Preferential Use of Central Metabolism In Vivo Reveals a Nutritional Basis for Polymicrobial Infection

The human genitourinary tract is a common anatomical niche for polymicrobial infection and a leading site for the development of bacteremia and sepsis. Most uncomplicated, community-acquired urinary tract infections (UTI) are caused by Escherichia coli, while another bacterium, Proteus mirabilis, is more often associated with complicated UTI. Here, we report that uropathogenic E. coli and P. mirabilis have divergent requirements for specific central pathways in vivo despite colonizing and occupying the same host environment. Using mutants of specific central metabolism enzymes, we determined glycolysis mutants lacking pgi, tpiA, pfkA, or pykA all have fitness defects in vivo for P. mirabilis but do not affect colonization of E. coli during UTI. Similarly, the oxidative pentose phosphate pathway is required only for P. mirabilis in vivo. In contrast, gluconeogenesis is required only for E. coli fitness in vivo. The remarkable difference in central pathway utilization between E. coli and P. mirabilis during experimental UTI was also observed for TCA cycle mutants in sdhB, fumC, and frdA. The distinct in vivo requirements between these pathogens suggest E. coli and P. mirabilis are not direct competitors within host urinary tract nutritional niche. In support of this, we found that co-infection with E. coli and P. mirabilis wild-type strains enhanced bacterial colonization and persistence of both pathogens during UTI. Our results reveal that complementary utilization of central carbon metabolism facilitates polymicrobial disease and suggests microbial activity in vivo alters the host urinary tract nutritional niche.     

Potential Probiotics Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 Co-Aggregate with Clinical Isolates of Proteus mirabilis and Prevent Biofilm Formation

A urinary tract infection (UTI) is a multi-factorial disease including cystitis, pyelonephritis, and pyelitis. After Escherichia coli, Proteus mirabilis is the most common UTI-associated opportunistic pathogen. Antibiotic resistance of bacteria and infection recurrence can be connected to biofilm formation by P. mirabilis. In this study, human and sheep isolates of P. mirabilis were investigated for antibiotic sensitivity using an antibiotic disk test. Co-aggregation of the tested potential probiotic bacilli, Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933, with the isolated pathogen was also evaluated. Then, the anti-biofilm activity of naturally derived metabolites, such as subtilin and subtilosin, in the bacilli-free supernatants was assessed against biofilms of P. mirabilis isolates. The isolated pathogens were sensitive to 30 μg of amikacin and 5 μg of ciprofloxacin but resistant to other tested antibiotics. After 24 h, auto-aggregation of B. amyloliquefaciens B-1895 was at 89.5% and higher than auto-aggregation of B. subtilis KATMIRA1933 (59.5%). B. amyloliquefaciens B-1895 strongly co-aggregated with P. mirabilis isolates from human UTIs. Cell-free supernatants of B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 showed higher antimicrobial activity against biofilms of P. mirabilis isolated from humans as compared with biofilms of sheep isolates. According to our knowledge, this is the first report evaluating the anti-biofilm activity of probiotic spore-forming bacilli against clinical and animal UTI isolates of P. mirabilis. Further studies are recommended to investigate the anti-biofilm activity and the mode of action for the antimicrobial substances produced by these bacilli, subtilosin and subtilin.

     

Antimicrobial activity of the acidophilic eukaryotic microalga Coccomyxa onubensis

