Microbiome precision editing: Using PEG as a selective fermentation initiator against methicillin‐resistant Staphylococcus aureus

Recent creation of a Unified Microbiome Initiative (UMI) has the aim of understanding how microbes interact with each other and with us. When pathogenic Staphylococcus aureus infects the skin, the interplay between S. aureus and skin commensal bacteria occurs. Our previous data revealed that skin commensal bacteria can mediate fermentation against the growth of USA300, a community‐acquired methicillin‐resistant S. aureus MRSA. By using a fermentation process with solid media on a small scale, we define poly(ethylene glycol) dimethacrylate (PEG‐DMA) as a selective fermentation initiator which can specifically intensify the probiotic ability of skin commensal Staphylococcus epidermidis bacteria. At least five short‐chain fatty acids including acetic, butyric and propionic acids with anti‐USA300 activities are produced by PEG‐DMA fermentation of S. epidermidis. Furthermore, the S. epidermidis‐laden PEG‐DMA hydrogels effectively decolonized USA300 in skin wounds in mice. The PEG‐DMA and its derivatives may become novel biomaterials to specifically tailor the human skin microbiome against invading pathogens.     

A novel cysteine‐free venom peptide with strong antimicrobial activity against antibiotics‐resistant pathogens from the scorpion Opistophthalmus glabrifrons

Antibiotic‐resistant bacteria, such as methicillin‐resistant Staphylococcus aureus and vancomycin‐resistant Enterococcus, pose serious threat to human health. The outbreak of antibiotic‐resistant pathogens in recent years emphasizes once again the urgent need for the development of new antimicrobial agents. Here, we discovered a novel antimicrobial peptide from the scorpion Opistophthalmus glabrifrons, which was referred to as Opisin. Opisin consists of 19 amino acid residues without disulfide bridges. It is a cationic, amphipathic, and α‐helical molecule. Protein sequence homology search revealed that Opisin shares 42.1–5.3% sequence identities to the 17/18‐mer antimicrobial peptides from scorpions. Antimicrobial assay showed that Opisin is able to potently inhibit the growth of the tested Gram‐positive bacteria with the minimal inhibitory concentration (MIC) values of 4.0–10.0 μM; in contrast, it possesses much lower activity against the tested Gram‐negative bacteria and a fungus. It is interesting to see that Opisin is able to strongly inhibit the growth of methicillin‐ and vancomycin‐resistant pathogens with the MICs ranging from 2.0 to 4.0 μM and from 4.0 to 6.0 μM, respectively. We found that at a concentration of 5 × MIC, Opisin completely killed all the cultured methicillin‐resistant Staphylococcus aureus. These results suggest that Opisin is a promising therapeutic candidate for the treatment of the antibiotic‐resistant bacterial infections. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Public transport as a reservoir of methicillin‐resistant staphylococci

Aims: The aim of this study was to explore the occurrence of methicillin‐resistant staphylococci in a large urban public transport system. Methods and Results: Samples were taken from hand rails, which passengers hold onto when they are standing. In total, 1400 swabs taken from 55 vehicles (trolleybuses, trams and buses) were examined. As many as 30·1% samples were positive for the presence of methicillin‐resistant coagulase‐negative staphylococci (MRCoNS), but none for methicillin‐resistant Staphylococcus aureus (MRSA). MRCoNS were isolated from all 55 vehicles. Nearly 50% of MRCoNS isolates displayed resistance not only to beta‐lactams, but at least to two or more other classes of antimicrobials as well. Conclusions: This study demonstrated widespread occurrence of MRCoNS on hand rails in public transport vehicles. MRSA was not detected. Significance and Impact of the Study: The recovery of methicillin‐resistant staphylococci from public transport system implies a potential risk for transmission of these bacteria in an out‐hospital environment.     

Sesquiterpenoids with Anti‐MDR Staphylococcus aureus Activities from Ferula ferulioides

Seven new sesquiterpenoids together with 21 known sesquiterpenoid derivatives were isolated from the medicinal plant Ferula ferulioides (Steud .) Korovin . Their structures were elucidated by comprehensive spectroscopic analyses and chemical transformations. The isolated compounds were evaluated for their antibacterial activities against a panel of bacteria including multidrug‐resistant (MDR) and methicillin‐resistant Staphylococcus aureus (MRSA), displaying minimum inhibitory concentration (MIC) values in the range of 0.5–128 mg/l.     

