis, and S. saprophyticus. MRSA strains successfully colonize the urinary tract of female CBA mice with selective induction of inflammation in the kidneys but not the bladder. MRSA mutants lacking CopL, a copper-binding cell surface lipoprotein, and the ACME genomic region containing copL, exhibit decreased fitness in the mouse urinary tract compared to parental strains. Copper sensitivity assays, cell-associated copper and iron content, and bioavailability of iron during copper stress demonstrate that homeostasis of copper and iron is interlinked in S. aureus. Importantly, relative fitness of the MRSA mutant lacking the ACME region is further decreased in mice that receive supplemental copper compared to the parental strain. In summary, copper is mobilized to the urinary tract during UTI caused by Gram-positive pathogens, and copper resistance is a fitness factor for MRSA during UTI. IMPORTANCE Urinary tract infection (UTI) is an extremely common infectious condition affecting people throughout the world. Increasing antibiotic resistance in pathogens causing UTI threatens our ability to continue to treat patients in the clinics. Better understanding of the host-pathogen interface is critical for development of novel interventional strategies. Here, we sought to elucidate the role of copper in host-Staphylococcus aureus interaction during UTI. Our results reveal that copper is mobilized to the urine as a host response in patients with UTI. Our findings from the murine model of UTI demonstrate that copper resistance is involved in the fitness of methicillin-resistant S. aureus (MRSA) during interaction with the host. We also establish a critical link between adaptation to copper stress and iron homeostasis in S. aureus.

Eleutheroside K Isolated from Acanthopanax henryi (Oliv.) Harms Inhibits the Expression of Virulence-Related Exoproteins in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus (S.) aureus (MRSA) is a representative pathogen that produces numerous virulence factors involving manifold cytotoxins and exotoxins. The present study was designed to investigate the influence of Eleutheroside K (ETSK), a single compound isolated from the leaves of Acanthopanax (A.) henryi (Oliv.) Harms, on the exotoxins secreted by MRSA. The transcription and translation of the exotoxins (α-hemolysin and staphylococcal enterotoxins) related to virulence in S. aureus were determined via quantitative RT-PCR and western blot analysis. The effect of ETSK on the production of tumor necrosis factor (TNF)-α was evaluated using enzyme-linked immunosorbent assay. As a result, ETSK at sub-MIC concentrations could reduce the protein expression of α-hemolysin and enterotoxin, and the expression of genes that regulate virulence factors was also inhibited. In addition, the TNF-inducing activity of S. aureus was attenuated by ETSK in a dose-dependent manner. These results revealed that ETSK not only reduced the protein and gene expression levels of related exotoxins but also suppressed the ability of S. aureus to induce macrophages to release cytokines. This study indicated that the inhibition of MRSA infection by ETSK may be achieved by reducing the virulence of S. aureus and highlighted the potential of ETSK as an innovative strategy for the prevention and treatment of MRSA infections.

Transcriptome Analysis and Weighted Gene Co-expression Network Reveal Multitarget-Directed Antibacterial Mechanisms of Benzyl Isothiocyanate against Staphylococcus aureus.

Staphylococcus aureus can cause many diseases and has a strong tendency to develop resistance to multiple antibiotics. In this study, benzyl isothiocyanate (BITC) was shown to have an excellent inhibitory effect on S. aureus ATCC25923 and methicillin-resistant S. aureus strains, with a minimum inhibitory concentration of 10 µg/mL. Under a scanning electron microscope, shrinkage and lysis of the cellular envelope were observed when exposed to BITC, and a bactericidal mode of BITC against S. aureus was further confirmed through flow cytometry. Additionally, the RNA profiles of S. aureus cells exposed to BITC indicated a violent transcriptional response to BITC. Through Kyoto Encyclopedia of Genes and Genomes analysis, it was found that many pathways involving bacterial survival were significantly affected, such as RNA degradation, oxidative phosphorylation, arginine biosynthesis, and so forth. A gene co-expression network was constructed using weighted gene co-expression network analysis, and six biologically meaningful co-expression modules and 125 hub genes were identified from the network. Among them, EfeB, GroES, SmpB, and Lsp were possibly targeted by BITC, leading to the death of S. aureus. Our results indicated a great potential of BITC to be applied in food safety and pharmaceuticals, highlighting its multitarget-directed bactericidal effects on S. aureus.

In vitro efficacy of Azadirachta indica leaf extract against methicillin resistant Staphylococci isolated from skin infection.

