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Simple Summary: Current immunotherapies are ineffectively designed toward single-target inhibition in a complex multifactored process. Moreover, available therapeutics are incapable of meeting the needs of the expanding cancer patient population due to resistance, recurrence, immune-related adverse events, or patients that do not express the intended target. This study proposes Master Regulators of Immune Evasion (MR-IE) inhibition as a more effective systemic therapeutic strategy that targets multiple immune evasion molecules to reinstate immune recognition and the elimination of tumors. Moreover, this study also identifies and ranks ideal candidate MR-IEs for each cancer type associated with patient mortality. This study has been validated in cholangiocarcinoma. This investigation offers MR-IE as a new avenue of therapeutic targets that can be validated and developed as an immunotherapeutic strategy for all cancer types. Moreover, we address that not all cancers would respond to the same target, and thus, we propose mortality-associated MR-IEs per cancer type. Background: Cancer immune evasion is a multifaceted process that synchronizes pro-tumoral immune infiltration, immunosuppressive inflammation, and inhibitory immune checkpoint expression (IC). Current immunotherapies combat this issue by reinstating immunosurveillance of tumors; however, it benefits a limited patient population. Thus, a more effective immunotherapeutic strategy is warranted to cater to specific patient populations. This investigation introduces a novel immunotherapeutic strategy via inhibition of master regulators of immune evasion (MR-IE). Methods: Samples of the TCGA Pan-Cancer Atlas transcriptomic data were subset and stratified based on IC and estimated immune cell infiltration. Transcriptomic analysis was conducted to unravel pathways associated with the immune evasion process. Transcription factor enrichment and survival analyses were conducted to identify and rank candidate MR-IEs per cancer type. Results: Inhibition of the top-ranking MR-IE candidate of cholangiocarcinoma (CCA), MYC, modulated the gene and protein expression of PD-L1. Moreover, pro-tumoral inflammatory markers, IFNA21 and CX3CL1, were downregulated, and anti-tumoral cytokines, IL-18 and IL-16, were upregulated. Lastly, MYC inhibition potentiated fourth-generation anti-folate receptor alpha (FRα) CAR-T cell therapy against CCA cells. Conclusions: Cumulatively, this study highlights the promise of MR-IE inhibition as a novel potent immunotherapeutic strategy for the treatment of CCA and offers a candidate list of MR-IEs per cancer type for further validation. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma (CCA) is a difficult-to-treat cancer, with limited therapeutic options and surgery being the only curative treatment. Standard chemotherapy involves gemcitabine-based therapies combined with cisplatin, oxaliplatin, capecitabine, or 5-FU with a dismal prognosis for most patients. Receptor tyrosine kinases (RTKs) are aberrantly expressed in CCAs encompassing potential therapeutic opportunity. Hence, 112 RTK inhibitors were screened in KKU-M213 cells, and ceritinib, an approved targeted therapy for ALK-fusion gene driven cancers, was the most potent candidate. Ceritinib's cytotoxicity in CCA was assessed using MTT and clonogenic assays, along with immunofluorescence, western blot, and qRT-PCR techniques to analyze gene expression and signaling changes. Furthermore, the drug interaction relationship between ceritinib and cisplatin was determined using a ZIP synergy score. Additionally, spheroid and xenograft models were employed to investigate the efficacy of ceritinib in vivo. Our study revealed that ceritinib effectively killed CCA cells at clinically relevant plasma concentrations, irrespective of ALK expression or mutation status. Ceritinib modulated multiple signaling pathways leading to the inhibition of the PI3K/Akt/mTOR pathway and activated both apoptosis and autophagy. Additionally, ceritinib and cisplatin synergistically reduced CCA cell viability. Our data show ceritinib as an effective treatment of CCA, which could be potentially explored in the other cancer types without ALK mutations. [ABSTRACT FROM AUTHOR]

