Faculty of Agricultural Sciences, Nutritional Sciences and Environmental Management Justus-Liebig-University Giessen, Germany and Central Laboratories Friedrichsdorf GmbH, Germany Effect of microbiologically active substances in powdered infant formula on the growth and detection of Cronobacter spp. Thesis submitted in partial fulfilment of the requirements for the degree of Doctor Nutritional Sciences (Dr. oec. troph.) Submitted by ZHU, Sha Frankfurt am Main, 2013 This Ph.D. work was approved by the committee (Faculty 09: Agricultural Sciences, Nutritional Sciences, and Environmental Management) of Justus-Liebig-University Giessen, as a thesis to award the Doctor degree of Nutritional Sciences (Dr. oec. troph.) 1. Supervisor: Professor Dr. Sylvia Schnell 2. Supervisor: Professor Dr. Uwe Wenzel Date of disputation: 16. Dec. 2013 Contents List of abbreviations I Chapter 1 Introduction ....................................................................................................... 1 1.1 Taxonomy............................................................................................................ 2 1.2 Biochemical Characteristics of Cronobacter ...................................................... 3 1.3 Epidemiology and Pathogenicity ........................................................................ 3 1.4 Production of Powdered Infant Formula, Contamination Routes and Prevalence of Cronobacter in Powdered Infant Formula ...................................................... 5 1.5 Methodology ....................................................................................................... 7 1.5.1 Conventional Microbiological Isolation of Cronobacter ................................... 7 1.5.1.1 Culture-based Isolation ....................................................................................... 7 1.5.1.2 Chromogenic and Fluorogenic Media for Cronobacter Differentiation ............. 8 1.5.1.3 Other Culture Method: Impedance method ........................................................ 9 1.5.2 Identification of Cronobacter ............................................................................. 9 1.5.2.1 Phenotypic Methods: Biochemical Identification Kits ....................................... 9 1.5.2.2 Genotypic Methods ........................................................................................... 10 1.6 Growth of Cronobacter in Reconstituted PIF ................................................... 13 1.7 Growth of Cronobacter in rehydrated PIF and Raw Materials as a Prerequisite for a Successful Detection ................................................................................ 15 1.8 Prevalence of non-Cronobacter Enterobacteriaceae in PIF and the related influence on Cronobacter Identification ........................................................... 17 Chapter 2 Thermal inactivationof Cronobacter spp. during rehydration of powdered infant formula with water of 70°C or higer ....................................................................... 27 Chapter 3 Growth inhibition of Cronobacter spp. strains in reconstituted powdered infant formula acidified with organic acids supported by natural stomach acidity ...... 40 Chapter 4 Rapid detection of Cronobacter spp. with a method combining impedance technology and rRNA based lateral flow assay ................................................................. 49 Chapter 5 Matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry, 16S rRNA gene sequencing, and API 32E for identification of Cronobacter spp.: A comparative study.............................................................................. 55 Chapter 6 Discussion ........................................................................................................ 62 6.1 Inactivation of Cronobacter in Reconstituted PIF ............................................ 63 6.2 Improvement of the Screening Procedure for the Cronobacter Detection from PIF ..................................................................................................................... 69 6.3 Chromogenic Isolation Media and Identification Systems for the Detection of Cronobacter ...................................................................................................... 76 Summary…….. ..................................................................................................................... 88 Zusammenfassung ................................................................................................................ 90 Addendum…. ....................................................................................................................... 93 Danksagung….. .................................................................................................................... 96 Curriculum Vitae ................................................................................................................. 97 Eidesstattliche Erklärung .................................................................................................... 98 LIST OF ABBREVIATIONS I List of Abbreviations AFNOR Association Française de Normalisation BHA Brain hearth infusion agar BHI Brain heart infusion BPW Buffered peptone water CAC Codex Alimentarius Commission CFU Colony forming unit CLF Central Laboratories Friedrichsdorf CSB Cronobacter screening broth DFI agar Druggan-Forsythe-Iversen agar DIN Deutsche Industrie Norm DNA Desoxyribonucleic acid DSMZ Deutsche Stammlung von Mikroorganismen und Zellkulturen DT Detection time EC European Commission EE Enterobacteriaceae Enrichment ESE Enterobacter sakazakii enrichment broth ESIA Enterobacter sakazakii isolation agar ESPM Enterobacter sakazakii chromogenic plating medium ESSB Enterobacter sakazakii selective broth f-AFLP fluorescent-labelled amplified fragment length polymorphism FAO Food and Agriculture Organization FDA Food and Drug Administration FIF Fermented infant formula FISH Fluorescence in situ hybridization FUF Follow-up formula h hour(s) HCl Hydrogen chloride LIST OF ABBREVIATIONS II HIV Human immunodeficiency virus ICMSF International Commission on Microbiological Specification for Foods ISO/TS International Standards Organization/Technical Specification LOD Limit of detection LPOS Lactoperoxidase system LST Lauryl sulphate tryptose MALDI-TOF MS Matrix-assisted laser desorption/ionization-time of flight mass spectrometry min minute(s) MLSA Multilocus sequence analysis NEC Necrotizing enterocolitis PCR Polymerase chain reaction PIF Powdered infant formula rRNA Ribosomal ribonucleic acid SCFA Short-chain fatty acid TSA Tryptic soy agar VRBG Violet red bile glucose WHO World Health Organization CHAPTER 1 1 Chapter 1 Introduction CHAPTER 1 2 The genus of Cronobacter spp. is an emerging opportunistic food borne pathogen, causing rare but serious infections of bacteraemia, meningitis and necrotizing enterocolitis in infants (Anonymous, 2006a; Anonymous, 2007b; Mullane et al., 2007). Contaminated powered infant formula (PIF) has been implicated as a most likely source of transmission of Cronobacter spp.. A microbiological association between consumption of PIF contaminated during manufacture and/or mishandling when reconstituted and a number of clinical outbreaks has been established (Van Acker et al., 2001). Understanding of biochemical characteristics and growth profiles of Cronobacter spp. is therefore essential for the investigation of inhibitive strategies against the growth of Cronobacter spp. as well as for the development of detection and identification methods of Cronobacter spp. in reconstituted PIF. 1.1 Taxonomy Cronobacter spp. are a group of gram-negative, motile, non-spore forming, facultative anaerobic bacteria. These organisms were originally referred to as “yellow-pigmented Enterobacter cloacae” until they were classified as a new species, Enterobacter sakazakii, within the genus Enterobacter (Farmer et al., 1980). In year 2007 and 2008, a further classification of E. sakazakii with the creation of a new genus, Cronobacter, has been proposed. The genus Cronobacter consists of six species: C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. genomospecies 1 and C. dublinensis which contains three subspecies dublinensis, lactaridi and lausannensis (Iversen et al., 2007; Iversen et al., 2008). This genus proposal was based on data from a polyphasic taxonomic study of an extensive collection of target strains in which full-length 16S rRNA sequencing, fluorescent-labelled Amplified Fragment Length Polymorphism (f-AFLP) fingerprinting, ribotyping, DNA-DNA hybridization and characterization of phenotypic profiles were applied. Recently Joseph et al. (2012) used 16S rRNA sequencing and a Multilocus Sequence Analysis (MLSA) of seven housekeeping genes to re-evaluate the diversity of the genus. Based on his data, a new species C. condimenti was identified and the original C. genomospecies 1 was replaced by C. CHAPTER 1 3 universalis. Cronobacter genus comprises currently seven species: C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. dublinensis, C. condimenti and C. universalis. 