On the true identity of Sergentomyia gemmea and description of a closely related species: Se. raynali n. sp.

Several species of Leishmania are responsible for leishmaniases in Thailand, although little is known about their transmission. Sergentomyia gemmea has been suspected several times to transmit Leishmania martiniquensis. Some captures carried out in Thailand and Lao People's Democratic Republic have emphasized the scarcity of Se. gemmea, comprising only 1% of the collected females. The sequencing of cytochrome B mtDNA of our specimens showed that our specimens are not grouped with other Se. gemmea previously deposited in GenBank. The latter are grouped with some Se. khawi and Se. hivernus that we processed in the present study. We suspect misidentifications and propose focusing on the most useful characters for identification of Se. gemmea based on the examination of type-specimens. The examination of the ascoids exhibiting anterior spurs is the most important one. However, we also describe Se. raynali n. sp. exhibiting comparable spurs but differing from Se. gemmea by its original cibarium. Finally, the vectorial role of Se. gemmea appears very questionable in the absence of new evidence.

Samplers for evaluation and quantification of ultra-low volume space sprays.

A field study was conducted to explore the suitability of 5 pesticide deposition samplers for airborne spray and ground deposits from ultra-low-volume (ULV) space sprays. Samplers included horizontally stretched stationary cotton ribbons at 2 heights, rotating ribbon, rotating Teflon slides, and filter paper. Slides were also used for droplet-size analysis. A set of 7 samplers of each type was placed at 1, 7, 15, 25, 40, 65, and 90 m from the spray line along the spray swath. Water and BVA13 oil with fluorescent dyes as tracers were sprayed with the use of a truck-mounted ULV sprayer at dusk and dawn. Results suggest that the horizontal and rotating cotton ribbons are best for quantification of airborne spray and filter paper is best for ground deposition collection. The rotating slide samplers only detected the BVA13 oil-based sprays.

The use of phages for the removal of infectious biofilms.

Biofilm formation occurs spontaneously on both inert and living systems and is an important bacterial survival strategy. In humans biofilms are responsible for many pathologies, most of them associated with the use of medical devices. A major problem of biofilms is their inherent tolerance to host defences and antibiotic therapies; there is therefore an urgent need to develop alternative ways to prevent and control biofilm-associated clinical infections. Several in vitro experiments have shown that phages are able to infect biofilm cells and that those phages inducing the production of depolymerases have an advantage since they can penetrate the inner layers of the biofilm by degrading components of the biofilm exopolymeric matrix. In practice clinically relevant biofilms and especially those associated with the use of medical devices can possibly be controlled for example by the topic application or the impregnation of the surface of the device with a phage solution. Another interesting approach has been the use of a phage encoding a phage polysaccharide lyase to treat Pseudomonas aeruginosa biofilms in cystic fibrosis patients by aerosol administration. All these strategies require prior identification of the phage and/or polysaccharide depolymerase capable of infecting the bacterial cells and degrading the polysaccharide within the biofilm, respectively. The biofilm organisms must therefore be isolated and screened against a bank of phages. This procedure is essential and raises important biotechnological challenges: the existence of a bank of phages well characterised (physiologically and genetically) whose efficacy in vivo has been tested and pharmacokinetics studied; the existence of economical and safe production protocols and purification methods (e.g. the presence of endotoxins in a phage preparation may compromise phage therapy). It is however important to consider the fact that the chances of getting a specific phage with a high lytic capability and preferential expressing a relevant exopolymer degrading enzyme is likely to be low. Genetically engineered phages can play an important role in this process. Phages can be genetically manipulated to alter their host range and to induce the production of depolymerases. It is therefore important to reinforce the application of synthetic biology to engineer phages able to efficiently degrade medical biofilms. It is also important to develop efficient methods of phage delivery and to study "in vivo" the phage performance against biofilms. It is still not clear how effective the biofilm can be in protecting the phages against the immune system. Efficient and economic phage production and purification protocols need also to be addressed before one can hope to use phage treatment to prevent or control infectious biofilms.

Antagonistic interactions amongst bacteriocin-producing enteric bacteria in dual species biofilms.

