Pseudomonas Aeruginosa Biofilm Research Articles

Safety and efficacy of a bacteriophage cocktail in an in vivo model of Pseudomonas aeruginosa sinusitis

Pseudomonas aeruginosa (PA) is a bacterial pathogen that frequently displays antibiotic resistance. Its presence within the sinuses of chronic rhinosinusitis sufferers is associated with poorer quality of life. Obligately lytic bacteriophages (phages) are viruses that infect, replicate within, and lyse bacteria, causing bacterial death. The aims of this study were to assess the safety and efficacy of a PA phage cocktail (CT-PA) in a sheep model of rhinosinusitis. The sheep rhinosinusitis model was adapted to simulate PA infection in sheep frontal sinuses. To assess efficacy, after a 7-day biofilm formation period, sheep received twice-daily frontal trephine flushes of CT-PA or saline for 1 week. Biofilm quantitation on frontal sinus mucosa was performed using LIVE/DEAD BacLight staining. To assess safety, sheep received twice-daily frontal trephine flushes of CT-PA or vehicle control for 3 weeks. Blood and fecal samples were collected throughout treatment. Histopathology of frontal sinus, lung, heart, liver, spleen, and kidney tissue was performed. Sinus cilia were visualized using scanning electron microscopy (SEM). The Efficacy arm showed a statistically significant reduction in biofilm biomass with all concentrations of CT-PA tested (P < 0.05). Phage presence in sinuses was maintained for at least 16hours after the final flush. All Safety arm sheep completed 3 weeks of treatment. Phage was detected consistently in feces and sporadically in blood and organ samples. Histology and SEM of tissues revealed no treatment-related damage. In conclusion, CT-PA was able to decrease sinus PA biofilm at concentrations of 108-1010 PFU/mL. No safety concerns were noted.

Electrical stimulation disrupts biofilms in a human wound model and reveals the potential for monitoring treatment response with volatile biomarkers.

Management of biofilm infections relies on time-consuming laboratory techniques and monitoring treatment by subjective clinical evaluations. Due to these limitations, there is a need to explore alternative strategies. The aims of this study were to assess the feasibility of using volatile organic compound (VOC) biomarkers to monitor treatment response and measure anti-biofilm efficacy of electrical stimulation (ES) in vitro and in human cutaneous wound biofilm models. Staphylococcus aureus (MSSA) and Pseudomonas aeruginosa (PA) biofilms were exposed to ES, ciprofloxacin, or both, with efficacy assessed and quantified by fluorescence staining, enumeration, metabolic assays, and biomass quantification; VOCs were measured by gas chromatography-mass spectrometry. In vitro MSSA and PA and ex vivo PA biofilms exposed to ES showed significantly reduced bacterial viability, metabolic activity, and biomass compared to controls (p < 0.05). There was significant variation in the relative abundance of VOCs in in vitro MSSA and PA and in ex vivo PA biofilms exposed to ES and antibiotic (p < 0.05). 2-methyl-1-propanol was associated with MSSA viability (R = 0.93, p < 0.05), biomass (R = 0.97, p < 0.05), and metabolic activity (R = 0.93, p < 0.05) and 3-methyl-1-butanol was associated with PA biomass (R = 0.93, p < 0.05). We showed that ES and VOC biomarkers are possible options for alternative nonpharmacological antimicrobial management of biofilms and noninvasive monitoring of wound infection treatment responses, respectively.

Challenges and Promises for Planning Future Clinical Research Into Bacteriophage Therapy Against Pseudomonas aeruginosa in Cystic Fibrosis. An Argumentative Review.

