Efficacy of the Combination of Tobramycin and a Macrolide in an InVitroPseudomonas aeruginosa Mature Biofilm Model

ABSTRACT Respiratory disease is the main cause of morbidity and mortality in patients with cystic fibrosis (CF). In particular, patients suffer from chronic infection due to biofilm formation by opportunistic Pseudomonas aeruginosa (32). Therefore, there is an urgent need to develop alternative ways to treat biofilm-associated clinical infections. The aim of this study was to compare the antimicrobial effects in vitro of the combinations tobramycin-clarithromycin and tobramycin-azithromycin against five P. aeruginosa biofilms and to establish the most effective combination. We performed a kinetic study over a period of 28 days of a twice-daily coadministration of the combinations tobramycin-clarithromycin and tobramycin-azithromycin on 12-day-old, mature P. aeruginosa biofilms formed on microplate pegs for 4 clinical isolates and one laboratory strain (PAO1) to simulate the treatment of CF patients with tobramycin inhalation solution (TOBI) through aerosolization. A synergy between tobramycin and clarithromycin was recorded for 3/5 biofilms, with a bacterial decrease of more than 5 log. Conversely, we found an antagonistic activity when 4 μg/ml tobramycin was administered with azithromycin at 2 μg/ml for P. aeruginosa PAO1 and with azithromycin at 2, 20, 50, 100, and 200 μg/ml for P. aeruginosa PYO1. Treatment with tobramycin at 4 μg/ml combined with clarithromycin at 200 μg/ml eradicated all five biofilms, while tobramycin-azithromycin at the same concentrations eradicated only three biofilms. Results of this study suggest that local administration of tobramycin and clarithromycin into the respiratory tract represents a better strategy than the combination tobramycin-azithromycin for the treatment of P. aeruginosa-associated pulmonary infections.

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Inhibitory effect of propafenone derivatives on pseudomonas aeruginosa biofilm and pyocyanin production

Srpski arhiv za celokupno lekarstvo ◽

10.2298/sarh180727102p ◽

2020 ◽

Vol 148 (3-4) ◽

pp. 196-202

Author(s):

Snjezana Petrovic ◽

Jasmina Basic ◽

Zoran Mandinic ◽

Dragana Bozic ◽

Marina Milenkovic ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Laboratory Strain ◽

Inhibitory Effect ◽

Negative Control ◽

Clinical Strains ◽

Control Value ◽

Essential Components ◽

Pyocyanin Production

Introduction/Objective. Biofilm and pyocyanin production are essential components of Pseudomonas aeruginosa virulence and antibiotic resistance. Our objective was to examine inhibitory effect of synthetized propafenone derivatives 3-(2-Fluorophenyl)- 1-(2- (2-hydroxy-3-propylamino-propoxy)-phenyl)-propan-1-one hydrochloride (5OF) and3-(2- Trifluoromethyl-phenyl)-1-(2-(2-hydroxy-3-propylamino-propoxy)-phenyl)-propan-1-one hydrochloride (5CF3) on biofilm and pyocyanin in Pseudomonas aeruginosa clinical strains. Methods. Effects were tested on nine clinical isolates and one control laboratory strain of P. aeruginosa. In vitro analysis of biofilm growing was performed by incubating bacteria (0.5 McFarland) with 5OF and 5CF3 (500?31.2 ?g/ml) and measuring optical density (OD) at 570 nm. Bacteria in medium without compounds were positive control. Blank medium (an uninoculated medium without test compounds) was used as negative control. Pyocyanin production was estimated by OD at 520 nm, after bacteria incubated with 5CF3 and 5OF (250 and 500 ?g/ml), treated with chloroform, and chloroform layer mixed with HCl. Results. A total of 500 ?g/ml of 5OF and 5CF3 completely inhibited biofilm formation in 10/10 and 4/10 strains, respectively. A total of 250 ?g/ml of 5OF and 5CF3 strongly inhibited biofilm formation in 7/10 strains, while inhibition with 125 ?g/ml of 5OF and 5CF3 was moderate. Lower concentrations had almost no effect on biofilm production. Pyocyanin production was reduced to less than 40% of the control value in 6/9, and less than 50% of the control in 7/9 strains with 500 ?g/ml of 5OF and 5CF3, respectively. At 250 ?g/ml 5OF and 5CF3, most strains had pyocyanin production above 50% of the control value. Conclusion. Synthetized propafenone derivatives, 5OF and 5CF3, inhibited biofilms and pyocyanin production of Pseudomonas aeruginosa clinical strains. Presented results suggest that propafenone derivatives are potential lead-compounds for synthesis of novel antipseudomonal drugs.

