FOOD MICROBIOLOGY AND ANALYSIS-ASSIGNMENT GROUP Cronobacter sakazakii GROUP MEMBERS: Hutama Tri Cita, Irimat Robert, Acolatse

Jude, Musabe Edwin Tinka, Ssepuuya Geoffrey Date of submission: 13th November, 2012 Introduction The producer of powdered infant formula (PIF) seeks a cost effective, rapid method, validated by a reputable organisation (e.g ISO) that gives reliable and acceptable results for the bacterium Cronobacter sakazakii in tandem with the EU regulation No 365/2010. The method should in addition be affordable and sustainable, economically, and technologically. 1. Cronobacter sakazakii: Description and occurrence. Cronobacter sakazakii is a Gram-negative, motile, peritrichous non-spore forming, facultative anaerobic bacterium, a member of the Enterobacteriaceae Family (FAO/WHO, 2008). Enterobacter sakazakii was initially known as yellow pigmented Enterobacter cloacae until 1980 when it was identified from the former, and in 2007, E. sakazakii was reclassified as Cronobacter sakazakii (Farber and Forsythe, 2008). The first cases of neonatal meningitis believed to have been caused by E. sakazakii were reported in 1958 (Farber and Forsythe, 2008). It is an opportunistic pathogen and cause of rare cases of meningitis, necrotizing enterocolitis, and sepsis in infants notably following the consumption of Powdered Infant Formula (Ordonez et al, 2012; WHO, 2008) and the case-fatality rate among infected neonates has been reported to be as high as 33% - 80% often resulting into death (Donelly, 2011; NCEZID, 2012). Despite being found and isolated in a number of foods, predominantly PIF, The natural habitat of E. sakazakii is currently unknown (Druddy, 2006). Abdesselam (2012) notes three major clinical manifestations seen in Cronobacter sp. infections as; necrotic enterocolitis, septicemia, meningitis and cerebritis Table 1. Growth and survival condition of Cronobacter Sakazakii summarized below (....,YEAR) Parameter Unit Temperature for growth 37 0C Range 6 45C Optimum 43C Generation time at 22C 37 44 minutes D-value at 60C 3.52 3.58 2. Virulence Little and incomplete information exists about the virulence and pathogenicity of Cronobacter sakazii (FAO, 2004; WHO, 2006; Forsythe, 2010; Abdesselam, 2012). Cronobacter spp. are noted for factors such as their desiccation resistance and production of exopolysaccharide (EPS), which may allow them to persist in dry environments (Iversen, et al.2009). The organism can invade human intestinal cells, replicate in macrophages and invade the blood-brain barrier, and this varies between species (Forsythe, 2010). Research done towards virulence of C.sakazakii showes that strains persisted or replicated in macrophages and showed moderate attachement and invasion of human endothelial cells, but maintained replication in macrophages and invasion of brain endothelial cells (WHO, 2008). 3. Which food products are involved A wide variety of food sources are implicated as food carriers for Cronobacter sakazakii including milk, cheese, kefir, tofu, meats, vegetables, rice, fermented bread, dried foods, tea, herbs and

spices), powdered infant formula (PIF) and powdered milk (PM) are the most common vehicles implicated (Ordonez et al, 2012; Iversen and Forsythe 2004? Reference ).

COMMISSION REGULATION (EU) No 365/2010 of 28 April 2010 amending Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs as regards Enterobacteriaceae in pasteurised milk and other pasteurised liquid dairy products and Listeria monocytogenes in food grade salt. Table 2. COMMISSION REGULATION (EU) No 365/2010 Food category Microorganisms Sampling plan (1) n C Limits (2) m M Analytical reference Method (3) Stage where the criterion applies

1.24 Dried infant formulae and dried dietary foods for Cronobacter special spp. medical (Enterobacter purposes sakazakii) intended for infants below 6 months of age (14)

30

Absence in 10 g

ISO/TS 22964

Products placed on the market during their shelf-life

(1) n = number of units comprising the sample; c = number of sample units giving values between m and M. (2) For points 2.2.7, 2.2.9 and 2.2.10 m = M. (3) The most recent edition of the standard shall be used.
4. Method: Use of a Chromogenic Agar; Chromocult Enterobacter Sakazaki Agar Its principle is based on the chromogenic detection of -D-glucosidase common to all Cronobacter sakazakii. Hydrolysis of the chromogenic substrate leads to the formation of typical blue to turquoise (blue to green) colonies of Cronobacter sakazakii while other bacteria grow colourless The method would require 24 hours after ,18 hours pre enrichment and 24 hours enrichment in buffered peptone water and mLST-Vancomycin medium respectively, this makes it a quick method compared to other commercial available agars, Chromocult Enterobacter Sakazaki Agar is also recommended by ISO 22964. Reasons for choosing method 1. It is a faster method compared to the ISO standard method and other commercially available methods as shown in the Annex table 1

2. It is a cheap method compared to other methods as shown in the Annex table 1 3. It is recommended by ISO 22964 Chromocult Enterobacter sakazaki Agar is supplied by Merck KGaA