Although early aggressive and prolonged treatment with specific antibiotics can extend survival in patients with cystic fibrosis (CF) colonized by opportunistic Pseudomonas aeruginosa (PA), antibiotics fail to eradicate the infecting multidrug-resistant (MDR) PA strains in CF. Century-long research has suggested treating patients with bacteriophages (phages, prokaryotic viruses) naturally hosted by bacteria. Although the only phage types used in therapy, lytic phages, lyse PA aggregated in biofilm matrix by depolymerase degrading enzymes, how they can effectively, safely, and persistently do so in patients with CF is unclear. Even though advanced techniques for formulating phage cocktails, training phages and collecting phage libraries have improved efficacy in vitro, whether personalized or ready-to-use therapeutic approaches or phages and antibiotics combined are effective and safe in vivo, and can reduce PA biofilms, remains debatable. Hence, to advance clinical research on phage therapy in clinical trials, also involving mucoid and non-mucoid multidrug-resistant PA in CF, and overcome problems in Western international regulations, we need reliable and repeatable information from experiments in vitro and in vivo on phage characterization, cocktail selection, personalized approaches, and phages combined with antibiotics. These findings, challenges, and promises prompted us to undertake this argumentative review to seek up-to-date information from papers describing lytic phage activity tested in vitro on PA laboratory strains, and PA strains from chronic infections including CF. We also reviewed in vivo studies on phage activity on pulmonary and non-pulmonary animal host models infected by laboratory or CF PA strains. Our argumentative review provides essential information showing that future phage clinical research in CF should use well-characterized and selected phages isolated against CF PA, tested in vitro under dynamic conditions in cocktails or combined with antibiotics, and in vivo on non-pulmonary and pulmonary host models infected with mucoid and non-mucoid CF MDR PA. Our findings should encourage pharmaceutical industries to conduct clinical trials in vitro and in vivo testing patented genomic engineered phages from phage libraries combined with antibiotics to treat or even prevent multidrug-resistant PA in CF, thus helping international regulatory agencies to plan future clinical research on phage therapy in CF.

Mitigation of a nitrate reducing Pseudomonas aeruginosa biofilm and anaerobic biocorrosion using ciprofloxacin enhanced by D-tyrosine

Pseudomonas aeruginosa (PA) is a ubiquitous microbe. It can form recalcitrant biofilms in clinical and industrial settings. PA biofilms cause infections in patients. They also cause biocorrosion of medical implants. In this work, D-tyrosine (D-tyr) was investigated as an antimicrobial enhancer for ciprofloxacin (CIP) against a wild-type PA biofilm (strain PAO1) on C1018 carbon steel in a strictly anaerobic condition. Seven-day biofilm prevention test results demonstrated that 2 ppm (w/w) D-tyr enhanced 30 ppm CIP by achieving extra 2-log sessile cell reduction compared with the 30 ppm CIP alone treatment. The cocktail of 30 ppm CIP + 2 ppm D-tyr achieved similar efficacy as the 80 ppm CIP alone treatment in the biofilm prevention test. Results also indicated that the enhanced antimicrobial treatment reduced weight loss and pitting corrosion. In the 3-hour biofilm removal test, the cocktail of 80 ppm CIP + 5 ppm D-tyr achieved extra 1.5-log reduction in sessile cell count compared with the 80 ppm CIP alone treatment. The cocktail of 80 ppm CIP + 5 ppm D-tyr achieved better efficacy than the 150 ppm CIP alone treatment in the biofilm removal test.

Synergistic Activity of Berberine with Azithromycin against Pseudomonas Aeruginosa Isolated from Patients with Cystic Fibrosis of Lung In Vitro and In Vivo

Background/Aims: Pseudomonas aeruginosa (PA) is one of the major opportunistic pathogens which can cause chronic lung infection of cystic fibrosis (CF). The formation of PA biofilm promotes CF development and restricts the antimicrobial efficacies of current antibiotics. Methods: The antimicrobial effects of azithromycin (AZM) and berberine (BER) alone and in combination were evaluated using microdilution method, checkerboard assay, time-kill test, qRT-PCR analysis and absorption method. The treatments of AZM and/or BER were further evaluated in an animal lung infection model via observing survival rate, bacterial burden and histopathology of lung, the levels of pro-/anti-inflammatory cytokines. Results: AZM-BER were demonstrated to be synergistic against ten clinical PA isolates as well as the standard reference PA ATCC27853, in which PA03 was the most susceptible isolate to AZM-BER with FICI of 0.13 and chosen for subsequent experiments. The synergism of AZM-BER was further confirmed against PA03 in time-kill test and scanning electron microscope (SEM) at their concentrations showing synergism. In PA03, we found that AZM-BER could significantly attenuate productions of a series of virulence factors including alginate, LasA protease, LasB protease, pyoverdin, pyocyanin, chitinase as well as extracellular DNA, and remarkably inhibit the levels of quorum sensing (QS) molecules and the expressions of lasI, lasR, rhlI, rhlR at 1/2×MIC, 1×MIC and 2×MIC. In the infection model, the mice survival were increased markedly, the inflammations of infected lungs were improved greatly along with reduced IL-6, IL-8 and ascended IL-10 at 0.8 mg/kg of AZM combined with 3.2 mg/kg of BER. Conclusion: BER might be a promising synergist to enhance the antimicrobial activity of AZM in vitro and in vivo.

