Mycoscience 59 (2018) 467e472 Contents lists available at ScienceDirect Application of an Environmental Phage-Based Assay Mycoscience (Sample6 Detect HL for the Detection of journal homepage: www.elsevier.com/locate/myc Listeria spp. in Ice Cream Full paper Elba Veronica Arias-Rios, Kristina Tenney, Tam Mai, Sam Anderson, Hamatispora, a new genus of aquatic fungi in Microthyriales isolated Ruth Marie Cantera, Jasmine M. Pando, Brandon Selover, Lourdes M. Nadala, from fallen leaves in Vietnam Seana K. Davidson, Mansour Samadpour Le Thi Hoang Yen a, *, Kaoru Yamaguchi b, Yasuhisa Tsurumi b, Duong Van Hop a, Katsuhiko Ando b Background: Dairy products are common sources of Listeria outbreaks, and early a Institute of Microbiology and Biotechnology, Vietnam National University, E2, 144 Xuan Thuy, Cau Giay, Ha Noi, Viet nam b detection of the pathogen is critical to prevent outbreaks of illnesses and financial losses Biological Resource Center, National Institute of Technology and Evaluation, 2-5-8, Kazusakamatari, Kisarazu, Chiba, 292-0818, Japan for dairy producers. Objective: This study aimed to evaluate Sample6 Detect HT/L for article info abstract effective detection of Listeria monocytoenes and L. innocua in ice cream. ethods: Performance of the Sample6 DETECT HT/L was compared with U.S. Food and Drug Article history: A new aquatic fungus was isolated from submerged, decaying leaves collected at Phu Quoc Island in Received 10 April 2017 Vietnam. The fungus produced hyaline, unique-shaped conidia consisting of a hook-shaped main axis Received in revised form and three arms at the helicoid part of the main axis. Based on its conidial development and morpho- Administration (FDA) Bacterioloical Analytical Manual (BAM) Chapter 10 method for 4 April 2018 logical characteristics, Hamatispora phuquocensis was newly introduced. Phylogenetic analyses based on Accepted 10 April 2018 LSU nrDNA sequences showed that it clusters with Microthyrium spp. and belongs to Microthyriales. detection of Listeria spp. in ice cream using an unpaired study design. Results: R2- Available online 11 June 2018 Furthermore, we generated ITS barcode for this hyphomycetous fungus. enriched samples tested with Sample6 Detect HT/L performed as well as the reference © 2018 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. Keywords: method at all time points tested from 15 to 24 h. R2 is a proprietary blend for use with the Freshwater Helico-staurospore test kit that helps with early detection. All the dPODC values (Sample6 Detect HT/L Phylogeny Taxonomy presumptive and confirmed results) equaled zero, indicating 100% concordance between the methods. Both Sample6 Detect HT/L and FDA BAM results showed low dPODC values, with confidence intervals indicating no significant differences between Sample6 1. Introduction described and illustrated as belonging to a new genus and its Detect HT/L and reference method results. Conclusions: Sample6 Detect HT/L is suitable phylogenetic positon of the new fungus is also discussed. During a survey of micro-fungi in Vietnam, an undescribed to detect Listeria spp. in ice cream, even with a 12 h enrichment. Sample6 Detect HT/L species was detected and isolated from fallen leaves. The shape of 2. Materials and methods demonstrated equivalent detection of L. monocytoenes and L. innocua from R2-enriched the conidium was characterized by a main axis and three arms. The main axis was shaped like a question mark and the three arms 2.1. Sampling, isolation and preservation samples as expected with 15 and 18 h enrichment when compared with the 24 h FDA developed from three different cells of the helicoid part of the main axis. Matsushima (1975) reported a fungus from decayed leaves of Fallen broad leaves were collected from the Suoi Tranh stream at BAM method for L. monocytoenes. Highlights: These results indicate that Sample6 Podocarpusm acrophyllus (Thunb.) Sweet collected in Kobe city, Phu Quoc Island, Vietnam in Nov. 2011. Samples were put in clean Detect HT/L, primarily developed for environmental samples, can be used to detect Hyogo Pref., Japan which possess similar morphology to ours and plastic bags with a zip and transported to the laboratory. The leaves described the fungus as “Fungus Imperfectus nonnominates, MFC- were rinsed in tap water, and were incubated in moist chambers. Listeria spp. in ice cream with less incubation time, resulting in faster detection. 