A Phenotypic And Genotypic Analysis Of The Antimicrobial Potential Of .
ORIGINAL RESEARCHpublished: 21 September 2016doi: 10.3389/fmicb.2016.01455A Phenotypic and Genotypic Analysisof the Antimicrobial Potential ofCultivable Streptomyces Isolatedfrom Cave Moonmilk DepositsMarta Maciejewska 1† , Delphine Adam 1† , Loïc Martinet 1 , Aymeric Naômé 1 ,Magdalena Całusińska 2 , Philippe Delfosse 2 , Monique Carnol 3 , Hazel A. Barton 4 ,Marie-Pierre Hayette 5 , Nicolas Smargiasso 6 , Edwin De Pauw 6 , Marc Hanikenne 7,8 ,Denis Baurain 8,9 and Sébastien Rigali 1*1Edited by:Learn-Han Lee,Monash University Malaysia Campus,MalaysiaReviewed by:Antoine Danchin,Institute of Cardiometabolismand Nutrition – Pitié-SalpêtrièreHospital, FranceDenis Axenov-Gribanov,Irkutsk State University, Russia*Correspondence:Sébastien Rigalisrigali@ulg.ac.be† Theseauthors have contributedequally to this work.Specialty section:This article was submitted toAntimicrobials, Resistanceand Chemotherapy,a section of the journalFrontiers in MicrobiologyReceived: 08 July 2016Accepted: 31 August 2016Published: 21 September 2016Citation:Maciejewska M, Adam D, Martinet L,Naômé A, Całusińska M, Delfosse P,Carnol M, Barton HA, Hayette M-P,Smargiasso N, De Pauw E,Hanikenne M, Baurain D and Rigali S(2016) A Phenotypic and GenotypicAnalysis of the Antimicrobial Potentialof Cultivable Streptomyces Isolatedfrom Cave Moonmilk Deposits.Front. Microbiol. 7:1455.doi: 10.3389/fmicb.2016.01455InBioS – Centre for Protein Engineering, Institut de Chimie B6a, University of Liège, Liège, Belgium, 2 EnvironmentalResearch and Innovation Department, Luxembourg Institute of Science and Technology, Belvaux, Luxembourg, 3 InBioS –Plant and Microbial Ecology, Botany B22, University of Liège, Liège, Belgium, 4 Department of Biology, University of Akron,Akron, OH, USA, 5 Department of Clinical Microbiology, University Hospital of Liège, Liège, Belgium, 6 MolSys Research Unit,Mass Spectrometry Laboratory, University of Liège, Liège, Belgium, 7 InBioS – Functional Genomics and Plant MolecularImaging, University of Liège, Liège, Belgium, 8 PhytoSYSTEMS, University of Liège, Liège, Belgium, 9 InBioS – EukaryoticPhylogenomics, University of Liège, Liège, BelgiumMoonmilk speleothems of limestone caves host a rich microbiome, among whichActinobacteria represent one of the most abundant phyla. Ancient medical textsreported that moonmilk had therapeutical properties, thereby suggesting that itsfilamentous endemic actinobacterial population might be a source of natural productsuseful in human treatment. In this work, a screening approach was undertaken in orderto isolate cultivable Actinobacteria from moonmilk of the Grotte des Collemboles inBelgium, to evaluate their taxonomic profile, and to assess their potential in biosynthesisof antimicrobials. Phylogenetic analysis revealed that all 78 isolates were exclusivelyaffiliated to the genus Streptomyces and clustered into 31 distinct phylotypes displayingvarious pigmentation patterns and morphological features. Phylotype representativeswere tested for antibacterial and antifungal activities and their genomes were mined forsecondary metabolite biosynthetic genes coding for non-ribosomal peptide synthetases(NRPSs), and polyketide synthases (PKS). The moonmilk Streptomyces collectionwas found to display strong inhibitory activities against a wide range of referenceorganisms, as 94, 71, and 94% of the isolates inhibited or impaired the growth ofGram-positive, Gram-negative bacteria, and fungi, respectively. Interestingly, 90% ofthe cave strains induced strong growth suppression against the multi-drug resistantRasamsonia argillacea, a causative agent of invasive mycosis in cystic fibrosis andchronic granulomatous diseases. No correlation was observed between the globalantimicrobial activity of an individual strain and the number of NRPS and PKS genespredicted in its genome, suggesting that approaches for awakening cryptic metabolitesbiosynthesis should be applied to isolates with no antimicrobial phenotype. Overall, ourwork supports the common belief that moonmilk might effectively treat various infectiousFrontiers in Microbiology www.frontiersin.org1September 2016 Volume 7 Article 1455