1.2 Biochemical Characteristics of Cronobacter Farmer et al. (1980) found that Cronobacter spp. (previously E. sakazakii) possessed biochemical reactions very similar to those of E. cloacae but Cronobacter spp. were D-sorbitol negative and produced yellow-pigmented colonies on Tryptic Soy Agar (TSA) at 25°C after 48 h. An additional distinguishing property for Cronobacter spp. is the Tween 80 esterase activity, which was positive in 97.3% of isolates (Aldova et al., 1983). Muytjens et al. (1984) investigated the enzymatic profiles of 129 Cronobacter spp. strains and 97 Enterobacter strains including E. cloacae, E. aerogenes and E. agglomerans and determined the presence of α-glucosidase as a major difference between Cronobacter spp. and other Enterobacter species. 1.3 Epidemiology and Pathogenicity Cronobacter spp. are considered as emerging opportunistic pathogens and have been identified as causes of bacteraemia, necrotizing enterocolitis and neonatal meningitis (Arseni et al., 1987; Bar-Oz et al., 2001; Giovannini et al., 2008; Mullane et al., 2007). Urmenyi and Franklin (1961) recorded the first isolation of Cronobacter spp. from a case of neonatal meningitis. Surveillance data of the Food and Agriculture Organization of the United Nations and World Health Organization (FAO/WHO) (Anonymous, 2008b) showed that since 1961 and up to 2008, approximately 120 documented cases of Cronobacter infections in infants and young children less than three years of age have been reported worldwide with at least 27 associated deaths, showing an average case-fatality rate of 22%. This leads to the International Commission on Microbiological Specification for Foods (ICMSF) (Anonymous, 2002a) ranking Cronobacter spp. as “Severe hazard for restricted populations, life threatening or substantial chronic sequelae or long duration.” An annual CHAPTER 1 4 incidence rate for Cronobacter invasive infections has been estimated to be 1 per 100,000 infants, whereas higher annual incidence rates among the low birth weight infants (< 2500 g) and very low birth weight infants (< 1500 g) of 8.7 and 9.4 per 100,000 infants respectively were reported (Anonymous, 2006a). The case-mortality rate caused by Cronobacter infection has been reported to be higher (50%) among premature or (very) low birth weight infants than full-term or infants with birth weight ≥ 2500 g (30%) (Lai, 2001). Although Cronobacter has caused diseases in all age groups, neonates under 28 days, particularly preterm-infants, low birth weight infants, immunocompromised or medically debilitated infants as well as infants with HIV-positive mothers are thought to be at the greatest risk for severe infection (Anonymous, 2007b). It has been concluded that infants during the first few weeks of life are particularly susceptible to meningitis rather than bacteraemia if exposed to Cronobacter. Data from 45 clinical cases have revealed that bacteraemia appears to occur at a later median age of 35 days of life in premature infants, whereas meningitis cases have been more frequently observed among full-term infants who generally develop symptoms before the age of one week. Infant with bacteraemia tend to fare better with a mortality of 10%. Conversely 44% of those with meningitis died and the majority of survivors experienced long-term neurological consequences (Anonymous, 2006a). The mechanism of pathogenicity and potential virulence factors of Cronobacter remain elusive. Pagotte et al. (2003) were the first to investigate the virulence factor in Cronobacter spp. using a suckling mouse model and found four out of 18 Cronobacter strains producing enterotoxin. Townsend et al. (2007) reported that Cronobacter strains persisted or replicated in macrophages and showed attachment and invasion of human endothelial cells to different extents, indicating a diversity of virulence among Cronobacter isolates. However its relevance to human infant infections has yet to be established. To date only strains of C. sakazakii, C. malonaticus and C. turicensis have been found to be associated with reported neonatal infections. Particularly a variety of C. sakazakii with sequence type ST4 causes more infant meningitis (Joseph and Forsythe, 2011). Despite the inter- and intra-species variation in virulence, no data currently show that any of the Cronobacter species does not CHAPTER 1 5 pose a risk to neonatal and infant health. Therefore, all species in the genus Cronobacter are considered to be pathogenic (Anonymous, 2008b). In the investigation performed by Pagotte et al. (2003), a lethal dose of 10 8 CFU on infant mouse by intraperitoneal injection has been concluded for all the 18 Cronobacter strains, whereas only two isolates caused death when given orally, however, this value does not necessarily reflect the dose-response in human neonates. Till now no evident data for an infectious dose associated with Cronobacter spp. is available. An infectious dose of 1000 CFU similar to that of Neisseria meningitidis and E. coli O157 has been proposed by Iversen and Forsythe (2003). The FAO/WHO (Anonymous, 2007b) estimated a linear dose-response relationship at low dose below 10,000 CFU. 1.4 Production of Powdered Infant Formula, Contamination Routes and Prevalence of Cronobacter in Powdered Infant Formula It has been internationally recognized that breast milk is the best source of nutrition to infants and young children. There are, however, occasions when breast milk is not available or where the mother is unable to breastfeed. In such cases, powdered formulae which are formulated to meet the special nutritional needs of infants represent one of the dietary options to replace the breast milk partially or totally (Anonymous, 2007c). Codex Alimentarius Commission (CAC) has divided powdered formulae into powdered infant formula (PIF), follow-up formula (FUF), human milk fortifiers and formula for special medical purposes. PIF is defined as a breast milk substitute specially manufactured to satisfy the nutritional requirements of infants as sole source of nutrition during the first months of life to the introduction of appropriate complementary feeding. FUF is defined as a food intended for use as a liquid part of the weaning diet for the infant from the 6 th month on and for young children (Anonymous, 2008a). PIF can be manufactured in a wet-mix process, a dry-blending process or a combined CHAPTER 1 6 process (Cordier, 2008). In the wet-mix process, all unprocessed raw materials as well as separately processed ingredients are handled in a liquid form, heat treated, dried and brought to the filling stage. The dry-mix procedure involves dry mixing of heat sensitive ingredients like vitamins, minerals, starch and carbohydrates into the powder after spray-drying. In the mix-process, the unprocessed raw materials and ingredients are processed in a liquid phase to obtain a base powder and other ingredients are added into the base powder and further blended. Due to the limitations of current technology, it is not possible to produce sterile PIF at a feasible price. PIF can be contaminated with low levels of pathogens e.g. Cronobacter spp. via intrinsic and extrinsic routes. Intrinsic contamination of PIF during the manufacturing process after pasteurization can occur either via addition of thermally sensitive plant-derived ingredients (e.g. vitamins, starch, protein and lecithin) or via transmission of materials in the processing environment such as aerosol, dust and water droplet (Iversen et al., 2009; Mullane et al., 2008). It is notable that although the standard pasteurization practices (71.6°C, 15 s or 74.4°C, 25 s) are sufficient to inactive Cronobacter spp, the organism may survive the spray drying (Arku et al., 2008). An extrinsic contamination of PIF can occur during the preparation and feeding when contaminated utensils such as spoons, blenders, bottles, teats are used (Bar-Oz et al., 2001; Simmons et al., 1989). Being a ubiquitous organism, Cronobacter spp. have been isolated from a wide variety of food, environmental and clinical sources (Cottyn et al., 2001; Farber, 2004; Gallagher and Ball, 1991; Gassem, 1999; Kandhai et al., 2004; Leclercq et al., 2002). The presence of Cronobacter in PIF is of particular concern. A clear epidemiological correlation between Cronobacter infections in infants and the consumption of contaminated PIF has been described (Biering et al., 1989; Clark et al., 1990). Together with Salmonella, Cronobacter spp. have been categorized as “A” organisms. PIF contaminated with “A” organisms has been both epidemiologically and microbiologically shown to be the source and vehicle of infection in infants (Anonymous, 2007b). Several studies have investigated the prevalence of Cronobacter spp. in PIF and other infant foods. Muytjens et al. (1988) examined 141 CHAPTER 1 7 different breast milk substitute powders from 35 countries and determined a contamination level of Cronobacter ranging from 0.36 to 66 CFU per 100 g. In a survey of 120 cans of Canadian infant formula samples, the prevalence of Cronobacter was 6.7%, with a contamination level of 0.36 CFU per 100 g (Nazarowec-White and Farber, 1997a). In a British survey, two samples (2.4%) were positive for Cronobacter among 82 PIF products purchased from retails (Iversen and Forsythe, 2004). Jongenburger et al. (2011) investigated 2291 samples of 1 g from a recalled batch and found a sporadic presence of Cronobacter cells in eight samples, with the two largest clusters containing 123 and 560 cells respectively. 