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Secondary active transporters shuttle substrates across eukaryotic and prokaryotic membranes, utilizing different electrochemical gradients. They are recognized as one of the antimicrobial efflux pumps among pathogens. While primary active transporters within the genome of C. difficile 630 have been completely cataloged, the systematical study of secondary active transporters remains incomplete. Here, we not only identify secondary active transporters but also disclose their evolution and role in drug resistance in C. difficile 630. Our analysis reveals that C. difficile 630 carries 147 secondary active transporters belonging to 27 (super)families. Notably, 50 (34%) of them potentially contribute to antimicrobial resistance (AMR). AMR-secondary active transporters are structurally classified into five (super)families: the p-aminobenzoyl-glutamate transporter (AbgT), drug/metabolite transporter (DMT) superfamily, major facilitator (MFS) superfamily, multidrug and toxic compound extrusion (MATE) family, and resistance-nodulation-division (RND) family. Surprisingly, complete RND genes found in C. difficile 630 are likely an evolutionary leftover from the common ancestor with the diderm. Through protein structure comparisons, we have potentially identified six novel AMR-secondary active transporters from DMT, MATE, and MFS (super)families. Pangenome analysis revealed that half of the AMR-secondary transporters are accessory genes, which indicates an important role in adaptive AMR function rather than innate physiological homeostasis. Gene expression profile firmly supports their ability to respond to a wide spectrum of antibiotics. Our findings highlight the evolution of AMR-secondary active transporters and their integral role in antibiotic responses. This marks AMR-secondary active transporters as interesting therapeutic targets to synergize with other antibiotic activity.

Abstract A poor outcome for cholangiocarcinoma (CCA) patients is still a clinical challenge. CCA is typically recognized by the desmoplastic nature, which accounts for its malignancy. Among various extracellular matrix proteins, laminin is the most potent inducer for CCA migration. Herein, we accessed the expression profiles of laminin gene family and explored the significance of the key laminin subunit on CCA aggressiveness. Of all 11 laminin genes, LAMA3, LAMA5, LAMB3 and LAMC2 were concordantly upregulated based on the analysis of multiple public transcriptomic datasets and also overexpressed in Thai CCA cell lines and patient tissues in which LAMA3A upregulated in the highest frequency (97%) of the cases. Differential expression genes (DEGs) analysis of low and high laminin signature groups revealed LAMA3 as the sole common DEG in all investigated datasets. Restratifying CCA samples according to LAMA3 expression indicated the association of LAMA3 in the focal adhesion pathway. Silencing LAMA3 revealed that it plays important roles in CCA cell proliferation, adhesion, migration and epithelial-to-mesenchymal transition. Taken together, this research signifies the roles of dysregulated ECM homeostasis in CCA malignancy and highlights, for the first time, the potential usage of LAMA3 as the diagnostic biomarker and the therapeutic target to tackle the CCA stromal.

Background: Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium that is commonly found in the Thai population. CCA has poor prognosis and a low survival rate due to the lack of early diagnosis methods and the limited effectiveness of current treatments. A number of oncogenic spliced-transcripts resulting from mRNA splicing errors have been reported in CCA, and aberrant mRNA splicing is suspected to be a key driver of this cancer type. The hyperphosphorylation of serine/arginine rich-splicing factors (SRSFs) by serine/arginine protein kinases (SRPKs) causes them to translocate to the nucleus where they facilitate gene splicing errors that generate cancer-related mRNA/protein isoforms. Methods: The correlation between SRPK expression and the survival of CCA patients was analyzed using data from The Cancer Genome Atlas (TCGA) dataset. The effect of SRPK inhibitors (SRPIN340 and SPHINX31) on two CCA cell lines (KKU-213A and TFK-1) was also investigated. The induction of cell death was studied by Calcein-AM/PI staining, AnnexinV/7AAD staining, immunofluorescence (IF), and Western blotting (WB). The phosphorylation and nuclear translocation of SRSFs was tracked by WB and IF, and the repair of splicing errors was examined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Results: High levels of SRPK1 and SRPK2 transcripts, and in particular SRPK1, correlated with shorter survival in CCA patients. SRPIN340 and SPHINX31 increased the number of dead and apoptotic cells in a dose-dependent manner. CCA also showed diffuse expression of cytoplasmic cytochrome C and upregulation of cleaved caspase-3. Moreover, SRSFs showed low levels of phosphorylation, resulting in the accumulation of cytoplasmic SRSF1. To link these phenotypes with aberrant gene splicing, the apoptosis-associated genes Bridging Integrator 1 (BIN1), Myeloid cell leukemia factor 1 (MCL-1) and B-cell lymphoma 2 (BCL2) were selected for further investigation. Treatment with SRPIN340 and SPHINX31 decreased anti-apoptotic BIN1+12A and increased pro-apoptotic MCL-1S and BCL-xS. Conclusions: The SRPK inhibitors SRPIN340 and SPHINX31 can suppress the phosphorylation of SRSFs and their nuclear translocation, thereby producing BIN1, MCL-1 and BCL2 isoforms that favor apoptosis and facilitate CCA cell death.

