Rapid Identification of Vibrio Species Isolated from the Southern Coastal Regions of Korea by MALDI-TOF Mass Spectrometry and Comparison of MALDI Sample Preparation Methods.

Vibrio species are generally recognized as pathogens predominant in seafood along coastal areas. The food industry has sought to develop efficient microbial detection methods. Owing to the limits of conventional methods, this study aimed to establish a rapid identification method for Vibrio isolated from Korea, based on matrix-assisted laser-desorption/ionization timeof- flight mass spectrometry (MALDI-TOF MS). Four different preparation procedures were compared to determine the appropriate means to pretreat Vibrio species, using 17 isolates and five reference strains. Extended direct transfer and full formic acid extraction methods using bacterial colonies on agar plates revealed very low identification rates. Formic acid and trifluoroacetic acid (TFA) extractions using bacterial broth cultures were also performed. All Vibrio isolates and reference strains prepared by TFA extraction were successfully identified to the species level (17/22, 77.3%) and to the genus level (5/22, 22.7%). Thus, TFA extraction was considered the most appropriate method to pretreat Vibrio species for MALDI-TOF MS. The remaining 33 isolates and two reference strains were prepared by TFA extraction and analyzed by MALDI-TOF MS. Overall, 50 isolates were identified to the species level (40/50, 80%) and to the genus level (10/50, 20%). All isolates were identified as 43 V. alginolyticus, six V. parahaemolyticus, and one V. vulnificus species. V. alginolyticus and V. parahaemolyticus were isolated from fish offal (87.5% and 12.5%, respectively), seawater (91.3%, 8.7%), and shellfish (62.5%, 37.5%), whereas V. alginolyticus and V. vulnificus were isolated from sediment (90.9% and 9.1%, respectively). This study established a reliable system of MALDI-TOF MS preparation and analysis for Vibrio identification.

A proteomic approach for rapid identification of Weissella species isolated from Korean fermented foods on MALDI-TOF MS supplemented with an in-house database.

Weissella are obligate heterofermentative lactic acid bacteria belonging to the Leuconostocaceae family. Some Weissella can be found in salted and fermented foods, such as kimchi and jeotgal, and plays an important role in the fermentation process. In the present study, for the first time, a rapid and accurate identification method for Weissella species from kimchi and jeotgal was developed based on MALDI-TOF MS, supplemented with an in-house database. Of the 135 Weissella spectra aligned with the MALDI bioTyper database, 56 isolates (41.5%) yielded no reliable identification results with low log scores (<1.7). After registering the spectra of six Weissella reference strains, all of the isolates were correctly identified, of which 113 (83.7%) and 22 (16.3%) were identified at the species and genus level, respectively. Moreover, a dendrogram generated by protein profiles of the different Weissella species clearly presented distinctive clusters, and PCA analysis separated the spectra of Weissella species into four clusters. In comparing food origins, different Weissella species were identified from two fermented foods. W. soli and W. cibaria were isolated from kimchi, while W. thailandensis and W. halotolerans were isolated from jeotgal. The results of our proteomic approach confirm that the MALDI bioTyper database, with our in-house Weissella database, is sufficient for Weissella identification. The MALDI-TOF MS method provides fast and reliable discrimination between different species in the genus Weissella and, therefore, will be useful for safety control in fish farms or in the production of fermented foods. This method can also be applied to the control of opportunistic pathogenic Weissella in human clinical infections.

Rapid and accurate identification of species of the genus Pediococcus isolated from Korean fermented foods by matrix-assisted laser desorption/ionization time-of-flight MS with local database extension.

Pediococci are halophilic lactic acid bacteria, within the family Lactobacillaceae, which are involved in the fermentation of various salted and fermented foods, such as kimchi and jeotgal. In this study, a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS method was developed for the rapid identification of species of the genus Pediococcus. Of the 130 Pediococcus spectra aligned with the Biotyper taxonomy database, 122 isolates (93.9 %) yielded log scores <1.7, which means they were not identifiable. After registering the spectra of 11 reference strains of the genus Pediococcus, all of the isolates were correctly identified, of which 84 (64.6 %) and 46 (35.4 %) were identified at the species and genus level, respectively. In comparing food origins, no relationship was found between the bacterial characteristics and food environment. We were able to produce a Biotyper system for identification of members of the genus Pediococcus with locally extended Pediococcus reference strains. The MALDI-TOF MS method is fast, simple and reliable for discriminating between species in the genus Pediococcus and therefore will be useful for quality control in determining the spoilage of alcoholic beverages or in the production of fermented food.

Development of a Rapid and Accurate Identification Method for Citrobacter Species Isolated from Pork Products Using a Matrix-Assisted Laser-Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

Previous detection methods for Citrobacter are considered time consuming and laborious. In this study, we have developed a rapid and accurate detection method for Citrobacter species in pork products, using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). A total of 35 Citrobacter strains were isolated from 30 pork products and identified by both MALDI-TOF MS and 16S rRNA gene sequencing approaches. All isolates were identified to the species level by the MALDI-TOF MS, while 16S rRNA gene sequencing results could not discriminate them clearly. These results confirmed that MALDI-TOF MS is a more accurate and rapid detection method for the identification of Citrobacter species.

Screening of Bacteriocin-producing Enterococcus faecalis Strains for Antagonistic Activities against Clostridium perfringens.

This study was conducted to isolate and characterize bacteriocin-producing bacteria against Clostridium perfringens (C. perfringens) from domestic animals to determine their usefulness as probiotics. Bacteriocin-producing bacteria were isolated from pig feces by the spot-on-lawn method. A total of 1,370 bacterial stains were isolated, and six were tentatively selected after identifying the inhibitory activity against the pathogenic indicator C. perfringens KCTC 3269 and KCTC 5100. The selected strains were identified as Enterococcus faecalis (E. faecalis) by 16s rRNA sequencing. Most of the isolated bacterial strains were resistant to 0.5% bile salts for 48 h and remained viable after 2 h at pH 3.0. Some E. faecalis also showed strong inhibitory activity against Listeria monocytogenes KCTC 3569, KCTC 3586 and KCTC 3710. In the present study, we finally selected E. faecalis AP 216 and AP 45 strain based on probiotic selection criteria such as antimicrobial activity against C. perfringens and tolerance to acid and bile salts. The bacteriocins of E. faecalis AP 216 and AP 45 strains were highly thermostable, showing anticlostridial activities even after incubation at 121℃ for 15 min. These bacteriocinproducing bacteria and/or bacteriocins could be used in feed manufacturing as probiotics as an alternative to antibiotics in the livestock industry.