Extremophilic microalgae are unexplored as a source of pharmaceuticals despite the fact that its biomass can be produced at large scale with low risk of contamination. A significant amount of antimicrobial activity was produced by extracts obtained from the eukaryotic acidophilic microalgae Coccomyxa onubensis in non‐polar solvents, such as hexane, diethyl ether, and chloroform or in weakly polar solvents, such as dichloromethane, against Gram‐negative and Gram‐positive bacteria, and also the yeast Candida albicans. The most effective activity was shown by chloroform extract against Escherichia coli S, Salmonella enterica, and Proteus mirabilis; hexane extract against P. mirabilis, Sa. enterica, and Ca. albicans; dichloromethane extract against Sa. enterica or diethyl ether extract against E. coli S and the Gram‐positive Staphylococcus aureus MB. The lowest minimum inhibitory concentration values were recorded against E. coli S (305 μg mL ^?1) and P. mirabilis (153 μg mL ^?1) (using chloroform extract) and against P. mirabilis (106 μg mL^?1) (using hexane extract). Fatty acids, but not carotenoids, seem to be involved in the antimicrobial activity of this microalga. However, further biochemical and biotechnological studies must be conducted in order to characterize and purify the bioactive principles from Co. onubensis for assessing its potential as a pharmaceutical source and feasibility of production.     

奇异变形杆菌毒力因子的研究进展

奇异变形杆菌是革兰阴性细菌,在自然界中广泛存在,具有特殊的群集运动能力,与临床关系密切,可在膀胱和肾脏中形成结石,在尿道留置管外表面、内腔中形成结晶生物膜,是引起复杂尿路感染的主要病原体,主要的毒力因子包括菌毛、黏附素、尿素酶、溶血素、金属摄取和免疫逃避等。综述了近年来有关奇异变形杆菌毒力因子的研究,为相关研究提供参考。     

Antimicrobial photodynamic activity of toluidine blue encapsulated in mesoporous silica nanoparticles against Pseudomonas aeruginosa and Staphylococcus aureus

In the present study, the antimicrobial and antibiofilm efficacy of toluidine blue (TB) encapsulated in mesoporous silica nanoparticles (MSN) was investigated against Pseudomonas aeruginosa and Staphylococcus aureus treated with antimicrobial photodynamic therapy (aPDT) using a red diode laser 670?nm wavelength, 97.65?J cm^?2 radiant exposure, 5?min). Physico-chemical techniques (UV-visible (UV-vis) absorption, photoluminescence emission, excitation, and FTIR) and high-resolution transmission electron microscopy (HR-TEM) were employed to characterize the conjugate of TB encapsulated in MSN (TB MSN). TB MSN showed maximum antimicrobial activities corresponding to 5.03 and 5.56 log CFU ml^?1 reductions against P. aeruginosa and S. aureus, respectively, whereas samples treated with TB alone showed 2.36 and 2.66 log CFU ml^?1 reductions. Anti-biofilm studies confirmed that TB MSN effectively inhibits biofilm formation and production of extracellular polymeric substances by P. aeruginosa and S. aureus.     

Serotyping of <Emphasis Type="Italic">Proteus mirabilis</Emphasis> clinical strains based on lipopolysaccharide O-polysaccharide and core oligosaccharide structures

The aim of this work was to serotype Proteus mirabilis urinary tract infection (UTI) strains based on chemically defined O-antigens with the use of two clinical collections from Sweden and Poland consisting of 99 and 24 UTI strains, respectively. A simple two-step serotyping scheme was proposed using enzyme immunoassay with heat-stable surface antigens of Proteus cells and immunoblotting with isolated lipopolysaccharides (LPSs). Using polyclonal anti-P. mirabilis rabbit antisera, 50 Swedish and 8 Polish strains were classified into serogroups O10, O38, O36, O30, O17, O23, O9, O40, O49, O27, O5, O13, O24, O14, and O33. From the Swedish strains, 10 belonged to serogroup O10 and five to each of serogroups O38, O36, and O9. Therefore, none of the O-serogroups was predominant. The majority of the serotyped clinical strains possess acidic O-antigens containing uronic acids and various acidic non-carbohydrate substituents. In immunoblotting, antisera cross-reacted with both O-antigen and core of LPSs. The core region of 19 LPSs bound a single serum, and that of 12 LPSs bound more than two sera. Following bioinformatic analysis of the available sequences, a molecular approach to the prediction of Proteus core oligosaccharide structures was proposed. The identification of the core type of P. mirabilis R110, derived from a serogroup O3 wild strain, using restriction fragments length polymorphism analysis of galacturonic acid transferase is shown as an example. In summary, the most frequent O-serogroups among P. mirabilis UTI stains were identified. The diversity of serological reactions of LPSs is useful for serotyping of P. mirabilis clinical isolates. A possible role of the acidic components of O-antigens in UTI is discussed.     