ABSTRACTA complex microbial community in the gut may prevent the colonization of enteric pathogens such as Salmonella. Some individual or a combination of species in the gut may confer colonization resistance against Salmonella. To gain a better understanding of the colonization resistance against Salmonella enterica, we isolated a library of 1,300 bacterial strains from feral chicken gut microbiota which represented a total of 51 species. Using a co-culture assay, we screened the representative species from this library and identified 30 species that inhibited Salmonella enterica subspecies enterica serovar Typhimurium in vitro. To improve the Salmonella inhibition capacity, from a pool of fast-growing species, we formulated 66 bacterial blends, each of which composed of 10 species. Bacterial blends were more efficient in inhibiting Salmonella as compared to individual species. The blend that showed maximum inhibition (Mix10) also inhibited other serotypes of Salmonella frequently found in poultry. The in vivo effect of Mix10 was examined in a gnotobiotic and conventional chicken model. The Mix10 consortium significantly reduced Salmonella load at day 2 post-infection in gnotobiotic chicken model and decreased intestinal tissue damage and inflammation in both models. Cell-free supernatant of Mix10 did not show Salmonella inhibition, indicating that Mix10 inhibits Salmonella through either nutritional competition, competitive exclusion, or through reinforcement of host immunity. Out of 10 species, 3 species in Mix10 did not colonize, while 3 species constituted more than 70% of the community. Two of these species were previously uncultured bacteria. Our approach could be used as a high-throughput screening system to identify additional bacterial sub-communities that confer colonization resistance against enteric pathogens and its effect on the host.IMPORTANCESalmonella colonization in chicken and human infections originating from Salmonella-contaminated poultry is a significant problem. Poultry has been identified as the most common food linked to enteric pathogen outbreaks in the United States. Since multi-drug-resistant Salmonella often colonize chicken and cause human infections, methods to control Salmonella colonization in poultry are needed. The method we describe here could form the basis of developing gut microbiota-derived bacterial blends as a microbial ecosystem therapeutic against Salmonella.

IntroductionBile duct cancer (cholangiocarcinoma, CCA) has a poor prognosis for patients, and despite recent advances in targeted therapies for other cancer types, it is still treated with standard chemotherapy. Anaplastic lymphoma kinase (ALK) has been shown to be a primary driver of disease progression in lung cancer, and ALK inhibitors are effective therapeutics in aberrant ALK-expressing tumors. Aberrant ALK expression has been documented in CCA, but the use of ALK inhibitors has not been investigated. Using CCA cell lines and close-to-patient primary cholangiocarcinoma cells, we investigated the potential for ALK inhibitors in CCA.MethodsALK, cMET, and ROS1 expression was determined in CCA patient tissue by immunohistochemistry and digital droplet polymerase chain reaction, and that in cell lines was determined by immunoblot and immunofluorescence. The effect on cell viability and mechanism of action of ALK, cMet, and ROS1 inhibitors was determined in CCA cell lines. To determine whether ceritinib could affect primary CCA cells, tissue was taken from four patients with biliary tract cancer, without ALK rearrangement, mutation, or overexpression, and grown in three-dimensional tumor growth assays in the presence or absence of humanized mesenchymal cells.ResultsALK and cMet but not ROS were both upregulated in CCA tissues and cell lines. Cell survival was inhibited by crizotinib, a c-met/ALK/ROS inhibitor. To determine the mechanism of this effect, we tested c-Met-specific and ALK/ROS-specific inhibitors, capmatinib and ceritinib, respectively. Whereas capmatinib did not affect cell survival, ceritinib dose-dependently inhibited survival in all cell lines, with IC50 ranging from 1 to 9 µM and co-treatments with gemcitabine and cisplatin further sensitized cells, with IC50 ranging from IC50 0.60 to 2.32 µM. Ceritinib did not inhibit cMet phosphorylation but did inhibit ALK phosphorylation. ALK was not mutated in any of these cell lines. Only ceritinib inhibited 3D growth of all four patient samples below mean peak serum concentration, in the presence and absence of mesenchymal cells, whereas crizotinib and capmatinib failed to do this. Ceritinib appeared to exert its effect more through autophagy than apoptosis.DiscussionThese results indicate that ceritinib or other ALK/ROS inhibitors could be therapeutically useful in cholangiocarcinoma even in the absence of aberrant ALK/ROS1 expression.

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.

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.

BackgroundNasopharyngeal 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.MethodsMicroarrays 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.ResultsOur 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.ConclusionOur 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.