AIMS: The objective of this study was to investigate the antagonistic interactions between bacteriocin-producing enteric bacteria in dual species biofilms and the interspecies interactions correlated with sensitivity to biocides. METHODS AND RESULTS: When compared with their single species counterparts, the dual species biofilms formed by bacteriocin-producing strains exhibited a decrease in biofilm size and an increase in sensitivity to the antimicrobial agents hypochlorite, triclosan and benzalkonium chloride. The five dual species biofilms studied all resulted in biofilms containing a mixture of the two strains. This was attributed to the spatial distribution of cells within the biofilm, with each strain forming its own microcolonies. The production of a bacteriocin also gave a strain a competitive advantage when interacting with a bacteriocin-sensitive strain within a biofilm, both in gaining a foothold in a new environment and in preventing the colonization of a potential competitor into a pre-established biofilm. CONCLUSIONS: It was concluded that bacteriocins might be used specifically for interacting with competing strains within a biofilm, as opposed to a planktonic, environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike planktonically grown bacteriocin-producing populations, where one strain will always be out-competed, bacteriocin-producing and bacteriocin-sensitive strains can coexist in biofilm communities, clearly demonstrating major differences between biofilm and planktonic competition. This paper highlights the importance of bacteriocin production in the development of biofilm communities.

The biofilm matrix--an immobilized but dynamic microbial environment.

The biofilm matrix is a dynamic environment in which the component microbial cells appear to reach homeostasis and are optimally organized to make use of all available nutrients. The major matrix components are microbial cells, polysaccharides and water, together with excreted cellular products. The matrix therefore shows great microheterogeneity, within which numerous microenvironments can exist. Although exopolysaccharides provide the matrix framework, a wide range of enzyme activities can be found within the biofilm, some of which will greatly affect structural integrity and stability.

Exopolysaccharides in biofilms, flocs and related structures.

In biofilms, flocs and similar multispecies microbial communities, exopolysaccharides (EPSs) are always present, frequently as the major component other than water. The EPSs vary widely in their composition, structure and properties and thus it is impossible to generalise about their contribution to biofilm or floc structure. Relatively few of the polymers obtained from biofilms and flocs have been adequately purified and analysed but such evidence as is so far available suggests that the polysaccharides closely resemble those synthesised by the corresponding planktonic bacteria. From a knowledge of the physical properties of these, it is now possible to present a reasonably accurate picture of some of the factors which they may contribute to the structure and stability of complex aggregates of micro-organisms in biofilms and flocs.

Structural determination of the acidic exopolysaccharide produced by a Pseudomonas sp. strain 1.15.

Pseudomonas strain 1.15 was isolated from a freshwater biofilm and shown to produce considerable amounts of an acidic polysaccharide which was investigated by methylation analysis, NMR spectroscopy and ionspray mass spectrometry (ISMS). The polysaccharide was depolymerised by a bacteriophage-associated endoglucosidase and by autohydrolysis, and the resulting oligosaccharides were investigated by NMR spectroscopy and mass spectrometry. The resulting data showed that the parent repeating unit of the 1.15 exopolysaccharide (EPS) is a branched hexasaccharide. The main chain is constituted of the trisaccharide -->4)-alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->3)-beta-D-Glcp- (1--> and the side chain alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->3)-alpha-D-Galp-(1-->is linked to O-3 of the first Fuc residue. The terminal non-reducing Gal carries a 1-carboxyethylidene acetal in the R configuration at the positions 4 and 6. Of the four different O-acetyl groups present in non-stoichiometric amounts, two were established to be on O-2 of the 3-linked Gal and on O-2 of the 4-linked Fuc.

Bacteriophage and associated polysaccharide depolymerases--novel tools for study of bacterial biofilms.

