Alhagi maurorum extract modulates quorum sensing genes and biofilm formation in Proteus mirabilis.

Proteus mirabilis (P. mirabilis) is a frequent cause of catheter-associated urinary tract infections. This study aims to investigate the anti-infective effect of Alhagi maurorum extract (AME), the traditional medicinal plant in the middle east, on the biofilm-forming P. mirabilis isolates. Hydroalcoholic extract and oil of A. maurorum were characterized by HPLC and GC-MS. The antiproliferative, anti-biofilm, and bactericidal activity of AME at various concentrations were assessed by turbidity, crystal violet binding, and agar well diffusion assays, respectively. The AME's effect on adhesion and quorum sensing (QS) were investigated by in vitro adhesion assay on cell culture and agar overlay assay using Janthinobacterium lividum (ATCC 12472) as a biosensor strain. In addition, the expression level of selected genes involved in QS and biofilm regulation were determined by quantitative Real-Time PCR. Furthermore, the bladder phantom model was created to evaluate the assays and investigate the catheter's calcium deposition. The most effective chemical compounds found in AME were tamarixetin, quercetin, and trans-anethole. Although AME did not inhibit swarming motility, it reduced biofilm production and exerted a concentration-dependent anti-adhesive and anti-QS activity against P. mirabilis. AME also downregulated the expression level of selected genes involved in biofilm formation and QS. This study showed that AME as a natural compound reduced biofilm formation of P. mirabilis by targeting virulence factor genes, quorum sensing, and other strategies that include preventing the adhesion of P. mirabilis to the cells. The results suggest that A. maurorum extract might have the potential to be considered for preventing UTIs caused by P. mirabilis.

Novel pathomechanism for spontaneous bacterial peritonitis: disruption of cell junctions by cellular and bacterial proteases.

OBJECTIVE: Spontaneous bacterial peritonitis (SBP) is a life-threatening complication of liver cirrhosis with a 1-year mortality of 66%. Bacterial translocation (BT) from the intestine to the mesenteric lymph nodes is crucial for the pathogenesis of SBP. DESIGN: Since BT presupposes a leaky intestinal epithelium, the integrity of mucus and epithelial cell junctions (E-cadherin and occludin) was examined in colonic biopsies from patients with liver cirrhosis and controls. SBP-inducing Escherichia coli (E. coli) and Proteus mirabilis (P. mirabilis) were isolated from ascites of patients with liver cirrhosis and co-cultured with Caco-2 cells to characterise bacteria-to-cell effects. RESULTS: SBP-derived E. coli and P. mirabilis led to a marked reduction of cell-to-cell junctions in a dose-dependent and time-dependent manner. This effect was enhanced by a direct interaction of live bacteria with epithelial cells. Degradation of occludin is mediated via increased ubiquitination by the proteasome. Remarkably, a novel bacterial protease activity is of pivotal importance for the cleavage of E-cadherin. CONCLUSION: Patients with liver cirrhosis show a reduced thickness of colonic mucus, which allows bacteria-to-epithelial cell contact. Intestinal bacteria induce degradation of occludin by exploiting the proteasome of epithelial cells. We identified a novel bacterial protease activity of patient-derived SBP-inducing bacteria, which is responsible for the cleavage of E-cadherin structures. Inhibition of this protease activity leads to stabilisation of cell junctions. Thus, targeting these mechanisms by blocking the ubiquitin-proteasome system and/or the bacterial protease activity might interfere with BT and constitute a novel innovative therapeutic strategy to prevent SBP in patients with liver cirrhosis.

Serum levels of immunoglobulin and complement in UTI of patients caused by Proteus mirabilis and using AgNPs as antiswarming.

The use of plant extracts represents a promising approach for the synthesis of silver nanoparticles (AgNPs). This study reports the low-cost, green synthesis of AgNPs using the extract of clove and black seeds. The biosynthesized AgNPs were confirmed and characterized by analysis of the spectroscopy profile of the UV-visible spectrophotometer. The purpose of the present study is to evaluate the inhibitory effect concentration (MIC) of AgNPs, clove, and black cumin seed extracts on the growth and swarming of P. mirabilis. Clinical isolates of P. mirabilis were isolated from patients suffering from urinary tract infections. Thirteen types of antibiotics were used in the present study to detect their ability to inhibit P. mirabilis's resistance. Immunological findings included the determination of serum levels of IgG, IgM, IgA and complement protein C3 and C4. Results showed that IgG and IgA concentrations significantly increased (1311.13 ± 72.54 and 279 ± 21.31) respectively in UTI patients in comparison to the healthy control group which was 1089.88 ± 37.33 and 117.611 ± 4.19 respectively, While IgM concentrations were increased non significantly in UTI patients (153.331 ± 6.45) in comparison to healthy control (145.2 ± 13.49). Complement components C3 showed a significant increase in UTI patients with mean values of 125.95 ± 6.22 compared to the control group with mean values of 55.191 ± 9.64, while C4 showed statically non-significant among UTI patients in comparison with the control group (35.195 ± 2.34 and 34.371 ± 1.22) respectively.

