Journal of Microbiology

Journal Articles

Eradication of drug-resistant Acinetobacter baumannii by cell-penetrating peptide fused endolysin: Jeonghyun Lim , Jaeyeon Jang , Heejoon Myung , Miryoung Song; J. Microbiol. 2022;60(8):859-866. Published online May 25, 2022; DOI: https://doi.org/10.1007/s12275-022-2107-y

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Abstract: Antimicrobial agents targeting peptidoglycan have shown successful results in eliminating bacteria with high selective toxicity. Bacteriophage encoded endolysin as an alternative antibiotics is a peptidoglycan degrading enzyme with a low rate of resistance. Here, the engineered endolysin was developed to defeat multiple drug-resistant (MDR) Acinetobacter baumannii. First, putative endolysin PA90 was predicted by genome analysis of isolated Pseudomonas phage PBPA. The His-tagged PA90 was purified from BL21(DE3) pLysS and tested for the enzymatic activity using Gram-negative pathogens known for having a high antibiotic resistance rate including A. baumannii. Since the measured activity of PA90 was low, probably due to the outer membrane, cell-penetrating peptide (CPP) DS4.3 was introduced at the N-terminus of PA90 to aid access to its substrate. This engineered endolysin, DS-PA90, completely killed A. baumannii at 0.25 μM, at which concentration PA90 could only eliminate less than one log in CFU/ml. Additionally, DS-PA90 has tolerance to NaCl, where the ~50% of activity could be maintained in the presence of 150 mM NaCl, and stable activity was also observed with changes in pH or temperature. Even MDR A. baumannii strains were highly susceptible to DS-PA90 treatment: five out of nine strains were entirely killed and four strains were reduced by 3–4 log in CFU/ml. Consequently, DS-PA90 could protect waxworm from A. baumannii-induced death by ~70% for ATCC 17978 or ~44% for MDR strain 1656-2 infection. Collectively, our data suggest that CPP-fused endolysin can be an effective antibacterial agent against Gramnegative pathogens regardless of antibiotics resistance mechanisms.

Application of fast expectation-maximization microbial source tracking to discern fecal contamination in rivers exposed to low fecal inputs: Youfen Xu , Ganghua Han , Hongxun Zhang , Zhisheng Yu , Ruyin Liu; J. Microbiol. 2022;60(6):594-601. Published online April 18, 2022; DOI: https://doi.org/10.1007/s12275-022-1651-9

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Abstract: Community-based microbial source tracking (MST) can be used to determine fecal contamination from multiple sources in the aquatic environment. However, there is little scientific information on its application potential in water environmental management. Here, we compared SourceTracker and Fast Expectation-maximization Microbial Source Tracking (FEAST) performances on environmental water bodies exposed to low fecal pollution and evaluated treatment effects of fecal pollution in the watershed utilizing community-based MST. Our results showed that FEAST overall outperformed SourceTracker in sensitivity and stability, and was able to discern multi-source fecal contamination (mainly chicken feces) in ambient water bodies exposed to low fecal inputs. Consistent with our previous PCR/qPCR-based MST assays, FEAST analysis indicates that fecal pollution has been significantly mitigated through comprehensive environmental treatment by the local government. This study suggests that FEAST can be a powerful tool for accurately evaluating the contribution of multi-source fecal contamination in environmental water, facilitating environmental management.

Vibrio parahaemolyticus cqsA controls production of quorum sensing signal molecule 3-hydroxyundecan-4-one and regulatessensing signal molecule 3-hydroxyundecan-4-one and regulates colony morphology: Kui Wu , Yangyun Zheng , Qingping Wu , Haiying Chen , Songzhe Fu , Biao Kan , Yongyan Long , Xiansheng Ni , Junling Tu; J. Microbiol. 2019;57(12):1105-1114. Published online November 4, 2019; DOI: https://doi.org/10.1007/s12275-019-9379-x

