Resistance to acetohydroxamate acquired by slow adaptive increases in urease in cultured tobacco cells

Urease activity of tobacco XD cells (1U cells) had undergone a 4-fold increase (4U cells) during a year of growth on urea (Skokut and Filner 1980 Plant Phvsiol 65: 995-1003). A clone of 4U cells gave rise to 12U cells during another year of growth on urea. The doubling time of 12U cells on urea is 2.2 days, compared to about 4 days for 1U cells, while 1U and 12U cells double in 2 days on nitrate. Acetohydroxamic acid (AHA), a specific inhibitor/reversible inactivator of jack bean urease, affects tobacco cell urease similarly. Fifty per cent inhibition of growth by AHA occurred at 20 micromolar in 1U cells growing on urea and at 165 micromolar in 12U cells growing on urea, but at 600 micromolar for either 1U or 12U cells growing on nitrate. When 12U cells were grown on urea with 100 micromolar AHA, extractable urease activity decreased 80% within 2.5 hours and remained at this level for 2 weeks; the doubling time increased to 3.7 days, and intracellular urea rose 2-fold, compared to 12U cells grown on urea without AHA. Urease of 12U cells inactivated by AHA in vivo could be reactivated to its pre-AHA level by incubation at 30 C after extraction and separation from free AHA. AHA inhibited incorporation of ¹⁵N from [¹⁵N]urea into Kjeldahl nitrogen in the cells, in spite of the increased intracellular urea. These results indicate that AHA acts primarily by inhibiting urease action, rather than by inhibition of formation of urease protein or of uptake of urea. Because 12U cells are 8 times more tolerant of AHA than 1U cells, it is likely that growth on urea in the presence of AHA should select strongly for cells with high urease.     

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.     

Chelator-Induced Dispersal and Killing of Pseudomonas aeruginosa Cells in a Biofilm

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.     

Influence of quorum sensing signal molecules on biofilm formation in <Emphasis Type="Italic">Proteus mirabilis</Emphasis> O18

The influence of basis of quorum sensing molecules on Proteus strains is much less known as compared to Pseudomonas or Escherichia. We have previously shown that a series of acylated homoserine lactones (acyl-HSL) does not influence the ureolytic, proteolytic, or hemolytic abilities, and that the swarming motility of Proteus mirabilis rods is strain specific. The aim of the presented study was to find out if the presence of a series of acyl-HSL influences biofilm formation of P. mirabilis laboratory strain belonging to O18 serogroup. This serogroup is characterized by the presence of a unique non-carbohydrate component, namely phosphocholine. Escherichia coli and P. mirabilis O18 strains used in this work contains cloned plasmids encoding fluorescent protein genes with constitutive gene expression. In mixed biofilms in stationary and continuous flow conditions, P. mirabilis O18 overgrow whole culture. P. mirabilis O18 strain has genetically proved a presence of AI–2 quorum sensing system. Differences in biofilm structure were observed depending on the biofilm type and culture methods. From tested acylated homoserine lactones (BHL, HHL, OHL, DHL, dDHL, tDHL), a significant influence had BHL on thickness, structure, and the amount of exopolysaccharides produced by biofilms formed by P. mirabilis O18 pDsRed₂.     

Virulence targeted inhibitory effect of linalool against the exclusive uropathogen Proteus mirabilis

Proteus mirabilis is one of the leading causes of catheter-associated UTIs (CAUTI) in individuals with prolonged urinary catheterization. Since, biofilm assisted antibiotic resistance is reported to complicate the treatment strategies of P. mirabilis infections, the present study was aimed to attenuate biofilm and virulence factor production in P. mirabilis. Linalool is a naturally occurring monoterpene alcohol found in a wide range of flowers and spice plants and has many biological applications. In this study, linalool exhibited concentration dependent anti-biofilm activity against crystalline biofilm of P. mirabilis through reduced production of the virulence enzyme urease that raises the urinary pH and drives the formation of crystals (struvite) in the biofilm. The results of q-PCR analysis unveiled the down regulation of biofilm/virulence associated genes upon linalool treatment, which was in correspondence with the in vitro bioassays. Thus, this study reports the feasibility of linalool acting as a promising anti-biofilm agent against P. mirabilis mediated CAUTI.     