Inhibition of swarming motility using in vitro hyperthermia.

Hyperthermia is a therapeutic technique in which body tissue is exposed to temperatures in the region of 40-45 °C to induce a physiological or biological effect. Swarming motility is an important virulence factor for Proteus mirabilis and Pseudomonas aeruginosa and swarming phenomenon is a coordinated multicellular movement of differentiated bacterial population over semi-solid surfaces. In this study, we aimed to investigate the inhibitory effect of hyperthermia on bacterial swarming motility using a modified thermobiogram method and show the potential of this thermal method to treat bacterial infections. Ten P. mirabilis and 10 P. aeruginosa clinical isolates were included in the study. Sheep blood agar (SBA) plates were prepared and inoculated with bacterial suspensions of clinical isolates. Inoculated SBA plates were incubated inside 2 different incubators; at 37 °C and 45 °C for 20 h. The diameter of bacterial growing zones (swarming diameters) were measured every 2 h and noted. Finally, Gram stains of the isolates were prepared for microscopic examination. Wilcoxon signed-rank test was used to compare the swarming inhibition rates of the isolates incubated at 37 °C and 45 °C. Regarding P. mirabilis species, a significant difference was found between two different temperatures (P = 0.0078). So, a temperature at the level of hyperthermia significantly inhibited the swarming motility of P. mirabilis isolates. In addition, transformation to coccus form was observed at 45 °C. We speculate that these findings might be useful for employing thermal therapies including hyperthermia method to treat infectious diseases caused by swarming bacterial pathogens in the future.

Decreased biofilm formation in Proteus mirabilis after short-term exposure to a simulated microgravity environment.

BACKGROUND: Microbes threaten human health in space exploration. Studies have shown that Proteus mirabilis has been found in human space habitats. In addition, the biological characteristics of P. mirabilis in space have been studied unconditionally. The simulated microgravity environment provides a platform for understanding the changes in the biological characteristics of P. mirabilis. OBJECTIVE: This study intends to explore the effect of simulated microgravity on P. mirabilis, the formation of P. mirabilis biofilm, and its related mechanism. METHODS: The strange deformable rods were cultured continuously for 14 days under microgravity simulated in high-aspect rotating vessels (HARVs). The morphology, growth rate, metabolism, and biofilm formation of the strain were measured, and the phenotypic changes of P. mirabilis were evaluated. Transcriptome sequencing was used to detect differentially expressed genes under simulated microgravity and compared with phenotype. RESULTS: The growth rate, metabolic ability, and biofilm forming ability of P. mirabilis were lower than those of normal gravity culture under the condition of simulated microgravity. Further analysis showed that the decrease of growth rate, metabolic ability, and biofilm forming ability may be caused by the downregulation of related genes (pstS, sodB, and fumC). CONCLUSION: The simulated microgravity condition enables us to explore the potential relationship between bacterial phenotype and molecular biology, thus opening up a suitable and constructive method for medical fields that have not been explored before. It provides a certain strategy for the treatment of P. mirabilis infectious diseases in space environment by exploring the microgravity of P. mirabilis.

Phospholipids and Fatty Acids Affect the Colonization of Urological Catheters by Proteus mirabilis.

