scholarly journals Synergistic action of phage phiIPLA-RODI and lytic protein CHAPSH3b: a combination strategy to target Staphylococcus aureus biofilms

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Ana Catarina Duarte ◽  
Lucía Fernández ◽  
Vincent De Maesschalck ◽  
Diana Gutiérrez ◽  
Ana Belén Campelo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Population ◽  
Ex Vivo ◽  
Viable Cell ◽  
Combination Strategy ◽  
Initial Exposure ◽  
Cell Counts ◽  
The Individual ◽  
Inert Surfaces ◽  
Time Kill

AbstractStaphylococcus aureus is considered a priority pathogen due to its increasing acquisition of antibiotic resistance determinants. Additionally, this microbe has the ability to form recalcitrant biofilms on different biotic and inert surfaces. In this context, bacteriophages and their derived lytic proteins may be a forward-looking strategy to help combat staphylococcal biofilms. However, these antimicrobials exhibit individual limitations that may be overcome by combining them with other compounds. This work investigates the combination of a phage-derived lytic protein, CHAPSH3b, and the virulent bacteriophage phiIPLA-RODI. The obtained results show the synergy between both antimicrobials for the treatment of 24-h-old S. aureus biofilms, with greater reductions in viable cell counts observed when phage and lysin are applied together compared to the individual treatments. Time-kill curves and confocal microscopy revealed that the fast antibacterial action of CHAPSH3b reduces the population up to 7 hours after initial exposure, which is subsequently followed by phage predation, limiting regrowth of the bacterial population. Moreover, at least 90% of bacteriophage insensitive mutants are susceptible to the lytic protein. Therefore, CHAPSH3b might help curtail the development of phage resistance during treatment. The combination of the lysin and phiIPLA-RODI also showed promising results in an ex vivo pig skin model of wound infection. Overall, the results of this study demonstrate that the combination of phage-derived lytic proteins and bacteriophages can be a viable strategy to develop improved antibiofilm products.

scholarly journals Response of Listeria monocytogenes to Disinfection Stress at the Single-Cell and Population Levels as Monitored by Intracellular pH Measurements and Viable-Cell Counts

2009 ◽  
Vol 75 (13) ◽  
pp. 4550-4556 ◽  
Author(s):  
Vicky G. Kastbjerg ◽  
Dennis S. Nielsen ◽  
Nils Arneborg ◽  
Lone Gram
Keyword(s):  
Listeria Monocytogenes ◽  
Single Cell ◽  
Intracellular Ph ◽  
Bacterial Population ◽  
Single Cells ◽  
Viable Cell ◽  
Population Subdivision ◽  
Single Cell Level ◽  
Cell Level ◽  
Cell Counts

ABSTRACT Listeria monocytogenes has a remarkable ability to survive and persist in food production environments. The purpose of the present study was to determine if cells in a population of L. monocytogenes differ in sensitivity to disinfection agents as this could be a factor explaining persistence of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level by measuring intracellular pH (pHi) over time by fluorescence ratio imaging microscopy. pHi values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pHi was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pHi 5 to 5.5) (P < 0.05). The protective effect of NaCl was reflected by viable-cell counts at a higher concentration of Incimaxx (0.0031%), where the salt-grown population survived better than the population grown without NaCl (P < 0.05). NaCl protected attached cells through drying but not during disinfection. This study indicates that a population of L. monocytogenes cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present.

scholarly journals Unpredictable Effects of Rifampin as an Adjunctive Agent in Elimination of Rifampin-Susceptible and -Resistant Staphylococcus aureus Strains Grown in Biofilms

2010 ◽  
Vol 54 (9) ◽  
pp. 3907-3912 ◽  
Author(s):  
Sander Croes ◽  
Patrick S. Beisser ◽  
Cees Neef ◽  
Cathrien A. Bruggeman ◽  
Ellen E. Stobberingh
Keyword(s):  
Staphylococcus Aureus ◽  
Genetic Background ◽  
Viable Cell ◽  
Viable Count ◽  
Cell Counts ◽  
Resistant Strains ◽  
Resistant Mutants ◽  
Static Conditions ◽  
Additional Value ◽  
Count Change

