Mirabilis Biofilm Formation Research Articles

Flavonoids Isolated From Delphinium semibarbatum Flowering Aerial Parts With Their Antibacterial, Antibiofilm, and Antiswarming Activity Against Proteus mirabilis and Staphylococcus aureus

Proteus mirabilis and Staphylococcus aureus are pathogens associated with CAUTIs. Understanding their significance is crucial for effective prevention and treatment. In this study, the Delphinium semibarbatum flavonoid‐rich fraction (DSF) was analyzed, and its antibacterial, antibiofilm, and antiswarming properties against these two bacterial species were evaluated. In the phytochemical analysis, four flavonoids, including kaempferol, 4′‐O‐methyl quercetin, quercetin, and kaempferol‐3‐O‐beta‐D‐glucopyranoside (K3G), were isolated. The minimum inhibitory concentration (MIC50) of DSF on P. mirabilis and S. aureus was 1000 μg/mL. Similarly, kaempferol, K3G, and 4′‐O‐methyl quercetin exhibited inhibitory effects of 20%, 13%, and 12%, respectively, at a concentration of 1000 μg/mL compared to the P. mirabilis control. However, the MIC50 of kaempferol and 4′‐O‐methyl quercetin for S. aureus were 125 μg/mL and 62.25 μg/mL, respectively. K3G exhibited an inhibitory effect of 37% at a concentration of 1000 μg/mL. DSF reduced biofilm at 1 mg/mL by 78% and 74% for P. mirabilis and S. aureus, respectively. Quercetin was the most effective in inhibiting P. mirabilis biofilm formation, with a 96% inhibition rate, followed by kaempferol and K3G, with 67% and 29%, respectively, at the same concentration. The best bioactive compound against biofilm inhibition of S. aureus was quercetin at 250 μg/mL, exhibiting 90% inhibition, followed by K3G, 4′‐O‐methyl quercetin, and kaempferol with 81%, 45%, and 33%, respectively, at a concentration of 125 μg/mL. In the swarming inhibition assay, DSF showed 78% inhibition at concentrations of 5 μg/mL. Similarly, all bioactive compounds showed 45% antiswarming activity at this concentration. The docking results of the isolated flavonoids on two target proteins, SarA and MrpH, showed that the presence of the methoxy group in the structure of 4′‐O‐methyl quercetin reduced its interaction with these two proteins. In the ADMET prediction, isolated flavonoids showed good predicted properties suitable for treating these two microbial species in urinary infection diseases.

Inhibitory activity of Mentha spicata oils on biofilms of Proteus mirabilis isolated from burns

Introduction and Aim: Proteus mirabilis is an opportunistic pathogen, infecting humans, through the release of endotoxins and enzymes such as urease, hemolysin, protease, DNase etc. One of the factors contributing to its virulence is its unusual ability to form crystalline biofilms. This study aimed to investigate the effect of Mentha spicata volatile oil on P. mirabilis biofilm formation. Materials and Methods: P. mirabilis was isolated from infected wound of burns of patients using conventional biochemical tests. Vitek 2-Compact System was used to confirm the diagnosis of bacterial isolates. The isolates were tested for their susceptibility to 11 antibiotics. Aqueous and alcoholic extracts as well as Volatile Oil and natural Menthol extracted from M. spicata were tested for their ability to inhibit biofilm formation by P. mirabilis. Results: In this study 16 out of the 45 burn injury samples were tested positive for P. mirabilis. Bacterial isolates were found to be resistant to the drugs Levofloxacin and Norfloxacin, with percentages of 10.2% and 11.8%, respectively. Majority of these isolates had the capacity to produce several virulence factors, including biofilm in variable amounts and the enzymes protease, hemolysin, DNase, and gelatinase. The volatile oil and natural component menthol extracted from M. spicata inhibited the formation of biofilm at increasing concentrations. Conclusion: The volatile oil and the natural menthol compound of M. spicata are effective in inhibiting biofilm formation by P. mirabilis.

