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Patients coinfected with respiratory syncytial virus (RSV) and bacteria have longer hospital stays, higher risk of intensive care unit admission, and worse outcomes. We describe a model of RSV line 19F/methicillin-resistant Staphylococcus aureus (MRSA) USA300 coinfection that does not impair viral clearance, but prior RSV infection enhances USA300 MRSA bacterial growth in the lung. The increased bacterial burden post-RSV correlates with reduced accumulation of neutrophils and impaired bacterial killing by alveolar macrophages. Surprisingly, reduced neutrophil accumulation is likely not explained by reductions in phagocyte-recruiting chemokines or alterations in proinflammatory cytokine production compared with mice infected with S. aureus alone. Neutrophils from RSV-infected mice retain their ability to migrate toward chemokine signals, and neutrophils from the RSV-infected lung are better able to phagocytize and kill S. aureus ex vivo on a per cell basis. In contrast, while alveolar macrophages could ingest USA300 post-RSV, intracellular bacterial killing was impaired. The RSV/ S. aureus coinfected lung promotes a state of overactivation in neutrophils, demonstrated by increased production of reactive oxygen species (ROS) that can drive formation of neutrophil extracellular traps (NETs), resulting in cell death. Mice with RSV/ S. aureus coinfection had increased extracellular DNA and protein in bronchoalveolar lavage fluid and histological evidence confirmed NETosis in vivo . Taken together, these data highlight that prior RSV infection can prime the overactivation of neutrophils leading to cell death that impairs neutrophil accumulation in the lung. Additionally, alveolar macrophage killing of bacteria is impaired post-RSV. Together, these defects enhance USA300 MRSA bacterial growth in the lung post-RSV., Competing Interests: The authors declare no conflict of interest.

The growth of pathogenic bacteria in moist and wet surfaces and tubing of medically relevant devices results in serious infections in immunocompromised patients. In this study, we investigated and demonstrated the successful implementation of a UV-C side-emitting optical fiber in disinfecting medically relevant pathogenic bacteria ( Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus [MRSA]) within tight channels of polytetrafluoroethylene (PTFE). PTFE is a commonly used material both in point-of-use (POU) water treatment technologies and medical devices (dental unit water line [DUWL], endoscope). For a 1-m-long PTFE channel, up to ≥6 log inactivation was achieved using a 1-m-long UV side-emitting optical fiber (SEOF) with continuous 16-h exposure of low UV-C radiation ranging from ~0.23 to ~29.30 μW/cm 2 . Furthermore, a linear model was used to calculate the inhibition zone constant (k`), which enables us to establish a correlation between UV dosage and the extent of inactivated surface area (cm 2 ) for surface-bound Escherichia coli on a nutrient-rich medium. The k` value for an irradiance ranging from ~150 to ~271.50 μW/cm 2 was calculated to be 0.564 ± 0.6 cm·cm 2 /mJ. This study demonstrated the efficacy of SEOFs for disinfection of medically relevant microorganisms present in medically and domestically relevant tight channels. The impact of the results in this study extends to the optimization of operational efficiency in pre-existing UV surface disinfection setups that currently operate at UV dosages exceeding the optimal levels.IMPORTANCEGermicidal UV radiation has gained global recognition for its effectiveness in water and surface disinfection. Recently, various works have illustrated the benefit of using UV-C side-emitting optical fibers (SEOFs) for the disinfection of tight polytetrafluoroethylene (PTFE) channels. This study now demonstrates its impact for disinfection of medically relevant organisms and introduces critical design calculations needed for its implementation. The flexible geometry and controlled emission of light in these UV-SEOFs make them ideal for light distribution in tight channels. Moreover, the results presented in this manuscript provide a novel framework that can be employed in various applications, addressing microbial contamination and the disinfection of tight channels., Competing Interests: The authors declare that Dr. Mariana Lanzarini-Lopes and Dr. Katrina Fitzpatrick are co-founders and majority owners of Optical Waters Inc, the company from which the Optostrand was purchased for use in this study.

Background: Infectious diseases represent a significant global strain on public health security and impact on socio-economic stability all over the world. The increasing resistance to the current antimicrobial treatment has resulted in the crucial need for the discovery and development of novel entities for the infectious treatment with different modes of action that could target both sensitive and resistant strains., Methods: Compounds were synthesized using the classical organic chemistry methods. Prediction of biological activity spectra was carried out using PASS and PASS-based web applications. Pharmacophore modeling in LigandScout software was used for quantitative modeling of the antibacterial activity. Antimicrobial activity was evaluated using the microdilution method. AutoDock 4.2 ® software was used to elucidate probable bacterial and fungal molecular targets of the studied compounds., Results: All compounds exhibited better antibacterial potency than ampicillin against all bacteria tested. Three compounds were tested against resistant strains MRSA, P. aeruginosa and E. coli and were found to be more potent than MRSA than reference drugs. All compounds demonstrated a higher degree of antifungal activity than the reference drugs bifonazole (6-17-fold) and ketoconazole (13-52-fold). Three of the most active compounds could be considered for further development of the new, more potent antimicrobial agents., Conclusion: Compounds 5b (Z)-3-(3-hydroxyphenyl)-5-((1-methyl-1 H -indol-3-yl)methylene)-2-thioxothiazolidin-4-one and 5g (Z)-3-[5-(1 H -Indol-3-ylmethylene)-4-oxo-2-thioxo-thiazolidin-3-yl]-benzoic acid as well as 5h (Z)-3-(5-((5-methoxy-1 H -indol-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)benzoic acid can be considered as lead compounds for further development of more potent and safe antibacterial and antifungal agents.

There is a need for new antimicrobial systems due to increased global resistance to current antimicrobials. Pomegranate rind extract (PRE) and Zn (II) ions both possess a level of antimicrobial activity and work has previously shown that PRE/Zn (II) in combination possesses synergistic activity against Herpes simplex virus and Micrococcus luteus . Here, we determined whether such synergistic activity extended to other, more pathogenic, bacteria. Reference strains of methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis , Escherichia coli , and Pseudomonas aeruginosa were cultured and subjected to challenge by PRE, Zn (II), or PRE + Zn (II), in time-kill assays. Data were obtained independently by two researchers using different PRE preparations. Statistically significant synergistic activity for PRE + Zn (II) was shown for all four bacterial strains tested compared to untreated controls, although the extent of efficacy and timescales varied. Zn (II) exerted activity and at 1 h, it was not possible to distinguish with PRE + Zn (II) combination treatment in all cases. PRE alone showed low activity against all four bacteria. Reproducible synergistic bactericidal activity involving PRE and Zn (II) has been confirmed. Potential mechanisms are discussed. The development of a therapeutic system that possesses demonstrable antimicrobial activity is supported which lends itself particularly to topical delivery applications, for example MRSA infections.

Five series of novel carbazole derivatives containing an aminoguanidine, dihydrotriazine, thiosemicarbazide, semicarbazide or isonicotinic moiety were designed, synthesised and evaluated for their antimicrobial activities. Most of the compounds exhibited potent inhibitory activities towards different bacterial strains (including one multidrug-resistant clinical isolate) and one fungal strain with minimum inhibitory concentrations (MICs) between 0.5 and 16 µg/ml. Compounds 8f and 9d showed the most potent inhibitory activities (MICs of 0.5-2 µg/ml). Furthermore, compounds 8b , 8d , 8f , 8k , 9b and 9e with antimicrobial activities were not cytotoxic to human gastric cancer cell lines (SGC-7901 and AGS) or a normal human liver cell line (L-02). Structure-activity relationship analyses and docking studies implicated the dihydrotriazine group in increasing the antimicrobial potency and reducing the toxicity of the carbazole compounds. In vitro enzyme activity assays suggested that compound 8f binding to dihydrofolate reductase might account for the antimicrobial effect.