ABSTRACT The use of rifampin as an adjunct in biofilm-associated infections is based on the ability to penetrate into biofilms and a presumed activity against dormant bacteria. Yet, its efficacy remains contradictory, and rifampin-resistant strains frequently emerge during therapy. Therefore, the efficacy against rifampin-susceptible and isogenic rifampin-resistant methicillin-susceptible Staphylococcus aureus (MSSA) strains was evaluated. Biofilms were generated under static conditions using MSSA with various genetic backgrounds. Oxacillin alone or with rifampin at various concentrations was subsequently added, and after 24 h biomass and viable cell counts were determined. Upon rifampin addition, interstrain variations in viable count change, ranging from a tendency toward antagonism to synergy, were observed among all strains tested, irrespective of the genetic background of the strain. Similar variations were observed in changes in biomass. The decrease in viable count upon rifampin addition was negatively correlated to formation of large amounts of biomass, since strains embedded by more biomass showed a diminished reduction in viable count. Rifampin (1 μg/ml) as adjunct to oxacillin achieved greater reductions in biomass produced by most rifampin-susceptible isolates, ranging from 17 to 54%, compared to 4% for oxacillin alone. In contrast, rifampin had no additional value in reduction of biomass of isogenic rifampin-resistant mutants. At subinhibitory concentrations of rifampin (0.008 μg/ml), none of the strains tested yielded an extra reduction in biomass that was ≥40%. In conclusion, the effects of rifampin as adjunct on biomass and viable count were unpredictable, and the use of rifampin against biofilm containing rifampin-resistant strains seems unwarranted.

scholarly journals Design and Selection of Engineered Lytic Proteins With Staphylococcus aureus Decolonizing Activity

2021 ◽  
Vol 12 ◽  
Author(s):  
Diana Gutiérrez ◽  
Lorena Rodríguez-Rubio ◽  
Patricia Ruas-Madiedo ◽  
Lucía Fernández ◽  
Ana Belén Campelo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Ex Vivo ◽  
Human Keratinocytes ◽  
Storage Conditions ◽  
Pig Skin ◽  
Low Protein ◽  
Staphylococcal Skin Infections ◽  
High Concentrations ◽  
Ph Range ◽  
Time Kill

Staphylococcus aureus causes various infections in humans and animals, the skin being the principal reservoir of this pathogen. The widespread occurrence of methicillin-resistant S. aureus (MRSA) limits the elimination and treatment of this pathogen. Phage lytic proteins have been proven as efficient antimicrobials against S. aureus. Here, a set of 12 engineered proteins based on endolysins were conceptualized to select the most optimal following a stepwise funnel approach assessing parameters including turbidity reduction, minimum inhibitory concentration (MIC), time-kill curves, and antibiofilm assays, as well as testing their stability in a broad range of storage conditions (pH, temperature, and ionic strength). The engineered phage lysins LysRODIΔAmi and ClyRODI-H5 showed the highest specific lytic activity (5 to 50 times higher than the rest), exhibited a shelf-life up to 6 months and remained stable at temperatures up to 50°C and in a pH range from 3 to 9. LysRODIΔAmi showed the lower MIC values against all staphylococcal strains tested. Both proteins were able to kill 6 log units of the strain S. aureus Sa9 within 5 min and could remove preformed biofilms (76 and 65%, respectively). Moreover, LysRODIΔAmi could prevent biofilm formation at low protein concentrations (0.15–0.6 μM). Due to its enhanced antibiofilm properties, LysRODIΔAmi was selected to effectively remove S. aureus contamination in both intact and disrupted keratinocyte monolayers. Notably, this protein did not demonstrate any toxicity toward human keratinocytes, even at high concentrations (22.1 μM). Finally, a pig skin ex vivo model was used to evaluate treatment of artificially contaminated pig skin using LysRODIΔAmi (16.5 μg/cm2). Following an early reduction of S. aureus, a second dose of protein completely eradicated S. aureus. Overall, our results suggest that LysRODIΔAmi is a suitable candidate as antimicrobial agent to prevent and treat staphylococcal skin infections.

