Identification of Microorganism Species in Leafy Vegetables Using MALDI-TOF MS (Matrix Assisted Laser Desorption / Ionization Mass Spectrometry) Technique

Abstract

The foodborne fungal and bacterial pathogens are increasingly associated with fresh fruit and vegetables. Bacterial colonization of fresh produce plants can occur to high levels on the external tissue but bacteria have also been detected within plant tissue. Leafy vegetables such as lettuce, spinach, cabbage, rocket, mint, parsley, dill, and leek were collected from different vegetable markets in the counties of Defne and Antakya of Hatay province. Growing media potato dextrose agar, nutrient agar and blood agar media were used for microbiological analysis. Their identification were performed based on phenotypic characters and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique. According to the results of MALDI-TOF MS analysis, when the distribution of microorganisms were examined, the most bacterial species were identified with 60 species at the species level, followed by 4 fungi and 1 yeast.   The most microorganism species were obtained from lettuce samples.   When the distribution of species level is examined; firstly, 9 species with Pseudomonas, 5 species with Lactobacillus, 4 species with Acinetobacter and 2 species with Klebsiella, Citrobacter, Enterobacter, Lysinibacillus were obtained and Pantoea one species Bacillus, one species Escherichia coli, one species followed by Exiguobacterium genera. When the colony density of microorganism species were examined, the most intensive colony was determined in the species belonging to the genus Pseudomonas and Acinetobacter. At least colony density was observed in fungi. In result, Salmonella sp, Klebsiella pneumoniae, Escherichia coli, Bacillus cereus, Klebsiella oxytoca species were found to be very dangerous species for human health.

Keywords

• MALDI-TOF MS,Microorganism,fresh vegetables,identification

Yaprağı Yenen Sebzelerdeki Mikroorganizma Türlerinin MALDI-TOF MS (Matris Destekli Lazer Desorpsiyon/İyonizasyon Uçuş Süresi Kütle Spektrometresi) Tekniği Kullanılarak Tanılanması

Öz

Gıda kaynaklı fungal ve bakteriyel patojenler giderek taze meyve ve sebzelerle ilişkilendirilmektedir. Taze üretilen bitkilerin bakteriyel kolonizasyonu, dış dokuda yüksek seviyelerde oluşabilir, ancak bitki dokusu içerisinde bakteriler de tespit edilmektedir. Hatay ilinin Defne ve Antakya ilçelerinde farklı sebze pazarlarından marul, ıspanak, lahana, roka, nane, maydanoz, dereotu, pırasa gibi yapraklı sebzeler toplanmıştır. Mikrobiyolojik analizler için patates dekstroz agar, nutrient agar ve kanlı agar besi yerleri kullanılmıştır. Bu mikroorganizmaların tanılanması, fenotipik karakterler ve matris destekli lazer desorpsiyon/iyonizasyon uçuş süresi kütle spektrometresi (MALDI-TOF MS) tekniğine dayalı olarak gerçekleştirilmiştir. MALDI-TOF MS analizi sonucuna göre mikroorganizmaların dağılımı incelendiğinde, tür düzeyinde 60 adet ile en fazla bakteri türleri tanılanırken, bunu 4 adet fungus ve 1 adet maya izlemiştir. En fazla mikroorganizma türü marul örneklerinden elde edilmiştir. Cins düzeyinde dağılımları incelendiğinde; 9 tür ile Pseudomonas başta gelirken, bunu 5 tür ile Lactobacillus, 4 tür ile Acinetobacter ve 2 tür ile Klebsiella, Citrobacter, Enterobacter, Lysinibacillus, Pantoea 1 tür Bacillus, 1 tür Escherichia coli, 1 tür Exiguobacterium cinsleri izlemiştir. Mikroorganizma türlerinin koloni yoğunlukları incelendiğinde, en yoğun koloni Pseudomonas ve Acinetobacter cinsine ait türlerde belirlenmiştir. En az koloni yoğunluğu funguslarda gözlenmiştir. Çalışma sonucunda Salmonella sp, Klebsiella pneumoniae, Escherichia coli, Bacillus cereus, Klebsiella oxytoca türleri insan sağlığı için en tehlikeli türler olarak bulunmuştur.

