In vitro bactericidal activity of diterpenoids isolated from Aframomum melegueta K.Schum against strains of Escherichia coli, Listeria monocytogenes and Staphylococcus aureus

Ethnopharmacological relevance: The ethnobotanical use of Aframomum melegueta in the treatment of urinary tract and soft tissue infection suggested that the plant has antimicrobial activity.

Materials and methods: To substantiate the folkloric claims, an acetone, 50:50 acetone:methanol and 2:1 chloroform:methanol extracts were tested against Escherichia coli K12; acetone extract and the fractions of acetone extracts were tested against Listeria monocytogenes. Bioassay-guided fractionation was performed on the extract using L. monocytogenes as the test organism to isolate the bioactive compounds which were then tested against all the other organisms.

Results: Four known labdane diterpenes (G3 and G5) were isolated for the first time from the rhizomes of A. melegueta and purified. These were tested against E. coli, L. monocytogenes, methicillin resistant Staphylococus aureus (MRSA) and S. aureus to determine antibacterial activity. The result showed that two compounds G3 and G5 exhibited more potent antibacterial activity compared to the current clinically used antibiotics ampicillin, gentamicin and vancomycin and can be potential antibacterial lead compounds. The structure of the labdane diterpenes were elucidated using nuclear magnetic resonance (NMR) spectroscopy and Mass spectrometry. A possible mode of action of the isolated compound G3 and its potential cytotoxicity towards mammalian cells were also discussed.

Conclusion: The results confirmed the presence of antibacterial compounds in the rhizomes of A. melegueta with a favourable toxicity profile which could be further optimized as antibacterial lead compounds.

Genotypic and antimicrobial characterisation of Propionibacterium acnes isolates from surgically excised lumbar disc herniations

The anaerobic skin commensal Propionibacterium acnes is an underestimated cause of human infections and clinical conditions. Previous studies have suggested a role for the bacterium in lumbar disc herniation and infection. To further investigate this, five biopsy samples were surgically excised from each of 64 patients with lumbar disc herniation. P. acnes and other bacteria were detected by anaerobic culture, followed by biochemical and PCR-based identification. In total, 24/64 (38%) patients had evidence of P. acnes in their excised herniated disc tissue. Using recA and mAb typing methods, 52% of the isolates were type II (50% of culture-positive patients), while type IA strains accounted for 28% of isolates (42% patients). Type III (11% isolates; 21% patients) and type IB strains (9% isolates; 17% patients) were detected less frequently. The MIC values for all isolates were lowest for amoxicillin, ciprofloxacin, erythromycin, rifampicin, tetracycline, and vancomycin (≤1 mg/L). The MIC for fusidic acid was 1-2 mg/L. The MIC for trimethoprim and gentamicin was 2 to ≥4  mg/L. The demonstration that type II and III strains, which are not frequently recovered from skin, predominated within our isolate collection (63%) suggests that the role of P. acnes in lumbar disc herniation should not be readily dismissed.

Isolating Informational Masking in Both Pure and Complex Tone Sequences

Objectives: Interference between a target and simultaneous maskers occurs both at the cochlear level through energetic masking and more centrally through informational masking (IM). Hence, quantifying the amount of IM requires a strict control of the energetic component. Presenting target and maskers on different sides (i.e., dichotically) reduces energetic masking but provides listeners with important lateralization cues that also drastically reduce IM. The main purpose of this study (Experiment 1) was to evaluate a "switch" manipulation aiming at restoring most of the IM despite dichotic listening. Experiment 2 was designed to investigate the source of the difficulty induced by this switching dichotic condition.

Design: In Experiment 1, the authors presented 60 normal-hearing young adults with a detection task in which a regularly repeating target was embedded in a randomly varying background masker. The authors evaluated spatial masking release induced by three different dichotic listening conditions in comparison with a diotic baseline. Dichotic stimuli were presented in either a nonswitching or a switching condition. In the latter case, the presentation sides of dichotic target and maskers alternated several times throughout 10 sec sequences. The impact of the number of switches on IM was investigated parametrically, with both pure and complex tone sequences. In Experiment 2, the authors compared performance of 13 young, normal-hearing listeners in a monotic and dichotic version of the rapidly switching condition, using pure-tone sequences.

Results: When target and maskers switched rapidly within sequences, IM was significantly stronger than in nonswitching dichotic sequences and was comparable with the masking effect induced by diotic sequences. Furthermore, Experiment 2 suggests that rapidly switching target and maskers prevent listeners from relying on lateralization cues inherent to the dichotic condition, hence preserving important amounts of IM.

