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Supplementary material16S rRNA gene sequence analysisFor the phylogenetic identification of each defined OTU, one colony was chosen for 16S rRNA gene sequencing. Colonies were picked, suspended in 50 ?l aqua dest. and heated to 95 °C for 10 minutes to release the genomic DNA (gDNA). 2 ?l of cell lysate were directly used for PCR. The PCR was performed with primer #27f 3’-AGA GTT TGA TCM TGG CTC AG -5’ and #1492R 3’-ACC TTG TTA CGA CTT-5’ (Lane, 1991). PCR conditions were as described in Hausdorf et al. (2011). PCR products were purified with the QIAquick PCR purification kit (Qiagen, Hilden), cloned into the pGEM-T plasmid (Promega, Mannheim, Germany), transformed into JM109 competent cells (Promega) according to the manufacturer’s guidelines and sequenced by GATC Biotech AG (Konstanz, Germany). Sequences were checked for chimeric sequences with the software MALLARD (Ashelford et al., 2006). The ARB software (Ludwig, 2004) and the SILVA database (Yarza et al., 2010) were used for the sequences alignment and the phylogenetic analyses using the Maximum Parsimony algorithm (Kolaczkowski & Thornton, 2004) for tree construction. Sequences obtained in this study were deposited in the Genbank (National Center for Biotechnology Information, NCBI, BA123456) under the accession numbers JQ845807- JQ845877.Diversity estimatorsDiversity and distribution of the isolated bacteria of the taken samples from the spinach-processing plant were analyzed by several different indices: To estimate the diversity of detected bacteria the Shannon index (H) was calculated with [H=-sum((ni/n)ln(ni/n))] where ni is the number of individuals of a taxon i and n the total number of organisms of all species (Wang et al., 2005). This index gives the proportional abundance of species and reacts sensitively to rare species. In order to describe the uniformity of the OTU distribution, the evenness [eH/S] was calculated, where S is the total number of species. These indices were calculated with the software PAST v1.72 (Hammer et al.2001). The rarefaction analysis, applying the software Analytic rarefaction (Holland, 2003) evaluates how the number of species within a sample changes with the number of individuals (Hughes et al., 2001) reflecting the OTU richness of a clone, or in our case, a library of isolates. The coverage was determined with Good?s formula [(1-(n/N))*100] whereby n is the number of phylotypes (OTU) represented by one isolate and N the total number of isolates (Good, 1953).Supplementary Table 1: Isolated colonies in each sample and corresponding diversity indices.Sample* Cultivable cells [log cfu l-1]No. of colonies analyzed by MALDI-TOF MSDefined OTUs per sampleEvennessCoverage [%]**Shannon diversity indexTW 0-----S07.871170.52862.18WW1878240.66872.81S1777180.51492.22WW2847130.53831.85S28.72470.80641.91WW37.8111130.36951.49S3763130.33851.52BW5830.96801.05S48.33890.71871.66*Order of rows represents the sequential order of operations in the processing plant. S0 = uncleaned spinach; TW = tap water; WW1 – WW3 = water from wash basins 1 - 3; S1 - S3 = spinach from wash basins 1 - 3; BW = water from blancher; S4 = sample of spinach after blanching; **coverage based on Good?s formulaSupplementary Table 2: Detected species as determined by MALDI-TOF MS and their occurrence (log cfu l-1) as established with the plate count method on different media. MediumASM+SS1Blood agarSample*S4S1WW1WW2WW3Arcobacter sp.60000Bacillus sp.00000Brevundimonas sp.00000Escherichia vulneris04000Microbacterium sp.00000Pantoea sp.00040Pseudomonas sp.04404Raoultella sp.00000Staphylococcus sp.00000MediumMarine agarSample*S0WW1S1WW2S2WW3S3BWS4Arcobacter sp.000000000Bacillus sp.4770000116Brevundimonas sp.000005000Escherichia vulneris000000000Microbacterium sp.007000000Pantoea sp.000800000Pseudomonas sp.000070700Raoultella sp.000800000Staphylococcus sp.009000000*Order of columns represents (left to right) the sequential order of operations in the processing plant. S0 = uncleaned spinach; WW1 – WW3 = water from wash basins 1 - 3; S1 - S3 = spinach from wash basins 1 - 3; BW = water from blancher; S4 = sample of spinach after blanching; ASM+SS1 = Arcobacter selective media + selective supplement 1.Supplementary Table 3: Comparison of microbial community structures of samples from a carrot-processing plant (Hausdorf et al., 2011), obtained by 16S rRNA gene sequence analysis, to diversities of samples from a spinach-processing plant obtained by MALDI-TOF MS (this study). Taxonomic affiliationCarrot wash water(16S rRNA gene clone library)Spinach wash water(MALDI-TOF MS)OTUClones [%]OTUIsolates [%]Actinobacteria--63Bacteroidetes 1714NDNDFirmicutes15142925Proteobacteria62714256Alphaproteobacteria1031<1Betaproteobacteria221032Epsilonproteobacteria2111<1Gammaproteobacteria28473754ND = not detectedSupplementary Figure 1: Examples of mass-spectral profiles of isolates from a spinach-washing plant. ................
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