Role of Proteus mirabilis flagella in biofilm formation

Proteus mirabilis(P. mirabilis) is a common etiological agent of urinary tract infections, particularly those associated with catheterization. P. mirabilis efficiently forms biofilms on different surfaces and shows a multicellular behavior called ‘swarming’, mediated by flagella. To date, the role of flagella in P. mirabilis biofilm formation has been under debate. In this study, we assessed the role of P. mirabilis flagella in biofilm formation using an isogenic allelic replacement mutant unable to express flagellin. Different approaches were used, such as the evaluation of cell surface hydrophobicity, bacterial motility and migration across catheter sections, measurements of biofilm biomass and biofilm dynamics by immunofluorescence and confocal microscopy in static and flow models. Our findings indicate that P. mirabilis flagella play a role in biofilm formation, although their lack does not completely avoid biofilm generation. Our data suggest that impairment of flagellar function can contribute to biofilm prevention in the context of strategies focused on particular bacterial targets.

A light-guiding urinary catheter for the inhibition of Proteus mirabilis biofilm formation.

Catheter-associated urinary tract infection (CAUTI) is a leading cause of hospital-acquired infections worldwide causing debilitating illness for patients as well as a significant financial and treatment burden on health services. CAUTI is linked with the build-up of biofilms on catheter surfaces which act as a reservoir for infection. Additionally, urease-producing bacteria such as Gram-negative Proteus mirabilis (PM), can form crystalline biofilms which encrust catheter surfaces ultimately leading to blockages which require immediate removal of the catheter. Currently there are limited treatments available to prevent the formation of biofilms by PM as well as other urinary tract infection causing bacteria. A novel concept for a light-guiding urinary catheter is presented where a silicone elastomer waveguide incorporated along the length of the catheter is used to irradiate the catheter surfaces with antimicrobial blue light (405 nm) to prevent biofilm formation in situ. The prototype device is mass producible while also easy to fabricate in a lab setting for research studies. The inhibitory effect of blue light on PM biofilm formation over a range of irradiances is described for the first time showing an LD90 at 192–345 J/cm2 and total inhibition at 1,700 J/cm2 In vitro studies show that the light-guiding catheter (LGC) prototypes exhibit a 98% inhibition in PM biofilm formation inside the catheter lumen at an average estimated irradiance of 30–50 mW/cm2 (324–540 J/cm2 fluence) showing that the concept is highly effective, promising to be a powerful and economical antimicrobial approach to prevent catheter associated biofilm development and blockage.

Lactobacillus spp. derived biosurfactants effect on expression of genes involved in Proteus mirabilis biofilm formation.

Nosocomial infections (NIs) have been defined as infections ocuurring shortly after hospitalization or discharging from the hospital. It is associated with increased morbidities and mortalities. Proteus mirabilis considered as the hospital-acquired pathogen. The purpose of this study was to investigate the effect of Lactobacillus acidophilus-derived biosurfactant on P. mirabilis biofilm formation and flhDC/rsmA expression level (P. mirabilis standard strain ATCC 7002 and urinary infection isolated P. mirabilis strains). One of the potential strategies for the prevention of nosocomial infections is the use of probiotics. L. acidophilus was selected as a probiotic strain to produce biosurfactants. A biosurfactant reduces the adhesion of strains to microtiter plate and glass slide surfaces due to the reduction of surface tension. By using Real time PCR quantitation method we showed that biosurfactant significantly reduced rsmA expression whereas increased flhDC expression in P. mirabilis isolates. Several properties of P. mirabilis cells (biofilm formation, adhesion, and gene expression) were changed after L. acidophilus- derived biosurfactant treatment. In this study we showed that biosurfacant treatment can pave the way for a possible control of biofilm development. Based on our findings, we suggest that the prepared biosurfactant may interfere with adhesion of P. mirabilis to catheters and other devices.

Antibiofilm Effects of Epigallocatechin Gallate Against Proteus mirabilis Wild-Type and Ampicillin-Induced Strains.