1.5 Methodology 1.5.1 Conventional Microbiological Isolation of Cronobacter 1.5.1.1 Culture-based Isolation The conventional culture–based procedure for detection of micro-organisms in foods comprises three basic steps: The first step is pre-enrichment in a non-selective medium which enables recovery of sub-lethally injured cells. The second step is selective enrichment containing compounds which promote the growth of the target micro-organisms to be isolated but are inhibitory to the majority of the background micro-organisms. The third step is streaking the selective broth onto selective solid media. In case of presumptive colonies on the agar, a confirmation step will be carried out. The initial method for isolation and detection of Cronobacter from PIF applied by Muytjens et al. (1988) and Nazarowec-White and Farber (1997a) comprised 1) pre-enrichment in Buffered Peptone Water (BPW), 2) enrichment in Enterobacteriaceae Enrichment (EE) broth, 3) streaking onto Violet Red Bile Glucose (VRBG) agar, 4) picking five Enterobacteriaceae colonies on TSA agar and incubation at 25°C for 48-72h, 5) confirmation of yellow-pigmented colonies on TSA with API 20E (bioMérieux, France). This method was CHAPTER 1 8 adopted by the U.S. Food and Drug Administration (FDA) with modification of pre-enrichment in distilled water (Anonymous, 2002b). Being a time-consuming procedure, the FDA method requires a minimum of 5 days to finish. Also no selective pre-enrichment and enrichment broths were involved in this method. It is likely that Cronobacter spp. are outgrown by background Enterobacteriaceae, leading to few Cronobacter colonies isolated on VRGB and a subsequently reduced chance of picking the target organisms onto TSA. In contrast the European Community (EC) regulation refers to International Standards Organization (ISO) standard method ISO/TS 22964 which consists of pre-enrichment in BPW, followed by selective enrichment in modified lauryl sulphate tryptose (mLST) broth with vancomycin, plating on selective differential media and biochemical characterization of typical colonies (Anonymous, 2006b). 1.5.1.2 Chromogenic and Fluorogenic Media for Cronobacter Differentiation Several media have been developed for a specific detection of Cronobacter from PIF. All of these selective media take advantage of the biochemical characteristic, the α-glucosidase activity of Cronobacter which was first described by Muytjens et al. (1988). A differential selective medium, OK medium, described by Oh and Kang (2004) used a fluorogenic substrate, 4-methyl-umbelliferyl-α-D-glucopyranoside with the fluorogen serving as indicator to detect α-glucosidase activity. Druggan-Forsythe-Iversen (DFI) agar contains the chromogenic substrate 5-bromo-4-chloro-3-indolyl-α-D-glucopyranoside (Iversen et al., 2004a). Being hydrolysed by α-glucosidase positive organism, bromo-choloro-indigo will be liberated from the substrate and typical blue-green colonies will be produced. In addition, sodium thiosulphate and ammonium iron citrate were incorporated in the DFI agar as hydrogen sulphide indicator to differentiate Cronobacter from H2S positive organisms like Proteus and Salmonella which appear black on the agar. The Enterobacter sakazakii isolation agar (ESIA) recommended in the ISO method has been commercially available by AES Chemunex France since 2005 and it is based on the same chromogenic agent as DFI agar. Restaino et al. (2006) described the R&F Enterobacter CHAPTER 1 9 sakazakii chromogenic plating medium (ESPM) which contains three sugars (sorbitol, D-arabitol and adonitol) as well as two chromogens (X-α-D-glucopyranoside and X-α-D-cellobioside), causing Cronobacter colonies to create blue-black or blue-grey colonies on medium. 1.5.1.3 Other Culture Method: Impedance method The impedance method is an alternative to the conventional microbiological methods. The term impedance refers to the electrical resistance that occurs in the alternating current circuit. Due to the microbial metabolism in the liquid culture medium, large molecules are broken down into smaller, electrically charged molecules. These changes in the molecular composition increase the conductivity of liquid nutrients and lower their electrical resistance. With the first application in clinical microbiology in 1975 (Wheeler and Goldschmidt, 1975), the technology has been widely used as a standardized rapid method in food microbiology in quality control laboratories in food industry, being recognized by numbers of official methods collections such as Association Française de Normalisation (AFNOR) (Anonymous, 2010a, 2010b) and Deutsche Industrie Norm (DIN NORM) (Anonymous, 1999, 2001, 2005). For detection and enumeration of several food relevant micro-organisms such as lactobacilli, E. coli, Clostridia and Salmonella, the impedance measurement has been assessed as a valid method (Colquhoun et al., 1995; Dromigny et al., 1997; Joosten et al., 1994; Lanzanova et al., 1993). To date the impedance technology has been optimized in its specificity for food relevant bacteria by the development of impedance specific media and the combination of impedance detection with immunological and molecular biological confirmation methods. 1.5.2 Identification of Cronobacter 1.5.2.1 Phenotypic Methods: Biochemical Identification Kits Traditional phenotypic identification is based upon biochemical pathway and carbon source utilization. For confirmation of presumptive Cronobacter spp. isolated from agars, various commercially available biochemical systems, including API 20E, ID 32E, VITEK 2 (bioMérieux, France) and Biolog GN2 (Biolog, USA) have been used. Iversen et al. (2006) CHAPTER 1 10 investigated the original 15 biotypes described by Farmer et al. (1980) in correlation with four clusters formed in 16S rRNA sequences and established a new biogroup 16. As indicated by Iversen et al. (2007), five Cronobacter species C. sakazakii, C. malonaticus, C. muytjensii, C. dublinenesis and C. turicensis can be distinguished using four key biochemical reactions indole, dulcitol, malonate and methyl-α-D-glucoside. 16 biogroups are distributed among Cronobacter species as following: Cronobacter sakazakii sp. nov. (Biogroup 1-4, 7, 8, 11 and 13) Cronobacter malonaticus sp. nov. (Biogroup 5, 9, 14) Cronobacter dublinensis subsp. (Biogroup 12) dublinensis subsp. nov. Cronobacter dublinensis subsp. (Biogroup 10) lausannensis subsp. nov. Cronobacter dublinensis subsp. (Biogroup 6) lactaridi subsp. nov. Cronobacter muytjensii sp. nov. (Biogroup 15) Cronobacter turicensis sp. nov. (Biogroup 16) Two strains belonging to Cronobacter universalis (formerly Cronobacter genomospecies 1) have not been associated with any specific biogroup. 1.5.2.2 Genotypic Methods 1.5.2.2.1 Polymerase Chain Reaction (PCR) To date several conventional and real time Polymerase Chain Reaction (PCR)-based systems that enable sensitive, specific and rapid end detection of Cronobacter from PIF, enrichment broths and media have been described. Targets for conventional PCR assays include e.g. the 16S rRNA gene (Hassan et al., 2007; Lehner et al., 2004), 1,6 α-gulcosidase gene (gluA) (Lehner et al., 2006b), outer membrane protein A (ompA) gene (Mohan Nair and Venkitanarayanan, 2006) and a gene encoding a zinc-containing metalloprotease (zpx) (Kothary et al., 2007). Seo and Brackett (2005) developed a quantitative real time PCR assay for Cronobacter detection targeting an internal segment of the dnaG gene within the CHAPTER 1 11 macromolecular synthesis operon. Liu et al. (2006) evaluated a real time PCR method based on the amplification of an internal transcribed spacer sequence of the 16S–23S rDNA, using a Taqman probe and SYBR Green simultaneously. 1.5.2.2.2 Fluoreszenz in situ Hybridization (FISH) Fluorescence in situ hybridization (FISH) is an alternative to the DNA-targeted PCR-based approaches for rapid bacterial detection. FISH has been widely used for microbial identification in medical diagnoses (Kempf et al., 2005; Russmann et al., 2001) and has also allowed a rapid detection and identification of organisms including Enterobacteriaceae, and Pseudomonas in drink water and food (Baudart et al., 2005; Kitaguchi et al., 2005). Based on the binding of fluorescently labelled single-stranded DNA-probes to specific regions on the ribosomal RNA of the bacteria, the FISH system detects only living cells (Amann et al., 1995; DeLong et al., 1989). Using the commercially available test system based on FISH, the VIT ® kit (Vermicon Identification Technology, Germany), presumptive Cronobacter isolates have been identified with 100% accuracy (Lehner et al., 2006a). The same test kit has been applied in the investigation of Sanjaq (2008), showing no false negative or false positive results in differentiating 101 Cronobacter isolates from 7 other Enterobacteriaceae species. 