Chelated iron sources are inhibitors of Pseudomonas aeruginosa biofilms and distribute efficiently in an in vitro model of drug delivery to the human lung

Aims:  To determine whether chelated sources of ferric iron were efficient inhibitors of biofilm formation in Pseudomonas aeruginosa and might be suitable for drug delivery to the lungs of cystic fibrosis (CF) patients via nebulization.
Methods and Results:  The response of P. aeruginosa biofilms to elevated iron concentrations in the form of eight structurally varied iron chelators in a microtitre plate assay for biofilm production was examined in the lab. Among these iron chelates, picolinic acid and acetohydroxamic acid-chelated iron were able to effectively thwart biofilm production in P. aeruginosa PA14 and in 20 clinical isolates of P. aeruginosa from a local hospital. The chelated iron sources showed excellent distribution in an Anderson cascade impactor model of particle size distribution in the human lung.
Conclusions:  Ferric picolinate and ferric acetohydroxamate are effective anti-biofilm compounds against both lab and clinical strains of P. aeruginosa and are readily nebulized into particles of suitable size for lung delivery.
Significance and Impact of the Study:  The data herein serve both to solidify the growing base of literature correlating high iron levels with biofilm inhibition in P. aeruginosa and to highlight the potential of these chelators as nebulized agents to combat biofilms of P. aeruginosa in CF patients.     

Conversion of volatile fatty acids into polyhydroxyalkanoate by Ralstonia eutropha

Aims:  The aims of this study were to optimize condensed corn solubles (CCS) as a medium for growth of Ralstonia eutropha and to determine the effects of individual volatile fatty acids (VFAs) on polyhydroxyalkanoate (PHA) production .
Methods and Results:  A CCS medium of concentration 240 g l⁻¹ with a carbon : nitrogen ratio of 50 : 1 was developed as the optimal medium. Cultures were grown in 1-l aerated flasks at 250 rev min⁻¹ at 30°C for 120 h. Comparable growth rates were observed in CCS vs a defined medium. At 48 h, VFAs were fed individually at different levels. Optimal levels of all the acids were determined to maximize PHA production. An overall comparison of the VFAs indicated that butyric and propionic acids provided the best results.
Conclusion:  An optimized CCS medium supported growth of R. eutropha . Butyric and propionic acids were the most efficient carbon sources to maximize PHA production when added at the 5 g l⁻¹ level.
Significance and Impact of the Study:  The study shows that a byproduct of ethanol industry can be effectively used as a low cost medium for PHA production, thus partly reducing the cost of commercialization of biopolymers.     

Rhamnolipid biosurfactants decrease the toxicity of chlorinated phenols to Pseudomonas putida DOT-T1E

Aims:  To investigate the effect of a mixture of rhamnolipid R1 and R2 biosurfactants produced by a Pseudomonas aeruginosa strain on the toxicity of phenol and chlorophenols to Pseudomonas putida DOT-T1E.
Methods and Results:  Toxicity was quantified by the effective concentration 50% (EC50), that is the concentration that causes a 50% inhibition of bacterial growth. The presence of 300 mg l⁻¹ rhamnolipids, that is at about twice their critical micelle concentration (CMC), increased the EC50 of phenol, 4-chlorophenol, 2,4-dichlorophenol and 2,4,5-trichlorophenol by about 12, 19, 32 and 40%, respectively, and consequently reduced the bioavailability and the freely dissolved concentration of the toxic phenolic compounds. The reduction was related to the phenols' octanol–water partition coefficients ( K _ow).
Conclusions:  The reduction in toxicity of the phenols can be explained by a combination of toxin accumulation in biosurfactant micelles and hydrophobic interactions of the phenols with rhamnolipid-based dissolved organic carbon.
Significance and Impact of the Study:  Results provide evidence that next to the effect of the micelle formation also hydrophobic interactions with rhamnolipid-based dissolved organic carbon affects the bioavailability of the phenols. Quantifying the effect of biosurfactants on the toxicity of hydrophobic compounds such as phenols thus appears to be a useful approach to assess their bioavailable equilibrium concentration.     