Use of Pistacia lentiscus mastic for sustained-release system of chlorocresol and benzoic acid for in vitro prevention of bacterial colonization of silicon urinary catheter

Urinary tract infections (UTI) are among the most common types of nosocomial infections. Patients with indwelling urinary catheters are at the highest risk of getting infections. A sustained-release method of chlorocresol and benzoic acid using a varnish of Pistacia lentiscus mastic was developed to prevent catheter colonization by Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Pseudomonas aeruginosa. Coatings of both antiseptics significantly reduced the number of colonizing bacteria on silicon urinary catheters for 72 h. Chlorocresol-coated catheters were significantly (P ≤ 0·05) more effective than benzoic acid. Except for the Pr. mirabilis, chlorocresol completely inhibited the colonization of catheters by the tested bacteria for 48 h. Nonetheless, the colonization of catheters by Pr. mirabilis was significantly reduced after 48 and 72 h by more than 3·5 logs. Although benzoic acid failed to completely inhibit bacterial growth, it significantly reduced the colonization of the catheters by all the tested bacteria by more than two logs for 72 h. The inhibition of colonization of catheters was confirmed by examining the tested catheters by scanning electron microscopy. The obtained results indicate the potential benefits of using mastic as a varnish for sustaining the release of chlorocresol and benzoic acid to prevent and reduce the colonization of urinary catheters by bacteria.     

Inhibition of bacterial adherence to rat bladder epithelial cells by human immune serum globulin

The ability of commerical human immune serum globulin (HISG) to inhibit the adherence of urinary tract infection isolates to rat bladder epithelial cells was investigated utilizing an in vitro adherence system. Significant decreases in adherence were noted when strains ofEscherichia coli, Klebsiella pneumoniae, Proteus mirabilis, andEnterobacter cloacae were tested against five HISG preparations. An enzyme-linked immunosorbent assay indicated that all five HISG preparations also contained antibodies against type-1 pili isolated fromKlebsiella pneumoniae. The presence of antibodies directed against a bacterial adhesin and the effectiveness of HISG in inhibiting the attachment of a wide range of urinary pathogens to bladder cells suggest that HISG may have practical therapeutic values in the prophylaxis of diseases where bacterial adherence is a prerequisite for the initiation of infection.     

Sorption of Methylene Blue by an Oscillatoria sp.–Dominated Cyanobacterial Mat

The ability of an Oscillatoria sp.–dominated cyanobacterial mat in sorbing methylene blue (MB), a cationic dye, was investigated using the batch contact method. The sorption of MB onto the powdered biomass was not significantly influenced by initial pH (2–10) and temperature (5–45°C) of the solution. MB sorption occurred slowly, requiring 1–8 h for the establishment of equilibrium. A slow attainment of equilibrium seems to be related with the large size of MB ions. The isotherm data of MB sorption by the mat biomass could effectively fit to Langmuir and Freundlich models. The maximum MB sorption capacity (q _max) of the test biomass was 78.43 mg g^?1, which changed little with variation in biomass concentration. Moreover, the test biomass could efficiently sorb MB from solution in presence of Na⁺, K⁺, and Ca²⁺, which usually occur at high concentrations in natural waters, and also in presence of Cd²⁺. These particular characteristics together with pH and temperature independence of the sorption process make the mat biomass an ideal MB sorbent.     