1.5.2.2.3 16S rRNA gene sequencing Much effort has been put on the development of genotypic tools for Cronobacter identification with a high discrimination power to the species and subspecies level. The 16S rRNA gene is by far the most common housekeeping genetic marker because the ribosomal small subunit is present universally among bacteria and it includes both hypervariable regions with species-specific variability where sequences have diverged over evolution and strongly conserved regions, which makes the 16S rRNA gene sequencing a highly useful tool to study bacterial phylogeny, ecology and taxonomy (Janda and Abbott, 2007; Weisburg et al., 1991). Genomic DNA is extracted, amplified with universal 16S rRNA primer and sequenced. The cycle sequencing reaction is a modification of the traditional Sanger method using base-specific, dideoxynucleotide-terminated chain elongation method with modification for the use of reverse transcriptase and RNA template (Lane et al., 1985; CHAPTER 1 12 Sanger et al., 1977). By comparing sequence data in gene databases in public or private domains, it is possible to analyse relationships between various organisms and to identify unknown micro-organisms. This molecular approach has been extensively used for identification and classification of environmental and clinical bacterial isolates (Clarridge, 2004; Drancourt et al., 2000; Vandamme et al., 1996). Using 16S rRNA gene sequencing, the type strain of Cronobacter was found to be closer to Citrobacter (C.) koseri (97.8% similar) than to E. cloacae (97%) although the latter has been found to share most phenotypic features with Cronobacter (Iversen et al., 2004c). Four phylogenetic clusters have been defined among 189 Cronobacter strains analysed with partial 16S rRNA gene sequencing and this helped to form the basis of the taxonomic re-classification of these organisms (Iversen et al., 2006). 1.5.2.2.4 Matrix-assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Currently, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has been introduced as a new method for phylogenetic classification and identification of bacteria on the basis of protein profiling. The concept of bacterial differentiation/identification by detection of protein mass patterns is based on the principle that genomic sequences of organisms coding for production of proteins are determinant for phylogenetic differences between organisms. Since proteins reflect the genomic differences, the mass spectra should be able to serve as a differentiation/classification vector for bacteria (Holland et al., 1996; Krishnamurthy and Ross, 1996; Krishnamurthy et al., 1996). Each mass spectrometer is composed of three units: an ion source to generate ion and transfer the analyte ions into the gas phase; a mass analyzer to separate ions by the mass-to charge ratio (m/z) and a detector for ion monitoring. In practice, bacterial samples are first subjected to a protein extraction method, embedded in small acidic molecules known as matrix such as α-cyano-4-hydroxycinnamic acid which desorbs laser energy and analyzed by mass spectrometry (Freiwald and Sauer, 2009). As a large majority of the bacterial proteins detected by this approach are of ribosomal origin, the generated masses of these ribosomal CHAPTER 1 13 proteins represent a specific fingerprint which can be used for phylogenetic classification and identification of bacteria. Based on comparison of protein biomarkers with high reproducibility and whole specific spectral fingerprints of protein peak patterns, MALDI-TOF MS identification system has been validated for foodborne pathogens such as Campylobacter, Clostridia, Cronobacter, Salmonella and Listeria (Alispahic et al., 2010; Barbuddhe et al., 2008; Dieckmann et al., 2008; Grosse-Herrenthey et al., 2008; Stephan et al., 2010). 1.6 Growth of Cronobacter in Reconstituted PIF Previous work by Nazarowec-White and Farber (1997a) showed that the growth temperature of eleven Cronobacter strains in Brain Heart Infusion (BHI) ranged from 5.5°C to 45°C. Iversen et al. (2004b) reported that the organism grew in reconstituted PIF from 6°C to 47°C, optimally at 37°C-43°C. According to Nazarowec-White and Farber (1997a), average generation times of Cronobacter strains were 40 min at room temperature of 23°C and 4.98 h at 10°C in three different formulae. Iversen et al. (2004b) found the mean generation times of six Cronobacter strains in reconstituted PIF were 13.7 h, 1.7 h and 22 min at 6°C, 21°C and 37°C. Lag times of Cronobacter spp. reported by Nazarowec-White and Farber (1997a) ranged from 9 to 47 h at 10°C and from 2 to 3 h at 23°C respectively. Lag times have been estimated to be 83 h at 10°C and 1.7 h at 37°C in average (Kandhai et al., 2006). Based on a low contamination level of 0.36 Cronobacter cells/100 g PIF reported by Muytjens et al. (1988) and a single feeding of 18 g PIF, an infectious dose of 1000 Cronobacter cells would be achieved in 9 days at 8°C, 7.9 days at 10°C, 17.9 h at 21°C and 7 h at 37°C, without taking into account possible inactivation of Cronobacter cells during PIF preparation, bacterial proliferation in infant stomach or cumulative exposure due to 4-6 feeding of infants in a 24 h period (Iversen and Forsythe, 2003). From the data shown in this theoretical calculation model, it is evident that reconstituted PIF contaminated with low levels of Cronobacter is unlikely to cause an infection in a healthy baby unless inappropriate preparation, handling and storage of feeding bottle including e.g. gross temperature abuse or CHAPTER 1 14 extrinsic contamination through poor hygiene preparation exist. The FAO/WHO established a quantitative risk assessment model for Cronobacter in PIF and concluded reconstitution temperatures of 40°C and 50°C, extended holding time at room temperature and long feeding periods as determining factors regarding enhanced risk of Cronobacter infection (Anonymous, 2006a). Therefore any control strategy with bacteriostatic or bacteriocidal properties in reconstituted PIF are believed to be capable of minimizing the infection risk. Heat treatment of food just prior to consumption has long been used as a primary tool to reduce the risk of infection caused by food borne pathogen. Edelson-Mammel and Buchanan (2004) investigated the effect of reconstituting PIF with water of different temperatures on Cronobacter inactivation and concluded that preparing PIF with water of 70°C or greater lead to a more than 4-log reduction in Cronobacter levels. Based on their findings, FAO/WHO (Anonymous, 2007c) recommended use of water ≥ 70°C to reduce the infection risk in infants associated with Cronobacter contamination. Besides thermal inactivation, there has been an increasing interest in introducing external hurdles via incorporation of various natural antimicrobials to control Cronobacter in PIF. Lactoferrin and nisin have been shown to have detectable antimicrobial activity against Cronobacter depending on concentration and temperature (Al-Nabulsi et al., 2009). Gurtler and Beuchat (2007) reported that lactoperoxidase inhibited the growth of Cronobacter in reconstituted PIF. Plant-derived essential oils including caprylic acid and trans-cinnamaldehyde have been determined to be effective agents in the suppression of Cronobacter in reconstituted infant formula as well as in the inhibition and inactivation of Cronobacter biofilms (Amalaradjou et al., 2009; Amalaradjou and Venkitanarayanan, 2011). A study of Kim et al. (2009) concluded that muscadine seed extracts, as rich sources of phenolic compounds and organic acids, displayed a strong antimicrobial activity against Cronobacter strains which was mainly caused by organic acids like tartaric, malic and tannic acid. Back et al. (2009) screened the inhibitory effects of eight organic acids and found propionic and acetic acid as most effective against Cronobacter in liquid foods including baby food. Commercially available acidified infant formula by direct addition of lactic acid in 1.4 g/100 g of dry composition has been shown to be effective in limiting Cronobacter (Kreb, 2010). Besides CHAPTER 1 15 direct addition of organic acids in PIF as an alternative acidification intervention has been developed through the fermentation of the basic ingredients of infant formula with lactic acid bacteria. In a study of Joosten and Lardeau (2004), a commercially available biologically acidified (fermented) infant formula showed a clear bacteriostatic effect on pathogenic bacteria including Cronobacter spp.. 1.7 Growth of Cronobacter in rehydrated PIF and Raw Materials as a Prerequisite for a Successful Detection Except for one method developed by AES Laboratories which directly starts with an enrichment in a