Carotenoid production by Xanthophyllomyces dendrorhous: use of pineapple juice as a production medium

Aims:  To select a carotenoid-hyperproducing mutant and to formulate pineapple juice as a carotenoid production medium.
Methods and Results:  Yeast strain of Xanthophyllomyces dendrorhous mutant GM 807 (derived from gamma irradiation) and the mutant n78 (from neutron irradiation) were selected based on higher carotenoid production in diluted pineapple juice as a medium. The selected strain was evaluated under various concentrations of pineapple juice. The results obtained in the study demonstrate that the mutant GM 807 enhanced production by 65·8% when using pineapple juice as medium at 10% dilution in place of yeast malt medium.
Conclusion:  Pineapple juice could be used as a sole medium for carotenoid production.
Significance and Impact of the Study:  Our results provide useful information for the design of production media for carotenoids to be used in various applications.     

Screening method to identify inhibitors of siderophore biosynthesis in the opportunistic fungal pathogen, Aspergillus fumigatus

Aims:  Aspergillus fumigatus is the most common cause of airborne mould infections in immunocompromised patients worldwide. Our aim was to develop a method to identify agents that inhibit siderophore biosynthesis because this pathway is unique to the fungus and is essential for virulence.
Methods and Results:  A high-throughput two-step screening assay was developed using 96-well plates in which fungal growth and siderophore production is assessed spectrophotometrically. If a compound inhibits growth only in iron-limited medium (screen 1), its effect on siderophore production is then determined (screen 2). The proof of concept was demonstrated using a known antifungal agent, amphotericin B, and a strain of A. fumigatus deficient in siderophore production.
Conclusions:  The two-stage screening method clearly identified growth defects in A. fumigatus related specifically to siderophore biosynthesis.
Significance and Impact of the Study:  The increasing incidence of life-threatening fungal infections has produced an urgent need for novel antifungal agents. The method described in this report will facilitate the identification of novel antifungal compounds that inhibit a pathway critical for A. fumigatus virulence and have a reduced probability of affecting host metabolism.     

The Effect of the Human Gut-Signalling Hormone, Norepinephrine, on the Growth of the Gastric Pathogen Helicobacter pylori

Background and Aims: Helicobacter pylori is an important human pathogen, infecting around half the population of the world. It has developed a number of refinements to allow it to persist in the human stomach. Catecholamine hormones have been shown to enhance growth of other bacterial species and are found in the gastric niche. We aimed to study growth enhancement of H. pylori by the human catecholamine hormones epinephrine and norepinephrine.
Methods: Growth studies were carried out in complex and defined media containing the hormones epinephrine, norepinephrine, and normetanephrine, the main host metabolite of norepinephrine. Bacterial density was measured by viable count or optical density. Intracellular ATP was measured using a bioluminescence assay technique.
Results: Both epinephrine and norepinephrine enhanced H. pylori growth in a dose-dependent strain-independent fashion, with norepinephrine being more effective than epinephrine. We showed a rapid (4 hours) dose-dependent effect on metabolic activity, as measured by intracellular ATP levels. We used a chemically defined medium to study mechanisms: chelation of ferric iron blocked H. pylori growth, which could be overcome by addition of norepinephrine. Disruption of the catechol group of norepinephrine abrogated its H. pylori- growth-promoting activity.
Conclusions: Norepinephrine stimulates growth of H. pylori under otherwise growth-restricted conditions, and this effect is related to the ability of norepinephrine to bind ferric iron. This supports the notion that norepinephrine may aid H. pylori persistence in the stomach.     

Optimization of a new heteropolysaccharide production by a native isolate of Leuconostoc sp. CFR-2181

Aim:  This work is aimed at optimizing the production of a new heteropolysaccharide (HePS) of Leuconostoc sp. CFR-2181 by standardizing the fermentation conditions in a low cost semi-synthetic medium.
Methods and Results:  Both nutritional and cultural parameters, such as carbon source and its concentration, initial pH of the exopolysaccharide (EPS) medium, fermentation temperature and fermentation period were optimized. Fermentation of the EPS medium (pH 6·7), containing sucrose at 5% (w/v) and 5% (v/v) inoculum, at 25 ° C resulted in maximum production of HePS (18·38 g l⁻¹) by the isolate in 4 h of fermentation.
Conclusions:  The isolate was found to produce good amount of HePS in just 4 h in a low cost semi-synthetic EPS medium.
Significance and Impact of the Study:  This is the first report on rapid production of HePS by any lactic culture, which can significantly reduce the cost of the EPS.     