Fluorescence in blue light (FLU) is involved in inactivation and localization of glutamyl‐tRNA reductase during light exposure

Fluorescent in blue light (FLU) is a negative regulator involved in dark repression of 5‐aminolevulinic acid (ALA) synthesis and interacts with glutamyl‐tRNA reductase (GluTR), the rate‐limiting enzyme of tetrapyrrole biosynthesis. In this study, we investigated FLU‘s regulatory function in light‐exposed FLU‐overexpressing (FLUOE) Arabidopsis lines and under fluctuating light intensities in wild‐type (WT) and flu seedlings. FLUOE lines suppress ALA synthesis in the light, resulting in reduced chlorophyll content, but more strongly in low and high light than in medium growth light. This situation indicates that FLU's impact on chlorophyll biosynthesis depends on light intensity. FLU overexpressors contain strongly increased amounts of mainly membrane‐associated GluTR. These findings correlate with FLU‐dependent localization of GluTR to plastidic membranes and concomitant inhibition, such that only the soluble GluTR fraction is active. The overaccumulation of membrane‐associated GluTR indicates that FLU binding enhances GluTR stability. Interestingly, under fluctuating light, the leaves of flu mutants contain less chlorophyll compared with WT and become necrotic. We propose that FLU is basically required for fine‐tuned ALA synthesis. FLU not only mediates dark repression of ALA synthesis, but functions also to control balanced ALA synthesis under variable light intensities to ensure the adequate supply of chlorophyll.     

Antigenic relatedness of Proteus mirabilis and Escherichia coli with rat tissues and human erythrocytes

The cross-reactions of rat kidney with P. mirabilis and E. coli, and of rat urinary bladder with P. mirabilis were established by precipitation, agglutination and immunofluorescence methods. One of the cross-reactive bacterial components was the lipopolysaccharide. Human erythrocytes of Blood-groups A, O and AB were agglutinated by antisera to P. mirabilis and E. coli. The role of antigenic relatedness in urinary tract infections is discussed.     

Rapid and Accurate Detection of Urinary Pathogens by Mobile IMS-Based Electronic Nose: A Proof-of-Principle Study

Urinary tract infection (UTI) is a common disease with significant morbidity and economic burden, accounting for a significant part of the workload in clinical microbiology laboratories. Current clinical chemisty point-of-care diagnostics rely on imperfect dipstick analysis which only provides indirect and insensitive evidence of urinary bacterial pathogens. An electronic nose (eNose) is a handheld device mimicking mammalian olfaction that potentially offers affordable and rapid analysis of samples without preparation at athmospheric pressure. In this study we demonstrate the applicability of ion mobility spectrometry (IMS) –based eNose to discriminate the most common UTI pathogens from gaseous headspace of culture plates rapidly and without sample preparation. We gathered a total of 101 culture samples containing four most common UTI bacteries: E. coli, S. saprophyticus, E. faecalis, Klebsiella spp and sterile culture plates. The samples were analyzed using ChemPro 100i device, consisting of IMS cell and six semiconductor sensors. Data analysis was conducted by linear discriminant analysis (LDA) and logistic regression (LR). The results were validated by leave-one-out and 5-fold cross validation analysis. In discrimination of sterile and bacterial samples sensitivity of 95% and specificity of 97% were achieved. The bacterial species were identified with sensitivity of 95% and specificity of 96% using eNose as compared to urine bacterial cultures. In conclusion: These findings strongly demonstrate the ability of our eNose to discriminate bacterial cultures and provides a proof of principle to use this method in urinanalysis of UTI.     