selective medium Enterobacter sakazakii selective broth (ESSB), all culture-based methods published to date including the FDA reference method and the ISO standard method involve an initial non-selective pre-enrichment step where PIF is diluted in a ratio of 1:10 in distilled water or in BPW and incubated at 37°C for 16-20 h (Fanning and Forsythe, 2008; Kandhai, 2010; Lampel and Chen, 2009). In addition, rapid methods such as PCR, real time PCR or impedance have to be combined with the pre-enrichment step as well due to the necessity for reconstitution of stressed Cronobacter cells in PIF and for acquisition of minimal cell numbers required for detection limit. The Cronobacter growth in reconstituted PIF during the pre-enrichment stage is hence an extremely important prerequisite for the whole detection, because the following isolation and confirmation steps would not make sense unless a sufficient growth of target micro-organism is guaranteed in the pre-enrichment broth. According to ISO/TS 22964:2006, 0.1 ml from the overnight pre-enrichment is applied in 10 ml mLST for further isolation. In order to make sure that at least 1 CFU Cronobacter would be included in this 0.1 ml aliquot, Cronobacter cells initially present at low levels in 100 g PIF must theoretically increase to a minimal density of 9000 CFU in 900 ml pre-enrichment broth after overnight incubation at 37°C. Conversely, no Cronobacter is detectable if this lowest detection limit in the pre-enrichment is not achieved. Using the same simplistic model of Iversen and Forsythe (2003) which assumed an average of 0.36 Cronobacter cells/100 g PIF, lag time of 2 h and doubling time of 0.5 h at CHAPTER 1 16 37°C, it can be calculated that 100 g PIF reconstituted in 900 ml water would need to be kept for approximately 7.5 h at 37°C before a cell number of Cronobacter of 9000 CFU is achieved. It implies that at ideal conditions a sufficient Cronobacter growth can be ensured during the pre-enrichment. In the reality, nevertheless, any non-target micro-organisms present in the sample that are able to grow in BPW or water might compete with Cronobacter during the pre-enrichment and further reduce the numbers of target colonies transferred into selective broth. In addition, special PIF products with antimicrobial agents have been shown to limit the Cronobacter growth. In commercially available biologically acidified infant formulae e.g. NAN PELARGON ® as well as in acidified formula supplemented directly with organic acid, concentration of Cronobacter cells after 6 h incubation at 30°C were restricted to 10 CFU/ml comparative to the initial spiking level, while in the non-acidified formula Cronobacter grew to as high as 10 5 CFU/ml (Kreb, 2010). In the investigation of Joosten and Lardeau (2004), similarly, Cronobacter levels over a period of 6 h at 37°C in rehydrated acidified formula were 1000-fold lower than that detected in non-acidified formula. Oshima et al. (2012) screened thirty-three antimicrobial agents including peptides, organic acids, organic acid esters and lactoperoxidase system (LPOS) etc. for their anti-Cronobacter activity and determined final numbers of C. sakazakii grown in reconstituted milk powder at 37°C after 8 h at a very low level of < 1 log CFU/ml in the presence of LPOS combined with nisin or lacticin, showing the most potential in Cronobacter inhibition, whereas in the controlling infant formula group without these additives a high number of Cronobacter in 7 log CFU/ml was detected. Although the antimicrobial hurdles added to PIF have been shown to be beneficial in protecting against Cronobacter overgrowth due to inhibitory interactions, there is risk of target micro-organism not reaching the required cell limit in the reconstitution stage, which could lead to a false negative outcome by detection. It is strongly emphasized that samples from which no Cronobacter is detectable do not necessarily indicate their microbiological safety. Therefore, the limited growth of Cronobacter in PIF or PIF additives must be taken into consideration when a method for Cronobacter detection is developed, validated or applied. CHAPTER 1 17 1.8 Prevalence of non-Cronobacter Enterobacteriaceae in PIF and the related influence on Cronobacter Identification Mutyjens et al. (1988) were the first to assess the microbiological quality of powdered substitutes for breast milk with regard to members of Enterobacteriaceae family. Enterobacter (E.) agglomerans has been found to be present in PIF most frequently with 27% of all isolates, followed by Enterobacter (E.) cloacae with 23% and Klebsiella (K.) pneumoniae with 10%. However the identity of E. agglomerans in this study is uncertain because since the revision of Enterobacter-Pantoea-Citrobacter group, the former E. agglomerans now encompasses both Pantoea spp. and Escherichia vulneris (Janda and Abbott, 1998). In another survey performed by Iversen and Forsythe (2004), a distribution of most common organisms for E. cloacae (25%) and Pantoea (P.) spp. (19%) was described. Estuningsih et al. (2006) found 24% P. agglomerans, 20% Escherichia hermanni, 16% E. cloacae and 6% K. pneumoniae in PIF products manufactured in Indonesia and Malaysia. E. cloacae and E. agglomerans were isolated from 15 infant food formula products in UK (Shaker et al., 2007). Popp et al. (2009) reported E. cloacae, K. pneumoniae, K. oxytoca and Pantoea spp. as most frequently isolated Enterobacteriaceae species from infant formula products. These available data allow the conclusion that besides Cronobacter spp., the most dominant Enterobacteriacea isolated from PIF included Enterobacter species, Pantoea species, and Klebsiella species. The available data do not indicate any relationship between Enterobacteriaceae and Cronobacter present in PIF samples. However neither is it possible to rule out a possible correlation with the available data. Cordier (2008) has shown that high levels of Enterobacteriaceae > 100 CFU/g are likely to represent an increased risk of product contamination with Cronobacter. Monitoring of Enterobacteriaceae is used as an indicator of hygienic status in production facilities (Anonymous, 2007b). As shown in the microbiological criteria for PIF applied by the European Commission, EC No 1441/2007 (Anonymous, 2007a), the detection of Enterobacteriaceae is a process hygienic criterion, while Salmonella and Cronobacter spp. serve as food safety criteria (Table 1). CHAPTER 1 18 Table 1. Current microbiological criteria for dried infant formula for infants up to 6 months of age and for formulae for special medical purposes of European Union Micro-organisms n c m M Enterobacteriaceae (10 g) 10 0 0/10 g NA Process hygienic parameter Cronobacter spp. (10 g) 30 0 0/30 g NA Food safety parameters Salmonella (25 g) 30 0 0/30 g NA n is the number of samples to be analyzed per lot; c is the number of samples allowed between “m” and “M” value; m is the microbiological limit separating good from marginally acceptable quality; M is the microbiological limit separating marginally acceptable from unacceptable quality. Based on findings of Guillaume-Gentil et al. (2005) and Nazarowec-White and Farber (1997b) that Cronobacter spp. showed a higher tolerance against osmotic stress and heat stress than most of other Enterbacteriaceae competitors except for four strains of E. hermanni, E. cloacae, K. oxytoca and K. pneumoniae, the new ISO specification for PIF ISO/TS 22964:2006 has been developed using mLST as the selective enrichment broth (Anonymous, 2006b). However, either using the selective enrichment media combined with elevated temperature in ISO method or using the non-differential FDA enrichment procedure, Enterobacteriaceae in different genera and species have been isolated from PIF, environmental samples or foods other than infant formula where competitive flora quantitatively occurred (Baumgartner et al., 2009; Iversen et al., 2009; Lehner et al., 2010; Shaker et al., 2007). CHAPTER 1 19 Objectives of the study Consumption of reconstituted PIF contaminated with Cronobacter spp. has been shown to be associated with a high risk of infection diseases in infants. The present work aimed to get more insight into the effect of using hot water with a temperature of 70°C or higher for PIF rehydration on the growth and survival of Cronobacter present in fermented and non-fermented infant formula (Chapter 2). As an alternative to thermal inactivation, the influence of organic acids as natural antimicrobial barrier on the growth of Cronobacter spp. in laboratory medium as well as in the reconstituted PIF was investigated. Particularly the anti-Cronobacter effect of most inhibitive organic acids was studied under natural infant stomach acidity (Chapter 3). Due to the antimicrobial compounds or competing flora in PIF, the growth of Cronobacter spp. in the pre-enrichment stage can be inhibited, which could influence principally the overall effectiveness of the Cronobacter detection method. A newly developed impedance method combined with rRNA lateral flow assay for detection of Cronobacter spp. in PIF was evaluated, with possible effect of organism’s history (healthy or stressed) and competing background flora assessed (Chapter 4). At last, various systems for the isolation, identification and differentiation of Cronobacter spp. from other micro-organisms with a common presence in PIF have been comparatively assessed in view of their sensitivity, selectivity, discrimination power, rapidity as well as convenience in performance (Chapter 5 and Addendum). References Aldova, E., Hausner, O., and Postupa, R. (1983). Tween-esterase activity in Enterobacter sakazakii. Zentralbl Bakteriol Mikrobiol Hyg A 256, 103-8. 