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Disparate Candida albicans Biofilm Formation in Clinical Lipid Emulsions Due to Capric Acid-Mediated Inhibition

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01394-19 ◽

2019 ◽

Vol 63 (11) ◽

Author(s):

Hubertine M. E. Willems ◽

Jeremy S. Stultz ◽

Molly E. Coltrane ◽

Jabez P. Fortwendel ◽

Brian M. Peters

Keyword(s):

Candida Albicans ◽

Biofilm Formation ◽

Bloodstream Infections ◽

Capric Acid ◽

Lipid Emulsions ◽

Biofilm Growth ◽

Content Type ◽

Biofilm Model ◽

Hypha Formation

ABSTRACT Receipt of parenteral nutrition (PN) remains an independent risk factor for developing catheter-related bloodstream infections (CR-BSI) caused by fungi, including by the polymorphic fungus Candida albicans, which is notoriously adept at forming drug-resistant biofilm structures. Among a variety of macronutrients, PN solutions contain lipid emulsions to supply daily essential fats and are often delivered via central venous catheters (CVCs). Therefore, using an in vitro biofilm model system, we sought to determine whether various clinical lipid emulsions differentially impacted biofilm growth in C. albicans. We observed that the lipid emulsions Intralipid and Omegaven both stimulated C. albicans biofilm formation during growth in minimal medium or a macronutrient PN solution. Conversely, Smoflipid inhibited C. albicans biofilm formation by approximately 50%. Follow-up studies revealed that while Smoflipid did not impair C. albicans growth, it did significantly inhibit hypha formation and hyphal elongation. Moreover, growth inhibition could be recapitulated in Intralipid when supplemented with capric acid—a fatty acid present in Smoflipid but absent in Intralipid. Capric acid was also found to dose dependently inhibit C. albicans biofilm formation in PN solutions. This is the first study to directly compare different clinical lipid emulsions for their capacity to affect C. albicans biofilm growth. Results derived from this study necessitate further research regarding different lipid emulsions and rates of fungus-associated CR-BSIs.

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Effects of vancomycin, daptomycin, and tigecycline on coagulase-negative staphylococcus biofilm and bacterial viability within biofilm: an in vitro biofilm model

Canadian Journal of Microbiology ◽

10.1139/cjm-2015-0855 ◽

2016 ◽

Vol 62 (9) ◽

pp. 735-743 ◽

Cited By ~ 5

Author(s):

Barcin Ozturk ◽

Necati Gunay ◽

Bulent M. Ertugrul ◽

Serhan Sakarya

Keyword(s):

Biofilm Formation ◽

Treatment Time ◽

Antimicrobial Effect ◽

Bacterial Biofilm ◽

Coagulase Negative Staphylococcus ◽

Biofilm Inhibition ◽

Major Barrier ◽

Biofilm Model ◽

Repeated Doses

Bacteria may hide in a hydrated polysaccharide matrix known as a biofilm. The structure of the bacterial biofilm renders phagocytosis difficult and increases antibiotic resistance. We hypothesized that repeated doses of antibiotics have an effect on bacteria within the biofilm and that it could inhibit or eradicate biofilm formation. Two clinical biofilm-positive coagulase-negative staphylococcus isolates were evaluated. The effects of antibiotics on preformed and nascent biofilm and on bacterial eradication within the biofilm were determined using different doses of vancomycin, daptomycin, and tigecycline for different durations in an in vitro biofilm model. Vancomycin neither penetrated the biofilm nor had any microbicidal effect on bacteria within the biofilm. Daptomycin had a microbicidal effect on bacteria within the biofilm but had no effect on biofilm inhibition and eradication (independent from dose and treatment time). Tigecycline inhibited and eradicated biofilm formation and had a microbicidal effect on bacteria within the biofilm. In conclusion, (i) biofilm formation appeared to be a major barrier to vancomycin activity, (ii) daptomycin had an antimicrobial effect on the bacteria within the biofilm but not on the biofilm burden, and (iii) tigecycline had effects both on bacteria within the biofilm and on biofilm burden. Thus, both tigecycline and daptomycin might be promising candidates for the treatment of biofilm infections.