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Abstract: In order to adapt to different environments, Vibrio parahaemolyticus employed a complicated quorum sensing system to orchestrate gene expression and diverse colony morphology patterns. In this study, the function of the putative quorum sensing signal synthase gene cqsA (VPA0711 in V. parahaemolyticus strain RIMD2210633 genome) was investigated. The cloning and expression of V. parahaemolyticus cqsA in Escherichia coli system induced the production of a new quorum sensing signal that was found in its culture supernatant. The signal was purified by high performance liquid chromatography
methods
and determined to be 3-hydroxyundecan- 4-one by indirect and direct mass spectra assays. The deletion of cqsA in RIMD2210633 changed V. parahaemolyticus colony morphology from the classical ‘fried-egg’ shape (thick and opaque in the center, while thin and translucent in the edge) of the wild-type colony to a ‘pancake’ shape (no significant difference between the centre and the edge) of the cqsAdeleted colony. This morphological change could be restored by complementary experiment with cqsA gene or the signal extract. In addition, the expression of opaR, a well-known quorum sensing regulatory gene, could be up-regulated by cqsA deletion. Our results suggested that V. parahaemolyticus used cqsA to produce 3-hydroxyundecan-4-one signal and thereby regulated colony morphology and other quorum sensing-associated behaviors.

Partial characteristics of hemolytic factors secreted from airborne Aspergillus and Penicillium, and an enhancement of hemolysis by Aspergillus micronesiensis CAMP-like factor via Staphylococcus aureus-sphingomyelinase: Sumonrat Kaveemongkonrat , Kwanjit Duangsonk , Jos Houbraken , Phimchat Suwannaphong , Nongnuch Vanittanakom Vanittanakom , Malee Mekaprateep; J. Microbiol. 2019;57(12):1086-1094. Published online November 4, 2019; DOI: https://doi.org/10.1007/s12275-019-9133-4

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Abstract: One of the advantages for initial survival of inhaled fungal spores in the respiratory tract is the ability for iron acquisition via hemolytic factor-production. To examine the ability of indoor Aspergillus and Penicillium affecting hemolysis, the secreted factors during the growth of thirteen strains from eight species were characterized in vitro for their hemolytic activity (HA) and CAMP-like reaction. The hemolytic index of HA on human blood agar of Aspergillus micronesiensis, Aspergillus wentii, Aspergillus westerdijkiae, Penicillium citrinum, Penicillium copticola, Penicillium paxilli, Penicillium steckii, and Penicillium sumatrense were 1.72 ± 0.34, 1.61 ± 0.41, 1.69 ± 0.16, 1.58 ± 0.46, 3.10 ± 0.51, 1.22 ± 0.19, 2.55 ± 0.22, and 1.90 ± 0.14, respectively. The secreted factors of an Aspergillus wentii showed high HA when grown in undernourished broth at 25°C at an exponential phase and were heat sensitive. Its secreted proteins have an estimated relative molecular weight over 50 kDa. Whereas, the factors of Penicillium steckii were secreted in a similar condition at a late exponential phase but showed low HA and heat tolerance. In a CAMP-like test with sheep blood, the synergistic hemolytic reactions between most tested mold strains and Staphylococcus aureus were identified. Moreover, the enhancement of α-hemolysis of Staphylococcus aureus could occur through the interaction of Staphylococcus aureus-sphingomyelinase and CAMP-like factors secreted from Aspergillus micronesiensis. Further studies on the characterization of purified hemolytic- and CAMP-like-factors secreted from Aspergillus wentii and Aspergillus micronesiensis may lead to more understanding of their involvement of hemolysis

Identification and characterization of a marine-derived chitinolytic fungus, Acremonium sp. YS2-2: Dawoon Chung , Kyunghwa Baek , Seung Seob Bae , Jaejoon Jung; J. Microbiol. 2019;57(5):372-380. Published online February 26, 2019; DOI: https://doi.org/10.1007/s12275-019-8469-0

