Bacteria/Mass Spectrometry Figure 5. Weighted pattern matching. Starting from a given pattern matching result, which cannot differentiate between closely related sub- species, a limited number of mass signals are selected for accurate identification. The flow-chart shows the combination of selected specific mass signals to differ important Erwinia sub-species. For example, an initial pattern matching analysis revealed two potential candidate strains, E. amylovora and E. pyrifoliae. The mass signals at 7696 m/z and the mass peaks at 8186, 8244, 8287 m/z are specific for E. pyrifoliae and E. amylovora rubus strains, respectively, which distinguish them from E. amylovora. The mass peak at 9554 m/z distinguished the North American type strain E. amylovora 273 from the other E. amylovora strains of American and European origin, which instead have an additional peak at 9510 m/z. For the automated weighted pattern analysis, specific values listed in Table 1 were assigned to these marker masses. This approach was applied to distinguish closely related Erwinia strains as shown in Table 2. doi:10.1371/journal.pone.0002843.g005 can undergo specific testing of a large variety of different bacteria, we will steadily increase the numbers of bacterial type strain reference spectra. Therefore, we will apply the procedure presented in conjunction with the use of analysis software and the mass spectra database to an effort with internationally approved reference stocks. Materials and Methods Tubes and tips were purchased from Eppendorf. All chemicals were of highest purity (HPLC-grade) and purchased from Sigma- Aldrich. The Erwinia strains came from various strain collections, others (described in ref. 3) and the E. coli K12-strains 1100 and W3350 were obtained from the collection of the JKI Dossenheim. The bacteria used for the reference database and the dendrogram were the following: E. amylovora CFBP1232 (T), E. amylovora Ea1/79 DSM 17948, E. amylovora 273 ATCC 49946, E. amylovora IL6 (rubus) (Lab collection JKI Dossenheim, isolated in Illinois, USA), E. amylovora MR1 (rubus) (Lab collection JKI Dossenheim, isolated in Michigan, USA), E. pyrifoliae 16/96 (T) DSM 12163, E. pyrifoliae 1/96 DSM 12162, E. pyrifoliae Ejp557 (Japan) (Lab collection JKI Dossenheim, isolated from Nashi pear, Hokkaido, Japan, 1994, A. Tanii), E. tasmaniensis 1/99 (T) DSM 17950, E. tasmaniensis 2/99 DSM 17949, E. billingiae Eb 660 (T) NCPPB660 and Eb 661 (T) NCPPB661, E. persicina CFBP3622 (T), E. rhapontici CFBP3618 (T), E. psidii CFBP3627 (T), Pectobacterium cacticida CFBP3628 (T), Brenneria quercini CFBP3617 (T), E. mallotivora CFBP2503 (T), E. Table 2. Weighted pattern-matching. toletana CFBP6631 (T), E. papayae CFBP5189 (T), E. tracheiphila CFBP2355 (T), E. coli 1100 (E. coli/ K-12, Lab collection JKI Dossenheim), E. coli W3350 (E. coli/ K-12, Lab collection JKI Dossenheim). Erwinia type strains are indicated (T); CFBP = Col- lection Franc¸aise des Bacte´ries Phytopathoge`nes; DSMZ = Ger- man Collection of Microorganisms and Cell Cultures; NCPPB = National Collection of Plant Pathogenic Bacteria (UK); ATCC = American Type Culture Collection; JKI = Julius Kuehn Institute. Infection of in vitro pear plants (micro-propagated plants): Pear leaves were wounded and inoculated with cells of the German E. amylovora strain Ea1/79. After incubating for 5 days, the infected pear plantlets displayed symptoms typical of fire blight infection, such as water soaking and necrosis accompanied by the production of bacterial ooze. We washed the bacteria from the plant surface with 1.5 ml water, centrifuged the samples at 10006 g for a minute, and decanted the liquid. Afterwards we suspended and inactivated the bacteria as is described below. Isolation of Erwinia spp. from necrotic wood of pear trees (from Carinthia): Fifty milligram of dark bark slices contaminated with bacteria were immersed in 1 ml water. After soaking for 15 minutes, samples were diluted, and 200 ml of that were plated on LB agar with cycloheximide (50 mg/ml). White colonies were assayed on semi-selective agar for E. amylovora by using PCR and DNA sequencing at the JKI Dossenheim. E. amylovora colonies were processed as blind samples for MALDI analysis at the Max- Planck-Institute for Molecular Genetics (Berlin). Isolate Infected pear blossoms (directly isolated) MSP Analysis Matching MO Erwinia amylovora IL6 (rubus) Erwinia amylovora CFBP1232 Erwinia amylovora 1/79 Score Matching MO 2.5 2.4 2.3 Weighted Analysis Erwinia amylovora CFBP1232 Erwinia amylovora IL6 (rubus) Erwinia pyrifoliae 16/96 Score 2.4 1.7 1.6 Weighted pattern-matching contributes to an increased accuracy of automated bacterial identification. For example, in the case of pears infected with E. amylovora (German strain Ea1/79), initial pattern matching analysis could not differentiate between different E. amylovora subspecies. Visual inspection of specific mass signals listed in Table 1 helped to unambiguously identify the correct sub-species. Software-supported weighted pattern matching leads to an accurate analysis of the correct subspecies, for example, the E. amylovora (type strain CFBP1232) was identified unambiguously. More information on the weighted pattern matching analysis is depicted in Figure 5 and in Table 3. doi:10.1371/journal.pone.0002843.t002 PLoS ONE | www.plosone.org 7 July 2008 | Volume 3 | Issue 7 | e2843