Changes in equine hindgut bacterial populations during oligofructose-induced laminitis

In the horse, carbohydrate overload is thought to play an integral role in the onset of laminitis by drastically altering the profile of bacterial populations in the hindgut. The objectives of this study were to develop and validate microbial ecology methods to monitor changes in bacterial populations throughout the course of experimentally induced laminitis and to identify the predominant oligofructose-utilizing organisms. Laminitis was induced in five horses by administration of oligofructose. Faecal specimens were collected at 8 h intervals from 72 h before to 72 h after the administration of oligofructose. Hindgut microbiota able to utilize oligofructose were enumerated throughout the course of the experiment using habitat-simulating medium. Isolates were collected and representatives identified by 16S rRNA gene sequencing. The majority of these isolates collected belonged to the genus Streptococcus, 91% of which were identified as being most closely related to Streptococcus infantarius ssp. coli. Furthermore, S. infantarius ssp. coli was the predominant oligofructose-utilizing organism isolated before the onset of lameness. Fluorescence in situ hybridization probes developed to specifically target the isolated Streptococcus spp. demonstrated marked population increases between 8 and 16 h post oligofructose administration. This was followed by a rapid population decline which corresponded with a sharp decline in faecal pH and subsequently lameness at 24-32 h post oligofructose administration. This research suggests that streptococci within the Streptococcus bovis/equinus complex may be involved in the series of events which precede the onset of laminitis in the horse.

Construction and analysis of a metagenomic library from an enhanced biological phosphorus removal biomass

The enhanced biological phosphorus removal (EBPR) process is regularly used for the treatment of wastewater, but suffers from erratic performance. Successful EBPR relies on the growth of bacteria called polyphosphate-accumulating organisms (PAOs), which store phosphorus intracellularly as polyphosphate, thus removing it from wastewater. Metabolic models have been proposed which describe the measured chemical transformations, however genetic evidence is lacking to confirm these hypotheses. The aim of this research was to generate a metagenomic library from biomass enriched in PAOs as determined by phenotypic data and fluorescence in situ hybridisation (FISH) using probes specific for the only described PAO to date, Candidatus Accumulibacter phosphatis. DNA extraction methods were optimised and two fosmid libraries were constructed which contained 93 million base pairs of metagenomic data. Initial screening of the library for 16S rRNA genes revealed fosmids originating from a range of non-pure-cultured wastewater bacteria. The metagenomic libraries constructed will provide the ability to link phylogenetic and metabolic data for bacteria involved in nutrient removal from wastewater. Keywords DNA extraction; EBPR; metagenomic library; 16S rRNA gene.

Detection and discrimination of herpes simplex viruses, Haemophilus ducreyi, Treponema pallidum, and Calymmatobacterium (Klebsiella) granulomatis from genital ulcers

Background. Genital ulcer disease (GUD) is commonly caused by pathogens for which suitable therapies exist, but clinical and laboratory diagnoses may be problematic. This collaborative project was undertaken to address the need for a rapid, economical, and sensitive approach to the detection and diagnosis of GUD using noninvasive techniques to sample genital ulcers. Methods. The genital ulcer disease multiplex polymerase chain reaction (GUMP) was developed as an inhouse nucleic acid amplification technique targeting serious causes of GUD, namely, herpes simplex viruses (HSVs), Haemophilus ducreyi, Treponema pallidum, and Klebsiella species. In addition, the GUMP assay included an endogenous internal control. Amplification products from GUMP were detected by enzyme linked amplicon hybridization assay (ELAHA). Results. GUMP-ELAHA was sensitive and specific in detecting a target microbe in 34.3% of specimens, including 1 detection of HSV-1, three detections of HSV-2, and 18 detections of T. pallidum. No H. ducreyi has been detected in Australia since 1998, and none was detected here. No Calymmatobacterium ( Klebsiella) granulomatis was detected in the study, but there were 3 detections during ongoing diagnostic use of GUMP-ELAHA in 2004 and 2005. The presence of C. granulomatis was confirmed by restriction enzyme digestion and nucleotide sequencing of the 16S rRNA gene for phylogenetic analysis. Conclusions. GUMP-ELAHA permitted comprehensive detection of common and rare causes of GUD and incorporated noninvasive sampling techniques. Data obtained by using GUMP-ELAHA will aid specific treatment of GUD and better define the prevalence of each microbe among at-risk populations with a view to the eradication of chancroid and donovanosis in Australia.