Platelet-derived growth factors (PDGFs) and PDGF receptors (PDGFRs) play essential roles in promoting cholangiocarcinoma (CCA) cell survival by mediating paracrine crosstalk between tumor and cancer-associated fibroblasts (CAFs), indicating the potential of PDGFR as a target for CCA treatment. Clinical trials evaluating PDGFR inhibitors for CCA treatment have shown limited efficacy. Furthermore, little is known about the role of PDGF/PDGFR expression and the mechanism underlying PDGFR inhibitors in CCA related to Opisthorchis viverrini (OV). Therefore, we examined the effect of PDGFR inhibitors in OV-related CCA cells and investigated the molecular mechanism involved. We found that the PDGF and PDGFR mRNAs were overexpressed in CCA tissues compared to resection margins. Notably, PDGFR-α showed high expression in CCA cells, while PDGFR-β was predominantly expressed in CAFs. The selective inhibitor CP-673451 induced CCA cell death by suppressing the PI3K/Akt/Nrf2 pathway, leading to a decreased expression of Nrf2-targeted antioxidant genes. Consequently, this led to an increase in ROS levels and the promotion of CCA apoptosis. CP-673451 is a promising PDGFR-targeted drug for CCA and supports the further clinical investigation of CP-673451 for CCA treatment, particularly in the context of OV-related cases. [ABSTRACT FROM AUTHOR]

Nasopharyngeal carcinoma (NPC) is a prevalent cancer in Southeast Asia, but effective treatment options remain limited, and chemotherapy has a high resistance rate. Asiatic acid (AA), a triterpenoid found in Centella asiatica, has shown anticancer activity in various cancers. Therefore, this study aims to investigate the anticancer effects and mechanisms of AA in NPC cell lines. The effects of AA on NPC cytotoxicity, apoptosis, and migration were determined in TW-01 and SUNE5-8F NPC cell lines. Western blot analysis was performed to evaluate the protein expression levels affected by AA. The role of AA in proliferation and migration was investigated in STAT3 and claudin-1 knockdown cells. AA inhibited NPC cell viability and migration and induced cell death by increasing cleaved caspase-3 expression. Moreover, AA inhibited STAT3 phosphorylation and reduced claudin-1 expression in NPC cells. Although knockdown of STAT3 or claudin-1 slightly reduced cell viability, it did not enhance the anti-proliferative effect of AA. However, knockdown of STAT3 or claudin-1 increased the anti-migratory effect of AA in NPC cells. These results suggest that AA can be a promising candidate for drug development against NPC. [ABSTRACT FROM AUTHOR]

Simple Summary: Cholangiocarcinoma (CCA) is an architecturally complex and highly heterogeneous tumour and is difficult to diagnose at early stages due to its late onset and asymptomatic nature. Recent studies have focused on the Fibroblast Growth Factor Receptors (FGFRs), a sub-family of Tyrosine Kinase Receptors (RTKs), as promising targets for therapy. Pemigatinib, a small-molecule inhibitor of FGFR, was the first FDA-approved targeted therapy drug for CCA patients with FGFR2 fusions who had failed first-line chemotherapy. However, only a limited cohort of patients benefit from this therapeutic strategy as the mutation does not necessarily correlate to the response. In this study, aberrant FGFR expression in CCA samples was found using a bioinformatics approach and further confirmed by immunohistochemistry. PD173074, a selective pan-FGFR inhibitor, was found to be sensitive to CCA cell lines with FGFR expression, suggesting that it can be used to suppress CCA cells even without the FGFR2 fusions. Moreover, correlation analysis of publicly available cohorts suggested the possibility of crosstalk amongst the FGFR and EGFR family of receptors and dual inhibition of PD173074 and erlotinib was found to be synergistic in CCA using cell lines and patient-derived complex models. Thus, this study suggests further clinical investigation of PD173074, as well as other FGFR inhibitors, to benefit a larger cohort of CCA patients and novel therapeutic strategies involving dual inhibition of FGFRs and EGFR. Cholangiocarcinoma (CCA) is an architecturally complex tumour with high heterogeneity. Discovery at later stages makes treatment challenging. However, the lack of early detection methodologies and the asymptomatic nature of CCA make early diagnosis more difficult. Recent studies revealed the fusions in Fibroblast Growth Factor Receptors (FGFRs), a sub-family of RTKs, as promising targets for targeted therapy for CCA. Particularly, FGFR2 fusions have been of particular interest, as translocations have been found in approximately 13% of CCA patients. Pursuing this, Pemigatinib, a small-molecule inhibitor of FGFR, became the first targeted therapy drug to be granted accelerated approval by the FDA for treating CCA patients harbouring FGFR2 fusions who have failed first-line chemotherapy. However, despite the availability of Pemigatinib, a very limited group of patients benefit from this treatment. Moreover, as the underlying mechanism of FGFR signalling is poorly elucidated in CCA, therapeutic inhibitors designed to inhibit this pathway are prone to primary and acquired resistance, as witnessed amongst other Tyrosine Kinase Inhibitors (TKIs). While acknowledging the limited cohort that benefits from FGFR inhibitors, and the poorly elucidated mechanism of the FGFR pathway, we sought to characterise the potential of FGFR inhibitors in CCA patients without FGFR2 fusions. Here we demonstrate aberrant FGFR expression in CCA samples using bioinformatics and further confirm phosphorylated-FGFR expression in paraffinised CCA tissues using immunohistochemistry. Our results highlight p-FGFR as a biomarker to guide FGFR-targeted therapies. Furthermore, CCA cell lines with FGFR expression were sensitive to a selective pan-FGFR inhibitor, PD173074, suggesting that this drug can be used to suppress CCA cells irrespective of the FGFR2 fusions. Finally, the correlation analysis utilising publicly available cohorts suggested the possibility of crosstalk amongst the FGFR and EGFR family of receptors as they are significantly co-expressed. Accordingly, dual inhibition of FGFRs and EGFR by PD173074 and EGFR inhibitor erlotinib was synergistic in CCA. Hence, the findings from this study provide support for further clinical investigation of PD173074, as well as other FGFR inhibitors, to benefit a larger cohort of patients. Altogether, this study shows for the first time the potential of FGFRs and the importance of dual inhibition as a novel therapeutic strategy in CCA. [ABSTRACT FROM AUTHOR]