Maciejewska et al.Antimicrobial Properties of Moonmilk Streptomyces Isolatesdiseases thanks to the presence of a highly diverse population of prolific antimicrobialproducing Streptomyces, and thus may indeed constitute a promising reservoir ofpotentially novel active natural compounds.Keywords: geomicrobiology, secondary metabolism, MLSA phylogeny, cryptic antibiotics, genome mininginter-/intraspecies communication (Sengupta et al., 2013), and ascues triggering adaptations, such as motility or biofilm formation(Linares et al., 2006), or can be used as alternative carbon and/ornitrogen sources (Dantas et al., 2008). Consequently, smallbioactive molecules expressed under nutrient-starved conditions,could be used as weapons, as signals, or as nutrient sources.Moonmilk is a comparatively rare speleothem in caveenvironments, where it forms as a thick calcite paste (similar inconsistency to toothpaste) up to 10s-of-centimeters in thicknessin passageways that receive significant airflow (Hill and Forti,1997; Borsato et al., 2000). The exact process of moonmilkformation remains in debate; however, the consistency, the highabundance of filamentous bacterial cells and members of theActinobacteria has led researchers to suggest a biogenic origin(Cañaveras et al., 2006; Rooney et al., 2010; Portillo and Gonzalez,2011). Despite its relative scarcity in caves, moonmilk has hadlong scientific interest due to its historical use as a medicaltreatment. Interestingly, moonmilk was used in human andanimal therapy since the Middle Ages (Reinbacher, 1994). Itscurative properties could be associated with the presence of thenumerous filamentous Actinobacteria, particularly Streptomyces,presumably producing bioactive molecules. Indeed, isolation ofStreptomyces with antimicrobial and antifungal activities has beenreported for moonmilk from the Bolshaya Oreshnaya Cave inSiberia (Axenov-Gribanov et al., 2016). The identification ofpotential novel compounds with broad-spectrum activities fromStreptomyces found in this karstic secondary deposit supports theidea of moonmilk being a great target for bioactivity screening,although to date very few studies have examined cultivablemoonmilk microbiome and the diversity of its metabolome asa possible reservoir of novel compounds. The fact that cavesare unique and still highly under-explored environment increasethe chances of finding novel organisms and consequently novelbioactive compounds that might be useful in the context of theglobal health problem of antibiotic resistance (Bush et al., 2011;Laxminarayan et al., 2013; Berendonk et al., 2015; Frère andRigali, 2016).In this work, we report the isolation and phylogenetic analysisof a collection of novel Streptomyces isolates from moonmilkdeposits, and assess their potential as producers of compoundswith antimicrobial and antifungal properties through in vitroscreening and genome mining approaches.INTRODUCTIONMembers of the phylum Actinobacteria can be found in allkinds of extreme environments (Saiz-Jimenez, 1999; Bull, 2010;Prieto-Davó et al., 2013; Zhang et al., 2014; Mohammadipanahand Wink, 2015; Shivlata and Satyanarayana, 2015). Theirsuccessful survival in severe conditions suggests broad adaptiveabilities that might be directly related to their very diverse andspecialized (secondary) metabolism. Natural small molecules,collectively termed the parvome (Davies and Ryan, 2012),apart from having essential ecological functions, possess a widerange of bioactivities, which are applicable for agro-industrialpurposes and human/animal therapy (Hopwood, 2007). Sincethe metabolome of soil-dwelling Actinobacteria, especially of themembers of the genus Streptomyces, has been widely exploited,leading to the multiple re-isolation of already known bioactivecompounds, the attention has been refocused toward unexploredand extreme environments, which can potentially be a sourceof novel species and consequently of novel molecules of interest(Cheeptham et al., 