Bacteriophage for three representative strains of Gram-negative biofilm bacteria have proved to be of widespread occurrence. Lytic bacteriophage have been isolated from local sewage for the bacterium 1.15, an exopolysaccharide (EPS)-producing pseudomonad found originally as a component of biofilms in a local river, and for two Enterobacter agglomerans strains from industrial biofilms. Representative examples of all three bacteriophage possess a relatively low burst size and on solid media, exhibit very large plaques surrounded by a wide halo (5-20 mm) indicative of polysaccharide depolymerase action. The bacteriophage are thus similar to other viruses for EPS-producing bacteria in inducing the synthesis of enzymes degrading the polymers which occlude the bacterial cell surface. In each preparation, the polysaccharase activity was associated both with sedimented phage particles and with the supernate of bacterial lysates. The enzymes have been partially purified and used to prepare polysaccharide digests in which the major products from each polysaccharide are the presumed repeat units of the polymers or oligomers of these. The soluble phage enzymes each degrade their substrate by acting as endo-glycanohydrolases. The phage and their associated enzymes thus provide very useful highly specific tools for studies of biofilms incorporating the bacterial host strains. Their potential applications in studies on bacterial biofilms are discussed.

Novel and established applications of microbial polysaccharides.

Microbial exopolysaccharides such as xanthan and dextran have been commercial products for many years; the search for new gelling agents has yielded gellan. Exopolysaccharides have many other novel properties to offer, and the discovery of immune modulation and tumouristasis by beta-D-glucans, and the use of bacterial cellulose in audio membranes and of hyaluronic acid in cosmetics provide some novel applications. Semisynthetic polymers and polysaccharides as sources of oligosaccharides and as enzyme substrates in the determination of enzyme specificity should further increase the interest in these macromolecules.

The role of exopolysaccharides in dual species biofilm development.

A plasmid encoding the green fluorescent protein (GFP) of Aequorea victoria was transformed into a biofilm-forming strain of Enterobacter agglomerans originally isolated from an industrial environment. The transformed strain, EntGFP, could then be identified in dual species biofilms by direct visualization, plate counts and quantitiative fluorescence measurements. A variety of cell constituents and products may be involved in the adhesion and accumulation process and exopolysaccharides (EPS) represent one of these factors. The involvement of EPS in the initial adhesion events and the role in dual species biofilm development was investigated. Cells of EntGFP and Klebsiella pneumoniae Gl interact forming biofilms more successfully in a mixture than in isolation. The co-resistance results in enhanced biofilm formation and increased resistance to disinfection. Microscopic examination showed that the two species were often closely juxtaposed in microcolonies, suggesting the interactions involve surface-associated macromolecules. Fluorescence was used to measure the adhesion of EntGFP cells to Kleb, pneumoniae Gl (Gl) EPS. The results showed EntGFP adhered better to Gl EPS that Ent EPS. Polysaccharde depolymerases isolated from a bacteriophage for Ent. agglomerans were used to degrade Ent EPS specifically. Following polysaccharase treatment, the adhaesion of EntGFP to Gl cells was reduced. This suggests both types of EPS mediate adhesion. The two types of EPS were dissolved in dimethylsulphoxide and when mixed, their viscosity increased, reaching a maximum after ∼+40 min. This may partially explain the increased protection of dual species biofilms from disinfectants. The depolymerases were used to treat dual species biofilms and this resulted in the effective removal of both species from the surface. This may suggest Ent contributes more EPS to the biofilm matrix. The EPS play an important role in EntGFP and Gl dual species biofilm formation both as adhesins and as the EPS interact, changing their physical properties.

The structure of the exopolysaccharide produced by the halophilic Archaeon Haloferax mediterranei strain R4 (ATCC 33500).

The halophilic Archaeon Haloferax mediterranei exudes into the growth medium a high molecular weight sulfated polysaccharide. The structure of the repeating unit of this polymer was determined by a combination of glycose, methylation, and sulfate analysis, periodate oxidation, and 1D and 2D NMR spectroscopic analysis of the native and periodate-oxidised/reduced polysaccharides. The location of the sulfate group was established from the 1H and 13C NMR data. The structure of the repeating unit of the polysaccharide may be written as [formula: see text]

Polysaccharide lyases.

Polysaccharide lyases are the products of various microorganisms, bacteriophage and some eukaryotes. All such enzymes cleave a hexose-1,4-alpha- or beta-uronic acid sequence by beta-elimination. They are in some examples, the only known type of enzymes degrading their polyanionic substrates. Although only a small number of these enzymes have been exhaustively studied, the pectin lyases of bacterial origin have proved to be of interesting crystal structure containing a parallel beta-helix domain. Alginate and heparin lyases may yield products with biotechnological potential.

