Structure and Assembly of the Proteus mirabilis Flagellar Motor by Cryo-Electron Tomography.

Proteus mirabilis is a Gram-negative Gammaproteobacterium and a major causative agent of urinary tract infections in humans. It is characterized by its ability to switch between swimming motility in liquid media and swarming on solid surfaces. Here, we used cryo-electron tomography and subtomogram averaging to reveal the structure of the flagellar motor of P. mirabilis at nanometer resolution in intact cells. We found that P. mirabilis has a motor that is structurally similar to those of Escherichia coli and Salmonella enterica, lacking the periplasmic elaborations that characterize other more specialized gammaproteobacterial motors. In addition, no density corresponding to stators was present in the subtomogram average suggesting that the stators are dynamic. Finally, several assembly intermediates of the motor were seen that support the inside-out assembly pathway.

First-time characterization of viable but non-culturable Proteus mirabilis: Induction and resuscitation.

Pathogenic bacteria can enter into a viable but non-culturable (VBNC) state under unfavourable conditions. Proteus mirabilis is responsible for dire clinical consequences including septicaemia, urinary tract infections and pneumonia, but is not a species previously known to enter VBNC state. We suggested that stress-induced P. mirabilis can enter a VBNC state in which it retains virulence. P. mirabilis isolates were incubated in extreme osmotic pressure, starvation, low temperature and low pH to induce a VBNC state. Resuscitation was induced by temperature upshift and inoculation in tryptone soy broth with Tween 20 and brain heart infusion broth. Cellular ultrastructure and gene expression were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR), respectively. High osmotic pressure and low acidity caused rapid entry into VBNC state. Temperature upshift caused the highest percentage of resuscitation (93%) under different induction conditions. In the VBNC state, cells showed aberrant and dwarf morphology, virulence genes and stress response genes (envZ and rpoS) were expressed (levels varied depending on strain and inducing factors). This is the first-time characterization of VBNC P. mirabilis. The ability of P. mirabilis pathogenic strains to enter a stress-induced VBNC state can be a serious public health threat.

Cell Shape and Population Migration Are Distinct Steps of Proteus mirabilis Swarming That Are Decoupled on High-Percentage Agar.

Swarming on rigid surfaces requires movement of cells as individuals and as a group of cells. For the bacterium Proteus mirabilis, an individual cell can respond to a rigid surface by elongating and migrating over micrometer-scale distances. Cells can form groups of transiently aligned cells, and the collective population is capable of migrating over centimeter-scale distances. To address how P. mirabilis populations swarm on rigid surfaces, we asked whether cell elongation and single-cell motility are coupled to population migration. We first measured the relationship between agar concentration (a proxy for surface rigidity), single-cell phenotypes, and swarm colony phenotypes. We find that cell elongation and single-cell motility are coupled with population migration on low-percentage hard agar (1% to 2.5%) and become decoupled on high-percentage hard agar (>2.5%). Next, we evaluate how disruptions in lipopolysaccharide (LPS), specifically the O-antigen components, affect responses to hard agar. We find that LPS is not essential for elongation and motility of individual cells, as predicted, and instead functions to broaden the range of agar concentrations on which cell elongation and motility are coupled with population migration. These findings demonstrate that cell elongation and motility are coupled with population migration under a permissive range of surface conditions; increasing agar concentration is sufficient to decouple these behaviors. Since swarm colonies cover greater distances when these steps are coupled than when they are not, these findings suggest that collective interactions among P. mirabilis cells might be emerging as a colony expands outwards on rigid surfaces.IMPORTANCE How surfaces influence cell size, cell-cell interactions, and population migration for robust swarmers like P. mirabilis is not fully understood. Here, we have elucidated how cells change length along a spectrum of sizes that positively correlates with increases in agar concentration, regardless of population migration. Single-cell phenotypes can be decoupled from collective population migration simply by increasing agar concentration. A cell's lipopolysaccharides function to broaden the range of agar conditions under which cell elongation and single-cell motility remain coupled with population migration. In eukaryotes, the physical environment, such as a surface matrix, can impact cell development, shape, and migration. These findings support the idea that rigid surfaces similarly act on swarming bacteria to impact cell shape, single-cell motility, and collective population migration.