Purpose: The production of alternative novel antimicrobial agents is considered an efficient way to cope with multidrug resistance among pathogenic bacteria. E50-52 and Ib-AMP4 antimicrobial peptides (AMPs) have illustrated great proven antibacterial effects. The aim of this study was recombinant production of these AMPs and investigation of their synergistic effects on methicillin-resistant Staphylococcus aureus (MRSA)., Method: At first, the codon optimized sequences of the Ib-AMP4 (UniProt: 024006 (PRO_0000020721), and E50-52 (UniProtKB: P85148) were individually ligated into the pET-32α vector and transformed into E. coli. After the optimization of production and purification steps, the MIC (Minimum inhibitory concentration), time kill and growth kinetic tests of recombinant proteins were determined against MRSA. Finally, the in vivo wound healing efficiency was tested., Results and Conclusion: The recorded MIC of recombinant Trx-Ib-AMP4, Trx-E50-52 against MRSA bacterium were 0.375 and 0.0875 mg/mL respectively. The combination application of the produced AMPs by the checkerboard method confirmed their synergic activity. The results of the time-kill showed sharply decrease of the number of viable cells with over five time reductions in log 10  CFU/mL by the combination of Trx-E50-52 and Trx-IbAMP4 at 2 × MIC within 240 min. The growth kinetic results confirmed the combination of Trx-E50-52 and Trx-IbAMP4 had much greater success in the reduction of over 50 % of MRSA suspensions' turbidity within the first hour. Wound healing assay and histological analysis of infected mice treated with Trx-Ib-AMP4 or Trx-E50-52 compared with those treated with a combination of Trx-Ib-AMP4 and Trx-E50-52 showed significant synergic effects., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Nettle (Urtica dioica L), as a plant rich in biologically active compounds, is one of the most important plants used in herbal medicine. Studies have shown that this plant has antioxidant, antiplatelet, hypoglycemic and hypocholesterolemia effects. In this study, we characterized three Alternaria endophytic fungi isolated from their host U. dioica. We hypothesized that these endophytic fungi can produce new bioactive metabolites, which may possess the bioactive property with potential application in the medical and pharmaceutical industries. The antibacterial activity was evaluated against reference and isolated strains, including Methicillin-Resistant Staphylococcus aureus. A wide range of antimicrobial activities similar to those measured in nettle leaves was detected especially for Alternaria sorghi. Furthermore, the highest antioxidant activity detected with DPPH free radical scavenging was measured for A. sorghi and nettle leaves ethyl acetate extracts. In addition, whereas catalase activity was similar in the three isolated fungi and nettle leaves, total thiol content and superoxide dismutase activity were significantly higher in leaves. A. sorghi showed the best activities compared to other isolated fungi. The characterization and further production of bioactive compounds produced by this endophyte should be investigated to fight bacteria and especially those that develop drug multi-resistance.

Novel complexes of type [Cu(N-N)(dmtp) 2 (OH 2 )](ClO 4 ) 2 ·dmtp (( 1 ) N-N: 2,2'-bipyridine; ( 2 ) L: 1,10-phenantroline and dmtp: 5,7-dimethyl-1,2,4-triazolo[1,5- a ]pyrimidine) were designed in order to obtain biologically active compounds. Complexes were characterized as mononuclear species that crystallized in the space group P-1 of the triclinic system with a square pyramidal geometry around the copper (II). In addition to the antiproliferative effect on murine melanoma B16 cells, complex ( 1 ) exhibited low toxicity on normal BJ cells and did not affect membrane integrity. Complex ( 2 ) proved to be a more potent antimicrobial in comparison with ( 1 ), but both compounds were more active in comparison with dmtp-both against planktonic cells and biofilms. A stronger antimicrobial and antibiofilm effect was noticed against the Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA). Both electron paramagnetic resonance (EPR) and Saccharomyces cerevisiae studies indicated that the complexes were scavengers rather than reactive oxygen species promoters. Their DNA intercalating capacity was evidenced by modifications in both absorption and fluorescence spectra. Furthermore, both complexes exhibited nuclease-like activity, which increased in the presence of hydrogen peroxide.

Background: This study investigated the effects of terpinen-4-ol on methicillin-resistant Staphylococcus aureus (MRSA) and its biofilm, and the possible mechanisms governing this effect., Results: We observed that terpinen-4-ol has good antibacterial activity and inhibits the formation of MRSA biofilm. The MIC and MBC values for terpinen-4-ol against S. aureus were 0.08% ~ 0.32%. And terpinen-4-ol at 0.32% could kill all bacteria and clear all biofilms. Untargeted metabolomic and transcriptomic analyses showed that terpinen-4-ol strongly inhibited DNA and RNA biosynthesis in MRSA at 2 h after treatment by affecting genes and metabolites related to purine and pyrimidine metabolic pathways. Some differential genes which play important roles in DNA synthesis and the production of eDNA from biofilm exposed to terpinen-4-ol was also significantly decreased compared with that of the control., Conclusions: Terpinen-4-ol has good antibacterial activity and significantly inhibits the formation of MRSA biofilm by inhibiting purine and pyrimidine metabolism., (© 2021. The Author(s).)

Wounds resulting from surgeries, implantation of medical devices, and musculoskeletal trauma result in pain and can also result in infection of damaged tissue. Up to 80% of these infections are due to biofilm formation either on the surface of implanted devices or on surrounding wounded tissue. Bacteria within a biofilm have intrinsic growth and development characteristics that allow them to withstand up to 1,000 times the minimum inhibitory concentration of antibiotics, demonstrating the need for new therapeutics to prevent and treat these infections. Cis-2-decenoic acid (C2DA) is known to disperse preformed biofilms and can prevent biofilm formation entirely for some strains of bacteria. Additionally, local anesthetics like bupivacaine have been shown to have antimicrobial effects against multiple bacterial strains. This study sought to evaluate hexanoic acid-treated electrospun chitosan membranes (HA-ESCM) as wound dressings that release C2DA and bupivacaine to simultaneously prevent infection and alleviate pain associated with musculoskeletal trauma. Release profiles of both therapeutics were evaluated, and membranes were tested in vitro against Methicillin-resistant Staphylococcus aureus (MRSA) to determine efficacy in preventing biofilm infection and bacterial growth. Results indicate that membranes release both therapeutics for 72 hr, and release profile can be tailored by loading concentration. Membranes were effective in preventing biofilm growth but were toxic to fibroblasts when loaded with 2.5 or 5 mg of bupivacaine., (© 2021 Wiley Periodicals LLC.)

Understating how antibiotic tolerance impacts subsequent resistance development in the clinical setting is important to identifying effective therapeutic interventions and prevention measures. This study describes a patient case of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia which rapidly developed resistance to three primary MRSA therapies and identifies genetic and metabolic changes selected in vivo that are associated with rapid resistance evolution. Index blood cultures displayed susceptibility to all (non-beta-lactam) antibiotics with the exception of trimethoprim/ sulfamethoxazole. One month after initial presentation, during the same encounter, blood cultures were again positive for MRSA, now displaying intermediate resistance to vancomycin and ceftaroline and resistance to daptomycin. Two weeks later, blood cultures were positive for a third time, still intermediate resistant to vancomycin and ceftaroline and resistant to daptomycin. Mutations in mprF and vraT were common to all multidrug resistant isolates whereas mutations in tagH, agrB and saeR and secondary mprF mutation emerged sequentially and transiently resulting in distinct in vitro phenotypes. The baseline mutation rate of the patient isolates was unremarkable ruling out the hypermutator phenotype as a contributor to the rapid emergence of resistance. However, the index isolate demonstrated pronounced tolerance to the antibiotic daptomycin, a phenotype that facilitates the subsequent development of resistance during antibiotic exposure. This study exemplifies the capacity of antibiotic-tolerant pathogens to rapidly develop both stable and transient genetic and phenotypic changes, over the course of a single patient encounter., Competing Interests: The authors have declared that no competing interests exist.

Enrichment culture (EC) remains gold standard for detecting MRSA colonisation, but molecular methods shorten turnaround time. The CE-marked automated Hologic Panther Fusion MRSA Assay (HPFM) is validated for nasal swabs. We compared HPFM with EC following an in-house PCR for detection of MRSA in nasal, pharyngeal, and perineal ESwabs. The same ESwabs were analysed using HPFM and inoculated in selective Tryptic Soy Broth (TSB) for overnight incubation. TSBs were screened by a PCR targeting nuc, femA, mecA, and mecC. Only samples with PCR results compatible with MRSA presence were inoculated onto 5% blood agar and chromogenic MRSA plates. HPFM detected MRSA in 103 of 132 EC positive samples indicating a sensitivity of 78.0% across sample types. When paired TSBs of 29 EC positive/HPFM negative samples were re-analysed by HPFM, MRSA was detected in 17/29 TSBs indicating that enrichment will increase the sensitivity of HPFM. HPFM analyses of cultured isolates from the remaining 12 EC positive/HPFM negative samples failed to detect orfX. HPFM reported the presence of MRSA in 22 samples where EC failed to identify MRSA. Fifteen of these ESwabs had been kept and direct culture without enrichment identified MRSA in seven samples. HPFM was useful for all sample sites. Compared to EC, the sensitivity of HPFM was limited because of lack of analytical sensitivity and failure to detect all MRSA variants. Failure of some MRSA-containing samples to enrich in cefoxitin-containing TSB indicates an unappreciated limitation of EC, which may lead to underestimation of the specificity of molecular assays., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The antibacterial and antioxidant packaging films were fabricated by incorporating licorice residue extracts (LREs) into oxidized starch (OS) films. The bioactive fraction (BF) was firstly obtained from LREs by using bioassay-guided isolation method. The BF showed potent anti-Gram (+) bacteria effects, especially against methicillin-resistant S. aureus (MRSA) with MIC of 32.5 μg/mL. The present results also indicated that the addition of BF could significantly decrease the moisture content, water vapor permeability, light transmittance of OS films. Notably, the antibacterial and antioxidant activities of OS films significantly enhanced with the concentration of BF increasing. Moreover, the films with the highest concentration of BF showed the lowest tensile strength (4.23 MPa) and the highest elongation at break (63.89%). Meanwhile, the bioactive films could release bioactive compounds such as licochalcone A and licochalcone B into the alcoholic and fatty food simulants. Taken together, the active OS films containing LREs have the potential for application in food packaging films, due to its potential against MRSA and antioxidant activity as well as good physicochemical properties., (Copyright © 2021. Published by Elsevier B.V.)