Background: Nasopharyngeal carcinoma (NPC) is a type of cancers that develops in the nasopharynx, the very upper part of the throat behind the nose. NPC is typically diagnosed in later stages of the disease and has a high rate of recurrence due to the location of the tumor growth site. In this study, we compared the gene expression profiles of NPC tissues from patients with and without recurrence to identify potential molecular biomarkers of NPC recurrence. Methods: Microarrays were used to analyze the expression of genes in 15 NPC tissues taken at the time of diagnosis and at the site of recurrence following therapeutic treatment. Pathway enrichment analysis was used to examine the biological interactions between the major differentially expressed genes. The target identified was then validated using immunohistochemistry on 86 NPC tissue samples. Results: Our data showed that the Wnt signaling pathway was enhanced in NPC tissues with recurrence. FZD10, a component of the Wnt signaling pathway, was significantly expressed in NPC tissues, and was significantly associated with NPC recurrence. Conclusion: Our study provides new insights into the pathogenesis of NPC and identifies FZD10 as a potential molecular biomarker for NPC recurrence. FZD10 may be a promising candidate for NPC recurrence and a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Background: Members of the Wnt signaling pathway have been shown to play a role in nasopharyngeal carcinoma (NPC) progression. Aim: The purpose of this study was to investigate WNT8B protein expression in NPC patients using tissue microarray (TMA) analysis and to evaluate its correlation with patient survival and clinical parameters. Methods: A total of 82 NPC cases, together with six normal nasopharyngeal tissue samples, were targeted to construct the TMA blocks. The WNT8B protein expression was evaluated by immunohistochemistry and its correlation to the clinicopathological features was investigated. Results: Sixty-two of 82 (75.6%) cases exhibited high WNT8B protein expression while 20/82 (24.4%) cases appeared to have low WNT8B expression. The univariate analysis revealed that systemic metastasis was associated with patient 5-year survival. The multivariate Cox proportional hazard regression analysis showed that WNT8B expression and systemic metastasis were significantly associated with the survival of NPC patients. Furthermore, there was no correlation found between the WNT8B protein expression and other clinicopathological parameters. Conclusion: Our results suggest that the expression of WNT8B is associated with NPC patients' survival and could serve as an independent prognostic factor for NPC patients. [ABSTRACT FROM AUTHOR]