IntroductionCholangiocarcinoma (CCA) is difficult to cure due to its ineffective treatment and advanced stage diagnosis. Thoroughly mechanistic understandings of CCA pathogenesis crucially help improving the treatment success rates. Using three-dimensional (3D) cell culture platform offers several advantages over a traditional two-dimensional (2D) culture as it resembles more closely to in vivo tumor. MethodsHere, we aimed to establish the 3D CCA spheroids with lowly (KKU-100) and highly (KKU-213A) metastatic potentials to investigate the CCA migratory process and its EMT-associated galectin-3 in the 3D setting. Results and discussionFirstly, the growth of lowly metastatic KKU-100 cells was slower than highly metastatic KKU-213A cells in both 2D and 3D systems. Hollow formation was observed exclusively inside the KKU-213A spheroids, not in KKU-100. Additionally, the migration activity of KKU-213A cells was higher than that of KKU-100 cells in both 2D and 3D systems. Besides, altered expression of galectin-3 were observed across all CCA culture conditions with substantial relocalization from inside the 2D cells to the border of spheroids in the 3D system. Notably, the CCA migration was inversely proportional to the galectin-3 expression in the 3D culture, but not in the 2D setting. This suggests the contribution of culture platforms to the alternation of the CCA cell migration process. ConclusionsThus, our data revealed that 3D culture of CCA cells was phenotypically distinct from 2D culture and pointed to the superiority of using the 3D culture model for examining the CCA cellular mechanisms, providing knowledges that are better correlated with CCA phenotypes in vivo.

Receptor-binding proteins (RBPs) are located at the viral tail and mediate the initial recognition of phage to a specific bacterial host. Phage RBPs have co-evolved with numerous types of host receptors resulting in the formation of a diverse assortment of cognate pairs of RBP-receptors that function during the phage attachment step. Although several Clostridioides difficile bacteriophages have been discovered, their RBPs are poorly described. Using homology analysis, putative prophage-tail structure (pts) genes were identified from the prophage genome of the C. difficile HN10 strain. Competition and enzyme-linked immunosorbent assays, using recombinant PtsHN10M, demonstrated the interaction of this Pts to C. difficile cells, suggesting a role as a phage RBP. Gel filtration and cross-linking assay revealed the native form of this protein as a homotrimer. Moreover, truncated variants indicated that the C-terminal domain of PtsHN10M was important for binding to C. difficile cells. Interaction of PtsHN10M was also observed to the low-molecular weight subunit of surface-layer protein A (SlpA), located at the outermost surface of C. difficile cells. Altogether, our study highlights the function of PtsHN10M as an RBP and potentially paves the way toward phage engineering and phage therapy against C. difficile infection.

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.

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.

ATP-binding cassette (ABC) transporters belong to one of the largest membrane protein superfamilies, which function in translocating substrates across biological membranes using energy from ATP hydrolysis. Currently, the classification of ABC transporters in Clostridioides difficile is not complete. Therefore, the sequence-function relationship of all ABC proteins encoded within the C. difficile genome was analyzed. Identification of protein domains associated with the ABC system in the C. difficile 630 reference genome revealed 226 domains: 97 nucleotide-binding domains (NBDs), 98 transmembrane domains (TMDs), 30 substrate-binding domains (SBDs), and one domain with features of an adaptor protein. Gene organization and transcriptional unit analyses indicated the presence of 78 ABC systems comprising 28 importers and 50 exporters. Based on NBD sequence similarity, ABC transporters were classified into 12 sub-families according to their substrates. Interestingly, all ABC exporters, accounting for 64% of the total ABC systems, are involved in antibiotic resistance. Based on analysis of ABC proteins from 49 C. difficile strains, the majority of core NBDs are predicted to be involved in multidrug resistance systems, consistent with the ability of this organism to survive exposure to an array of antibiotics. Our findings herein provide another step toward a better understanding of the function and evolutionary relationships of ABC proteins in this pathogen.