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Abstract: Chitin is the most abundant biopolymer in marine environments. To facilitate its utilization, our laboratory screened marine-derived fungal strains for chitinolytic activity. One chitinolytic strain isolated from seawater, designated YS2-2, was identified as Acremonium species based on morphological and phylogenetic analyses. Acremonium species are cosmopolitan fungi commonly isolated from both terrestrial and marine environments, but their chitinolytic activity is largely unknown. The extracellular crude enzyme of YS2-2 exhibited optimum chitinolytic activity at pH 6.0–7.6, 23–45°C, and 1.5% (w/v) NaCl. Degenerate PCR revealed the partial cDNA sequence of a putative chitinase gene, chiA, in YS2-2. The expression of chiA was dramatically induced in response to 1% (w/v) colloidal chitin compared to levels under starvation, chitin powder, and glucose conditions. Moreover, the chiA transcript levels were positively correlated with chitinolytic activities under various colloidal chitin concentrations, suggesting that ChiA mediates chitinolytic activity in this strain. Our results provide a basis for additional studies of marinederived chitinolytic fungi aimed at improving industrial applications.

Lytic KFS-SE2 phage as a novel bio-receptor for Salmonella Enteritidis detection: In Young Choi , Cheonghoon Lee , Won Keun Song , Sung Jae Jang , Mi-Kyung Park; J. Microbiol. 2019;57(2):170-179. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-8610-0

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Abstract: Since Salmonella Enteritidis is one of the major foodborne pathogens, on-site applicable rapid detection methods have been required for its control. The purpose of this study was to isolate and purify S. Enteritidis-specific phage (KFS-SE2 phage) from an eel farm and to investigate its feasibility as a novel, efficient, and reliable bio-receptor for its employment. KFS-SE2 phage was successfully isolated at a high concentration of (2.31 ± 0.43) × 1011 PFU/ml, and consisted of an icosahedral head of 65.44 ± 10.08 nm with a non-contractile tail of 135.21 ± 12.41 nm. The morphological and phylogenetic analysis confirmed that it belongs to the Pis4avirus genus in the family of Siphoviridae. KFS-SE2 genome consisted of 48,608 bp with 45.7% of GC content. Genome analysis represented KFS-SE2 to have distinctive characteristics as a novel phage. Comparative analysis of KFS-SE2 phage with closely related strains confirmed its novelty by the presence of unique proteins. KFS-SE2 phage exhibited excellent specificity to S. Enteritidis and was stable under the temperature range of 4 to 50°C and pH of 3 to 11 (P < 0.05). The latent time was determined to be 20 min. Overall, a new lytic KFS-SE2 phage was successfully isolated from the environment at a high concentration and the excellent feasibility of KFS-SE2 phage was demonstrated as a new bio-receptor for S. Enteritidis detection.

Review

[MINIREVIEW] Progress of analytical tools and techniques for human gut microbiome research: Eun-Ji Song , Eun-Sook Lee , Young-Do Nam; J. Microbiol. 2018;56(10):693-705. Published online September 28, 2018; DOI: https://doi.org/10.1007/s12275-018-8238-5

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Abstract: Massive DNA sequencing studies have expanded our insights and understanding of the ecological and functional characteristics of the gut microbiome. Advanced sequencing technologies allow us to understand the close association of the gut microbiome with human health and critical illnesses. In the future, analyses of the gut microbiome will provide key information associating with human individual health, which will help provide personalized health care for diseases. Numerous molecular biological analysis tools have been rapidly developed and employed for the gut microbiome researches; however, methodological differences among researchers lead to inconsistent data, limiting extensive share of data. It is therefore very essential to standardize the current
method
ologies and establish appropriate pipelines for human gut microbiome research. Herein, we review the methods and procedures currently available for studying the human gut microbiome, including fecal sample collection, metagenomic DNA extraction, massive DNA sequencing, and data analyses with bioinformatics. We believe that this review will contribute to the progress of gut microbiome research in the clinical and practical aspects of human health.

