Table I The 24 Currently Established Species within the Bcc Bcc species Type strain Source Year identifi ed and/or named Country/region Reference B. catarinensis DSM 103188T (BR 10601T) Native grassland soil 2017 Southern Brazilian (14) B. alpine PO-04-17-38= T DSM 28031= T LMG 28138 T Volcanic soils 2017 Pico de Orizaba (Mexico) (15) B. puraquae CAMPA 1040 T (=LMG 29660= T DSM 103137 T ) Hospital and soils 2017 Argentina (16) B. paludis MSh1(=DSM T 100703T= MCCC1K01245 T ) Surface peat 2016 Southeast Pahang, Malaysia (17) B. stagnalis LMG28156T (=CCUG65686T ) Soil and water 2015 Australia (18) B. territorii LMG 28158T (=CCUG65687T ) Soil and water sampling 2015 Territory of Australia (18) B. pseudomultivorans LMG 26883T(=CCUG 62895 T ) CF sputum 2013 USA (19) B. latens FIRENZE 3 T = LMG 24064T= CCUG 54555T CF sputum 2008 Italy (20) B. diffusa AU1075 T = LMG 24065T5CCUG 54558 T CF sputum 2008 USA (20) B. arboris ES0263AT= LMG 24066 T = CCUG 54561 T Morris Arboretum 2008 Philadelphia (PA, USA) (20) B. seminalis AU0475 T = LMG 24067T= CCUG 54564 T CF sputum 2008 USA (20) B. metallica AU0553 T = LMG 24068T= CCUG 54567 T CF sputum 2008 USA (20) B. ubonensis LMG 20358T Soil 2008 Thailand (20) B. dolosa LMG 18943(=CCUG T 47727 T ) Environment and CF sputum 2004 USA (21) B. cenocepacia LMG 16656 (=NCTC 13227, ATCC BAA-245, CCM 4899) CF sputum 2003 Edinburgh (UK) (22) B. anthina LMG 20980 (=CCUG 46047) Rhizosphere of a house-plant 2002 Nashville (TN, USA) (23) B. pyrrocinia LMG 14095 Soil 2002 USA (23) B. contaminans J2956 T (=LMG 23361T= CCUG 55526T ) Milk of a sheep with mastitis 2001 Spain (24) B. ambifaria LMG 19182(=CCUG T 44356 T ) Pea plants 2001 Wisconsin (USA) (25) B. stabilis LMG 14294 CF sputum 2000 Leuven (Belgium) (26) B. multivorans LMG 13010 CF sputum 1997 Brussels (Belgium) (27) B. lata 383 T (=ATCC 17760 T = LMG 22485T= CCUG 55525T ) Forest soil 1966 Trinidad (24) B. vietnamiensis TVV75 (=LMG 10929) Rice rhizosphere soil 1995 Vietnam (28) B. cepacia Burkholder (=717-ICPB 25, =ATCC 25416, =NCTC 10743) Rot of onion bulbs 1950 New York State (USA) (29) BURKHOLDERIA CEPACIA COMPLEX IN PERSONAL CARE PRODUCTS 135
JOURNAL OF COSMETIC SCIENCE 136 carried out investigation of Bcc susceptibility to the common preservatives used in PCPs. The aim of this study was to reveal insights into the diversity of Bcc isolates from PCPs and the susceptibility to different preservatives commonly used. MATERIALS AND METHODS BACTERIAL STRAINS A total of 25 B. cepacia complex isolates were obtained from contaminated PCPs, includ- ing creams, shampoos, lotions, and conditioners, and stored for a period of 2 years (2015– 2017) (Table II). B. cepacia ATCC 25416 was used as a reference strain in the study. All strains were grown in Tryptone soya agar (TSA Oxoid™ Thermo Fisher Scientifi c, Waltham, MA) and incubated for 24–48 h at 37°C (±1°C). Strains were stored at -80°C (±1°C) with 20% glycerol until used. All Bcc samples were prepared in duplicate. ANTIMICR OBIALS 2-methyl-4-isothiazolin-3-one (MIT), consisting of a 3:1 (volume:volume) ratio of 5-chloro-2-methyl-4-isothiazolin-3-one (cMIT) and MIT at a fi nal concentration of 14% (m/vol) total active ingredient, was obtained from Guangdong Demei Biology Technology Co., Ltd (Guangzhou, China). Dimethoxy dimethyl hydantoin (DMDMH 55%, m/vol) and methyl 4-hydroxybenzoate (MH) were purchased from Sigma-Aldrich (St. Louis, MO). ISOLATION AND IDENTIFICATION OF STRAINS One gram of recovered PCPs was introduced onto the lecithin tween-80 nutrient agar plates (Guagdong Huankai Microbial Sci. & Tech. Co., Ltd., Guangzhou, China), the samples were streaked using a sterile loop, and the plates incubated for 18–24 h at 37°C. Gram staining and microscopic observation were performed on all isolated strains. Strains were identifi ed by the API 20NE system (API-bioMérieux, La Balme les Grottes, France) according to the manufacturer’s directions. Genomic DNA was isolated using a genomic DNA extraction kit from TaKaRa (Tokyo, Japan) according to the manufacturer’s instructions for bacterial cells. PCR was performed using primer pairs for BCR1 (5′-TGACCGCCGAGAAGAGCAA-3′) and BCR2 (5′-CTCTTCTTCGTCCATCGCCTC-3′) in a Mastercycler® 5330 (Eppendorf AG, Hamburg, Germany) and run for 35 thermal cycles of 94°C (4 min), 58°C (30 s), and 72°C (30 s). A 10-min elongation step was included in the fi nal cycle (38). The reaction products were separated and detected on an ABI PRISM Genetic Analyzer 3100 (Applied Biosystems, Foster City, CA). Sequencing was performed by Huada Gene Technology Co. Ltd (Shenzhen, China). The resulting sequen ces were assessed for similarities among known sequences using the Basic Local Alignment Tool at the National Center for Biotechnology Information (NCBI, NIH, Bethesda, MD). Multiple nucleotide sequence alignments of the recA gene were constructed using CLUSTAL X (Dublin, Ireland). Phylogenetic and molecular evolutionary
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