Novel bioemulsifiers from microorganisms for use in foods.

The main objective of this study was to test the range of microorganisms for production of extracellular, high molecular weight emulsifiers for potential use in foods. A standard emulsification assay developed specifically for assessing food emulsifiers was used to examine 24 extracellular microbial products from bacteria, yeasts and algae. Of the 24 products tested, nine had emulsification ability that was as good as and eight had emulsifying properties that were better than those of the commonly used food emulsifiers gum arabic and carboxymethylcellulose. The eight good producer organisms included the yeasts Candida utilis, Candida valida, Hansenula anomala, Rhodospiridium diobovatum and Rhodotorula graminis, the red alga Porphiridium cruentum, and the bacteria Klebsiella spp. and Acinetobacter calcoaceticus. Of these, C. utilis was selected for further study due to the excellent emulsification properties of its extracellular products and food-grade status of the organism. Crude preparations of the bioemulsifier from C. utilis exhibited low viscosity and had a carbohydrate content of over 80%. Preliminary trials showed that the bioemulsifier from this organism had potential for use in salad cream.

Gellan lyases--novel polysaccharide lyases.

A number of bacterial strains capable of degrading the bacterial exopolysaccharide gellan have been isolated by standard enrichment procedures. They include several pink-pigmented Gram-negative rod-shaped bacteria. A red-pigmented Gram-positive bacillus earlier found to degrade the exopolysaccharide xanthan from Xanthomonas campestris also showed slight gellanase activity. All the Gram-negative bacteria are non-fermentative, motile and amylase-producing. The gellan degradation in each case is due to eliminase-type enzymes (lyases) which appear to be extracellular enzymes cleaving the sequence... beta-D-glucosyl-(1-->4)-beta-D-glucuronosyl... in the tetrasaccharide repeat unit of the substrate polysaccharides. Although in some isolates these enzymes appear to be exo-acting, it appears from the loss in viscosity of the alternative substrate deacetylated rhamsan that they are predominantly endoenzymes. The enzyme activity is inducible: it is almost absent from glucose-grown cells. Associated with the 'gellanase' activity, all the Gram-negative bacterial isolates possess intracellular alpha-L-rhamnosidase and beta-D-glucosidase activities apparently located in the periplasm. The enzymes are highly specific and fail to cause significant degradation of most of the other bacterial exopolysaccharides which have been shown to be structurally related to gellan. As well as acting on gellan, they exert similar degradative activity against the chemically deacylated form of polysaccharide S194 (rhamsan gum), which is effectively a gentiobiosylated form of gellan. The enzymes only have relatively slight activity against the natural, acylated gellan-like polysaccharides from the bacteria now designated as strains of Sphingomonas paucimobilis.

Structure-function relationships in microbial exopolysaccharides.

Sufficient well-characterized microbial exopolysaccharides are now available to permit extensive studies on the relationship between their chemical structure and their physical attributes. This is seen even in homopolysaccharides with relatively simple structures but is more marked when greater differences in structure exist, as are found in several heteropolysaccharides. The specific and sometimes unique properties have, in the case of several of these polymers, provided a range of commercial applications. The existence of "families" of structurally related polysaccharides also indicates the specific role played by certain structures and substituents; the characteristics of several of these microbial polysaccharide families will be discussed here. Thus, microbial exopolysaccharides frequently carry acyl groups which may profoundly affect their interactive properties although these groups often have relatively little effect on solution viscosity. Xanthan with or without acylation shows marked differences in synergistic gelling with plant gluco- and galacto-mannans, although the polysaccharides with different acylation patterns show similar viscosity. Similarly "gelrite" from the bacterium originally designated as Auromonas (Pseudomonas)elodea is of greater potential value after deacetylation, when it provides a valuable gelling agent, than it is as a viscosifier in the natural acylated form. The Klebsiella type 54 polysaccharide only forms gels when it, too, has been chemically deacetylated to give a structure equivalent to the Enterobacter XM6 polymer. Both these polysaccharides form gels due to the enhanced interaction with cations following deacylation and to the conformation adopted after removal of the acyl groups. Recent work in our laboratory suggests that deacetylation of certain bacterial alginates also significantly increases ion binding by these polysaccharides, making them more similar in their properties to algal alginates even although the alginates from some Pseudomonas species lack poly-L-guluronic acid sequences. The existence within families of polysaccharides of types in which monosaccharides are altered within a specific structure, or with varying side-chains, also gives an indication of the way in which such substituents affect the physical properties of the polymers in aqueous solution.

