Clostridium difficile is recognized as a problematic pathogen, causing severe enteric diseases including antibiotic-associated diarrhea and pseudomembranous colitis. The emergence of antibiotic resistant C. difficile has driven a search for alternative anti-infection modalities. A promising strategy for controlling bacterial infection includes the use of bacteriophages and their gene products. Currently, knowledge of phages active against C. difficile is still relatively limited by the fact that the isolation of phages for this organism is a technically demanding method since bacterial host themselves are difficult to culture. To isolate and characterize phages specific to C. difficile, a genotoxic agent, mitomycin C, was used to induce temperate phages from 12 clinical isolates of C. difficile. Five temperate phages consisting of ΦHR24, ΦHN10, ΦHN16-1, ΦHN16-2, and ΦHN50 were successfully induced and isolated. Spotting assays were performed against a panel of 92 C. difficile isolates to screen for susceptible bacterial hosts. The results revealed that all the C. difficile phages obtained in this work displayed a relatively narrow host range of 0–6.5% of the tested isolates. Electron microscopic characterization revealed that all isolated phages contained an icosahedral head connected to a long contractile tail, suggesting that they belonged to the Myoviridae family. Restriction enzyme analysis indicated that these phages possess unique double-stranded DNA genome. Further electron microscopic characterization revealed that the ΦHN10 absorbed to the bacterial surface via attachment to cell wall, potentially interacting with S-layer protein. Bacteriophages isolated from this study could lead to development of novel therapeutic agents and detection strategies for C. difficile.

The phytoaccumulation and phytotoxicity of heavy metals, cadmium (Cd), and chromium (Cr) on a common duckweed, Wolffia globosa, were studied. W. globosa were cultured in 3% Hoagland's nutrient medium, which was supplemented with 1, 2, 4, and 8 mg/L of Cd and Cr and were separately harvested after 3, 6, 9, and 12 days. The accumulation of Cd and Cr in W. globosa showed significant increases when the exposure time and metal concentration were increased. The effects of Cd and Cr on the biomass productivity and total chlorophyll content in W. globosa indicated that there were significant decreases in the biomass productivity and total chlorophyll content when the exposure time and metal concentration were increased.

Antibiotic resistance is a major concern in Clostridium difficile, the causative agent of antibiotic-associated diarrhea. Reduced susceptibility to first- and second-line agents is widespread, therefore various attempts have been made to seek alternative preventive and therapeutic strategies against this pathogen. In this work, the antimicrobial properties of asiatic acid were evaluated against C. difficile. Asiatic acid displayed substantial inhibitory effects on 19 C. difficile isolates collected from different sources with minimal inhibitory concentrations ranging from 10 to 20 μg/ml. Time kill analysis and minimal bactericidal concentration revealed potential bactericidal activity of this compound. Asiatic acid induced membrane damages and alterations in morphological ultrastructure in C. difficile, thereby causing the leakage of intracellular substances. Moreover, asiatic acid also displayed an inhibitory effect on cell motility, but did not interfere with biofilm formation and spore germination. Analysis of drug combination showed no synergistic effect between asiatic acid and vancomycin/metronidazole. Altogether, asiatic acid exhibited strong antimicrobial activity against vegetative cells and could serve as an alternative resource for tackling C. difficile.

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Chlamydia is a known pathogen in both saltwater and freshwater crocodiles. However, the exact species/strain has not been clearly identified. In this study, we successfully cultivated Siamese crocodile Chlamydia in McCoy cells at a temperature of 30°C. Electron microscopy; phylogeny based on nine conserved taxonomically informative markers, on ompA, or on seven housekeeping genes; and whole-genome sequencing and analysis of the isolate confirmed the identity of the isolate as a new member of the genus Chlamydia, a new species that we name Chlamydia crocodili.