1585”. Subsequently, Nawawi (1985) reported the fungus from After 3e7 d, each leaf was stamped on low carbon agar (LCA) (Miura foam sample in Malaysia. Goncz€ ol€ and Revay� (2006) reported a & Kudo, 1970). A single spore on the LCA was isolated by a Sker- _______________________________________ similarly shaped conidium from draining rainwater from intact man's micromanipulator under a light microscope to obtain the Abstract of an article in Journal of AOAC International 102: 1-6 (2019). leaves of Picea abies (L.) H. Karst in Romania and identified the pure culture. Cultures have been deposited to the Vietnam Type fungus as Tricladiella pluvialis (Ando & Tubaki, 1984). Hosoya and Culture Collection, Institute of Microbiology and Biotechnology, Lourdes M. Nadala: Participant of the 15th UM, 1987-1988. Tanaka (2007) surveyed freshwater hyphomycetes in Yakushima Vietnam National University, Ha Noi, Vietnam (VTCC) and to the Island, Kagoshima Pref., Japan and reported a conidium like Mat- National Institute of Technology and Evaluation, Biological Re- sushima's fungus. The conidial shape was unique and the fungus sources Center, Chiba, Japan (NBRC). appeared to be a hitherto undescribed species which could not be accommodated into any a genus. In this study, the fungus is 2.2. Morphological study The isolates were cultured at 25 �C on a potato carrot agar me- * Corresponding author. dium (PCA, extract from 20 g/L potato, extract from 20 g/L carrot, 2% E-mail address: [email protected] (L.T.H. Yen). agar), LCA and potato dextrose agar (PDA, Nissui, Japan) for https://doi.org/10.1016/j.myc.2018.04.004 1340-3540/© 2018 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. Reproduced from Mycoscience 59: 467e472 (2018). Duong Van Hop: Participant of the 15th UM, 1987-1988. 362 363 468 L.T.H. Yen et al. / Mycoscience 59 (2018) 467e472 morphological observations. Observations were made under a dif- shaped with a long tail bearing lateral branches; lateral branches ferential interference contrast microscope (DIC: Axioplan 2, Zeiss, straight-shaped develop from different cells of the helicoids part of Jena, Germany) and a scanning electron microscope (JSM-6060: the main axis. JEOL, Tokyo, Japan). For SEM observation, a small piece (ca. 5 5 mm) of the colony was cut and fixed with 1% OsO4 aq. sol. at Hamatispora phuquocensis L.T.H. Yen, K. Yamag. & K. Ando, sp. � room temperature for 2 h, dehydrated in an ethanol series and nov. Figs. 1e3. finally substituted with isoamyl acetate. After critical point drying MycoBank no.: MB 822832. (HCP-2: Hitachi, Tokyo, Japan) and coating with platinum- Type: VIETNAM, Phu Quoc Island, Kien Giang province, from palladium (JUC-5000: JEOL), the specimens were observed under fallen leaves of an unidentified deciduous broad-leaved tree in the the SEM at 15 kV. Suoi Tranh stream, 5 Nov 2011, leg. L.T.H. Yen (Holotype, VTCCF-H- 1219; ex-holotype culture, VTCCF-1219 NBRC 111195; isotype, ¼ 2.3. Molecular phylogeny NBRC H-521261; ex-isotype culture, VTCCF-1219 NBRC 111195). ¼ Gene sequences ex-holotype: LC064073 (LSU) and LC064074 2.3.1. DNA extraction, PCR and sequencing (ITS). Small pieces of a colony (3 3 mm) grown on malt extract agar Etymology: The epithet phuquocensis refers to the location � (MEA) medium at 25 �C for 10 d were put into 2 mL Cryo tubes. DNA where the fungus was discovered. was extracted using the PrepMan™ Ultra Sample Preparation Re- Mycelium composed of branched, septate, hyaline to olivaceous agent (Applied Biosystems, Foster City, CA, USA). PCR was per- brown, smooth, 1.2e2 mm wide hyphae. Sexual morph: Undeter- formed by using KOD-Plus Kit (Toyobo, Osaka, Japan), following the mined. Asexual morph: Conidiophore reduced to conidiogenous cells. manufacturer's protocol. The nrDNA large subunit region (LSU D1/ Conidiogenous cells holoblastic, solitary, hyaline and simple, pro- D2) was amplified with primers NL1 and NL4 (O'Donnell, 1993). To duced on hyphae, 8e15 1.5e2 mm. Conidia multi-septate, consist- � amply the ITS region, the combination ITS1 and ITS4 (White, Bruns, ing of a main axis and 3(e5) arms; the main axis comprised of two Lee, & Taylor, 1990) were used. Amplification of the DNA fragments parts: a helicoid part and a tail part, constricted at the joint portion of was performed using the GeneAmp PCR System 9700 (Applied the helicoid and the tail part; the helicoid part 3(e5)-celled, 7e10 mm Biosystems) under the following thermal cycling programme: an in diam; the tail part 5e6-celled, 35e40 1.8e2.5 mm. The arms � initial denaturation at 94 �C for 2 min, 35 cycles of denaturation at developing from each cellof the helicoid partof the main axis; the first 94 C for 15 s, annealing at 56 C for 30 s, extension at 68 C for arm (4e)5(e6)-celled, 35e40 1.8e2.5 mm; the second arm 4e5- � � � � 1 min 30 s, a final extension at 68 C for 10 min, and a 16 C soak. celled, 25e35 1.8e2.5 mm; the third arm 3e4-celled, � � � PCR products were checked by agarose gel electrophoresis, and 20e30 1.8e2.5 mm; constricted at the base of the first and second � were purified by using AMPureKit (Agencourt Biosciences, Beverly, arms, not constricted at the base of the third arm. MA, USA). Sequencing reactions were performed by using the Big Cultural characteristics: The isolate (VTCCF-1219 NBRC ¼ Dye Terminator V.3.1 Cycle Sequencing Kit (Applied Biosystems) 111195) grows slowly on PCA, LCA and PDA, with a blight grey, black and the primers of the PCR.