Proteus mirabilis is an opportunistic pathogen associated with nosocomial infections and foodborne diseases. The resistance and biofilm formation of P. mirabilis have been a great concern. In this study a multidrug-resistant P. mirabilis strain 012 was exposed to a lethal dose of ampicillin (10 mg/mL, 2.5-fold minimal bactericidal concentration) for 24 h at 37°C. After resuscitation and isolation, five variant isolates were selected and subjected to ampicillin induction by repeatedly streaking on ampicillin-containing plates (10 mg/mL) for at least three times. In biofilm formation assays by using crystal violet staining, we found that the variant strains had enhanced biofilm-forming abilities. (-)-epigallocatechin-3-gallate (EGCG) at a minimum inhibitory concentration (MIC) (256 μg/mL) significantly reduced the biofilm formation of all variant strains and the wild-type strain (p < 0.01). Sub-MIC of EGCG (128 μg/mL) suppressed the biofilms of wild-type and two variants. However, it stimulated the biofilms of the other three variants. The antibiofilm effects of EGCG against the wild-type strain were further confirmed by confocal laser scanning microscopy. Scanning electron microscopy revealed that EGCG induced variants to form more fibrous structures. Our results revealed that a lethal dose of antibiotic exposure increased antibiotic resistance and biofilm formation of P. mirabilis. EGCG may be used as a promising antibiofilm agent to prevent the P. mirabilis biofilm formation in the food industry. However, the sub-MIC of EGCG is not effective and will not be applied.

Anti-biofilm activity of LC-MS based Solanum nigrum essential oils against multi drug resistant biofilm forming P. mirabilis.

Urinary tract infections are second most important diseases worldwide due to the increased amount of antibiotic resistant microbes. Among the Gram negative bacteria, P. mirabilis is the dominant biofilm producer in urinary tract infections next to E. coli. Biofilm is a process that produced self-matrix of more virulence pathogens on colloidal surfaces. Based on the above fact, this study was concentrated to inhibit the P. mirabilis biofilm formation by various in-vitro experiments. In the current study, the anti-biofilm effect of essential oils was recovered from the medicinal plant of Solanum nigrum, and confirmed the available essential oils by liquid chromatography-mass spectroscopy analysis. The excellent anti-microbial activity and minimum biofilm inhibition concentration of the essential oils against P. mirabilis was indicated at 200 µg/mL. The absence of viability and altered exopolysaccharide structure of treated cells were showed by biofilm metabolic assay and phenol–sulphuric acid method. The fluorescence differentiation of P. mirabilis treated cells was showed with more damages by confocal laser scanning electron microscope. Further, more morphological changes of essential oils treated cells were differentiated from normal cells by scanning electron microscope. Altogether, the results were reported that the S. nigrum essential oils have anti-biofilm ability.

MrpH, a new class of metal-binding adhesin, requires zinc to mediate biofilm formation.

Proteus mirabilis, a Gram-negative uropathogen, is a major causative agent in catheter-associated urinary tract infections (CAUTI). Mannose-resistant Proteus-like fimbriae (MR/P) are crucially important for P. mirabilis infectivity and are required for biofilm formation and auto-aggregation, as well as for bladder and kidney colonization. Here, the X-ray crystal structure of the MR/P tip adhesin, MrpH, is reported. The structure has a fold not previously described and contains a transition metal center with Zn2+ coordinated by three conserved histidine residues and a ligand. Using biofilm assays, chelation, metal complementation, and site-directed mutagenesis of the three histidines, we show that an intact metal binding site occupied by zinc is essential for MR/P fimbria-mediated biofilm formation, and furthermore, that P. mirabilis biofilm formation is reversible in a zinc-dependent manner. Zinc is also required for MR/P-dependent agglutination of erythrocytes, and mutation of the metal binding site renders P. mirabilis unfit in a mouse model of UTI. The studies presented here provide important clues as to the mechanism of MR/P-mediated biofilm formation and serve as a starting point for identifying the physiological MR/P fimbrial receptor.