Structure and Antibacterial Activity of Brominated Diterpenes from the Red Alga Sphaerococcus coronopifolius

Four new tetracyclic brominated diterpenes, 1 – 4 , were isolated from the organic extract of Sphaerococcus coronopifolius, collected from the rocky coasts of Corfu Island. The structures of the new natural products, as well as their relative configurations, were elucidated on the basis of extensive spectral analyses, including 2D‐NMR experiments. The isolated metabolites were evaluated for their antibacterial activity against a panel of bacteria including multidrug‐resistant (MDR) and methicillin‐resistant Staphylococcus aureus (MRSA) with MIC values in the range of 16–128 μg/ml.     

Acute necrotic stomatitis (noma) associated with methicillin-resistant Staphylococcus aureus infection in a newly acquired rhesus macaque (Macaca mulatta)

Backgroud A newly acquired rhesus macaque was suffering from rapid destruction of the left cheek caused by necrotizing stomatitis. Methods To restore reconstructive surgery and intensive care with antibiotics, wound protection, wound healing agents, and debridement were applied. Results Staphylococcus aureus and Enterococcus faecalis were isolated from the culture of the lesion, and the antibiotic susceptibility test revealed methicillin‐resistant Staphylococcus aureus infection. Vancomycin and ampicillin‐sulbactam effectively treated the bacterial infections, and reconstructive surgery was performed once the infection was cleared. Topical application of recombinant human epidermal growth factor (rhEGF) was useful to treat exposed wound of the noma lesion. Conclusions Simian noma associated with methicillin‐resistant Staphylococcus aureus (MRSA) had not previously been reported in non‐human primates. Although noma associated with MRSA is hard to cure because of its rapid and destructive progress, the aggressive therapy used in this study led to the successful resolution of an acute necrotic stomatitis lesion in a rhesus macaque.     

Ioniols I and II,Tetracyclic Diterpenes with Antibacterial Activity,from Sphaerococcus coronopifolius

Two naturally occurring diterpenes featuring unprecedented tetracyclic skeletons, ioniols I and II ( 1 and 2 , resp.), along with two previously reported metabolites 3 and 4 , were isolated from the organic extract of Sphaerococcus coronopifolius collected from the rocky coasts of Corfu island in the Ionian Sea. The structures of the new natural products, as well as their relative configuration, were elucidated on the basis of extensive spectral analysis, including 2D‐NMR experiments. The isolated metabolites were evaluated for their antibacterial activity against a panel of Staphylococcus aureus strains, which included multidrug‐resistant (MDR) and methicillin‐resistant Staphylococcus aureus (MRSA) strains.     

Volatile Oils from the Aerial Parts of Eremophila maculata and Their Antimicrobial Activity

The essential oils isolated from the fresh flowers, fresh leaves, and both fresh and air‐dried stems of Eremophila maculata (Scrophulariaceae) were characterized by GC‐FID and GC/MS analyses. Sabinene was the major component in most of the oils, followed by limonene, α‐pinene, benzaldehyde, (Z)‐β‐ocimene, and spathulenol. The leaf and flower essential oils showed antibacterial and antifungal activity against five Gram‐positive and four Gram‐negative bacterial strains, multi‐resistant clinical isolates from patients, i.e., methicillin‐resistant Staphylococcus aureus (MRSA), as well as two yeasts. Minimum inhibitory concentrations (MICs) and minimum microbicidal concentrations (MMCs) were between 0.25 and 4 mg/ml.     

Norfloxacin and ursolic acid: in vitro association and postantibiotic effect against Staphylococcus aureus

Aims: We investigated the effectiveness in vitro of the association between norfloxacin (NOR) and ursolic acid (UA) against Staphylococcus aureus. Methods and Results: The minimal inhibitory concentrations (MICs), the minimal bactericidal concentrations, the bacterial killing and the postantibiotic effect (PAE) of NOR and UA were determined both singly and in combination. A synergistic interaction was observed against Staph. aureus ATCC 29213: the mean PAEs were 3 h for NOR, ?1·2 h for UA (1 × MIC) and 2·0 h for UA (2 × MIC). Synergism was observed with longer PAEs and postantibiotic sub‐MIC effects after NOR/UA exposure. UA was also active against clinical isolates and methicillin‐resistant Staph. aureus. Conclusions: The application of antimicrobial combinations may address the rising resistance to established classes of both systemic and topical agents. Significance and Impact of the Study: In vitro interactions between NOR and UA may contribute to the development of novel topical agents for the treatment of skin infections as well as for topical formulations.     

