Surang Chankhamhaengdecha

Endophytic Actinomycetes: A Novel Source of Potential Acyl Homoserine Lactone Degrading Enzymes

Several Gram-negative pathogenic bacteria employ N-acyl-L-homoserine lactone (HSL) quorum sensing (QS) system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE) results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9%) and 68 (51.5%) of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at 151.30 ± 3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified as Streptomyces based on 16S   rRNA gene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain in HSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6- to C12-HSL in which C10HSL is the most favorable substrate for this enzyme. In an in vitro pathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused by Pectobacterium carotovorum ssp. carotovorum as demonstrated. To our knowledge, this is the first report of HSL-acylase activity derived from an endophytic Streptomyces.

Inactivation of Clostridium difficile spores by microwave irradiation.

Spores are a potent agent for Clostridium difficile transmission. Therefore, factors inhibiting spores have been of continued interest. In the present study, we investigated the influence of microwave irradiation in addition to conductive heating for C. difficile spore inactivation in aqueous suspension. The spores of 15 C. difficile isolates from different host origins were exposed to conductive heating and microwave irradiation. The complete inhibition of spore viability at 10(7) CFU/ml was encountered following microwave treatment at 800 W for 60 s, but was not observed in the conductive-heated spores at the same time-temperature exposure. The distinct patterns of ultrastructural alterations following microwave and conductive heat treatment were observed and the degree of damages by microwave was in the exposure time-dependent manner. Microwave would therefore be a simple and time-efficient tool to inactivate C. difficile spores, thus reducing the risk of C. difficile transmission.

Evaluation of multiplex PCR with enhanced spore germination for detection of Clostridium difficile from stool samples of the hospitalized patients.

Clostridium difficile poses as the most common etiologic agent of nosocomial diarrhea. Although there are many diagnostic methods to detect C. difficile directly from stool samples, the nucleic acid-based approach has been largely performed in several laboratories due to its high sensitivity and specificity as well as rapid turnaround time. In this study, a multiplex PCR was newly designed with recent accumulated nucleotide sequences. The PCR testing with various C. difficile ribotypes, other Clostridium spp., and non-Clostridium strains revealed 100% specificity with the ability to detect as low as ~22 genomic copy number per PCR reaction. Different combinations of sample processing were evaluated prior to multiplex PCR for the detection of C. difficile in fecal samples from hospitalized patients. The most optimal condition was the non-selective enrichment at 37°C for 1 h in brain heart infusion broth supplemented with taurocholate, followed by the multiplex PCR. The detection limit after sample processing was shown as being 5 spores per gram of fecal sample. Two hundred and thirty-eight fecal samples collected from the University affiliated hospital were analyzed by the enrichment multiplex PCR procedure. The results suggested that the combination of sample processing with the high-performance detection method would be applicable for routine diagnostic use in clinical setting.

Insights into drug resistance mechanisms in Clostridium difficile

The incidence of Clostridium difficile infection has been elevated and becoming common in hospitals worldwide. Although antibiotics usually serve as the primary treatment for bacterial infection including C. difficile infection, limitations and failures have been evident due to drug resistance. Antibiotic resistance in C. difficile has been recognized as one of the most important factors to promote the infection and increase the level of severity and the recurrence rate. Several outbreaks in many countries have been linked to the emergence of hypervirulent drug-resistant strains. This pathogen harbours various mechanisms against the actions of antibiotics. The present study highlights three main drug-resistant strategies in C. difficile including drug inactivation, target modification and efflux pump. Other mechanisms that potentially contribute to drug-resistant traits in this organism are also discussed.

CD2068 potentially mediates multidrug efflux in Clostridium difficile

Clostridium difficile is a major cause of antibiotic-associated diarrhea and the treatment thereof becomes more difficult owing to a rise of multidrug resistant strains. ATP-binding cassette (ABC) transporters are known to play a crucial role in the resistance to multiple antibiotics. In this study, the potential contribution of an ABC transporter in C. difficile multidrug resistance was investigated. The expression level of the cd2068 gene in C. difficile encoding an ABC transporter was up-regulated following the exposure to certain antibiotics compared to the control cells. Heterologous expression of CD2068 in Escherichia coli revealed that it mediated the efflux of fluorescent substrates and conferred resistance to multiple drugs. The CD2068-associated ATPase activity in membrane vesicles was also stimulated by various antibiotics. Furthermore, the insertional inactivation of the cd2068 gene in C. difficile led to a significant increase in susceptibility to antibiotics, which could be genetically complemented, supporting that CD2068 was directly associated to the drug resistance. These results demonstrate the potential role for the ABC transporter CD2068 in the resistance mechanism against multiple drugs in C. difficile.

Gut Microbial succession during conventionalization of germfree chicken

A gnotobiotic chicken model was developed to study the succession of intestinal microflora from hatching to 18 days of age. Intestinal samples were collected from a local population of feral chickens and administered orally to germ-free 3 day old chicks. Animals were enthanized on 0, 9 and 18 days of age and intestinal samples were collected and subjected to genomic analysis. The five most prevalent phyla were Bacteroidetes (45.73±3.35%), Firmicutes (36.47±2.60%), Proteobacteria (8.28±0.91%), Actinobacteria (5.09±0.52%), and Spriochetes (2.10±0.38%). Principle coordinate analysis indicated the 0, 9 day and 18 day variables clustered together and the microbial communities changed temporally. The Morista-Horn index values ranged from 0.72 to 1, indicating the communities at 0, 9 or 18 days were more similar than dissimilar. The predicted functional profiles of the microbiomes of 0, 9 and 18 days were also similar. These results indicate the gnotobiotic chicks stably maintain the phylogentic diversity and predicted metabolic functionality of the inoculum community. Importance The domestic chicken is the cornerstone of animal agriculture worldwide with a flock population exceeding 40 billion birds/year. It serves as the economically valuable source of protein globally. Microbiome of poultry has important effects on chicken growth, feed conversion, immune status and pathogen resistance. The significance of our research is in developing a gnotobiotic chicken model to study chicken gut microbiota function. Our experimental model shows that young germfree chicks are able to colonize diverse set of gut bacteria. Therefore, besides using this model to study mechanisms of gut microbiota interactions in the chicken gut, our model could be also used for applied aspects such as determining the safety and efficacy of new probiotic strains derived from chicken gut microbiota.

Characterization of Bacteriophages Infecting Clinical Isolates of Clostridium difficile

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.

Antimicrobial Effect of Asiatic Acid Against Clostridium difficile Is Associated With Disruption of Membrane Permeability.

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.