Effect of culturing processes and copper addition on laccase production by the white-rot fungus Fomes fomentarius MUCL 35117

Aim:  To produce high laccase activities from the white-rot fungus Fomes fomentarius .
Methods and Results:  Different culturing methods, viz, cell immobilization on stainless steel sponges and plastic material and solid-state fermentation (SSF) using wheat bran as substrate were used for laccase production by the white-rot fungus F. fomentarius . The SSF study expresses the highest laccase activities, nearly to 6400 U l⁻¹ after 13 days of laboratory flasks cultivation. When the wheat bran medium was supplemented with 2 mmol l⁻¹ copper sulfate, laccase activity increased by threefold in comparison to control cultures, reaching 27 864 U l⁻¹. With the medium thus optimized, further experiments were performed in a 3 l fixed-bed bioreactor (working volume 1·5 l) leading to a laccase activity of about 6230 U l⁻¹ on day 13.
Conclusions:  The results obtained clearly showed the superiority of wheat bran for laccase production over stainless steel sponges and plastic material. Supplementing the wheat bran solid medium with 2 mmol l⁻¹ copper sulfate allowed obtaining high activities at flask scale. The system was scaled to fixed-bed laboratory reactor.
Significance and Impact of the Study:  The high enzyme production along with the low-cost of the substrate, showed the suitability of the system F. fomentarius – SSF for industrial purposes.     

Investigation of bacteria with polyketide synthase genes and antimicrobial activity isolated from South China Sea sponges

Aims:  To obtain bacteria with PKS (polyketide synthase) genes and antimicrobial activity from sponges.
Methods and Results:  Eighteen bacteria with KS (ketosynthase) genes were identified by polymerase chain reaction (PCR) screening of 98 isolates from South China Sea sponges, Stelletta tenuis , Halichondria rugosa , Dysidea avara and Craniella australiensis . 16S rRNA gene-based B last analysis indicated that 15 isolates belonged to the phylum Firmicutes , among which 14 isolates were closely related to genus Bacillus , and 1 to Staphylococcus lentus . Two isolates were identified as actinomycetes, and one as Alcaligenes sp. in the phylum Proteobacteria . The 18 KS domains belong to trans-AT type I PKS and match PKS of marine bacterial symbionts. The 18 bacteria exhibited broad-spectrum antimicrobial activities against fungi, gram-positive and gram-negative bacteria. A 21·8-kb PKS gene cluster fragment containing five modules was isolated from the Staphylococcus lentus isolate A75 by screening of a fosmid library.
Conclusions:  The PKS gene diversity and different antimicrobial spectra indicate the potential of bacteria associated with South China Sea sponges for diverse polyketide production.
Significance and Impact of the Study:  Combined with bioactivity assay the PKS gene-based approach can be applied to efficient screening of strains of pharmaceutical value and the prediction of related compounds.     

Statistical optimization of medium components for extracellular protease production by an extreme haloarchaeon, Halobacterium sp. SP1(1)

Aims:  Optimization of medium components for extracellular protease production by Halobacterium sp. SP1(1) using statistical approach.
Methods and Results:  The significant factors influencing the protease production as screened by Plackett–Burman method were identified as soybean flour and FeCl₃. Response surface methodology such as central composite design was applied for further optimization studies. The concentrations of medium components for higher protease production as optimized using this approach were (g l⁻¹): NaCl, 250; KCl, 2; MgSO₄, 10; tri-Na-citrate, 1·5; soybean flour, 10 and FeCl₃, 0·16. This statistical optimization approach led to production of 69·44 ± 0·811 U ml⁻¹ of protease.
Conclusions:  Soybean flour and FeCl₃ were identified as important factors controlling the production of extracellular protease by Halobacterium sp. SP1(1). The statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 3·9-fold increase in extracellular protease production.
Significance and Impact of the Study:  The present study is the first report on statistical optimization of medium components for production of haloarchaeal protease. The study also explored the possibility of using extracellular protease produced by Halobacterium sp. SP1(1) for various applications like antifouling coatings and fish sauce preparation using cheaper raw material.     

