Sains Malaysiana 43(12)(2014): 1855–1863

Incidence of Cronobacter sakazakii in Powdered Infant Formula Milk Available in Malaysia

(Kehadiran Cronobacter sakazakii dalam Susu Tepung Rumusan Bayi yang Terdapat di Malaysia)

NORRAKIAH ABDULLAH SANI¹*, MASOMEH GHASSEM¹, ABDUL SALAM BABJI¹,

UMA PRIYA KUPUSAMY² & NORIZAN JAAFAR²

¹School of Chemical Sciences and Food Technology, Faculty of Science and Technology

Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

²Chemistry Department of Malaysia, 46661 Petaling Jaya, Selangor, Malaysia

Received: 4 August 2011/Accepted: 30 September 2014

ABSTRACT

Enterobacter sakazakii previously known as 'yellow-pigmented E. cloacae' has been classified as a new genus 'Cronobacter' based on taxonomic analysis and geno-and phenotypic evaluation. This pathogenic organism has been associated with rare form of infant meningitis and necrotizing enterocolitis (NEC) with high mortality rate (40-80%). Some cases have been linked to the consumption of contaminated powdered infant formula milk (PIF). The objective of this study was to determine the presence of Cronobacter spp. in PIF sold in Malaysia. A selective chromogenic agar, Brilliance Enterobacter sakazakii (DFI, Oxoid), was used for detection of Cronobacter strains. Presumptive Cronobacter isolates were identified using biochemical tests (API 20E and MicrogenTM) and molecular assays (SYBR Green Real-time PCR and 16S ribosomal DNA sequencing). All presumptive Cronobacter strains produced typical blue-green colonies and non-Cronobacter strains produced yellow colonies on Brilliance Enterobacter sakazakii agar (DFI formulation). A total of 12 presumptive isolates were selected from DFI agar and identified with biochemical and molecular tests. The results indicated prevalence of 12.5% C. sakazakii contamination from 72 PIF samples. Molecular detection methods such as Real-time PCR and 16S rDNA proved to have higher identification percentage compared to the biochemical tests. In this study, it was observed that molecular assays were suitable means for sensitive identification of Cronobacter strains in PIF samples.

Keywords: Biochemical test; chromogenic media; Cronobacter sakazakii; enterobacteriaceae; Real time-PCR

ABSTRAK

Enterobacter sakazakii yang sebelum ini dikenali sebagai 'E. cloacae berpigmen kuning' telah diklasifikasikan sebagai genus baru 'Cronobacter' berdasarkan analisis takson serta semakan secara fenotip dan genotip. Organisma patogen ini dikaitkan dengan penyakit meningitis pada bayi dan enterokolitis nekrosis (NE) dengan kadar kematian yang tinggi (40-80%). Sesetengah kes dikaitkan dengan pengambilan susu tepung rumusan bayi (PIF) yang telah tercemar. Objektif kajian ini adalah untuk menentukan kehadiran Cronobacter spp. di dalam PIF di Malaysia. Suatu agar pilihan kromogenik, Brilliance Enterobacter sakazakii (DFI, Oxoid) telah digunakan bagi mengenal pasti pencilan Cronobacter. Pencilan Cronobacter jangkaan telah dikenal pasti dengan menggunakan ujian biokimia (API 20E dan MicrogenTM) dan asai molekul (SYBR Hijau Masa-nyata PCR dan penjujukan 16S DNA ribosom). Semua pencilan Cronobacter jangkaan telah menghasilkan koloni berwarna biru kehijauan manakala pencilan bukan Cronobacter menghasilkan koloni berwarna kuning di atas agar Brilliance Enterobacter sakazakii (DFI formulation). Sejumlah 12 pencilan jangkaan telah dipilih daripada agar DFI dan dikenal pasti dengan menggunakan ujian biokimia dan molecular. Hasil kajian menunjukkan kontaminasi lazim oleh C. sakazakii daripada 72 sampel PIF adalah sebanyak 12.5%. Kaedah pengesanan molekular seperti Masa-nyata PCR dan 16S rDNA telah menunjukkan peratus pengenalpastian yang lebih tinggi berbanding dengan hasil ujian biokimia. Kajian ini menunjukkan pengasaian secara molekular adalah suatu cara yang sesuai dan sensitif bagi mengenal pasti pencilan Cronobacter dalam sampel PIF.

