Clinprotools v 2

The method of MALDI-TOF MS analysis followed that of our previous study^{17 (link)}. The strains were cultured at 37 °C for 16-24 h using 5% sheep blood agar. We performed ethanol-formic acid protein extraction from grown colonies for preparation of the MALDI-TOF MS analysis and used Bruker Bacterial Test Standard (Bruker Daltonik, Bremen, Germany) for calibration. MALDI-TOF MS analysis was performed using MALDI Biotyper (Bruker Daltonik). Spectra obtained by MALDI-TOF MS analysis were used for comparison of the spectrum of the ST131 with that of the non-ST131 using ClinProTools v2.2 (Bruker Daltonik), and peaks specific to ST131 were searched. We analysed spectra with Peak Statistic Calculation, which includes the Wilcoxon rank sum test. We analysed each isolate three times, and the peaks that were observed at least two of the three times in Peak Statistic Calculation were extracted as reproducible specific peaks of ST131. For the specific peaks judged to be significant by ClinProTools, peaks within the range of ±400 ppm were confirmed by FlexAnalysis v3.4 software (Bruker Daltonik) for each strain. The settings used for MALDI-TOF MS analysis were linear positive mode, 20-Hz laser frequency, 20-kV acceleration voltage, 18.5-kV IS2 voltage, 250-ns extraction delay, and 2000–20,000 m/z range.

The MS spectra were then exported to flex Analysis v3.3, ClinProTools v2.2 and MALDI-Biotyper v3.0. (Bruker Daltonics) software for data processing (smoothing, baseline subtraction, peak picking). The quality of MS spectra was evaluated by visualization of spectra obtained from the four spots for each sample with the flex Analysis v3.3 software (Bruker Daltonics). Cluster analyses (MSP dendrogram) and principal component analysis (PCA) were performed to verify intra-species reproducibility and inter-species specificity as well as variability within different castes (soldiers and workers) of the same species. Cluster analyses were performed based on the comparison of the MSPs given by the MALDI-Biotyper v3.0. software and grouped according to the mass profile of the proteins (i.e., their mass signals and intensities) and it reflects how tick specimens are related to each other. The setting parameters were as follows: distance measure by correlation, linkage by average; the score threshold value for a single organism was 300 (arbitrary unit) and for related organisms was 0 (arbitrary unit).

Five morphologically and ITS2 PCR-confirmed female mosquitoes of each species were processed for creation of the database. The spectra were processed using the Flex Analysis software for peak smoothening and baseline subtraction. The spectra generated from four spots of the same specimen were compared using the ClinProTools v.2.2 software (Bruker Daltoniks) to check for reproducibility of the spectra. Out of these four spectra, the one having maximum number of peaks with > 50% intensity was selected. These selected spectra from all five representative specimens were used for creation of the database using Biotyper v.3.0. The same was done for all the nine species included in the study.