In this cross-sectional study, the isolation and identification of Methicillin Resistant Staphylococcus aureus (MRSA) and Methicillin Resistant S. epidermidis (MRSE) was described from skin infections (n=100). Initial isolation was done by conventional procedures followed by amplification/ sequence analysis of 16S rRNA. Methicillin resistance was determined using cefoxitin discs and resistant isolates were screened for mec-A gene followed by Minimum Inhibitory Concentrations (MIC) determination of vancomycin. In second phase, we investigated extract of Azadirachta indica leaves using Fourier Transformed Infrared Spectroscopy (FTIR-Spectroscopy) and investigated in vitro activity. Initially, total of 28 Staphylococci were identified. 16S rRNA gene sequence confirmed S. aureus (22), S. epidermidis (3) and S. saprophyticus (3) isolates. Cefoxitin discs showed (7/22) MRSA, (3/3) (MRSE) and none of the methicillin resistant S. saprophyticus. MRSA and MRSE isolates showed presence of mec-A gene. However, all isolates were sensitive to vancomycin MIC (0.5-2µg/mL) and sensitive to Linezolid. FTIR-Spectroscopy of A. indica indicated the presence of azadirachtin and nimbolinin. The mean zone of inhibition was measured 14.23±1.37 and 13.66±0.70 against MRSA and MRSE isolates, respectively. Altogether, MRSA and MRSE is significant public health concern. However, vancomycin and linezolid were found effective and extract of A. indica showed in vitro effects.

Identification of a novel gene argJ involved in arginine biosynthesis critical for persister formation in Staphylococcus aureus.

Staphylococcus aureus can cause both acute and recurrent persistent infections such as peritonitis, endocarditis, abscesses, osteomyelitis, and chronic wound infections. Effective therapies to treat persistent disease are paramount. However, the mechanisms of S. aureus persistence are poorly understood. In this study, we performed a comprehensive and unbiased high-throughput mutant screen against a transposon-insertion mutant library of S. aureus USA300 and focused on the role of argJ encoding an acetyltransferase in the arginine biosynthesis pathway, whose transposon insertion caused a significant defect in persister formation using multiple drugs and stresses. Genetic complementation and arginine supplementation restored persistence in the argJ transposon insertion mutant while generation of mutations on the active site of the ArgJ protein caused a defect in persistence. Quantitative RT-PCR analysis showed that the genes encoded in the arg operon were over-expressed under drug stressed conditions and in stationary phase cultures. In addition, the argJ mutant had attenuated virulence in both mouse and C. elegans. Our studies identify a new mechanism of persistence mediated by arginine metabolism in S. aureus. These findings provide not only novel insights about the mechanisms of S. aureus persistence but also offer novel therapeutic targets that may help to develop more effective treatment of persistent S. aureus infections.

Eleucanainones A and B: Two Dimeric Structures from the Bulbs of Eleutherine americana with Anti-MRSA Activity.

Two naphthoquinone-derived heterodimers with unprecedented carbon skeletons, eleucanainones A (1) and B (2), were isolated from the bulbs of Eleutherine americana. Their structures were elucidated by comprehensive spectroscopic methods. The structures of 1 and 2 were determined to be the first examples of dibenzofuran- and naphthalenone-containing naphthoquinone dimers. Compound 1 exhibited significant anti-MRSA activity in vitro with minimum inhibitory concentration (MIC) values of 0.78 µg/mL by downregulation of basal expression of agrA, cidA, icaA and sarA in methicillin-resistant S. aureus (MRSA).

Interactions of Tea-Derived Catechin Gallates with Bacterial Pathogens.

Green tea-derived galloylated catechins have weak direct antibacterial activity against both Gram-positive and Gram-negative bacterial pathogens and are able to phenotypically transform, at moderate concentrations, methicillin-resistant Staphylococcus aureus (MRSA) clonal pathogens from full ß-lactam resistance (minimum inhibitory concentration 256-512 mg/L) to complete susceptibility (~1 mg/L). Reversible conversion to susceptibility follows intercalation of these compounds into the bacterial cytoplasmic membrane, eliciting dispersal of the proteins associated with continued cell wall peptidoglycan synthesis in the presence of ß-lactam antibiotics. The molecules penetrate deep within the hydrophobic core of the lipid palisade to force a reconfiguration of cytoplasmic membrane architecture. The catechin gallate-induced staphylococcal phenotype is complex, reflecting perturbation of an essential bacterial organelle, and includes prevention and inhibition of biofilm formation, disruption of secretion of virulence-related proteins, dissipation of halotolerance, cell wall thickening and cell aggregation and poor separation of daughter cells during cell division. These features are associated with the reduction of capacity of potential pathogens to cause lethal, difficult-to-treat infections and could, in combination with ß-lactam agents that have lost therapeutic efficacy due to the emergence of antibiotic resistance, form the basis of a new approach to the treatment of staphylococcal infections.

Baicalin suppress growth and virulence-related factors of methicillin-resistant Staphylococcus aureus in vitro and vivo.

