Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni

Campylobacter jejuni is naturally transformable but is selective in the DNA used in transformation. Natural transformation allows for rapid adaptation and provides a means for DNA repair, both of which enhance bacterial fitness. Our work demonstrates that the methylation status of C. jejuni DNA is critical for transformation; methylation of a single 6-bp sequence in proximity to the transforming DNA is sufficient to allow nontransforming DNA to transform C. jejuni. Our data argue against previously described means for DNA discrimination; specifically, there is no evidence that a restriction enzyme recognizes the unmodified 6-bp sequence, and no evidence of a typical DNA uptake sequence. Therefore, we have identified a distinct DNA discrimination mechanism in Campylobacter

Development of the Tonsil Microbiome in Pigs and Effects of Stress on the Microbiome

Tonsils, lympho-epithelial tissues located at the junction of the oropharynx and nasopharynx, play a key role in surveillance, colonization, and persistence of inhaled and ingested pathogens. In pigs, the tonsils are a reservoir for numerous bacteria and viruses, including host-specific pathogens and potential zoonotic pathogens as well as commensal organisms. However, there are no in depth studies of the development of the tonsillar microbiome in pigs, or any mammal, over time. The goal of this study was to follow the development of the tonsil microbiome in healthy pigs from birth to market weight. Samples were collected using tonsil brushes from 16 piglets (4 each from 4 sows) at newborn, 1, 2, 3, and 4 weeks of age, and from 8 of those piglets at 6, 8, 10, 12, 16, and 19 weeks of age. Bacterial DNA was isolated from each sample and 16S rDNA genes were amplified and sequenced. Sequence analysis showed that members of the Streptococcaceae, Pasteurellaceae, and Moraxellaceae were present at all time points and represent the three most abundant families identified. Other community members appeared transiently or increased or decreased significantly with disruption events or stress. We observed four significant shifts in the tonsil community that coincided with well-defined disruption events: weaning plus addition of Carbadox plus movement to the nursery at week 3, removal of Carbadox and addition of Tylan at week 5, removal of Tylan and habitat change at week 9, and habitat change at week 16. Weaning triggered a bloom of Streptococcaeae and decrease of Moraxellaceae. The shift from Carbadox to Tylan led to reduction in Proteobacteria and Streptococcaceae but an increase in other Firmicutes, accompanied by a dramatic increase in community richness. Cessation of Tylan coincided with a return to a less rich community, and a bloom in Clostridiales. The final shift in habitat was accompanied by a decrease in Clostridiales and increase in Proteobacteria. The tonsillar microbiome of older pigs resembled the previously described mature core tonsillar microbiome. This study demonstrates a temporal succession in the development of the pig tonsillar microbiome, and significant community shifts that correlate with disruption events

Development of the tonsillar microbiome in pigs from newborn through weaning

Abstract Background Porcine tonsils are lympho-epithelial tissues, colonized by numerous bacteria and viruses, that act as a reservoir for both host-specific pathogens and zoonotic pathogens with a high potential of transmission to humans. There are no existing studies describing the development of the tonsillar microbiome. We sequenced 16S rRNA genes from tonsillar samples of pigs to follow the development of the microbial communities from birth through weaning. Samples derived from sows were also analyzed to determine potential sources for the tonsil microbiome in piglets. Results The composition of the newborn piglet tonsil microbiome could be differentiated by litter and had strong similarity to the sow teat skin as well as sow vaginal microbiome. The tonsil microbiome in these young piglets was mainly dominated by members of the Pasteurellaceae, Moraxellaceae, and Streptococcaceae families, while there were some transient members of the microbiome that were abundant at specific times, such as Staphylococcaceae in newborns and Fusobacteriaceae and Leptotrichiaceae in weeks 2 and 3. The microbiome initially differed between litters but over the following 3 weeks the communities of different litters converged in composition and then diverged in week 4 due to a combination of changes and stresses associated with weaning, including a shift from milk to a solid diet, in-feed Carbadox® and room change. Conclusions A significant portion of the tonsil microbiome was acquired either at birth from the sow vaginal tract or within a few hours post-birth from the sow teat skin. Our data demonstrate a temporal succession in the development of the pig tonsillar microbiome through the first weeks of life, with a convergence in the composition of the microbiome in all piglets by 3 weeks of age. The combination of management practices associated with weaning coincided with dramatic shifts in the tonsillar microbiome

Branched-Chain Amino Acids Are Required for the Survival and Virulence of Actinobacillus pleuropneumoniae in Swine▿

In Actinobacillus pleuropneumoniae, which causes porcine pleuropneumonia, ilvI was identified as an in vivo-induced (ivi) gene and encodes the enzyme acetohydroxyacid synthase (AHAS) required for branched-chain amino acid (BCAA) biosynthesis. ilvI and 7 of 32 additional ivi promoters were upregulated in vitro when grown in chemically defined medium (CDM) lacking BCAA. Based on these observations, we hypothesized that BCAA would be found at limiting concentrations in pulmonary secretions and that A. pleuropneumoniae mutants unable to synthesize BCAA would be attenuated in a porcine infection model. Quantitation of free amino acids in porcine pulmonary epithelial lining fluid showed concentrations of BCAA ranging from 8 to 30 μmol/liter, which is 10 to 17% of the concentration in plasma. The expression of both ilvI and lrp, a global regulator that is required for ilvI expression, was strongly upregulated in CDM containing concentrations of BCAA similar to those found in pulmonary secretions. Deletion-disruption mutants of ilvI and lrp were both auxotrophic for BCAA in CDM and attenuated compared to wild-type A. pleuropneumoniae in competitive index experiments in a pig infection model. Wild-type A. pleuropneumoniae grew in CDM+BCAA but not in CDM−BCAA in the presence of sulfonylurea AHAS inhibitors. These results clearly demonstrate that BCAA availability is limited in the lungs and support the hypothesis that A. pleuropneumoniae, and potentially other pulmonary pathogens, uses limitation of BCAA as a cue to regulate the expression of genes required for survival and virulence. These results further suggest a potential role for AHAS inhibitors as antimicrobial agents against pulmonary pathogens