Development of a simple, rapid and specific assay for the simultaneous detection of Campylobacter spp. and Salmonella spp. based on duplex loop-mediated isothermal amplification (d-LAMP), combined with lateral-flow biosensor (LFB) is reported herein. LAMP amplicons of both pathogens were simultaneously amplified and specifically differentiated by LFB. The specificity of the d-LAMP-LFB was evaluated using a set of 68 target and 12 non-target strains, showing 100% inclusivity and exclusivity. The assay can simultaneously detect Campylobacter and Salmonella strains as low as 1 ng and 100 pg genomic DNA per reaction, respectively. The lowest inoculated detection limits for Campylobacter and Salmonella species in artificially contaminated chicken meat samples were 103 CFU and 1 CFU per 25 grams, respectively, after enrichment for 24 h. Furthermore, compared to culture-based methods using field chicken meat samples, the sensitivity, specificity and accuracy of d-LAMP- LFB were 95.6% (95% CI, 78.0%-99.8%), 71.4% (95% CI, 29.0%-96.3%) and 90.0% (95% CI, 73.4%-97.8%), respectively. The developed d-LAMP-LFB assay herein shows great potentials for the simultaneous detection of the Campylobacter and Salmonella spp. and poses a promising alternative approach for detection of both pathogens with applications in food products. [ABSTRACT FROM AUTHOR]

Purpose The potential of members of the epidermal growth factor receptor (ErbB) family as drug targets in cholangiocarcinoma (CCA) has not been extensively addressed. Although phase III clinical trials showed no survival benefits of erlotinib in patients with advanced CCA, the outcome of the standard-of-care chemotherapy treatment for CCA, gemcitabine/cisplatin, is discouraging so we determined the effect of other ErbB receptor inhibitors alone or in conjunction with chemotherapy in CCA cells. Materials and Methods ErbB receptor expression was determined in CCA patient tissues by immunohistochemistry and digitaldroplet polymerase chain reaction, and in primary cells and cell lines by immunoblot. Effects on cell viability and cell cycle distribution of combination therapy using ErbB inhibitors with chemotherapeutic drugs was carried out in CCA cell lines. 3D culture of primary CCA cells was then adopted to evaluate the drug effect in a setting that more closely resembles in vivo cell environments. Results CCA tumors showed higher expression of all ErbB receptors compared with resection margins. Primary and CCA cell lines had variable expression of erbB receptors. CCA cell lines showed decreased cell viability when treated with chemotherapeutic drugs (gemcitabine and 5-fluorouracil) but also with ErbB inhibitors, particularly afatinib, and with a combination. Sequential treatment of gemcitabine with afatinib was particularly effective. Co-culture of CCA primary cells with cancer-associated fibroblasts decreased sensitivity to chemotherapies, but sensitized to afatinib. Conclusion Afatinib is a potential epidermal growth factor receptor targeted drug for CCA treatment and sequential treatment schedule of gemcitabine and afatinib could be explored in CCA patients. [ABSTRACT FROM AUTHOR]

A Gram-positive, coccobacillus, white raised and circular with an entire edge colony, and obligately anaerobic bacterium, strain SW178 was isolated from the cecum content of feral chickens in Brookings, South Dakota, USA. The most closely related strain based on 16S rRNA gene sequence analysis of strain SW178 was Mediterraneibacter torques ATCC 27756T (Ruminococcus torques ATCC 27756T) with 96.94% similarity. The genome of strain SW178 is 3.18 Mbp with G+C content of 46.9 mol%. The optimal temperature and pH for growth in modified brain heart infusion (BHI-M) medium were 45 °C and pH 7.5, respectively. The sole carbon sources of the strain were dextrin, L-fucose, D-galacturonic, α-D-glucose, L-rhamnose and D-sorbitol. The primary cellular fatty acids were C14: 0, C16: 0 and C16: 0 dimethyl acetal (DMA). Based on the genotypic and phenotypic comparison, we proposed that strain SW178 belong to the genus Mediterraneibacter in the family Lachnospiraceae as a novel species, in which the name Mediterraneibacter catenae is proposed. The type strain is SW178 (= DSM 109242T = CCOS 1886T). [ABSTRACT FROM AUTHOR]

