Lactobacilli antagonize the growth, motility, and adherence of Brachyspira pilosicoli: a potential intervention against avian intestinal spirochetosis.

Avian intestinal spirochetosis (AIS) results from the colonization of the ceca and colorectum of poultry by pathogenic Brachyspira species. The number of cases of AIS has increased since the 2006 European Union ban on the use of antibiotic growth promoters, which, together with emerging antimicrobial resistance in Brachyspira, has driven renewed interest in alternative intervention strategies. Probiotics have been reported as protecting livestock against infection with common enteric pathogens, and here we investigate which aspects of the biology of Brachyspira they antagonize in order to identify possible interventions against AIS. The cell-free supernatants (CFS) of two Lactobacillus strains, Lactobacillus reuteri LM1 and Lactobacillus salivarius LM2, suppressed the growth of Brachyspira pilosicoli B2904 in a pH-dependent manner. In in vitro adherence and invasion assays with HT29-16E three-dimensional (3D) cells and in a novel avian cecal in vitro organ culture (IVOC) model, the adherence and invasion of B. pilosicoli in epithelial cells were reduced significantly by the presence of lactobacilli (P < 0.001). In addition, live and heat-inactivated lactobacilli inhibited the motility of B. pilosicoli, and electron microscopic observations indicated that contact between the lactobacilli and Brachyspira was crucial in inhibiting both adherence and motility. These data suggest that motility is essential for B. pilosicoli to adhere to and invade the gut epithelium and that any interference of motility may be a useful tool for the development of control strategies.

The intestinal spirochete Brachyspira pilosicoli attaches to cultured Caco-2 cells and induces pathological changes.

BACKGROUND: Brachyspira pilosicoli is an anaerobic spirochete that has received relatively little study, partly due to its specialized culture requirements and slow growth. This bacterium colonizes the large intestine of various species, including humans; typically, a dense layer of spirochete cells may be found intimately attached by one cell end to the surface of colonic enterocytes. Colonized individuals may develop colitis, but the mechanisms involved are not understood. The current study aimed to develop an in vitro model to investigate this process. METHODOLOGY/PRINCIPAL FINDINGS: Four strains of B. pilosicoli were incubated at a high multiplicity of infection with monolayers of a human colonic adenocarcinoma cell line (Caco-2 cells). One strain isolated from a pig (95/1000) and one from a human (WesB) attached to the monolayers. Colonization increased with time, with the Caco-2 cell junctions being the initial targets of attachment. By electron microscopy, individual spirochete cells could be seen to have one cell end invaginated into the Caco-2 cell membranes, with the rest of the spirochete draped over the Caco-2 cell surface. After 6 h incubation, the monolayer was covered with a layer of spirochetes. Colonized monolayers demonstrated a time-dependent series of changes: staining with labelled phalloidin identified accumulation of actin at the cell junctions; ZO-1 staining revealed a loss of Caco-2 tight junction integrity; and Hoechst staining showed condensation and fragmentation of nuclear material consistent with apoptosis. Using quantitative reverse transcription PCR, the colonized monolayers demonstrated a significant up-regulation of interleukin-1beta (IL-1beta) and IL-8 expression. B. pilosicoli sonicates caused significant up-regulation of IL-1beta, TNF-alpha, and IL-6, but culture supernatants and non-pathogenic Brachyspira innocens did not alter cytokine expression. CONCLUSIONS/SIGNIFICANCE: The changes induced in the Caco-2 cells provide evidence that B. pilosicoli has pathogenic potential, and give insights into the likely in vivo pathogenesis.

Typhlitis caused by intestinal Serpulina-like bacteria in domestic guinea pigs (Cavia porcellus).

Between January 1992 and December 1996, Serpulina-like bacteria were demonstrated in intestinal tract lesions from 37 of 88 guinea pigs submitted to the University of Ghent in Ghent, Belgium, for necropsy because of disease and death from different unknown causes. All infected animals had a history of sudden death with minimal introductory clinical signs. Occasionally, they produced yellow, slimy feces or showed nervous signs, but the condition always had a fatal outcome within 24 h. When larger colonies of guinea pigs were involved, the disease spread very rapidly unless treatment with ronidazole was initiated. Lesions consisted of a catarrhal or hemorrhagic inflammation of the colon and cecum (typhlitis). Electron microscopy demonstrated the presence of large numbers of Serpulina-like organisms adhering to the cecal mucosae of these animals. Attempts to isolate the agents failed. The organisms did not stain by an immunofluorescence technique for the detection of Serpulina hyodysenteriae. The present data provide evidence that intestinal Serpulina-like organisms can be important as a cause of disease in guinea pigs.

Comparative distribution and taxonomic value of cellular fatty acids in thirty-three genera of anaerobic gram-negative bacilli.

Cellular fatty acid profiles were determined for species in 33 genera of anaerobic gram-negative bacilli and were confirmed to be a useful taxonomic tool. Most of the genera could be differentiated by visual inspection of their profiles. The three genus pairs that were most difficult to distinguish visually (Bacteroides and Prevotella, Pectinatus and Megamonas, and Serpulina and Bilophila) and the species of these genera were differentiated by the MIDI (Microbial ID, Inc.) identification system. Similarities in cellular fatty acid profiles may be correlated with similarities in other phenotypic characteristics, but more often there is no other obvious phenotypic relationship. Although medium components may not change the constituents detected or the ratios among the constituents detected for some species, identical medium changes may result in vast differences in the profiles obtained with other species. Thus, if a worker wishes to compare profiles of various taxa, it is essential that the same cultural and analytical conditions be used.