Influence of acetyl and pyruvate substituents on the solution properties of xanthan polysaccharide.

Xanthan, an exocellular polysaccharide produced by the plant pathogenic bacterium Xanthomonas campestris has been the subject of considerable interest in recent years because of its unusual rheological properties in solution ('weak gel') and consequent range of applications. The polymer consists of a cellulosic backbone with trisaccharide side chains linked to alternate backbone residues; acetyl and pyruvate substituents are carried in variable amounts on these side chains. In this study a series of xanthans differing in the percentage of substituent groups and in molecular weight range have been prepared by culturing a variety of different strains of X. campestris. All of the xanthans have been characterized by a range of physicochemical techniques. In particular, the intrinsic viscosities at low shear rates, and at a range of ionic strengths, have been determined and the geometric persistence lengths evaluated by the Smidsrød-Haug method. Intensity light scattering measurements have been made using the procedure of Coviello and co-workers to promote molecular dispersion. Despite significant differences in the acetyl and pyruvate contents, the molecular weight vs mean square radius behaviour of our samples did not differ substantially from each other or from those reported for other xanthan samples in the literature. The persistence length, determined by the method of Schmidt et al. (120 +/- 8 nm) was also, within experimental error, the same for all the samples measured. These values differed considerably from those calculated from the ionic strength dependence of intrinsic viscosity (the Smidsrød-Haug method) was reported by Tinland and Rinaudo and calculated for our samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term storage of xanthan in seawater at elevated temperature: physical dimensions and chemical composition of degradation products.

Commercial xanthan and xanthan from Xanthomonas strain 646 produced in the laboratory have been subjected to heat treatment for various periods of time in oxygen depleted, high salinity, aqueous solutions. Both the viscosity and the carbohydrate content decreased with increasing incubation time at a specified temperature. The losses increased with increasing temperature. Data from electron micrographs and dialysable sugar content indicate that random cleavage of the double-stranded xanthan chain is the main mechanism responsible for the decreasing viscosity. Removal of pyruvate and acetate substituents on the side chains was apparently not related to the change in physical dimensions. The mannose/glucose ratio in the non-diffusible fraction decreased with incubation time, apparently not related to change in physical dimensions. Electron micrographs showed that one of the samples appeared as highly aggregated in the native condition. After 1 month at 80 degrees C, we observed that the aggregates had dissolved and that the viscosity had increased fivefold. This suggests that heat treatment can be used to avoid microgels and to obtain higher viscosifying power of the native xanthan.

Monoclonal antibodies reacting with the exopolysaccharide xanthan from Xanthomonas campestris.

We have prepared murine hybridomas secreting monoclonal antibodies against the exopopolysaccharide xanthan from Xanthomonas campestris pv. campestris 646 after fusing NSO myeloma cells and spleen cells from BALB/c mice immunized with xanthan. Four hybridomas, secreting antibodies designated A6 (IgM kappa), B3 (IgM kappa), D1 (IgM kappa), and D3 (IgG2A kappa), were selected for further studies. All antibodies reacted with a range of different xanthans. Competition studies using variants of the exopopolysaccharide as competitors suggested that specificity was mainly against the side-chain. One of the antibodies (B3) appeared to require the fully acylated side-chain with the pyruvylated terminal mannose as the immunodominant part. The three others were assumed to be directed against the nonsubstituted trisaccharide with the inner mannose-glucuronic acid being immunodominant. None of the antibodies reacted with cellulose (the xanthan backbone). Using immunoblotting techniques on nitrocellulose paper both a mixture of monoclonal antibodies, and also polyclonal ascitic fluid, could detect xanthan quantities of approximately 0.1 microgram.