Antibacterial activity in the sea urchin Echinus esculentus

The studies described in this thesis fall into two parts: 1. A survey of the normal bacterial flora of the common British sea urchin, Echinus esculentus, in comparison with the bacterial flora of seawater and sand from the same locality. 2. An investigation of the antibacterial activity in E. esculentus. This formed the main part of the work. In part 1, the normal bacterial flora, of the sea urchin was examined with isolates from the coelomic fluid, the peristomial membrane and the gut. Aerobic heterotrophic organisms from these sites were identified by a scheme based on that of Shewan, Hobbs and Hodgkiss (1960). The main genera identified were PseudomonasVibrio, Aeromonas, Flavobacterium, Acinetobacter and Moraxella. A few Gram-positive bacteria were also isolated. Of 188 urchins examined, two-thirds had sterile coelomic fluid and it is likely that organisms found there had been introduced by damage to the animal and do not form a permanent indigenous flora. In part 2, initial experiments showed that urchins were capable of clearing, within 24 h, large doses of marine bacteria which had been injected into the coelomic cavity. This indicated that sea urchins possess an efficient antibacterial mechanism. A procedure was developed to examine in vitro the coelomic fluid of sea urchins for antibacterial activity. As test bacterium in these experiments a marine pseudomonad, strain 111, was chosen bacause it produced characteristic black, agar-digesting colonies on marine 2216E agar which were not readily confused with contaminating bacteria. A non-bactericidal control fluid was included in all tests. This consisted of the boiled supernatant of coelomic fluid which was considered to be nutritionally and ionically equivalent to coelomic fluid, and which allowed growth of the test bacterium. Strain 111 incubated in coelomic fluid for 48 h WS'S usually reduced to less than 5% of its initial viable count, whereas in the control fluid the bacteria multiplied. Coelomocytes clot almost immediately when coelomic fluid is withdrawn from urchins but this appeared to have no effect on in vitro antibacterial activity. In vitro the fluid from all 188 urchins studied showed antibacterial activity. The activity was temperature-dependent (optimum

Genetic aspects of antibiotic resistance, haemolysin and bacteriocin production in enterococci

A previous survey of enterococci had identified five strains of Streptococcus faecalis (K55 and SB94) - two subspecies liquefaciens (K60 and K88) and one zymogenes (K87) - and two S. faecium strains (K46 and SB69) which were resistant to tetracycline and streptomycin but susceptible to gentamicin. All the S. faecalis strains and K46 were in addition resistant to erythromycin but only the S. faecium strains were penicillin and ampicillin resistant. The minimal inhibitory concentrations of a further six antibiotics were determined. These values confirmed that in S. faecalis strains, erythromycin resistance was accompanied by resistance to lincomycin and pristinamycin IA, a phenotype typical of macrolide - lincosamide - streptogramin B - type (MLS) antibiotics resistance. The erythromycin resistant K46 however, although resistant to lincomycin, was pristinamycin susceptible and so the basis of resistance is unknown. S. faecalis K60, K87 and SB94 were resistant to kanamycin and neomycin as was S. faecium K46 but all strains were susceptible to spectinomycin. The phenotypes were consistent with resistance mediated by enzymic modification of streptomycin with adenyltransferase (6) and of kanamycin and neomycin with phosphotransferase (3') (5")-III. Erythromycin and tetracycline resistances were expressed constitutively in all strains. Only one S. faecalis (K88) was found to be chloramphenicol resistant and as is typical of Gram-positive bacteria, resistance was inducible. The ability to produce bacteriocin was restricted to beta-haemolytic strain K87 and to strain SB94. Subsequent results indicated that strain K87 probably produced more than one bacteriocin, the activity of which was repressed in the parental strain but which, in derivatives, could be enhanced by the presence of streptomycin. Evidence for the location of resistance, haemolysin and bacteriocin genes was sought from study of the transfer characteristics and stability of markers and from examination of the plasmid content of parental strains and their derivatives. The well characterised S. faecalis subspecies zymogenes strain DS5 (Clewell et al., 1982b) was included for comparison in transfer and curing experiments. All the S. faecalis strains aggregated in response to a cell free filtrate of a plasmid free recipient strain JH2-1, indicating the presence of at least one conjugative plasmid although the low transfer frequencies of most resistance genes in broth matings suggested that response was not necessarily encoded by antibiotic resistance plasmids. Transfer of beta-haemolytic activity and all resistance markers was observed after broth matings but the range of transfer frequencies between strains was wide. Furthermore, the incidence of transfer could be variable particularly in the transfer of DS5 erythromycin resistance and all K87 antibiotic resistances which seemed to be dependent on the production of active donor bacteriocin. Matings of S. faecalis strains carried out on membrane filters were only marginally more efficient in terms of transfer frequencies but were superior with regard to reproducibility of transfer. No antibiotic resistance transfer from S. faecium donors was observed after broth matings and only SB69 tetracycline resistance transferred after filter mating at very low frequency. Several resistance determinants and those encoding β-haemolysin were found to be capable of retransfer indicative of association with genes specifying conjugative ability. Analysis of transconjugant phenotypes revealed that the tetracycline resistance gene of K55, the streptomycin resistance gene of K88 and β-haemolytic activities were always transferred alone but some resistance markers were usually co-transferred with other donor markers. (Abstract shortened by ProQuest.

Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifs

Common to all of the nitrate nitrite porter family are two conserved motifs in transmembrane helices 5 and 11 termed NS (nitrate signature) 1 and NS2. Although perfectly conserved substrate-interacting arginine residues have been described in transmembrane helices 2 and 8, the role of NSs has not been investigated. In the present study, a combination of structural modelling of NrtA (nitrate transporter from Aspergillus nidulans) with alanine scanning mutagenesis of residues within and around the NSs has been used to shed light on the probable role of conserved residues in the NSs. Models show that Asn 168 in NS1 and Asn 459 in NS2 are positioned approximately midway within the protein at the central pivot point in close proximity to the substrate-binding residues Arg 368 and Arg 87 respectively, which lie offset from the pivot point towards the cytoplasmic face. The Asn 168 /Arg 368 and Asn 459 /Arg 87 residue pairs are relatively widely separated on opposite sides of the probable substrate translocation pore. The results of the present study demonstrate the critical structural contribution of several glycine residues in each NS at sites of close helix packing. Given the relative locations of Asn 168 /Arg 368 and Asn 459 /Arg 87 pairs, the validity of the models and possible role of the NSs together with the substrate-binding arginine residues are discusse

Genetic characterization of morphologically variant strains of Paracoccidioides brasiliensis

Molecular characterization of Paracoccidioides brasiliensis variant strains that had been preserved under mineral oil for decades was carried out by random amplified polymorphic DNA analysis (RAPD). On P. brasiliensis variants in the transitional phase and strains with typical morphology, RAPD produced reproducible polymorphic amplification products that differentiated them. A dendrogram based on the generated RAPD patterns placed the 14 P. brasiliensis strains into five groups with similarity coefficients of 72%. A high correlation between the genotypic and phenotypic characteristics of the strains was observed. A 750 bp-RAPD fragment found only in the wild-type phenotype strains was cloned and sequenced. Genetic similarity analysis using BLASTx suggested that this RAPD marker represents a putative domain of a hypothetical flavin-binding monooxygenase (FMO)-like protein of Neurospora crassa.FiocruzBritish Council Progra

Methoxyflurane: a review with emphasis on its role in dental practice

Methoxyflurane was developed as an anaesthetic agent and introduced into clinical practice in 1960. It soon became evident that it possessed analgesic properties that other drugs did not. Due to toxicity concerns, it lost favour in general anaesthesia and had been largely abandoned by the late 1970's. The manufacturer withdrew it in 1999, and the Food and Drug Administration in the United States did not renew its license in 2005. It has also been withdrawn by the European Union. However, it continues to be used in Australasia, primarily as an inhaled self-administered analgesic by emergency services immediately following trauma. It has become attractive for use in dental practice, likely due to its effectiveness as an analgesic and its additional sedative qualities. Its acceptance is controversial as its use in dentistry is largely elective. Despite its good safety record in analgesic doses, adverse reactions have been recorded. Practitioners should be well aware of risks associated with its use before considering administration, and carefully assess whether or not there are equally good alternative options that do not the carry the same risks. Methoxyflurane is reviewed below with an emphasis on its use in dental practice.Angus Kingon, Tami Yap, Carmelo Bonanno, Paul Sambrook, and Michael McCulloug

Genetic characterization of morphologically variant strains of Paracoccidioides brasiliensis