ABSTRACT Endolysin is a phage-encoded cell-wall hydrolase which degrades the peptidoglycan layer of the bacterial cell wall. The enzyme is often expressed at the late stage of the phage lytic cycle and is required for progeny escape. Endolysins of bacteriophage that infect Gram-positive bacteria often comprises two domains: a peptidoglycan hydrolase and a cell-wall binding domain (CBD). Although the catalytic domain of endolysin is relatively well-studied, the precise role of CBD is ambiguous and remains controversial. Here, we focus on the function of endolysin CBD from a recently isolated Clostridioides difficile phage. We found that the CBD is not required for lytic activity, which is strongly prevented by the surface layer of C. difficile. Intriguingly, hidden Markov model analysis suggested that the endolysin CBD is likely derived from the CWB2 motif of C. difficile cell-wall proteins but possesses a higher binding affinity to bacterial cell-wall polysaccharides. Moreover, the CBD forms a homodimer, formation of which is necessary for interaction with the surface saccharides. Importantly, endolysin diffusion and sequential cytolytic assays showed that CBD of endolysin is required for the enzyme to be anchored to post-lytic cell-wall remnants, suggesting its physiological roles in limiting diffusion of the enzyme, preserving neighboring host cells, and thereby enabling the phage progeny to initiate new rounds of infection. Taken together, this study provides an insight into regulation of endolysin through CBD and can potentially be applied for endolysin treatment against C. difficile infection. IMPORTANCE Endolysin is a peptidoglycan hydrolase encoded in a phage genome. The enzyme is attractive due to its potential use as antibacterial treatment. To utilize endolysin for the therapeutic propose, understanding of the fundamental role of endolysin becomes important. Here, we investigate the function of cell-wall binding domain (CBD) of an endolysin from a C. difficile phage. The domain is homologous to a cell-wall associating module of bacterial cell-wall proteins, likely acquired during phage-host coevolution. The interaction of CBD to bacterial cell walls reduces enzyme diffusion and thereby limits cell lysis of the neighboring bacteria. Our findings indicate that the endolysin is trapped to the cell-wall residuals through CBD and might serve as an advantage for phage replication. Thus, employing a CBD-less endolysin might be a feasible strategy for using endolysin for the treatment of C. difficile infection.

Abstract Background Chemoresistance is an obstacle to the successful treatment of nasopharyngeal carcinoma (NPC). Lapatinib is a targeted tyrosine kinase inhibitor therapeutic drug also used to treat NPC, but high doses are often required to achieve a result. To investigate the mechanism for the development of Lapatinib resistance, we characterised a number of NPC cell lines to determine the role of FOXO3 and sirtuins in regulating NPC resistance. Methods Sulforhodamine B (SRB) assays, Clonogenic assays, Protein extraction, quantification and western blotting, RT qPCR, Co-immunoprecipitation assay. Results To explore novel treatment strategies, we first characterized the Lapatinib-sensitivity of a panel of NPC cell lines by SRB and clonogenic cytotoxic assays and found that the metastatic NPC (C666–1 and 5-8F) cells are highly resistant whereas the poorly metastatic lines (6-10B, TW01 and HK-1) are sensitive to Lapatinib. Western blot analysis of the Lapatinib-sensitive 6-10B and resistant 5-8F NPC cells showed that the expression of phosphorylated/inactive FOXO3 (P-FOXO3;T32), its target FOXM1 and its regulator SIRT2 correlate negatively with Lapatinib response and sensitivity, suggesting that SIRT2 mediates FOXO3 deacetylation to promote Lapatinib resistance. In agreement, clonogenic cytotoxic assays using wild-type and foxo1/3/4 −/− mouse embryonic fibroblasts (MEFs) showed that FOXO1/3/4-deletion significantly attenuates Lapatinib-induced cytotoxicity, confirming that FOXO proteins are essential for mediating Lapatinib response. SRB cell viability assays using chemical SIRT inhibitors (i.e. sirtinol, Ex527, AGK2 and AK1) revealed that all SIRT inhibitors can reduce NPC cell viability, but only the SIRT2-specific inhibitors AK1 and AGK2 further enhance the Lapatinib cytotoxicity. Consistently, clonogenic assays demonstrated that the SIRT2 inhibitors AK1 and AGK2 as well as SIRT2-knockdown increase Lapatinib cytotoxicity further in both the sensitive and resistant NPC cells. Co-immunoprecipitation studies showed that besides Lapatinib treatment, SIRT2-pharmaceutical inhibition and silencing also led to an increase in FOXO3 acetylation. Importantly, SIRT2 inhibition and depletion further enhanced Lapatinib-mediated FOXO3-acetylation in NPC cells. Conclusion Collectively, our results suggest the involvement of SIRT2-mediated FOXO3 deacetylation in Lapatinib response and sensitivity, and that SIRT2 can specifically antagonise the cytotoxicity of Lapatinib through mediating FOXO3 deacetylation in both sensitive and resistant NPC cells. The present findings also propose that SIRT2 can be an important biomarker for metastatic and Lapatinib resistant NPC and that targeting the SIRT2-FOXO3 axis may provide novel strategies for treating NPC and for overcoming chemoresistance.

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).

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.

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.