Anahtar Kelimeler

• MALDI-TOF MS,Mikroorganizma,Taze sebze,Tanı

References

1. Alam, MS. Feroz, F. Rahman, H. Das, K.K. Noor, R. (2015). Microbiological contamination sources of freshly cultivated vegetables, Nutrition & Food Science Vol. 45 No. 4, pp. 646-658.
2. Ayhan, T., Ufuk, B., and Sine, O.T. (2011). Efficacy of Different Decontamination Treatment on Microbial Population of Leafy Vegetables. Journal of Geographic Information and Decision Analysis. 36(1): 9-13.
3. Barak, J.D., Schroeder, B.K., 2012. Interrelationships of Food Safety and Plant Pathology: The Life Cycle of Human Pathogens on Plants. Annual Review of Phytopathology 50: 241-266.
4. Beuchat, LR. (2002). Ecological factors influencing survival and growth of human pathogens on raw fruits and vegetables. Microbes and Infection. 4: 413-423.
5. Carolis, E. D., Posteraro, B., Lass-Flo, C., Vella, A., Florio, A. R., Torelli, R., Girmenia, C., Colozza, C., Tortorano, A. M., Sanguinetti M., Fadda, G., (2012). Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clinic Microbiology Infect. 18: 475–484.
6. Chalupová, J., Raus, M., Sedlářová, M., Šebela, M., (2014). Identification of fungal microorganisms by MALDI-TOF mass spectrometry. Biotechnology Advances 32:230–241.
7. Gauthier, G.M., Keller, N.P., (2013). Crossover fungal pathogens: The biology and pathogenesis of fungi capable of crossing kingdoms to infect plants and humans. Fungal Genetics and Biology 61: 146–157.
8. Hasnain S, Rehman Y, Mahmood S (2015) Cross-Kingdom Pathogenicity across Plants and Human Beings. J Bacteriol Parasitol 6:e124. doi:10.4172/2155-9597.1000e124

9. Hausdorf L., Mundt K., Winzer, M., Cordes, C., Fröhling, A., Schlüter, O., Klocke, M., (2013). Characterization of the cultivable microbial community in a spinach-processing plant using MALDI-TOF MS. Food Microbiology. Food Microbiology 34 (2013) 406-411.
10. Kara, M., Uysal, A.,Soylu, S., Kurt, Ş., Soylu, E.M., (2017). Identification of Plant-Associated Microorganisms Employing MALDI TOF Mass Spectrometry as a Rapid Detection Technique. International Conference on Agriculture, Forest, Food Sciences and Technologies (ICAFOF), 15-17 May, 2017. Nevşehir, p. 1111.
11. Kirzinger, MW., Nadarasah, G., Stavrinides, J. (2011). Insights into crosskingdom plant pathogenic bacteria. Genes 2: 980-997.
12. Lund, B.M., Baird-Parker, T.C., and Gould, G.W. 2000. “The Microbiological Safety And Quality of Food.”Volume I. An Aspen Publictions, Maryland,
13. Mahmood, S., Rehman, Y., Hasnain, S., (2015). Cross-Kingdom Pathogenicity across Plants and Human Beings. J Bacteriol Parasitology 6: 4. doi:10.4172/2155-9597.1000-124.
14. Mellmann, A., Harmsen, D., Cummings, CA., Zentz, EB., Leopold, SR., (2011). Prospective Genomic Characterization of the German Enterohemorrhagic Escherichia coli O104:H4 Outbreak by Rapid Next Generation Sequencing Technology. Microbiology 105 (6), 1729-1743.
15. Roberts, T.A., Pitt, J.I., Farkas, J., and Graw, F.H. (1998). “Microorganisms in food. Microbial ecology of food commodities.” Blackie Academic & Professional, UK.
16. Sengun, I.Y., Karapinar, M.,( 2004). Effectiveness of lemon juice, vinegar and their mixture in the elimination of Salmonella typhimurium on carrots (Daucus carota L.). International Journal of Food Microbiology., 96: 301-305.
17. Sharma, K., Goss, E.M., Dickstein, E.R., Smith, M.E., Johnson, J.A., (2014). Exserohilum rostratum: characterization of a cross-kingdom pathogen of plants and humans. Plos One 9: 108691.
18. Sivapalasingam, S., Friedman, C. R., Cohen, L., Tauxe, R. V., 2004. Fresh produce: a growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997. J. Food Prot. 67(10):2342-2353.
19. Uysal, A., Kurt, Ş., Soylu E.M., Kara M., Soylu S., (2018). Evaluation of the Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) Mass Spectrometry for Identification of Some Plant Fungal Pathogenic Species. International Agricultural Science Congress. 09-12 May Van/Turkey.
20. Vandekinderen, I., Van Camp, J., Devlieghere, F., Veramme, K., Denon, Q., Ragaert, P., De Meulenaer, B. (2008). Effect of decontamination agents on the microbial population, sensorial quality, and nutrient content of grated carrots (Daucus carota L.). Journal of Agricultural Food Chemistry.56: 5723-5731.
21. Wright, K. M., Chapman, S., McGeachy, K., Humphris, S., Campbell, E., Toth, I. K., and Holden, N. J. (2013). The endophytic lifestyle of Escherichia coli O157:H7: Quantification and internal localization in roots. Phytopathology 103:333-340.