The impact of ethanol extract of propolis on biofilm forming by Proteus Mirabilis strains isolated from chronic wounds infections.

Alcoholic propolis extracts may be used to eliminate microbes in mucous membranes and skin inflammations and in wound infections. The aim of this study was an assessment of the ethanol extract of propolis (EEP) activity against biofilm formation by P. mirabilis. Six clinical strains of P. mirabilis isolated from patients with chronic wound infection, and one reference strain of P. mirabilis ATCC 29906 were used. Biofilm was formed in 96-well plate. In order to evaluate the effect of EEP at a concentration range of 1.56-100 mg/mL on the forming and mature biofilm, P. mirabilis cells were released by sonication. In this study the effectiveness of 25-100 mg/mL of EEP on the forming P. mirabilis biofilm and concentrations of 25-50 mg/mL of EEP on formed biofilm has been demonstrated. Our results suggest the possibility of using the EEP in treatment of chronic wound infection caused by P. mirabilis.

Identification of small molecule compounds active against Staphylococcus aureus and Proteus mirabilis.

Staphylococcus aureus is a human pathogen rapidly becoming a serious health problem due to ease of acquiring antibiotic resistance. To help identify potential new drug candidates effective against the pathogen, a small focused library was screened for inhibition of bacterial growth against several pathogens, including S. aureus. At least one of the compounds, Compound 10, was capable of blocking bacterial growth of S. aureus in a test tube with IC50 = 140 ±â€¯30 µM. Another inhibitor, Compound 7, was bacteriostatic against S. aureus with IC50 ranging from 33 to 150 µM against 3 different strains. However, only Compound 7 was bactericidal against P. mirabilis as examined by electron microscopy. Human cell line toxicity studies suggested that both compounds had small effect on cell growth at 100 µM concentration as examined by MTT assay. Analysis of compounds' structures showed lack of similarity to any known antibiotics and bacteriostatics, potentially offering the inhibitors as an alternative to existing solutions in controlling bacterial infections for selected pathogens.

Anaerobic choline metabolism in microcompartments promotes growth and swarming of Proteus mirabilis.

Gammaproteobacteria are important gut microbes but only persist at low levels in the healthy gut. The ecology of Gammaproteobacteria in the gut environment is poorly understood. Here, we demonstrate that choline is an important growth substrate for representatives of Gammaproteobacteria. Using Proteus mirabilis as a model, we investigate the role of choline metabolism and demonstrate that the cutC gene, encoding a choline-trimethylamine lyase, is essential for choline degradation to trimethylamine by targeted mutagenesis of cutC and subsequent complementation experiments. Proteus mirabilis can rapidly utilize choline to enhance growth rate and cell yield in broth culture. Importantly, choline also enhances swarming-associated colony expansion of P. mirabilis under anaerobic conditions on a solid surface. Comparative transcriptomics demonstrated that choline not only induces choline-trimethylamine lyase but also genes encoding shell proteins for the formation of bacterial microcompartments. Subsequent analyses by transmission electron microscopy confirmed the presence of such novel microcompartments in cells cultivated in liquid broth and hyper-flagellated swarmer cells from solid medium. Together, our study reveals choline metabolism as an adaptation strategy for P. mirabilis and contributes to better understand the ecology of this bacterium in health and disease.

Biofilms, flagella, and mechanosensing of surfaces by bacteria.