Effect of traditionally used herb Pedalium murex L. and its active compound pedalitin on urease expression – For the management of kidney stone

Pedalium murex L. is a medicinal herb that has been used for the treatment of diseases related to kidney in the traditional system of medicine. The current study aims to study the effect of ethyl acetate extract of P. murex (EAEP) and its fractionated compound pedalitin against urease production and UreC gene expression in Proteus mirabilis. The selected reference strain Proteus mirabilis (MTCC 425) and the isolates culture of Proteus mirabilis were subjected to study the antibacterial efficacy of P. murex. Expression analysis of P. mirabilis urease gene was successfully done by QPCR. The ethyl acetate extract effectively inhibit the reference Proteus mirabilis and bacterial isolates of Proteus mirabilis in the clinical samples studied. EAEP has showed more potent activity (56.7%) against urease enzyme and pedalitin also exhibited potent activity (30.1%). Using qPCR, the expression of UreC gene of P. mirabilis was controlled by EAEP and also its bioactive compound pedalitin. The present study clearly demonstrated the potency of P. murex in controlling the growth of pathogenic P. mirabilis and to control the expression of urease enzyme production as well as to restrict the urease gene expression in P. mirabilis.     

Regulation of urease gene expression by Streptococcus salivarius growing in biofilms

The metabolism of urea by urease enzymes of oral bacteria profoundly influences oral biofilm pH homeostasis and oral microbial ecology. The purpose of this study was to gain insight into the regulation of expression of the low pH-inducible urease genes in populations of Streptococcus salivarius growing in vitro in biofilms and to explore whether urease regulation or the levels of urease expression in biofilm cells differed significantly from planktonic cells. Two strains of S. salivarius harbouring urease promoter fusions to a chloramphenicol acetyltransferase (cat) gene were used: PurelCAT, containing a fusion to the full-length, pH-sensitive promoter; or Pureldelta100CAT, a constitutively derepressed deletion derivative of the urease gene promoter. The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium with or without pH control. Both CAT and urease activities in biofilms were measured at 'quasi-steady state' and after a 25mM glucose pulse. The results showed that CAT expression in PurelCAT was repressed at relatively neutral pH values, and that expression could be induced by acidic pH after carbohydrate challenge. Biofilms of PurelCAT grown at low pH, without buffering, had about 20-fold higher CAT levels, and only a modest further induction could be elicited with carbohydrate pulsing. The levels of CAT in biofilms of PurelCAT grown in buffered medium were slightly higher than those reported for planktonic cells cultured at pH 7.0, and the levels of CAT in Purel-CAT growing at low pH or after induction were similar to those reported for fully induced planktonic cells. CAT activity in Pureldelta100CAT was constitutively high, regardless of growth conditions. Interestingly, urease activity detected in biofilms of the parent strain, S. salivarius 57.1, could be as much as 130-fold higher than that reported for fluid chemostat cultures grown under similar conditions. The higher level of urease activity in biofilms was probably caused by the accumulation of the stable urease enzyme within biofilm cells, low pH microenvironments and the growth phase of populations of cells in the biofilm. The ability of S. salivarius biofilm cells to upregulate urease expression in response to pH gradients and to accumulate greater quantities of urease enzyme when growing in biofilms may have a significant impact on oral biofilm pH homeostasis and microbial ecology in vivo. Additionally, S. salivarius carrying the pH-sensitive urease gene promoter fused to an appropriate reporter gene may be a useful biological probe for sensing biofilm pH in situ.     

