Enantiomeric glycosylated cationic block co-beta-peptides eradicate Staphylococcus aureus biofilms and antibiotic-tolerant persisters

Abstract The treatment of bacterial infections is hindered by the presence of biofilms and metabolically inactive persisters. Here, we report the synthesis of an enantiomeric block co-beta-peptide, poly(amido-D-glucose)-block-poly(beta-L-lysine), with high yield and purity by one-shot one-pot anionic-ring opening (co)polymerization. The co-beta-peptide is bactericidal against methicillin-resistant Staphylococcus aureus (MRSA), including replicating, biofilm and persister bacterial cells, and also disperses biofilm biomass. It is active towards community-acquired and hospital-associated MRSA strains which are resistant to multiple drugs including vancomycin and daptomycin. Its antibacterial activity is superior to that of vancomycin in MRSA mouse and human ex vivo skin infection models, with no acute in vivo toxicity in repeated dosing in mice at above therapeutic levels. The copolymer displays bacteria-activated surfactant-like properties, resulting from contact with the bacterial envelope. Our results indicate that this class of non-toxic molecule, effective against different bacterial sub-populations, has promising potential for the treatment of S. aureus infections.

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Garlic Extract (Allium sativum L.) Effectively Inhibits Staphylococcus aureus and Escherichia coli by Invitro Test

Tropical Health and Medical Research ◽

10.35916/thmr.v0i0.22 ◽

2020 ◽

Vol 2 (2) ◽

pp. 61-68

Author(s):

Agnina Listya Anggraini ◽

Ratih Dewi Dwiyanti ◽

Anny Thuraidah

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Bacterial Infections ◽

Allium Sativum ◽

Antibacterial Properties ◽

Herbal Medicines ◽

Garlic Extract ◽

Dilution Method ◽

Initial Stage

Infection is a disease caused by the presence of pathogenic microbes, including Staphylococcus aureus and Escherichia coli. Garlic (Allium sativum L.) has chemical contents such as allicin, alkaloids, flavonoids, saponins, tannins, and steroids, which can function as an antibacterial against Staphylococcus aureus and Escherichia coli. This study aims to determine the antibacterial properties of garlic extract powder against Staphylococcus aureus and Escherichia coli. This research is the initial stage of the development of herbal medicines to treat Staphylococcus aureus and Escherichia coli infections. The antibacterial activity test was carried out by the liquid dilution method. The concentrations used were 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL and 70 mg/mL. The results showed that the Minimum Inhibitory Concentration (MIC) against Staphylococcus aureus and Escherichia coli was 40 mg/mL and 50 mg / mL. Minimum Bactericidal Concentration (MBC) results for Staphylococcus aureus and Escherichia coli are 50 mg/mL and 70 mg/mL. Based on the Simple Linear Regression test, the R2 value of Staphylococcus aureus and Escherichia coli is 0.545 and 0.785, so it can be concluded that there is an effect of garlic extract powder on the growth of Staphylococcus aureus and Escherichia coli by 54.5% and 78.5%. Garlic (Allium sativum L.) extract powder has potential as herbal medicine against bacterial infections but requires further research to determine its effect in vivo.

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A novel application of bubble-eye strain of Carassius auratus for ex vivo fish immunological studies

Scientific Reports ◽

10.1038/s41598-021-89882-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hiroto Nakajima ◽

Atsushi Miyashita ◽

Hiroshi Hamamoto ◽

Kazuhisa Sekimizu

Keyword(s):

Inflammatory Cytokines ◽

Immune Cells ◽

Ex Vivo ◽

Large Bubble ◽

Suitable Model ◽

Bacterial Cells ◽

Functional Impairments ◽

Gene Expressions ◽

Pro Inflammatory Cytokines

AbstractIn this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at elevated temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and elevated rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

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Rabbit model of Staphylococcus aureus implant-associated spinal infection

Disease Models & Mechanisms ◽

10.1242/dmm.045385 ◽

2020 ◽

Vol 13 (7) ◽

pp. dmm045385

Author(s):

Oren Gordon ◽

Robert J. Miller ◽

John M. Thompson ◽

Alvaro A. Ordonez ◽

Mariah H. Klunk ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Bone Remodeling ◽