Formation of a bacterial biofilm is a developmental process that begins when a cell attaches to a surface, but how does a bacterial cell know it is on or near a surface in the first place? The phase of this 'swim-or-stick' switch is determined by a sensory transduction mechanism referred to as surface sensing, which involves the rotating bacterial flagellum. This review explores six bacterial species as models of flagellar mechanosensing of surfaces to understand the current state of our knowledge and the challenges that lie ahead. A common link between these bacteria is a requirement for the proper function of the flagellar motor stators that channel ions into the cell to drive flagellar rotation. Conditions that affect ion flow act as a signal that, ultimately, controls the master transcriptional regulatory circuits controlling the flagellar hierarchy and biofilm formation.

Evaluation of environmental scanning electron microscopy for analysis of Proteus mirabilis crystalline biofilms in situ on urinary catheters.

Proteus mirabilis is a common cause of catheter-associated urinary tract infections and frequently leads to blockage of catheters due to crystalline biofilm formation. Scanning electron microscopy (SEM) has proven to be a valuable tool in the study of these unusual biofilms, but entails laborious sample preparation that can introduce artefacts, undermining the investigation of biofilm development. In contrast, environmental scanning electron microscopy (ESEM) permits imaging of unprocessed, fully hydrated samples, which may provide much insight into the development of P. mirabilis biofilms. Here, we evaluate the utility of ESEM for the study of P. mirabilis crystalline biofilms in situ, on urinary catheters. In doing so, we compare this to commonly used conventional SEM approaches for sample preparation and imaging. Overall, ESEM provided excellent resolution of biofilms formed on urinary catheters and revealed structures not observed in standard SEM imaging or previously described in other studies of these biofilms. In addition, we show that energy-dispersive X-ray spectroscopy (EDS) may be employed in conjunction with ESEM to provide information regarding the elemental composition of crystalline structures and demonstrate the potential for ESEM in combination with EDS to constitute a useful tool in exploring the mechanisms underpinning crystalline biofilm formation.

Observation of multicellular spinning behavior of Proteus mirabilis by atomic force microscopy and multifunctional microscopy.

This study aimed to observe the multicellular spinning behavior of Proteus mirabilis by atomic force microscopy (AFM) and multifunctional microscopy in order to understand the mechanism underlying this spinning movement and its biological significance. Multifunctional microscopy with charge-coupled device (CCD) and real-time AFM showed changes in cell structure and shape of P. mirabilis during multicellular spinning movement. Specifically, the morphological characteristics of P. mirabilis, multicellular spinning dynamics, and unique movement were observed. Our findings indicate that the multicellular spinning behavior of P. mirabilis may be used to collect nutrients, perform colonization, and squeeze out competitors. The movement characteristics of P. mirabilis are vital to the organism's biological adaptability to the surrounding environment.

Inhibition of development, swarming differentiation and virulence factors in Proteus mirabilis by an extract of Lithrea molleoides and its active principle (Z,Z)-5-(trideca-4',7'-dienyl)-resorcinol.

Antibacterial activity of Lithrea molleoides extract against Proteus mirabilis has been previously reported by our group. In the present study, the compound (Z,Z)-5-(trideca-4',7'-dienyl)-resorcinol (1) was isolated as its responsible active principle. The effects of the compound obtained and of L. molleoides extract on P. mirabilis growth and virulence factors were evaluated. Compound 1 showed MIC and MBC values of 4000 µg/ml. It was found that the extract, at four times the MIC, produced complete killing of the uropathogen at 2h from the beginning of the experiment, while the alkylresorcinol, at four times the MIC, produced the same effect after 24 h. Hemolysis was adversely affected in treatments with both products at 8 µg/ml, while hemagglutination was not altered. The whole extract induced complete autoaggregation of P. mirabilis at 2000 µg/ml, while compound 1 at the same concentration did not show this property. Swarming motility was delayed in treatments with the extract and with 1 at 1000 and 8 µg/ml, respectively, at 8h from the beginning of the assay. Complete inhibition of the phenomenon was still observed after 24 h when compound 1 was added at 125 µg/ml. These findings offer the possibility of new classes of antimicrobial medicines to tackle infections caused by P. mirabilis.

Lipopolysaccharide interaction is decisive for the activity of the antimicrobial peptide NK-2 against Escherichia coli and Proteus mirabilis.