Conclusions: This paradigm thus provides an original tool to isolate IM in signal and maskers having overlapping spectra.

Culture Age, temperature, and pH affect the polyol and trehalose contents of fungal propagules

The growth and conidial physiology of the entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus were studied under different conditions. The effects of culture age (up to 120 days), temperature (5 to 35°C), and pH (2.9 to 11.1) were determined. Growth was optimal at pH 5 to 8 for each isolate and between 20 and 35°C, depending on the isolate. The predominant polyol in conidia was mannitol, with up to 39, 134, and 61 mg g of conidia-1 for B. bassiana, M. anisopliae, and P. farinosus, respectively. Conidia of M. anisopliae contained relatively small amounts of lower-molecular-weight polyols and trehalose (less than 25 mg g-1 in total) in all treatments. Conidia of B. bassiana and P. farinosus contained up to 30, 32, and 25 mg of glycerol, erythritol, and trehalose, respectively, g-1, depending on the treatment. Conidia of P. farinosus contained unusually high amounts of glycerol and erythritol at pH 2.9. The apparent effect of pH on gene expression is discussed in relation to the induction of a water stress response. To our knowledge, this is the first report of polyols and trehalose in fungal propagules produced over a range of temperature or pH. Some conditions and harvesting times were associated with an apparent inhibition of synthesis or accumulation of polyols and trehalose. This shows that culture age and environmental conditions affect the physiological quality of inoculum and can thereby determine its potential for biocontrol.

Molecular surveillance for carbapenemase genes in carbapenem-resistant Pseudomonas aeruginosa in Australian patients with cystic fibrosis.

Summary: The aim of this study was to assess the prevalence of acquired carbapenemase genes amongst carbapenem non-susceptible Pseudomonas aeruginosa isolates in Australian patients with cystic fibrosis (CF). Cross-sectional molecular surveillance for acquired carbapenemase genes was performed on CF P. aeruginosa isolates from two isolate banks comprising: (i) 662 carbapenem resistant P. aeruginosa isolates from 227 patients attending 10 geographically diverse Australian CF centres (2007-2009), and (ii) 519 P. aeruginosa isolates from a cohort of 173 adult patients attending one Queensland CF clinic in 2011. All 1189 P. aeruginosa isolates were tested by polymerase chain reaction (PCR) protocols targeting ten common carbapenemase genes, as well the Class 1 integron intI1 gene and the aadB aminoglycoside resistance gene. No carbapenemase genes were identified among all isolates tested. The intI1 and aadB genes were frequently detected and were significantly associated with the AUST-02 strain (OR 24.6, 95% CI 9.3-65.6; p < 0.0001) predominantly from Queensland patients. Despite the high prevalence of carbapenem resistance in P. aeruginosa in Australian patients with CF, no acquired carbapenemase genes were detected in the study, suggesting chromosomal mutations remain the key resistance mechanism in CF isolates. Systematic surveillance for carbapenemase-producing P. aeruginosa in CF by molecular surveillance is ongoing.

Within-host evolution of Burkholderia pseudomallei over a twelve-year chronic carriage infection

UNLABELLED: Burkholderia pseudomallei causes the potentially fatal disease melioidosis. It is generally accepted that B. pseudomallei is a noncommensal bacterium and that any culture-positive clinical specimen denotes disease requiring treatment. Over a 23-year study of melioidosis cases in Darwin, Australia, just one patient from 707 survivors has developed persistent asymptomatic B. pseudomallei carriage. To better understand the mechanisms behind this unique scenario, we performed whole-genome analysis of two strains isolated 139 months apart. During this period, B. pseudomallei underwent several adaptive changes. Of 23 point mutations, 78% were nonsynonymous and 43% were predicted to be deleterious to gene function, demonstrating a strong propensity for positive selection. Notably, a nonsense mutation inactivated the universal stress response sigma factor RpoS, with pleiotropic implications. The genome underwent substantial reduction, with four deletions in chromosome 2 resulting in the loss of 221 genes. The deleted loci included genes involved in secondary metabolism, environmental survival, and pathogenesis. Of 14 indels, 11 occurred in coding regions and 9 resulted in frameshift mutations that dramatically affected predicted gene products. Disproportionately, four indels affected lipopolysaccharide biosynthesis and modification. Finally, we identified a frameshift mutation in both P314 isolates within wcbR, an important component of the capsular polysaccharide I locus, suggesting virulence attenuation early in infection. Our study illustrates a unique clinical case that contrasts a high-consequence infectious agent with a long-term commensal infection and provides further insights into bacterial evolution within the human host.