Proteus mirabilis-mediated CAUTIs are usually initiated by the adherence of bacteria to a urinary catheter surface. In this paper, three isolates of different origin and exhibiting different adhesion abilities were investigated in search of any changes in lipidome components which might contribute to P. mirabilis adhesion to catheters. Using GC-MS and LC-MS/MS techniques, 21 fatty acids and 27 phospholipids were identified in the examined cells. The comparison of the profiles of phospholipids and fatty acids obtained for catheter-attached cells and planktonic cells of the pathogens indicated C11:0 and PE 37:2 levels as values which could be related to P. mirabilis adhesion to a catheter, as well as cis C16:1, PE 32:0, PE 33:0, PE 38:2, PG 33:1, PG 34:0, PE 30:1, PE 32:1 and PG 30:2 levels as values which could be associated with cell hydrophobicity. Based on DiBAC4 (3) fluorescence intensity and an affinity to p-xylene, it was found that the inner membrane depolarization, as well as strong cell-surface hydrophobicity, were important for P. mirabilis adhesion to a silicone catheter. A generalized polarization of Laurdan showed lower values for P. mirabilis cells attached to the catheter surface than for planktonic cells, suggesting lower packing density of membrane components of the adherent cells compared with tightly packed, stiffened membranes of the planktonic cells. Taken together, these data indicate that high surface hydrophobicity, fluidization and depolarization of P. mirabilis cell membranes enable colonization of a silicone urinary catheter surface.

Proteus mirabilis Employs a Contact-Dependent Killing System against Competing Enterobacteriaceae.

Many bacterial species employ systems for interference competition with other microorganisms. Some systems are effective without contact (e.g., through secretion of toxins), while other systems (e.g., type VI secretion system [T6SS]) require direct contact between cells. Here, we provide the initial characterization of a novel contact-dependent competition system for Proteus mirabilis. In neonatal mice, a commensal P. mirabilis strain apparently eliminated commensal Escherichia coli. We replicated the phenotype in vitro and showed that P. mirabilis efficiently reduced the viability of several Enterobacteriaceae species but not Gram-positive species or yeast cells. Importantly, P. mirabilis strains isolated from humans also killed E. coli. A reduction of viability occurred from early stationary phase to 24 h of culture and was observed in shaking liquid media as well as on solid media. Killing required contact but was independent of T6SS, which is the only contact-dependent killing system described for P. mirabilis. Expression of the killing system was regulated by osmolarity and components secreted into the supernatant. Stationary-phase P. mirabilis culture supernatant itself did not kill but was sufficient to induce killing in an exponentially growing coculture. In contrast, killing was largely prevented in media with low osmolarity. In summary, we provide the initial characterization of a potentially novel interbacterial competition system used by P. mirabilis. IMPORTANCE The study of bacterial competition systems has received significant attention in recent years. These systems are important in a multitude of polymicrobial environments and collectively shape the composition of complex ecosystems like the mammalian gut. They are also being explored as narrow-spectrum alternatives to specifically eliminate problematic pathogenic species. However, only a small fraction of the estimated number of interbacterial competition systems has been identified. We discovered a competition system that is novel for Proteus mirabilis. Inspired by an observation in infant mice, we confirmed in vitro that P. mirabilis was able to efficiently kill several Enterobacteriaceae species. This killing system might represent a new function of a known competition system or even a novel system, as the observed characteristics do not fit with described contact-dependent competition systems. Further characterization of this system might help understand how P. mirabilis competes with other Enterobacteriaceae in various niches.

An investigation of the impact of triclosan adaptation on Proteus mirabilis clinical isolates from an Egyptian university hospital.

Antibiotic resistance is a main threat to the public health. It is established that the overuse and misuse of antibiotics are highly contributing to antibiotic resistance. However, the impact of nonantibiotic antimicrobial agents like biocides on antibiotic resistance is currently investigated and studied. Triclosan (TCS) is a broad-spectrum antibacterial agent widely used as antiseptic and disinfectant. In this study, we aimed to evaluate the effect of exposure of Proteus mirabilis clinical isolates to sublethal concentrations of TCS on their antibiotic susceptibility, membrane characteristics, efflux activity, morphology, and lipid profile. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of TCS were determined for 31 P. mirabilis clinical isolates. The tested isolates were adapted to increasing sublethal concentrations of TCS. The MICs of 16 antibiotics were determined before and after adaptation. Membrane characteristics, efflux activity, ultrastructure, and lipid profile of the tested isolates were examined before and after adaptation. Most adapted P. mirabilis isolates showed increased antibiotic resistance, lower membrane integrity, lower outer and inner membrane permeability, and higher membrane depolarization. Nonsignificant change in membrane potential and lipid profile was found in adapted cells. Various morphological changes and enhanced efflux activity was noticed after adaptation. The findings of the current study suggest that the extensive usage of TCS at sublethal concentrations could contribute to the emergence of antibiotic resistance in P. mirabilis clinical isolates. TCS could induce changes in the bacterial membrane properties and increase the efflux activity and in turn decrease its susceptibility to antibiotics which would represent a public health risk.