Antimicrobial activity Study of triclosan-loaded WBPU on Proteus mirabilis in vitro

To evaluate the antimicrobial activity study of triclosan-loaded waterborne polyurethanes (WBPU) on Proteus mirabilis in vitro. Inhibition zone assays on petri plates with triclosan-loaded WBPU samples were used to test its antimicrobial activity on Proteus mirabilis. Models of the catheterized bladder supplied with artificial urine infected with Proteus mirabilis were employed to confirm the antimicrobial activity of triclosan-loaded WBPU. Bacteria colony counting, pH of the residual urine at each time point and catheter blockage time were recorded. Confocal laser scanning microscopy, scanning electron microscopy and encrustation deposits dry weighing were used for evaluating the biofilm formation. Inhibition zones formed in the triclosan-loaded WBPU groups in a dose-response manner (the radius for samples with 1, 0.1 and 0.01mg triclosan were 9.93±1.08, 6.07±0.54 and 2.47±0.25mm, P<0.001). The bacterial growth in the triclosan group was markedly inhibited, which was almost undetectable after 12h of bladder running. Residual urine pH in the control group increased significantly in comparison with the triclosan group (9.50±0.04 vs. 6.17±0.01 at 24h, P<0.001). The presence of triclosan-loaded WBPU decreased catheter encrustations and markedly postponed the catheter blockage time, as well as suppressed the Proteus mirabilis biofilm formation (33.9±13.9mg vs. 1.4±1.5mg, P=0.016). Triclosan-loaded WBPU significantly inhibited Proteus mirabilis' growth and biofilm formation, indicating the promising antibacterial effects on Proteus mirabilis in vitro. Further efforts are under way that involves coating the material onto the urinary catheters and in vivo studies.

Development of a Phage Cocktail to Control Proteus mirabilis Catheter-associated Urinary Tract Infections

Proteus mirabilis is an enterobacterium that causes catheter-associated urinary tract infections (CAUTIs) due to its ability to colonize and form crystalline biofilms on the catheters surface. CAUTIs are very difficult to treat, since biofilm structures are highly tolerant to antibiotics. Phages have been used widely to control a diversity of bacterial species, however, a limited number of phages for P. mirabilis have been isolated and studied. Here we report the isolation of two novel virulent phages, the podovirus vB_PmiP_5460 and the myovirus vB_PmiM_5461, which are able to target, respectively, 16 of the 26 and all the Proteus strains tested in this study. Both phages have been characterized thoroughly and sequencing data revealed no traces of genes associated with lysogeny. To further evaluate the phages’ ability to prevent catheter’s colonization by Proteus, the phages adherence to silicone surfaces was assessed. Further tests in phage-coated catheters using a dynamic biofilm model simulating CAUTIs, have shown a significant reduction of P. mirabilis biofilm formation up to 168 h of catheterization. These results highlight the potential usefulness of the two isolated phages for the prevention of surface colonization by this bacterium.

Wild mushroom extracts as inhibitors of bacterial biofilm formation.

Microorganisms can colonize a wide variety of medical devices, putting patients in risk for local and systemic infectious complications, including local-site infections, catheter-related bloodstream infections, and endocarditis. These microorganisms are able to grow adhered to almost every surface, forming architecturally complex communities termed biofilms. The use of natural products has been extremely successful in the discovery of new medicine, and mushrooms could be a source of natural antimicrobials. The present study reports the capacity of wild mushroom extracts to inhibit in vitro biofilm formation by multi-resistant bacteria. Four Gram-negative bacteria biofilm producers (Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Acinetobacter baumannii) isolated from urine were used to verify the activity of Russula delica, Fistulina hepatica, Mycena rosea, Leucopaxilus giganteus, and Lepista nuda extracts. The results obtained showed that all tested mushroom extracts presented some extent of inhibition of biofilm production. Pseudomonas aeruginosa was the microorganism with the highest capacity of biofilm production, being also the most susceptible to the extracts inhibition capacity (equal or higher than 50%). Among the five tested extracts against E. coli, Leucopaxillus giganteus (47.8%) and Mycenas rosea (44.8%) presented the highest inhibition of biofilm formation. The extracts exhibiting the highest inhibitory effect upon P. mirabilis biofilm formation were Sarcodon imbricatus (45.4%) and Russula delica (53.1%). Acinetobacter baumannii was the microorganism with the lowest susceptibility to mushroom extracts inhibitory effect on biofilm production (highest inhibition—almost 29%, by Russula delica extract). This is a pioneer study since, as far as we know, there are no reports on the inhibition of biofilm production by the studied mushroom extracts and in particular against multi-resistant clinical isolates; nevertheless, other studies are required to elucidate the mechanism of action.