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An Overview of Biological and Computational Methods for Designing Mechanism-Informed Anti-biofilm Agents

Frontiers in Microbiology ◽

10.3389/fmicb.2021.640787 ◽

2021 ◽

Vol 12 ◽

Author(s):

Andy Y. An ◽

Ka-Yee Grace Choi ◽

Arjun S. Baghela ◽

Robert E. W. Hancock

Keyword(s):

Antibiotic Resistance ◽

Small Molecules ◽

In Silico ◽

Biofilm Formation ◽

Stringent Response ◽

Antibiotic Resistant ◽

Biologically Relevant ◽

Biofilm Model

Bacterial biofilms are complex and highly antibiotic-resistant aggregates of microbes that form on surfaces in the environment and body including medical devices. They are key contributors to the growing antibiotic resistance crisis and account for two-thirds of all infections. Thus, there is a critical need to develop anti-biofilm specific therapeutics. Here we discuss mechanisms of biofilm formation, current anti-biofilm agents, and strategies for developing, discovering, and testing new anti-biofilm agents. Biofilm formation involves many factors and is broadly regulated by the stringent response, quorum sensing, and c-di-GMP signaling, processes that have been targeted by anti-biofilm agents. Developing new anti-biofilm agents requires a comprehensive systems-level understanding of these mechanisms, as well as the discovery of new mechanisms. This can be accomplished through omics approaches such as transcriptomics, metabolomics, and proteomics, which can also be integrated to better understand biofilm biology. Guided by mechanistic understanding, in silico techniques such as virtual screening and machine learning can discover small molecules that can inhibit key biofilm regulators. To increase the likelihood that these candidate agents selected from in silico approaches are efficacious in humans, they must be tested in biologically relevant biofilm models. We discuss the benefits and drawbacks of in vitro and in vivo biofilm models and highlight organoids as a new biofilm model. This review offers a comprehensive guide of current and future biological and computational approaches of anti-biofilm therapeutic discovery for investigators to utilize to combat the antibiotic resistance crisis.

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Contribution of Bacillomycin D in Bacillus amyloliquefaciens SQR9 to Antifungal Activity and Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.02645-12 ◽

2012 ◽

Vol 79 (3) ◽

pp. 808-815 ◽

Cited By ~ 96

Author(s):

Zhihui Xu ◽

Jiahui Shao ◽

Bing Li ◽

Xin Yan ◽

Qirong Shen ◽

...

Keyword(s):

Biofilm Formation ◽

Bacillus Amyloliquefaciens ◽

Root Colonization ◽

Antagonistic Activity ◽

Vital Role ◽

Content Type ◽

Reverse Transcription Pcr ◽

Bacillomycin D

ABSTRACTBacillus amyloliquefaciensstrains are capable of suppressing soilborne pathogens through the secretion of an array of lipopeptides and root colonization, and biofilm formation ability is considered a prerequisite for efficient root colonization. In this study, we report that one of the lipopeptide compounds (bacillomycin D) produced by the rhizosphere strainBacillus amyloliquefaciensSQR9 not only plays a vital role in the antagonistic activity againstFusarium oxysporumbut also affects the expression of the genes involved in biofilm formation. When the bacillomycin D and fengycin synthesis pathways were individually disrupted, mutant SQR9M1, which was deficient in the production of bacillomycin D, only showed minor antagonistic activity againstF. oxysporum, but another mutant, SQR9M2, which was deficient in production of fengycin, showed antagonistic activity equivalent to that of the wild-type strain ofB. amyloliquefaciensSQR9. The results fromin vitro, rootin situ, and quantitative reverse transcription-PCR studies demonstrated that bacillomycin D contributes to the establishment of biofilms. Interestingly, the addition of bacillomycin D could significantly increase the expression levels ofkinCgene, but KinC activation is not triggered by leaking of potassium. These findings suggest that bacillomycin D contributes not only to biocontrol activity but also to biofilm formation in strainB. amyloliquefaciensSQR9.

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An in vitro biofilm model associated to dental implants: Structural and quantitative analysis of in vitro biofilm formation on different dental implant surfaces

Dental Materials ◽

10.1016/j.dental.2014.07.008 ◽

2014 ◽

Vol 30 (10) ◽

pp. 1161-1171 ◽

Cited By ~ 49

Author(s):

M.C. Sánchez ◽

A. Llama-Palacios ◽

E. Fernández ◽

E. Figuero ◽

M.J. Marín ◽

...