Phosphatidylglycerol is a widely used mimetic to study the effects of AMPs (antimicrobial peptides) on the bacterial cytoplasmic membrane. However, the antibacterial activities of novel NK-2-derived AMPs could not be sufficiently explained by using this simple model system. Since the LPS (lipopolysaccharide)-containing outer membrane is the first barrier of Gram-negative bacteria, in the present study we investigated interactions of NK-2 and a shortened variant with viable Escherichia coli WBB01 and Proteus mirabilis R45, and with model membranes composed of LPS isolated from these two strains. Differences in net charge and charge distribution of the two LPS have been proposed to be responsible for the differential sensitivity of the respective bacteria to other AMPs. As imaged by TEM (transmission electron microscopy) and AFM (atomic force microscopy), NK-2-mediated killing of these bacteria was corroborated by structural alterations of the outer and inner membranes, the release of E. coli cytoplasma, and the formation of unique fibrous structures inside P. mirabilis, suggesting distinct and novel intracellular targets. NK-2 bound to and intercalated into LPS bilayers, and eventually induced the formation of transient heterogeneous lesions in planar lipid bilayers. However, the discriminative activity of NK-2 against the two bacterial strains was independent of membrane intercalation and lesion formation, which both were indistinguishable for the two LPS. Instead, differences in activity originated from the LPS-binding step, which could be demonstrated by NK-2 attachment to intact bacteria, and to solid-supported LPS bilayers on a surface acoustic wave biosensor.

The Dienes phenomenon: competition and territoriality in Swarming Proteus mirabilis.

When two different strains of swarming Proteus mirabilis encounter one another on an agar plate, swarming ceases and a visible line of demarcation forms. This boundary region is known as the Dienes line and is associated with the formation of rounded cells. While the Dienes line appears to be the product of distinction between self and nonself, many aspects of its formation and function are unclear. In this work, we studied Dienes line formation using clinical isolates labeled with fluorescent proteins. We show that round cells in the Dienes line originate exclusively from one of the swarms involved and that these round cells have decreased viability. In this sense one of the swarms involved is dominant over the other. Close cell proximity is required for Dienes line formation, and when strains initiate swarming in close proximity, the dominant Dienes type has a significant competitive advantage. When one strain is killed by UV irradiation, a Dienes line does not form. Killing of the dominant strain limits the induction of round cells. We suggest that both strains are actively involved in boundary formation and that round cell formation is the result of a short-range killing mechanism that mediates a competitive advantage, an advantage highly specific to the swarming state. Dienes line formation has implications for the physiology of swarming and social recognition in bacteria.

A study of the structure of the crystalline bacterial biofilms that can encrust and block silver Foley catheters.

The aim of this study was to examine the structure of the crystalline bacterial biofilms that encrust and block silver/hydrogel-coated latex catheters. Scanning electron microscopy was used to examine the crystalline deposits that were found encrusting catheters obtained from six patients undergoing long-term catheterization in a community setting. Large populations of bacilli and cocci were seen on all catheters developing on a basal foundation layer of crystalline material. These observations show that in patients prone to catheter encrustation, crystalline material formed in the urine can cover the surfaces of silver catheters. Extensive bacterial biofilms then develop on the crystals, shielded from the underlying silver. It is suggested that if antimicrobials are to be incorporated into catheters to prevent encrustation, they must diffuse out from the catheter surface and reduce the viable cell populations of the urease producing bacteria that elevate the urinary pH and trigger crystal formation.

An explanatory model to validate the way water activity rules periodic terrace generation in Proteus mirabilis swarm.

This paper explains the biophysical principles which, according to us, govern the Proteus mirabilis swarm phenomenon. Then, this explanation is translated into a mathematical model, essentially based on partial differential equations. This model is then implemented using numerical methods of the finite volume type in order to make simulations. The simulations show most of the characteristics which are observed in situ and in particular the terrace generation.