Aim: To investigate the epidemiology of S. aureus and MRSA nasal carriage among people with diabetes at the Korle Bu Teaching Hospital in Accra, including the prevalence, predictors of carriage, and antibiotic resistance., Methodology: This study was cross-sectional, involving 300 diabetes patients and 106 non-diabetic individuals. Swab specimens of the nares were obtained from the participants and bacteriologically-cultured. Identification and characterization of S. aureus and MRSA were based on standard bacteriological methods; antimicrobial susceptibility testing was by the Kirby-Bauer method., Results: The prevalence of staphylococcal carriage, the diabetes group relative to the non-diabetes group, were 31.0% and 10.4% (S. aureus), and 3.3% and 0.0% (MRSA). Presence of diabetes predisposed to S. aureus carriage, but not MRSA nor coagulase-negative staphylococci (CoNS) carriage (OR = 3.88; p < 0.0001). Colonization with CoNS was protective of S. aureus (OR = 0.039, p < 0.001) and MRSA (OR = 0.115, p = 0.043) colonization among the diabetics. The antimicrobial resistance patterns recorded among the S. aureus isolated from the diabetic individuals relative to the non-diabetics were as follows: penicillin (95% vs. 91%), tetracycline (37% vs. 27%), cotrimoxazole (30% vs. 36%), erythromycin (17% vs. 0%), norfloxacin (13% vs. 0%), clindamycin (12% vs. 0%), gentamicin (9% vs. 0%), fusidic acid (10% vs. 9%), linezolid (4% vs. 0%), and rifampicin (5% vs. 0%). The proportion of multidrug resistant S. aureus was 41% (n = 38) in the diabetes group and 0% in the non-diabetes group; this difference was statistically significant (p = 0.01)., Conclusions: The presence of diabetes predisposed the participants to S. aureus carriage by almost four folds, but not MRSA carriage. Colonization with CoNS was protective of S. aureus and MRSA carriage in the diabetes group. Finally, linezolid remains a good therapeutic agent for anti-MRSA therapy., Competing Interests: FCNK and M-MO are affiliated with FleRhoLife Research Consult, but this firm didnot provide financial support nor play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The host defense peptide caerin 1.9 was originally isolated from skin secretions of an Australian tree frog and inhibits the growth of a wide range of bacteria in vitro . In this study, we demonstrated that caerin 1.9 shows high bioactivity against several bacteria strains, such as Staphylococcus aureus, Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus haemolyticus in vitro . Importantly, unlike the antibiotic Tazocin, caerin 1.9 does not induce bacterial resistance after 30 rounds of in vitro culture. Moreover, caerin 1.1, another peptide of the caerin family, has an additive antibacterial effect when used together with caerin 1.9. Furthermore, caerin 1.1 and 1.9 prepared in the form of a temperature-sensitive gel inhibit MRSA growth in a skin bacterial infection model of two murine strains. These results indicate that caerin 1.1 and 1.9 peptides could be considered an alternative for conventional antibiotics. IMPORTANCE Antibiotic-resistant bacteria cause severe problems in the clinic. We show in our paper that two short peptides isolated from an Australian frog and prepared in the form of a gel are able to inhibit the growth of antibiotic-resistant bacteria in mice, and, unlike antibiotics, these peptides do not lead to the development of peptide-resistant bacteria strains.

Objective: To evaluate the efficacy of topical mupirocin in reducing Staphylococcus aureus colonization in infants in the neonatal intensive care unit (NICU)., Study Design: A prospective double-blind randomized controlled trial of mupirocin vs placebo in S aureus-colonized infants was conducted in a tertiary care NICU between October 2016 and December 2019. Weekly universal active surveillance with polymerase chain reaction screening identified colonized infants. Colonized infants received a 5-day course of mupirocin (mupirocin group) or petroleum jelly (control group). Repeat courses were given for additional positive screens., Results: A total of 216 infants were enrolled; 205 were included in data analyses. Primary decolonization was more successful for mupirocin-treated infants (86 of 104 [83%]) than for controls (20 of 101; 20%) (P < .001). Although recurrent S aureus colonization occurred frequently (59 of 81 [73%] mupirocin-treated and 26 of 33 [79%] controls), subsequent decolonization remained more successful for mupirocin-treated infants than for controls (38 of 49 [78%] vs 2 of 21 [10%]; P < .001). Subgroup analyses of infants of ≤30 weeks' gestational age yielded similar results; decolonization occurred more often in mupirocin-treated infants compared with control infants (63 of 76 [83%] vs 13 of 74 [18%]; P < .001). Bacterial sterile site infections tended to be less frequent in mupirocin-treated infants compared with controls (2 of 104 [2%] vs 8 of 101 [8%]; P = .057). No invasive S aureus infections occurred in mupirocin-treated infants, but 50% of infections in controls were from S aureus, and 1 resulted in death., Conclusions: Universal active surveillance and targeted treatment with topical mupirocin is a successful decolonization strategy for NICU infants and may prevent S aureus infection. However, S aureus colonization frequently recurs, necessitating repeat treatment., Trial Registration: Clinicaltrials.gov: NCT02967432., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) is an emerging cause of hospital-associated urinary tract infections (UTI), especially in catheterized individuals. Despite being rare, MRSA UTI are prone to potentially life-threatening exacerbations such as bacteremia that can be refractory to routine antibiotic therapy. To delineate the molecular mechanisms governing MRSA urinary pathogenesis, we exposed three S. aureus clinical isolates, including two MRSA strains, to human urine for 2 h and analyzed virulence characteristics and changes in gene expression. The in vitro virulence assays showed that human urine rapidly alters adherence to human bladder epithelial cells and fibronectin, hemolysis of sheep red blood cells (RBCs), and surface hydrophobicity in a staphylococcal strain-specific manner. In addition, transcriptome sequencing (RNA-Seq) analysis of uropathogenic strain MRSA-1369 revealed that 2-h-long exposure to human urine alters MRSA transcriptome by modifying expression of genes encoding enzymes catalyzing metabolic pathways, virulence factors, and transcriptional regulators. In summary, our results provide important insights into how human urine specifically and rapidly alters MRSA physiology and facilitates MRSA survival in the nutrient-limiting and hostile urinary microenvironment. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is an uncommon cause of urinary tract infections (UTI) in the general population. However, it is important to understand MRSA pathophysiology in the urinary tract because isolation of MRSA in urine samples often precedes potentially life-threatening MRSA bacteremia. In this report, we describe how exposure to human urine alters MRSA global gene expression and virulence. We hypothesize that these alterations may aid MRSA in acclimating to the nutrient-limiting, immunologically hostile conditions within the urinary tract leading to MRSA UTI.

Multiresistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) cause serious postoperative infections. A skin tolerant far-UVC (< 240 nm) irradiation system for their inactivation is presented here. It uses UVC LEDs in combination with a spectral filter and provides a peak wavelength of 233 nm, with a full width at half maximum of 12 nm, and an irradiance of 44 µW/cm 2 . MRSA bacteria in different concentrations on blood agar plates were inactivated with irradiation doses in the range of 15-40 mJ/cm 2 . Porcine skin irradiated with a dose of 40 mJ/cm 2 at 233 nm showed only 3.7% CPD and 2.3% 6-4PP DNA damage. Corresponding irradiation at 254 nm caused 15-30 times higher damage. Thus, the skin damage caused by the disinfectant doses is so small that it can be expected to be compensated by the skin's natural repair mechanisms. LED-based far-UVC lamps could therefore soon be used in everyday clinical practice to eradicate multiresistant pathogens directly on humans., (© 2021. The Author(s).)

Despite intensified efforts to develop an effective antibiotic, S. aureus is still a major cause of mortality and morbidity worldwide. The multidrug resistance of bacteria has considerably increased the difficulties of scientific research and the concomitant emergence of resistance is to be expected. In this study we have investigated the in vitro activity of 15 ethanol extracts prepared from Moroccan medicinal plants traditionally used for treatment of skin infections. Among the tested species I. viscosa , C. oxyacantha , R. tinctorum , A. herba alba, and B. hispanica showed moderate anti-staphylococcal activity. However, R. alaternus showed promising growth-inhibitory effects against specific pathogenic bacteria especially methicillin-susceptible Staphylococcus aureus Panton-Valentine leucocidin positive (MSSA-PVL) and methicillin-resistant S. aureus (MRSA). The bioguided fractionation of this plant using successive chromatographic separations followed by nuclear magnetic resonance (NMR) and mass spectrometry (MS) including EIMS and HREIMS analysis yielded the emodin ( 1 ) and kaempferol ( 2 ). Emodin being the most active with MICs ranging between 15.62 and 1.95 µg/mL and showing higher activity against the tested strains in comparison with the crude extract, its mechanism of action and the structure-activity relationship were interestingly discussed. The active compound has not displayed toxicity toward murine macrophage cells. The results obtained in the current study support the traditional uses of R. alaternus and suggest that this species could be a good source for the development of new anti-staphylococcal agents.