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P., and Hill, C. (2012). Efficacy of organic acids, bacteriocins, and the lactoperoxidase system in inhibiting the growth of Cronobacter spp. in rehydrated infant formula. J Food Prot 75, 1734-42. Pagotto, F. J., Nazarowec-White, M., Bidawid, S., and Farber, J. M. (2003). Enterobacter sakazakii: infectivity and enterotoxin production in vitro and in vivo. J Food Prot 66, 370-5. Popp, A., Iversen, C., Fricker-Feer, C., Gschwend, K., and Stephan, R. (2009). Identification of Enterobacteriaceae isolates from raw ingredients, environmental samples and products of an infant formula processing plant. Arch Lebensmittelhyg 60, 92-7. Restaino, L., Frampton, E. W., Lionberg, W. C., and Becker, R. J. (2006). A chromogenic plating medium for the isolation and identification of Enterobacter sakazakii from foods, food ingredients, and environmental sources. J Food Prot 69, 315-22. Russmann, H., Kempf, V. A., Koletzko, S., Heesemann, J., and Autenrieth, I. B. (2001). 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Enterobacter sakazakii infections in neonates associated with intrinsic contamination of a powdered infant formula. Infect Control Hosp Epidemiol 10, 398-401. Stephan, R., Ziegler, D., Pfluger, V., Vogel, G., and Lehner, A. (2010). Rapid genus- and species-specific identification of Cronobacter spp. by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 48, 2846-51. Townsend, S. M., Hurrell, E., Gonzalez-Gomez, I., Lowe, J., Frye, J. G., Forsythe, S., and Badger, J. L. (2007). Enterobacter sakazakii invades brain capillary endothelial cells, persists in human macrophages influencing cytokine secretion and induces severe brain pathology in the neonatal rat. Microbiology CHAPTER 1 26 153, 3538-47. Urmenyi, A. M., and Franklin, A. W. (1961). Neonatal death from pigmented coliform infection. Lancet 1, 313-5. Van Acker, J., De Smet, F., Muyldermans, G., Bougatef, A., Naessens, A., and Lauwers, S. (2001). Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula. J Clin Microbiol 39, 293-7. Vandamme, P., Pot, B., Gillis, M., de Vos, P., Kersters, K., and Swings, J. (1996). Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 60, 407-38. Weisburg, W. G., Barns, S. M., Pelletier, D. A., and Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697-703. Wheeler, T. G., and Goldschmidt, M. C. (1975). Determination of bacterial cell concentrations by electrical measurements. J Clin Microbiol 1, 25-9. CHAPTER 2 27 Chapter 2 Thermal inactivation of Cronobacter spp. during rehydration of powdered infant formula with water of 70°C or higher CHAPTER 2 28 Abstract The presence of Cronobacter spp. in powered infant formula (PIF) has been associated to outbreaks of rare, but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in newborns. This study was undertaken to explore the influence of reconstitution water of 70°C or higher on survival of Cronobacter spp. in PIF. A higher final temperature was obtained in plastic baby bottles than glass baby bottles after PIF was mixed with water of the same temperature. An average reduction in Cronobacter levels of 3.6 log was achieved if contaminated PIF was prepared with water at 80°C in plastic bottles. The inhibitory effect on Cronobacter in fermented infant formula (FIF) was reduced by reconstituting the formula with water of 80°C. Reconstitution temperature of 80°C did not lead to a greater reduction in vitamin C concentration in PIF compared to lower reconstitution temperatures of 20°C and 40°C. Introduction Cronobacter species, formerly known as Enterobacter sakazakii, are Gram-negative, rod-shaped pathogenic bacteria of the family Enterobactericeae. These organisms have been implicated as causes of life-threatening infections like meningitis, necrotizing enterocolitis and bacteremia predominantly in infants < 4 weeks of age with a mortality rate of 20%-50% (Anonymous, 2007a; Clark et al. 1990; Lehner and Stephan, 2004). Powdered infant formula (PIF) is not a sterile product. Contamination of PIF can occur intrinsically during the manufacturing process or from extrinsic sources through contaminated utensils (e.g. spoons, blenders, teats, bottles) (Anonymous, 2007b). PIF contaminated with Cronobacter spp. has been microbiologically and epidemiologically shown to be the vehicle and source of infection in infants. Heat treatment of food just prior to consumption has been used as a primary tool to reduce the risk associated with foodborne pathogens (Edelson-Mammel and Buchanan, 2004). FAO/WHO (Anonymous, 2007b) recommended reconstitution of PIF with water at 70°C or higher to reduce the potential risk associated with Cronobacter contamination. However, heating infant milk to ≥ 70°C raises other concerns such as loss of heat-sensitive nutrients and an increased risk of scalding. Besides, the high reconstitution CHAPTER 2 29 temperature may be inadequate for some PIF products containing probiotics because they could be killed by water of ≥ 70°C (Anonymous, 2006). This study was undertaken to evaluate the effect of preparing PIF and FIF with water ≥ 70°C on survival of Cronobacter. Materials and Methods Temperature of rehydrated PIF in baby bottles prepared with water at 70°C and 80°C The tap water was first boiled in an electric kettle. The freshly boiled water was left at the room temperature to cool down to 80°C and 70°C respectively. According to the preparation instruction, 90 ml water were added to sterilized baby bottles made of glass and of plastic respectively. Three scoops (13.5 g) of PIF were added to the water. In the same manner, five scoops (22.5 g) PIF were dissolved in 150 ml water in glass and plastic baby bottles. The bottles were caped and gently agitated by hand at room temperature for 10 seconds. The temperature of rehydrated PIF in baby bottles was measured and three independent trials were performed. Survival of Cronobacter strain in PIF prepared with water of 80°C Commercially available PIF products were first screened for absence of Cronobacter spp. with ISO/TS 22964. 90 ml water of 80°C were poured in to a sterilized plastic baby bottle prior to adding 3 scoops of PIF. The rehydrated PIF was inoculated with 1 ml fresh overnight culture of C. sakazakii CLF2688 at levels of 10 8 , 10 7 , 10 6 and 10 5 and 10 4 CFU/90 ml respectively. The bottle was left at room temperature for 2 min and then cooled under the running tap water to 37°C. The C. sakazakii count in the bottle was determined by spread-plate technique using Brilliance TM E. sakazakii chromogenic agar Druggan-Forsythe-Iversen (DFI) formulation (Oxoid, UK). Three independent trials were performed. Impact of reconstitution temperature of 80°C on the antibacterial effect of FIF FIF in different fermentation levels (0%, 30%, 50% and 100%) were applied in this test. A non-fermented neutral PIF starter product served as reference. In the first group, 1-10 CFU CHAPTER 2 30 C. sakazakii (CLF2688) were added to 90 ml reconstituted FIF and PIF which were prepared with water of 40°C in a plastic baby bottle. To investigate the effect of a higher reconstitution temperature on the inhibitory activity of FIF against Cronobacter, 90 ml FIF and PIF were first prepared with water at 80°C and left at room temperature for 2 min. After being cooled down to 40°C under the running tap water, the infant formulae were artificially spiked with 1 ml C. sakazakii (CLF2688) in 1-10 CFU/90 ml of diets. Inoculated infant formulae in both groups were stored at room temperature for 24 h. The Cronobacter growth was followed by enumeration with DFI chromogenic agar after 5 and 24 h. Three independent trials were performed. Vitamin C concentration in PIF prepared with water of 20°C, 40°C and 80°C 12.20 g commercially available PIF product were weighed in the flask and filled with 100 ml water of 20°C, 40°C and 80°C respectively. The flask was well shaken and stored at a household fridge temperature of 9°C for 8 h. The concentration of vitamin C in rehydrated PIF was determined with a potentiometric method on titrator (Mettler TOLEDO T50, Germany) using 2,6-dichchlorophenolindophenol at 0 h, 4 h and 8 h. Statistic analysis Data were analyzed with Student’s t test using Microsoft ® Excel 2003 software. Significant differences are presented at a 95% confidence level (p < 0.05). Results Compared with initial water temperatures of 70°C and 80°C added to bottles, the temperatures