Defining the "core microbiome" of the microbial communities in the tonsils of healthy pigs.

BACKGROUND: Porcine tonsils are the colonization site for many pathogenic as well as commensal microorganisms and are the primary lymphoid tissue encountered by organisms entering through the mouth or nares. The goal of this study was to provide an in-depth characterization of the composition and structure of the tonsillar microbial communities and to define the core microbiome in the tonsils of healthy pigs, using high throughput bar-coded 454-FLX pyrosequencing. RESULTS: Whole tonsils were collected at necropsy from 12 16-week-old finisher pigs from two healthy herds. Tonsil brushes were also used to collect samples from four of these animals. Bacterial DNA was isolated from each sample, amplified by PCR with universal primers specific for the bacterial 16S rRNA genes, and the PCR products sequenced using pyrosequencing. An average of 13,000 sequences were generated from each sample. Microbial community members were identified by sequence comparison to known bacterial 16S rRNA gene sequences.The microbiomes of these healthy herds showed very strong similarities in the major components as well as distinct differences in minor components. Pasteurellaceae dominated the tonsillar microbiome in all animals, comprising ~60% of the total, although the relative proportions of the genera Actinobacillus, Haemophilus, and Pasteurella varied between the herds. Also found in all animals were the genera Alkanindiges, Peptostreptococcus, Veillonella, Streptococcus and Fusobacterium, as well as Enterobacteriaceae and Neisseriaceae. Treponema and Chlamydia were unique to Herd 1, while Arcanobacterium was unique to Herd 2.Tonsil brushes yielded similar results to tissue specimens, although Enterobacteriaceae and obligate anaerobes were more frequently found in tissue than in brush samples, and Chlamydia, an obligately intracellular organism, was not found in brush specimens. CONCLUSIONS: We have extended and supported our previous studies with 16S clone libraries, using 16S rRNA gene pyrosequencing to describe the microbial communities in tonsils of healthy pigs. We have defined a core microbiome, dominated by Pasteurellaceae, in tonsil specimens, and have also demonstrated the presence of unique minor components of the tonsillar microbiome present in each herd. We have validated the use of non-invasive tonsil brushes, in comparison to tonsil tissue, which will facilitate future studies.

Membrane topology and DNA-binding ability of the Streptococcal CpsA protein.

Many streptococcal pathogens require a polysaccharide capsule for survival in the host during systemic infection. The highly conserved CpsA protein is proposed to be a transcriptional regulator of capsule production in streptococci, although the regulatory mechanism is unknown. Hydropathy plots of CpsA predict an integral membrane protein with 3 transmembrane domains and only 27 cytoplasmic residues, whereas other members of the LytR_cpsA_psr protein family are predicted to have a single transmembrane domain. This unique topology, with the short cytoplasmic domain, membrane localization, and large extracellular domain, suggests a novel mechanism of transcriptional regulation. Therefore, to determine the actual membrane topology of CpsA, specific protein domains were fused to beta-galactosidase or alkaline phosphatase. Enzymatic assays confirmed that the predicted membrane topology for CpsA is correct. To investigate how this integral membrane protein may be functioning in regulation of capsule transcription, purified full-length and truncated forms of CpsA were used in electrophoretic mobility shift assays to characterize the ability to bind the capsule operon promoter. Assays revealed that full-length, purified CpsA protein binds specifically to DNA containing the capsule promoter region. Furthermore, the large extracellular domain is not required for DNA binding, but all cytoplasmic regions of CpsA are necessary and sufficient for specific binding to the capsule operon promoter. This is the first demonstration of a member of this protein family interacting with its target DNA. Taken together, CpsA, as well as other members of the LytR_cpsA_psr protein family, appears to utilize a unique mechanism of transcriptional regulation.

Microbial communities in the tonsils of healthy pigs.

The tonsils of mammals such as humans and pigs are colonized with an extensive microbiota and are frequently the site for asymptomatic carriage of bacterial pathogens. The goal of this study was to determine the composition of the microbial community of the tonsils in healthy pigs. Tonsils were collected from eight pigs from two different healthy herds. Samples of the tonsils from each pig were used for culture dependent and culture independent identification of the microbial community. Aerobic cultivation identified Pasteurella multocida, Actinobacillus spp., Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus suis, Streptococcus dysgalactiae, and Escherichia coli from ≥ 50% of the pigs in both herds. For culture independent studies, microbial community members were identified by 16S rRNA sequences using the Ribosomal Database Project Pipeline programs developed at Michigan State University. Dominant genera identified by 16S rRNA analysis in pigs from both herds included Actinobacillus, Haemophilus, Pasteurella, Porphyromonas, Fusobacterium, Bacteroides, and Prevotella. These genera were detected in nearly every pig regardless of herd. In contrast, there was an asymmetric distribution of minor genera between the two herds, suggesting herd-specific differences in the microbial communities. In addition, we demonstrated primer bias between two frequently used forward primers when targeting the tonsillar community. Our results suggest that the major bacterial community members found in porcine tonsils are the same regardless of herd, while the minor species are unique to each herd. This is the first analysis using 16S rRNA sequence libraries of the composition of microbial communities in the porcine upper respiratory tract.

Analysis of the polysaccharide capsule of the systemic pathogen Streptococcus iniae and its implications in virulence.

Systemic pathogens have developed numerous strategies for evading the defenses of the host, permitting dissemination and multiplication in various tissues. One means of survival in the host, particularly in the bloodstream, has been attributed to the ability to avoid phagocytosis via capsular polysaccharide. To further define the virulence capacity of Streptococcus iniae, a zoonotic pathogen with the ability to cause severe systemic disease in both fish and humans, we performed an analysis of the capsule locus. The initial analysis included cloning and sequencing of the capsule synthesis operon, which revealed an approximately 21-kb region that is highly homologous to capsule operons of other streptococci. A genetic comparison of S. iniae virulent strain 9117 and commensal strain 9066 revealed that the commensal strain does not have the central region of the capsule operon composed of several important capsule synthesis genes. Four 9117 insertion or deletion mutants with mutations in the beginning, middle, or end of the capsule locus were analyzed to determine their capsule production and virulence. Virulence profiles were analyzed for each mutant using three separate criteria, which demonstrated the attenuation of each mutant in several tissue environments. These analyses also provided insight into the different responses of the host to each mutant strain compared to a wild-type infection. Our results demonstrate that capsule is not required for all host environments, while excess capsule is also not optimal, suggesting that for an "ideal" systemic infection, capsule production is most likely regulated while the bacterium is in different environments of the host.