Background: Melittin is one of the most studied antimicrobial peptides, and several in vitro experiments have demonstrated its antibacterial efficacy. However, there is evidence showing melittin has non-promising effects such as cytotoxicity and hemolysis. Therefore, concerns about unwanted collateral toxicity of melittin lie ahead in the path toward its clinical development. With these considerations, the present study aimed to fill the gap between in vitro and in vivo studies., Methods: In the first step, in vitro toxicity profile of melittin was assessed using cytotoxicity and hemolysis tests. Next, a maximum intraperitoneal (i.p.) sub-lethal dose was determined using BALB/c mice. Besides toxicity, antimicrobial efficacy of melittin against extensively drug-resistant (XDR) Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), and KPC-producing Klebsiella pneumonia (KPC-KP) pathogens were tested using both in vitro and in vivo methods., Results: Melittin showed extensive hemolysis (HD 50  = 0.44 µg/mL), and cytotoxicity (IC 50  = 6.45 µg/mL) activities with i.p. LD 50 value of 4.98 mg/kg in BALB/c mice. In vitro antimicrobial evaluation showed melittin MIC range from 8 to 32 µg/mL for the studied pathogens. Treatment of infected mice with repeated sub-lethal doses of melittin (2.4 mg/kg) displayed no beneficial effect on their survival and peritoneal bacterial loads., Conclusions: These results indicate that melittin at its safe dose could not exhibit antimicrobial activity, which hinders its application in clinical practice., (© 2021. The Author(s).)

The increase in the number of deaths from infections caused by multidrug-resistant bacteria and cancer diseases highlights the need for new molecules with biological activity. Actinobacteria represent a potential source of new compounds, as these microorganisms have already produced a great diversity of clinically employed antibiotics. Endophytes from unexplored biomes, such as the Pantanal (the largest wetland in the world), can be a source of new molecules. Hymenachne amplexicaulis is among the unexplored native plants of the Pantanal in terms of its endophytic community. This plant is considered a weed in other countries due to its ability to adapt and compete with native plants, and there is evidence to suggest that the endophytic community of H. amplexicaulis plays an important role in this competitiveness. To explore its therapeutic potential, the present study isolated, identified (using partial sequence of the 16S rDNA) and bioprospected H. amplexicaulis endophytic actinobacteria. Ten isolates belonging to the genera Streptomyces, Microbispora, Leifsonia, and Verrucosispora were obtained from root fragments. The susceptibility profile of the isolates to the different classes of antibiotics was evaluated, with 80 % of the isolates showing resistance to the antibiotics Nalidixic Acid, Ampicillin, Chloramphenicol, Oxacillin, and Rifampicin. To assess antibacterial and antitumor activities, methanolic extracts were obtained by fermentation in SG culture medium at 36 °C at 180 rpm for 10 days. The extract produced from the S. albidoflavus CMRP4854 isolate was the only one to show activity against the Gram-negative bacterium Acinetobacter baumanii. Due to the great clinical importance of this pathogen and the difficulty in obtaining active compounds against it, the CMRP4854 isolate should be further investigated for the identification of active compounds and mode of action. We also emphasize the results obtained by the extract of the isolates Streptomyces albidoflavus CMRP4852 and Verrucosispora sp. CMRP4860 that presented antibacterial effect against Methicilin-resistant Staphylococcus aureus (MRSA) (MIC: 1.5 μg/mL and 13 μg/mL, respectively) and Vancomycin-resistant Enterococcus (VRE) (MIC: 40 μg/mL for both extracts). Extracts (200 μg/mL) of these two endophytes also showed selective cytotoxicity action against murine B16-F10 melanoma cells. However, the CMRP4852 extract also affected the density of normal cells. Due to these results, the crude extract of isolate CMRP4860 Verrucosispora sp., which was the only one that presented cytotoxicity and reduced cell density only in tumor cells, was selected for subsequent analysis involving scale-up fermentation of the CMRP4860 resulting in 9 fractions that were tested against both bacteria and tumor cells, with particular fractions showing promise and meriting further investigation. Taken together, the results of this study not only show for the first time that the endophytic community of H. amplexicaulis actinobacteria can produce secondary metabolites that potentially possess important antibacterial and cytotoxic properties, but also reinforce the pressing need to conserve biomes such as the Brazilian Pantanal., (Copyright © 2021. Published by Elsevier GmbH.)

A key characteristic of Staphylococcus aureus infections, and one that also varies phenotypically between clones, is that of biofilm formation, which aids in bacterial persistence through increased adherence and immune evasion. Though there is a general understanding of the process of biofilm formation - adhesion, proliferation, maturation and dispersal - the tightly orchestrated molecular events behind each stage, and what drives variation between S. aureus strains, has yet to be unravelled. Herein we measure biofilm progression and dispersal in real-time across the five major S. aureus CDC-types (USA100-USA500) revealing adherence patterns that differ markedly amongst strains. To gain insight into this, we performed transcriptomic profiling on these isolates at multiple timepoints, compared to planktonically growing counterparts. Our findings support a model in which eDNA release, followed by increased positive surface charge, perhaps drives initial abiotic attachment. This is seemingly followed by cooperative repression of autolysis and activation of poly-N-acetylglucosamine (PNAG) production, which may indicate a developmental shift in structuring the biofilm matrix. As biofilms mature, diminished translational capacity was apparent, with 53 % of all ribosomal proteins downregulated, followed by upregulation of anaerobic respiration enzymes. These findings are noteworthy because reduced cellular activity and an altered metabolic state have been previously shown to contribute to higher antibiotic tolerance and bacterial persistence. In sum, this work is, to our knowledge, the first study to investigate transcriptional regulation during the early, establishing phase of biofilm formation, and to compare global transcriptional regulation both temporally and across multiple clonal lineages.

Prodigiosin, a red linear tripyrrole pigment, has long been recognised for its antimicrobial property. However, the physiological contribution of prodigiosin to the survival of its producing hosts still remains undefined. Hence, the aim of this study was to investigate the biological role of prodigiosin from Serratia marcescens, particularly in microbial competition through its antimicrobial activity, towards the growth and secreted virulence factors of four clinical pathogenic bacteria (methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa) as well as Staphylococcus aureus and Escherichia coli. Prodigiosin was first extracted from S. marcescens and its purity confirmed by absorption spectrum, high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrophotometry (LC-MS/MS). The extracted prodigiosin was antagonistic towards all the tested bacteria. A disc-diffusion assay showed that prodigiosin is more selective towards Gram-positive bacteria and inhibited the growth of MRSA, S. aureus and E. faecalis and Gram-negative E. coli. A minimum inhibitory concentration of 10 μg/μL of prodigiosin was required to inhibit the growth of S. aureus, E. coli and E. faecalis whereas > 10 μg/μL was required to inhibit MRSA growth. We further assessed the effect of prodigiosin towards bacterial virulence factors such as haemolysin and production of protease as well as on biofilm formation. Prodigiosin did not inhibit haemolysis activity of clinically associated bacteria but was able to reduce protease activity for MRSA, E. coli and E. faecalis as well as decrease E. faecalis, Salmonella Typhimurium and E. coli biofilm formation. Results of this study show that in addition to its role in inhibiting bacterial growth, prodigiosin also inhibits the bacterial virulence factor protease production and biofilm formation, two strategies employed by bacteria in response to microbial competition. As clinical pathogens were more resistant to prodigiosin, we propose that prodigiosin is physiologically important for S. marcescens to compete against other bacteria in its natural soil and surface water environments., Competing Interests: The authors have declared that no competing interests exist.

Antibiotics are widely used for treatment of bacterial infections, and their overuse has contributed to microbial resistance. Currently, an alternative antibiotic-free therapy for inactivating bacteria is of great interest. Black phosphorus (BP), a biocompatible and nontoxic rising-star two-dimensional layered material, has gained remarkable interest in many bioapplications including biosensing, cancer therapy, drug delivery, and also antibacterial treatment. However, BP nanosheets suffer from instability in ambient environments due to rapid oxidation and degradation. To address this issue, BP nanosheets were modified with quaternized chitosan (QCS) by electrostatic adsorption to prepare a BP-QCS composite for photothermal/pharmaco treatment of bacterial infection. The BP-QCS has obviously enhanced solubility and chemical stability in aqueous suspensions. We have demonstrated that under near-infrared (NIR) irradiation, the BP-QCS can synergistically inactivate more than 95% methicillin-resistant Staphylococcus aureus ( S . aureus ) (MRSA) and Escherichia coli within 10 min with a dose of only 75 μg/mL in vitro. Meanwhile, the BP-QCS composite under NIR can synergistically inactivate 98% S. aureus in vivo . Furthermore, the BP-QCS suspensions at effective antibacterial concentrations have negligible cytotoxicity and in vivo toxicity.