Meganema perideroedes gen. nov., sp nov., a filamentous alphaproteobacterium from activated sludge

An industrial wastewater treatment plant at Grindsted, Denmark, has suffered from bulking problems for several years caused by filamentous bacteria. Five strains were isolated from the sludge by micromanipulation, Phylogenetic analysis of the 16S rRNA gene sequences showed that the strains formed a monophyletic cluster in the Alphaproteobacteria, and they were phenotypically different from their closest relatives and from all hitherto known filamentous bacteria described (closest relative Brevundimonas vesicularis ATCC 11426(T), 89(.)8% sequence similarity). In pure culture, the cells (1(.)5-2(.)0 mu m) in filaments are Gram-negative and contain polyphosphate and polyhydroxyalkanoates. The optimum temperature for growth is 30 degrees C and the strains grow in 2 % NaCl and are oxidase- and catalase-positive. Ubiquinone 10 is the major quinone. The major fatty acid (C-18: 1 omega 7c) and smaller amounts of unsaturated fatty acids, 3-hydroxy fatty acids with a chain length of 16 and 18 carbon atoms and small amounts of 10-methyl-branched fatty acids with 18 carbon atoms (C-19: 0 10-methyl) affiliated the strains with the Methylobacterium/Xanthobacter group in the Alphaproteobacteria. The G + C content of the DNA is 42(.)9 mol% (for strain Gr1(T)). The two most dissimilar isolates by 16S rRNA gene comparison (Gr1(T) and Gr10; 97(.)7 % identical) showed 71(.)5 % DNA-DNA relatedness. Oligonucleotide probes specific for the pure cultures were designed for fluorescence in situ hybridization and demonstrated that two filamentous morphotypes were present in the Grindsted wastewater treatment plant. It is proposed that the isolates represent a new genus and species, Meganema perideroedes gen. nov., sp. nov. The type strain of Meganema perideroedes is strain Gr1(T) (=DSM 15528(T) =ATCC BAA-740(T)).

The detection of environmental autoinducing peptide quorum-sensing genes from an uncultured Clostridium sp in landfill leachate reactor biomass

Aims: To elucidate whether a dominant uncultured clostridial (Clostridium thermocellum-like) species in an environmental sample (landfill leachate), possesses an autoinducing peptide (AIP) quorum-sensing (QS) gene, although it may not be functional. Methods and Results: A modified AIP accessory gene regulator (agr)C PCR protocol was performed on extracted DNA from a landfill leachate sample (also characterized by 16S rRNA gene cloning) and the PCR products were cloned, sequenced and phylogenetically analysed. It appeared that two agrC gene phylotypes existed, most closely related to the C. thermocellum agrC gene, differing by only 1 bp. Conclusions: It is possible to specifically identify and characterize the agrC AIP QS gene from uncultured Firmicutes (C. thermocellum-like) bacteria derived from environmental (landfill leachate) sample. Significance and Impact of the Study: This is the first successful attempt at identifying AIP QS genes from a cellulolytic environment (landfill). The agrC gene was identified as being most closely related to the C. thermocellum agrC gene, the same bacterium identified as being dominant, according to 16S rRNA gene cloning and subsequently fluorescence in situ hybridization analyses, in the same biomass.

Isolates of 'Candidatus Nostocoida limicola' Blackall et al. 2000 should be described as three novel species of the genus Tetrasphaera, as Tetrasphaera jenkinsii sp nov., Tetrasphaera vanveenii sp nov and Tetrasphaera veronensis sp nov.