Attaching NorA efflux pump inhibitors to methylene blue enhances antimicrobial photodynamic inactivation of Escherichia coli and Acinetobacter baumannii in vitro and in vivo

Resistance of bacteria to antibiotics is a public health concern worldwide due to the increasing failure of standard antibiotic therapies. Antimicrobial photodynamic inactivation (aPDI) is a promising non-antibiotic alternative for treating localized bacterial infections that uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species and kill microbes. Phenothiazinium photosensitizers like methylene blue (MB) and toluidine blue O are hydrophobic cations that are naturally expelled from bacterial cells by multidrug efflux pumps, which reduces their effectiveness. We recently reported the discovery of a NorA efflux pump inhibitor-methylene blue (EPI-MB) hybrid compound INF55-(Ac)en–MB that shows enhanced photodynamic inactivation of the Gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) relative to MB, both in vitro and in vivo. Here, we report the surprising observation that INF55-(Ac)en–MB and two related hybrids bearing the NorA efflux pump inhibitors INF55 and INF271 also show enhanced aPDI activity in vitro (relative to MB) against the Gram-negative bacteria Escherichia coli and Acinetobacter baumannii, despite neither species expressing the NorA pump. Two of the hybrids showed superior effects to MB in murine aPDI infection models. The findings motivate wider exploration of aPDI with EPI-MB hybrids against Gram-negative pathogens and more detailed studies into the molecular mechanisms underpinning their activity.     

Native flagellin does not protect mice against an experimental Proteus mirabilis ascending urinary tract infection and neutralizes the protective effect of MrpA fimbrial protein

Proteus mirabilis expresses several virulence factors including MR/P fimbriae and flagella. Bacterial flagellin has frequently shown interesting adjuvant and protective properties in vaccine formulations. However, native P. mirabilis flagellin has not been analyzed so far. Native P. mirabilis flagellin was evaluated as a protective antigen and as an adjuvant in co-immunizations with MrpA (structural subunit of MR/P fimbriae) using an ascending UTI model in the mouse. Four groups of mice were intranasally treated with either MrpA, native flagellin, both proteins and PBS. Urine and blood samples were collected before and after immunization for specific antibodies determination. Cytokine production was assessed in immunized mice splenocytes cultures. Mice were challenged with P. mirabilis, and bacteria quantified in kidneys and bladders. MrpA immunization induced serum and urine specific anti-MrpA antibodies while MrpA coadministered with native flagellin did not. None of the animals developed significant anti-flagellin antibodies. Only MrpA-immunized mice showed a significant decrease of P. mirabilis in bladders and kidneys. Instead, infection levels in MrpA-flagellin or flagellin-treated mice showed no significant differences with the control group. IL-10 was significantly induced in splenocytes of mice that received native flagellin or MrpA-flagellin. Native P. mirabilis flagellin did not protect mice against an ascending UTI. Moreover, it showed an immunomodulatory effect, neutralizing the protective role of MrpA. P. mirabilis flagellin exhibits particular immunological properties compared to other bacterial flagellins.     

Photodynamic antimicrobial chemotherapy by liposome-encapsulated water-soluble photosensitizers

Photodynamic antimicrobial chemotherapy is an alternative method for killing bacterial cells in view of the increasing problem of multi-antibiotic resistance. We examined the effect of three water-soluble photosensitizers (PhS): methylene blue (MB), neutral red (NR) and rose bengal (RB) on Gram-positive and Gram-negative bacteria. We compared the efficacy of PhS in their free form and encapsulated in liposomal formulations against various bacterial strains, and determined conditions for the effective use of encapsulated PhS. We found that all three PhS were able to eradicate the Gram-positive microbes Staphylococcus aureus and Sarcina lutea; and MB and RB were effective against St. epidermidis. In the case of the Gram-negative species, MB and RB were cytotoxic against the Shigella flexneri, NR-inactivated Escherichia coli and Salmonella para B, and BR was effective in killing Pseudomonas aeruginosa. None of the examined PhS showed activity against Klebsiella pneumoniae. MB and NR enclosed in liposomes gave a stronger antimicrobial effect than free PhS for all tested prokaryotes, whereas encapsulation of RB led to no increase in its activity. We suggest that encapsulation of PhS can increase the photoinactivation of bacteria.     