Purpose: The growing prevalence of multidrug-resistant (MDR) bacteria makes it clinically urgent to develop an agent able to detect and treat infections simultaneously. Silver has served as a broad-spectrum antimicrobial since ancient times but suffers from major challenges such as moderate antimicrobial activity, nonspecific toxicity, and difficulty to be visualized in situ . Here, we propose a new photoactive silver nanoagent that relies on a photosensitizer-triggered cascade reaction to liberate Ag + on bacterial surfaces exclusively, allowing the precise killing of MDR bacteria. Additionally, the AgNP core acts as a backgroundless surface-enhanced Raman scattering (SERS) substrate for imaging the distribution of the nanoagents on bacterial surfaces and monitoring their metabolic dynamics in the infection sites. Methods: In this strategy, the photoactive antibacterial AgNP was decorated with photosensitizers (Chlorin e6, Ce6) and Raman reporter (4-Mercaptobenzonitrile, 4-MB) to provide new opportunities for clinically monitoring and fighting MDR bacterial infections. Upon 655 nm laser activation, the Ce6 molecules produce ROS efficiently, triggering the rapid release of Ag + from the AgNP core to kill bacteria. Poly[4-O-(α-D-glucopyranosyl)-D-glucopyranose] (GP) was introduced as bacteria-specific targeting ligands. SERS spectra of the prepared GP-Ce6/MB-AgNPs were recorded after injecting for 0.5, 4, 8, 12, 24, and 48 h to track the dynamic metabolism of the nanoagents and thus guiding the antibacterial therapy. Results: This new antimicrobial strategy exerts a dramatically enhanced antibacterial activity. The in vitro antibacterial efficiencies of this non-antibiotic technique were up to 99.6% against Methicillin-resistant Staphylococcus aureus (MRSA) and 98.8% against Escherichia coli (EC), while the in vivo antibacterial efficiencies for MRSA- and Carbapenem-resistant Pseudomonas aeruginosa (CRPA)-infected mice models were 96.8% and 93.6%, respectively. Besides, backgroundless SERS signal intensity of the wound declined to the level of normal tissue until 24 h, indicating that the nanoagents had been completely metabolized from the infected area. Conclusion: Given the backgroundless monitoring ability, high antibacterial efficacy, and low toxicity, the photoactive cascading agents would hold great potential for MDR-bacterial detection and elimination in diverse clinical settings., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Cellulose nanofibers (CNF) isolated from plant biomass have attracted considerable interests in polymer engineering. The limitations associated with CNF-based nanocomposites are often linked to the time-consuming preparation methods and lack of desired surface functionalities. Herein, we demonstrate the feasibility of preparing a multifunctional CNF-zinc oxide (CNF-ZnO) nanocomposite with dual antibacterial and reinforcing properties via a facile and efficient ultrasound route. We characterized and examined the antibacterial and mechanical reinforcement performances of our ultrasonically induced nanocomposite. Based on our electron microscopy analyses, the ZnO deposited onto the nanofibrous network had a flake-like morphology with particle sizes ranging between 21 to 34 nm. pH levels between 8-10 led to the formation of ultrafine ZnO particles with a uniform size distribution. The resultant CNF-ZnO composite showed improved thermal stability compared to pure CNF. The composite showed potent inhibitory activities against Gram-positive (methicillin-resistant Staphylococcus aureus (MRSA)) and Gram-negative Salmonella typhi ( S. typhi ) bacteria. A CNF-ZnO-reinforced natural rubber (NR/CNF-ZnO) composite film, which was produced via latex mixing and casting methods, exhibited up to 42% improvement in tensile strength compared with the neat NR. The findings of this study suggest that ultrasonically-synthesized palm CNF-ZnO nanocomposites could find potential applications in the biomedical field and in the development of high strength rubber composites.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have reached epidemic proportions globally. Therefore, there is an urgent need for a continuous supply of antibiotics to combat the problem. In this study, bacteria initially identified as species belonging to the Bacillus amyloliquefaciens operational group were re-identified based on the housekeeping gene, gyrB. Cell-free supernatants (CFS) from the strains were used for antimicrobial tests using the agar well diffusion assay against MRSA and various types of pathogenic bacteria. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and physicochemical characteristics of the CFS were determined. Based on gyrB sequence analysis, five strains (PD9, B7, PU1, BP1 and L9) were identified as Bacillus velezensis. The CFS of all B. velezensis strains showed broad inhibitory activities against Gram-negative and -positive as well as MRSA strains. Strain PD9 against MRSA ATCC 33742 was chosen for further analysis as it showed the biggest zone of inhibition (21.0 ± 0.4 mm). The MIC and MBC values obtained were 125 μl/ml. The crude antimicrobial extract showed bactericidal activity and was stable at various temperatures (40-80°C), pH (4-12), surfactants (Tween 20, Tween 80, SDS and Triton X-100) and metal ions (MgCI2, NaCI2, ZnNO3 and CuSO4) when tested. However, the crude extract was not stable when treated with proteinase K. All these properties resembled the characteristics of peptides. The antimicrobial compound from the selected strain was purified by using solvent extraction method and silica gel column chromatography. The purified compound was subjected to High Performance Liquid Chromatography which resulted in a single peak of the anti-MRSA compound being detected. The molecular weight of the anti-MRSA compound was determined by using SDS-PAGE and zymogram. The size of the purified antimicrobial peptide was approximately ~ 5 kDa. The antimicrobial peptide produced from B. velezensis strain PD9 is a promising alternative to combat the spread of MRSA infections in the future., Competing Interests: NO authors have competing interests.

Methicillin-resistant Staphylococcus aureus (MRSA) with reduced sensitivity to vancomycin (VAN) has caused many clinical cases of VAN treatment failure, but the molecular mechanism underlying the reduced sensitivity to VAN is still unclear. We isolated a heterogeneous VAN-intermediate Staphylococcus aureus (hVISA), which was also a MRSA strain with reduced sensitivity to VAN. To investigate the molecular mechanism underlying the reduced sensitivity to VAN exhibited by the hVISA strain, we compared the hVISA strain with a VAN-sensitive MRSA strain, known as the N315 strain. The images captured by transmission electron microscopy showed that the cell wall of the hVISA strain was significantly thicker than that of the N315 strain (36·72 ± 1·04 nm vs 28·15 ± 1·25 nm, P < 0·05), and the results of real-time quantitative PCR analysis suggested that the expression levels of the cell wall thickness related genes (glmS, vraR/S, sgtB, murZ and PBP4) of the hVISA strain were significantly higher than those of the N315 strain (P < 0·05). In conclusion, this study indicated that the upregulation of the expression of the genes related to cell wall synthesis might be the molecular mechanism underlying the cell wall thickening of the hVISA strain and might be related to its resistance to VAN., (© 2021 The Authors. Letters in Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Polymicrobial biofilms consisting of fungi and bacteria are frequently formed on endotracheal tubes and may contribute to development of ventilator associated pneumonia (VAP) in critically ill patients. This study aimed to determine the role of early Candida albicans biofilms in supporting dual-species (dual-kingdom) biofilm formation with respiratory pathogens in vitro, and investigated the effect of targeted antifungal treatment on bacterial cells within the biofilms. Dual-species biofilm formation between C. albicans and three respiratory pathogens commonly associated with VAP (Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus) was studied using quantitative PCR. It was shown that early C. albicans biofilms enhanced the numbers of E. coli and S. aureus (including methicillin resistant S. aureus; MRSA) but not P. aeruginosa within dual-species biofilms. Transwell assays demonstrated that contact with C. albicans was required for the increased bacterial cell numbers observed. Total Internal Reflection Fluorescence microscopy showed that both wild type and hyphal-deficient C. albicans provided a scaffold for initial bacterial adhesion in dual species biofilms. qPCR results suggested that further maturation of the dual-species biofilm significantly increased bacterial cell numbers, except in the case of E.coli with hyphal-deficient C. albicans (Ca&#95;gcn5Δ/Δ). A targeted preventative approach with liposomal amphotericin (AmBisome®) resulted in significantly decreased numbers of S. aureus in dual-species biofilms, as determined by propidium monoazide-modified qPCR. Similar results were observed when dual-species biofilms consisting of clinical isolates of C. albicans and MRSA were treated with liposomal amphotericin. However, reductions in E. coli numbers were not observed following liposomal amphotericin treatment. We conclude that early C. albicans biofilms have a key supporting role in dual-species biofilms by enhancing bacterial cell numbers during biofilm maturation. In the setting of increasing antibiotic resistance, an important and unexpected consequence of antifungal treatment of dual-species biofilms, is the additional benefit of decreased growth of multi-drug resistant bacteria such as MRSA, which could represent a novel future preventive strategy., Competing Interests: The authors have declared that no competing interests exist.