Keyword(s):

Quantitative Analysis ◽

Dental Implants ◽

Dental Implant ◽

Biofilm Formation ◽

Biofilm Model

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Reduced Vancomycin Susceptibility in anIn VitroCatheter-Related Biofilm Model Correlates with Poor Therapeutic Outcomes in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02073-12 ◽

2013 ◽

Vol 57 (3) ◽

pp. 1447-1454 ◽

Cited By ~ 42

Author(s):

Wessam Abdelhady ◽

Arnold S. Bayer ◽

Kati Seidl ◽

Cynthia C. Nast ◽

Megan R. Kiedrowski ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Wall Thickness ◽

Biofilm Formation ◽

Methicillin Resistant ◽

Content Type ◽

Biofilm Model ◽

Mrsa Strains

ABSTRACTStaphylococcus aureusis the most common cause of endovascular infections, including catheter sepsis and infective endocarditis (IE). Vancomycin (VAN) is the primary choice for treatment of methicillin-resistantS. aureus(MRSA) infections. However, high rates of VAN treatment failure in MRSA infections caused by VAN-susceptible strains have been increasingly reported. Biofilm-associated MRSA infections are especially prone to clinical antibiotic failure. The present studies examined potential relationships between MRSA susceptibility to VAN in biofilmsin vitroand nonsusceptibility to VAN in endovascular infectionin vivo. Using 10 “VAN-susceptible” MRSA bloodstream isolates previously investigated for VAN responsiveness in experimental IE, we studied the mechanism(s) of suchin vivoVAN resistance, including: (i) VAN binding to MRSA organisms; (ii) the impact of VAN on biofilm formation and biofilm composition; (iii) VAN efficacy in anin vitrocatheter-related biofilm model; (iv) effects on cell wall thickness. As a group, the five strains previously categorized as VAN nonresponders (non-Rsp) in the experimental IE model differed from the five responders (Rsp) in terms of lower VAN binding, increased biofilm formation, higher survival in the presence of VAN within biofilms in the presence or absence of catheters, and greater biofilm reduction upon proteinase K treatment. Interestingly, sub-MICs of VAN significantly promoted biofilm formation only in the non-Rsp isolates. Cell wall thickness was similar among all MRSA strains. These results suggest that sublethal VAN levels that induce biofilm formation and reduce efficacy of VAN in thein vitrocatheter-associated biofilms may contribute to suboptimal treatment outcomes for endovascular infections caused by “VAN-susceptible” MRSA strains.

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Role of Tobramycin in the Induction and Maintenance of Viable but Non-Culturable Pseudomonas aeruginosa in an In Vitro Biofilm Model

Antibiotics ◽

10.3390/antibiotics9070399 ◽

2020 ◽

Vol 9 (7) ◽

pp. 399

Author(s):

Gianmarco Mangiaterra ◽

Nicholas Cedraro ◽

Salvatore Vaiasicca ◽

Barbara Citterio ◽

Roberta Galeazzi ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Laboratory Strain ◽

Specific Pattern ◽

Clinical Strain ◽

Biofilm Model ◽

Lung Infections ◽

The Difference ◽

Infection Recurrence

The recurrence of Pseudomonas aeruginosa (PA) biofilm infections is a major issue in cystic fibrosis (CF) patients. A pivotal role is played by the presence of antibiotic-unresponsive persisters and/or viable but non-culturable (VBNC) forms, whose development might be favored by subinhibitory antibiotic concentrations. The involvement of tobramycin and ciprofloxacin, widely used to treat CF PA lung infections, in the abundance of VBNC cells was investigated in PA biofilms models. In vitro biofilms of the laboratory strain PAO1-N and the clinical strain C24 were developed and starved by subculture for 170 days in a non-nutrient (NN) broth, unsupplemented or supplemented with one-quarter minimal inhibitory concentration (MIC) of tobramycin or ciprofloxacin. VBNC cells abundance, estimated as the difference between total live (detected by qPCR and flow cytometry) and colony forming unit (CFU) counts, showed a strain- and drug-specific pattern. A greater and earlier abundance of VBNC PAO1-N cells was detected in all conditions. Exposure of the C24 strain to NN and NN + ciprofloxacin induced only a transient VBNC subpopulation, which was more abundant and stable until the end of the experiment in tobramycin-exposed biofilms. The same response to tobramycin was observed in the PAO1-N strain. These findings suggest that low tobramycin concentrations might contribute to PA infection recurrence by favoring the development of VBNC forms.