after PIF-mixing decreased in all bottle/serving volumes combinations (Table 1). However for both 90 ml and 150 ml serving volumes, the temperature in rehydrated PIF dropped to a less extend in bottles made of plastic than that of glass. Besides, a more rapid decrease in temperature after PIF-mixing was observed in smaller serving volume of 90 ml than in larger volume of 150 ml in glass bottles, while the final temperatures in plastic bottles did not differ between rehydration quantities. CHAPTER 2 31 Table 1. Temperature of rehydrated infant formula after hot water of 70°C and 80°C was added to glass and plastic baby bottles containing PIF PIF reconstitution temperature Water quantity 70°C 80°C Glass bottle Plastic bottle Glass bottle Plastic bottle 90 ml 54°C 62°C 63°C 70°C 150 ml 58°C 62°C 65°C 70°C As higher final temperatures were reached in PIF rehydrated with water of 80°C than 70°C, the water of 80°C was used for preparing PIF in the following tests. With rehydration temperature of 80°C, the observed Cronobacter cell reduction in plastic bottles ranged from 3.5 log to 4.4 log when initial counts exceeded 10 6 CFU/90 ml, whereas in glass bottles a higher Cronobacter surviving number was obtained at initial inoculums of 10 8 , 10 7 and 10 6 CFU. Especially at higher inoculums of 10 8 and 10 7 CFU Cronobacter cells, the thermal inactivation effect of preparing PIF with water at 80°C was very pronounced in plastic bottles: a 200 to 300 fold higher C. sakazakii counts surviving in glass bottles were obtained than in plastic ones. At lower inoculum of 10 6 CFU, the difference in Cronobacter survival between bottles of two materials was minimal, while at a contamination level of 10 5 CFU, Cronobacter counts dropped in both kinds of bottles to 10 2 CFU. For an inoculum of 10 4 CFU/90 ml PIF, the reduction was greater than 2 log and this is below the lowest detection limit of 1 CFU/ml corresponding to limit of detection (LOD) of 1.9 log CFU/90 ml (Table 2). CHAPTER 2 32 Table 2. Survival of C. sakazakii (CLF2688) during the rehydration of PIF with water of 80°C at different initial contamination levels Surviving Cronobacter (log CFU/90 ml) in Mean ± SD Initial inoculum of Cronobacter in infant formula (log CFU/90 ml) Glass bottle Plastic bottle 8 6.2 ± 0.3 3.7 ± 0.3 7 5.2 ± 0.2 2.8 ± 0.1 6 3.1 ± 0.1 2.5 ± 0.3 5 2.2 ± 0.5 2.5 ± 0.2 4 < 1.9 a < 1.9 a a Lower limit of detection: 1 CFU/ml = 90 CFU/90 ml (log 90 CFU/90 ml = 1.9) After 5 h incubation at room temperature, the C. sakazakii populations were between 2 and 3 log CFU/ml in all products but in the 100% fermented FIF, regardless of reconstitution temperatures. In the FIF fermented to 100%, a slight but significant reduction in Cronobacter growth by 0.7 log was shown for reconstitution with 40°C when compared with the same product treated previously with water of 80°C (Fig. 1). On the other hand, among all products rehydrated with an ambient water temperature of 40°C as recommended by manufacturer, only limited inhibitory effect was shown in FIF fermented to 100% and no antibacterial effect was visible in formulae fermented to 30% and 50% compared with non-fermented FIF or reference PIF. In the group rehydrated with water of 80°C, the target micro-organism was able to grow to ≥ 7 log CFU/ml in all products within 24 h incubation, while a significant cell reduction ranging from 1.5 log to 2.4 log was shown in FIF fermented in 30%, 50% and 100% when prepared with water of 40°C (Fig. 2). The pH values at 0 h were 6.70, 6.25, 6.00, 5.60 and 6.80 for FIF fermented to 0%, 30%, 50%, 100% as well as in the control PIF respectively. After 5 h incubation slightly enhanced pH values were obtained for all products. After 24 h incubation decreased pH values of 4.16, 4.15 and 4.30 were obtained in FIF fermented to 30%, 50% and 100%. No obvious pH drop was observed in non-fermented FIF and reference product. CHAPTER 2 33 0 1 2 3 4 0% 30% 50% 100% PIF Ref erence L o g C F U /m l P IF 40°C 80°C Figure 1. Growth of C. sakazakii (CLF2688) (log CFU/ml) in FIF/PIF hydrated with 40°C and 80°C after 5 h incubation at 22°C with an initial inoculum of 1-10 CFU Cronobacter/90 ml. Value with * is significantly different (p < 0.05) with remaining values without *. * CHAPTER 2 34 4 5 6 7 8 9 0% 30% 50% 100% PIF Ref erence L o g C F U /m l P IF 40°C 80°C Figure 2. Growth of C. sakazakii (CLF2688) (log CFU/ml) in FIF/PIF hydrated with 40°C and 80°C after 24 h incubation at 22°C with an initial inoculum of 1-10 CFU Cronobacter/90 ml. Values with * are significantly different (p < 0.05) with remaining values without *. The three values with * revealed no significant differences with each other (p > 0.05). As shown in Fig. 3, the vitamin C concentrations in rehydrated PIF were at the beginning 14.9, 14.5, 14.6 mg/100 ml and were reduced to 10.7, 10.2 and 10.9 mg/100 ml for preparation water temperature of 20°C, 40°C and 80°C respectively after 8 h storage at 9°C, which indicates that the vitamin C reduction caused by a high preparation temperature did not differ from that at lower temperatures. * * * CHAPTER 2 35 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 0 4 8 time (h) V it C c o n c e n tr a ti o n ( m g /1 0 0 m l) 40°C 20°C 80°C Figure 3. Vitamin C concentration in PIF rehydrated with water of 20°C, 40°C and 80°C over 8 h at a household fridge temperature. Discussion In Germany, use of water at 40°C or 50°C for reconstituting PIF is recommended by the manufacturers for products such as Milumil, Aptamil, Hipp and Alete. However, FAO/WHO (Anonymous, 2006) performed a risk assessment model for Cronobacter in PIF and concluded that PIF reconstitution with water at 40°C and 50°C is highly associated with an increased risk of Cronobacter infection. Edelson-Mammel and Buchanan (2004) reported that using water at 70°C or greater for PIF preparation was able to cause a more than 4-log reduction in Cronobacter levels and could therefore significantly reduce the risk of infection. In addition, Chen et al. (2009) showed less survival of Cronobacter cells in reconstitution water ≥ 70°C in larger volumes than in smaller volumes because a higher temperature was maintained for a longer time in larger volumes, exhibiting a more pronounced lethality effect. In the present work, temperature differences between PIF prepared in smaller and larger volumes were only observed in feeding bottles made of glass. It is known that lower final temperatures achieved in PIF after reconstitution water is added allow a better survival of Cronobacter cells (Chen et al., 2009; Kim and Park, 2007). Due to the fact that younger CHAPTER 2 36 infants often receive liquid formula prepared in a smaller quantity per feeding than older infants and the younger babies are more vulnerable to Cronobacter infections than older babies (Anonymous, 2006), the consumption of contaminated PIF in a smaller serving volume could therefore pose a high risk of Cronobacter infection particularly to infants at younger age. As shown in Table 1, the highest final temperature of 70°C was achieved in PIF prepared with water of 80°C in plastic bottle regardless of serving volumes, while the lowest final temperature of 54°C after PIF-mixing was obtained in 90 ml PIF rehydrated with water of 70°C in glass bottle. This might indicate that the plastic bottle can hold heat for longer period of time than glass one probably due to the high capacity of glass material for heat absorption. The temperature drop in PIF after it was mixed with hot water has been documented in various previous studies (Chen et al., 2009; Edelson-Mammel and Buchanan, 2004; Kim and Park, 2007). However, no information about the material of baby bottles used was given in details: while only a rough description as “feeding bottles” or “baby bottles” was given in publications of Edelson-Mammel and Buchanan (2004) and Kim and Park (2007), Chen et al. (2009) did not mention at all in what kind of container the PIF was reconstituted and the growth of Cronobacter was determined. From the data presented in Table 1, it is evident that the material of baby bottles represents an important determinant for temperature drop in rehydrated PIF impacting the Cronobacter survival. In the present work, rehydration of 90 ml PIF in 80°C hot water resulted in greater than 3 log and 2 log reduction in the Cronobacter strain tested in plastic and glass bottle respectively (Table 2). By contrast, in the pilot study of Edelson-Mammel and Buchanan (2004), a stronger effect of reconstitution water of 80°C on Cronobacter inactivation in PIF of ≥ 4 log has been reported. However it must be noted that in their investigation a higher volume of formula (180 ml) was prepared using 25.5 g PIF contributing to decrease of temperature in a less extent and further a lower Cronobacter survival. In the study of Chen et al. (2009), Cronobacter cell inactivation of greater than 4 log by using water of 80°C was observed on two pre-selected heat-sensitive target strains inoculated in larger serving volumes. Last but not least, different