scholarly journals Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 438 ◽  
Author(s):  
Nanna Olsen ◽  
Elowine Thiran ◽  
Tobias Hasler ◽  
Thomas Vanzieleghem ◽  
Georgios Belibasakis ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Laser Scanning ◽  
Combined Treatment ◽  
Viable Cell ◽  
Laser Scanning Microscopy ◽  
Enzyme Treatment ◽  
Confocal Laser ◽  
Cell Counts ◽  
Glass Surfaces ◽  
Individual Enzyme

Staphylococcus aureus is an important pathogen and biofilm former. Biofilms cause problems in clinics and food production and are highly recalcitrant to antibiotics and sanitizers. Bacteriophage endolysins kill bacteria by degrading their cell wall and are therefore deemed promising antimicrobials and anti-biofilm agents. Depolymerases targeting polysaccharides in the extracellular matrix have been suggested as parts of a multi-enzyme approach to eradicate biofilms. The efficacy of endolysins and depolymerases against S. aureus biofilms in static models has been demonstrated. However, there is a lack of studies evaluating their activity against biofilms grown under more realistic conditions. Here, we investigated the efficacy of the endolysin LysK and the poly-N-acetylglucosamine depolymerase DA7 against staphylococcal biofilms in static and dynamic (flow cell-based) models. LysK showed activity against multiple S. aureus strains, and both LysK and DA7 removed static and dynamic biofilms from polystyrene and glass surfaces at low micromolar and nanomolar concentrations, respectively. When combined, the enzymes acted synergistically, as demonstrated by crystal violet staining of static biofilms, significantly reducing viable cell counts compared to individual enzyme treatment in the dynamic model, and confocal laser scanning microscopy. Overall, our results suggest that LysK and DA7 are potent anti-biofilm agents, alone and in combination.

Assay of Enterocin AS-48 for Inhibition of Foodborne Pathogens in Desserts

2009 ◽  
Vol 72 (8) ◽  
pp. 1654-1659 ◽  
Author(s):  
PILAR MARTINEZ VIEDMA ◽  
HIKMATE ABRIOUEL ◽  
NABIL BEN OMAR ◽  
ROSARIO LUCAS LÓPEZ ◽  
EVA VALDIVIA ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Listeria Monocytogenes ◽  
Bacillus Cereus ◽  
Proteolytic Activity ◽  
Foodborne Pathogens ◽  
Viable Cell ◽  
Inoculum Size ◽  
Cell Counts

Enterocin AS-48 was tested against Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes in different kinds of desserts. The highest activity against S. aureus was detected in baker cream. However, in yogurt-type soy-based desserts and in gelatin pudding, AS-48 (175 arbitrary units [AU]/g) reduced viable cell counts of S. aureus by only 1.5 to 1.8 log units at most. The efficacy of AS-48 in puddings greatly depended on inoculum size, and viable S. aureus counts decreased below detection levels within 24 h for inocula lower than 4 to 5.5 log CFU/g. For L. monocytogenes, bacteriocin concentrations of 52.5 to 87.5 AU/g reduced viable counts below detection levels and avoided regrowth of survivors. The lowest activity was detected in yogurt-type desserts. For B. cereus, viable cell counts were reduced below detection levels for bacteriocin concentrations of 52.5 AU/g in instant pudding without soy or by 175 AU/g in the soy pudding. In gelatin pudding, AS-48 (175 AU/g) reduced viable cell counts of B. cereus below detection levels after 8 h at 10°C or after 48 h at 22°C. Bacteriocin addition also inhibited gelatin liquefaction caused by the proteolytic activity of B. cereus.