Bacterial-induced pH shifts link individual cell physiology to macroscale collective behavior.

Bacteria have evolved a diverse array of signaling pathways that enable them to quickly respond to environmental changes. Understanding how these pathways reflect environmental conditions and produce an orchestrated response is an ongoing challenge. Herein, we present a role for collective modifications of environmental pH carried out by microbial colonies living on a surface. We show that by collectively adjusting the local pH value, Paenibacillus spp., specifically, regulate their swarming motility. Moreover, we show that such pH-dependent regulation can converge with the carbon repression pathway to down-regulate flagellin expression and inhibit swarming in the presence of glucose. Interestingly, our results demonstrate that the observed glucose-dependent swarming repression is not mediated by the glucose molecule per se, as commonly thought to occur in carbon repression pathways, but rather is governed by a decrease in pH due to glucose metabolism. In fact, modification of the environmental pH by neighboring bacterial species could override this glucose-dependent repression and induce swarming of Paenibacillus spp. away from a glucose-rich area. Our results suggest that bacteria can use local pH modulations to reflect nutrient availability and link individual bacterial physiology to macroscale collective behavior.

Pathogenesis of Proteus mirabilis in Catheter-Associated Urinary Tract Infections.

Proteus mirabilis (PM) is a Gram-negative rod-shaped bacterium and widely exists in the natural environment, and it is most noted for its swarming motility and urease activity. PM is the main pathogen causing complicated urinary tract infections (UTIs), especially catheter-associated urinary tract infections. Clinically, PM can form a crystalline biofilm on the outer surface and inner cavity of the urethral indwelling catheter owing to its ureolytic biomineralization. This leads to catheter encrustation and blockage and, in most cases, is accompanied by urine retention and ascending UTI, causing cystitis, pyelonephritis, and the development of bladder or kidney stones, or even fatal complications such as septicemia and endotoxic shock. In this review, we discuss how PM is mediated by a catheter into the urethra, bladder, and then rose to the kidney causing UTI and the main virulence factors associated with different stages of infection, including flagella, pili or adhesins, urease, hemolysin, metal intake, and immune escape, encompassing both historical perspectives and current advances.

Anti-Biofilm Effect of Octenidine and Polyhexanide on Uropathogenic Biofilm-Producing Bacteria.

BACKGROUND: A catheter allowing a release of antibacterial substances such as antiseptics into the bladder could be a new way of preventing biofilm formation and subsequent catheter-associated urinary tract infections. METHODS: Minimal inhibitory and bactericidal concentration (MIC/MBC) determinations in cation-adjusted Mueller-Hinton broth and artificial urine were performed for 4 antiseptics against 3 uropathogenic biofilm producers, Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis. Furthermore, effects of octenidine and polyhexanide against catheter biofilm formation were determined by quantification of biofilm-producing bacteria. RESULTS: Sodium hypochlorite showed MIC/MBC values between 200 and 800 mg/L for all strains tested. Triclosan was efficient against E. coli and P. mirabilis (MIC ≤2.98 mg/L) but ineffective against P. aeruginosa. Octenidine and polyhexanide showed antibacterial activity against all 3 species tested (MIC 1.95-7.8 and 3.9-31.25 mg/L). Both octenidine and polyhexanide were able to prevent biofilm formation on catheter segments in a concentration dependent manner. Furthermore, adding 250 mg/L of each biocide disrupted biofilms formed by E. coli and P. mirabilis, whereas even 500 mg/L was not sufficient to completely destroy P. aeruginosa biofilms. CONCLUSION: Octenidine- and polyhexanide-containing antiseptics showed a broad effect against typical uropathogenic biofilm producers even in high dilutions. This study provides a basis for further investigation of the potential of octenidine and polyhexanide as prophylaxis or treatment of catheter biofilms.

Diffusion of antibiotics through a biofilm in the presence of diffusion and absorption barriers.