Design,synthesis, molecular docking,anti-Proteus mirabilis and urease inhibition of new fluoroquinolone carboxylic acid derivatives

New hydroxamic acid, hydrazide and amide derivatives of ciprofloxacin in addition to their analogues of levofloxacin were prepared and identified by different spectroscopic techniques. Some of the prepared compounds revealed good activity against the urease splitting bacteria, Proteus mirabilis. The urease inhibitory activity was investigated using indophenol method. Most of the tested compounds showed better activity than the reference acetohydroxamic acid (AHA). The ciprofloxacin hydrazide derivative 3a and levofloxacin hydroxamic acid 7 experienced the highest activity (IC₅₀ = 1.22 μM and 2.20 μM, respectively). Molecular docking study revealed high spontaneous binding ability of the tested compounds to the active site of urease.     

In vitro activity of vancomycin, quinupristin/dalfopristin, and linezolid against intact and disrupted biofilms of staphylococci

 

Shed cells or disrupted parts of the biofilm may enter the circulation causing serious and very hard to treat biofilm-associated infections. The activity of antimicrobial agents against the shed cells/disrupted biofilms is largely unknown.

Methods

We studied the in vitro susceptibility of intact and disrupted biofilms of thirty clinical isolates of methicillin-resistant and methicillin–susceptible Staphylococcus aureus (MRSA and MSSA) and Staphylococcus epidermidis to vancomycin, quinupristin/dalfopristin, and linezolid and compared it to that of the suspended (planktonic) cells.

Results

Bacteria in the disrupted biofilms were as resistant as those in the intact biofilms at the minimum inhibitory concentrations of the antibiotics. At higher concentrations, bacteria in the disrupted biofilms were significantly (P < 0.001) less resistant than those in the intact biofilms but more resistant than the planktonic cells. Quinupristin/dalfopristin showed the best activity against cells of the disrupted biofilms at concentrations above MICs and vancomycin, at 500 and 1,000 μg/ml, was significantly more active against the biofilms of MRSA and S. epidermidis

Conclusion

The difficulty of treating biofilm-associated infections may be attributed not only to the difficulty of eradicating the biofilm focus but also to the lack of susceptibility of cells disrupted from the biofilm to antimicrobial agents.     

Surface Percolation for Soil Improvement by Biocementation Utilizing In Situ Enriched Indigenous Aerobic and Anaerobic Ureolytic Soil Microorganisms

The use of biocementation via microbially induced carbonate precipitation (MICP) for improving the mechanical properties of weak soils in the laboratory has gained increased attention in recent years. This study proposes an approach for applying biocementation in situ, by combining the surface percolation of nutrients and cementation solution (urea/CaCl₂) with in situ cultivation of indigenous soil urease positive microorganisms under non-sterile conditions. The enrichment of indigenous ureolytic soil bacteria was firstly tested in batch reactors. Using selective conditions (i.e., pH of 10 and urea concentrations of 0.17 M), highly active ureolytic microorganisms were enriched from four diverse soil samples under both oxygen-limited (anoxic) and oxygen-free (strictly anaerobic) conditions, providing final urease activities of more than 10 and 5 U/mL, respectively. The enrichment of indigenous ureolytic soil microorganisms was secondly tested in pure silica sand columns (300 and 1000 mm) for biocementation applications using the surface percolation approach. By applying the same selective conditions, the indigenous ureolytic soil microorganisms with high urease activity were also successfully enriched for both the fine and coarse sand columns. However, the in situ enriched urease activity was highly related to the dissolved oxygen of the percolated growth medium. The results showed that the in situ cultivated urease activity may produce non-clogging cementation over the entire 1000-mm columns, with unconfined compressive strength varying between 850–1560 kPa (for coarse sand) and 150–700 kPa (for fine sand), after 10 subsequent applications of cementation solution. The typically observed loss of ureolytic activity during the repeated application of the cementation solution was recovered by providing more growth medium under selective enrichment conditions, enabling the in situ enriched ureolytic microorganisms to increase in numbers and urease activity in such a way that continued cementation was possible.     

