Influence of Ampicillin Resistant Cronobacter Sakazakii on the Stability and Expression of gfpuv in Mutant Cells

YUSNITA WAHYUNI SILITONGA, RATIH DEWANTI HARIYADI, SITI NURJANAH, M. NIZAR HANAFIAH NASUTION

Abstract


Construction of Cronobacter sakazakii with pGFPuv could facilitate the tracking of the bacteria in foodstuff. GFPuv plasmids are completed with ampicillin resistant gene for easy selection. Previous studies indicated that ampicillin resistant bacteria could not be made into GFPuv mutants, but the reason was unknown. The objectives of this study were to investigate the performance of C. sakazakii GFPuv mutants based on their stability and GFP expression and to understand the correlation between ampicillin resistance traits in the wild types with the success of GFPuv mutants construction. The results showed that the ampicillin resistance level of C. sakazakii isolates varied. Of the 12 isolates studied, 8 isolates (66.6 %) were susceptible ((FWHc3, FWHb6, FWHb15, YRw3, E2, E4, E6, and E9), two (16.66%) had intermediate resistance to ampicillin (Desb10, E7), and the other two (16.6 %) were ampicillin resistant (FWHd1, E1). The ampicillin sensitive C. sakazakii could be labeled with pGFPuv and their plasmid stability were most stable (E2). The ampicillin resistant C. sakazakii isolates also could be labeled with pGFPuv but the resulting mutants were unstable in the cell and could not express the GFPuv. The high resistance isolate could not express the GFPuv (E7 and FWHd1). Characterization of isolate resistance on antibiotic marker of plasmid is required to assure successfulness and stability of bacteria labelling process.

Keywords


Cronobacter sakazakii, GFPuv, Mutant,Stability, Ampicillin resistance

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References


Akasaka N, Astuti W, Ishii Y, Hidese R, Sakoda H, Fujiwara R. 2015 Change in the plasmid copy number in acetic acid bacteria in response to growth phase and acetic acid consentration. J. Biosci. Bioeng. 119(6): 661-668.

Al-Nabulsi AA, Osaili TM, Elabedeen NOZ. Jaradat ZW, Shaker R, Khaerallah R, Tarazi YH, Holley AH. 2011. Inpact of environmental stress desiccation, acidity, alkalinity, heat or cold on antibiotic susceptibility of Cronobacter sakazakii. Int. J. Food Microbiol. 146: 137-143.

Ash RJ, Mauck B, Morgan M. 2002. Antibiotic resistance of Gram-negative bacteria in rivers, United States. Emerg. Infec. Dis. 8(7): 713-716.

Crameri A, Whitehorn EA, Tate E, Willem PC, Stemmer WPC. 1996. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol. 14: 315-319.

Clontech. Certificate of Analysis pGFPuv. http://www.clontech.com [14 sept 2013].

Dewanti-Hariyadi R, Larasati F, Nuraida L. 2012. Survival of Cronobacter sakazakii in skim milk during spray drying, storage and reconstitution. J. Technol. Food Industry. 23(2): 186-192.

Estuningsih S, Kress C, Hassan AA, Akinden O, Schneider E, Usleber E. 2006. Enterobacteriaceae in dehydrated powdered infant formula manufactured in Indonesia and Malaysia. J. Food Protect. 69(12): 3013-3017.

Gitapratiwi D, Dewanti-Hariyadi R, Hidayat SH. 2012. Genetic relatedness of Cronobacter spp. (Enterobacter sakazakii) isolated from dried food products in Indonesia. Int. Food Res. J. 19(4): 1745-149.

Haddix PL, Paulsen ET, Werner TF. 2000. Measurement of mutation to antibiotic resistance: ampicillin resistance in Serratia marcescens. Bioscene. 26(1): 17-21.

Hamdani FW. 2012. Evaluation of genetic diversity of local isolates Cronobacter spp. (Enterobacter sakazakii) from dried foods. MS thesis, Bogor Agricultural University, Bogor, Indonesia.