IMPORTANCE: Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we document within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. Our study provides insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens.

Burkholderia cepacia complex epidemiology in persons with cystic fibrosis from Australia and New Zealand

The Burkholderia cepacia complex (Bcc) is a group of significant opportunistic respiratory pathogens which affect people with cystic fibrosis. In this study, we sought to ascertain the epidemiology and geographic species distribution of 116 Bcc isolates collected from people with CF in Australia and New Zealand. We performed a combination of recA-based PCR, amplified rDNA restriction analysis (ARDRA), pulsed-field gel electrophoresis and repetitive extragenic palindromic PCR on each isolate. Each Burkholderia cenocepacia isolate was also screened by PCR for the presence of the B. cepacia epidemic strain marker. One hundred and fourteen isolates were assigned to a species using recA-based PCR and ARDRA. B. cenocepacia, B. multivorans and B. cepacia accounted for 45.7%, 29.3% and 11.2% of the isolates, respectively. Strain analysis of B. cenocepacia revealed that 85.3% of the isolates were unrelated. One related B. cenocepacia strain was identified amongst 15 people. Whilst full details of person-to-person contact was not available, all patients attended CF centres in Queensland (Qld) and New South Wales (NSW). Although person-to-person transmission of B. cenocepacia strains has occurred in Australia, the majority of CF-related Bcc infections in Australia and New Zealand are most likely acquired from the environment.

Burkholderia pseudomallei:another emerging pathogen in cystic fibrosis

BACKGROUND: Burkholderia pseudomallei is an important cause of acute fulminant pneumonia and septicaemia in tropical regions of northern Australia and south east Asia. Subacute and chronic forms of the disease also occur. There have been three recent reports of adults with cystic fibrosis (CF) who presumably acquired B pseudomallei infection during extended vacations or residence in either Thailand or northern Australia.

METHODS: The clinical course, molecular characteristics, serology and response to treatment are described in four adult CF patients infected with B pseudomallei. Polymerase chain reaction (PCR) based methods were used to confirm B pseudomallei and exclude B cepacia complex. Genotyping was performed using randomly amplified polymorphic DNA (RAPD) PCR and pulsed field gel electrophoresis (PFGE).

RESULTS: Four patients are described with a mean duration of infection of 32 months. All but one patient lived in tropical Queensland. Two patients (with the longest duration of infection) deteriorated clinically and one subsequently died of respiratory failure. Both responded to intravenous treatment specifically targeting B pseudomallei. Another patient suffered two severe episodes of acute bronchopneumonia following acquisition of B pseudomallei. Eradication of the organism was not possible in any of the cases. PFGE of a sample isolate from each patient revealed the strains to be unique and RAPD analysis showed retention of the same strain within an individual over time.

CONCLUSIONS: These findings support a potential pathogenic role for B pseudomallei in CF lung disease, producing both chronic infection and possibly acute bronchopneumonia. Identical isolates are retained over time and are unique, consistent with likely environmental acquisition and not person to person spread. B pseudomallei is emerging as a significant pathogen for patients with CF residing and holidaying in the tropics.

Multiplex Touchdown PCR for Rapid Typing of the Opportunistic Pathogen Propionibacterium acnes.

The opportunistic human pathogen Propionibacterium acnes is comprised of a number of distinct phylogroups, designated types IA1, IA2, IB, IC, II and III, that vary in their production of putative virulence factors, inflammatory potential, as well as biochemical, aggregative and morphological characteristics. Although Multilocus Sequence Typing (MLST) currently represents the gold standard for unambiguous phylogroup classification, and individual strain identification, it is a labour and time-consuming technique. As a consequence, we have developed a multiplex touchdown PCR assay that will, in a single reaction, confirm species identity and phylogeny of an isolate based on its pattern of reaction with six primer sets that target the 16S rRNA (all isolates), ATPase (type IA1, IA2, IC), sodA (type IA2, IB), atpD (type II) and recA (type III) housekeeping genes, as well as a Fic family toxin gene (type IC). When applied to 312 P. acnes isolates previously characterised by MLST, and representing type IA1 (n=145), IA2 (n=20), IB (n=65), IC (n=7), II (n=45) and III (n=30), the multiplex displayed 100% sensitivity and 100% specificity for the detection of isolates within each targeted phylogroup. No cross-reactivity with isolates from other bacterial species was observed. The multiplex assay will provide researchers with a rapid, high-throughput and technically undemanding typing method for epidemiological and phylogenetic investigations. It will facilitate studies investigating the association of lineages with various infections and clinical conditions, as well as a pre-screening tool to maximise the number of genetically diverse isolates selected for downstream, higher resolution sequence-based analyses.