Background: The microbial community residing in the animal gastrointestinal tract play a crucial role in host health. Because of the high complexity of gut microbes, many microbes remain unclassified. Deciphering the role of each bacteria in health and diseases is only possible after its culture, identification, and characterization. During the culturomics study of feral chicken cecal sample, we cultured a possible novel strain SW165T. Methods: For the possible novel strain SW165T, phenotypic characterization was performed using colony morphology, Gram staining, growth in different temperature and pH and motility. Biochemical assays included carbon source utilization, enzymatic activity, cellular fatty acids and short chain fatty acid production. 16S rRNA sequencing and whole genome sequencing and comparison was performed for genetic analysis. Results: This strain was isolated from cecal content of feral chickens in Brookings, South Dakota, USA. Phylogenetic analyses based on 16S rRNA gene sequence revealed that the closest valid neighbor was Olsenella profusa DSM 13989T (96.33% similarity) within the family Atopobiaceae. Cells were Gram-strain-positive and obligately anaerobic bacilli in chains. The optimum temperature and pH for the growth of the microorganism were 37-45oC and pH 6.0-7.5 respectively. This strain produced acetic acid as the primary fermentation product. Major fatty acids were C12:0, C14:0, C14:0 DMA and summed feature 1 (C13:1 at 12-13 and C14:0 aldehyde). Strain SW165T exhibited a genome size of 2.43 Mbp with a G+C content of 67.59 mol%, which is the second highest G+C content among members of the genus Olsenella. The digital DNA-DNA hybridization and OrthoANI values between SW165T and DSM 13989T were only 17.6 ± 5.3 and 74.35%, respectively. Conclusion: Based on the phenotypic, biochemical, and genomic analyses, we propose the new species of the genus Olsenella, and name it Olsenella lakotia SW165T sp. nov., (=DSM 107283 =CCOS 1887) as the type strain.

Background: The microbial community residing in the animal gastrointestinal tract play a crucial role in host health. Because of the high complexity of gut microbes, many microbes remain unclassified. Deciphering the role of each bacteria in health and diseases is only possible after its culture, identification, and characterization. During the culturomics study of feral chicken cecal sample, we cultured a possible novel strain SW165T. Methods: For the possible novel strain SW165T, phenotypic characterization was performed using colony morphology, Gram staining, growth in different temperature and pH and motility. Biochemical assays included carbon source utilization, enzymatic activity, cellular fatty acids and short chain fatty acid production. 16S rRNA sequencing and whole genome sequencing and comparison was performed for genetic analysis. Results: This strain was isolated from cecal content of feral chickens in Brookings, South Dakota, USA. Phylogenetic analyses based on 16S rRNA gene sequence revealed that the closest valid neighbor was Olsenella profusa DSM 13989T (96.33% similarity) within the family Atopobiaceae. Cells were Gram-strain-positive and obligately anaerobic bacilli in chains. The optimum temperature and pH for the growth of the microorganism were 37-45oC and pH 6.0-7.5 respectively. This strain produced acetic acid as the primary fermentation product. Major fatty acids were C12:0, C14:0, C14:0 DMA and summed feature 1 (C13:1 at 12-13 and C14:0 aldehyde). Strain SW165T exhibited a genome size of 2.43 Mbp with a G+C content of 67.59 mol%, which is the second highest G+C content among members of the genus Olsenella. The digital DNA-DNA hybridization and OrthoANI values between SW165T and DSM 13989T were only 17.6 ± 5.3 and 74.35%, respectively. Conclusion: Based on the phenotypic, biochemical, and genomic analyses, we propose the new species of the genus Olsenella, and name it Olsenella lakotia SW165T sp. nov., (=DSM 107283 =CCOS 1887) as the type strain.

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.

Baicalein, a prominent flavonoid from the indigenous herbal plant Scutellaria baicalensis Georgi, possesses broad-spectrum anticancer activities. However, the biological effects of baicalein on nasopharyngeal carcinoma (NPC) and its underlying mechanisms remain unclarified. Thus, in this study, we examined the effects of baicalein on NPC cell lines and investigated the corresponding molecular mechanism through transcriptome profiling. In the study, four NPC cell lines were treated with various concentrations of baicalein at different time points. Cellular toxicity and proliferative inhibition of baicalein were examined by MTT assay. Metastatic phenotypes of NPC cells were investigated by wound healing, transwell, and adhesion assays. Additionally, microarray experiments were performed to determine the cellular pathways affected by baicalein. The expression and localization of the integrin β8 were validated by western immunoblotting and immunofluorescence. Our results revealed that baicalein exhibited its cytotoxicity and antiproliferative activity on all tested NPC cell lines. It also significantly inhibited metastatic phenotypes at sub-lethal concentrations. Transcriptomic analysis showed that baicalein significantly affected the focal adhesion pathway in NPC, where integrin β8 was greatly diminished. Thus, the present study results suggested that baicalein inhibits the metastatic phenotypes of NPC cells by modulating integrin β8, one of the major molecules in a focal adhesion pathway.

Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies that primarily originate from the bile duct. Tumor heterogeneity is a prime characteristic of CCA and considering the scarcity of approved targeted therapy drugs, this makes precision oncology impractical in CCA. Stratifying patients based on their molecular signature and biomarker-guided therapy may offer a conducive solution. Receptors tyrosine kinases (RTK) are potential targets for novel therapeutic strategies in CCA as RTK signaling is dysregulated in CCA. This study aims to identify targetable RTK profile in CCA using a bioinformatic approach. We discovered that CCA samples could be grouped into molecular subtypes based on the gene expression profile of selected RTKs (RTK25). Using the RTK25 gene list, we discovered five distinct molecular subtypes of CCA in this cohort. Tyrosine kinase inhibitors that target each RTK profile and their subsequent molecular signatures were also discovered. These results suggest that certain RTKs correlate with each other, indicating that tailored dual inhibition of RTKs may be more favorable than monotherapy. The results from this study can direct future investigative attention towards validating this concept in in vivo and in vitro systems. Ultimately, this will facilitate biomarker-guided clinical trials for the successful approval of novel therapeutic options in CCA.

In recent decades, the incidence of Clostridioides difficile infection (CDI) has remained high in both community and health-care settings. With the increasing rate of treatment failures and its ability to form spores, an alternative treatment for CDI has become a global priority. We used the microdilution assay to determine minimal inhibitory concentrations (MICs) of vancomycin and teicoplanin against 30 distinct C. difficile strains isolated from various host origins. We also examined the effect of drugs on spore germination and outgrowth by following the development of OD600. Finally, we confirmed the spore germination and cell stages by microscopy. We showed that teicoplanin exhibited lower MICs compared to vancomycin in all tested isolates. MICs of teicoplanin ranged from 0.03–0.25 µg/mL, while vancomycin ranged from 0.5–4 µg/mL. Exposure of C. difficile spores to broth supplemented with various concentrations of antimicrobial agents did not affect the initiation of germination, but the outgrowth to vegetative cells was inhibited by all test compounds. This finding was concordant with aberrant vegetative cells after antibiotic treatment observed by light microscopy. This work highlights the efficiency of teicoplanin for treatment of C. difficile through prevention of vegetative cell outgrowth.

Clostridioides difficile has been recognized as a life-threatening pathogen that causes enteric diseases, including antibiotic-associated diarrhea and pseudomembranous colitis. The severity of C. difficile infection (CDI) correlates with toxin production and antibiotic resistance of C. difficile. In Thailand, the data addressing ribotypes, toxigenic, and antimicrobial susceptibility profiles of this pathogen are scarce and some of these data sets are limited. In this study, two groups of C. difficile isolates in Thailand, including 50 isolates collected from 2006 to 2009 (THA group) and 26 isolates collected from 2010 to 2012 (THB group), were compared for toxin genes and ribotyping profiles. The production of toxins A and B were determined on the basis of toxin gene profiles. In addition, minimum inhibitory concentration of eight antibiotics were examined for all 76 C. difficile isolates. The isolates of the THA group were categorized into 27 A−B+CDT− (54%) and 23 A-B-CDT- (46%), while the THB isolates were classified into five toxigenic profiles, including six A+B+CDT+ (23%), two A+B+CDT− (8%), five A−B+CDT+ (19%), seven A−B+CDT− (27%), and six A−B−CDT− (23%). By visually comparing them to the references, only five ribotypes were identified among THA isolates, while 15 ribotypes were identified within THB isolates. Ribotype 017 was the most common in both groups. Interestingly, 18 unknown ribotyping patterns were identified. Among eight tcdA-positive isolates, three isolates showed significantly greater levels of toxin A than the reference strain. The levels of toxin B in 3 of 47 tcdB-positive isolates were significantly higher than that of the reference strain. Based on the antimicrobial susceptibility test, metronidazole showed potent efficiency against most isolates in both groups. However, high MIC values of cefoxitin (MICs 256 μg/mL) and chloramphenicol (MICs ≥ 64 μg/mL) were observed with most of the isolates. The other five antibiotics exhibited diverse MIC values among two groups of isolates. This work provides evidence of temporal changes in both C. difficile strains and patterns of antimicrobial resistance in Thailand.

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 A gnotobiotic Gallus gallus (chicken) model was developed to study the dynamics of intestinal microflora from hatching to 18 days of age employing metagenomics. Intestinal samples were collected from a local population of feral chickens and administered orally to germfree 3-day-old chicks. Animals were euthanized on days 9 and 18 postinoculation, and intestinal samples were collected and subjected to metagenomic analysis. On day 18, the five most prevalent phyla were Bacteroidetes (43.03 ± 3.19%), Firmicutes (38.51 ± 2.67%), Actinobacteria (6.77 ± 0.7%), Proteobacteria (6.38 ± 0.7%), and Spirochaetes (2.71 ± 0.55%). Principal-coordinate analysis showed that the day 18 variables clustered more closely than the day 9 variables, suggesting that the microbial communities had changed temporally. The Morista-Horn index values ranged from 0.7 to 1, indicating that the communities in the inoculum and in the day 9 and day 18 samples were more similar than dissimilar. The predicted functional profiles of the microbiomes of the inoculum and the day 9 and day 18 samples were also similar (values of 0.98 to 1). These results indicate that the gnotobiotic chicks stably maintained the phylogenetic 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 an economically valuable source of protein globally. The microbiome of poultry has important effects on chicken growth, feed conversion, immune status, and pathogen resistance. The aim of our research was to develop a gnotobiotic chicken model appropriate for the study chicken gut microbiota function. Our experimental model shows that young germfree chicks are able to colonize diverse sets of gut bacteria. Therefore, besides the use of this model to study mechanisms of gut microbiota interactions in the chicken gut, it could be also used for applied aspects such as determining the safety and efficacy of new probiotic strains derived from chicken gut microbiota.