Hwang GL, Rahman MA, Razak SA, Sohm F, Farahmand H, Smith A, Brooks, Maclean N. 2003. Isolation and characterisation of tilapia ?-actin promoter and comparison of its activity with carp ?-actin promoter. Biochim. Biophys. Acta. 1625: 11-18.

Iversen C, Forsythe S. 2003. Risk profile of Enterobacter sakazakii, an emergent pathogen associated with infant milk formula. Trends. Food Sci. Tech. 14: 443-454.

Kim K, Jang SS, Kim SK, Park JH, Heu S, Ryu S. 2008. Prevalence and genetic diversity of Enterobacter sakazakii in ingredients of infant food. Int. J. Food Microbiol. 122: 196-203.

Kuzina LV, Peloquin JJ, Vacek DC, Miller TA. 2001. Isolation and identification of bacteria associated with adult laboratory Mexican fruit flies, Anastrepha ludens (Diptera: Tephritidae). Curr. Microbiol. 42: 290-294.

Lee YD, Park JH, Chang H. 2012. Detection antibiotic and biofilm formation of Cronobacter spp. from various foods in Korea. Food Control. 24: 225-230.

Li Y, Chen Q, Zhao J, Jiang H, Lu F, Bie X, Lu Z. 2014. Isolation, identification and antimicrobial resistance of Cronobacter spp. isolated from various foods in China. Food Control. 37: 109-144.

Ma L, Zang G, Doyle MP. 2011. Green fluorescent protein labeling of Listeria, Salmonella and Escherichia coli O157:H7 for safety related studies. PLoS ONE 6: e18083.

Meutia YR, Dewanti-Hariyadi R, Estuningsih S. 2009. The effect of reconstitution temperature for local isolates of Enterobacter sakazakii (Cronobacter sp.) from powdered infant formula and weaning food. J. Agro-Based Industry. 26(1): 22-30.

Noor SM, Poeloengan. pp. 2008. 367-371 in: The sensitivity of Enterobacter sakazakii to antibiotic. National proposed prospect dairy industry to free trade 2020. April 21, Bogor, Indonesia. Indonesian Research Center for Veterinary Science, Bogor, Indonesia.

Nurjanah S, Dewanti-Hariyadi R, Estuningsih S, Suhartono MT. 2014. Stability andgrowth characteristics of GFPuv-Labeled Cronobacter sakazakii isolated from foods. Food Sci. Biotechnol. 23(5): 1491-1496.

Nurjanah S, Sulistyanti ST, Dewanti-Hariyadi R. 2015. Aplication of GFPuv labeled Cronobacter sakazakii for evaluation of its survival during cornstarch processing. Word Eng. Technol. 3: 1-6.

Rahman MA, Hwang GL, Razak SA, Sohm F, Maclean N. 2000. Copy number related transgene expression and mosaic somatic expression in hemizygous and homozygous transgenic tilapia (Oreochromis niloticus). Transgenic Res. 9(6): 417- 427.

Sambrook J, Russel DW. 2001. Molecular cloning: a laboratory manual. 3th ed. Cold Spring Harbor Laboratory Press, New York.

Seftiono H. 2012. Thermal inactivation kinetics of Cronobacter spp. (Enterobacter sakazakii) in infant formula and buffer system with various aw and pH. MS thesis, Bogor Agricultural University, Bogor, Indonesia.

Shimomura O. 2005. The discovery of aequerin and green fluorescent protein. J Microscopy. 217: 3-15.

Stock I, Wiedemann B. 2002. Natural antibiotic susceptibility of Enterobacter amnigenus, Enterobacter cancerogenus, Enterobacter gergoviae, Enterobacter sakazakii strains. Clin. Microbiol. Infect. 8: 564-578.

Zhang S, Zhao B, Liu X, Gao Z, Huang X. 2013. Construction of new GFP vector and its use for Fusarium oxysporum transformation. Saudi J. Biol. Sci. 20: 23-27


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