Evidence that the 36kb plasmid of Brachyspira hyodysenteriae contributes to virulence

Swine dysentery (SD) results from infection of the porcine large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Recently the genome of virulent Australian B. hyodysenteriae strain WA1 was sequenced, and a 36. kilobase (kb) circular plasmid was identified. The plasmid contained 31 genes including six rfb genes that were predicted to be involved with rhamnose biosynthesis, and others associated with glycosylation. In the current study a set of PCRs was developed to amplify portions of nine of the plasmid genes. When used with DNA extracted from virulent strain B204, PCR products were generated, but no products were generated with DNA from avirulent strain A1. Analysis of the DNA using pulsed field gel electrophoresis (PFGE) identified a plasmid band in strains WA1 and B204, but not in strain A1. These results demonstrate that strain A1 does not contain the plasmid, and suggests that lack of the plasmid may explain why this strain is avirulent. To determine how commonly strains lacking plasmids occur, DNA was extracted from 264 Australian field isolates of B. hyodysenteriae and subjected to PCRs for three of the plasmid genes. Only one isolate (WA400) that lacked the plasmid was identified, and this absence was confirmed by PFGE analysis of DNA from the isolate and further PCR testing. To assess its virulence, 24 pigs were experimentally challenged with cultures of WA400, and 12 control pigs were challenged with virulent strain WA1 under the same conditions. Significantly fewer (P= 0.03) of the pigs challenged with WA400 became colonised and developed SD (13/24; 54%) compared to the pigs infected with WA1 (11/12; 92%). Gross lesions in the pigs colonised with WA400 tended to be less extensive than those in pigs colonised with WA1, although there were no obvious differences at the microscopic level. The results support the likelihood that plasmid-encoded genes of B. hyodysenteriae are involved in colonisation and/or disease expression

Taxonomic Features and Comparison of the Gut Microbiome from Two Edible Fungus-Farming Termites (Macrotermes falciger, M. natalensis) Harvested in the Vhembe District of Limpopo, South Africa

Background Termites are an important food resource for many human populations around the world, and are a good supply of nutrients. The fungus-farming ‘higher’ termite members of Macrotermitinae are also consumed by modern great apes and are implicated as critical dietary resources for early hominins. While the chemical nutritional composition of edible termites is well known, their microbiomes are unexplored in the context of human health. Here we sequenced the V4 region of the 16S rRNA gene of gut microbiota extracted from the whole intestinal tract of two Macrotermes sp. soldiers collected from the Limpopo region of South Africa. Results Major and minor soldier subcastes of M. falciger exhibit consistent differences in taxonomic representation, and are variable in microbial presence and abundance patterns when compared to another edible but less preferred species, M. natalensis. Subcaste differences include alternate patterns in sulfate-reducing bacteria and methanogenic Euryarchaeota abundance, and differences in abundance between Alistipes and Ruminococcaceae. M. falciger minor soldiers and M. natalensissoldiers have similar microbial profiles, likely from close proximity to the termite worker castes, particularly during foraging and fungus garden cultivation. Compared with previously published termite and cockroach gut microbiome data, the taxonomic representation was generally split between termites that directly digest lignocellulose and humic substrates and those that consume a more distilled form of nutrition as with the omnivorous cockroaches and fungus-farming termites. Lastly, to determine if edible termites may point to a shared reservoir for rare bacterial taxa found in the gut microbiome of humans, we focused on the genus Treponema. The majority of Treponemasequences from edible termite gut microbiota most closely relate to species recovered from other termites or from environmental samples, except for one novel OTU strain, which clustered separately with Treponema found in hunter-gatherer human groups. Conclusions Macrotermes consumed by humans display special gut microbial arrangements that are atypical for a lignocellulose digesting invertebrate, but are instead suited to the simplified nutrition in the fungus-farmer diet. Our work brings to light the particular termite microbiome features that should be explored further as avenues in human health, agricultural sustainability, and evolutionary research

A novel approach to probe host-pathogen interactions of bovine digital dermatitis, a model of a complex polymicrobial infection