Journal Articles

Proposal of three novel species of soil bacteria, Variovorax ureilyticus, Variovorax rhizosphaerae, and Variovorax robiniae, in the family Comamonadaceae: Tuan Manh Nguyen , Ngoc Hoang Trinh , Jaisoo Kim; J. Microbiol. 2018;56(7):485-492. Published online June 14, 2018; DOI: https://doi.org/10.1007/s12275-018-8025-3

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Abstract: Three novel bacterial strains (UCM-2T, UCM-G28T, and UCM-G35T) were obtained while isolating soil bacteria for the development of antibiotics. Cells of these strains were Gram-negative, non-spore forming, motile by means of a single flagellum, and rod shaped. In all strains, the predominant isoprenoid quinone was ubiquinone-8 (Q-8). Cells contained C16:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), and C17:0 cyclo as the major fatty acids, and C10:0 3-OH as the major hydroxy fatty acid. The polar lipid profiles of the three novel strains were dominated by diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. The genomic DNA G + C contents of strains UCM-2T, UCM-G28T, and UCMG35T were 67.5, 65.9, and 66.4 mol%, respectively. Phylogenetic analyses based on 16S rRNA sequences showed that strain UCM-2T was most closely related to Variovorax soli NBRC 106424T, whereas strains UCM-G28T and UCM-G35T were most similar to Variovorax ginsengisoli Gsoil 3165T. Values indicating DNA-DNA hybridization between the novel isolates and closely related species in the genus Variovorax were lower than the 70% cut-off point. These phenotypic, chemotaxonomic, and phylogenetic data indicate that the three isolates should be classified as new members of the genus Variovorax, for which the names Variovorax ureilyticus sp. nov., Variovorax rhizosphaerae sp. nov., and Variovorax robiniae sp. nov. are proposed. The type strains are UCM-2T (= KACC 18899T = NBRC 112306T), UCMG28T (= KACC 18900T = NBRC 112307T), and UCM-G35T (= KACC 18901T = NBRC 112308T), respectively.

Promising cellulolytic fungi isolates for rice straw degradation: Diana Catalina Pedraza-Zapata , Andrea Melissa Sánchez-Garibello , Balkys Quevedo-Hidalgo , Nubia Moreno-Sarmiento , Ivonne Gutiérrez-Rojas; J. Microbiol. 2017;55(9):711-719. Published online September 2, 2017; DOI: https://doi.org/10.1007/s12275-017-6282-1

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Abstract: The objective of this study was to evaluate the potential of eight fungal isolates obtained from soils in rice crops for straw degradation in situ. From the initial eight isolates, Pleurotus ostreatus T1.1 and Penicillium sp. HC1 were selected for further characterization based on qualitative cellulolytic enzyme production and capacity to use rice straw as a sole carbon source. Subsequently, cellulolytic, xylanolytic, and lignolytic (Pleurotus ostreatus) activity on carboxymethyl cellulose, oat xylan, and rice straw with different nitrogen sources was evaluated. From the results obtained it was concluded both isolates are capable to produce enzymes necessary for rice straw degradation. However, their production is dependent upon carbon and nitrogen source. Last, it was established that Pleurotus ostreatus T1.1 and Penicillium sp. HC1 capability to colonize and mineralize rice straw, in mono-and co-culture, without affecting nitrogen soil content.

Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus: Van-Trinh Luu , Hye Yun Moon , Jee Youn Hwang , Bo-Kyu Kang , Hyun Ah Kang; J. Microbiol. 2017;55(8):655-664. Published online July 28, 2017; DOI: https://doi.org/10.1007/s12275-017-7218-5

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Abstract: Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARSbased plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNVCP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.