The enormous spread of Staphylococcus aureus infections through biofilms is a major concern in hospital-acquired infections. Biofilm formation by S. aureus on any surface is facilitated by adjusting its redox status. This organism is a facultative anaerobe shift more towards reductive conditions by enhancing nitrogen metabolism where glutamine synthesis plays a key role. Glutamine is synthesized by glutamine synthetase (GS) encoded by the glnA gene. The gene was amplified by PCR from the chromosomal DNA of S. aureus , sequenced (HQ329146.1), and cloned. The pure recombinant GS exhibited K m of 11.06 ± 0.05 mmol·L -1 for glutamate and 2.4 ± 0.03 mmol·L -1 for ATP. The glnA gene sequence showed a high degree of variability with its human counterpart, while it was highly conserved in bacteria. Structural analysis revealed that the GS structure of S. aureus showed close homology with other Gram-positive bacteria and exhibited a high degree of variation with Escherichia coli GS. In the present study, we observed the increased presence of GS activity in multidrug-resistant strains of S. aureus with elevated biofilm units, grown in brain heart infusion broth; among them methicillin-resistant strains S. aureus LMV 3, 4, and 5 showed higher biofilm units. All these results explain the important role of glutamine biosynthesis with elevated biofilm units in the pathogenesis of S. aureus .

In S. aureus biofilms, bacteria are embedded in a matrix of extracellular polymeric substances (EPS) and are highly tolerant to antimicrobial drugs. We thus sought to identify non-antibiotic substances with broad-spectrum activity able to destroy the EPS matrix and enhance the effect of antibiotics on embedded biofilm bacteria. Among eight substances tested, subtilisin A (0.01 U/mL) and calcium gluconate (CaG, Ca 2+ 1.25 mmol/L) significantly reduced the biomass of biofilms formed by at least 21/24 S. aureus isolates. Confocal laser scanning microscopy confirmed that they both eliminated nearly all the proteins and PNAG from the matrix. By contrast, antibiotics alone had nearly no effect on biofilm biomass and the selected one (oxytetracycline-OTC) could only slightly reduce biofilm bacteria. The combination of OTC with CaG or subtilisin A led to an additive reduction (average of 2 log 10 CFU/mL) of embedded biofilm bacteria on the isolates susceptible to OTC (MBC < 10 μg/mL, 11/24). Moreover, these two combinations led to a reduction of the embedded biofilm bacteria higher than 3 log 10 CFU/mL for 20-25% of the isolates. Further studies are now required to better understand the factors that cause the biofilm produced by specific isolates (20-25%) to be susceptible to the combinations.

Fatty acids-assisted superparamagnetic maghemite (γ-Fe 2 O 3 ) NPs was biologically synthesized using extract of polyherbal drug Liv52 (L52E). The NPs were characterized by UV-vis spectroscopy, FT-IR, SEM, TEM, EDX, XRD and VSM. The major biological molecules present in L52E analysed by GC-MS were saturated fatty acids (palmitic acid 21.95%; stearic acid 13.99%; myristic acid 1.14%), monounsaturated fatty acid (oleic acid 18.43%), polyunsaturated fatty acid (linoleic acid 20.45%), and aromatic phenol (cardanol monoene 11.92%) that could imply in bio-fabrication and stabilization of γ-Fe 2 O 3 NPs. The FT-IR spectra revealed involvement of carboxylic group of fatty acids, amide group of proteins and hydroxyl group of phenolic compounds that acts as reducing and capping agents. The synthesized NPs were used to investigate their antimicrobial, antibiofilm activity against P. aeruginosa, MRSA and C. albicans and anticancer activity on colon cancer cells (HCT-116) for biomedical applications. Further, molecular docking study was performed to explore the interaction of Fe 2 O 3 NPs with major cell wall components i.e., peptidoglycan and mannoproteins. The docking studies revealed that Fe 2 O 3 interacted efficiently with peptidoglycan and mannoproteins and Fe 2 O 3 get accommodated into catalytic cleft of mannoprotein. Due to magnetic property, the biological activity of γ-Fe 2 O 3 can be further enhanced by applying external magnetic field alone or in amalgamation with other therapeutics drugs., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

There have been relatively few studies which support a link between Ganoderma boninense, a phytopathogenic fungus that is particularly cytotoxic and pathogenic to plant tissues and roots, and antimicrobial compounds. We previously observed that liquid-liquid extraction (LLE) using chloroformmethanol-water at a ratio (1:1:1) was superior at detecting antibacterial activities and significant quantities of antibacterial compounds. Herein, we demonstrate that antibacterial secondary metabolites are produced from G. boninense mycelia. Antibacterial compounds were monitored in concurrent biochemical and biophysical experiments. The combined methods included high performance thin-layer chromatography (HPTLC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR) spectroscopy. The antibacterial compounds derived from mycelia with chloroform-methanol extraction through LLE were isolated via a gradient solvent elution system using HPTLC. The antibacterial activity of the isolated compounds was observed to be the most potent against Staphylococcus aureus ATCC 25923 and multidrug-resistant S. aureus NCTC 11939. GC-MS, HPLC, and FTIR analysis confirmed two antibacterial compounds, which were identified as 4,4,14α-trimethylcholestane (m/z = 414.75; lanostane, C 30 H 54 ) and ergosta-5,7,22-trien-3β-ol (m/z = 396.65; ergosterol, C 28 H 44 O). With the aid of spectroscopic evaluations, ganoboninketal (m/z = 498.66, C 30 H 42 O 6 ), which belongs to the 3,4-seco-27-norlanostane triterpene family, was additionally characterized by 2D-NMR analysis. Despite the lack of antibacterial potential exhibited by lanostane; both ergosterol and ganoboninketal displayed significant antibacterial activities against bacterial pathogens. Results provide evidence for the existence of bioactive compounds in the mycelia of the relatively unexplored phytopathogenic G. boninense, together with a robust method for estimating the corresponding potent antibacterial secondary metabolites.

Multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of hospital-acquired and community infections and pose a challenge to the human health care system. Therefore, it is important to find new drugs that show activity against these bacteria, both in monotherapy and in combination with other antimicrobial drugs. Gliotoxin (GT) is a mycotoxin produced by Aspergillus fumigatus and other fungi of the Aspergillus genus. Some evidence suggests that GT shows antimicrobial activity against S. aureus in vitro, albeit its efficacy against multidrug-resistant strains such asMRSA or vancomycin-intermediate S. aureus (VISA) strainsis not known. This work aimedto evaluate the antibiotic efficacy of GT as monotherapy or in combination with other therapeutics against MRSA in vitro and in vivo using a Caenorhabditis elegans infection model.

Daptomycin (DAP) is one of the most potent antibiotics used for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Due to an increase in its administration for combating MRSA infections, DAP non-susceptible (DAP-NS) MRSA strains have recently been reported in clinical settings. The presence of single nucleotide polymorphisms (SNPs) in the multiple peptide resistance factor (mprF) gene is the most frequently reported cause for the evolution of DAP-NS MRSA strains; however, there are some variations of SNPs that could lead to DAP-NS. In this study, we used two clinical MRSA strains, including DAP susceptible (DAP-S) and DAP-NS, isolated from the same patient at different time points. We introduced T345I SNP to mprF of the DAP-S MRSA strain using the gene exchange method with pIMAY vector. Further, we investigated the phenotype of the mutant strain, including drug susceptibility, cell surface positive charge, and growth speed. The mutant strain exhibited (i) resistance to DAP, (ii) up-regulation of positive surface charge, (iii) slower growth speed, and (iv) thickened cell walls. Hence, the SNP in mprF may have caused an up-regulation in MprF function, with a subsequent increase in positive surface charge. Cumulatively, these results demonstrated that the T345I amino acid substitution in mprF represents one of the primary causes of DAP-NS in MRSA strains., Competing Interests: The authors have declared that no competing interests exist.

This research was aimed at the preparation of a hybrid film based on a layered silicate saponite (Sap) with the immobilized photosensitizer phloxine B (PhB). Sap was selected because of its high cation exchange capacity, ability to exfoliate into nanolayers, and to modify different surfaces. The X-ray diffraction of the films confirmed the intercalation of both the surfactant and PhB molecules in the Sap film. The photosensitizer retained its photoactivity in the hybrid films, as shown by fluorescence spectra measurements. The water contact angles and the measurement of surface free energy demonstrated the hydrophilic nature of the hybrid films. Antimicrobial effectiveness, assessed by the photodynamic inactivation on hybrid films, was tested against a standard strain and against methicillin-resistant bacteria of Staphylococcus aureus (MRSA). One group of samples was irradiated (green LED light; 2.5 h) and compared to nonirradiated ones. S. aureus strains manifested a reduction in growth from 1-log 10 to over 3-log 10 compared to the control samples with Sap only, and defects in S. aureus cells were proven by scanning electron microscopy. The results proved the optimal photo-physical properties and anti-MRSA potential of this newly designed hybrid system that reflects recent progress in the modification of surfaces for various medical applications.