Chronic lung infection by Pseudomonas aeruginosa biofilm is cured by L-Methionine in combination with antibiotic therapy.

Bacterial biofilms are associated with 80–90% of infections. Within the biofilm, bacteria are refractile to antibiotics, requiring concentrations >1,000 times the minimum inhibitory concentration. Proteins, carbohydrates and DNA are the major components of biofilm matrix. Pseudomonas aeruginosa (PA) biofilms, which are majorly associated with chronic lung infection, contain extracellular DNA (eDNA) as a major component. Herein, we report for the first time that L-Methionine (L-Met) at 0.5 μM inhibits Pseudomonas aeruginosa (PA) biofilm formation and disassembles established PA biofilm by inducing DNase expression. Four DNase genes (sbcB, endA, eddB and recJ) were highly up-regulated upon L-Met treatment along with increased DNase activity in the culture supernatant. Since eDNA plays a major role in establishing and maintaining the PA biofilm, DNase activity is effective in disrupting the biofilm. Upon treatment with L-Met, the otherwise recalcitrant PA biofilm now shows susceptibility to ciprofloxacin. This was reflected in vivo, in the murine chronic PA lung infection model. Mice treated with L-Met responded better to antibiotic treatment, leading to enhanced survival as compared to mice treated with ciprofloxacin alone. These results clearly demonstrate that L-Met can be used along with antibiotic as an effective therapeutic against chronic PA biofilm infection.

WS10.7 The role of C-type lectins in the recognition of Pseudomonas aeruginosa

Colonization and persistent lung infection by Pseudomonas aeruginosa (PA), is an important cause of morbidity and mortality in Cystic Fibrosis patients. PA persists inside the lungs by forming drug-resistant biofilms. Biofilms are enclosed in extracellular polymeric substance (EPS) which functions as a sticky matrix that protects biofilms from harsh environment. Polysaccharide synthesis locus (psl) is an important mannose rich matrix component that promotes biofilm formation. Studies showed that psl reduces the phagocytic activity of immune cells and complement activation. Incubation of PA biofilms with labelled purified human monocytes and macrophages indicate that macrophages, unlike monocytes attached to PA biofilms. Cell surface receptors expressed by monocytes and macrophages were compared and, as previously described, high mannose receptor (MR) expression was found in macrophages. MR is a carbohydrate binding receptor that plays a crucial role in innate immunity. MR binds mannose-rich carbohydrates through C-type lectin-like domains (CTLD) 4 to 7. Using a recombinant protein containing the mannose binding region of MR (CTLD 4–7-Fc) it was found that MR can directly interact with PA biofilms and that this binding is increased when psl expression is increased. We hypothesize that MR expressed by macrophages and dendritic cells might contribute to PA recognition through psl. Future work will explore the capability of immune mammalian C-type lectins to recognise PA biofilms and purified psl and elucidate the role of psl in modulating the recognition of PA by immune cells using PA strains expressing different combinations of extracellular polysaccharides.

An in situ Raman spectroscopy-based microfluidic "lab-on-a-chip" platform for non-destructive and continuous characterization of Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa biofilm was cultivated and characterized in a microfluidic "lab-on-a-chip" platform coupled with confocal Raman microscopy in a non-destructive manner. Biofilm formation could be quantified by this label-free platform and correlated well with confocal laser scanning microscopy. This Raman-microfluidic platform could also discriminate biofilms at different developmental stages.

Bacteria present in carotid arterial plaques are found as biofilm deposits which may contribute to enhanced risk of plaque rupture.

