Proteotyping of Campylobacter jejuni by MALDI-TOF MS and Strain Solution Version 2 Software.

Identification of microorganisms by MALDI-TOF MS has become a popular method in the past 20 years. Strain Solution ver. 2 software appended with MALDI-TOF MS enables accurate discrimination of serotypes and strains beyond the genus and species level by creating a theoretical mass-based database. In this study, we constructed a theoretical mass database with the validated biomarkers to proteotype Campylobacter jejuni. Using 10 strains belonging to Campylobacter spp. available from culture collections and 41 Campylobacter jejuni strains isolated from humans and foods, the ribosomal protein subunits L36, L32, S14, L24, L23, L7/L12, and S11 could be selected as the effective biomarkers for the proteotyping of C. jejuni at MALDI-TOF MS. An accurate database of their theoretical mass-based values was constructed by matching these gene DNA sequences and the observed mass peaks. We attempted to automatically classify 41 strains isolated from nature using this database and Strain Solution ver. 2 software, and 38 strains (93%) were correctly classified into the intended group based on the theoretical mass-based values. Thus, the seven biomarkers found in this study and Strain Solution ver. 2 are promising for the proteotyping of C. jejuni by MALDI-TOF MS.

Simultaneous Discrimination of Cereulide-Producing Bacillus cereus and Psychrotolerant B. cereus Group by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry.

ABSTRACT: Cereulide-producing Bacillus cereus, which causes foodborne illnesses with vomiting, and psychrotolerant B. cereus group strains such as Bacillus mycoides, which can grow at ≥7°C and cause spoilage of refrigerated foods, are significant concerns for the food industry. Rapid and simple methods to discriminate the cereulide-producing B. cereus and psychrotolerant B. cereus group strains from other B. cereus group strains are needed. We developed a novel, rapid, and simple method with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis for simultaneous discrimination of these two groups from other B. cereus group strains. A potassium adduct of cereulide was used to detect cereulide-producing B. cereus, and three ribosomal subunit proteins (L30, S16, and S20) were used to detect psychrotolerant B. cereus group. A total of 51 B. cereus group strains were analyzed by MALDI-TOF MS. The biomarkers allowed successful discrimination of 16 cereulide-producing B. cereus and 15 psychrotolerant B. cereus group strains from other B. cereus group strains. The results showed that this MALDI-TOF MS analysis allows simultaneous discrimination of cereulide-producing B. cereus and psychrotolerant B. cereus group strains from other B. cereus group strains. This efficient method has the potential to be a valuable tool for ensuring food safety.

Discrimination of psychrotolerant Bacillus cereus group based on MALDI-TOF MS analysis of ribosomal subunit proteins.

Psychrotolerant species of the Bacillus cereus group, Bacillus mycoides and Bacillus weihenstephanensis, can grow at ≥ 7 °C and are significant concerns for the food industry due to their ability to cause spoilage of refrigerated food. In addition to that, some strains of B. weihenstephanensis can produce emetic toxin, namely cereulide, which is known to cause vomiting. Therefore, rapid and simple methods to discriminate psychrotolerant B. cereus group species are crucial. Here, matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and the S10-spc-alpha operon gene encoded ribosomal protein mass spectrum (S10-GERMS) method were used to discriminate psychrotolerant species of the B. cereus group based on a set of four ribosomal subunit proteins (S10, S16, S20 and L30). A total of 36 strains of B. cereus group were cultured on LB agar, and analyzed by MALDI-TOF MS. The four biomarkers successfully discriminated 12 strains of psychrotolerant species from mesophilic species of the B. cereus group. Furthermore, the four biomarkers also classified some Bacillus thuringiensis strains. MALDI-TOF MS analysis using the S10-GERMS method allowed simple and rapid discrimination of psychrotolerant species of the B. cereus group from other mesophilic species. This method has a possibility to enable manufacturers and distributors of refrigerated foods to control psychrotolerant species of the B. cereus group effectively.

Improved MALDI-MS method for the highly sensitive and reproducible detection of biomarker peaks for the proteotyping of Salmonella serotypes.