Ex Vivo ◽

Rabbit Model ◽

Pedicle Screws ◽

Lumbar Vertebra ◽

Wound Dehiscence ◽

Spinal Infection ◽

Positron Emission

ABSTRACTPost-surgical implant-associated spinal infection is a devastating complication commonly caused by Staphylococcus aureus. Biofilm formation is thought to reduce penetration of antibiotics and immune cells, contributing to chronic and difficult-to-treat infections. A rabbit model of a posterior-approach spinal surgery was created, in which bilateral titanium pedicle screws were interconnected by a plate at the level of lumbar vertebra L6 and inoculated with a methicillin-resistant S.aureus (MRSA) bioluminescent strain. In vivo whole-animal bioluminescence imaging (BLI) and ex vivo bacterial cultures demonstrated a peak in bacterial burden by day 14, when wound dehiscence occurred. Structures suggestive of biofilm, visualized by scanning electron microscopy, were evident up to 56 days following infection. Infection-induced inflammation and bone remodeling were also monitored using 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) and computed tomography (CT). PET imaging signals were noted in the soft tissue and bone surrounding the implanted materials. CT imaging demonstrated marked bone remodeling and a decrease in dense bone at the infection sites. This rabbit model of implant-associated spinal infection provides a valuable preclinical in vivo approach to investigate the pathogenesis of implant-associated spinal infections and to evaluate novel therapeutics.

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TSPphg Lysin from the Extremophilic Thermus Bacteriophage TSP4 as a Potential Antimicrobial Agent against Both Gram-Negative and Gram-Positive Pathogenic Bacteria

Viruses ◽

10.3390/v12020192 ◽

2020 ◽

Vol 12 (2) ◽

pp. 192 ◽

Cited By ~ 6

Author(s):

Feng Wang ◽

Xinyu Ji ◽

Qiupeng Li ◽

Guanling Zhang ◽

Jiani Peng ◽

...

Keyword(s):

Bacterial Infections ◽

Pathogenic Bacteria ◽

Infection Model ◽

Bacterial Cells ◽

Skin Damage ◽

Gram Positive ◽

Antibiotic Resistant ◽

Gram Negative

New strategies against antibiotic-resistant bacterial pathogens are urgently needed but are not within reach. Here, we present in vitro and in vivo antimicrobial activity of TSPphg, a novel phage lysin identified from extremophilic Thermus phage TSP4 by sequencing its whole genome. By breaking down the bacterial cells, TSPphg is able to cause bacteria destruction and has shown bactericidal activity against both Gram-negative and Gram-positive pathogenic bacteria, especially antibiotic-resistant strains of Klebsiella pneumoniae, in which the complete elimination and highest reduction in bacterial counts by greater than 6 logs were observed upon 50 μg/mL TSPphg treatment at 37 °C for 1 h. A murine skin infection model further confirmed the in vivo efficacy of TSPphg in removing a highly dangerous and multidrug-resistant Staphylococcus aureus from skin damage and in accelerating wound closure. Together, our findings may offer a therapeutic alternative to help fight bacterial infections in the current age of mounting antibiotic resistance, and to shed light on bacteriophage-based strategies to develop novel anti-infectives.

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Efficacy of Bacteriophages Against Staphylococcus aureus Isolates from Bovine Mastitis

Pharmaceuticals ◽

10.3390/ph13030035 ◽

2020 ◽

Vol 13 (3) ◽

pp. 35 ◽

Cited By ~ 2

Author(s):

Isabel Titze ◽

Tatiana Lehnherr ◽

Hansjörg Lehnherr ◽

Volker Krömker

Keyword(s):

Staphylococcus Aureus ◽

Host Range ◽

Bacterial Infections ◽

Plaque Assay ◽

Bovine Milk ◽

Bovine Mastitis ◽

Raw Milk ◽

Pasteurized Milk

The lytic efficacy of bacteriophages against Staphylococcus aureus isolates from bovine milk was investigated in vitro, regarding possible applications in the therapy of udder inflammation caused by bacterial infections (mastitis). The host range of sequenced, lytic bacteriophages was determined against a collection of 92 Staphylococcus (S.) aureus isolates. The isolates originated from quarter foremilk samples of clinical and subclinical mastitis cases. A spot test and a subsequent plaque assay were used to determine the phage host range. According to their host range, propagation and storage properties, three phages, STA1.ST29, EB1.ST11, and EB1.ST27, were selected for preparing a bacteriophage mixture (1:1:1), which was examined for its lytic activity against S. aureus in pasteurized and raw milk. It was found that almost two thirds of the isolates could be lysed by at least one of the tested phages. The bacteriophage mixture was able to reduce the S. aureus germ density in pasteurized milk and its reduction ability was maintained in raw milk, with only a moderate decrease compared to the results in pasteurized milk. The significant reduction ability of the phage mixture in raw milk promotes further in vivo investigation.