Application of luminescent biosensors for monitoring the degradation and toxicity of BTEX compounds in soils

Aims:  To assess the changes in acute toxicity and biodegradation of benzene, toluene, ethylbenzene and xylene (collectively referred to as BTEX) compounds in soil over time and compare the performances of biological and chemical techniques.
Methods and Results:  Biological methods ( lux -based bacterial biosensors, basal respiration and dehydrogenase activity) were related to changes in the concentration of the target compounds. There was an initial increase in toxicity determined by the constitutively expressed biosensor, followed by a continual reduction as degradation proceeded. The biosensor with the BTEX-specific promoter was most induced when BTEX concentrations were highest. The treatment with nutrient amendment had a significant increase in microbial activity, while the sterile control produced the lowest level of degradation.
Significance and Impact of the Study:  Luminescent biosensors were able to monitor changes in contaminant toxicity and bioavailability in aqueous extracts from BTEX-impacted soils as degradation proceeded. The integration of biological tests with chemical analysis enables a fuller understanding of the biodegradation processes occurring at their relative rates.
Conclusions:  The biological methods were successfully used in assessing the performance of different treatments for enhancing natural attenuation of BTEX from contaminated soils. While, chemical analysis showed biodegradation of parent BTEX compounds in biologically active soils, the biosensor assays reported on changes in bioavailability and potentially toxic intermediate fractions as they estimated the integrative effect of contaminants.     

Optimization of medium constituents for improved chitinase production by Paenibacillus sp. D1 using statistical approach

Aims:  Statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1.
Methods and Results:  Urea, K₂HPO₄, chitin and yeast extract were identified as significant components influencing chitinase production by Paenibacillus sp. D1 using Plackett–Burman method. Response surface methodology (central composite design) was applied for further optimization. The concentrations of medium components for improved chitinase production were as follows (g l⁻¹): urea, 0·33; K₂HPO₄, 1·17; MgSO₄, 0·3; yeast extract, 0·65 and chitin, 3·75. This statistical optimization approach led to the production of 93·2 ± 0·58 U ml⁻¹ of chitinase.
Conclusions:  The important factors controlling the production of chitinase by Paenibacillus sp. D1 were identified as urea, K₂HPO₄, chitin and yeast extract. Statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 2·56-fold increase in chitinase production.
Significance and Impact of the Study:  The present investigation provides a report on statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Paenibacillus species are gram-positive, spore-forming bacteria with several PGPR and biocontrol potentials. However, only few reports concerning mycolytic enzyme production especially chitinases are available. Chitinase produced by Paenibacillus sp. D1 represents new source for biotechnological and agricultural use.     

Response of Wolfiporia cocos to iron availability: alterations in growth, expression of cellular proteins, Fe3+-reducing activity and Fe3+-chelators production

Aims:  The main objective of this study was to evaluate the behaviour of the brown-rot fungus Wolfiporia cocos under differential iron availability.
Methods and Results:  W. cocos was grown under three differential iron conditions. Growth, catecholate and hydroxamate production, and mycelial and extracellular Fe³⁺-reducing activities were determined. Iron starvation slowed fungal growth and accelerated pH decline. Some mycelial proteins of low molecular weight were repressed under iron restriction, whereas others of high molecular weight showed positive iron regulation. Mycelial ferrireductase activity decreased as culture aged, while Fe³⁺-reducing activity of low molecular reductants constantly increased. Hydroxamates production suffered only limited iron repression, whereas catecholates production showed to be more iron repressible.
Conclusions:  W. cocos seems to possess more than one type of iron acquisition mechanism; one involving secretion of organic acids and ferrireductases and/or extracellular reductants, and another relying on secretion of catecholates and hydroxamates chelators.
Significance and Impact of the Study:  This paper is the first to report the kinetic study of brown-rot fungus grown under differential iron availability, and the information provided here contributes to address more traditional problems in protecting wood from brown decay, and also makes a contribution in the general area of the physiology of brown-rot fungi.     