Kata kunci: Cronobacter sakazakii; enterobacteriaceae; Masa-nyata PCR; medium kromogenik; ujian biokimia

REFERENCES

Bar-Oz, B., Preminger, A., Peleg, O., Block, C. & Arad, I. 2001. Enterobacter sakazakii in the newborn. Acta-Paediatrica 90: 356-358.

Bornemann, R., Zerr, D.M., Heath, J., Koehler, J., Grandjean, M., Pallipamu, R. & Duchin, J. 2002. An outbreak of Salmonella serotype Saint Paul in a children's hospital. Infection Control and Hospital Epidemiology 23: 671-676.

Bowen, A.B. & Braden, C.R. 2006. Invasive Enterobacter sakazakii disease in infants. Emerging Infectious Diseases 12: 1185-1189.

Brenner, D.J. & Farmer, J.J. 2005. Order XIII. “Enterobacteriales”, In Bergey´s Manual of Systematic Bacteriology, Volume 2, Part B, edited by Brenner, D.J., Krieg, N.R. & Staley, J.T. New York: Springer. pp. 587-850.

Caubilla-Barron, J. & Forsythe, S. 2007. Dry stress and survival time of Enterobacter sakazakii and other Enterobacteriaceae. Journal of Food Protection 70: 2111-2117.

Cawthorn, D.M., Botha, S. & Witthuhn, R.S. 2008. Evaluation of different methods for the detection and identification of Enterobacter sakazakii isolated from South African infant formula milks and the processing environment. International Journal of Food Microbiology 127: 129-138.

Fanning, S. & Forsythe, S.J. 2007. Isolation and identification of Enterobacter sakazakii. Chapter 2 in Emerging Issues in Food Safety. In Enterobacter sakazakii, edited by Farber, J.M. & Forsythe, S.J. Washington, DC: American Society for Microbiology. pp. 27-59.

Food and Agriculture Organization /World Health Organization (FAO/WHO). 2006. Meeting on Enterobacter sakazakii and Salmonella in powdered infant formula, Rome. Food And Agriculture Organization/World Health Organization http:// www.who.int/foodsafety/publications/ micro/mra6/en/.

Food and Agriculture Organization/World Health Organization (FAO/WHO). 2004. Enterobacter sakazakii and other microorganisms in powdered infant formula: Microbiological Risk Assessment. Series, No. 6. ISBN: 924156265)United Nations. Available from: <http://www.who.int/ foodsafety/ micro/meetings/feb2004/en/>.

Food and Drug Administration (FDA). 2002. Isolation and enumeration of Enterobacter sakazakii from dehydrated powdered infant formula. Available at: (http://www.cfsan. fda.gov/ ~comm/mmesakaz.html2002).

Forsythe, S.J. 2005. Enterobacter sakazakii and other bacteria in powdered infant milk formula. Maternal and Child Nutrition 1: 44-50.

Friedemann, M. 2007. Enterobacter sakazakii in food and beverages (other than infant formula and milk powder). International Jounal of Food Microbiology 116: 1-10.

Ganeswire, A.R., Thong, K.L. & Puthucheary, S.D. 2003. Nosocomial outbreak of Enterobacter gergoviae bacteraemia in a neonatal intensive care unit. The Journal of Hospital Infection 53: 292-296.

Guillaume-Gentil, O., Sonnard, V., Khandai, M.C., Marugg, J.D. & Joosten, H. 2005. A simple and rapid cultural method for detection of Enterobacter sakazakii in environmental samples. Journal of Food Protection 68: 64-69.

International Organization for Standardization (ISO). 2006. Milk and milk products-detection of Enterobacter sakazakii. ISO/TS 22964:2006 and IDF/RM 210:2006. International Organization for Standardization, Geneva, Switzerland.

Iversen, C. & Forsythe, S.J. 2007. Comparison of media for the isolation of Enterobacter sakazakii. Applied and Environmental Microbiology 73: 48-52.

Iversen, C. & Forsythe, S.J. 2004. Isolation of Enterobacter sakazakii and other Enterobacteriaceae from powdered infant formula milk and related products. Food Microbiology 21: 771-777.