We propose a model of antibiotic diffusion through a bacterial biofilm when diffusion and/or absorption barriers develop in the biofilm. The idea of this model is: We deduce details of the diffusion process in a medium in which direct experimental study is difficult, based on probing diffusion in external regions. Since a biofilm has a gel-like consistency, we suppose that subdiffusion of particles in the biofilm may occur. To describe this process we use a fractional subdiffusion-absorption equation with an adjustable anomalous diffusion exponent. The boundary conditions at the boundaries of the biofilm are derived by means of a particle random walk model on a discrete lattice leading to an expression involving a fractional time derivative. We show that the temporal evolution of the total amount of substance that has diffused through the biofilm explicitly depends on whether there is antibiotic absorption in the biofilm. This fact is used to experimentally check for antibiotic absorption in the biofilm and if subdiffusion and absorption parameters of the biofilm change over time. We propose a four-stage model of antibiotic diffusion in biofilm based on the following physical characteristics: whether there is absorption of the antibiotic in the biofilm and whether all biofilm parameters remain unchanged over time. The biological interpretation of the stages, in particular their relation with the bacterial defense mechanisms, is discussed. Theoretical results are compared with empirical results of ciprofloxacin diffusion through Pseudomonas aeruginosa biofilm, and ciprofloxacin and gentamicin diffusion through Proteus mirabilis biofilm.

Molecular Mechanisms Contributing Bacterial Infections to the Incidence of Various Types of Cancer.

Cancer causes a major health concern worldwide due to high incidence and mortality rates. To accomplish this purpose, the Scopus, PubMed, and Web of Science databases were searched using the keywords bacteria and cancer. Most of published research addressed several different factors that induced cancer, such as toxins, medications, smoking, and obesity. Nonetheless, few studies are dealing with cancer induction via bacterial infection. In addition, mechanisms of cancer induction via bacterial infections are not well understood. Therefore, in this review, we will shed light on different bacteria that induced cancer via different molecular mechanisms. Among the bacterial infection that induced cancer, Helicobacter pylori was the first recognized bacteria which caused gastric cancer and might be also linked to extragastric cancer in humans. H. pylori has been associated with adenocarcinoma in the distal stomach by its ability to cause severe inflammations. It has been found that inflammations induced cancer via different mechanisms including induction of cell proliferation and production of high levels of free radicals. Recently, free radicals were found to induce and cause various types of cancer. Salmonella typhi has been found to be associated with gallbladder carcinoma (GBC). Also, intercellular infection of lungs with Chlamydia pneumoniae was found to contribute as one of the ethological factors of lung cancer. Moreover, infection of the urinary tract with Staphylococcus aureus, Klebsiella spp., and Proteus mirabilis has been found to cause bladder cancer. These microorganisms produce a high level of N-nitrosamines which are metabolically activated leading to the generation of alkylating agents that damage DNA and other macromolecules. It is concluded that a certain bacterium is linked with induction of a specific type of cancer via different molecular and biochemical mechanisms as discussed in the text in details. This infection could potentially affect human health in different ways. In addition, it is important to know the possible factors involved in cancer induction for better treatment of cancer patients.

Tumble Suppression Is a Conserved Feature of Swarming Motility.

Many bacteria use flagellum-driven motility to swarm or move collectively over a surface terrain. Bacterial adaptations for swarming can include cell elongation, hyperflagellation, recruitment of special stator proteins, and surfactant secretion, among others. We recently demonstrated another swarming adaptation in Escherichia coli, wherein the chemotaxis pathway is remodeled to decrease tumble bias (increase run durations), with running speeds increased as well. We show here that the modification of motility parameters during swarming is not unique to E. coli but is shared by a diverse group of bacteria we examined-Proteus mirabilis, Serratia marcescens, Salmonella enterica, Bacillus subtilis, and Pseudomonas aeruginosa-suggesting that increasing run durations and speeds are a cornerstone of swarming.IMPORTANCE Bacteria within a swarm move characteristically in packs, displaying an intricate swirling motion in which hundreds of dynamic rafts continuously form and dissociate as the swarm colonizes an increasing expanse of territory. The demonstrated property of E. coli to reduce its tumble bias and hence increase its run duration during swarming is expected to maintain and promote side-by-side alignment and cohesion within the bacterial packs. In this study, we observed a similar low tumble bias in five different bacterial species, both Gram positive and Gram negative, each inhabiting a unique habitat and posing unique problems to our health. The unanimous display of an altered run-tumble bias in swarms of all species examined in this investigation suggests that this behavioral adaptation is crucial for swarming.

Transposon Insertion Site Sequencing of Providencia stuartii: Essential Genes, Fitness Factors for Catheter-Associated Urinary Tract Infection, and the Impact of Polymicrobial Infection on Fitness Requirements.