Antimicrobial and immunomodulatory properties of PGLa-AM1, CPF-AM1, and magainin-AM1: Potent activity against oral pathogens

Cationic amphipathic α-helical peptides are intensively studied classes of host defence peptides (HDPs). Three peptides, peptide glycine-leucine-amide (PGLa-AM1), caerulein-precursor fragment (CPF-AM1) and magainin-AM1, originally isolated from norepinephrine-stimulated skin secretions of the African volcano frog Xenopus amieti (Pipidae), were studied for their antimicrobial and immunomodulatory activities against oral and respiratory pathogens. Minimal effective concentrations (MECs), determined by radial diffusion assay, were generally lower than minimal inhibitory concentrations (MICs) determined by microbroth dilution. PGLa-AM1 and CPF-AM1 were particularly active against Streptococcus mutans and all three peptides were effective against Fusobacterium nucleatum, whereas Enterococcus faecalis and Candida albicans proved to be relatively resistant micro-organisms. A type strain of Pseudomonas aeruginosa was shown to be more susceptible than the clinical isolate studied. PGLa-AM1 displayed the greatest propensity to bind lipopolysaccharide (LPS) from Escherichia coli, P. aeruginosa and Porphyromonas gingivalis. All three peptides showed less binding to P. gingivalis LPS than to LPS from the other species studied. Oral fibroblast viability was unaffected by 50. μM peptide treatments. Production of the pro-inflammatory cytokine IL-8 by oral fibroblasts was significantly increased following treatment with 1 or 10. μM magainin-AM1 but not following treatment with PGLa-AM1 or CPF-AM1. In conclusion, as well as possessing potent antimicrobial actions, the X. amieti peptides bound to LPS from three human pathogens and had no effect on oral fibroblast viability. CPF-AM1 and PGLa-AM1 show promise as templates for the design of novel analogues for the treatment of oral and dental diseases associated with bacteria or fungi.

The British conservative party and Europe:The choosing of John Major

This article provides an empirical analysis of voting behaviour in the second ballot of the 1990 Conservative leadership contest that resulted in John Major becoming party leader and prime minister. Seven hypotheses of voting behaviour are generated from the extant literature relating voting to socio-economic variables (occupational and educational background), political variables (parliamentary experience, career status, age and electoral marginality) and ideological variables (drawn from survey data on MPs' positions on economic, European and moral issues). These hypotheses are tested using data on voting intentions gathered from published lists of MPs' declarations, interviews with each of the leadership campaign teams, and correspondence with MPs. Bivariate relationships are presented, followed by logistic regression analysis to isolate the unique impact that each variable had on voting. This shows that educational background, parliamentary experience and (especially) attitudes to Europe were the key factors determining voting. The importance of Europe in the contest is particularly instructive: the severe problems for Major's leadership which were caused by the issue can be attributed to, and understood in the context of, the 1990 contest in which he became leader.

Characterisation of a novel panel of polymorphic microsatellite loci for the liver fluke, Fasciola hepatica, using a next generation sequencing approach

The liver fluke, Fasciola hepatica is an economically important pathogen of sheep and cattle and has been described by the WHO as a re-emerging zoonosis. Control is heavily reliant on the use of drugs, particularly triclabendazole and as a result resistance has now emerged. The population structure of F. hepatica is not well known, yet it can impact on host-parasite interactions and parasite control with drugs, particularly regarding the spread of triclabendazole resistance. We have identified 2448 potential microsatellites from 83Mb of F. hepatica genome sequence using msatfinder. Thirty-five loci were developed and optimised for microsatellite PCR, resulting in a panel of 15 polymorphic loci, with a range of three to 15 alleles. This panel was validated on genomic DNA from 46 adult F. hepatica; 38 liver flukes sourced from a Northwest abattoir, UK and 8 liver flukes from an established isolate (Shrewsbury; Ridgeway Research). Evidence for null alleles was found at four loci (Fh_1, Fh_8, Fh_13 and Fh_14), which showed markedly higher levels of homozygosity than the remaining 11 loci. Of the 38 liver flukes isolated from cattle livers (n=10) at the abattoir, 37 genotypes were identified. Using a multiplex approach all 15 loci could be amplified from several life cycle stages that typically yield low amounts of DNA, including metacercariae, the infective life cycle stage present on pasture, highlighting the utility of this multiplex microsatellite panel. This study reports the largest panel of microsatellite markers available to date for population studies of F. hepatica and the first multiplex panel of microsatellite markers that can be used for several life cycle stages.