Effects of the Essential Oil from Origanum vulgare L. on Survival of Pathogenic Bacteria and Starter Lactic Acid Bacteria in Semihard Cheese Broth and Slurry

2016 ◽  
Vol 79 (2) ◽  
pp. 246-252 ◽  
Author(s):  
GEANY TARGINO de SOUZA ◽  
RAYSSA JULLIANE de CARVALHO ◽  
JOSSANA PEREIRA de SOUSA ◽  
JOSEAN FECHINE TAVARES ◽  
DONALD SCHAFFNER ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Essential Oil ◽  
Pathogenic Bacteria ◽  
Viable Cell ◽  
Inhibitory Effects ◽  
Origanum Vulgare ◽  
Cell Counts ◽  
Cheese Slurry

ABSTRACT This study assessed the inhibitory effects of the essential oil from Origanum vulgare L. (OVEO) on Staphylococcus aureus, Listeria monocytogenes, and a mesophilic starter coculture composed of lactic acid bacteria (Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris) in Brazilian coalho cheese systems. The MIC of OVEO was 2.5 μl/ml against both S. aureus and L. monocytogenes and 0.6 μl/ml against the tested starter coculture. In cheese broth containing OVEO at 0.6 μl/ml, no decrease in viable cell counts (VCC) of both pathogenic bacteria was observed, whereas the initial VCC of the starter coculture decreased approximately 1.0 log CFU/ml after 24 h of exposure at 10°C. OVEO at 1.25 and 2.5 μl/ml caused reductions of up to 2.0 and 2.5 log CFU/ml in S. aureus and L. monocytogenes ,respectively, after 24 h of exposure in cheese broth. At these same concentrations, OVEO caused a greater decrease of initial VCC of the starter coculture following 4 h of exposure. Higher concentrations of OVEO were required to decrease the VCC of all target bacteria in semisolid coalho cheese slurry compared with cheese broth. The VCC of Lactococcus spp. in coalho cheese slurry containing OVEO were always lower than those of pathogenic bacteria under the same conditions. These results suggest that the concentrations of OVEO used to control pathogenic bacteria in semihard cheese should be carefully evaluated because of its inhibitory effects on the growth of starter lactic acid cultures used during the production of the product.

scholarly journals In VitroAntibacterial and Time-Kill Evaluation of theErythrina caffraThunb. Extract against Bacteria Associated with Diarrhoea

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Olufunmiso Olusola Olajuyigbe ◽  
Anthony Jide Afolayan
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Count ◽  
Micrococcus Luteus ◽  
Ethanolic Extract ◽  
Viable Cell ◽  
Viable Cell Count ◽  
Proteus Vulgaris ◽  
Gastrointestinal Infections ◽  
Time Kill

The antibacterial activities of stem bark ethanolic extract ofErythrina caffraThunb. against bacteria in diarrhoea was determinedin vitroby the agar diffusion and dilution, macrobroth dilution, and time-kill assay methods. The result showed that the extract produced inhibition zones ranging between15±1.0 mm and23±1.0 mm, and the bacteria were susceptible at concentrations ranging between ≤100 and ≤1000 μg/mL. While the MICs of the extract ranged between 39.1 and 625 μg/mL, and the MBCs ranged between 78.1 and 625μg/mL, the MICs ofMicrococcus luteus,Proteus vulgarisCSIR 0030,Enterococcus faecalisKZN, andStaphylococcus aureusOK3were less than 100 μg/mL, and the mechanisms of antibiosis indicated that the crude ethanolic extract was highly bactericidal against the entire test bacteria isolates. In the time-kill assay, the averagelogreduction of the viable cell count ranged between0.916log 10and1.851log 10 cfu/mL on incubating the bacteria for 4 h at the MICs, while the reduction ranged between0.183log 10and1.105log 10 cfu/mL after 8 h of incubation. Incubating the bacteria for 4 h at 2 × MICs resulted in the reduction of the viable cell count to between −0.264log 10and0.961log 10 cfu/mL, while the averagelogreduction ranged between −3.968log 10and −0.425log 10 cfu/mL after 8 h of incubation withMicrococcus luteus,Proteus vulgarisCSIR 0030, andStaphylococcus aureusOK3being the most highly affected bacteria. The result showed that the extract exhibited broader-spectrum antibacterial activity and justifies the use ofErythrina caffrain the folkloric medicine for treating gastrointestinal infections in South Africa.