Providencia stuartii is a common cause of polymicrobial catheter-associated urinary tract infection (CAUTI), and yet literature describing the molecular mechanisms of its pathogenesis is limited. To identify factors important for colonization during single-species infection and during polymicrobial infection with a common cocolonizer, Proteus mirabilis, we created a saturating library of ∼50,000 transposon mutants and conducted transposon insertion site sequencing (Tn-Seq) in a murine model of CAUTI. P. stuartii strain BE2467 carries 4,398 genes, 521 of which were identified as essential for growth in laboratory medium and therefore could not be assessed for contribution to infection. Using an input/output fold change cutoff value of 20 and P values of <0.05, 340 genes were identified as important for establishing single-species infection only and 63 genes as uniquely important for polymicrobial infection with P. mirabilis, and 168 genes contributed to both single-species and coinfection. Seven mutants were constructed for experimental validation of the primary screen that corresponded to flagella (fliC mutant), twin arginine translocation (tatC), an ATP-dependent protease (clpP), d-alanine-d-alanine ligase (ddlA), type 3 secretion (yscI and sopB), and type VI secretion (impJ). Infection-specific phenotypes validated 6/7 (86%) mutants during direct cochallenge with wild-type P. stuartii and 3/5 (60%) mutants during coinfection with P. mirabilis, for a combined validation rate of 9/12 (75%). Tn-Seq therefore successfully identified genes that contribute to fitness of P. stuartii within the urinary tract, determined the impact of coinfection on fitness requirements, and added to the identification of a collection of genes that may contribute to fitness of multiple urinary tract pathogens.IMPORTANCEProvidencia stuartii is a common cause of polymicrobial catheter-associated urinary tract infections (CAUTIs), particularly during long-term catheterization. However, little is known regarding the pathogenesis of this organism. Using transposon insertion site sequencing (Tn-Seq), we performed a global assessment of P. stuartii fitness factors for CAUTI while simultaneously determining how coinfection with another pathogen alters fitness requirements. This approach provides four important contributions to the field: (i) the first global estimation of P. stuartii genes essential for growth in laboratory medium, (ii) identification of novel fitness factors for P. stuartii colonization of the catheterized urinary tract, (iii) identification of core fitness factors for both single-species and polymicrobial CAUTI, and (iv) assessment of conservation of fitness factors between common uropathogens. Genomewide assessment of the fitness requirements for common uropathogens during single-species and polymicrobial CAUTI thus elucidates complex interactions that contribute to disease severity and will uncover conserved targets for therapeutic intervention.

A CpxR-Regulated zapD Gene Involved in Biofilm Formation of Uropathogenic Proteus mirabilis.

Proteus mirabilis, a frequent uropathogen, forms extensive biofilms on catheters that are infamously difficult to treat. To explore the mechanisms of biofilm formation by P. mirabilis, we performed in vivo transposon mutagenesis. A mutant with impaired biofilm formation was isolated. The mutant was found to have Tn5 inserted in the zapD gene, encoding an outer membrane protein of the putative type 1 secretion system ZapBCD. zapBCD and its upstream zapA gene, encoding a protease, constitute an operon under the control of CpxR, a two-component regulator. The cpxR mutant and zapA mutant strains also had a biofilm-forming defect. CpxR positively regulates the promoter activities of zapABCD, cpxP, and cpxR An electrophoretic mobility shift assay revealed that CpxR binds zapA promoter DNA. The loss of zapD reduced CpxR-regulated gene expression of cpxR, zapA, cpxP, and mrpA, the mannose-resistant Proteus-like (MR/P) fimbrial major subunit gene. The restoration of biofilm formation in the zapD mutant with a CpxR-expressing plasmid reinforces the idea that CpxR-mediated gene expression contributes to zapD-involved biofilm formation. In trans expression of zapBCD from a zapBCD-expressing plasmid also reestablished the biofilm formation ability of the cpxR mutant to a certain level. The zapD and cpxR mutants had significantly lower protease activity, adhesion, and autoaggregation ability and production of exopolysaccharides and extracellular DNA (eDNA) than did the wild type. Finally, we identified copper as a signal for CpxR to increase biofilm formation. The loss of cpxR or zapD abolished the copper-mediated biofilm upshift. CpxR was required for copper-induced expression of zapA and cpxR Taken together, these data highlight the important role of CpxR-regulated zapD in biofilm formation and the underlying mechanisms in P. mirabilis.