Despite differences in their morphologies, comparative analyses of 16S rRNA gene sequences revealed high levels of similarity (> 94 %) between strains of the filamentous bacterium 'Candidatus Nostocoida limicola' and the cocci Tetrasphaera australiensis and Tetrasphaera japonica and the rod Tetrasphaera elongata, all isolated from activated sludge. These sequence data and their chemotaxonomic characters, including cell wall, menaquinone and lipid compositions and fingerprints of their 16S-23S rRNA intergenic regions, support the proposition that these isolates should be combined into a single genus containing six species, in the family Intrasporangiaceae in the Actinobacteria. This suggestion receives additional support from DNA-DNA hybridization data and when partial sequences of the rpoC1 gene are compared between these strains. Even though few phenotypic characterization data were obtained for these slowly growing isolates, it is proposed, on the basis of the extensive chemotaxonomic and molecular evidence presented here, that 'Candidatus N. limicola' strains Ben 17, Ben 18, Ben 67, Ben 68 and Ben 74 all be placed into the species Tetrasphaera jenkinsii sp. nov. (type strain Ben 74(T) = DSM 17519(T) = NCIMB 14128(T)), 'Candidatus N. limicola' strain Ben 70 into Tetrasphaera vanveenii sp. nov. (type strain Ben 70(T) = DSM 17518(T) = NCIMB 14127(T)) and 'Candidatus N. limicola' strains Ver 1 and Ver 2 into Tetrasphaera veronensis sp. nov. (type strain Ver 1(T) = DSM 17520(T) = NCIMB 14129(T)).

A spirochete isolated from a case of severe virulent ovine foot disease is closely related to a Treponeme isolated from human periodontitis and bovine digital dermatitis

The isolation of spirochetes from severe ovine foot disease has been reported recently by our research group. In this study we describe the preliminary classification of this spirochete based on nucleotide sequence analysis of the PCR-amplified 16S rRNA gene. Phylogenetic analysis of this sequence in comparison with other previously reported 16S rRNA gene sequences showed that the spirochete belonged to the treponemal phylotype Treponema vincentii which has been associated with bovine digital dermatitis and human periodontal disease. Further work is required to define the common virulence determinants of these closely related treponemes in the aetiology of these tissue destructive diseases.

Spirochaetes and other bacterial species associated with bovine digital dermatitis

The 16S rRNA genes from spirochaetes associated with digital dermatitis of British cattle were amplified by polymerase chain reaction from digital dermatitis lesion biopsies using one universal and one treponeme-specific primer. Two treponemal sequences were identified both of which shared a high degree of homology with the oral pathogen Treponema denticola (98%). Two further 16S rRNA gene sequences were obtained and shared similarity to Bacteroides levii (99%) and Mycoplasma hyopharyngis (98%). Polymerase chain reaction with T. denticola-specific primers amplified a potential virulence gene from digital dermatitis lesions which shared a high degree of homology to the 46-kDa haemolysin gene of T. denticola. The significance of the presence of organisms in digital dermatitis lesions of the bovine foot which are closely related to oral pathogens is discussed.

A new phylogenetic group of Propionibacterium acnes

Immunofluorescence microscopy-based identification of presumptive Propionibacterium acnes isolates, using the P. acnes-specific mAb QUBPa3, revealed five organisms with an atypical cellular morphology. Unlike the coryneform morphology seen with P. acnes types I and II, these isolates exhibited long slender filaments (which formed large tangled aggregates) not previously described in P. acnes. No reaction with mAbs that label P. acnes types IA (QUBPa1) and II (QUBPa2) was observed. Nucleotide sequencing of the 16S rRNA gene (1484 bp) revealed the isolates to have between 99.8 and 99.9 % identity to the 16S rRNA gene of the P. acnes type IA, IB and II strains NCTC 737, KPA171202 and NCTC 10390, respectively. Analysis of the recA housekeeping gene (1047 bp) did reveal, however, a greater number of conserved nucleotide polymorphisms between the sequences from these isolates and those from NCTC 737 (98.9 % identity), KPA171202 (98.9 % identity) and NCTC 10390 (99.1 % identity). Phylogenetic investigations demonstrated that the isolates belong to a novel recA cluster or lineage distinct from P. acnes types I and II. We now propose this new grouping as P. acnes type III. The prevalence and clinical importance of this novel recA lineage amongst isolates of P. acnes remains to be determined.