2013; Claverías et al., 2015; Mohammadipanahand Wink, 2015; Liao et al., 2016).The geological isolation of caves from surface processesmakes them a unique niche, not only to study microbialinteractions and adaptations to extreme oligotrophy, but alsoto screen for potentially novel bioactive compounds. Although,the most common Actinobacteria reported from caves belongto Pseudonocardiaceae and Nocardiaceae families (Stomeo et al.,2008; Porca et al., 2012; Quintana et al., 2013; Riquelmeet al., 2015), many investigations have identified cultivablemembers of the genus Streptomyces, which are the mostprolific antimicrobial producers (Cañaveras et al., 1999; Grothet al., 1999; Cheeptham et al., 2013; Nimaichand et al., 2015;Axenov-Gribanov et al., 2016). The presence of Actinobacteriaas dominant members of microbial ecosystems in caves ispuzzling, as these bacteria, particularly Streptomyces are oftenpresented as major protagonists in the recycling of the residualplant biomass in nutrient-rich soil environments (Hodgson,2000). Nonetheless, 99% of allochtonous carbon enteringcaves, primarily with drip water, contains soil-derived dissolvedorganic carbon in the form of partially degraded plant andfungal polymers, which can be catabolized by the enzymaticarsenals of Streptomyces (Saiz-Jimenez and Hermosin, 1999;Simon et al., 2007; Barton, 2015). Additionally, highly prolificand diversified secondary metabolism could be a driving forcein the dominance of these species in oligotrophic environments,through which they could shape microbiomes thanks to theirspecialized metabolites, such as metal-chelators for acquiringtrace metals, and antibiotics to prevent nutrient-exclusion bycompetitive species (Bhullar et al., 2012). Antibiotics do notexclusively prevent microbial growth, but are also known to act asFrontiers in Microbiology www.frontiersin.orgMATERIALS AND METHODSSite Description and SamplingThe cave called Grotte des Collemboles (Springtails’ Cave)located in Comblain-au-Pont in Belgium is a shallow ( 20 m), 70 m long fissure cave, formed in the upper Viséan limestone2September 2016 Volume 7 Article 1455
Maciejewska et al.Antimicrobial Properties of Moonmilk Streptomyces IsolatesFIGURE 1 Cave map of the Grotte des Collemboles and visualization of the moonmilk deposit sampling points. Location of the Grotte des Collemboles(Springtails’ Cave) in Comblain-au-Pont (Liège, Belgium) and cave map with general view and close up of the moonmilk deposits from the different collection points(COL).(Figure 1), with an average annual temperature of 11.5 C.Due to cave protection policies and location on private property,specific location details and access information is available toother researchers upon request. Within the cave, white to brownorange (presumably from iron-oxide precipitates) moonmilkdeposits are found on the walls within the first 20 m of the cavein the first narrow chamber located at the entrance of the caveas well as in the narrow passages leading deeper into the cave(Figure 1). Samples used in this work were aseptically collectedin January 2012 from three moonmilk deposits (Figure 1). Softmoonmilk speleothem was scratched with sterile scalpels fromthe wall in the first chamber, adjacent to the cave entrance(COL4) and the walls in a narrow passage after the first chamber(COL1, COL3; Figure 1). Samples were collected into falcontubes, transferred to the laboratory, freeze-dried on a VirTisBenchtop SLC Lyophilizer (SP Scientific, Warminster, PA, USA)and stored at 20 C.sample from each collection point was suspended in 0.25Xstrength Ringer’s solution supplemented with 0.001% Tween80. Resulting moonmilk suspensions were serially diluted inPBS and inoculated in duplicates on ISP media (Shirling andGottlieb, 1966), starch nitrate (SN) medium (Gauze’s mediumNo.1; Waksman and Lechevalier, 1961), B-4 agar (Boquet et al.,1973), and minimal medium (Kieser et al., 2000) with 1% chitin(MMch). Isolation media were supplemented with nalidixic acid(75 µg/ml) and nystatin (50 µg/ml) to suppress the growth ofGram-negative bacteria and fungi, respectively. After 1 monthof incubation at 17 C, colony forming units (CFUs) wereenumerated and 129 isolates were selected. After two rounds ofsubcultivation, 78 isolates were recovered as purified strains andsubsequently preserved both on ISP2 slopes at 4 C and as 25%glycerol mycelium stock at 20 C.Isolation of Cultivable ActinobacterialSpeciesDNA Extraction, Genome Sequencing,and Gene Selection fromMoonmilk-Derived IsolatesSelective isolation of Streptomyces species from moonmilk wascarried out by a serial dilution method as described previously(Maciejewska et al., 2015). 