NMR-based metabolomics reveals the metabolite profiles of Vibrio parahaemolyticus under ferric iron stimulation: Jun Zhou , Chenyang Lu , Dijun Zhang , Chennv Ma , Xiurong Su; J. Microbiol. 2017;55(8):628-634. Published online July 28, 2017; DOI: https://doi.org/10.1007/s12275-017-6551-z

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Abstract: Vibrio parahaemolyticus is a halophilic bacterium endemic to coastal areas, and its pathogenicity has caused widespread seafood poisoning. In our previous research, the protein expression of V. parahaemolyticus in Fe3+ medium was determined using isobaric tags for relative and absolute quantitation (iTRAQ). Here, nuclear magnetic resonance (NMR) was used to detect changes in the V. parahaemolyticus metabolome. NMR spectra were obtained using methanol-water extracts of intracellular metabolites from V. parahaemolyticus under various culture conditions, and 62 metabolites were identified, including serine, arginine, alanine, ornithine, tryptophan, glutamine, malate, NAD+, NADP+, oxypurinol, xanthosine, dCTP, uracil, thymine, hypoxanthine, and betaine. Among these, 21 metabolites were up-regulated after the stimulation of the cells by ferric iron, and 9 metabolites were down-regulated. These metabolites are involved in amino acid and protein synthesis, energy metabolism, DNA and RNA synthesis and osmolality. Based on these results, we conclude that Fe3+ influences the metabolite profiles of V. parahaemolyticus.

Crystal structure of the inactive state of the receiver domain of Spo0A from Paenisporosarcina sp. TG-14, a psychrophilic bacterium isolated from an Antarctic glacier: Chang Woo Lee , Sun-Ha Park , Sung Gu Lee , Seung Chul Shin , Se Jong Han , Han-Woo Kim , Hyun Ho Park , Sunghwan Kim , Hak Jun Kim , Hyun Park , HaJeung Park , Jun Hyuck Lee; J. Microbiol. 2017;55(6):464-474. Published online March 9, 2017; DOI: https://doi.org/10.1007/s12275-017-6599-9

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Abstract: The two-component phosphorelay system is the most pre-valent mechanism for sensing and transducing environ-mental signals in bacteria. Spore formation, which relies on the two-component phosphorelay system, enables the long- term survival of the glacial bacterium Paenisporosarcina sp. TG-14 in the extreme cold environment. Spo0A is a key re-sponse regulator of the phosphorelay system in the early stage of spore formation. The protein is composed of a regu-latory N-terminal phospho-receiver domain and a DNA- binding C-terminal activator domain. We solved the three- dimensional structure of the unphosphorylated (inactive) form of the receiver domain of Spo0A (PaSpo0A-R) from Paenisporosarcina sp. TG-14. A structural comparison with phosphorylated (active form) Spo0A from Bacillus stearo-thermophilus (BsSpo0A) showed minor notable differences. A molecular dynamics study of a model of the active form and the crystal structures revealed significant differences in the α4 helix and the preceding loop region where phosphorylation occurs. Although an oligomerization study of PaSpo0A-R by analytical ultracentrifugation (AUC) has shown that the protein is in a monomeric state in solution, both crosslinking and crystal-packing analyses indicate the possibility of weak dimer formation by a previously undocumented mechanism. Collectively, these observations provide insight into the me-chanism of phosphorylation-dependent activation unique to Spo0A.

Azohydromonas riparia sp. nov. and Azohydromonas ureilytica sp. nov. isolated from a riverside soil in South Korea: Tuan Manh Nguyen , Jaisoo Kim; J. Microbiol. 2017;55(5):330-336. Published online January 26, 2017; DOI: https://doi.org/10.1007/s12275-017-6519-z