Chemical composition of Enterococcus faecalis in biofilm cells initiated from different physiologic states

Enterococcus faecalis is a ubiquitous bacterium of the gut that is observed in persistent periradicular infections. Its pathogenicity is associated with biofilm formation and the ability to survive under nutrient-poor (starvation) conditions. However, characteristics of chemical composition of biofilm cells developed by starved E. faecalis cells remain poorly understood. In this study, E. faecalis cells in exponential, stationary, and starvation phases were prepared and separately cultured to form biofilms. Confocal laser scanning microscopy was performed to verify biofilm formation. Raman microscopy was used to investigate the chemical composition of cells within the biofilms. Compared to cells in exponential or stationary phase, starved cells developed biofilms with fewer culturable cells (P?E. faecalis.     

Novel Insights into the Proteus mirabilis Crystalline Biofilm Using Real-Time Imaging

The long-term use of indwelling catheters results in a high risk from urinary tract infections (UTI) and blockage. Blockages often occur from crystalline deposits, formed as the pH rises due to the action of urease-producing bacteria; the most commonly found species being Proteus mirabilis. These crystalline biofilms have been found to develop on all catheter materials with P. mirabilis attaching to all surfaces and forming encrustations. Previous studies have mainly relied on electron microscopy to describe this process but there remains a lack of understanding into the stages of biofilm formation. Using an advanced light microscopy technique, episcopic differential interference contrast (EDIC) microscopy combined with epifluorescence (EF), we describe a non-destructive, non-contact, real-time imaging method used to track all stages of biofilm development from initial single cell attachment to complex crystalline biofilm formation. Using a simple six-well plate system, attachment of P. mirabilis (in artificial urine) to sections of silicone and hydrogel latex catheters was tracked over time (up to 24 days). Using EDIC and EF we show how initial attachment occurred in less than 1 h following exposure to P. mirabilis. This was rapidly followed by an accumulation of an additional material (indicated to be carbohydrate based using lectin staining) and the presence of highly elongated, motile cells. After 24 h exposure, a layer developed above this conditioning film and within 4 days the entire surface (of both catheter materials) was covered with diffuse crystalline deposits with defined crystals embedded. Using three-dimensional image reconstruction software, cells of P. mirabilis were seen covering the crystal surfaces. EDIC microscopy could resolve these four components of the complex crystalline biofilm and the close relationship between P. mirabilis and the crystals. This real-time imaging technique permits study of this complex biofilm development with no risk of artefacts due to sample manipulation. A full understanding of the stages and components involved in crystalline encrustation formation will aid in the development of new protocols to manage and ultimately prevent catheter blockage.     

The crystal structure of Sporosarcina pasteurii urease in a complex with citrate provides new hints for inhibitor design

Urease, the enzyme that catalyses the hydrolysis of urea, is a virulence factor for a large number of ureolytic bacterial human pathogens. The increasing resistance of these pathogens to common antibiotics as well as the need to control urease activity to improve the yield of soil nitrogen fertilization in agricultural applications has stimulated the development of novel classes of molecules that target urease as enzyme inhibitors. We report on the crystal structure at 1.50-Å resolution of a complex formed between citrate and urease from Sporosarcina pasteurii, a widespread and highly ureolytic soil bacterium. The fit of the ligand to the active site involves stabilizing interactions, such as a carboxylate group that binds the nickel ions at the active site and several hydrogen bonds with the surrounding residues. The citrate ligand has a significantly extended structure compared with previously reported ligands co-crystallized with urease and thus represents a unique and promising scaffold for the design of new, highly active, stable, selective inhibitors.     