250 mg of lyophilized moonmilkIn order to screen for the genes of interest, which would enableto identify moonmilk derived isolates, to perform phylogeneticanalysis, as well as to investigate strains antimicrobial properties,Frontiers in Microbiology www.frontiersin.org3September 2016 Volume 7 Article 1455
Maciejewska et al.Antimicrobial Properties of Moonmilk Streptomyces Isolatesde novo genome sequencing was carried out. DNA frompurified strains was extracted with GenElute Bacterial GenomicDNA Kit (Sigma-Aldrich, St. Louis, MO, USA) accordingto manufacturer’s instructions from liquid LB (Luria-Bertani;Difco, BD, Franklin Lakes, NJ, USA) cultures incubatedat 28 C. The genomic libraries of moonmilk isolates wereconstructed using Nextera XT kit (Illumina, Inc., San Diego,CA, USA). Library concentrations and mean fragment lengthswere measured by Qubit fluorometer (Invitrogen, Grand Island,NY, USA) and Agilent Bioanalyzer (Agilent Technologies,Santa Clara, CA, USA), respectively. De novo sequencingwith 2 250 bp and 2 300 bp reads configuration wascarried out on the Illumina MiSeq platform (Illumina, Inc.,San Diego, CA, USA) at the Luxembourg Institute of Scienceand Technology. Complete genomes were assembled from rawsequence data with SPAdes v.3.6.2 (Bankevich et al., 2012)using the “careful” option, and the quality of the assemblieswas subsequently assessed with QUAST v2.3 (Gurevich et al.,2013).To infer the evolutionary relationships between moonmilkstrains and their closest relatives, as well as between caveisolates themselves, 16S rRNA-based phylogeny was combinedwith multilocus sequence analysis (MLSA). For this purpose,along with the 16S rRNA gene, five additional housekeepinggenes were selected, namely atpD, gyrB, recA, rpoB, and trpB(Han et al., 2012). In order to identify these genes withinmoonmilk genomes, the corresponding nucleotide sequences(16S rRNA) and protein translations (atpD, gyrB, recA, rpoB,and trpB) were retrieved from the NCBI web portal forthree reference strains: Streptomyces peucetius AS 4.1799,Streptomyces pristinaespiralis ATCC 25486 and Streptomycesvenezuelae ATCC 10712 (Supplementary Table S1). Corealignments were built using MAFFT v7.273 (Katoh andStandley, 2013) with default parameters, then enriched inthe corresponding sequences from moonmilk genomes usingthe software “42” (D. Baurain, to be published elsewhere),which mines genomic contigs for orthologous genes andaligns the (translated) identified sequences on their closestrelatives. Enriched alignments were then refined by hand usingthe ED program of the MUST software package (Philippe,1993). Finally, protein sequences were turned into nucleotidesequences using the software “1331” (D. Baurain, to bepublished elsewhere), which uses a protein alignment as aguide to generate the corresponding nucleotide alignment fromgenomic contigs. The sequences of the five protein codinghousekeeping genes for all the moonmilk isolates were depositedin GenBank and the corresponding accession numbers are givenin Supplementary Table S2, while Table 1 and SupplementaryTable S3 list the NCBI accession numbers of the 16S rRNA genesequences.In order to profile the potential of moonmilk isolatesto biosynthesize secondary metabolites, the genes coding fortype I, type II, and type III polyketide synthases (PKS-I,PKS-II, and PKS-III) and non-ribosomal peptide synthetases(NRPS) were recovered from their genomes using antiSMASHv3.0.4 (Weber et al., 2015). Due to the fragmented natureof the genome assemblies (ranging from 318 contigs longerFrontiers in Microbiology www.frontiersin.orgthan 1 kb for MM23 to 1416 contigs for MM59) and tothe large size of the modular NRPS and PKS-I genes (over40 kb, Wang et al., 2014), the probability of finding completecoding sequences decreases together with the contig length.Therefore, counting the number of genes or clusters split acrossseveral shorter contigs would result in an overestimation ofthe total amount of such genes. To palliate the absence offully assembled chromosomes while still collecting meaningfulstatistics, we decided to apply a cut-off on the length of thecontigs selected for analysis (minimum length of 10 kb) andto consider NRPS and PKS-I gene sequences only when theydisplayed adenylation and acyltransferase domains, respectively.These domains, which are the highly selective gatekeeperenzymes for the incoming monomeric building blocks, arerequired for the initiation and elongation modules of theNRPS/PKS-I clusters. The number of predicted genes ofeach category for each individual phylotype is compared totheir respective mean antimicrobial activities against Grampositive, Gram-negative bacteria, and fungi. The accessionnumbers of NRPS and PKS-I/II/III genes are listed inSupplementary Table S4.Phylogenetic AnalysisTo carry out phylogenetic analysis, in each nucleotidealignment of the six selected housekeeping genes (seeabove), the sequences from the three reference strains usedto mine the moonmilk genomes were removed. Then,positions with missing character states in 5 moonmilkisolates were removed. Finally, the trimmed alignmentswere concatenated into a single (MLSA) supermatrix of10,632 nucleotides for 70 isolates using SCaFoS v1.30k(Roure et al., 2007). For 16S rRNA phylogenies, the closestrelatives to the moonmilk isolates were recovered by BLASTsearches using full-length 16S rRNA sequences, along withthe Streptomyces isolates from a moonmilk deposit in Siberia(Axenov-Gribanov et al., 2016). For the eight isolates forwhich the genomes were not sequenced (MM9, MM32,MM39, MM55, MM73, MM88, MM90, MM93), nearlyfull-length 16S rRNA sequences were obtained using PCRprimers and conditions as previously reported (Maciejewskaet al., 2015). The 78-moonmilk strain alignment of the16S rRNA was further processed using the software “twoscalp” (D. Baurain, to be published elsewhere) to integrate35 additional sequences, corresponding to the 27 (nonredundant) best BLAST hits, 7 Siberian moonmilk sequences,and the sequence of Saccharopolyspora erythraea, used as theoutgroup.Both the MLSA supermatrix and the 16S rRNA alignmentwere submitted to phylogenetic inference using the rapidbootstrap analysis of RAxML v8.1.17 (Stamatakis, 2014; 100pseudoreplicates) under the model GTR I 0 4 . The resultingMLSA and 16S rRNA trees were first formatted in FigTree v1.4.21then further arranged using Inkscape v0.912 . Patristic distancesbetween moonmilk isolates were derived from the MLSA /https://inkscape.org/September 2016 Volume 7 Article 1455
Maciejewska et al.Antimicrobial Properties of Moonmilk Streptomyces IsolatesTABLE 1 The closest relatives, phylogenetic affiliations, phylotype clustering, and isolation origin of the 31 representative moonmilk isolates.IsolateClosest relatives16S rRNA identity% (gaps)AccessionnumberOrigin of theclosest relativesCOLMdPhylotype16S MLSAMM1S. sp. CFMR 7 strain CFMR-7/S.fulvissimus DSM 4059399.1 (4)/99.1 (6)KU714864Plant(rubber)/unknownCOL3SNI IMM3Un. bacterium clone Md-54/Un.bacterium clone 10–35599.8 (0)/99.8 (0)KU714892Soil/soilCOL3SNII IIMM5S. scabiei BCCO 10 524/S.europaeiscabiei 08-46-04-2 (#50)99.7 (0)/99.4 (2)KU714904Both plant (potato)COL3SNIII IIIMM6S. sp. Mg1/S. sp. SXY1099.7 (0)/99.8 (0)KU714910Glacier soil(Alaska)/soilCOL3SNIV IVMM7S. sp. NEAU-spg16/S. sp. A4299.6 (0)/99.9 (0)KU714915Soil/soilCOL3SNV VMM10S. sp. NEAU-QHHV11/S. sp.