Staphylococcus aureus is a Gram-positive bacterium responsible for a variety of mild to life-threatening infections including bone infections such as osteomyelitis. This bacterium is able to invade and persist within non-professional phagocytic cells such as osteoblasts. In the present study, four different S. aureus strains, namely, 2SA-ST239-III (ST239), 5SA-ST5-II (ST5), 10SA-ST228-I (ST228), and 14SA-ST22-IVh (ST22), were tested for their ability to modulate cell viability in MG-63 osteoblast-like cells following successful invasion and persistence. Methicillin-sensitive S. aureus (MSSA) ATCC-12598-ST30 (ST30) was used as control strain. Despite being proven that ST30, ST239, and ST22 have a similar ability to internalize and persist in MG-63 osteoblast-like cells under our experimental conditions, we demonstrated that the observed decrease in cell viability was due to the different behavior of the considered strains, rather than the number of intracellular bacteria. We focused our attention on different biochemical cell functions related to inflammation, cell metabolism, and oxidative stress during osteoblast infections. We were able to show the following: (1) ST30 and ST239 were the only two clones able to persist and maintain their number in the hostile environment of the cell during the entire period of infection; (2) ST239 was the only clone able to significantly increase gene expression (3 and 24 h post-infection (p.i.)) and protein secretion (24 h p.i.) of both interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in MG-63 osteoblast-like cells; (3) the same clone determined a significant up-regulation of the transforming growth factorbeta 1 (TGF-β1) and of the metabolic marker glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs at 24 h p.i.; and (4) neither the MSSA nor the four methicillin-resistant S. aureus (MRSA) strains induced oxidative stress phenomena in MG-63 cells, although a high degree of variability was observed for the different clones with regard to the expression pattern of nuclear factor E2-related factor 2 (Nrf2) and its downstream gene heme oxygenase 1 (HO-1) activation. Our results may pave the way for an approach to S. aureus -induced damage, moving towards individualized therapeutic strategies that take into account the differences between MSSA and MRSA as well as the distinctive features of the different clones. This approach is based on a change of paradigm in antibiotic therapy involving a case-based use of molecules able to counteract pro-inflammatory cytokines activity such as selective cytokine signaling inhibitors (IL-6, TNF-α).

&lt;b&gt;Background and Objective:&lt;/b&gt; Antibacterial resistance is one of the top global public health problems. The use of natural substances, which can enhance the antibacterial activity of currently used medications, is a promising alternative to oppose antibacterial resistance. The pharmacological activities of lupinifolin, a prenylated flavanone isolated from stems of &lt;i&gt;Derris reticulata&lt;/i&gt; Craib., against growth and biofilm formation of &lt;i&gt;Streptococcus mutans&lt;/i&gt; and &lt;i&gt;Staphylococcus aureus&lt;/i&gt; have been previously documented. Nonetheless, interactions between lupinifolin and other antibacterial agents have not been determined. This study aimed to investigate the effects of lupinifolin in combinations with some antibacterial agents, specifically ampicillin, cloxacillin or vancomycin, against &lt;i&gt;S. mutans&lt;/i&gt;, Methicillin-Sensitive &lt;i&gt;S. aureus&lt;/i&gt; (MSSA) and Methicillin-Resistant &lt;i&gt;S. aureus&lt;/i&gt; (MRSA). &lt;b&gt;Materials and Methods:&lt;/b&gt; The checkerboard assay was performed to determine the antibacterial activity of lupinifolin plus the testing antibacterial agents. The Fractional Inhibitory Concentration Index (FICI) was calculated to indicate the interaction between lupinifolin and the antibacterial agent tested. &lt;b&gt;Results:&lt;/b&gt; Lupinifolin exerted the synergistic activity when using in combination with ampicillin or cloxacillin against MSSA with the FICIs of &lt;u&gt;<&lt;/u&gt;0.5. The potential synergistic effect was also observed with lupinifolin plus ampicillin or cloxacillin against MRSA. However, the combination of lupinifolin plus vancomycin resulted in no interaction against MRSA. The combined effects of lupinifolin and ampicillin or cloxacillin against &lt;i&gt;S. mutans&lt;/i&gt; were somewhat ambiguous with the borderline values of FICI of 0.5156 and 0.5625, respectively. &lt;b&gt;Conclusion:&lt;/b&gt; Lupinifolin potentially plays a role as an antibacterial intensifier against some pathogenic gram-positive bacteria, particularly MSSA and MRSA. Nonetheless, further experiments are required to explain the precise mechanism of synergy.

Background: Mature lysostaphin (~28-kDa Lss) from Staphylococcus simulans proves effective in killing methicillin-resistant Staphylococcus aureus (MRSA) which is endemic in hospitals worldwide. Lss is Zn 2+ -dependent endopeptidase, but its bacteriolytic activity could be affected by exogenously added Zn 2+ ., Objective: To gain greater insights into structural and functional impacts of Zn 2+ and Ni 2+ on Lss-induced bioactivity., Methods: Lss purified via immobilized metal ion-affinity chromatography was assessed for bioactivity using turbidity reduction assays. Conformational change of metal ion-treated Lss was examined by circular dichroism and intrinsic fluorescence spectroscopy. Co-sedimentation assay was performed to study interactions between Zn 2+ -treated Lss and S. aureus peptidoglycans. Metal ionbinding prediction and intermolecular docking were used to locate an extraneous Zn 2+ -binding site., Results: A drastic decrease in Lss bioactivity against S. aureus and MRSA was revealed only when treated with Zn 2+ , but not Ni 2+ , albeit no negative effect of diethyldithiocarbamate-Zn 2+ -chelator on Lss-induced bioactivity. No severe conformational change was observed for Lss incubated with exogenous Zn 2+ or Ni 2+ . Lss pre-treated with Zn 2+ efficiently bound to S. aureus cell-wall peptidoglycans, suggesting non-interfering effect of exogenous metal ions on cell-wall targeting (CWT) activity. In silico analysis revealed that exogenous Zn 2+ , but not Ni 2+ , preferably interacted with a potential extraneous Zn 2+ -binding site (His253, Glu318 and His323) placed near the Zn 2+ -coordinating Lssactive site within the catalytic (CAT) domain., Conclusion: Our present data signify the adverse influence of exogenous Zn 2+ ions on Lss-induced staphylolytic activity through the exclusive presence within the CAT domain of an extraneous inhibitory Zn 2+ -binding site, without affecting the CWT activity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

This study aims to isolate and identify the structure of antibacterial compounds having potent activity on methicillin-resistant Staphylococcus aureus (MRSA) from marine actinomycetes, and also to identify their mode of action. Lactoquinomycin A (LQM-A) (compound 1 ) and its derivatives ( 2 - 4 ) were isolated from marine-derived Streptomyces bacillaris strain MBTC38, and their structures were determined using extensive spectroscopic methods. These compounds showed potent antibacterial activities against Gram-positive bacteria, with MIC values of 0.06-4 μg/mL. However, the tested compounds exhibited weak inhibitory activity against Gram-negative bacteria, although they were effective against Salmonella enterica (MIC = 0.03-1 μg/mL). LQM-A exhibited the most significant inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA) (MIC = 0.25-0.5 μg/mL), with a low incidence of resistance. An in vivo dual-reporter assay designed to distinguish between compounds that inhibit translation and those that induce DNA damage was employed to assess the mode of action of LQM-A. LQM-A-induced DNA damage and did not inhibit protein synthesis. The gel mobility shift assay showed that LQM-A switched plasmid DNA from the supercoiled to relaxed form in a time- and concentration-dependent manner. These data suggest that LQM-A intercalated into double-stranded DNA and damaged DNA repair.

Aim: The purpose of this study was to evaluate the antimicrobial activity of the anesthetic etomidate against strains of MRSA and biofilms. Materials & methods: The antibacterial effect of etomidate was assessed by the broth microdilution method. To investigate the probable action mechanism of the compound flow cytometry techniques were used. Results: MRSA strains showed MIC equal to 500 and 1000 μg/ml of etomidate. Four-fifths (80%) of the tested MRSA strains demonstrated synergistic effect with oxacillin. Etomidate also showed activity against MRSA biofilm at concentration of 250 μg/ml. Cytometric analysis revealed that the cells treated with etomidate leading to cell death, probably by apoptosis. Conclusion: Etomidate showed antibacterial activity against MRSA.