The rapid identification and classification of pathogenic microorganisms, including Salmonella enterica, is important for the surveillance and prevention of foodborne diseases. Matrix-assisted laser desorption\ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been shown to be an effective tool for the rapid identification of microorganisms. In a previous report, a mass database consisting of 12 biomarker proteins, S8, L15, L17, L21, L25, S7, superoxide dismutase (SodA), peptidylprolyl cis-trans isomerase C, Gns, YibT, YaiA, and YciF, was introduced for the serotyping of S. enterica via MALDI-MS (Applied Microbiology and Biotechnology, 2017, 101, 8557-8569). However, the reproducibility of peak detection of biomarkers such as SodA at m\z 23 000 was poor. We report here an optimized MALDI-MS method for detecting these biomarkers with high sensitivity and reproducibility. The issue was solved by controlling the bacterial concentration at 1 × 10 to 1 × 102 MFU (3 × 106 to 3 × 107 CFU\µL, as calculated from the MFU), using the colony suspension supernatant obtained by centrifugation, and using matrix additives such as methylenediphosphonic acid and N-decyl-ß-D-maltopyranoside. We propose that the method including the above steps is one of the best for detecting biomarkers with high sensitivity and reproducibility.

Screening for human immunodeficiency virus using a newly developed fourth generation lateral flow immunochromatography assay.

BACKGROUND: High sensitivity for detection of HIV-1 p24 antigen allows for early detection of primary HIV-1 infections. OBJECTIVES: To evaluate the detection sensitivity and specificity of the Daina Screen® HIV Combo assay using clinical specimens in Japan where the pretest probability (prevalence) is low. STUDY DESIGN: We screened 17,373 preoperative outpatient blood samples using 4th generation lateral flow immunochromatography Daina Screen® HIV Combo assay for simultaneously detecting anti-HIV-1/2 and HIV-1 p24 antigen. RESULTS: Of the samples tested, 24 were positive for HIV-1 p24 antigen and 49 for HIV-1/2 antibody. Of the 49 samples, 36 were WB and HIV-1 RNA negative, 10 were WB and HIV-1 RNA positive, and 3 were WB positive, HIV-1 RNA negative, and in-house HIV-1 proviral DNA positive. RT-PCR revealed that of the 24 samples that were p24 antigen positive, one sample was HIV-1 RNA positive, which was reconfirmed using an in-house HIV-1 provirus DNA assay. From the 17,300 HIV-1 p24 antigen and anti-HIV-1/2 negative samples, pools containing 10 negative samples each were tested for HIV-1 by RT-PCR; all results were negative. CONCLUSION: The Daina Screen® HIV Combo assay had a sensitivity and specificity of 100% and 99.7%, respectively, which sufficiently detected HIV infection in the cohort.

Reduced-port endo-laparoscopic surgery using umbilical zigzag incision for concomitant operations: A case series.

INTRODUCTION: Recently, use of reduced-port surgery has become widespread; however, it is a difficult operation. Hachisuka et al. developed a method called the umbilical zigzag incision, which enlarges the fascial incision using only an umbilical skin incision. We believe this method will be feasible for concomitant laparoscopic surgery. We report our surgical techniques for concomitant laparoscopic surgery using an umbilical zigzag incision. METHODS: The patient who should receive more than 2 places of abdominal operation were indicated this procedure. In all cases, we made the zigzag incision in the umbilical region. After the linea alba and peritoneum were incised, the wound retractor was inserted through the incision, which enlarges the fascial opening. GelPoint was attached to the wound retractor and the operation was started. RESULTS: We could create a fascial opening of up to 6 cm with the umbilical zigzag incision, which improves the triangulation of forceps and reduces interference among the trocars. The trocars in the center of the abdomen could be utilized for almost all intraperitoneal operations. This procedure was especially useful in cases that included lymph node dissection because dissection of a malignant tumor is a delicate procedure. Furthermore, extraction of specimens and anastomosis went very smoothly because the fascial incision was large enough such that no extension of the incision was needed. No early or late postoperative complications occurred in any case. Postoperative wounds were clear and therefore patient satisfaction levels were high. CONCLUSION: Umbilical zigzag incision may be feasible especially in concomitant laparoscopic surgery.

Rapid Generation of Monoclonal Antibodies from Single B Cells by Ecobody Technology.