Cholangiocarcinoma (CCA), a group of malignancies that originate from the biliary tract, is associated with a high mortality rate and a concerning increase in worldwide incidence. In Thailand, where the incidence of CCA is the highest, the socioeconomic burden is severe. Yet, treatment options are limited, with surgical resection being the only form of treatment with curative intent. The current standard-of-care remains adjuvant and palliative chemotherapy which is ineffective in most patients. The overall survival rate is dismal, even after surgical resection and the tumor heterogeneity further complicates treatment. Together, this makes CCA a significant burden in Southeast Asia. For effective management of CCA, treatment must be tailored to each patient, individually, for which an assortment of targeted therapies must be available. Despite the increasing numbers of clinical studies in CCA, targeted therapy drugs rarely get approved for clinical use. In this review, we discuss the shortcomings of the conventional clinical trial process and propose the implementation of a novel concept, co-clinical trials to expedite drug development for CCA patients. In co-clinical trials, the preclinical studies and clinical trials are conducted simultaneously, thus enabling real-time data integration to accurately stratify and customize treatment for patients, individually. Hence, co-clinical trials are expected to improve the outcomes of clinical trials and consequently, encourage the approval of targeted therapy drugs. The increased availability of targeted therapy drugs for treatment is expected to facilitate the application of precision medicine in CCA.

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.

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.

Cholangiocarcinoma (CCA) is an aggressive biliary tract malignancy arising from the epithelial bile duct. The lack of early diagnostic biomarkers as well as therapeutic measures results in severe outcomes and poor prognosis. Thus, effective early diagnostic, prognostic, and therapeutic biomarkers are required to improve the prognosis and prolong survival rates in CCA patients. Recent advancement in omics technologies combined with the integrative experimental and clinical validations has provided an insight into the underlying mechanism of CCA initiation and progression as well as clues towards novel biomarkers. This work highlights the discovery and validation of molecular markers in CCA identified through omics approaches. The possible roles of these molecules in various cellular pathways, which render CCA carcinogenesis and progression, will also be discussed. This paper can serve as a reference point for further investigations to yield deeper understanding in the complex feature of this disease, potentially leading to better approaches for diagnosis, prognosis, and therapeutics.

Nasopharyngeal carcinoma (NPC) is an uncommon cancer, which has a distinctive ethnic and geographic distribution. Etiology of NPC is considered to be related with a complex interaction of environmental and genetic factors as well as Epstein-Barr virus infection. Since NPC is located in the silent painless area, the disease is usually therefore diagnosed at the advanced stages; hence early detection of NPC is difficult. Furthermore, understanding in molecular pathogenesis is still lacking, pondering the identification of effective prognostic and diagnostic biomarkers. Dysregulation of signaling molecules in intracellular signal transduction, which regulate cell proliferation, apoptosis, and adhesion, underlines the basis of NPC pathogenesis. In this paper, the molecular signaling pathways in the NPC are discussed for the holistic view of NPC development and progression. The important insights toward NPC pathogenesis may offer strategies for identification of novel biomarkers for diagnosis and prognosis.

The impact of Clostridium difficile infection (CDI) on healthcare is becoming increasingly recognized as it represents a major cause of nosocomial diarrhea. A rising number of CDI cases and outbreaks have been reported worldwide. Here, we developed the pig ileal-ligated loop model for semi-quantitative analysis comparing temporal differential proteomes in C. difficile following in vivo incubation with in vitro growth using isobaric tags for relative and absolute quantification (iTRAQ). Proteins retrieved from the in vitro cultures and the loop contents after 4, 8, and 12 h in vivo incubation were subjected to in-solution digestion, iTRAQ labeling, two-dimensional liquid chromatography/tandem mass spectrometry and statistical analyses. From a total of 1152 distinct proteins identified in this study, 705 proteins were available for quantitative measures at all time points in both biological and technical replicates; 109 proteins were found to be differentially expressed. With analysis of clusters of orthologous group and protein-protein network interactions, we identified the proteins that might play roles in adaptive responses to the host environment, hence enhancing pathogenicity during CDI. This report represents the quantitative proteomic analysis of C. difficile that demonstrates time-dependent protein expression changes under conditions that mimic in vivo infection and identifies potential candidates for diagnostic or therapeutic measures.