A molecular basis for polyketide biosynthesis in marine dinoflagellates with emphasis on Karenia brevis

Polyketides derived from dinoflagellates are among the most complex and unique structures identified to date. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). No studies of the biosynthesis of dinoflagellate derived polyketides at the genomic level have been reported to date. Nine strains representing seven different species of dinoflagellates were screened for the presence of type I and type II polyketide synthases (PKS) by PCR and RT-PCR. Seven of the nine strains yielded products that were homologous with known and putative type I polyketide synthases. In each case, the presence of a PKS gene was correlated with the presence of bacteria in the cultures as identified by amplification of the bacterial 16S rRNA gene. However, residual phylogenetic signals, resistance to methylation sensitive restriction enzymes and the lack of hybridization to bacterial isolates support a dinoflagellate origin for most of these genes. ^ A more detailed analysis of Karenia brevis, a toxic marine dinoflagellate endemic to the Gulf of Mexico, also supports the hypothesis that dinoflagellates have polyketide synthase genes. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. PKS encoding genes amplified from K. brevis culture were found to be similar to PKS genes from the closely related protist, Cryptosporidium parvum. This suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. This dissertation reports the localization of these PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate. ^

Changes in Community Structure of Sediment Bacteria Along the Florida Coastal Everglades Marsh–Mangrove–Seagrass Salinity Gradient

Community structure of sediment bacteria in the Everglades freshwater marsh, fringing mangrove forest, and Florida Bay seagrass meadows were described based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) patterns of 16S rRNA gene fragments and by sequencing analysis of DGGE bands. The DGGE patterns were correlated with the environmental variables by means of canonical correspondence analysis. There was no significant trend in the Shannon–Weiner index among the sediment samples along the salinity gradient. However, cluster analysis based on DGGE patterns revealed that the bacterial community structure differed according to sites. Not only were these salinity/vegetation regions distinct but the sediment bacteria communities were consistently different along the gradient from freshwater marsh, mangrove forest, eastern-central Florida Bay, and western Florida Bay. Actinobacteria- and Bacteroidetes/Chlorobi-like DNA sequences were amplified throughout all sampling sites. More Chloroflexi and members of candidate division WS3 were found in freshwater marsh and mangrove forest sites than in seagrass sites. The appearance of candidate division OP8-like DNA sequences in mangrove sites distinguished these communities from those of freshwater marsh. The seagrass sites were characterized by reduced presence of bands belonging to Chloroflexi with increased presence of those bands related to Cyanobacteria, γ-Proteobacteria, Spirochetes, and Planctomycetes. This included the sulfate-reducing bacteria, which are prevalent in marine environments. Clearly, bacterial communities in the sediment were different along the gradient, which can be explained mainly by the differences in salinity and total phosphorus.

Multiplex PCR for Assessment of Tetracycline Resistance (tet) Genes in Antibiotic-Resistant Soil Bacteria and the Ecological Implications

Antibiotics are becoming increasingly prevalent in bacterial communities due to clinical and agricultural misuse and overuse in their environment. As exposure increases, so does the incidence of microbial resistance. Such is the case with bacterial resistance to tetracyclines, a phenotype often acquired through the horizontal gene transfer of tet genes between bacteria. The objective of this project was to analyze the bacterial diversity of tet resistance genes in soil from Miami-Dade County. Bacterial isolates were Gram-stained and the Kirby-Bauer antibiotic disk diffusion test was performed to determine each bacterium’s degree of resistance. The 16S rRNA gene from antibiotic-resistant isolates was amplified by PCR and sequenced to identify the isolates. All isolates’ tet genes were amplified by multiplex PCR, sequenced, and compared. Among eight isolates, three distinct species were positively identified based on their 16S rRNA sequences and four distinct tet genes were identified, though all tested susceptible to tetracycline via the Kirby-Bauer test. This project clarifies some aspects of the ecology of antibiotic resistance genes, their natural ecological function and the potential for the expansion of intrinsic multi-antibiotic resistance into new ecosystems and/or hosts.