ABSTRACTAtherosclerosis, a disease condition resulting from the buildup of fatty plaque deposits within arterial walls, is the major underlying cause of ischemia (restriction of the blood), leading to obstruction of peripheral arteries, congestive heart failure, heart attack, and stroke in humans. Emerging research indicates that factors including inflammation and infection may play a key role in the progression of atherosclerosis. In the current work, atherosclerotic carotid artery explants from 15 patients were all shown to test positive for the presence of eubacterial 16S rRNA genes. Density gradient gel electrophoresis of 5 of these samples revealed that each contained 10 or more distinct 16S rRNA gene sequences. Direct microscopic observation of transverse sections from 5 diseased carotid arteries analyzed with a eubacterium-specific peptide nucleic acid probe revealed these to have formed biofilm deposits, with from 1 to 6 deposits per thin section of plaque analyzed. A majority, 93%, of deposits was located proximal to the internal elastic lamina and associated with fibrous tissue. In 6 of the 15 plaques analyzed, 16S rRNA genes from Pseudomonas spp. were detected. Pseudomonas aeruginosa biofilms have been shown in our lab to undergo a dispersion response when challenged with free iron in vitro. Iron is known to be released into the blood by transferrin following interaction with catecholamine hormones, such as norepinephrine. Experiments performed in vitro showed that addition of physiologically relevant levels of norepinephrine induced dispersion of P. aeruginosa biofilms when grown under low iron conditions in the presence but not in the absence of physiological levels of transferrin.

G385 RATNO – Reducing Antibiotic Tolerance using Nitric Oxide in Cystic Fibrosis: report of a proof of concept clinical trial

AimsTo investigate whether low dose Nitric Oxide (NO) enhances antibiotic therapy through disruption of Pseudomonas aeruginosa (PA) bacteria in biofilms in patients with Cystic Fibrosis (CF).BackgroundBacterial biofilms present a major...

Structure‐Based Optimization of the Terminal Tripeptide in Glycopeptide Dendrimer Inhibitors of Pseudomonas aeruginosa Biofilms Targeting LecA

The galactopeptide dendrimer GalAG2 ((β-Gal-OC6H4CO-Lys-Pro-Leu)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2) binds strongly to the Pseudomonas aeruginosa (PA) lectin LecA, and it inhibits PA biofilms, as well as disperses already established ones. By starting with the crystal structure of the terminal tripeptide moiety GalA-KPL in complex with LecA, a computational mutagenesis study was carried out on the galactotripeptide to optimize the peptide-lectin interactions. 25 mutants were experimentally evaluated by a hemagglutination inhibition assay, 17 by isothermal titration calorimetry, and 3 by X-ray crystallography. Two of these tripeptides, GalA-KPY (dissociation constant (K(D))=2.7 μM) and GalA-KRL (K(D)=2.7 μM), are among the most potent monovalent LecA ligands reported to date. Dendrimers based on these tripeptide ligands showed improved PA biofilm inhibition and dispersal compared to those of GalAG2, particularly G2KPY ((β-Gal-OC6H4CO-Lys-Pro-Tyr)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2). The possibility to retain and even improve the biofilm inhibition in several analogues of GalAG2 suggests that it should be possible to fine-tune this dendrimer towards therapeutic use by adjusting the pharmacokinetic parameters in addition to the biofilm inhibition through amino acid substitutions.

Pseudomonas aeruginosa biofilm aggravates skin inflammatory response in BALB/c mice in a novel chronic wound model

Chronic wounds are presumed to persist in the inflammatory state, preventing healing. Emerging evidence indicates a clinical impact of bacterial biofilms in soft tissues, including Pseudomonas aeruginosa (PA) biofilms. To further investigate this, we developed a chronic PA biofilm wound infection model in C3H/HeN and BALB/c mice. The chronic wound was established by an injection of seaweed alginate-embedded P. aeruginosa PAO1 beneath a third-degree thermal lesion providing full thickness skin necrosis, as in human chronic wounds. Cultures revealed growth of PA, and both alginate with or without PAO1 generated a polymorphonuclear-dominated inflammation early after infection. However, both at days 4 and 7, there were a more acute polymorphonuclear-dominated and higher degree of inflammation in the PAO1 containing group (p < 0.05). Furthermore, PNA-FISH and supplemented DAPI staining showed bacteria organized in clusters, resembling biofilms, and inflammation located adjacent to the PA. The chronic wound infection showed a higher number of PAO1 in the BALB/c mice at day 4 after infection as compared to C3H/HeN mice (p < 0.006). In addition, a higher concentration of interleukin-1beta in the chronic wounds of BALB/c mice was observed at day 7 (p < 0.02), despite a similar number of bacteria in the two mouse strains. The present study succeeded in establishing a chronic PA biofilm infection in mice. The results showed an aggravating impact of local inflammation induced by PA biofilms. In conclusion, our findings indicate that improved infection control of chronic wounds reduces the inflammatory response and may improve healing.

