The effect of N-acetylcysteine in a combined antibiofilm treatment against antibiotic-resistant Staphylococcus aureus

2020 ◽  
Vol 75 (7) ◽  
pp. 1787-1798
Author(s):  
Arthika Manoharan ◽  
Theerthankar Das ◽  
Gregory S Whiteley ◽  
Trevor Glasbey ◽  
Frederik H Kriel ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Cleavage ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Combination Treatments ◽  
Resazurin Assay ◽  
Biofilm Architecture ◽  
Biofilm Disruption ◽  
The Uk

Abstract Background The WHO declared Staphylococcus aureus as a ‘pathogen of high importance’ in 2017. One-fifth of all bloodstream-related infections in Australia and 12 000 cases of bacteraemia in the UK (2017–18) were caused by the MRSA variant. To address the need for novel therapies, we investigated several permutations of an innovative combination therapy containing N-acetylcysteine (NAC), an antibiotic and an enzyme of choice in eradicating MRSA and MSSA biofilms. Methods Biofilm viability (resazurin assay) and colony count methods were used to investigate the effect of NAC, antibiotics and enzymes on S. aureus biofilm disruption and killing. The effects of NAC and enzymes on the polysaccharide content of biofilm matrices were analysed using the phenol/sulphuric acid method and the effect of NAC on DNA cleavage was determined using the Qubit fluorometer technique. Changes in biofilm architecture when subjected to NAC and enzymes were visualized using confocal laser scanning microscopy (CLSM). Results NAC alone displayed bacteriostatic effects when tested on planktonic bacterial growth. Combination treatments containing 30 mM NAC resulted in ≥90% disruption of biofilms across all MRSA and MSSA strains with a 2–3 log10 decrease in cfu/mL in treated biofilms. CLSM showed that NAC treatment drastically disrupted S. aureus biofilm architecture. There was also reduced polysaccharide production in MRSA biofilms in the presence of NAC. Conclusions Our results indicate that inclusion of NAC in a combination treatment is a promising strategy for S. aureus biofilm eradication. The intrinsic acidity of NAC was identified as key to maximum biofilm disruption and degradation of matrix components.

scholarly journals Individual or Combined Effects of Meropenem, Imipenem, Sulbactam, Colistin, and Tigecycline on Biofilm-Embedded Acinetobacter baumannii and Biofilm Architecture

2016 ◽  
Vol 60 (8) ◽  
pp. 4670-4676 ◽  
Author(s):  
Yung-Chih Wang ◽  
Shu-Chen Kuo ◽  
Ya-Sung Yang ◽  
Yi-Tzu Lee ◽  
Chun-Hsiang Chiu ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Laser Scanning ◽  
Bactericidal Effect ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Roughness Coefficient ◽  
Content Type ◽  
Biofilm Thickness ◽  
Carbapenem Resistant ◽  
Biofilm Architecture

ABSTRACTAcinetobacter baumanniibiofilms are difficult to eradicate. We investigated the effects of meropenem (2 mg/liter), imipenem (2 mg/liter), sulbactam (4 mg/liter), colistin (2 mg/liter), and tigecycline (2 mg/liter), alone or in combination, on biofilm-embedded carbapenem-resistant and carbapenem-susceptibleA. baumannii(CRAb and CSAb, respectively) cells, as well as on the architecture of the biofilms.A. baumanniiATCC 15151 (Ab15151) and its OXA-82-overproducing transformant, along with two clinical CSAb and two clinical CRAb isolates of differing clonalities, were used. The minimal bactericidal concentrations for biofilm-embedded cells of the six tested isolates were >50-fold those of their planktonic cells. When used individually, meropenem exhibited a higher killing effect than the other four antimicrobials on biofilm-embedded CSAb cells in the colony biofilm assay. For two clinical CRAb isolates, meropenem plus sulbactam or sulbactam plus tigecycline showed >100-fold the bactericidal effect exhibited by these agents used alone after 48 h of treatment. The effect of antimicrobials on the architecture of Ab15151 biofilm emitting green fluorescence was determined by confocal laser scanning microscopy using COMSTAT software. Significant decreases in the maximum biofilm thickness were observed after exposure to meropenem and imipenem. Meropenem plus sulbactam significantly decreased the biomass and mean thickness and increased the roughness coefficient of biofilms, but sulbactam plus tigecycline only decreased the maximum and mean biofilm thickness compared to any of these agents used alone. Meropenem was active against biofilm-embedded CSAb, whereas meropenem plus sulbactam exhibited synergism against biofilm-embedded CRAb and caused significantly more damage to the biofilm architecture than did any of the agents used alone.

scholarly journals High-Throughput Screening for Small-Molecule Inhibitors of Staphylococcus epidermidis RP62a Biofilms