Development of a novel artificial medium based on utilization of algal photosynthetic metabolites by symbiotic heterotrophs

Aims:  (i) Quantitative and qualitative analyses of photosynthetic metabolites of Chlorella sorokiniana and elucidation of the mechanism of their utilization by algal symbionts. (ii) Development of artificial medium that imitates photoautotroph–heterotroph interaction and investigation of its suitability for isolation of novel microbes from the environment.
Methods and Results:  Various components, including free dissolved carbohydrates, nitrogenous compounds and vitamin, were detected and together contributed 11·1% (as carbon content) of the total photosynthetic metabolites in the medium. Utilization of these photosynthetic metabolites in algal culture broth by algal symbionts was studied. Many symbionts showed specific utilization patterns. A novel artificial extracellular released organic carbon medium, which imitated the nutritional conditions surrounding algae, was developed based on the pattern of utilization of the algal metabolites by the symbiotic heterotrophs. About 42·9% of the isolates were closely related to photoautotrophic-dependent and oligotrophic bacteria.
Conclusions:  With the novel artificial medium, it was possible to selectively isolate some bacterial strains.
Significant and Impact of the Study:  Synthetic bacterial growth medium is an important and basic tool for bacterial isolation from environmental samples. The current study shows that preferential separation of typical bacterial subset can be achieved by using artificial medium that mimics photosynthetic metabolites.     

Adaptation to flavomycin in the ruminal bacterium, Prevotella bryantii

Aims:  The recent EU ban of growth-promoting antibiotics in animal production was based on fears concerning antibiotic resistance being transmitted to human pathogens. This paper explores the adaptation mechanism of a common ruminal bacterium, Prevotella bryantii , to one of the banned compounds, flavomycin (flavophospholipol).
Methods and Results:  Growth in the presence of flavomycin (2 and 20  μ g ml⁻¹) was characterized by a concentration-dependent increase in the length of the lag phase, which decreased after previous flavomycin exposure. From growth patterns on solid medium, decreased sensitivity appeared to be due to a whole-population adaptation. Proteomic analysis indicated upregulation of three native proteins occurred following flavomycin adaptation. Further analysis of two of these proteins resulted in no database matches, suggesting that they may be species-specific. Flavomycin adaptation also resulted in co-adaptation to bacitracin and vancomycin.
Conclusions:  Adaptation of P. bryantii to flavomycin, which also resulted in co-adaptation to bacitracin and vancomycin, may involve an increased availability of undecaprenyl pyrophosphate.
Significance and Impact of the Study:  The use of flavomycin, and similar growth-promoting antibiotics, in animal production may prompt adaptive responses in ruminal bacteria which can significantly change their antibiotic sensitivity.     

Chemical defence of Battus philenor larvae against attack by the parasitoid Trogus pennator

Abstract 1. Parasitoids in the genus Trogus (Hymenoptera: Ichneumonidae) attack the larvae of swallowtail butterflies (Lepidoptera: Papilionidae). Only two of the three major tribes of the subfamily Papilioninae are attacked although species of all three tribes commonly occur together. The tribe Troidini is relatively free of parasitoids of any kind, and it has been proposed that the aristolochic acids sequestered by troidines protect them from parasitism.
2. The responses of T. pennator (Fabricius) to the sympatric troidine Battus philenor (Linnaeus) were examined. Three hypotheses that could explain why this wasp does not parasitise B. philenor were considered. (1) Battus philenor does not produce compounds used by the wasp to locate hosts. (2) The larval integument contains compounds that deter attack. (3) The parasitoid offspring cannot survive in B. philenor .
3. The first hypothesis was not supported as the frass of B. philenor larvae was found to act as a searching arrestant comparable to the frass of a host species.
4. The second hypothesis was supported. The B. philenor larvae were rejected when the wasps examined them using their antennae, and ethanolic washes of B. philenor cuticle deterred attack by T. pennator when applied to otherwise acceptable host larvae. Bioassays of fractions of the ethanolic wash and of pure aristolochic acids established that aristolochic acids were at least partly responsible for the deterrent effect.
5. The third hypothesis was also supported. Larvae of B. philenor attacked by the parasitoids developed into butterflies.
6. These results indicate that both behavioural and physiological barriers, the former attributable at least in part to sequestered compounds and the latter of unknown mechanism, prevent T. pennator from parasitising B. philenor .     