Toxicity of common biocides used in aquaculture to embryos and larvae of Brachymystax tsinlingensis Li

Brachymystax tsinlingensis Li is a threatened fish species endemic to China. With the problems of environmental factors and seeding breeding diseases, it is important to further improve the efficiency of seeding breeding and the basis of resource protection. This study investigated the acute toxicity of copper, zinc and methylene blue (MB) on hatching, survival, morphology, heart rate (HR) and stress behaviour of B. tsinlingensis. Eggs (diameter: 3.86 ± 0.07 mm, weight: 0.032 ± 0.004 g) of B. tsinlingensis were selected randomly from artificial propagation and developed from eye-pigmentation-stage embryos to yolk-sac stage larvae (length: 12.40 ± 0.02 mm, weight: 0.03 ± 0.001 g) and exposed to different concentrations of Cu, Zn and MB for 144 h in a series of semi-static toxicity tests. The acute toxicity tests indicated that the 96-h median lethal concentration (LC₅₀) values of the embryos and larvae were 1.71 and 0.22 mg l⁻¹ for copper and 2.57 and 2.72 mg l⁻¹ for zinc, respectively, whereas the MB LC₅₀ after 144-h exposure for embryos and larvae were 67.88 and 17.81 mg l⁻¹, respectively. The safe concentrations of copper, zinc and MB were 0.17, 0.77 and 6.79 mg l⁻¹ for embryos and 0.03, 0.03 and 1.78 mg l⁻¹ for larvae, respectively. Copper, zinc and MB treatments with concentrations greater than 1.60, 2.00 and 60.00 mg l⁻¹, respectively, led to a significantly low hatching rate and significantly high embryo mortality (P < 0.05), and copper and MB treatments with concentrations greater than 0.2 and 20 mg l⁻¹ led to significantly high larvae mortality (P < 0.05). Exposure to copper, zinc and MB resulted in developmental defects, including spinal curvature, tail deformity, vascular system anomalies and discolouration. Moreover, copper exposure significantly reduced the HR of larvae (P < 0.05). The embryos exhibited an obvious change in behaviour, converting from the normal behaviour of emerging from the membrane head first to emerging tail first, with probabilities of 34.82%, 14.81% and 49.07% under copper, zinc and MB treatments, respectively. The results demonstrated that the sensitivity of yolk-sac larvae to copper and MB was significantly higher than that of embryos (P < 0.05) and that B. tsinlingensis embryos or larvae might be more resistant to copper, zinc and MB than other members of the Salmonidae family, which benefits their resource protection and restoration.     

Gemifloxacin can partially overcome quinolone resistance of H. pylori with gyrA mutation in Taiwan

Backgrounds: The levofloxacin resistance caused by gyrA gene mutation is rising rapidly to limit wide application for Helicobacter pylori eradication. We investigated whether gemifloxacin has a superior antimicrobial activity to levofloxacin against H. pylori. Materials and Methods: Forty‐four consecutive clinical H. pylori isolates with levofloxacin resistance and 80 randomly selected levofloxacin‐sensitive controls were tested for gemifloxacin sensitivity by E‐test. The resistance to levofloxacin or gemifloxacin was defined as minimal inhibitory concentration (MIC) >1 mg/L. The clinical features and GyrA mutation patterns checked by direct sequencing were also analyzed to assess its association with the H. pylori gemifloxacin resistance. Results: All levofloxacin‐sensitive H. pylori isolates were sensitive to gemifloxacin. Eight strains (18.2%) resistant to levofloxacin could be still sensitive to gemifloxacin. Gemifloxacin achieved a 5‐time lower in MIC levels against levofloxacin‐resistant isolates. Nearly all levofloxacin‐resistant isolates (97.7%, 43/44) had GyrA mutation at amino acid position 87 or 91. Double mutation sites may play dual roles in quinolone resistance, as N87K plus H57Y or D91N plus V77A mutations showed high‐level resistance to both quinolones; whereas D91Y plus A97V or D91N plus A97V mutations showed low level levofloxacin resistance to become sensitive to gemifloxacin. In H. pylori isolates with single N87K, D91Y or D91N mutation, near 20% was gemifloxacin‐sensitive and levofloxacin‐resistant. The gemifloxacin‐resistant rate of H. pylori was higher in patients with gastric ulcer than in those without (p <.05). Conclusion: Gemifloxacin is superior to levofloxacin in antimicrobial activity against clinical H. pylori isolates, and even overcome some levofloxacin resistance.     