Surface disinfection properties of the combination of an antimicrobial peptide,ranalexin, with an endopeptidase,lysostaphin, against methicillin‐resistant Staphylococcus aureus (MRSA)

Aims: To characterize the antibacterial synergy of the antimicrobial peptide, ranalexin, used in combination with the anti‐staphylococcal endopeptidase, lysostaphin, against methicillin‐resistant Staphylococcus aureus (MRSA), and to assess the combination’s potential as a topical disinfectant or decolonizing agent for MRSA. MRSA causes potentially lethal infections, and pre‐operative patients colonized with MRSA are often treated with chlorhexidine digluconate and mupirocin cream to eradicate carriage. However, chlorhexidine is unsuitable for some patients, and mupirocin resistance is increasingly encountered, indicating new agents are required. Methods and Results: Using an ex vivo assay, ranalexin and lysostaphin tested in combination reduced viable MRSA on human skin to a greater extent than either compound individually. The combination killed bacteria within 5 min and remained effective and synergistic even in high salt and low pH conditions. Conclusions: The combination is active against MRSA on human skin and under conditions that may be encountered in sweat. Significance and Impact of the Study: Although the exact mechanism of activity remains unresolved, considering its specific spectrum of activity, fast killing kinetics and low likelihood of resistance arising, the combination of ranalexin with lysostaphin warrants consideration as a new agent to eradicate nasal and skin carriage of Staph. aureus, including MRSA.     

Synergistic activity of 1-(1-naphthylmethyl)-piperazine with ciprofloxacin against clinically resistant Staphylococcus aureus, as determined by different methods

Aims: To evaluate the interaction of 1‐(1‐naphthylmethyl)‐piperazine (NMP) and ciprofloxacin (CPFX) in vitro against fluoroquinolone (FQ)‐resistant clinical isolates of methicillin‐resistant Staphylococcus aureus (MRSA). Methods and Results: The in vitro interaction of NMP and CPFX in 12 FQ‐resistant clinical isolates of MRSA was assessed using a checkerboard microdilution method. In the study, a synergistic antimicrobial effect between NMP and CPFX was observed in all 12 FQ‐resistant strains tested, as determined by the fractional inhibitory concentration index (FICI), and in 10 strains using ΔE models. No antagonistic activity was observed in any of the strains tested. These positive interactions were also confirmed using the time–killing test and agar diffusion assay for the selected strain, MRSA 1862; synergistic activity was observed when NMP was combined with the first‐line antimicrobial agent CPFX against Staph. aureus. Conclusions: Synergistic activity between NMP and CPFX against clinical isolates of FQ‐resistant Staph. aureus was observed in vitro. Significance and Impact of the Study: This report might provide alternative methods to reduce the resistance of Staph. aureus to CPFX.     

Effects of Piper cubeba L. essential oil on methicillin‐resistant Staphylococcus aureus: an AFM and TEM study

The increasing prevalence of antibiotic‐resistant bacteria is creating a real challenge for health care systems worldwide, making the development of novel antibiotics a necessity. In addition to the development of new antibiotics, there is an urgent need for in‐depth characterization of the mechanisms of bacterial resistance toward new drugs. Here, we used essential oils extracted in our laboratory from Piper cubeba against methicillin‐resistant Staphylococcus aureus ATCC 43300, one of the most prominent antibiotic‐resistant bacteria. Effects of the essential oils extracted from P cubeba on bacteria were mainly evaluated using 2 powerful microscopy techniques: atomic force microscopy and transmission electron microscopy. High‐resolution atomic force microscopy images of the cells were obtained close to their native environment by immobilizing the cells on porous Polyether sulfone membranes, which were prepared in our laboratory with a wide range and distribution of pore sizes and depth. Inhibition zones (mm) and minimum inhibitory concentrations were determined. Two different concentrations of the oil were used to treat the cells: 50 μg/mL minimum inhibitory concentration and 25 μg/mL. The 50 μg/mL oil solution caused severe damage to the bacterial cells at microscopic levels while the 25 μg/mL solution showed no effects compared to the control. However, at nanoscopic levels, the 25 μg/mL oil solution caused significant changes in the cell wall, which could potentially impair bacterial activities. These results were also confirmed by transmission electron microscopy micrographs. Our results indicate that the extract has a good biological activity against methicillin‐ and oxacillin‐resistant S aureus and that it acts on the cell wall and plasma (cytoplasmic) membrane.     