Prevention of Biofilm Formation by Polyquaternary Polymer

Objective: Biofilm formation has been linked to otologic implant infection and extrusion. The purpose of this study was to determine if a microbicidal polyquaternary polymer (PP) could prevent biofilm development by common otopathogens, Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). Method: Polyurethane tubes (20 per treatment) with and without a PP were briefly exposed to plasma or saline, then to SA or PA. Tubes were incubated in growth media. After 4 days, antibiotics were added to kill planktonic bacteria. Biofilm formation was assessed by bacterial counts and scanning electron microscopy. Results: SA biofilm was reduced by 8 logs on tubes with the PP compared to the control tubes ( P &lt; .0001). PA biofilm formation was also less on tubes with the PP ( P &lt; .0001), but the reduction was only 1 log. Plasma promoted SA and PA biofilm on the control tubes ( P &lt; .0001) but not on the tubes with the PP ( P = .07 and .40 for SA and PA, respectively). Conclusion: Incorporation of a microbicidal PP into polyurethane dramatically inhibits SA biofilm formation. Further research is warranted to evaluate the efficacy and safety of this technology in otologic implants.

Methylglyoxal: (active agent of manuka honey) in vitro activity against bacterial biofilms

Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) biofilms are associated with poor chronic rhinosinusitis (CRS) disease control following surgery. Manuka honey (MH) has been shown to be both an effective in vitro treatment agent for SA and PA biofilms and nontoxic to sinonasal respiratory mucosa. Methylglyoxal (MGO) has been reported to be the major antibacterial agent in MH. The effect of this agent against SA and PA biofilms has yet to be reported. Our objective was to determine the in vitro effect of MGO against biofilms of SA and PA, via in vitro testing of MGO against bacterial biofilms. An established biofilm model was used to determine the effective concentration (EC) of MGO against 10 isolates of methicillin-resistant SA (MRSA) and PA. The EC of MGO was also determined against planktonic (free-swimming) MRSA and PA. For MRSA, the EC against planktonic organisms was a concentration of 0.08 mg/mL to 0.3 mg/mL whereas against the biofilm MRSA isolates, the EC ranged from 0.5 mg/mL to 3.6 mg/mL. For PA, the EC against planktonic organisms was a concentration of 0.15 mg/mL to 1.2 mg/mL for planktonic organisms whereas against the biofilm PA isolates, the EC ranged from 1.8 mg/mL to 7.3 mg/mL. MGO, a component of MH, is an effective antimicrobial agent against both planktonic and biofilm MRSA and PA organisms in vitro.

Disruption of Contact Lens–AssociatedPseudomonas aeruginosaBiofilms Formed in the Presence of Neutrophils

To evaluate the capacity of neutrophils to enhance biofilm formation on contact lenses by an infectious Pseudomonas aeruginosa (PA) corneal isolate. Agents that target F-actin and DNA were tested as a therapeutic strategy for disrupting biofilms formed in the setting of neutrophils in vitro and for limiting the infectious bioburden in vivo. Biofilm formation by infectious PA strain 6294 was assessed in the presence of neutrophils on a static biofilm plate and on unworn etafilcon A soft contact lenses. A d-isomer of poly(aspartic acid) was used alone and with DNase to reduce biofilm formation on test contact lenses. The gentamicin survival assay was used to determine the effectiveness of the test compound in reducing subsequent intracellular bacterial load in the corneal epithelium in a contact lens infection model in the rabbit. In a static reactor and on hydrogel lenses, PA biofilm density was enhanced 30-fold at 24 hours in the presence of neutrophils (P < 0.0001). The combination of DNase and anionic poly(aspartic acid) reduced the PA biofilms formed in the presence of activated neutrophils by 79.2% on hydrogel contact lenses (P < 0.001). An identical treatment resulted in a 41% reduction in internalized PA in the rabbit corneal epithelium after 24 hours (P = 0.03). These results demonstrate that PA can exploit the presence of neutrophils to form biofilm on contact lenses within a short time. Incorporation of F-actin and DNA represent a mechanism for neutrophil-induced biofilm enhancement and are targets for available agents to disrupt pathogenic biofilms formed on contact lenses and as a treatment for established corneal infections.