Observations on the development of the crystalline bacterial biofilms that encrust and block Foley catheters.

The care of many patients undergoing long-term bladder catheterisation is complicated when the flow of urine through the catheter is blocked by encrustation. The problem results from infection by urease-producing bacteria, especially Proteus mirabilis, and the subsequent formation of crystalline biofilms on the catheter. The aim of this study was to discover how P. mirabilis initiates the development of these crystalline biofilms. The early stages in the formation of the biofilms were observed on a range of Foley catheters in a laboratory model of the catheterised bladder. Scanning electron micrographs revealed that when all-silicone, silicone-coated latex, hydrogel-coated latex, hydrogel/silver-coated latex and nitrofurazone silicone catheters were inserted into bladder models containing P. mirabilis and alkaline urine, their surfaces were rapidly coated with a microcrystalline foundation layer. X-ray microanalysis showed that this material was composed of calcium phosphate. Bacterial colonisation of the foundation layer followed and by 18h the catheters were encrusted by densely populated crystalline P. mirabilis biofilms. These observations have important implications for the development of encrustation-resistant catheters. In the case of silver catheters for example, bacterial cells can attach to the crystalline foundation layer and continue to grow, protected from contact with the underlying silver. If antimicrobials are to be incorporated into catheters to prevent encrustation, it is important that they diffuse into the urine and prevent the rise in pH that triggers crystal formation.

[Antagonistic effect of bacteriocinogenic Lactobacillus acidophilus on Klebsiella pneumoniae, Citrobacter freundii and Proteus mirabilis cells].

Data of the ultrastructural cellular changes of conditionally pathogenic enterobacteria, including K. pneumoniae, C. freundii and P. mirabilis cells impacts to bacteriocin-producing L. acidophilus are presented. Enterobacteria in response to the bacteriocinogenic effect of lactobacilli are manifestated by expressive destructions of sensitive to pore formation bacteriocin cells. Various morphological types of enterobacteria cells with increase of involution, lysing and resting forms are revealed. The specific ultrastructural changes of enterobacteria cells which evidencing the significant destructive processes of the cells membranes are detected. The destabilization of cellular wall in expansion periplasmic spaces and appearance of the ultrastructural reorganization of bacterial cells nucleoid also are registrated. Revealing the mechanism of lactobacilli secreted bacteriocin action to conditionally pathogenic enterobacteria might provide new ways to select the effective highly antagonistic probiotic strains.

Ultrastructure of Proteus mirabilis swarmer cell rafts and role of swarming in catheter-associated urinary tract infection.

Proteus mirabilis is a common cause of catheter-associated urinary tract infection (C-UTI). It blocks indwelling urethral catheters through the formation of extensive crystalline biofilms. The obstruction of urine flow can induce episodes of pyelonephritis, septicemia, and shock. P. mirabilis exhibits a type of motility referred to as swarming, in which multicellular rafts of elongated, hyperflagellated swarmer cells form and move rapidly in concert over solid surfaces. It has been suggested that swarming is important in the pathogenesis of C-UTI. In this study we generated a set of stable transposon mutants deficient in swarming and used them to assess the role of swarming in the migration of P. mirabilis over urinary catheters. Swarming was found to be essential for migration over all-silicone catheters. Swarming-deficient mutants were attenuated in migration over hydrogel-coated latex catheters, but those capable of swimming motility were able to move over and infect these surfaces. A novel vapor fixation technique for the preparation of specimens and scanning electron microscopy were used to resolve the ultrastructure of P. mirabilis multicellular rafts. The flagellar filaments of P. mirabilis were found to be highly organized during raft migration and were interwoven in phase to form helical connections between adjacent swarmer cells. Mutants lacking these novel organized structures failed to swarm successfully. We suggest that these structures are important for migration and formation of multicellular rafts. In addition, the highly organized structure of multicellular rafts enables P. mirabilis to initiate C-UTI by migration over catheter surfaces from the urethral meatus into the bladder.