DETERMINATION OF METHYLENE BLUE BIOSORPTION BY Rhizopus arrhizus IN THE PRESENCE OF SURFACTANTS WITH DIFFERENT CHEMICAL STRUCTURES

Methylene blue (MB) biosorption properties of Rhizopus arrhizus were investigated in the presence of surfactants. The effects of cationic and anionic surfactants on MB removal by dead biomass (1 g L^?1) were determined. MB removal was tested as a function of initial pH (2–12), contact time (5–1440 min), and dye (37.4–944.7 mg L^?1) and surfactant (0–10 mM) concentrations. The opposite charged anionic surfactant dodecylbenzenesulfonic acid sodium salt (DBS) enhanced sorption of cationic MB by biomass dramatically. Maximum biosorption capacity was 471.5 mg g^?1 at pH 8 with 0.5 mM DBS at 944.7 mg L^?1 MB concentration. The surfactant-stimulated fungal decolorization method may provide a highly efficient, inexpensive, and time-saving procedure in biological wastewater treatment technologies.     

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.     

Ozone treatment affects pigment precursor metabolism in pine seedlings

Five‐week‐old seedlings of Pinus halepensis Mill. and Pinus brutia Ten. were exposed to air polluted with ozone (O₃) (250 nl l^?1, 12 h day^?1 for 4 days) or to ambient air containing ca 10–20 nl l^?1 O₃, in the light (180 μmol m^?2 s^?1 photosynthetic photon flux density [PPFD], 12 h day^?1) and then fed for 24 h in the light (100 μmol m^?2 s^?1 PPFD) with various radioactive precursors of chlorophyll (Chl) and carotene biosynthesis: 5‐[4‐¹⁴C]‐aminolevulinic acid (¹⁴C‐ALA), l ‐[¹⁴C(U)]‐glutamic acid (¹⁴C‐Glu), or d ,l ‐[2‐¹⁴C]‐mevalonic acid (¹⁴C‐MVA). Pigments were then extracted from cotyledons and fully expanded needles. Chl a and carotene were separated by thin‐layer chromatography and high‐performance liquid chromatography and their specific activities were determined. ¹⁴C‐ALA and ¹⁴C‐Glu labels were incorporated into Chl a and carotene. Exposure to O₃ did not inhibit incorporation of ¹⁴C‐ALA into Chl a molecules, but hydrolysis of Chl a showed that O₃ inhibited phytol labelling of Chl a. Labelling of carotene was also inhibited by O₃, but not when ¹⁴C‐MVA was used as the label. These data suggest that O₃ treatment inhibits (directly or indirectly) the biosynthesis of isoprenoids from products of ALA and Glu metabolism in the plastid, but not from MVA in the cytosol. This inhibition was more prominent when ¹⁴C‐ALA was used as the label than when ¹⁴C‐Glu was the labelling precursor. A significant increase in pheophorbide a, a tetrapyrrole component of Chl a labelling, and a concomitant decrease in phytol labelling was observed following incubation of O₃‐treated pine seedlings with ¹⁴C‐ALA and ¹⁴C‐Glu. Stronger inhibition of carotene biosynthesis and activation of Chl a tetrapyrrole labelling by ¹⁴C‐ALA (in comparison with ¹⁴C‐Glu) indicated that exposure to O₃ inhibits the conversion of ALA to Glu as the first step in ALA catabolism. These results also suggested a more intensive Glu metabolism (in comparison with ALA) for carotene biosynthesis in the cytosol, as well as cooperation between two pathways of isopentenyl diphosphate biosynthesis.