In Vitro Biofilm Activity of Non-Candida albicans Candida Species

Candidosis has been attributed to C. albicans; however, infections caused by non-Candida albicans Candida (NCAC) species are increasingly being recognised. The ability of Candida to grow as a biofilm is an important feature that promotes both infection and persistence in the host. The biofilms’ activity is significant since high activity might be associated with enhanced expression of putative virulence factors, whilst in contrast low activity has previously been suggested as a mechanism for resistance of biofilm cells to antimicrobials. The aim of this study was to determine the metabolic activity of in vitro biofilms formed by different clinical isolates of NCAC species. The in situ total metabolic activity of C. parapsilosis, C. tropicalis and C. glabrata biofilms was determined using 2,3-(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay, and the number of cultivable cells was also established by CFU (colony forming unit) counts. The biofilm structure was assessed by scanning electron microscopy (SEM). Results showed that total biofilm metabolic activity was species and strain dependent. C. glabrata exhibited the lowest biofilm metabolic activity despite having the highest number of biofilm cultivable cells. Similarly, the metabolic activity of resuspended C. glabrata biofilm and planktonic cells was lower than that of the other species. This study demonstrates the existence of intrinsic activity differences amongst NCAC species, which could have important implications in terms of species relative virulence. Furthermore, the absence of an obvious correlation, between cultivable cells number and total biofilm activity, raises the question about which parameter is the most appropriate for the in vitro assessment of biofilms and their potential clinical significance.     

Purpurin Triggers Caspase-Independent Apoptosis in Candida dubliniensis Biofilms

Candida dubliniensis is an important human fungal pathogen that causes oral infections in patients with AIDS and diabetes mellitus. However, C. Dubliniensis has been frequently reported in bloodstream infections in clinical settings. Like its phylogenetically related virulent species C. albicans, C. Dubliniensis is able to grow and switch between yeast form and filamentous form (hyphae) and develops biofilms on both abiotic and biotic surfaces. Biofilms are recalcitrant to antifungal therapies and C. Dubliniensis readily turns drug resistant upon repeated exposure. More than 80% of infections are associated with biofilms. Suppression of fungal biofilms may therefore represent a viable antifungal strategy with clinical relevance. Here, we report that C. dubliniensis biofilms were inhibited by purpurin, a natural anthraquinone pigment isolated from madder root. Purpurin inhibited C. dubliniensis biofilm formation in a concentration-dependent manner; while mature biofilms were less susceptible to purpurin. Scanning electron microscopy (SEM) analysis revealed scanty structure consisting of yeast cells in purpurin-treated C. dubliniensis biofilms. We sought to delineate the mechanisms of the anti-biofilm activity of purpurin on C. Dubliniensis. Intracellular ROS levels were significantly elevated in fungal biofilms and depolarization of MMP was evident upon purpurin treatment in a concentration-dependent manner. DNA degradation was evident. However, no activated metacaspase could be detected. Together, purpurin triggered metacaspase-independent apoptosis in C. dubliniensis biofilms.     

Role of interspecies interactions in dual-species biofilms developed in vitro by uropathogens isolated from polymicrobial urinary catheter-associated bacteriuria

Most catheter-associated urinary tract infections are polymicrobial. Here, uropathogen interactions in dual-species biofilms were studied. The dual-species associations selected based on their prevalence in clinical settings were Klebsiella pneumoniae–Escherichia coli, E. coli–Enterococcus faecalis, K. pneumoniae–E. faecalis, and K. pneumoniae–Proteus mirabilis. All species developed single-species biofilms in artificial urine. The ability of K. pneumoniae to form biofilms was not affected by E. coli or E. faecalis co-inoculation, but was impaired by P. mirabilis. Conversely, P. mirabilis established a biofilm when co-inoculated with K. pneumoniae. Additionally, E. coli persistence in biofilms was hampered by K. pneumoniae but not by E. faecalis. Interestingly, E. coli, but not K. pneumoniae, partially inhibited E. faecalis attachment to the surface and retarded biofilm development. The findings reveal bacterial interactions between uropathogens in dual-species biofilms ranged from affecting initial adhesion to outcompeting one bacterial species, depending on the identity of the partners involved.     

Ureolytic nitrification at low pH by Nitrosospira spec.

An ureolytic ammonium-oxidizing chemolithotroph belonging to the genus Nitrosospira was shown to nitrify at pH 4.5 in a pH-stat with urea as a substrate. With ammonium as the sole substrate nitrification did not occur at pH values below 5.5. Nitrosomonas europaea ATCC 19718 and Nitrosospira briensis ATCC 25971 did not possess urease activity. The results indicate that in acid soils nitrification by ureolytic ammonium-oxidizing chemolithotrophs may not be restricted to microsites of neutral pH.