2013 ◽  
Vol 18 (7) ◽  
pp. 820-829 ◽  
Author(s):  
Warunya Panmanee ◽  
Deborah Taylor ◽  
Chloe J. A. Shea ◽  
Hong Tang ◽  
Sandra Nelson ◽  
...  
Keyword(s):  
High Throughput ◽  
Staphylococcus Epidermidis ◽  
High Throughput Screening ◽  
Laser Scanning ◽  
Bacterial Biofilm ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Response Curves ◽  
Resazurin Assay ◽  
Biofilm Bacteria

High-throughput screening (HTS) of 42 865 compounds was performed to identify compounds that inhibit formation of or kill Staphylococcus epidermidis RP62a biofilms. Three biological processes were assayed, including (1) growth of planktonic/biofilm bacteria, (2) assessment of metabolically active biofilm bacteria using a resazurin assay, and (3) assessment of biofilm biomass by crystal violet staining. After completing the three tiers (primary screening, hit confirmation, and dose-response curves), 352 compounds (representing ~0.8%) were selected as confirmed hit compounds from the HTS assay. The compounds were divided into groups based on their effectiveness on S. epidermidis biofilm properties. The majority of these affected both inhibition and killing of bacterial biofilm cultures. Only 16 of the confirmed hit compounds that have either an AC50 lower than 10 µM and/or Sconst ≥70 from those processed were selected for further study by confocal laser scanning microscopy (CLSM). The CLSM was used to evaluate the confirmed hit compounds on (1) inhibition of biofilm formation and (2) killing of preexisting S. epidermidis biofilms. Taken together, with further testing (e.g., disease-related conditions), such compounds may have applications as broad antimicrobial/antibiofilm use for prophylactic or therapeutic intervention to combat infections in surgical and intensive care clinics and battlefield settings.

scholarly journals Staphylococcus aureus Interferes with Streptococci Spatial Distribution and with Protein Expression of Species within a Polymicrobial Oral Biofilm

Antibiotics ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 116
Author(s):  
Etyene Schnurr ◽  
Pune N. Paqué ◽  
Thomas Attin ◽  
Paolo Nanni ◽  
Jonas Grossmann ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Laser Scanning ◽  
Bacterial Species ◽  
Fusobacterium Nucleatum ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Oral Diseases ◽  
Streptococcus Oralis ◽  
Oral Environment ◽  
Associated Species

We asked whether transient Staphylococcus aureus in the oral environment synergistically interacts with orally associated bacterial species such as Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans, and Veillonella dispar (six-species control biofilm 6S). For this purpose, four modified biofilms with seven species that contain either the wild type strain of the S. aureus genotype (USA300-MRSA WT), its isogenic mutant with MSCRAMM deficiency (USA300-MRSA ΔMSCRAMM), a methicillin-sensitive S. aureus (ST72-MSSA-) or a methicillin-resistant S. aureus (USA800-MRSA) grown on hydroxyapatite disks were examined. Culture analyses, confocal-laser-scanning microscopy and proteome analyses were performed. S. aureus strains affected the amount of supragingival biofilm-associated species differently. The deletion of MSCRAMM genes disrupted the growth of S. aureus and the distribution of S. mutans and S. oralis within the biofilms. In addition, S. aureus caused shifts in the number of detectable proteins of other species in the 6S biofilm. S. aureus (USA300-MRSA WT), aggregated together with early colonizers such as Actinomyces and streptococci, influenced the number of secondary colonizers such as Fusobacterium nucleatum and was involved in structuring the biofilm architecture that triggered the change from a homeostatic biofilm to a dysbiotic biofilm to the development of oral diseases.

scholarly journals Subinhibitory Concentrations of Mupirocin Stimulate Staphylococcus aureus Biofilm Formation by Upregulating cidA

2020 ◽  
Vol 64 (3) ◽  
Author(s):  
Ye Jin ◽  
Yinjuan Guo ◽  
Qing Zhan ◽  
Yongpeng Shang ◽  
Di Qu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Multidrug Resistant ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Content Type ◽  
Subinhibitory Concentrations ◽  
Biofilm Development