Microwave irradiation for shortening the processing time of samples of flagellated bacteria for scanning electron microscopy.

Microwave irradiation (MWI) has been applied to the development of rapid methods to process biological samples for scanning electron microscopy (SEM). In this paper we propose two simple and quick techniques for processing bacteria (Proteus mirabilis and Vibrio mimicus) for SEM using MWI. In the simplest methodology, the bacteria were placed on a cover-glass, air-dried, and submitted to conductivity stain. The reagent used for the conductivity stain was the mordant of a light microscopy staining method (10 ml of 5% carbolic acid solution, 2 g of tannic acid, and 10 ml of saturated aluminum sulfate 12-H2O). In the second method the samples were double fixed (glutaraldehyde and then osmium), submitted to conductivity stain, dehydrated through a series of ethanol solutions of increasing concentration, treated with hexamethyldisilazine (HMDS), and dried at 35 degrees C for 5 minutes. In both methods the steps from fixation to treatment with HMDS were done under MWI for 2 minutes in an ice-water bath, in order to dissipate the heat generated by the MWI. Although both techniques preserve bacterial morphology adequately, the latter, technique showed the best preservation, including the appearance of flagella, and that process was completed in less than 2 hours at temperatures of MWI between 4 to 5 degrees C.

Proteus mirabilis fimbriae (PMF) are important for both bladder and kidney colonization in mice.

Proteus mirabilis expresses different types of fimbriae simultaneously. Several fimbrial types have been described and their role in the colonization of the urinary tract is under study. Previously, P. mirabilis fimbriae (PMF) have been shown to be associated with bacterial colonization of the lower urinary tract but not of the kidneys. In this study, a pmfA mutant was generated and used in several in vivo and in vitro studies. Two different urinary tract infection models in the mouse and two in vitro assays of bacterial adhesion to uroepithelial cells were performed. Expression of PmfA in a collection of P. mirabilis strains of different sources was also assessed. The results shown here indicate that PMF are involved in both bladder and kidney colonization by P. mirabilis and that these fimbriae are widely distributed among P. mirabilis isolates from different origins since all strains tested expressed PmfA.

Swarming of Proteus mirabilis over ureteral stents: a comparative assessment.

BACKGROUND AND PURPOSE: Encrustation on indwelling ureteral stents is commonly related to the presence of urease-producing bacteria that elevate the pH of the urine through the hydrolysis of urea, resulting in the precipitation of calcium and magnesium salts. Using a model previously shown to measure accurately the ability of Proteus mirabilis to swarm over catheter surfaces (Eur J Clin Microbiol Infect Dis 1999;18:206), we investigated the ability of this organism to swarm over three ureteral stents with potential encrustation-resistance properties. MATERIALS AND METHODS: Three commercially available ureteral stents were selected for evaluation: a low surface-energy stent, a hydrogel-coated stent, and a silicone stent. Ten-microliter aliquots of a 4-hour culture of P. mirabilis 296 in Trypticase soya (TSA) broth was inoculated 5 mm from a 1-cm channel cut out from TSA plates. Ten-millimeter stent sections were placed as bridges across the central channel adjacent to the inocula. Time to pathogen crossing was measured. RESULTS: The mean time (+/- SD) to pathogen migration across the three test materials was 15.9 +/- 6.1, 19.8 +/- 9.5, and 29.7 +/- 14.3 hours for the low surface-energy, hydrogel-coated, and silicone stents, respectively. Statistical analysis revealed significant differences between the crossing times of the low surface-energy (P = 0.001) and hydrogel-coated (P = 0.034) stents compared with silicone but not between the low surface-energy and hydrogel-coated stents (P = 0.387). CONCLUSION: Migration of P. mirabilis 296 across silicone stents was significantly reduced compared with low surface-energy and hydrogel-coated stents. These findings suggest that P. mirabilis may have a lower affinity for silicone stents, which may translate into a reduced risk of infection with P. mirabilis and associated stent encrustation.