Cytotoxicity and Green Fluorescent Protein labeling of Cronobacter spp. to study its behaviour during corn drying
Suhartono, Maggy T.
MetadataShow full item record
Cronobacter spp. (previously known as Enterobacter sakazakii) is a group of Gram-negative bacteria consisted of six species, including Cronobacter sakazakii and Cronobacter muytjensii (Iversen et al. 2008). Cronobacter spp. is regarded as an emerging foodborne pathogen that causes meningitis, bacteremia and necrotizing enterocolitis in certain groups of infants as a consequence of contaminated infant formulae consumption. Pathogenesis of Cronobacter is caused by several virulence factors, including its toxins. Medium free cells of Cronobacter spp. have been reported to cause alteration of Vero cells morphology due to its cytotoxic activity. In Indonesia, Cronobacter spp. have been isolated (Estuningsih et al. 2006, Meutia et al. 2008, Gitapratiwi et al. 2012 and Hamdani 2012). All of the isolates were obtained from dried food products; i.e powdered infant formula, weaning food, rice flour, tapioca, cornstarch, sugar, cocoa powder and dried spices, suggesting that they were able to survive drying. The data reported by FAO-WHO (2004) showed that the ingredient added through dry mixing process was one of the sources of Cronobacter spp. contaminant in powder infant formulae. Current researches supported this issue, Dewanti-Hariyadi et al. (2010) have isolated Cronobacter spp. from cornstarch and Fiegen (2010) have isolated the pathogen form cornstarch and other ingredients (lecithin, potato starch, rice starch and wheat starch). It is important to understand Cronobacter spp. survival during drying due to its presence in dried food products. Maize was chosen as a food model in this study due to its widespread use as raw materials for dried food products. For specific detection and enumeration, a Green Fluorescent Protein (GFP) was introduced into Cronobacter cells as a marker. GFP is a protein from jellyfish (Aequorea Victoria) which can fluoresce and its gene has been inserted into a plasmid (pGFP) as a vector. The pGFPuv is a GFP variant plasmid having fluorescence signal 45-fold greater than a standard pGFP and this was achieved by DNA shuffling. The mutant can grow in ampicillin containing medium and was seen as a green fluorescents colony. The objectives of this study were to evaluate cytotoxic activity of food originated isolates of C. sakazakii and C. muytjensii and to evaluate their survival, colonization and penetration during maize drying. This research was conducted in three steps: 1. In Vitro analysis of cytotoxic activity of C. sakazakii and C. muytjensii using Vero cell and haemolytic activity on blood agar medium; 2. Construction of GFP-labeled C. sakazakii and C. muytjensii, study of their stability and growth characteristics; 3. Use of the mutant to study the survival, colonization and penetration of C. sakazakii during maize drying. As much as nineteen C. sakazakii isolates dan one C. muytjensii isolate obtained from previous research were used for this study originated from cornstarch, tapioca, flour, infant milk formulae, weaning food and powder dried spices. Twenty isolates of C. sakazakii and C. muytjensii originated from food have different cytotoxic activity of their toxins. Thirteen out of 20 isolates (65% samples) were confirmed as cytotoxic positive including one isolate C. muytjensii while 7 isolates were grouped as nontoxic. Five isolates with the highest cytotoxic activity were C. sakazakii FWHb6, E2, FWHb15, FWHc3 and FWHd16 (80%, 78%, 76%, 73% and 70% respectively relative to control Salmonella Typhimurium 100%), while C. muytjensii FWHd11 belong to moderate group. Haematoxylin Eosin staining showed that a number of Vero cells experienced cytoplasm degradation due to cell degeneration and some of underwent nucleic matter condensation due to the cell death. All isolates showed haemolytic activity after 72 h incubation. C. sakazakii and C. muytjensii isolates took up gfp-containing plasmid into their cell and expressed this gene, except for the ampicillin resistant isolate. Analysis of plasmid stability showed that all of mutants could maintain the plasmid for ca. 20 generations without negatively affecting the growth rates. C. sakazakii FWHd16 mutant was the most stable. Both of C. sakazakii and C. muytjensii mutants showed nearly identical growth curves as compared to the wild type parental isolates in lag, log and stationary phases. The bacterial growth rates (μmax) of mutants and their parental isolates were similar. From this study, the labeled Cronobacter (YRt2a and FWHd16 isolates) were found stable can be applied for studying its survival, colonization and penetration during maize drying. The mutants were easy to be detected and differentiated from ampicillin resistant micro flora due to its ability to fluoresce under UV light. The population of both isolates decreased during drying. The toxic isolate FWHd16 showed better survival than the non-toxic isolate YRt2a for all temperatures, and were still presence after drying. Microscope examination showed colonization of both isolates on maize surface. These mutants were able to penetrate to the inner side of the grain by entering injured surface or pores at the tip cap of the maize.