A Staphylococcus aureus (S.aureus) was isolated from pigs suffered in pneumonia that can't be cured by antibiotic such as methicillin and vancomycin. It was demonstrated that baicalin, an active natural compound extracted from the traditional Chinese medicinal, possess antimicrobial activity. In the present study, we evaluate it efficacy in vitro and vivo against this isolated methicillin-resistant S.aureus (MRSA). Our findings demonstrated that baicalin can inhibit S. aureus growth in a dose-dependent manner and attenuate the biofilm formation. Scanning electron microscopies showed that cell membrane was damaged and accompany with contents leaks after treated with high concentration of baicalin. In addition, baicalin exerted inhibitory effects on the expression of S.aureus virulence-related factors. Moreover, baicalin treated mice had enhanced survival after a lethal dose of S.aureus infection compared with untreated mice. Simultaneously, the pathological tissue damage and bacterium burden were decrease in baicalin treated mice. These data demonstrated that baicalin displayed a high effectiveness in vitro and vivo against MRSA infection, suggesting that baicalin may potentially be used to treat MRSA infection.

Natural Flavones from Morus alba against Methicillin-Resistant Staphylococcus aureus via Targeting the Proton Motive Force and Membrane Permeability.

The emergence and rapid spread of methicillin-resistant Staphylococcus aureus (MRSA) critically requires alternative therapeutic options. New antibacterial drugs and strategies are urgently needed to combat MRSA-associated infections. Here, we investigated the antibacterial activity of flavones from Morus alba and the potential mode of action against MRSA. Kuwanon G, kuwanon H, mulberrin, and morusin displayed high efficiency in killing diverse MRSA isolates. On the basis of structure-activity analysis, the cyclohexene-phenyl ketones and isopentenyl groups were critical to increase the membrane permeability and to dissipate the proton motive force. Meanwhile, mechanistic studies further showed that kuwanon G displayed rapid bactericidal activity in vitrowith difficulty in developing drug resistance. Kuwanon G targeted phosphatidylglycerol and cardiolipin in the cytoplasmic membrane through the formation of hydrogen bonds and electrostatic interactions. Additionally, kuwanon G promoted wound healing in a mouse model of MRSA skin infection. In summary, these results indicate that flavones are promising lead compounds to treat MRSA-associated infections through disrupting the proton motive force and membrane permeability.

Lysozyme-Assisted Photothermal Eradication of Methicillin-Resistant Staphylococcus aureus Infection and Accelerated Tissue Repair with Natural Melanosome Nanostructures.

Patients often face the challenge of antibiotic-resistant bacterial infections and lengthy tissue reconstruction after surgery. Herein, human hair-melanosome derivatives (HHMs), comprising keratins and melanins, are developed using a simple "low-temperature alkali heat" method for potentially personalized therapy. The mulberry-shaped HHMs have an average width of ∼270 nm and an average length of ∼700 nm, and the negatively charged HHMs can absorb positively charged Lysozyme (Lyso) to form the HHMs-Lyso composites through electrostatic interaction. These naturally derived biodegradable nanostructures act as exogenous killers to eliminate methicillin-resistant Staphylococcus aureus (MRSA) infection with a high antibacterial efficacy (97.19 ± 2.39%) by synergistic action of photothermy and "Lyso-assisted anti-infection" in vivo. Additionally, HHMs also serve as endogenous regulators of collagen alpha chain proteins through the "protein digestion and absorption" signaling pathway to promote tissue reconstruction, which was confirmed by quantitative proteomic analysis in vivo. Notably, the 13 upregulated collagen alpha chain proteins in the extracellular matrix (ECM) after HHMs treatment demonstrated that keratin from HHMs in collagen-dependent regulatory processes serves as a notable contributor to augmented wound closure. The current paradigm of natural material-tissue interaction regulates the cell-ECM interaction by targeting cell signaling pathways to accelerate tissue repair. This work may provide insight into the protein-level pathways and the potential mechanisms involved in tissue repair.

Tokiinshi, a traditional Japanese medicine (Kampo), suppresses Panton-Valentine leukocidin production in the methicillin-resistant Staphylococcus aureus USA300 clone.

It is necessary to develop agents other than antimicrobials for the treatment of Staphylococcus aureus infections to prevent the emergence of antimicrobial-resistant strains. Particularly, anti-virulence agents against the Panton-Valentine leukocidin (PVL)-positive methicillin-resistant S. aureus (MRSA), USA300 clone, is desired due to its high pathogenicity. Here, we investigated the potential anti-virulence effect of Tokiinshi, which is a traditional Japanese medicine (Kampo) used for skin diseases, against the USA300 clone. A growth inhibition assay showed that a conventional dose (20 mg/ml) of Tokiinshi has bactericidal effects against the clinical USA300 clones. Notably, the growth inhibition effects of Tokiinshi against S. epidermidis strains, which are the major constituents of the skin microbiome, was a bacteriostatic effect. The data suggested that Tokiinshi is unlikely to affect skin flora of S. epidermidis. Furthermore, PVL production and the expression of its gene were significantly suppressed in the USA300 clone by a lower concentration (5 mg/ml) of Tokiinshi. This did not affect the number of viable bacteria. Moreover, Tokiinshi significantly suppressed the expression of the agrA gene, which regulates PVL gene expression. For the first time, our findings strongly suggest that Tokiinshi has the potential to attenuate the virulence of the USA300 clone by suppressing PVL production via agrA gene suppression.