Background: Polymicrobial infections represent a great challenge for the clarification of disease etiology and the development of comprehensive diagnostic or therapeutic tools, particularly for fastidious and difficult-to-cultivate bacteria. Using bovine digital dermatitis (DD) as a disease model, we introduce a novel strategy to study the pathogenesis of complex infections. Results: The strategy combines meta-transcriptomics with high-density peptide-microarray technology to screen for in vivo-expressed microbial genes and the host antibody response at the site of infection. Bacterial expression patterns supported the assumption that treponemes were the major DD pathogens but also indicated the active involvement of other phyla (primarily Bacteroidetes). Bacterial genes involved in chemotaxis, flagellar synthesis and protection against oxidative and acidic stress were among the major factors defining the disease. Conclusions: The extraordinary diversity observed in bacterial expression, antigens and host antibody responses between individual cows pointed toward microbial variability as a hallmark of DD. Persistence of infection and DD reinfection in the same individual is common; thus, high microbial diversity may undermine the host's capacity to mount an efficient immune response and maintain immunological memory towards DD. The common antigenic markers identified here using a high-density peptide microarray address this issue and may be useful for future preventive measures against DD.Fil: Marcatili, Paolo. Technical University of Denmark; DinamarcaFil: Nielsen, Martin W.. Technical University of Denmark; DinamarcaFil: Sicheritz Ponten, Thomas. Technical University of Denmark; DinamarcaFil: Jensen, Tim K.. Technical University of Denmark; DinamarcaFil: Schafer Nielsen, Claus. Schafer-N ApS; DinamarcaFil: Boye, Mette. Hospital of Southern Jutland; DinamarcaFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Klitgaard, Kirstine. Technical University of Denmark; Dinamarc

Impact of anaerobic digestion and centrifugation/decanting processes in bacterial communities fractions

The work upon which this paper is based on was co-financed by COGERSA, S.A.U. (Projects FUO-EM-138-15, FUO-139-16), IDEPA (Project IDE/2015/000245), Spanish MINECO (Project CTM2015-63864-R) and FEDER funds from European Union. Technical assistance from the Scientific-Technical Services of the University of Oviedo is gratefully acknowledged

Intestinal spirochaetes of the genus Brachyspira share a partially conserved 26 kilobase genomic region with Enterococcus faecalis and Escherichia coli

Anaerobic intestinal spirochaetes of the genus Brachyspira include both pathogenic and commensal species. The two best-studied members are the pathogenic species B. hyodysenteriae (the aetiological agent of swine dysentery) and B. pilosicoli (a cause of intestinal spirochaetosis in humans and other species). Analysis of near-complete genome sequences of these two species identifi ed a highly conserved 26 kilobase (kb) region that was shared, against a background of otherwise very little sequence conservation between the two species. PCR amplification was used to identify sets of contiguous genes from this region in the related Brachyspira species B. intermedia, B. innocens, B. murdochii, B. alvinipulli, and B. aalborgi, and demonstrated the presence of at least part of this region in species from throughout the genus. Comparative genomic analysis with other sequenced bacterial species revealed that none of the completely sequenced spirochaete species from different genera contained this conserved cluster of coding sequences. In contrast, Enterococcus faecalis and Escherichia coli contained high gene cluster conservation across the 26 kb region, against an expected background of little sequence conservation between these phylogenetically distinct species. The conserved region in B. hyodysenteriae contained five genes predicted to be associated with amino acid transport and metabolism, four with energy production and conversion, two with nucleotide transport and metabolism, one with ion transport and metabolism, and four with poorly characterised or uncertain function, including an ankyrin repeat unit at the 5’ end. The most likely explanation for the presence of this 26 kb region in the Brachyspira species and in two unrelated enteric bacterial species is that the region has been involved in horizontal gene transfer

Taxonomy of anaerobic digestion microbiome reveals biases associated with the applied high throughput sequencing strategies

In the past few years, many studies investigated the anaerobic digestion microbiome by means of 16S rRNA amplicon sequencing. Results obtained from these studies were compared to each other without taking into consideration the followed procedure for amplicons preparation and data analysis. This negligence was mainly due to the lack of knowledge regarding the biases influencing specific steps of the microbiome investigation process. In the present study, the main technical aspects of the 16S rRNA analysis were checked giving special attention to the approach used for high throughput sequencing. More specifically, the microbial compositions of three laboratory scale biogas reactors were analyzed before and after addition of sodium oleate by sequencing the microbiome with three different approaches: 16S rRNA amplicon sequencing, shotgun DNA and shotgun RNA. This comparative analysis revealed that, in amplicon sequencing, abundance of some taxa (Euryarchaeota and Spirochaetes) was biased by the inefficiency of universal primers to hybridize all the templates. Reliability of the results obtained was also influenced by the number of hypervariable regions under investigation. Finally, amplicon sequencing and shotgun DNA underestimated the Methanoculleus genus, probably due to the low 16S rRNA gene copy number encoded in this taxon