Nasopharyngeal carcinoma (NPC) is one of the rare cancers in western countries but predominant in Southeast Asian countries including Thailand. One major cause for failure of NPC chemotherapeutic treatments is reportedly correlated with the elevation of cancer stem cell (CSC) fractions. Thus, this present study aims to investigate the effect of cisplatin (CDDP) treatment on the enrichment of cancer stem-like cells (CSCs) and its associated signaling pathway in EBV-negative NPC cells. Cisplatin-pretreated 5-8F NPC cells (5-8F CDDP) were first generated by treating the cells with 0.5 μM cisplatin for 48 h. After the instant treatment, 5-8F CDDP showed increased IC50 values, demonstrating a decrease in CDDP sensitization. Besides, the proportion of NPC cells with cancer stem-like phenotypes comprising side population (SP), key stemness-related gene expressions including SOX2, ALDH1, CD24 was significantly enhanced. Additionally, 5-8F CDDP displayed the upregulation of β-catenin gene, suggesting its association with the CSC-initiating mechanism. Furthermore, a tankyrase inhibitor for Wnt/β-catenin pathway, XAV939, substantially reduced CSCs and retrieved the cisplatin sensitivity in 5-8F CDDP. This confirms that the Wnt/β-catenin signaling is accountable for rising of the CSC population in EBV-negative NPC. Finally, the combined treatment of CDDP and XAV939 exhibited lower 5-8F CDDP cell viability compared to the treatment of CDDP alone, suggesting the reversal of cisplatin sensitization. In conclusion, the enhancement of CSCs in 5-8F NPC cells caused by the instant cisplatin treatment is initially mediated through the upregulation of β-catenin and activation of Wnt/β-catenin signaling pathway. As a result, a primary chemotherapeutic treatment with closely monitoring the targeted Wnt/β-catenin signaling pathway could potentially prevent the development of CSCs and improve the treatment efficiency in NPC. [ABSTRACT FROM AUTHOR]

Clostridium perfringens is a key anaerobic pathogen causing food poisoning. Definitive detection by standard culture method is time-consuming and labor intensive. Current rapid commercial test kits are prohibitively expensive. It is thus necessary to develop rapid and cost-effective detection tool. Here, loop-mediated isothermal amplification (LAMP) in combination with a lateral-flow biosensor (LFB) was developed for visual inspection of C. perfringens-specific cpa gene. The specificity of the developed test was evaluated against 40 C. perfringens and 35 other bacterial strains, which showed no cross-reactivity, indicating 100% inclusivity and exclusivity. LAMP-LFB detection limit for artificially contaminated samples after enrichment for 16 h was 1–10 CFU/g sample, which was comparable to the commercial real-time PCR kit. The detection performance of LAMP-LFB was also compared to culture-based method using 95 food samples, which revealed the sensitivity (SE), specificity (SP) and Cohen's kappa coefficient (κ) of 88.0% (95% CI, 75.6%-95.4%), 95.5% (95% CI, 84.8%-99.4%) and 0.832 (95% CI, 0.721–0.943), respectively. Area under the receiver operating characteristic (ROC) curve was 0.918 (95% CI, 0.854–0.981), indicating LAMP-LFB as high relative accuracy test. In conclusion, LAMP-LFB assay is a low-cost qualitative method and easily available for routine detection of C. perfringens in food samples, which could serve as an alternative to commercial test kit. [ABSTRACT FROM AUTHOR]

Thermophilic microorganisms and their enzymes have been utilized in various industrial applications. In this work, we isolated and characterized thermophilic anaerobic bacteria with the cellulose and hemicellulose degrading activities from a tropical dry deciduous forest in northern Thailand. Out of 502 isolated thermophilic anaerobic soil bacteria, 6 isolates, identified as Thermoanaerobacterium sp., displayed an ability to utilize a wide range of oligosaccharides and lignocellulosic substrates. The isolates exhibited significant cellulase and xylanase activities at high temperature (65°C). Among all isolates, Thermoanaerobacterium sp. strain R63 exhibited remarkable hydrolytic properties with the highest cellulase and xylanase activities at 1.15 U/mg and 6.17 U/mg, respectively. Extracellular extract of Thermoanaerobacterium sp. strain R63 was thermostable with an optimal temperature at 65°C and could exhibit enzymatic activities on pH range 5.0–9.0. Our findings suggest promising applications of these thermoanaerobic bacteria and their potent enzymes for industrial purposes. [ABSTRACT FROM AUTHOR]