Persisters of Klebsiella pneumoniae and Proteus mirabilis: A Common Phenomenon and Different Behavior Profiles.

Persisters of infectious agents are capable of surviving antibiotic treatment so the emergence of these subpopulations need to be overcome. In this study, we aimed to isolate, characterize and inhibit persister subpopulation in two clinical isolates Klebsiella pneumoniae and Proteus mirabilis. Different behavior profiles between the two isolates could be observed. The results of dose-dependent killing curve revealed that 2.3% (Klebsiella pneumoniae) versus 1.3% (Proteus mirabilis) persister cells could be recovered using 500 and 30 ug/ml ciprofloxacin, respectively. Upon resuscitation, persister cells exhibited only 65% versus 30% percentage growth and 5 versus 7 times cell elongation relative to Klebsiella pneumoniae and Proteus mirabilis, respectively. The levels of persister cells to ciprofloxacin of Klebsiella pneumoniae were dramatically decreased by about 79, 92, 97 and 83% in average by pre-exposure to hyperosmotic stress, temperature, different pHs, and hydrogen peroxide, respectively, while those of Proteus mirabilis were minimally decreased with corresponding reduction percentages of about 12%, 24 & 25%, and 0%. Regarding combating persisters, Klebsiella pneumoniae showed different response as compared to Proteus mirabilis. Among the tested sugars, the highest reduction of Klebsiella pneumoniae persister cells was obtained with pre-priming with sucrose while for Proteus mirabilis persister cells, the highest reduction was obtained with pre-priming with glucose. Using sodium salicylate with ciprofloxacin could eradicate persisters of Klebsiella pneumoniae at any tested concentration while for Proteus mirabilis it caused some reduction in persister cells at certain concentrations. Complete eradication of persisters was obtained by combining silver nitrate with ciprofloxacin for each test isolate.

Proteus mirabilis causing cellulitis in broiler chickens.

Given the need to understand the virulence profile of Proteus mirabilis isolates from cellulitis in broiler chickens and their ability to cause lesions, the present study aimed to characterize genotypically and phenotypically the virulence profiles of two strains of P. mirabilis isolated from cellulitis in broilers, as well as to evaluate their ability to experimentally reproduce the lesions in vivo. The strain with the highest virulence potential (LBUEL-A33) possessed mrpA, pmfA, ucaA, atfA (fimbriae), zapA, ptA (proteases), hpmA (hemolysin), and ireA (siderophore) genes, formed a very strong biofilm, and expressed the pattern of aggregative adhesion and cytotoxicity in Vero cells. The strain with the lowest virulence potential (LBUEL-A34) did not present the pmfA and ucaA genes, but expressed the pattern of aggregative adhesion, formed a strong biofilm, and did not show cytotoxicity. Both strains developed cellulitis in an animal model within 24 h post-inoculation (PI), and the degree of lesions was not significantly altered up to 120 h PI. The LBUEL-A33 strain was also inoculated in combination with an avian pathogenic Escherichia coli (APEC 046), and the lesions showed no significant changes from the individual inoculation of these two strains. Histological analysis showed that the LBUEL-A33 strain developed characteristic cellulitis lesions. Thus, both strains of P. mirabilis isolated in our study have several virulence factors and the ability to develop cellulitis in broilers.

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.

Altered metabolomic profile of dual-species biofilm: Interactions between Proteus mirabilis and Candida albicans.

In this study, we aimed to determine the interspecies interactions between Proteus mirabilis and Candida albicans. Mono and dual-species biofilms were grown in a microtiter plate and metabolomic analysis of the biofilms was performed. The effects of togetherness of two species on the expression levels of candidal virulence genes and urease and swarming activities of P.mirabilis were investigated. The growth of C.albicans was inhibited by P.mirabilis whereas the growth and swarming activity of P.mirabilis were increased by C.albicans. The inhibition of Candida cell growth was found to be biofilm specific. The alteration was not detected in urease activity. The expressions of EFG1, HWP1 and SAP2 genes were significantly down-regulated, however, LIP1 was upregulated by P.mirabilis. In the presence of P.mirabilis carbonhydrates, amino acids, polyamine and lipid metabolisms were altered in C.albicans. Interestingly, the putrescine level was increased up to 230 fold in dual-species biofilm compared to monospecies C.albicans biofilm. To our knowledge, this is the first study to investigate the impact of each microbial pathogen on the dual microbial environment by integration of metabolomic data.