Detection and quantification of methicillin‐resistant Staphylococcus aureus (MRSA) clones in retail meat products

Aims: The objective of the study was to determine the prevalence of methicillin‐resistant Staphylococcus aureus (MRSA) contamination of retail meat and to determine the level of contamination. Methods and Results: Pork (pork chops and ground pork), ground beef and chicken (legs, wings and thighs) were purchased at retail outlets in four Canadian provinces and tested for the presence of methicillin‐resistant Staph. aureus using qualitative and quantitative methods. MRSA was isolated from 9·6% of pork, 5·6% of beef and 1·2% of chicken samples (P = 0·0002). Low levels of MRSA were typically present, with 37% below the detection threshold for quantification and <100 CFU g^?1 present in most quantifiable samples. All isolates were classified as Canadian epidemic MRSA‐2 (CMRSA‐2) by pulsed field gel electrophoresis (PFGE), with two different PFGE subtypes, and were spa type 24/t242. Conclusions: MRSA contamination of retail meat is not uncommon. While CMRSA‐2, a human epidemic clone, has been found in pigs in Canada, the lack of isolation of livestock‐associated ST398 was surprising. Significance and Impact of the Study: The relevance of MRSA contamination of meat is unclear but investigation is required because of the potential for exposure from food handling. Sources of contamination require investigation because these results suggest that human or animal sources could be involved.     

Anti-biofilm potential of phenolic acids: the influence of environmental pH and intrinsic physico-chemical properties

Phenolic acids are a particular group of small phenolic compounds which have exhibited some anti-biofilm activity, although the link between their activity and their intrinsic pH is not clear. Therefore, the present work examined the anti-biofilm activity (inhibition of biomass and metabolic activity) of phenolic acids in relation to the environmental pH, as well as other physico-chemical properties. The results indicate that, while Escherichia coli was not inhibited by the phenolic acids, both methicillin resistant Staphylococcus aureus and methicillin resistant Staphylococcus epidermidis were susceptible to the action of all phenolic acids, with the pH playing a relevant role in the activity: a neutral pH favored MRSE inhibition, while acidic conditions favored MRSA inhibition. Some links between molecular polarity and size were associated only with their potential as metabolic inhibitors, with the overall interactions hinting at a membrane-based mechanism for MRSA and a cytoplasmic effect for MRSE.     

Anti‐biofilm activity of ultrashort cinnamic acid peptide derivatives against medical device‐related pathogens

The threat of antimicrobial resistance has placed increasing emphasis on the development of innovative approaches to eradicate multidrug‐resistant pathogens. Biofilm‐forming microorganisms, for example, Staphylococcus epidermidis and Staphylococcus aureus, are responsible for increased incidence of biomaterial infection, extended hospital stays and patient morbidity and mortality. This paper highlights the potential of ultrashort tetra‐peptide conjugated to hydrophobic cinnamic acid derivatives. These peptidomimetic molecules demonstrate selective and highly potent activity against resistant biofilm forms of Gram‐positive medical device‐related pathogens. 3‐(4‐Hydroxyphenyl)propionic)‐Orn‐Orn‐Trp‐Trp‐NH₂ displays particular promise with minimum biofilm eradication concentration (MBEC) values of 125 µg/ml against methicillin sensitive (ATCC 29213) and resistant (ATCC 43300) S. aureus and activity shown against biofilm forms of Escherichia coli (MBEC: 1000 µg/ml). Kill kinetics confirms complete eradication of established 24‐h biofilms at MBEC with 6‐h exposure. Reduced cell cytotoxicity, relative to Gram‐positive pathogens, was proven via tissue culture (HaCaT) and haemolysis assays (equine erythrocytes). Existing in nature as part of the immune response, antimicrobial peptides display great promise for exploitation by the pharmaceutical industry in order to increase the library of available therapeutic molecules. Ultrashort variants are particularly promising for translation as clinical therapeutics as they are more cost‐effective, easier to synthesise and can be tailored to specific functional requirements based on the primary sequence allowing factors such as spectrum of activity to be varied. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Detection of methicillin-resistant Staphylococcus pseudintermedius with commercially available selective media

Aims: Commercially available selective media for methicillin‐resistant Staphylococcus aureus (MRSA) were tested for the detection and isolation of methicillin‐resistant Staphylococcus pseudintermedius (MRSP). Methods and Results: Five different screening agars [mannitol salt agar with oxacillin and BD BBL? Chromagar? MRSA (BD Diagnostics); chromID? MRSA agar (bioMérieux); Oxacillin resistance screening agar base (ORSAB); and Brilliance MRSA agar (Oxoid)] were analysed for the detection of MRSP. Bacteria that may be isolated together with MRSP and may grow on the screening agars were included in the study to determine possible interference with the growth of MRSP. MRSP grew well on all selective media except on BD BBL? Chromagar? MRSA (BD Diagnostics) and chromID? MRSA agar (bioMérieux), on which a low to moderate growth rate was noted. Conclusions: ORSAB (Oxoid) and Brilliance MRSA agar (Oxoid) are most suitable for the detection and isolation of MRSP from clinical material. Significance and Impact of the Study: The importance of MRSP in veterinary medicine is increasing. Diagnostic systems are needed to detect MRSP carrier as soon as possible. This study provides information about selected MRSA screening agars for the detection of MRSP to the clinical microbiologists.     