(Acc.Nr.D63866)99.1 (4)/98.6 (7)KU714865Soil/soilCOL3SNVI VIMM12S. sanglieri A14/S. sp. ME03-5656.2c99.8 (0)/99.6 (0)KU714878Soil/plant (potato)COL3SNVII VIIMM13S. turgidiscabies ATCC 700248/S.turgidiscabies WI04-05A98.8 (4)/98.7 (4)KU714882Both plant (potato)COL3SNVIII VIIIMM14S. anulatus strain 173826/S. anulatusstrain 173541100 (0)/100 (0)KU714883Both unknownCOL3SNIX IXMM17S. sp. Mg1/S. sp. SXY1099.7 (0)/99.8 (0)KU714885Glacier soil(Alaska)/soilCOL3SNIV XXVIMM19S. sp. NEAU-spg16/S. sp. A4299.6 (0)/99.9 (0)KU714887Soil/soilCOL3SNV XXVIIMM21Un. bacterium clone Md-54/Un.bacterium clone 10–35599.7 (0)/99.7 (0)KU714888Soil/soilCOL3SNXI XIMM23Un. bacterium clone Md-54/Un.bacterium clone 10–35599.8 (0)/99.8 (0)KU714890Soil/soilCOL3SNII XXVIIIMM24S. sp. ME02-6979.3a/S. sp. 1C-HV898.3 (5)/98.4 (4)KU714891Plant(potato)/animals(ants)COL3SNXII XIIMM44Un. bacterium clone Md-54/Un.bacterium clone 10–35599.7 (0)/99.7 (0)KU714900Soil/soilCOL3SNXI XXIXMM48S. sp. HBUM171258/S. sp. Mg199.9 (1)/99.6 (0)KU714903Unknown/glaciersoil (Alaska)COL3MMchXIII XIIIMM59S. sp. ID05-8D/S. sp. ID01-6.2a99.5 (1)/99.4 (1)KU714909Both plant (potato)COL3MMchIII XXXMM68S. turgidiscabies ATCC 700248/S.turgidiscabies WI04-05A99.0 (2)/99.0 (2)KU714913Both plant (potato)COL3B-4XIV XIVMM90S. sp. AA58/S. sp. AS4099.5 (4)/99.4 (5)KU714925Soil/soilCOL1ISP4XV –MM99S. fulvissimus DSM 40593/S. sp.ME02-6987.2c99.7 (2)/99.7 (2)KU714928Unknown/plant(potato)COL1ISP6XVI XVIMM100S. sanglieri A14/S. sp. ME03-5656.2c99.9 (0)/99.5 (0)KU714866Soil/plant (potato)COL1B-4XVII XVIIMM104S. scopuliridis strain SCSIO ZJ46/S. sp.AK02-1a99.2 (0)/99.0 (0)KU714869Deep sea/plant(potato)COL3ISP6XVIII XVIIIMM105S. finlayi strain CB00817/S. olivoviridisstrain S399.4 (6)/99.3 (5)KU714870Soil/animals(earthworm)COL3ISP6XIX XIXMM106S. rishiriensis strain 1706/S. fimbriatusstrain cfcc315599.0 (0)/98.8 (1)KU714871Soil/unknownCOL3ISP1XX XXMM107S. pristinaespiralis strain HCCB10218/S. sp. NEAU-bt1098.8 (2)/98.8 (0)KU714872Soil/soilCOL3ISP1XXI XXIMM108S. sp. SXY66/S. sp. 1H-TWYE2100 (0)/99.3 (2)KU714873Soil/animals (ants)COL3ISP7XXII XXIIMM109S. lunaelactis MM109T /S. lunaelactisMM15100 (0)/99.9 (0)KM207217.2Cave/caveCOL3ISP7X XMM111S. sp. 1H-TWYE2/S. sp. SXY6699.7 (0)/99.5 (2)KU714875Animals (ants)/soilCOL4ISP6XXIII XXIIIMM117S. sp. PAMC26508/S. pratensis ATCC3333199.7 (0)/99.7 (0)KU714876Antarctic lichen/soilCOL4ISP7XXIV XXIVMM122S. sp. PAMC26508/S. pratensis ATCC33331100 (0)/100 (0)KU714879Antarctic lichen/soilCOL4B-4IX XXXIMM128S. sp. ZLN234/S. sp. SXY6699.9 (0)/99.0 (4)KU714881Glacier soil(Arctic)/soilCOL4SNXXV XXVB-4, B-4 agar medium; MMch, minimal medium with 1% chitin; ISP, International Streptomyces Project medium; SN, starch nitrate medium; COL, moonmilk collectionsite; Md, isolation medium; Un., uncultured. Symbols: , isolates not included in the MLSA; T, Type strain (Maciejewska et al., 2015).Frontiers in Microbiology www.frontiersin.org5September 2016 Volume 7 Article 1455
Maciejewska et al.Antimicrobial Properties of Moonmilk Streptomyces Isolates20 g) plates and incubated for 10 days at 28 C. The agar wascollected, poured into a flask with an equal volume of ethylacetate ( 300 ml) and agitated overnight at room temperaturefor metabolites extraction. Ethyl acetate was collected and piecesof agar were removed by centrifugation (25 min at 4000 rpm)before the solvent was evaporated on a rotary evaporator (IKARV10 digital, VWR, Radnor, PA, USA). The dried crude extractwas resuspended in 4 ml of pure methanol high pressureliquid chromatography (HPLC Barker UHPLC grade). Prior tofractionation by HPLC, the antifungal activity of the total extractwas assessed by a disk diffusion assay on a yeast peptone dextrose(YPD; peptone 20 g; yeast extract 10 g; glucose 20 g; agar 15 g)agar plate inoculated with Saccharomyces cerevisiae (ATCC 9763;with a cotton swab dipped in a 0.25–0.27 OD625 LB suspension).The full extract was then fractionated by HPLC (Waters, Milford,MA, USA) using a Waters 2695 Separations Module (Alliance)with a Waters 2998 Photodiode Array Detector coupled to aWaters Fraction Collector WFC III. The methanol extract wasanalyzed on a Nucleodur C18ec column (2.0 mm 150 mm,5 µm particle size, Macherey-Nagel) at a column temperatureof 40 C. Extract separation was achieved by increasing theacetonitrile (Barker, HPLC far UV grade)/water (milliQ filtratedon 0.22 µm) 0.05% trifluoroacetic acid (TFA, Sequencinggrade; Thermo Fisher Scientific, San Jose, CA, USA), ratio (from0 to 62.5% of acetonitrile during 30 min, then from 62.5 to100% of acetonitrile during 8 min) at a 300 µl/min flow rate.Online UV absorption measurement was performed from 190to 800 nm. Data were analyzed using Empower 3 software(Waters, Milford, MA, USA). The obtained extract fractions weresubsequently tested for antifungal activities by a disk diffusionassay as described above.using the TREEPLOT program of the MUST software package(Philippe, 1993).Antimicrobial Activity ScreeningAntimicrobial activities of one representative of each phylotypededuced from the MLSA, together with MM90 (representing 16Sphylotype XV) were tested using the cross-streak method ontwo (antifungal test) to five (antibacterial test) different culturemedia: Mueller Hinton Agar (MHA) (Difco, BD, Franklin Lakes,NJ, USA), Tryptic Soy Agar (TSA) (tryptone 15 g, soybean meal5 g, NaCl 5 g, agar 15 g; pH 7.3), ISP media No. 7 (Shirlingand Gottlieb, 1966), starch nitrate (SN) medium (Waksmanand Lechevalier, 1961), and minimal medium (Kieser et al.,2000) supplemented with 25 mM N-acetylglucosamine (MM GlcNAc). Each moonmilk strain was independently inoculatedfrom the mycelium stock as a single line in the center of thesquare plate and incubated for 7 days at 28 C, before beingcross-streaked with bacterial or fungal reference strains.Antibacterial activities were tested against a range of Grampositive and Gram-negative bacteria, including Escherichiacoli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853),Citrobacter freundii (ATCC 43864), Klebsiella pneumoniae(ATCC 13883), Bacillus subtilis (ATCC 19659), Staphylococcusaureus (ATCC 25923), and Micrococcus luteus (ATCC 9341).Each tested bacteria was cross-streaked perpendicular to thegrowth of the moonmilk isolate at the distance of 2 cm fromone another from a suspension prepared according to EUCASTrecommendations (EUCAST, 2015). Briefly, each inoculum wasmade from the overnight-grown plate of each pathogen in solidLB (Difco, BD, Franklin Lakes, NJ, USA) at 37 C by suspendingseveral morphologically similar colonies in LB broth (Difco, BD,Franklin Lakes, NJ, USA) until the OD625 nm reached 0.08–0.13, corresponding to the McFarland 0.5 standard. The referencebacterial strains were cross-streaked with a cotton swab, the platesincubated overnight at 37 C, and the activities determined bymeasurement of the inhibition zone (in cm).Antifungal activities were tested against a range of pathogenicfungi including Candida albicans (ATCC 10231), C. albicans(azole-resistant routine strain from the National ReferenceCenter for Mycoses, 13-160409-5014), referred as C. albicans‘R,’ Aspergillus fumigatus (Neqas 1210), Rasamsonia argillacea(Neqas 1872), Penicillium chrysogenum (Neqas 2068), andTrichophyton mentagrophytes (Neqas 1208). Each fungalstrain was suspended in water at the density equivalent to0.5 McFarland, and the obtained fungal suspension wasperpendicularly cross-streaked against moonmilk isolates with adistance of 4 cm from one another. The plates were incubated at37 C, for up to 4 days, and the measurements of inhibition zones(in cm) were recorded every day.Compound Identification byUltra-Performance LiquidChromatography-Tandem MassSpectrometry (UPLC-MS/MS)Fractions displaying antifungal activities revealed by the diskdiffusion assay were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Briefly, compoundswere separated by reverse-phase chromatography using UltraPerformance Liquid chromatography (UPLC IClass, Waters)using a Nucleodur C18ec column (2.0 mm 150 mm,5 µm particle size, Macherey-Nagel). Elution was achieved byincreasing the acetonitrile/water (milliQ filtrated on 0.22 µm) 0.05% trifluoroacetic acid ratio (from 0 to 62.5% during30 min, then from 62.5 to 100% during 8 min) at a 300 µl/minflow rate. On-line UV absorption measurement was performedat 210 and 265 nm and the chromatography system wasfinally coupled to a Q Exactive Plus hybrid QuadrupoleOrbitrap Mass Spectrometer (Thermo Fisher Scientific,San Jose, CA, USA), operated in positive ion mode andprogrammed for data-dependent acquisitions. Survey scanswere acquired at mass resolving power of 140,000 FWHM(full width at half maximum) from 100 to 1500 m/z (1 106ions accumulation target). The five most intense ions werethen selected to perform MS/MS experiments by HigherMM99 Isolate Antifungal AgentsExtraction and Purification by HighPressure Liquid Chromatography (HPLC)Streptomyces sp. MM99 was inoculated on 15 Glucose Yeastand Malt medium (GYM; glucose 4 g; yeast extract 4 g; maltextract 10 g; casein enzymatic hydrolysate 1 g; NaCl 2 g; agarFrontiers in Microbiology www.frontiersin.org6September 2016 Volume 7 Article 1455
Maciejewska et al.Antimicrobial Properties of Moonmilk Streptomyces IsolatesEnergy Collision Dissociation (HCD) fragmentations usingstepped normalized collision energy (NCE; 21,2; 25; 28) within2 amu isolation windows (resolution 17500, 1 105 ionsaccumulation target). A dynamic exclusion was enabled for10 s. Data were analyzed using Xcalibur v2.2 (Thermo FisherScientific, San Jose, CA,
Analysis of the Antimicrobial Potential of Cultivable Streptomyces Isolated from Cave Moonmilk Deposits. Front. Microbiol. 7:1455. doi: 10.3389/fmicb.2016.01455 A Phenotypic and Genotypic Analysis of the Antimicrobial Potential of Cultivable Streptomyces Isolated from Cave Moonmilk Deposits Marta Maciejewska 1†, Delphine Adam 1†, Loïc .
Faculty of Veterinary Medicine Department of Bacteriology, Mycology and Immunology Name: Amal Ibrahem Attia Mansour Title of thesis: Phenotypic and genotypic characterization of Pseudomonas aeruginosa from different sources. For the degree of master in veterinary medicine science (Bacteriology, Mycology and Immunology) Supervisors:
Zeeshan Qureshi, Abid Ullah Shah, Haziq Hussain, Nasir Azam, Maria Zaib, Irsa Zubair, Zara Azam-Assessment of Modus Operandi for Phenotypic and Genotypic Recognition of Salmonella Species EUROPEAN ACADEMIC RESEARCH - Vol. IV, Issue 8 / November 2016 6319 infec
4 Quality control process and participants Genotypic data that passed initial quality control at CIDR were released to the Quality Assurance/Quality Control (QA/QC) analysis team at the UWGCC, the study investigator's team and dbGaP. These data were analyzed by all four groups and the results were discussed in periodic conference calls.
Quality Control Report for Genotypic Data University of Washington February 5, 2015 Project: Genetics of Orofacial Clefts and Related Phenotypes . 4 Quality control process and participants4 5 Sample and participant number and composition5 6 Annotated vs. genetic sex5 7 Chromosomal anomalies5 8 Relatedness 7 9 Population structure 8
Genotypic and allelic data are reported as number with percent in parentheses, respectively. The χ2 tests were used for statistical analyses. Table 2. Allelic and genotypic distribution frequencies for c.-206 T A genetic polymorphism of RNF41 gene in the CHD patients and non-CHD control subjects.
Aug 28, 2009 · which systems of quality control and quality assurance (QC/QA) are essential. This article describes a system of QC/QA for GWAS with a focus on the genotypic data. We define QC as steps taken to monitor and control the quality of a product as it is being produced, while QA is defined as a post-production review of product quality. In
Genomic Selection (Genome-wide Marker Assisted Selection) 1. Reference Population 3. Genomic Selection 2. Data Analysis 4. Selected Animals Animals with genotypic and phenotypic information - QC and data processing - Prediction model: Young animals (selection candidates) Prediction of genetic merit using marker information Superior animals
API 653 Tank Inspection, Repair, Alteration and Reconstruction, 3rd 2005 American Petroleum Institute USA Current Inspection, repair, modification and reconstruction of tanks built edition incorporating addendum 1 to API 650 or API 12C and 2 4 . Standard Title Year Publishing body Country Status Primary focus BS EN 14015 Specification for the design and 2004 European Europe Current Design and .