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Enhancement in Site-Specific Delivery of Carvacrol against Methicillin Resistant Staphylococcus aureus Induced Skin Infections Using Enzyme Responsive Nanoparticles: A Proof of Concept Study

Pharmaceutics ◽

10.3390/pharmaceutics11110606 ◽

2019 ◽

Vol 11 (11) ◽

pp. 606 ◽

Cited By ~ 8

Author(s):

Maria Mir ◽

Naveed Ahmed ◽

Andi Dian Permana ◽

Aoife Maria Rodgers ◽

Ryan F. Donnelly ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Methicillin Resistant Staphylococcus Aureus ◽

Ex Vivo ◽

Infection Model ◽

Skin Infections ◽

Site Specific ◽

Methicillin Resistant ◽

Hydrogel Matrix ◽

Skin Retention

Methicillin resistant Staphylococcus aureus (MRSA) induced skin infections have become a challenging problem due to the escalating antibiotic resistance. Carvacrol (CAR) has been reported to be effective against MRSA. However, due to its characteristics, CAR exhibits low skin retention. In this study, CAR was formulated into site-specific nanoparticle (NPs) delivery system using poly(ε-caprolactone) (PCL), following incorporation into a hydrogel matrix to facilitate dermal delivery. The release study exhibited significantly higher release of CAR from PCL NPs in the presence of bacterial lipase, highlighting its potential for differential delivery. Moreover, encapsulation of CAR in PCL NPs resulted in a two-fold increase in its anti-MRSA activity. Dermatokinetic studies revealed that the NPs loaded hydrogel was able to enhance skin retention of CAR after 24 h (83.29 ± 3.15%), compared to free CAR-loaded hydrogel (0.85 ± 0.14%). Importantly, this novel approach exhibited effective antimicrobial activity in an ex-vivo skin infection model. Hence, these findings have proven the concept that the loading of CAR into a responsive NPs system can lead to sustained antimicrobial effect at the desired site, and may provide a novel effective approach for treatment of MRSA induced skin infections. However, further studies must be conducted to investigate in-vivo efficacy of the developed system in an appropriate infection model.

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The purine biosynthesis regulator PurR moonlights as a virulence regulator in Staphylococcus aureus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904280116 ◽

2019 ◽

Vol 116 (27) ◽

pp. 13563-13572 ◽

Cited By ~ 9

Author(s):

William E. Sause ◽

Divya Balasubramanian ◽

Irnov Irnov ◽

Richard Copin ◽

Mitchell J. Sullivan ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factors ◽

Ex Vivo ◽

Purine Biosynthesis ◽

In Vivo Studies ◽

Human Neutrophils ◽

Infection Models ◽

Genes Encoding ◽

Virulence Regulator

The pathogen Staphylococcus aureus colonizes and infects a variety of different sites within the human body. To adapt to these different environments, S. aureus relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in S. aureus remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator purR resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the purR mutant was still highly virulent in the presence of mutations that disrupt PurR’s canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of purR. Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that S. aureus toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the purR mutant. Thus, S. aureus repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis.

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RNA III Inhibiting Peptide Inhibits In Vivo Biofilm Formation by Drug-Resistant Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.6.1979-1983.2003 ◽

2003 ◽

Vol 47 (6) ◽

pp. 1979-1983 ◽

Cited By ~ 85

Author(s):

Andrea Giacometti ◽

Oscar Cirioni ◽

Yael Gov ◽

Roberto Ghiselli ◽

Maria Simona Del Prete ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Quorum Sensing ◽

Medical Devices ◽

Biofilm Formation ◽

Bacterial Infections ◽

Drug Resistant ◽

Dacron Graft

ABSTRACT Staphylococcus aureus is a prevalent cause of bacterial infections associated with indwelling medical devices. RNA III inhibiting peptide (RIP) is known to inhibit S. aureus pathogenesis by disrupting quorum-sensing mechanisms. RIP was tested in the present study for its ability to inhibit S. aureus biofilm formation in a rat Dacron graft model. The activity of RIP was synergistic with those of antibiotics for the complete prevention of drug-resistant S. aureus infections.

