A total of 167 B.pseudomallei were collected from clinical samples of melioidosis patients in different regions of Hainan. In addition, 7 B.thailandensis isolates were acquired from the soil in parts of Hainan, including Sanya (4 strains), Wanning (2 strains) and Ledong (1 strain). The reference strains of B.thailandensis(ATCC700388), B.cepacia (ATCC25416) and B.cenocepacia (ATCC25608) were all purchased from Beijing Zhongyuan company. Strains of B. cepacia, B.multivorans and B.gladioli were all obtained from clinical isolates of Hainan General Hospital, accounting for one strain each. A total of 30 representative B.pseudomallei isolates from different regions of Hainan were selected as the established strains.
VITEK2 Compact biochemical identification
After recovering all 180 strains stored at -80°C, they were inoculated with quality-control strain Enterobacter cloacae ATCC700323 onto the Columbia Sheep Blood Agar Plate (CBA)(ZhengZhou AnTu Biological Company,ZhengZhou City,China), MacConkey Agar Plate (MAC)(Zheng Zhou AnTu Biological Company,ZhengZhou City,China), and Influenza Blood Chocolate Plate (Vancomycin Chocolate Agar Plate, CHA,Zheng Zhou AnTu Biological Company,ZhengZhou City,China) and incubated in 5% CO2 at 35°C for 18-24 h. Vitek 2 compact(BioMerieux,French) VT2.R 7.01 of the GN(BioMerieux,French) card was used to identify the colonies grown on three different media, respectively, and the specific operation was conducted according to the standard operating procedure in the instrument manual.
Molecular biological identification
Using 16s rRNA gene sequencing
The identification of 13 strains (8 strains of B. thailandensis, 2 strains of B. cepacia, 1 strain of B. cenocepacia,1 strain of B. multivorans and 1 strain of B. gladioli) used in this study was confirmed at the species level using the primers described by Brett et al.[7-9]. The PCR products were sent to Tianyihui Yuan Company (guangzhou,china) for sequencing, and the sequences were searched against the GenBank database using the BLAST algorithm(https://blast.ncbi.nlm.nih.gov/Blast.cgi).
MLST sequencing and typing of B.pseudomallei pathogenic strains
The specific methods of MLST analysis referred in detail to the B.pseudomallei MLST research scheme established by Godoy et al.[10]. The sequencing results of alleles on seven housekeeping genes of each B.pseudomallei pathogenic strain were compared with gene sequences in the MLST database to find their respective allele numbers. The composition of all allele numbers was the B.pseudomallei sequence type (ST).
MLST sequencing and typing of B. thailandensis[10]
A total of seven isolates of B. thailandensis other than the reference strain (ATCC700388) were recovered and their chromosomal DNA was extracted, followed by PCR amplification and sequencing on seven alleles (ace, dtB, gmhD, lepA, lipA, narK, ndh) . TaKaRa LA Taq with GC buffer system and 50 μl reaction volumes were used for PCR. Amplification conditions: 95℃ for 2 min; 30 cycles of 94℃ for 40 s, 58℃ for 40 s, and 72℃ for 40 s; 72℃ for 7min. PCR products were sequenced bidirectionally.The sequencing results were collated and submitted to the MLST database (http://pubmlst.org/bpseudomallei/) to obtain the allele sequence number and determine the ST of strains.
MALDI-TOF MS acquisition
All of the isolates were inoculated onto the Columbia Sheep Blood Agar Plate (CBA), and 30 established strains were incubated onto the Columbia Sheep Blood Agar Plate (CBA), MacConkey Agar Plate (MAC) and Influenza Blood Chocolate Plate (Vancomycin Chocolate Agar Plate, CHA). All of the plates were inoculated in 5% CO2 at 37°C for 24 h. The 30 established strains were from different regions of Hainan island. The monoclonal colonies were picked up by 1 μl volume of sterile inoculation loop and uniformly spread on the target plate. After drying, 1 μl of CHCA matrix solution was added, and then dried at room temperature to form crystals. The VITEK MS(BioMerieux,French) was calibrated, and then mass spectra data were collected and analyzed as well as identified through SARAMS Premium software. After the spectrum acquisition, the data were transferred from a VITEK MS acquisition station to the Saramis analysis server.
Selection of reference spectra
A total of 100 mass spectra were collected for each established strain, and an average spectrum was generated for the 100 original spectra. SARAMS Premium software was used to transfer the average mass spectrum of B.pseudomallei bacteria from each sample and remove the debased spectra, and then aggregate these mass spectra to generate the reference spectra.
Creation of SuperSpectra
According to the specificity, intensity, mass-to-charge ratio (m/z) and other comprehensive parameters of the peak, a SuperSpectra containing all of the characteristic peaks was created. The original Spectra of the SuperSpectra should meet the following conditions: (1) The similarity between different strains of the same species should be 65% or more; (2) the original peak number of the database establishment spectra was controlled between 100 and 200; (3) select 39 specific masses (the common rate of each specific masses was ≥80%); (4) the weight of the obtained specific masses was 31, and the sum of the weights was 31 × 39 = 1209 < 1400; (5) the error range of mass number (%) was 0.0013-0.0746 < 0.08; (6) the absolute peak and relative peak intensity (%) range: 0.30-43.5 > 0. The SuperSpectra were activated for subsequent automated identification at the species level after it have been created.
Verification of SuperSpectra for B.pseudomallei isolates
To assess the capability and stability of the newly created SuperSpectra, external validation was performed for the remaining 150 validation isolates. Of the 150 strains, 137 were B.pseudomallei strains and their closely related species (8 strains of B. thailandensis, 2 strains of B. cepacia, 1 strain of B. cenocepacia, 1 strain of B. multivorans and 1 strain of B. gladioli).The 150 spectra were imported into SARAMS Premium software for pattern matching with the newly created SuperSpectrum database and the obtained identification results were analyzed for evaluating the clinical identification capabilities of the VITEK MS super mass spectrum of B.pseudomallei .
MALDI-TOF MS dendrogram
To construct the dendrogram, Flexanalysis software (version 3.4) was used to adjust the baseline and smoothness of the spectra. Cluster analysis was performed based on comparison the similarity of Main Spectra Projection by calculated the pattern matching.