incubation time could be responsible for the observed differences in Cronobacter survival rate as well. In the present study the heat in PIF prepared with hot CHAPTER 2 37 water of 80°C was kept for 2 min before Cronobacter counts were determined, whereas the bottles were hold for 10 min and analyzed for Cronobacter by Edelson-Mammel and Buchanan (2004). A relative low level of Cronobacter cells of 0.36 to 66 CFU Cronobacter/100 g PIF has been determined by Muytjens et al. (1988). This implies that preparing reconstituted formula using the best option presented here (80°C in plastic bottle) is likely to result in a high probability that a serving would not contain this micro-organism. Concerns have been raised in using water of ≥ 70°C to prepare PIF due to possible loss of nutrient in product or inactivation of probiotic added in specialized infant formula (Anonymous, 2006). Fermented infant formula is infant formula fermented with lactic acid-producing bacteria e.g. Bifidobacterium and Streptococcus during the production process. The FIF in the present work is based on a mixture of fully fermented PIF and non-fermented PIF product. The fermentation levels of FIF refer to the percentage of fully fermented product in the whole mixture. FIF usually contains low numbers of viable bacteria (< 10 3 CFU/g dry weight) in the final product due to the inactivation of the fermenting bacteria by physical treatment. Clinical trials have shown that some FIF could reduce the occurrence or severity of infectious diarrhea in infants which is likely to be associated with its bacteriostatic effect (Brunser et al., 1989; Joosten and Lardeau, 2004; Thibault et al., 2004). It has been concluded that the additional benefits of FIF result from the remaining bacterial components such as cell membrane or bacterial DNA, and/or bacterial metabolites such as organic acids or protein with enzyme activity rather than surviving intact living bacteria (Agostoni, et al., 2007; Ludwig, et al., 2012). As shown in Fig. 2, the FIF fermented to 30%, 50% and 100% showed an inhibition effect on Cronobacter growth in 24 h if rehydrated with water at 40°C. The cell reduction might be caused by the relatively lower pH values in these products after 24 h incubation. No inhibitive effect was demonstrated in FIF when it was prepared with 80°C water, indicating the thermal sensitivity of the compositions produced by fermentation. On the other hand, the inhibition effect of FIF is very limited. In FIF with lower fermentation levels of 30% and 50%, Cronobacter grew to higher than 100 CFU/ml equivalent to 9000 CFU/90 ml diet within 5 h at room temperature of 22°C if the PIF was reconstituted with water of 40°C CHAPTER 2 38 according to recommendation. 5 h is regarded as a critical timeframe in consumer practices where an extended feeding or holding can really occur. Most importantly, the cell concentration of 9000 CFU/90 ml exceeds the infectious dose of 1000 CFU estimated by Iversen and Forsythe (2003) and is very close to the infectious level of 10,000 CFU proposed by FAO/WHO (Anonymous, 2007a). In 24 h Cronobacter grew to a cell density of between 10 5 and 10 6 CFU/ml in FIF treated with water of 40°C, posing a high risk of infection. By comparison, the alternative Cronobacter inactivation strategy by using water of ≥ 70°C for reconstitution which should provide virtually instantaneous inactivation of this micro-organism is presumed to kill all Cronobacter cells present initially at low level in infant formula and is therefore likely to control the risk more effectively. Reduction of vitamin C ranging from 5.6% to 65.6% in four particular formulae due to preparation with boiling water has been noted in the report of the FAO/WHO expert meeting (Anonymous, 2006; Anonymous, 2007b). To solve this problem, dry formulae actually contain higher levels of vitamin C than labelled. After reconstituting with boiling water, the vitamin C levels in the four formulae mentioned above still exceeded the can label or were higher than the minimum recommendation (8 mg vitamin C/100 calories) required by the CAC Codex Standard for Infant Formula (Anonymous, 1981). Based on data in Fig. 4, the vitamin C concentration in reconstituted PIF decreased over the incubation duration at refrigerator temperature. However the reduction of vitamin C is not directly related to the enhanced water temperature. Rehydration of PIF with 80°C water resulted in a slighter vitamin C reduction of 18% and 25% at 4 h and 8 h respectively than reductions achieved with 20°C (21% and 30%) and 40°C (18% and 28%). References Agostoni, C., Goulet, O., Kolacek, S., Koletzko, B., Moreno, L., Puntis, J., Rigo, J., Shamir, R., Szajewska, H., and Turck, D. (2007). Fermented infant formulae without live bacteria. J Pediatr Gastroenterol Nutr 44, 392-7. Anonymous. (1981). CAC. Codex Standard for Infant Formula. (Codex Stan 71-1981). http://www.codexalimentarius.net/web/standard_list do?langen=en http://www.codexalimentarius.net/web/standard_list%20do?langen=en CHAPTER 2 39 Anonymous. (2006). FAO/WHO. Enterobacter sakazakii and Salmonella in powdered infant formula: meeting report. MRA Series 10. http://www.who.int/foodsafety/publications/micro/mra10.pdf. Anonymous. (2007a). FAO/WHO. Enterobacter sakazakii and other microorganisms in powdered infant formula: meeting report. MRA Series 6. http://www.who.int/foodsafety/publications/micro/mra6/en/. Anonymous. (2007b). FAO/WHO. Safety preparation storage and handling of powdered infant formula – Guidelines. http://www.who.int/foodsafety/publications/micro/pif2007/en/. Brunser, O., Araya, M., Espinoza, J., Guesry, P. R., Secretin, M. C., and Pacheco, I. (1989). Effect of an acidified milk on diarrhoea and the carrier state in infants of low socio-economic stratum. Acta Paediatr Scand 78, 259-64. Chen, P. C., Zahoor, T., Oh, S. W., and Kang, D. H. (2009). Effect of heat treatment on Cronobacter spp. in reconstituted, dried infant formula: preparation guidelines for manufacturers. Lett Appl Microbiol 49, 730-37. Clark, N. C., Hill, B. C., O’Hara, C. M., Steingrimsson, O., and Cooksey, R. C. (1990). Epidemiologic typing of Enterobacter sakazakii in two neonatal nosocomial outbreaks. Diagn Micro Infect Dis 13, 467-472. Edelson-Mammel, S. G., and Buchanan, R. L. (2004). Thermal inactivation of Enterobacter sakazakii in rehydrated infant formula. J Food Prot 67, 60-63. Iversen, C., and Forsythe, S. J. (2003). Risk profile of Enterobacteri sakazakii, an emergent pathogen associated with infant milk formula. Trends Food Sci Tech 14, 443-454. Joosten, H., and Lardeau, A. (2004). Enhanced microbiological safety of acidified infant formulas tested in vitro. S Afr J Clin Nutr 17, 87-92. Kim, S. H., and Park, J. H. (2007). Thermal resistance and inactivation of Enterobacter sakazakii isolates during rehydration of powdered infant formula. J Microbiol Biotechnol 17, 364-8. Lehner, A., and Stephan, R. (2004). Microbiological, Epidemiological, and Food safety aspects of Enterobacter sakazakii. J Food Prot 67, 2850-57. Ludwig, T., Huybers, S., Abrahamse, E., and Bouritius, H. (2012). Fermented infant formula. Patent Application EP20110164461. Muytjens, H. L., Roelofs, W. H., and Jaspar, G. H. J. (1988). Quality of powdered substitutes for breast milk with regard to members of the family Enterobacteriaceae. J Clin Microbiol 26, 743-46. Thibault, H., Aubert-Jacquin, C., and Goult, O. (2004). Effects of long-term consumption of a fermented infant formula (with Bifidobacteriium breve c50 and Streptococcus thermophilus 065) on acute diarrhea in healthy infants. J Pediatr Gastroenterol Nutr 39, 147-152. http://www.who.int/foodsafety/publications/micro/mra10.pdf http://www.who.int/foodsafety/publications/micro/mra6/en/ http://www.who.int/foodsafety/publications/micro/pif2007/en/ CHAPTER 3 40 Chapter 3 Growth inhibition of Cronobacter spp. strains in reconstituted powdered infant formula acidified with organic acids supported by natural stomach acidity S. Zhu, S. Schnell, M. Fischer Food Microbiology 35 (2013) 121-128 CHAPTER 3 41 CHAPTER 3 42 CHAPTER 3 43 CHAPTER 3 44 CHAPTER 3 45 CHAPTER 3 46 CHAPTER 3 47 CHAPTER 3 48 CHAPTER 4 49 Chapter 4 Rapid detection of Cronobacter spp. with a method combining impedance technology and rRNA based lateral flow assay S. Zhu, S. Schnell, M. Fischer International Journal of Food Microbiology 159 (2012) 54-58 CHAPTER 4 50 CHAPTER 4 51 CHAPTER 4 52 CHAPTER 4 53 CHAPTER 4 54 CHAPTER 5 55 Chapter 5 Matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry, 16S rRNA gene sequencing, and API 32E for identification of Cronobacter spp.: A comparative study S. Zhu, S. Schnell, M. Fischer Journal of Food Protection 74 (2011) 2182-2187 CHAPTER 5 56 CHAPTER 5 57 CHAPTER 5 58 CHAPTER 5 59 CHAPTER 5 60 CHAPTER 5 61 CHAPTER 6 62 Chapter 6 Discussion CHAPTER 6 63 6.1 Inactivation of Cronobacter in Reconstituted PIF PIF is not a sterile product. Being a nutrient-rich medium, reconstituted PIF provides a good substrate for growth of Cronobacter spp. (Nazarowec-White and Farber, 1997b). Once