Anti-biofilm activity of Quercus infectoria G. Olivier against methicillin-resistant Staphylococcus aureus

Aims: To establish the effect of Quercus infectoria G. Olivier extract and its main constituent, tannic acid, on staphylococcal biofilm and their anti‐biofilm mechanisms. Methods and Results: Anti‐biofilm activity of the plant materials on clinical isolated of methicillin‐resistant Staphylococcus aureus and methicillin‐susceptible Staph. aureus was employed using a crystal violet‐stained microtiter plate method. The extract at minimum inhibitory concentration (MIC; 0·25 mg ml^?1) was significantly reduced the biofilm formation of the isolates (P < 0·05). The effect on staphylococcal cell surface hydrophobicity (CSH) of the test compounds was investigated as a possible mode of action of the anti‐biofilm activity. The hydrophobicity index of all the bacterial isolates increased following treatment with supra‐MIC, MIC and sub‐MIC of the extract and tannic acid. Observation of the treated bacterial cells by electron microscopy revealed that the test compounds caused clumps of partly divided cocci with thickened and slightly rough cell wall. Conclusions: The results indicated that Q. infectoria extract and tannic acid affected staphylococcal biofilm formation and their effect on bacterial CSH and cell wall may involve in the anti‐biofilm activity. Significance and Impact of the Study: This evidence highlighted the anti‐biofilm potency of the natural products and clarified their anti‐biofilm mechanisms.     

Aureonitols A and B,Two New C13‐Polyketides from Chaetomium globosum,an Endophytic Fungus in Salvia miltiorrhiza

Two new C₁₃‐polyketides, aureonitols A and B ( 1 and 2 ), along with five known compounds ( 3 – 7 ), were isolated from the solid fermentation culture of the plant endophytic fungus Chaetomium globosum from the aerial parts of Salvia miltiorrhiza. The structures and absolute configurations of 1 and 2 were determined by comprehensive spectroscopic data analysis and computed methods. Compound 5 was found to display the remarkable antimicrobial activities against four multidrug‐resistant bacteria (Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, and Staphylococcus epidermidis) with MIC values of 3.13–6.25 μg/mL (ciprofloxacin: 0.78–1.56 μg/mL), and also against all tested fungal strains with MIC values of 3.13–25 μg/mL (ketoconazole: 0.78–12.50 μg/mL).     

Antibacterial activity of hinokitiol against both antibiotic‐resistant and ‐susceptible pathogenic bacteria that predominate in the oral cavity and upper airways

Hinokitiol, a component of the essential oil isolated from Cupressaceae, possesses antibacterial and antifungal activities and has been used in oral care products. In this study, the antibacterial activities of hinokitiol toward various oral, nasal and nasopharyngeal pathogenic bacteria, including Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, methicillin‐resistant and ‐susceptible Staphylococcus aureus, antibiotic‐resistant and ‐susceptible Streptococcus pneumoniae, and Streptococcus pyogenes were examined. Growth of all these bacterial strains was significantly inhibited by hinokitiol, minimal inhibitory concentrations of hinokitiol against S. mutans, S. sobrinus, P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, methicillin‐resistant S. aureus, methicillin‐susceptible S. aureus, antibiotic‐resistant S. pneumoniae isolates, antibiotic‐susceptible S. pneumoniae, and S. pyogenes being 0.3, 1.0, 1.0, 30, 0.5, 50, 50, 30, 0.3–1.0, 0.5, and 0.3 μg/mL, respectively. Additionally, with the exception of P. gingivalis, hinokitiol exerted bactericidal effects against all bacterial strains 1 hr after exposure. Hinokitiol did not display any significant cytotoxicity toward the human gingival epithelial cell line Ca9‐22, pharyngeal epithelial cell line Detroit 562, human umbilical vein endothelial cells, or human gingival fibroblasts, with the exception of treatment with 500 μg/mL hinokitiol, which decreased numbers of viable Ca9‐22 cells and gingival fibroblasts by 13% and 12%, respectively. These results suggest that hinokitiol exhibits antibacterial activity against a broad spectrum of pathogenic bacteria and has low cytotoxicity towards human epithelial cells.