Single B cell sampling following to direct gene amplification and transient expression in animal cells has been recognized as powerful monoclonal antibodies (mAbs) screening strategies. Here we report Ecobody technology which allows mAbs screening from single B cells in two days This technology uses Escherichia coli cell-free protein synthesis (CFPS) for mAb expression. In the CFPS step, we employed our original techniques: (1) 'Zipbody' as a modified Fab (fragment of antigen binding) format, in which the active Fab formation is facilitated by adhesive leucine zipper peptides fused at the C-termini of the light and heavy chains; and (2) an N-terminal SKIK peptide tag that can markedly increase protein production. By the Ecobody technology, we demonstrated rapid screening of antigen specific mAbs from immunized rabbits and Epstein-Barr Virus infected human B cells. We further obtained rabbit mAbs in E. coli expression system yielding to 8.5 mg of purified proteins from 1 L bacterial culture.

Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation.

We report a rapid and cost-effective monoclonal antibody screening method from single animal B cells using reverse transcription (RT)-PCR and Escherichia coli cell-free protein synthesis (CFPS), which allows evaluation of antibodies within 2 working days. This process is named "Ecobody technology". The method includes strategies to isolate B cells that specifically bind an antigen from the peripheral blood of immunised animals, and single-cell RT-PCR to generate DNA fragments of the VH and VL genes, followed by CFPS for production of fragments of antigen binding (Fab). In the CFPS step, we employed our techniques: 1) 'Zipbody' as a method for producing Fab, in which the association of heavy and light chains is facilitated by adhesive leucine zipper peptides fused at the C-termini of the Fab; and 2) an N-terminal SKIK peptide tag that can increase protein expression levels. Using Ecobody technology, we obtained highly-specific monoclonal antibodies for the antigens Vibrio parahaemolyticus and E. coli O26. The anti-V. parahaemolyticus Zipbody mAb was further produced in E. coli strain SHuffle T7 Express in inclusion bodies and refolded by a conventional method, resulting in significant antigen-binding activity (K D = 469 pM) and productivity of 8.5 mg purified antibody/L-culture.

Application of proteotyping Strain Solution™ ver. 2 software and theoretically calculated mass database in MALDI-TOF MS typing of Salmonella serotype.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based microbial identification is a popular analytical method. Strain Solution proteotyping software available for MALDI-TOF MS has great potential for the precise and detailed discrimination of microorganisms at serotype- or strain-level, beyond the conventional mass fingerprinting approaches. Here, we constructed a theoretically calculated mass database of Salmonella enterica subspecies enterica consisting of 12 biomarker proteins: ribosomal proteins S8, L15, L17, L21, L25, and S7, Mn-cofactor-containing superoxide dismutase (SodA), peptidyl-prolyl cis-trans isomerase C (PPIase C), and protein Gns, and uncharacterized proteins YibT, YaiA, and YciF, that can allow serotyping of Salmonella. Strain Solution ver. 2 software with the novel database constructed in this study demonstrated that 109 strains (94%), including the major outbreak-associated serotypes, Enteritidis, Typhimurium, and Infantis, could be correctly identified from others by colony-directed MALDI-TOF MS using 116 strains belonging to 23 kinds of typed and untyped serotypes of S. enterica from culture collections, patients, and foods. We conclude that Strain Solution ver. 2 software integrated with the accurate mass database will be useful for the bacterial proteotyping by MALDI-TOF MS-based microbial classification in the clinical and food safety fields.

N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae.

Despite advances in microbial protein expression systems, low production of proteins remains a great concern for some genes. Here we report that the insertion of a short peptide tag, consisting of Ser-Lys-Ile-Lys (SKIK), adjacent to the start codon of genes encoding difficult-to-express proteins can increase protein expression in Escherichia coli and Saccharomyces cerevisiae. Protein expression levels of a mouse monoclonal antibody (mAb), rabbit mAbs obtained from clonal B cells, and an artificially designed peptide were significantly increased simply by the addition of the SKIK tag in E. coli systems. In particular, a ∼30-fold increase in protein production was observed for the mouse mAb, and the artificially designed peptide band became detectable in sodium dodecyl sulfate-poly acrylamide gel electrophoresis after coomassie brilliant blue staining or western blotting on adding the SKIK tag. The tag also increased the expression of tagged proteins in S. cerevisiae and an E. coli cell-free protein synthesis system. Although the mechanism of high protein expression on addition of the tag is unclear, our findings offer great benefits to biotechnology research and industry.