Molecular characterization of Paracoccidioides brasiliensis variant strains that had been preserved under mineral oil for decades was carried out by random amplified polymorphic DNA analysis (RAPD). On P. brasiliensis variants in the transitional phase and strains with typical morphology, RAPD produced reproducible polymorphic amplification products that differentiated them. A dendrogram based on the generated RAPD patterns placed the 14 P. brasiliensis strains into five groups with similarity coefficients of 72%. A high correlation between the genotypic and phenotypic characteristics of the strains was observed. A 750 bp-RAPD fragment found only in the wild-type phenotype strains was cloned and sequenced. Genetic similarity analysis using BLASTx suggested that this RAPD marker represents a putative domain of a hypothetical flavin-binding monooxygenase (FMO)-like protein of Neurospora crassa.FiocruzBritish Council Progra

Entomopathogenic Fungus as a Biological Control for an Important Vector of Livestock Disease: The Culicoides Biting Midge

BACKGROUND: The recent outbreak of bluetongue virus in northern Europe has led to an urgent need to identify control measures for the Culicoides (Diptera: Ceratopogonidae) biting midges that transmit it. Following successful use of the entomopathogenic fungus Metarhizium anisopliae against larval stages of biting midge Culicoides nubeculosus Meigen, we investigated the efficacy of this strain and other fungi (Beauveria bassiana, Isaria fumosorosea and Lecanicillium longisporum) as biocontrol agents against adult C. nubeculosus in laboratory and greenhouse studies. METHODOLOGY/FINDINGS: Exposure of midges to 'dry' conidia of all fungal isolates caused significant reductions in survival compared to untreated controls. Metarhizium anisopliae strain V275 was the most virulent, causing a significantly decrease in midge survival compared to all other fungal strains tested. The LT(50) value for strain V275 was 1.42 days compared to 2.21-3.22 days for the other isolates. The virulence of this strain was then further evaluated by exposing C. nubeculosus to varying doses (10(8)-10(11) conidia m(-2)) using different substrates (horse manure, damp peat, leaf litter) as a resting site. All exposed adults were found to be infected with the strain V275 four days after exposure. A further study exposed C. nubeculosus adults to 'dry' conidia and 'wet' conidia (conidia suspended in 0.03% aq. Tween 80) of strain V275 applied to damp peat and leaf litter in cages within a greenhouse. 'Dry' conidia were more effective than 'wet' conidia, causing 100% mortality after 5 days. CONCLUSION/SIGNIFICANCE: This is the first study to demonstrate that entomopathogenic fungi are potential biocontrol agents against adult Culicoides, through the application of 'dry' conidia on surfaces (e.g., manure, leaf litter, livestock) where the midges tend to rest. Subsequent conidial transmission between males and females may cause an increased level of fungi-induced mortality in midges thus reducing the incidence of disease

Microbial degradation of furanic compounds: biochemistry, genetics, and impact

Microbial metabolism of furanic compounds, especially furfural and 5-hydroxymethylfurfural (HMF), is rapidly gaining interest in the scientific community. This interest can largely be attributed to the occurrence of toxic furanic aldehydes in lignocellulosic hydrolysates. However, these compounds are also widespread in nature and in human processed foods, and are produced in industry. Although several microorganisms are known to degrade furanic compounds, the variety of species is limited mostly to Gram-negative aerobic bacteria, with a few notable exceptions. Furanic aldehydes are highly toxic to microorganisms, which have evolved a wide variety of defense mechanisms, such as the oxidation and/or reduction to the furanic alcohol and acid forms. These oxidation/reduction reactions constitute the initial steps of the biological pathways for furfural and HMF degradation. Furfural degradation proceeds via 2-furoic acid, which is metabolized to the primary intermediate 2-oxoglutarate. HMF is converted, via 2,5-furandicarboxylic acid, into 2-furoic acid. The enzymes in these HMF/furfural degradation pathways are encoded by eight hmf genes, organized in two distinct clusters in Cupriavidus basilensis HMF14. The organization of the five genes of the furfural degradation cluster is highly conserved among microorganisms capable of degrading furfural, while the three genes constituting the initial HMF degradation route are organized in a highly diverse manner. The genetic and biochemical characterization of the microbial metabolism of furanic compounds holds great promises for industrial applications such as the biodetoxifcation of lignocellulosic hydrolysates and the production of value-added compounds such as 2,5-furandicarboxylic acid