Drug resistance in Clostridioides difficile becomes a public health concern worldwide, especially as the hypervirulent strains show decreased susceptibility to the first-line antibiotics for C. difficile treatment. Therefore, the simultaneous discovery and development of new compounds to fight this pathogen are urgently needed. In order to determinate new drugs active against C. difficile, we identified ticagrelor, utilized for the prevention of thrombotic events, as exhibiting potent growth-inhibitory activity against C. difficile. Whole-cell growth inhibition assays were performed and compared to vancomycin and metronidazole, followed by determining time-kill kinetics against C. difficile. Activities against biofilm formation and spore germination were also evaluated. Leakage analyses and electron microscopy were applied to confirm the disruption of membrane structure. Finally, ticagrelor's ability to synergize with vancomycin and metronidazole was determined using checkerboard assays. Our data showed that ticagrelor exerted activity with a MIC range of 20–40 µg/mL against C. difficile. This compound also exhibited an inhibitory effect on biofilm formation and spore germination. Additionally, ticagrelor did not interact with vancomycin nor metronidazole. Our findings revealed for the first time that ticagrelor could be further developed as a new antimicrobial agent for fighting against C. difficile. [ABSTRACT FROM AUTHOR]

Disturbances in cholesterol homeostasis of the bile duct epithelium, including transport interruption and the hyperaccumulation of intracellular cholesterol can lead to the initiation and progression of cholangiocarcinoma (CCA). Statins, which are lipid-lowering drugs, have been previously documented to exhibit anti-cancer properties. The role of statins in CCA cell cholesterol transport through the expression and function of ATP-binding cassette (ABC) A1 and ABCG1 was investigated in the current study. In four CCA cell lines, ABCA1 and ABCG1 expression was identified. However, neither ABCG5 nor ABCG8 expression was observed. Immunocytochemistry revealed that the expression of ABCA1 was localized in the proximity of the nucleus, while ABCG1 was more dispersed throughout the cytoplasm of KKU-100 cells. A cholesterol efflux assay was performed using bodipy cholesterol, and the translocation of cholesterol via ABCA1 and ABCG1 to Apo-A1 and high density lipoprotein was confirmed, respectively. Simvastatin and atorvastatin demonstrated the inhibitory effects on CCA cell viability. A reduction in intracellular lipid level and a lower expression of ABCA1 and ABCG1 were observed in KKU-100 cells under simvastatin treatment. The pre-exposure of KKU-100 cells to cholesterol diminished the statin effect. Furthermore, when KKU-100 cells were pre-loaded with cholesterol, ABCA1 and ABCG1-mediated exports were unaffected even though they were treated with simvastatin. The results of the current study indicated the limitations of the use of statin in CCA therapy, particularly under hypercholesterolemia conditions. [ABSTRACT FROM AUTHOR]

Andrographolide, a bioactive phytochemical from Andrographis paniculata , is emerging as a promising anticancer agent against various cancers. This study aims to investigate anticancer activities of andrographolide against cholangiocarcinoma (CCA) and to understand the underlying mechanism. The anti-proliferative activity of andrographolide was evaluated in a range of cholangiocarcinoma (CCA) cell lines including HuCCA-1, KKU-100, KKU-M213, and RMCCA-1. The anti-migration activity and the corresponding mechanism were studied in highly metastatic KKU-M213 cells. The results indicated that andrographolide significantly inhibited the proliferation of CCA cells with the 50% inhibitory growth concentration (IC50) of ∼120 µM. Andrographolide also inhibited CCA cell migration and invasion. Our further explorations demonstrated that andrographolide decreased the expression of claudin-1, a major tight junction protein, while it up-regulated the expression of Snail, a transcriptional repressor of claudin-1. Moreover, andrographolide induced the phosphorylation of Jun N-terminus kinase (JNK) and p-38 Mitogen-activated protein kinase (MAPK). Treatment with the p-38-specific inhibitor recovered the claudin-1 expression and migration ability of CCA cells. This work demonstrated the potential anticancer effects of andrographolide, indicating that andrographolide could inhibit CCA cell migration via suppression of claudin-1 through the activation of p-38 MAPK signaling pathway. This compound would be useful for development of alternative therapeutic agent for CCA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]