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Abstract: White and pale yellow coloured bacteria were isolated from the riverside soil, Daejeon, South Korea, and were designated UCM-11T, UCM-F25, and UCM-80T. We found that all strains were able to reduce nitrate, and the cells were aerobic and motile. The DNA G+C contents of UCM-11T, UCM-F25, and UCM-80T were between 68.9 to 71.2 mol% and the main ubiquinone was observed as Q-8. Based on16S rRNA gene sequences, strains UCM-11T and UCM-F25 were found to closely match with Azohydromonas australica IAM 12664T (98.48–98.55%), and the strain UCM-80T was the closest match with Azohydromonas lata IAM 12599T (98.34%). The presence of summed feature 3 (C16:1ω7c and/or C16:1ω6c), C16:0, summed feature 8 (C18:1ω7c and/or C18:1ω6c) as well as twokinds of hydroxyfatty acids consisting of C10:0 3-OH and C12:0 2-OH, and branched fatty acids containing C16:0 iso and C17:0 cyclo were detected in all the strains. Phosphatidy-lethanolamine was a major polar lipid. DNA–DNA related-ness confirmed UCM-11T, UCM-F25 and UCM-80T as novel members of the genus Azohydromonas. Based on the mor-phological, physiological, biochemical and genotypic char-acteristics, we suggest that strains UCM-11T, UCM-F25, and UCM-80T represent novel species within the genus Azohy-dromonas. The names Azohydromonas riparia sp. nov., and Azohydromonas ureilytica sp. nov. are proposed for the type strains UCM-11T (=KACC 18570T =NBRC 111646T) and UCM-80T (=KACC 18576T =NBRC 111658T), respectively.

Alteration in the ultrastructural morphology of mycelial hyphae and the dynamics of transcriptional activity of lytic enzyme genes during basidiomycete morphogenesis: Elena Vetchinkina , Maria Kupryashina , Vladimir Gorshkov , Marina Ageeva , Yuri Gogolev , Valentina Nikitina; J. Microbiol. 2017;55(4):280-288. Published online January 26, 2017; DOI: https://doi.org/10.1007/s12275-017-6320-z

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Abstract: The morphogenesis of macromycetes is a complex multilevel process resulting in a set of molecular-genetic, physiological- biochemical, and morphological-ultrastructural changes in the cells. When the xylotrophic basidiomycetes Lentinus edodes, Grifola frondosa, and Ganoderma lucidum were grown on wood waste as the substrate, the ultrastructural morphology of the mycelial hyphal cell walls differed considerably between mycelium and morphostructures. As the macromycetes passed from vegetative to generative development, the expression of the tyr1, tyr2, chi1, chi2, exg1, exg2, and exg3 genes was acti-vated. These genes encode enzymes such as tyrosinase, chi-tinase, and glucanase, which play essential roles in cell wall growth and morphogenesis.

Reovirus safety study for proliferation and differentiation of human adipose-derived mesenchymal stem cells: Jeong-Soo Park , Manbok Kim; J. Microbiol. 2017;55(1):75-79. Published online December 30, 2016; DOI: https://doi.org/10.1007/s12275-017-6542-0

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Abstract: Naturally occurring reoviruses are live replication-proficient viruses specifically infecting human cancer cells while sparing the normal counterparts. Stem cells can be highly susceptible to viral infection due to their innate high proliferation potential and other active signaling pathways of cells that might be involved in viral tropism. In the previous study, we showed that reoviruses could adversely affect murine embryonic stem cells’ integrity in vitro and in vivo. Oncolytic viruses, delivered systemically face many hurdles that also impede their localization and infection of, metastatic tumors, due to a variety of immune and physical barriers. To overcome such hurdles to systemic delivery, several studies supported the idea that certain types of cells, including mesenchymal stem cells, might play a role as cell carriers for oncolytic viruses. Thus, it would be interesting to examine whether human adult stem cells such as human adipose-derived mesenchymal stem cells could be saved by the reoviral challenge. In this study, we report that biological activities such as proliferation and multipotency of human adipose-derived stem cells are not affected by wild-type reovirus challenge as evidenced by survival, osteogenic and adipogenic differentiation potential assays following treatment with reoviruses. Therefore, unlike murine embryonic stem cells, our study strongly suggests that human adipose-derived adult stem cells could be spared in vivo during wild-type reoviral anti-cancer therapeutics in a clinical setting. Furthermore, the results support the possible clinical use of human adipose-derived stem cells as an effective cell carrier of oncolytic reovirus to maximize their tumor tropism and anti-tumor activity.

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