Development of 3D architecture of uropathogenic Proteus mirabilis batch culture biofilms—A quantitative confocal microscopy approach

This work studies the development of the 3D architecture of batch culture P. mirabilis biofilms on the basis of morpho-topological descriptors calculated from confocal laser scanning microscopy (CLSM) stacks with image processing routines. A precise architectonical understanding of biofilm organization on a morpho-topological level is necessary to understand emergent interactions with the environment and the appearance of functionally different progeny swarmer cells. P. mirabilis biofilms were grown on glass coverslips for seven days on LB broth and subjected to in situ immunofluorescence. Confocal image stacks were deconvolved prior to segmentation of regions of interest (ROI) that identify individual bacteria and extracellular material, followed by 3D reconstruction and calculation of different morpho-topological key descriptors.Results showed that P. mirabilis biofilm formation followed a five stage process: (i) reversible adhesion to the surface characterized by slow growth, presence of elongated bacteria, and absence of extracellular material, (ii) irreversible bacterial adhesion concomitant to decreasing elongation, and the beginning of extracellular polymer production, (iii) accelerated bacterial growth concomitant to continuously decreasing elongation and halting of extracellular polymer production, (iv) maturation of biofilm defined by maximum bacterial density, volume, minimum elongation, maximum extracellular material, and highest compaction, and (v) decreased bacterial density and extracellular material through detachment and dispersion. Swarmer cells do not play a role in P. mirabilis biofilm formation under the applied conditions. Our approach sets the basis for future studies of 3D biofilm architecture using dynamic in vivo models and different environmental conditions that assess clinical impacts of P. mirabilis biofilm.

Proteus Mirabilis Biofilm Protection Against Struvite Crystal Dissolution and its Implications in Struvite Urolithiasis

Proteus mirabilis biofilm formation, struvite (MgNH4PO4 · 6H2O) crystal formation and dissolution in an artificial urine mixture were monitored using computer-enhanced microscopy (CEM) and a 1 × 3mm. glass flow cell. Image analysis showed that P. mirabilis biofilm formation did not occur to any extent at macroenvironment flow rates greater than two mL/h (equivalent to a microenvironment flow rate of <5μm./sec). Essentially, cells attached to glass surfaces, grew slowly and divided. Daughter cells were generally released directly into the medium where they could then presumably colonize other regions. Microcolonies formed by the adhesion of aggregates of cells from the medium, and over time grew into biofilms. Struvite crystallization due to urease activity and pH elevation above neutrality, was preceded by the deposition of organic matter on the glass surface, followed by the appearance of a number of tiny (one to two μm.) crystals. Crystals forming within a biofilm at low dilution rates took on a characteristic twinned or “X-shaped” appearance (crystal habit) indicative of a rapid growth rate. Those forming outside the biofilm took on a more tabular appearance reflecting their slower growth. When the macroenvironment flow rate of artificial urine (initial pH 5.8) in the glass flow cell was increased from two mL/h to four mL/h, struvite crystals not associated with biofilms dissolved within five to 10 min. Crystals entrapped within the P. mirabilis biofilm withstood flow rates up to 200mL/h presumably due to the maintenance of an alkaline Mg-saturated microenvironment within the biofilm. These observations may suggest a mechanism by which struvite calculi can grow in spite of neutral or acidic urine pH and resist mild acidification therapy.