ABSTRACT Previous studies have shown that the administration of antibiotics at subinhibitory concentrations stimulates biofilm formation by the majority of multidrug-resistant Staphylococcus aureus (MRSA) strains. Here, we investigated the effect of subinhibitory concentrations of mupirocin on biofilm formation by the community-associated (CA) mupirocin-sensitive MRSA strain USA300 and the highly mupirocin-resistant clinical S. aureus SA01 to SA05 isolates. We found that mupirocin increased the ability of MRSA cells to attach to surfaces and form biofilms. Confocal laser scanning microscopy (CLSM) demonstrated that mupirocin treatment promoted thicker biofilm formation, which also correlated with the production of extracellular DNA (eDNA). Furthermore, quantitative real-time PCR (RT-qPCR) results revealed that this effect was largely due to the involvement of holin-like and antiholin-like proteins (encoded by the cidA gene), which are responsible for modulating cell death and lysis during biofilm development. We found that cidA expression levels significantly increased by 6.05- to 35.52-fold (P < 0.01) after mupirocin administration. We generated a cidA-deficient mutant of the USA300 S. aureus strain. Exposure of the ΔcidA mutant to mupirocin did not result in thicker biofilm formation than that in the parent strain. We therefore hypothesize that the mupirocin-induced stimulation of S. aureus biofilm formation may involve the upregulation of cidA.

scholarly journals Photodynamic Action of LED-Activated Curcumin againstStaphylococcus aureusInvolving Intracellular ROS Increase and Membrane Damage

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Yuan Jiang ◽  
Albert Wingnang Leung ◽  
Heyu Hua ◽  
Xiancai Rao ◽  
Chuanshan Xu
Keyword(s):  
Staphylococcus Aureus ◽  
Membrane Permeability ◽  
Blue Light ◽  
Laser Scanning ◽  
Membrane Damage ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Photodynamic Action ◽  
Intracellular Ros

Aim. To investigate the effect of photodynamic action of LED-activated curcumin on cell viability, membrane permeability, and intracellular reactive oxygen species ofStaphylococcus aureus.Methods.Staphylococcus aureuswas incubated with the different concentrations of curcumin for 60 min and then irradiated by blue light with the wavelength of 470 nm and with light dose of 3 J/cm2. The colony forming unit assay was used to investigate photocytotoxicity of curcumin onStaphylococcus aureus, confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) for assaying membrane permeability, FCM analysis with DCFH-DA staining for measuring the intracellular ROS level, and transmission electron microscopy (TEM) for observing morphology and structure.Results. Blue light-activated curcumin significantly killedStaphylococcus aureusin a curcumin dose-dependent manner. TEM observed remarkable structural damages inS. aureusafter light-activated curcumin. More red fluorescence of PI dye was found inS. aureustreated by blue light-activated curcumin than in those of the controlled bacterial cells. Intracellular ROS increase was observed after light-activated curcumin.Conclusion. Blue light-activated curcumin markedly damaged membrane permeability, resulting in cell death ofStaphylococcus aureusand highlighted that intracellular ROS increase might be an important event in photodynamic killing ofStaphylococcus aureusin the presence of curcumin.

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.

scholarly journals Role of Exopolysaccharides in Pseudomonas aeruginosa Biofilm Formation and Architecture

2011 ◽  
Vol 77 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Aamir Ghafoor ◽  
Iain D. Hay ◽  
Bernd H. A. Rehm
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Bacterial Biofilm ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Content Type ◽  
Biofilm Architecture

ABSTRACTPseudomonas aeruginosais an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslAΔalg8overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture ofP. aeruginosa.

scholarly journals Synergistic Antibiofilm Effects of Ultrasound and Phenyllactic Acid against Staphylococcus aureus and Salmonella enteritidis

Foods ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2171
Author(s):  
Jiaojiao Zhang ◽  
Debao Wang ◽  
Jinyue Sun ◽  
Zhilan Sun ◽  
Fang Liu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Respiratory Chain ◽  
Laser Scanning ◽  
Salmonella Enteritidis ◽  
Membrane Integrity ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Bacterial Cells ◽  
Phenyllactic Acid ◽  
Cell Membrane Integrity

This study evaluated the effect of the combination of ultrasound and phenyllactic acid (PLA) on inactivating Staphylococcus aureus and Salmonella enteritidis biofilm cells and determined the possible antibiofilm mechanism. S. aureus and S. enteritidis biofilm cells were separately treated with ultrasound (US, 270 W), phenyllactic acid (PLA, 0.5% and 1%), and their combination (US + 0.5% PLA, and US + 1% PLA) for 5, 10, 20, 30, and 60 min. Biofilm inactivation, polysaccharide, and respiratory chain dehydrogenase assays were conducted. US and PLA had a synergistic effect on inactivating bacterial cells in S. aureus and S. enteritidis biofilms. The combination of US and PLA significantly decreased the contents of soluble and insoluble polysaccharides and the activity of respiratory chain dehydrogenase in the biofilm cells compared to the single treatment. Confocal laser scanning microscopy, scanning electron microscopy, and intracellular adenosine-triphosphate (ATP) analyses indicated that the combination of US and PLA seriously destroyed the cell membrane integrity of the S. aureus and S. enteritidis biofilms and caused the leakage of intracellular ATP. These findings demonstrated the synergistic antibiofilm effect of US combined with PLA and offered a research basis for its application in the food industry.

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