scholarly journals Bacteriophage-Antibiotic Combination Strategy: an Alternative against Methicillin-Resistant Phenotypes of Staphylococcus aureus

2020 ◽  
Vol 64 (7) ◽  
Author(s):  
Razieh Kebriaei ◽  
Katherine Lev ◽  
Taylor Morrisette ◽  
Kyle C. Stamper ◽  
Jacinda C. Abdul-Mutakabbir ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Membrane Vesicle ◽  
Phage Resistance ◽  
Combination Strategy ◽  
Methicillin Resistant ◽  
Content Type ◽  
Potential Impact ◽  
Mrsa Strains ◽  
Time Kill ◽  
Antibiotic Combination

ABSTRACT Comparative time-kill experiments with Staphylococcus aureus bacteriophage (phage) Sb-1 alone and phage-antibiotic combinations (PACs) against two methicillin-resistant S. aureus (MRSA) strains have shown synergy with both daptomycin-phage and vancomycin-phage combinations. PACs prevented development of phage resistance and demonstrated bactericidal activity for all triple combinations. In addition, the extracellular membrane vesicle (MV) formation and the potential impact of phage on MV suppression were examined. Our results demonstrate the potential of PAC for combating MRSA infections.

scholarly journals Bacteriophage AB-SA01 Cocktail in Combination with Antibiotics against MRSA-VISA Strain in an In Vitro Pharmacokinetic/Pharmacodynamic Model

2020 ◽  
Vol 65 (1) ◽  
pp. e01863-20
Author(s):  
Razieh Kebriaei ◽  
Katherine L. Lev ◽  
Kyle C. Stamper ◽  
Susan M. Lehman ◽  
Sandra Morales ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Ex Vivo ◽  
Pharmacodynamic Model ◽  
Bacterial Eradication ◽  
Methicillin Resistant ◽  
Content Type ◽  
Bacteriophage Resistance ◽  
Potential Activity ◽  
Time Kill

ABSTRACTThis study aimed to test the efficacy of bacteriophage-antibiotic combinations (BACs) in vitro in 24-h time-kill settings and in ex vivo simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic models for 96 h. BACs prevented the development of bacteriophage resistance, while some bacteriophage resistance emerged in bacteriophage-alone treatments. In addition, BACs resulted in an enhancement of bacterial eradication in SEV models. Our findings support the potential activity of BAC therapy for combating serious methicillin-resistant Staphylococcus aureus (MRSA) infections.

Exebacase, in Addition to Daptomycin against MRSA

2021 ◽  
Author(s):  
Razieh Kebriaei ◽  
Kyle C. Stamper ◽  
Katherine L. Lev ◽  
Taylor Morrisette ◽  
Jacinda C. Abdul-Mutakabbir ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Bactericidal Activity ◽  
Ex Vivo ◽  
Lytic Activity ◽  
Vegetation Model ◽  
Methicillin Resistant ◽  
Initial Inoculum ◽  
Mrsa Strains ◽  
Time Kill

Exebacase is a lysin (cell wall hydrolase) with direct lytic activity against Staphylococcus aureus including methicillin-resistant S. aureus (MRSA). Time kill analysis experiments illustrated bactericidal activity of exebacase-daptomycin, against MRSA strains MW2 and 494. Furthermore, exebacase in addition to daptomycin (10, 6 and 4 mg/kg/d) in a two-compartment ex-vivo pharmacokinetic/pharmacodynamic simulated endocardial vegetation model with humanized doses resulted in reductions of 6.01, 4.99 and 2.81 log 10 CFU/g (from initial inoculum) against MRSA strain MW2.

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