Ethnopharmacological Relevance: Cochlospermum regium, known as "algodãozinho", is an important plant belonging to Brazilian biodiversity used in traditional medicine to treat infections, wounds and skin conditions., Aim of the Study: To assess the effects of aqueous and ethanolic extracts from C. regium leaves on methicillin-resistant Staphylococcus aureus planktonic cells and biofilm formation., Material and Methods: The phytochemical characterization of the extracts was carried out by quantification of flavonoids, phenols and tannins and HPLC-DAD. Minimum inhibitory concentrations, cell viability, and enzyme activity inhibition were determined in planktonic cells exposed to C. regium extracts. The effect of the extracts on biofilms was assessed by quantifying colony-forming units (CFUs) and the extracellular matrix, and by visualizing the biofilm structure using scanning electron microscopy., Results: Leaf extract contents showed high concentration of phenols and the gallic and ellagic acids were identified. The extracts showed potent antimicrobial activities at concentrations ranging from 62.5-250 μg/mL, and decreased coagulase activity. In addition, the extracts prevented biofilm formation, and the aqueous extract completely inhibited its formation., Conclusions: C. regium extracts stand out as promising alternative treatments for the prevention and treatment of methicillin-resistant Staphylococcus aureus infections., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Over the past years, short anti-microbial peptides have drawn growing attention in the research and trade literature because they are usually capable of killing a broad spectrum of pathogens by employing unique mechanisms of action. This study aimed to evaluate the anti-bacterial effects of a previously designed peptide named PVP towards the clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) in vitro. Secondary structure, cytotoxicity, and membrane-permeabilizing effects of the peptide were also assessed. PVP had a tendency to adopt alpha-helical conformation based upon structural predictions and circular dichroism spectroscopy (in 50% trifluoroethanol). The peptide showed MIC values ranging from 1 to 16 µg/mL against 10 strains of MRSA. In contrast to ciprofloxacin and gentamicin, PVP at sub-lethal concentration (1 µg/mL) did not provoke the development of peptide resistance after 14 serial passages. Remarkably, 1 h of exposure to 4 × MBC of PVP (8 µg/mL) was sufficient for total bacterial clearance, whereas 4 × MBC of vancomycin (8 µg/mL) failed to totally eradicate bacterial cells, even after 8 h. PVP showed negligible cytotoxicity against human dermal fibroblasts at concentrations required to kill the MRSA strains. The results of flow cytometric analysis and fluorescence microscopy revealed that PVP caused bacterial membrane permeabilization, eventually culminating in cell death. Owing to the potent anti-bacterial activity, fast bactericidal kinetics, and negligible cytotoxicity, PVP has the potential to be used as a candidate antibiotic for the topical treatment of MRSA infections.

Knee joint infection following total knee arthroplasty (TKA) is a serious condition and the treatments are complicated. The intra-articular solvent exchange-induced in situ forming matrix is of interest for modulating the release of antibiotics with a high drug concentration and a long period of exposed time at the target site. Stearic acid (S) and lauric acid (L) at various ratios were used as matrix formers by dissolving them in biocompatible solvents such as N-methyl pyrrolidone (NMP) and dimethyl sulfoxide (DMSO). Their matrix formation behaviors in phosphate buffer (pH7.4) and hyaluronic acid (HA) solution were evaluated. Also, the density, viscosity, injectability, solvent diffusion, in vitro degradability and drug release using the dialysis tube method were investigated. The L:S ratio of 1:1 in DMSO exhibited rapid matrix formation and a remarkably low viscosity (7.67±0.03 cp) with acceptable injectability (0.608±0.027N and 0.867±0.010N through 18-G and 27-G, respectively). Vancomycin HCl (V)-loaded L/S in situ forming matrix still provided ease of injection (1.079±0.215N and 1.230±0.145N through 18-G and 27-G needle, respectively) with fatty acid matrix formation after solvent exchange within 1min, whilst V sustainably released over 6days. It also presented effective antimicrobial activities against standard Staphylococcus aureus and methicillin-resistant Staphylococcus aureus strains. Therefore, V-loaded solvent exchange-induced in situ forming matrix using L and S as the matrix formers may be a potential local delivery system for treating knee joint infections occurring after TKA in the future., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest with this investigation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study evaluates the photosensitizing effectiveness of sodium copper chlorophyllin, a natural green colorant commonly used as a food additive (E-141ii), to inactivate methicillin-sensitive and methicillin-resistant Staphylococcus aureus under red-light illumination. Antimicrobial photodynamic inactivation (aPDI) was tested on a methicillin-sensitive reference strain (ATCC 25923) and a methicillin-resistant Staphylococcus aureus strain (GenBank accession number Mh087437) isolated from a clinical sample. The photoinactivation efficacy was investigated by exposing the bacterial strains to different E-141ii concentrations (0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 µM) and to red light (625 nm) at 30 J cm -2 . The results showed that E-141ii itself did not prevent bacterial growth for all tested concentrations when cultures were placed in the dark. By contrast, E-141ii photoinactivated both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) under red-light illumination. However, different dose responses were observed for MSSA and MRSA. Whilst the MSSA growth was inhibited to the detection limit of the method with E-141ii at 2.5 µM, >10 µM concentrations were required to inhibit the growth of MRSA. The data also suggest that E-141ii can produce reactive oxygen species (ROS) via Type I reaction by electron transfer from its first excited singlet state to oxygen molecules. Our findings demonstrate that the tested food colorant has great potential to be used in aPDI of MRSA.

To combat escalating levels of antibiotic resistance, novel strategies are developed to address the everlasting demand for new antibiotics. This study aimed at investigating amicoumacin antibiotics from the desert-derived Bacillus subtilis PJS by using the modern MS/MS-based molecular networking approach. Two new amicoumacins, namely hetiamacin E ( 1 ) and hetiamacin F ( 2 ), were finally isolated. The planar structures were determined by analysis of extensive NMR spectroscopic and HR-ESI-MS data, and the absolute configurations were concluded by analysis of the CD spectrum. Hetiamacin E ( 1 ) showed strong antibacterial activities against methicillin-sensitive and resistant Staphylococcus epidermidis at 2-4 µg/mL, and methicillin-sensitive and resistant Staphylococcus aureus at 8-16 µg/mL. Hetiamacin F ( 2 ) exhibited moderate antibacterial activities against Staphylococcus sp. at 32 µg/mL. Both compounds were inhibitors of protein biosynthesis demonstrated by a double fluorescent protein reporter system.

Functionality of the accessory gene regulator (agr) quorum sensing system is an important factor promoting either acute or chronic infections by the notorious opportunistic human and veterinary pathogen Staphylococcus aureus. Spontaneous alterations of the agr system are known to frequently occur in human healthcare-associated S. aureus lineages. However, data on agr integrity and function are sparse regarding other major clonal lineages. Here we report on the agr system functionality and activity level in mecC-carrying methicillin resistant S. aureus (MRSA) of various animal origins (n = 33) obtained in Europe as well as in closely related human isolates (n = 12). Whole genome analysis assigned all isolates to four clonal complexes (CC) with distinct agr types (CC599 agr I, CC49 agr II, CC130 agr III and CC1943 agr IV). Agr functionality was assessed by a combination of phenotypic assays and proteome analysis. In each CC, isolates with varying agr activity levels were detected, including the presence of completely non-functional variants. Genomic comparison of the agr I-IV encoding regions associated these phenotypic differences with variations in the agrA and agrC genes. The genomic changes were detected independently in divergent lineages, suggesting that agr variation might foster viability and adaptation of emerging MRSA lineages to distinct ecological niches.

Endolysins have been studied intensively as an alternative to antibiotics. In this study, endolysin derived from a phage which infects methicillin-resistant Staphylococcus aureus (MRSA) was cloned and expressed in Escherichia coli pET28a. Initially, the endolysin was cloned using Bam HI/ Xho I, resulting in expression of a recombinant endolysin which was expressed in inclusion bodies. While solubilization was successful, the protein remained nonfunctional. Recloning the endolysin using Nco I/ Xho I resulted in expression of soluble and functional proteins at 18°C. The endolysin was able to form halo zones on MRSA plates and showed a reduction in turbidity of MRSA growth. Therefore, cloning strategies should be chosen carefully even in an established expression system as they could greatly affect the functionality of the expressed protein.

Semisynthetic γ-mangostin derivative LS02 is a novel cationic amphiphilic peptidomimetic antimicrobial agent containing a hydrophobic scaffold and three hydrophilic and positively charged residues of arginine. LS02 showed low in vitro toxicity, potent activities against Gram-positive bacteria including MRSA (MIC = 1.56-6.25 μg/mL) and avoidance of drug resistance. The mode of action studies indicated that LS02 killed bacteria by disrupting bacterial cell membranes. LS02 not only exhibited good water solubility, low hemolytic activity and cell cytotoxicity, but also displayed excellent in vitro and in vivo antibacterial activity, indicating its great potential of being a lead compound as a novel membrane-active antibacterial agent capable of combating bacterial resistance., Competing Interests: Declaration of competing interest Authors declare no conflicts of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)