(Table 1) Individual codes, haplotype codes, GenBank accession numbers for DNA sequences and sample locality of the analysed Betamorpha fusiformis specimens from the ANDEEP III expedition

Based on our current knowledge about population genetics, phylogeography and speciation, we begin to understand that the deep sea harbours more species than suggested in the past. Deep-sea soft-sediment environment in particular hosts a diverse and highly endemic invertebrate fauna. Very little is known about evolutionary processes that generate this remarkable species richness, the genetic variability and spatial distribution of deep-sea animals. In this study, phylogeographic patterns and the genetic variability among eight populations of the abundant and widespread deep-sea isopod morphospecies Betamorpha fusiformis [Barnard, K.H., 1920. Contributions to the crustacean fauna of South Africa. 6. Further additions to the list of marine isopods. Annals of the South African Museum 17, 319-438] were examined. A fragment of the mitochondrial 16S rRNA gene of 50 specimens and the complete nuclear 18S rRNA gene of 7 specimens were sequenced. The molecular data reveal high levels of genetic variability of both genes between populations, giving evidence for distinct monophyletic groups of haplotypes with average p-distances ranging from 0.0470 to 0.1440 (d-distances: 0.0592-0.2850) of the 16S rDNA, and 18S rDNA p-distances ranging between 0.0032 and 0.0174 (d-distances: 0.0033-0.0195). Intermediate values are absent. Our results show that widely distributed benthic deep-sea organisms of a homogeneous phenotype can be differentiated into genetically highly divergent populations. Sympatry of some genotypes indicates the existence of cryptic speciation. Flocks of closely related but genetically distinct species probably exist in other widespread benthic deep-sea asellotes and other Peracarida. Based on existing data we hypothesize that many widespread morphospecies are complexes of cryptic biological species (patchwork hypothesis).

Inter-Species Microbial Sharing in Rural Madagascar: A Study of Environmental Influences on the Skin Microbiome

The skin is home to trillions of microbes, many of which are recently implicated in immune system regulation and various health conditions (33). The skin is continuously exposed to the outside environment, inviting microbial transfer between human skin and the people, animals, and surfaces with which an individual comes into contact. Thus, the aim of this study is to assess how different environmental exposures influence skin microbe communities, as this can strengthen our understanding of how microbial variation relates to health outcomes. This study investigated the skin microbial communities of humans and domesticated cattle living in rural Madagascar. The V3 region of the 16S rRNA gene was sequenced from samples of zebu (the domesticated cattle of Madagascar), zebu owners, and non-zebu owners. Overall, human armpits were the least diverse sample site, while ankles were the most diverse. The diversity of zebu samples was significantly different from armpits, irrespective of zebu ownership (one-way ANOVA and Tukey’s HSD, p<0.05). However, zebu owner samples (from the armpit, ankle forearm, and hand) were more similar to other zebu owner samples than they were to zebu, yet no more similar to other zebu owner samples than they were to non-zebu owner samples (unweighted UniFrac distances, p<0.05). These data suggest a lack of a microbial signature shared by zebu owners and zebu, though further taxonomic analysis is required to explain the role of additional environmental variables in dictating the microbial communities of various samples sites. Understanding the magnitude and directionality of microbial sharing has implications for a breadth of microbe-related health outcomes, with the potential to explain mosquito host preference and mitigate the threats of vector-borne diseases.

The cellular lipids of Romboutsia.

We have examined the lipids of three isolates, Romboutsia lituseburensis, Romboutsia ilealis, and Romboutsia sp. strain FRIFI, of the newly described genus Romboutsia by two-dimensional thin-layer chromatography (2D-TLC) and by liquid chromatography/mass spectrometry (LC/MS). We have found three phospholipids, phosphatidylglycerol (PG), cardiolipin and phosphatidic acid in all three species. A fourth phospholipid, lysyl-PG, was found in R. lituseburensis and strain FRIFI. Polyprenyl-phosphates were identified in the lipid extracts of all three species. Three glycolipids, mono-, di- and tri-hexosyldiacylglycerol, were common to all three species. An additional glycolipid, tetrahexosyl-diacylglycerol was identified in strain FRIFI. Acylated trihexosyldiacylglycerol and acyl-tetrahexosydiacylglycerol were also found in R. ilealis and strain FRIFI. Remarkably, no alk-1-enyl ether lipids (plasmalogens) were present in Romboutsia as distinct from bacteria of the related genus Clostridium in which these ether lipids are common. We have compared the lipidome of Romboutsia with that recently described for Clostridium difficile, which has plasmalogens, no lysyl-PG, and no tetrahexosyl-diacylglycerol. According to 16S rRNA gene sequencing, Romboutsia spp. and C. difficile are closely related (>95% sequence identity).