Tabel 3. Protocol used when using Chromocult Enterobacter Sakazaki Agar Pre-enrichment of 10 g sample in 90 ml BPW 18 h 2 h at 37C Selective-enrichment in mLST-Vancomycin medium 0.1ml from cultured BPW into 10 ml mLST-Vancomycin medium 24 2 h at 45 0.5C Isolation on ChromoCult Enterobacter sakazakii Agar Streak a loop full cultured mLST-Vancomycin medium on a plate of chromogenic agar 24 h at 44 1C

Sample preparation

Detection

Counting and confirmation

Blue-green colonies indicate presence of Cronobacter. sakazakii, no further confirmation required

In absence of Cronobacter sakazakii

Note: Pre-enrichment and Enrichment is important during sample preparation. Pre-enrichment is important in resuscitation of sub lethally injured cells; the high temperature reached in preparation of infant formula usually kills Cronobacter sakazakii. The enrichment step allows selective growth Cronobacter sakazakii before it can be identified by Chromocult Enterobacter Sakazaki Agar.

REFERENCES [Follow Harvard system, need somebody to fill the uncomplete reference (date/citated website) ] Abdesselam, K. 2012. Pathogenesis of Cronobacter species: Enterotoxin production, adhesion and invasion of the blood brain barrier.M.Sc, University of Ottawa. Communicable Disease Control Manual. 2012. Enterobacter sakazakii invasive disease. Epidemiology in New Zealand. http://www.health.govt.nz/.../cd-manual-enterobactersakazakii-invasive-di. [accessed: DD/MM/YYYY] Donnelly, M.T. 2011. Two cases of invasive enterobacter sakazakii infection in infants treated in Missouri hospitals. Missouri Departement of Health & Senior Services Druddy, D., Mullane, R., Quinn, T., Wall, P.G., Fanning S. 2006. Enterobacter sakazakii: An Emerging Pathogen in Powdered Infant Formula. Food Safety CID 42 FAO. 2004. EPIDEMIOLOGYAND PUBLIC HEALTH ASPECTS : Organisms of concern, Enterobacter sakazakii . http://www.fao.org/docrep/007/y5502e/y5502e07.htm2. [accessed : DD/MM/YYYY] FAO/WHO. 2008. Enterobacter Sakazakii (Cronobacter spp.) in powdered follow up formula. Meeting Report. Geneva, Switzerland. Pages 1-2 Farber and Forsythe. 2008. Emerging issues in food safety, Enterobacter sakazakii.American Society for Microbiology publishers, U.S.A. chap 1 Forsythe, S., 2010. Cronobacter species. Oxoid: Culture Vol 31 no.1 Iversen, C., Lehner, A., Healy, B., Fanning, S., Stephan, R., 2009.Comparison of virulence risk factors in Cronobacter and novel Enterobacter species. 1st International Conference on Cronobacter (Enterobacter sakazakii) http://abeetle.tripod.com/posters.pdf [accessed : DD/MM/YYYY] Merck. 2012. Merck microbiology manual 12th edition; Chromocult Enterobacter sakazakii Agar, Selective medium for the detection of Enterobacter sakazakii in milk powder and powdered infant formula. http://?????? Merck. 2012. Merck Cromocult, High safety at a low price. Chromocult Enterobacter sakazakii Agar for cost effective and safe detection of E. Sakazakii in food http://???? NCEZID. 2012. CDC Update: Investigation of Cronobacter Infections Among Infants in the United States. http://www.cdc.gov/foodsafety/diseases/cronobacter/investigation.html [accessed: DD/MM/YYYY] Ordonez, A.A., Begley M., Hill C. 2012. Polymorphisms in rpoS and heterogeneity in stress tolerance in natural isolates of Cronobacter sakazakii. American Society for Microbiology

Rapidmicrobilology. 2007. Merck's New Chromogenic Medium for Detection of Enterobacter sakazakii, http://www.rapidmicrobiology.com/news/1054h11.php [accessed, 10/11/2012] WHO. 2006. Enterobacter Sakazakii and Salmonella in Powdered Infant Formula: Meeting Report. WHO, Geneva, Switzerland. Pg.6

Entis. P, 2011. Profiling Cronobacter sakazakii (Enterobacter sakazakii). http://efoodalert.net/2011/12/25/profiling-cronobacter-sakazakii-enterobacter-sakazakii/ No sentences citated to this journal

ANNEX

Table 1. Comparison of commercially available media used in detection Cronobacter sakazaki No 1. Company Bio rad laboratories Agar Rapid Sakazaki Agar (Rapid testing) Quantity 20 plate Price (USD) 67.0 Comments Can be done rapidly within 24 but would require confirmation Only one enrichment step required Used in the normal ISO standard reference procedure and would take about 72 hours The Agar is presumptive and would require further confirmation Allows a fast and reliable detection within only 24 hours with no further confirmation step Would be used for confirmation if other chromogenic agars where used in isolation of C.sakazaki

2.

Conda Laboratories

Enterobacter Sakazaki Isolation Agar (for ISO protocol) Chrom ID sakazaki Agar Chromocult Enterobacter Sakazaki Agar API set

500 g

1,374

3.

Biomerieux Inc.

20 plates

60.05

Merck

500g

1029

Biomerieux Inc.

25 pieces

196.35