Laboratory diagnostics of Brachyspira species

Bacteria of the genus Brachyspira are intestinal spirochaetes that can cause diarrhoea and mortality in pigs. Laboratory diagnostics of Brachyspira species is essential for confirming clinical diagnosis, for providing data for optimal treatment and for surveillance of the bacteria in individual animals or herds. The aims of the present thesis were to evaluate presently used, and develop new, laboratory diagnostic techniques for Brachyspira species, to describe the pathogenic and epidemiological features and antimicrobial susceptibility of selected Brachyspira isolates, and to evaluate DNA-based epidemiological and phylogenetic tools. In study I, seeded samples were sent to laboratories in northern Europe to assess their ability to detect and identify Brachyspira species and test antimicrobial susceptibility. In study II, a PCR system was set up and compared with traditional culture and biochemical tests; and in study III, well-described Brachyspira isolates were analysed by five molecular typing methods. In the last two studies Brachyspira isolates from animals other than pigs were included. An atypical isolate detected in study II was further characterised together with identical or similar isolates (study IV) and a new genetic typing method for the entire genus of Brachyspira species was tested and evaluated (study V). Overall, the results indicated that laboratory diagnostics of Brachyspira species is difficult. Of the tested genetic typing methods, the nox gene and MLST sequence analyses showed the highest taxonomic resolutions, and randomly amplified polymorphic DNA showed the highest discriminatory power for the isolates studied. A high genetic variability was observed within the Brachyspira genus. This genetic variability could make it difficult to rely solely on DNA-based methods for detection and identification of brachyspiras. A new unique group of isolates pathogenic to pigs was detectable only by the combined use of culture, biochemical tests and PCR. The provisional name "B. suanatina" was suggested for this type of isolates, of which some were found in mallards. By MLST data analysed by e-BURST, a close evolutionary relationship was identified for "B. suanatina" and B. hyodysenteriae isolates recovered from pigs and mallards. In conclusion, these studies show that the most reliable method for laboratory diagnostics of Brachyspira species is culture and biochemical tests, used together with at least one DNA-based method

Systematic identification of gene families for use as markers for phylogenetic and phylogeny- driven ecological studies of bacteria and archaea and their major subgroups

With the astonishing rate that the genomic and metagenomic sequence data sets are accumulating, there are many reasons to constrain the data analyses. One approach to such constrained analyses is to focus on select subsets of gene families that are particularly well suited for the tasks at hand. Such gene families have generally been referred to as marker genes. We are particularly interested in identifying and using such marker genes for phylogenetic and phylogeny-driven ecological studies of microbes and their communities. We therefore refer to these as PhyEco (for phylogenetic and phylogenetic ecology) markers. The dual use of these PhyEco markers means that we needed to develop and apply a set of somewhat novel criteria for identification of the best candidates for such markers. The criteria we focused on included universality across the taxa of interest, ability to be used to produce robust phylogenetic trees that reflect as much as possible the evolution of the species from which the genes come, and low variation in copy number across taxa. We describe here an automated protocol for identifying potential PhyEco markers from a set of complete genome sequences. The protocol combines rapid searching, clustering and phylogenetic tree building algorithms to generate protein families that meet the criteria listed above. We report here the identification of PhyEco markers for different taxonomic levels including 40 for all bacteria and archaea, 114 for all bacteria, and much more for some of the individual phyla of bacteria. This new list of PhyEco markers should allow much more detailed automated phylogenetic and phylogenetic ecology analyses of these groups than possible previously.Comment: 24 pages, 3 figure

Variation in hemolytic activity of Brachyspira hyodysenteriae strains from pigs

Brachyspira hyodysenteriae is the primary cause of swine dysentery, which is responsible for major economic losses to the pig industry worldwide. The hemolytic activity of 10 B. hyodysenteriae strains isolated from stools of pigs with mild to mucohemorrhagic diarrhea was compared and seven hemolysis associated genes were sequenced. Hemolysis induced by these strains varied from strong to near absent. One weakly hemolytic B. hyodysenteriae strain showed sequence changes in five hemolysis associated genes (tlyA, tlyB, hemolysin III, hemolysin activation protein and hemolysin III channel protein) resulting in amino acid substitutions. The occurrence of weakly hemolytic strains identifiable as B. hyodysenteriae should be taken into account in swine dysentery diagnostics. The presence of these strains may affect herd dysentery status, with great impact on a farms trading opportunities