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Impact of Bicarbonate-β-Lactam Exposures on Methicillin-Resistant Staphylococcus aureus (MRSA) Gene Expression in Bicarbonate-β-Lactam-Responsive vs. Non-Responsive Strains

Genes ◽

10.3390/genes12111650 ◽

2021 ◽

Vol 12 (11) ◽

pp. 1650

Author(s):

Selvi C. Ersoy ◽

Blake M. Hanson ◽

Richard A. Proctor ◽

Cesar A. Arias ◽

Truc T. Tran ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Methicillin Resistant Staphylococcus Aureus ◽

Ex Vivo ◽

In Vivo Models ◽

Methicillin Resistant ◽

Genetic Perturbations ◽

Functional Relevance

Methicillin-resistant Staphylococcus aureus (MRSA) infections represent a difficult clinical treatment issue. Recently, a novel phenotype was discovered amongst selected MRSA which exhibited enhanced β-lactam susceptibility in vitro in the presence of NaHCO3 (termed ‘NaHCO3-responsiveness’). This increased β-lactam susceptibility phenotype has been verified in both ex vivo and in vivo models. Mechanistic studies to-date have implicated NaHCO3-mediated repression of genes involved in the production, as well as maturation, of the alternative penicillin-binding protein (PBP) 2a, a necessary component of MRSA β-lactam resistance. Herein, we utilized RNA-sequencing (RNA-seq) to identify genes that were differentially expressed in NaHCO3-responsive (MRSA 11/11) vs. non-responsive (COL) strains, in the presence vs. absence of NaHCO3-β-lactam co-exposures. These investigations revealed that NaHCO3 selectively repressed the expression of a cadre of genes in strain 11/11 known to be a part of the sigB-sarA-agr regulon, as well as a number of genes involved in the anchoring of cell wall proteins in MRSA. Moreover, several genes related to autolysis, cell division, and cell wall biosynthesis/remodeling, were also selectively impacted by NaHCO3-OXA exposure in the NaHCO3-responsive strain MRSA 11/11. These outcomes provide an important framework for further studies to mechanistically verify the functional relevance of these genetic perturbations to the NaHCO3-responsiveness phenotype in MRSA.

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Predilection for developing a hematogenous orthopaedic implant-associated infection in older versus younger mice

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-021-02594-0 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

John M. Thompson ◽

Alyssa G. Ashbaugh ◽

Yu Wang ◽

Robert J. Miller ◽

Julie E. Pickett ◽

...

Keyword(s):

At Risk ◽

Staphylococcus Aureus ◽

Mouse Model ◽

Ex Vivo ◽

Kirschner Wire ◽

Age Groups ◽

Orthopaedic Implant ◽

X Ray ◽

Risk Patients

Abstract Background The pathogenesis of hematogenous orthopaedic implant-associated infections (HOIAI) remains largely unknown, with little understanding of the influence of the physis on bacterial seeding. Since the growth velocity in the physis of long bones decreases during aging, we sought to evaluate the role of the physis on influencing the development of Staphylococcus aureus HOIAI in a mouse model comparing younger versus older mice. Methods In a mouse model of HOIAI, a sterile Kirschner wire was inserted retrograde into the distal femur of younger (5–8-week-old) and older (14–21-week-old) mice. After a 3-week convalescent period, a bioluminescent Staphylococcus aureus strain was inoculated intravenously. Bacterial dissemination to operative and non-operative legs was monitored longitudinally in vivo for 4 weeks, followed by ex vivo bacterial enumeration and X-ray analysis. Results In vivo bioluminescence imaging and ex vivo CFU enumeration of the bone/joint tissue demonstrated that older mice had a strong predilection for developing a hematogenous infection in the operative legs but not the non-operative legs. In contrast, this predilection was less apparent in younger mice as the infection occurred at a similar rate in both the operative and non-operative legs. X-ray imaging revealed that the operative legs of younger mice had decreased femoral length, likely due to the surgical and/or infectious insult to the more active physis, which was not observed in older mice. Both age groups demonstrated substantial reactive bone changes in the operative leg due to infection. Conclusions The presence of an implant was an important determinant for developing a hematogenous orthopaedic infection in older but not younger mice, whereas younger mice had a similar predilection for developing periarticular infection whether or not an implant was present. On a clinical scale, diagnosing HOIAI may be difficult particularly in at-risk patients with limited examination or other data points. Understanding the influence of age on developing HOIAI may guide clinical surveillance and decision-making in at-risk patients.

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