The Expression of Iron-repressible Outer Membrane Proteins in Helicobacter pylori and Its Association with Iron Deficiency Anemia

Background: Helicobacter pylori infection is known to be a cause of iron deficiency anemia (IDA) that is unresponsive to iron supplements. H. pylori bind iron to a specific receptor by iron-repressible outer membrane proteins (IROMPs) under conditions of restricted iron.
Materials and Methods: We compared the expression of IROMPs from strains of H. pylori under both iron-restricted and iron-supplemented conditions to determine the difference between strains with and without IDA. One standard strain, two clinical strains, and three IDA strains were cultured; and then the IROMPs were extracted under iron-restricted and iron-supplemented conditions. We used SDS-PAGE to compare the expression of the IROMPs from each strain.
Results:  IROMPs were found in IDA strains under iron-restricted conditions and their molecular sizes were estimated to be 56, 48, 41, and 37 kDa. In the iron-repleted media, the IROMPs were no longer present.
Conclusion: In the iron-depleted state, specific H. pylori strains associated with IDA demonstrated an advantage in iron acquisition due to a higher expression of IROMPs. Our results can explain in part why some patients with H. pylori infection are more prone to develop clinical IDA under restricted iron conditions in the host.     

The evaluation of human papillomavirus genotyping in cervical liquid-based cytology specimens; using the Roche Linear Array HPV genotyping assay

Objective:  To ascertain the usefulness of the Roche Linear Array human papillomavirus (HPV) genotyping assay for assessing HPV genotypes in liquid-based cytology (LBC) samples and to evaluate this methodology within a cytopathology laboratory. These tests are of importance as persistent infection with high-risk HPV genotypes is considered a causal factor in the development of cervical cancer.
Methods:  A total of 175 cervical LBC samples were tested using the Roche Linear Array HPV genotyping test. The suitability of the assay use in routine cytopathology laboratory was considered. HPV genotypes were matched to the cervical cytology results, which included negative, borderline nuclear abnormalities, mild, moderate and severe dyskaryosis.
Results:  The assay could be applied to screening samples with the combined result available at the reporting stage. There were no test failures. All samples used after cytological analysis had sufficient DNA for testing. The results were reproducible and easily read and there was concordance of results between biomedical scientists. The results of the assay showed co-infection with multiple HPV genotypes was common in both high-grade and low-grade cytology samples. The percentage of HPV+ samples in the normal cytology samples (although in this grouping the number of samples was low) was 37%. In the cytology samples reported as severe dyskaryosis the HPV genotypes most commonly found were HPV16 and HPV51.
Conclusion:  The assay was able to detect multiple HPV infection with a wide range of genotypes in LBC samples sent for routine cytological analysis. It would be suitable for use in a cytopathology laboratory. The results of the assay show that the genotype profile has some variation from other geographical regions, and more work is needed to determine population prevalence, to ascertain the impact of the HPV vaccine, to evaluate test for cure and HPV triage management.     

Production of conjugated linoleic acid by probiotic Lactobacillus acidophilus La-5

Aims:  To study the ability of the probiotic culture Lactobacillus acidophilus La-5 to produce conjugated linoleic acid (CLA), which is a potent anti-carcinogenic agent.
Methods and Results:  The conversion of linoleic acid to CLA was studied both by fermentation in a synthetic medium and by incubation of washed cells. Accumulation of CLA was monitored by gas chromatography analysis of the biomass and supernatants. While the fermentation conditions applied may not be optimal to observe CLA production in growing La-5 cells, the total CLA surpassed 50% of the original content in the washed cells after 48 h under both aerobic and micro-aerobic conditions. The restriction of oxygen did not increase the yield, but favoured the formation of trans, trans isomers.
Conclusions:  The capability of L. acidophilus La-5 to produce CLA is not dependant on the presence of milk fat or anaerobic conditions. Regulation of CLA production in this strain needs to be further investigated to exploit the CLA potential in fermented foods.
Significance and Impact of the study:  Knowledge gained through the conditions on the accumulation of CLA would provide further insight into the fermentation of probiotic dairy products. The capacity of the nongrowing cells to produce CLA is also of great relevance for the emerging nonfermented probiotic foods.