Analysis of Bacterial Communities during Clostridium difficile Infection in the Mouse.

Clostridium difficile infection (CDI) is a major cause of health care-associated disease. CDI initiates with ingestion of C. difficile spores, germination in the gastrointestinal (GI) tract, and then colonization of the large intestine. The interactions between C. difficile cells and other bacteria and with host mucosa during CDI remain poorly understood. Here, we addressed the hypothesis that, in a mouse model of CDI, C. difficile resides in multicellular communities (biofilms) in association with host mucosa. To do this, we paraffin embedded and then sectioned the GI tracts of infected mice at various days postinfection (p.i.). We then used fluorescent in situ hybridization (FISH) with 16S rRNA probes targeting most bacteria as well as C. difficile specifically. The results revealed that C. difficile is present as a minority member of communities in the outer (loose) mucus layer, in the cecum and colon, starting at day 1 p.i. To generate FISH probes that identify bacteria within mucus-associated communities harboring C. difficile, we characterized bacterial populations in the infected mouse GI tract using 16S rRNA gene sequence analysis of bacterial DNA prepared from intestinal content. This analysis revealed the presence of genera of several families belonging to Bacteroidetes and Firmicutes. These data suggest that formation of multispecies communities associated with the mucus of the cecum and colon is an important early step in GI tract colonization. They raise the possibility that other bacterial species in these communities modulate the ability of C. difficile to successfully colonize and, thereby, cause disease.

Alterations of lung microbiota in a mouse model of LPS-induced lung injury.

Acute lung injury (ALI) and the more severe acute respiratory distress syndrome are common responses to a variety of infectious and noninfectious insults. We used a mouse model of ALI induced by intratracheal administration of sterile bacterial wall lipopolysaccharide (LPS) to investigate the changes in innate lung microbiota and study microbial community reaction to lung inflammation and barrier dysfunction induced by endotoxin insult. One group of C57BL/6J mice received LPS via intratracheal injection (n = 6), and another received sterile water (n = 7). Bronchoalveolar lavage (BAL) was performed at 72 h after treatment. Bacterial DNA was extracted and used for qPCR and 16S rRNA gene-tag (V3-V4) sequencing (Illumina). The bacterial load in BAL from ALI mice was increased fivefold (P = 0.03). The community complexity remained unchanged (Simpson index, P = 0.7); the Shannon diversity index indicated the increase of community evenness in response to ALI (P = 0.07). Principal coordinate analysis and analysis of similarity (ANOSIM) test (P = 0.005) revealed a significant difference between microbiota of control and ALI groups. Bacteria from families Xanthomonadaceae and Brucellaceae increased their abundance in the ALI group as determined by Metastats test (P < 0.02). In concordance with the 16s-tag data, Stenotrohomonas maltophilia (Xanthomonadaceae) and Ochrobactrum anthropi (Brucellaceae) were isolated from lungs of mice from both groups. Metabolic profiling of BAL detected the presence of bacterial substrates suitable for both isolates. Additionally, microbiota from LPS-treated mice intensified IL-6-induced lung inflammation in naive mice. We conclude that the morbid transformation of ALI microbiota was attributed to the set of inborn opportunistic pathogens thriving in the environment of inflamed lung, rather than the external infectious agents.

Spore formation and toxin production in Clostridium difficile biofilms.

The ability to grow as a biofilm can facilitate survival of bacteria in the environment and promote infection. To better characterize biofilm formation in the pathogen Clostridium difficile, we established a colony biofilm culture method for this organism on a polycarbonate filter, and analyzed the matrix and the cells in biofilms from a variety of clinical isolates over several days of biofilm culture. We found that biofilms readily formed in all strains analyzed, and that spores were abundant within about 6 days. We also found that extracellular DNA (eDNA), polysaccharide and protein was readily detected in the matrix of all strains, including the major toxins A and/or B, in toxigenic strains. All the strains we analyzed formed spores. Apart from strains 630 and VPI10463, which sporulated in the biofilm at relatively low frequencies, the frequencies of biofilm sporulation varied between 46 and 65%, suggesting that variations in sporulation levels among strains is unlikely to be a major factor in variation in the severity of disease. Spores in biofilms also had reduced germination efficiency compared to spores obtained by a conventional sporulation protocol. Transmission electron microscopy revealed that in 3 day-old biofilms, the outermost structure of the spore is a lightly staining coat. However, after 6 days, material that resembles cell debris in the matrix surrounds the spore, and darkly staining granules are closely associated with the spores surface. In 14 day-old biofilms, relatively few spores are surrounded by the apparent cell debris, and the surface-associated granules are present at higher density at the coat surface. Finally, we showed that biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole then do cells cultured in liquid media. Taken together, our data suggest that C. difficile cells and spores in biofilms have specialized properties that may facilitate infection.

A safe and effective plant gene switch system for tissue-specific induction of gene expression in Arabidopsis thaliana and Brassica juncea.

The ability to regulate spatial and temporal expression of genes is a useful tool in biotechnology as well as studies of functional genomics. Such regulation can provide information concerning the function of a gene in a developmental context while avoiding potential harmful effects due to constitutive overexpression of the gene. A GUS gene construct that uses the ecdysone receptor-based chemically inducible system and several different tissue-specific promoters was introduced into the model plant Arabidopsis thaliana and into the crop plant Brassica juncea. Here we describe the results of studies showing that this system provides both temporal and spatial control of transgene expression, and confirm that this system is useful for tissue-specific and temporal induction of gene expression in A. thaliana and B. juncea.

Expression of single-chain antibody-barstar fusion in plants.

We successfully cloned and expressed a single-chain antibody (425scFv), that is directed to human epidermal growth factor receptor HER1 (EGFR) in transgenic tobacco plants as a fusion with bacterial barstar gene (425scFv-barstar). Plant-produced recombinant 425scFv-barstar was recovered using barstar-barnase system. Based on barstar-barnase affinity, during purification of the plant-produced 425scFv-barstar, we generated bispecific scFv-antibody heterodimers from individual single-chain fragments initially produced in different host systems with binding activity to both HER1 and HER2/neu tumor antigens. We demonstrated by flow cytometry and indirect immunofluorescent microscopy that both the components of heterodimer retain its specific cell-binding activity.