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Isolation and Characterisation of Probiotics for Antagonising Cariogenic Bacterium Streptococcus mutans and Preventing Biofilm Formation

10.20944/preprints201610.0102.v1 ◽

2016 ◽

Author(s):

Pei-Pei Lin ◽

You-Miin Hsieh ◽

Cheng-Chih Tsai

Keyword(s):

Antimicrobial Activity ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Antimicrobial Properties ◽

Antagonistic Activity ◽

Lactobacillus Pentosus ◽

Chewing Gums ◽

Dependent Cell ◽

Cariogenic Bacterium

Consumption of refined foods and beverages high in sugar make the teeth susceptible to the formation of biofilm, and lead to dental caries and diseases of the oral cavity such as periodontitis and periodontal disease. The aim of the present study was to determine the ability of selected probiotics to inhibit growth and biofilm formation by the cariogenic bacterium Streptococcus mutans in vitro. We screened strains of latic acid bacteria (LAB) (n=120) from the Bioresources Collection and Research Center (BCRC), saliva of healthy adults and infant stool. The antimicrobial activity of LAB in vitro was evaluated by agar spot culture and co-culture of the S. mutans strains. We determined the effect of heating and dilution factors (2- and 4-fold) on the antagonistic activity of LAB. Antagonistic substances in the spent culture suspensions (SCS) of LAB were precipitated by extraction with ammonium sulphate and chloroform to characterise the protein and lipophilic fractions. Results of co-culturing show that the SCS of the three LAB strains (Lactobacillus pentosus 13-1, 13-4 and Lactobacillus crispatus BCRC 14618) subjected to heat treatment showed significantly high antimicrobial activity. We found that substances produced by L. pentosus 13-4 which have the potential to exhibit antimicrobial properties might be lipophilic proteins. Additionally, we infer that the mechanism of reducing biofilm formation by Lactobacillus strains is associated with sucrose-dependent cell–cell adhesion and the gtfC level of glucosyltransferases (Gtfs) in the biofilm. Native LAB strains screened in our study may be used in chewing gums and other processed foods for preventing tooth decay.

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Assessment of Inhibitory Effects of Fluoride-Coated Tubes on Biofilm Formation by Using the In Vitro Dental Unit Waterline Biofilm Model

Applied and Environmental Microbiology ◽

10.1128/aem.00610-08 ◽

2008 ◽

Vol 74 (19) ◽

pp. 5958-5964 ◽

Cited By ~ 15

Author(s):

Toshiaki Yabune ◽

Satoshi Imazato ◽

Shigeyuki Ebisu

Keyword(s):

Biofilm Formation ◽

Polyvinylidene Fluoride ◽

In Vitro Model ◽

Bacterial Species ◽

Biofilm Model ◽

Dental Unit Waterlines ◽

Vitro Model ◽

Methylobacterium Mesophilicum ◽

The One

ABSTRACT This study aimed to establish an in vitro model to simulate biofilms formed in dental unit waterlines (DUWLs) and to investigate the ability of polyvinylidene fluoride (PVDF)-coated tubes to inhibit biofilm formation using this model. The water and biofilm samples were obtained from DUWLs which had been clinically used for 2.5 years, and the predominant bacteria were identified. A conventional polyurethane tube was incubated for 24 to 96 h in the mixed flora of isolated bacteria, and the optimal incubation conditions to simulate a clinically formed biofilm were determined by observation with a scanning electron microscope. Biofilm formation on a PVDF-coated tube was observed using this in vitro model, and the adherence of different bacterial species to conventional and PVDF-coated tubes was assessed. Sphingomonas paucimobilis, Acinetobacter haemolytics, and Methylobacterium mesophilicum were predominantly isolated from contaminated DUWLs. Incubation of the polyurethane tube with the mixed flora containing these three species for 96 h resulted in the formation of a mature biofilm similar to the one clinically observed. The PVDF-coated tube was significantly less adhesive to all three bacterial species than the polyurethane tube (P < 0.05 by the Mann-Whitney U test), and the attachment of small amounts of rods was observed even after incubation with the mixed flora for 96 h. In conclusion, an in vitro biofilm model was obtained by using a mixed flora of bacteria isolated from DUWLs, and the PVDF-coated tube was found to be effective in preventing biofilm formation using this model.

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