Pseudomonas Biofilm Formation after Haemophilus Infection

Tympanostomy tube (TT) biofilm formation may lead to refractory otorrhea and occlusion. Biofilms are commonly composed of multiple microbial species. One species may promote or inhibit biofilm formation by other species.The aim of this study was to determine if Haemophilus influenzae(HI) promotes the development of Pseudomonas aeruginosa(PA) biofilm on TTs. Controlled, in vitro. Academic research laboratory. Fluoroplastic TTs (20 per group) were exposed to plasma, allowed to dry, and cultured with HI for 7 days. TTs were either gas sterilized or treated for 24 hours with 10 or 3000 μg/mL ciprofloxacin. Half of the TTs from each treatment group underwent bacterial counts or scanning electron microscopy. The remainder, as well as TTs not exposed to HI, were cultured with PA for 4 days and treated with gentamicin to kill planktonic PA. Biofilm formation was quantified with bacterial counts. TTs treated with ciprofloxacin 3000 μg/mL had lower HI counts than TTs treated with 10 μg/mL (P = .0001), but viable HI persisted. PA biofilm formation on TTs with prior HI biofilm and treated with ciprofloxacin 10 μg/mL or gas sterilization was not different than TTs without HI. Less PA biofilm formed on TTs with HI treated with 3 mg/mL ciprofloxacin(P = .002). HI biofilm does not promote PA biofilm formation on TTs. Use of high-dose ototopical therapy to clear HI may reduce subsequent PA biofilm formation.

Effectiveness of honey on Staphylococcus aureus and Pseudomonas aeruginosa biofilms

Objectives Biofilms formed by Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) have been shown to be an important factor in the pathophysiology of chronic rhinosinusitis (CRS). As well, honey has been used as an effective topical antimicrobial agent for years. Our objective is to determine the in vitro effect of honey against biofilms produced by PA and SA. Study Design In vitro testing of honey against bacterial biofilms. Methods We used a previously established biofilm model to assess antibacterial activity of honey against 11 methicillin-susceptible SA (MSSA), 11 methicillin-resistant SA (MRSA), and 11 PA isolates. Honeys were tested against both planktonic and biofilm-grown bacteria. Results Honey was effective in killing 100 percent of the isolates in the planktonic form. The bactericidal rates for the Sidr and Manuka honeys against MSSA, MRSA, and PA biofilms were 63-82 percent, 73-63 percent, and 91-91 percent, respectively. These rates were significantly higher ( P < 0.001) than those seen with single antibiotics commonly used against SA. Conclusion Honey, which is a natural, nontoxic, and inexpensive product, is effective in killing SA and PA bacterial biofilms. This intriguing observation may have important clinical implications and could lead to a new approach for treating refractory CRS.

A High-Throughput In Vitro Model Illustrating Potential Microbiological Interactions During Treatment of Pseudomonas Aeruginosa Biofilm Associated Infections

Objective: Amend a real-time, high-throughput method of bacterial growth detection for use as a model of biofilm response to co-administered pharmaceuticals during the treatment of devise associated infections. Background: Biofilms are the root etiology for chronic infections, particularly in regard to infections in patients with implanted medical devices. Calcium channel blockers (CCBs) are used for control of hypertension and angina and are commonly prescribed to elderly patients. We address potential interference of commonly prescribed CCBs with levofloxacin for treatment of Pseudomonas aeruginosa biofilms. Methods: Inoculum of 1–3×106CFU∕mL in the log phase were seeded into each well of a polystyrene plate. Biofilms developed over 6h at 37°C, was washed and medium containing various CCBs plus levofloxacin was added to the biofilm. OD measurements were obtained at 1h intervals over 90h at 37°C. Changes in turbidity were kinetically measured with a vertical photometer with a wide-band filter. Results: Mibefradil and diltiazem appear to be strongly antagonistic toward levofloxacin where both of them decrease antibiofilm effect of levofloxacin and they encourage the selection of resistant mutants from biofilm. Discussion: Implanted medical devices are quite common and are subjected to biofilm infections. Increasing multi-drug resistance underscores the need to conserve current antibiotics by judicious use. This necessitates consideration of evidence regarding antagonistic or synergistic activity of commonly prescribed drugs of different classes toward commonly used antibiotics. The combinations described here show vital and previously unreported effects of some CCBs when co-prescribed with levofloxacin on Pseudomonas aeruginosa biofilm.