Microarray-based comparative genome hybridisations (CGH) and genome sequencing of Clostridium difficile isolates have shown that the genomes of this species are highly variable. To further characterize their genome variation, we employed integration of data from CGH, genome sequencing and putative cellular pathways. Transcontinental strain comparison using CGH data confirmed the emergence of a human-specific hypervirulent cluster. However, there was no correlation between total toxin production and hypervirulent phenotype, indicating the possibility of involvement of additional factors towards hypervirulence. Calculation of C. difficile core and pan genome size using CGH and sequence data estimated that the core genome is composed of 947 to 1,033 genes and a pan genome comprised of 9,640 genes. The reconstruction, annotation and analysis of cellular pathways revealed highly conserved pathways despite large genome variation. However, few pathways such as tetrahydrofolate biosynthesis were found to be variable and could be contributing to adaptation towards virulence such as antibiotic resistance.

Background: The microbial community residing in the animal gastrointestinal tract play a crucial role in host health. Because of the high complexity of gut microbes, many microbes remain unclassified. Deciphering the role of each bacteria in health and diseases is only possible after its culture, identification, and characterization. During the culturomics study of feral chicken cecal sample, we cultured a possible novel strain SW165 T. Methods: For the possible novel strain SW165 T, phenotypic characterization was performed using colony morphology, Gram staining, growth in different temperature and pH and motility. Biochemical assays included carbon source utilization, enzymatic activity, cellular fatty acids and short chain fatty acid production. 16S rRNA sequencing and whole genome sequencing and comparison was performed for genetic analysis. Results: This strain was isolated from cecal content of feral chickens in Brookings, South Dakota, USA. Phylogenetic analyses based on 16S rRNA gene sequence revealed that the closest valid neighbor was Olsenella profusa DSM 13989 T (96.33% similarity) within the family Atopobiaceae. Cells were Gram-strain-positive and obligately anaerobic bacilli in chains. The optimum temperature and pH for the growth of the microorganism were 37-45 oC and pH 6.0-7.5 respectively. This strain produced acetic acid as the primary fermentation product. Major fatty acids were C 12:0, C 14:0, C 14:0 DMA and summed feature 1 (C 13:1 at 12-13 and C 14:0 aldehyde). Strain SW165 T exhibited a genome size of 2.43 Mbp with a G+C content of 67.59 mol%, which is the second highest G+C content among members of the genus Olsenella. The digital DNA-DNA hybridization and OrthoANI values between SW165 T and DSM 13989 T were only 17.6 ± 5.3 and 74.35%, respectively. Conclusion: Based on the phenotypic, biochemical, and genomic analyses, we propose the new species of the genus Olsenella, and name it Olsenella lakotia SW165 T sp. nov., (=DSM 107283 =CCOS 1887) as the type strain.

Background: The microbial community residing in the animal gastrointestinal tract play a crucial role in host health. Because of the high complexity of gut microbes, many microbes remain unclassified. Deciphering the role of each bacteria in health and diseases is only possible after its culture, identification, and characterization. During the culturomics study of feral chicken cecal sample, we cultured a possible novel strain SW165 T. Methods: For the possible novel strain SW165 T, phenotypic characterization was performed using colony morphology, Gram staining, growth in different temperature and pH and motility. Biochemical assays included carbon source utilization, enzymatic activity, cellular fatty acids and short chain fatty acid production. 16S rRNA sequencing and whole genome sequencing and comparison was performed for genetic analysis. Results: This strain was isolated from cecal content of feral chickens in Brookings, South Dakota, USA. Phylogenetic analyses based on 16S rRNA gene sequence revealed that the closest valid neighbor was Olsenella profusa DSM 13989 T (96.33% similarity) within the family Atopobiaceae. Cells were Gram-strain-positive and obligately anaerobic bacilli in chains. The optimum temperature and pH for the growth of the microorganism were 37-45 oC and pH 6.0-7.5 respectively. This strain produced acetic acid as the primary fermentation product. Major fatty acids were C 12:0, C 14:0, C 14:0 DMA and summed feature 1 (C 13:1 at 12-13 and C 14:0 aldehyde). Strain SW165 T exhibited a genome size of 2.43 Mbp with a G+C content of 67.59 mol%, which is the second highest G+C content among members of the genus Olsenella. The digital DNA-DNA hybridization and OrthoANI values between SW165 T and DSM 13989 T were only 17.6 ± 5.3 and 74.35%, respectively. Conclusion: Based on the phenotypic, biochemical, and genomic analyses, we propose the new species of the genus Olsenella, and name it Olsenella lakotia SW165 T sp. nov., (=DSM 107283 =CCOS 1887) as the type strain.