Heating Mechanisms for Intermittent Loops in Active Region Cores from AIA/SDO EUV Observations

We investigate intensity variations and energy deposition in five coronal loops in active region cores. These were selected for their strong variability in the AIA/SDO 94 Å intensity channel. We isolate the hot Fe XVIII and Fe XXI components of the 94 Å and 131 Å by modeling and subtracting the "warm" contributions to the emission. HMI/SDO data allow us to focus on "inter-moss" regions in the loops. The detailed evolution of the inter-moss intensity time series reveals loops that are impulsively heated in a mode compatible with a nanoflare storm, with a spike in the hot 131 Å signals leading and the other five EUV emission channels following in progressive cooling order. A sharp increase in electron temperature tends to follow closely after the hot 131 Å signal confirming the impulsive nature of the process. A cooler process of growing emission measure follows more slowly. The Fourier power spectra of the hot 131 Å signals, when averaged over the five loops, present three scaling regimes with break frequencies near 0.1 min–1 and 0.7 min–1. The low frequency regime corresponds to 1/f noise; the intermediate indicates a persistent scaling process and the high frequencies show white noise. Very similar results are found for the energy dissipation in a 2D "hybrid" shell model of loop magneto-turbulence, based on reduced magnetohydrodynamics, that is compatible with nanoflare statistics. We suggest that such turbulent dissipation is the energy source for our loops

Distinguishing Species of Geoemyclid and Trionychid Turtles from Shell Fragments:Evidence from the Pleistocene at Niah Caves, Sarawak

Fragments of chelonian carapace and plastral dermal plates are well-represented from archaeological sites in the world's dry and wet tropics. However, although these bones are easily identified at an element level, few archaeological reports have explored the potential of using features of form and surface sculpturing as a way to refine that identification to genus or species. The ability to achieve such a refinement would benefit environmental and human subsistence strategy models alike. The objective of the current paper was to isolate recurrent and readily visible surface characteristics on the dermal plates from a selection of commonly occurring Southeast Asian hard- and soft-shelled turtles. Using these criteria, analysis is made of the chelonian assemblage from pre- and post-Last Glacial Maximum (LGM) cultural deposits in the West Mouth of Niah Cave. Copyright (C) 2009 John Wiley & Sons, Ltd.

Genome analysis of a simultaneously predatory and prey-independent, novel Bdellovibrio bacteriovorus from the River Tiber, supports in silico predictions of both ancient and recent lateral gene transfer from diverse bacteria

BACKGROUND: Evolution equipped Bdellovibrio bacteriovorus predatory bacteria to invade other bacteria, digesting and replicating, sealed within them thus preventing nutrient-sharing with organisms in the surrounding environment. Bdellovibrio were previously described as "obligate predators" because only by mutations, often in gene bd0108, are 1 in ~1x10(7) of predatory lab strains of Bdellovibrio converted to prey-independent growth. A previous genomic analysis of B. bacteriovorus strain HD100 suggested that predatory consumption of prey DNA by lytic enzymes made Bdellovibrio less likely than other bacteria to acquire DNA by lateral gene transfer (LGT). However the Doolittle and Pan groups predicted, in silico, both ancient and recent lateral gene transfer into the B. bacteriovorus HD100 genome.

RESULTS: To test these predictions, we isolated a predatory bacterium from the River Tiber- a good potential source of LGT as it is rich in diverse bacteria and organic pollutants- by enrichment culturing with E. coli prey cells. The isolate was identified as B. bacteriovorus and named as strain Tiberius. Unusually, this Tiberius strain showed simultaneous prey-independent growth on organic nutrients and predatory growth on live prey. Despite the prey-independent growth, the homolog of bd0108 did not have typical prey-independent-type mutations. The dual growth mode may reflect the high carbon content of the river, and gives B. bacteriovorus Tiberius extended non-predatory contact with the other bacteria present. The HD100 and Tiberius genomes were extensively syntenic despite their different cultured-terrestrial/freshly-isolated aquatic histories; but there were significant differences in gene content indicative of genomic flux and LGT. Gene content comparisons support previously published in silico predictions for LGT in strain HD100 with substantial conservation of genes predicted to have ancient LGT origins but little conservation of AT-rich genes predicted to be recently acquired.

CONCLUSIONS: The natural niche and dual predatory, and prey-independent growth of the B. bacteriovorus Tiberius strain afforded it extensive non-predatory contact with other marine and freshwater bacteria from which LGT is evident in its genome. Thus despite their arsenal of DNA-lytic enzymes; Bdellovibrio are not always predatory in natural niches and their genomes are shaped by acquiring whole genes from other bacteria.