Iversen, C., Druggan, P., Schumacher, S., Lehner, A., Feer, C., Joosten, H. & Stephan, R. 2008. Development of a novel screening method for the isolation of Cronobacter spp. (Enterobacter sakazakii). Applied and Environmental Microbiology 74: 2550-2553.

Iversen, C., Lehner, A., Mullane, N., Bidlas, E., Cleenwerck, I., Marugg, J., Fanning, S., Stephan, R. & Joosten, H. 2007. The taxonomy of Enterobacter sakazakii: Proposal of a new genus Cronobacter gen. nov. and descriptions of Cronobacter sakazakii comb. nov., Cronobacter sakazakii subsp. sakazakii, comb. nov., Cronobacter sakazakii subsp. malonaticus subsp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter duplinensis sp. nov. and Cronobacter genomospecies I. BMC Evolutionary Biology 7: 64.

Iversen, C., Druggan, P. & Forsythe, S.J. 2004. A selective differential medium for Enterobacter sakazaki. International Journal of Food Microbiology 96: 133-139.

Keyser, M., Witthuhn, R.C., Ronquest, L.C. & Britz, T.J. 2003. Treatment of winery effluent with upflow anaerobic sludge blanket (UASB)-granular sludges enriched with Enterobacter sakazakii. Biotechnology Letter 25: 1893-1898.

Lai, K.K. 2001. Enterobacter sakazakii infections among neonates, infants, children and adults: Case reports and a review of the literate. Medicine Baltimore 80(2): 113-122.

Lehner, A., Nitzsche, S., Breeuwer, P., Diep, B., Thelen, K. & Stephen, R. 2006. Comparison of two chromogenic media and evaluation of two molecular based identification systems for Enterobacter sakazakii detection. BMC Microbiology 6(15): 1-8.

Liu, Y., Cai, X., Zhang, X., Gao, Q., Yang, X., Zheng, Z., Luo, M. & Huang, X. 2006. Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula. Journal of Microbiological Methods 65: 21-31.

Loiwal, V., Kumar, A., Gupta, P., Gomber, S. & Ramachandran, V. 1999. Enterobacter aerogenes outbreak in a neonatal intensive care unit. Pediatrics International Journal 41(2): 157-161.

Mullane, N.R., Iversen, C., Healy, B., Walsh, C., Whyte, P., Wall, P.G., Quinn, T. & Fanning, S. 2007. Enterobacter sakazakii, an emerging bacterial pathogen with implications for infant health. Minerva Pediatrica 59: 137-148.

Muytjens, H.L., Roelofs, W.H. & Jaspar, G.H. 1988. Quality of powdered substitutes for breast milk with regard to members of the family Enterobacteriaceae. Journal of Clinical Microbiology 26: 743-746.

Olsen, S.J., Bishop, R., Brenner, F.W., Roels, T.H., Bean, N., Tauxe, R.V. & Slutsker, L. 2001. The changing epidemiology of Salmonella: Trends in serotypes isolated from humans in the United States, 1987-1997. Journal of Infectious Diseases 183: 753-761.

Ponchel, F., Toomes, C., Bransfield, K., Fong, T.L., Douglas, S.H., Field, S.L., Bell, S.M., Combaret, V., Puisieux, A., Mighell, A.J., Robinson, P.A., Inglehearn, C.F., Isaacs, J.D. & Markham, A.F. 2003. Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotechnology 3: 1-13.

Powell, J., Bureau, M.A., Pare, C., Gaildry, M.L., Cabana, D. & Patriquin, H. 1980. Necrotizing enterocolitis. Epidimic following an outbreak of Enterobacter cloacae type 3305573 in a neonatal intensive care unit. American Journal of Disease of Children 134(12): 1152-1154.

Thurm, V. & Gericke, B. 1994. Identification of infant food as a vehicle in a nosocomial outbreak of Citrobacter freundii: Epidemiological subtyping by allozyme, whole cell protein and antibiotic resistance. Journal of Applied Bacteriology 76: 553-558.

Witthuhn, R.C., Kemp, F. & Britz, T.J. 2006. Isolation and PCR detection of Enterobacter sakazakii in South African food products, specifically infant formula milks. World Journal of Microbiology and Biotechnology 23: 151-157.

Zhu, F., Snezna, R., Thomas, L.K. 2005. Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real time PCR. International Journal of Food Microbiology 99: 47-57.

*Corresponding author; email: norrasani@ukm.edu.my