INTRODUCTIONBackground of the StudyBreastfeeding remains the gold standard for infant feeding. It offers numerous benefits not only to the mother but more importantly to the infant. The World Health Organization (WHO) recommends, as part of the strategy to promote Infant and Young Child Feeding, that breastfeeding should be started early, within one hour after birth; should be exclusive for the first six months and continued with the introduction of complementary feeding at around six months of age until one to two years old and beyond as long as mutually acceptable to the infant and mother (WHO-UNICEF, 2003). Despite these recommendations, very few infants are being exclusively breastfed for the first six months especially in developing countries (33% in 1995 and 39% in 2010) (Xiaodong et. al., 2012). In the Philippines, the rate of exclusive breastfeeding is as low as 27% (National Statistics Office, 2013). Mothers resort to the giving of breast milk substitutes in the form of infant formulas. Reasons of the caregivers are varied ranging from not enough milk (31%); working (17%); nipples and breasts ache (17%); child does not want to breastfeed (11%); child is sick (11%); mother is sick (9%) (National Statistics Office, 2003). Although breastfeeding should be advocated according to the WHO, there are some reasons why breastfeeding may not be possible and infant formulas can be given but these are few compared to the numerous benefits that breastfeeding has to offer. For infants, these include those who will require special formula for medical reasons such as the presence of certain inborn errors of metabolism (galactosemia, maple syrup urine disease, phenylketonuria); infants for whom breast milk remains the best feeding option but who may need other food in addition to breast milk for a limited period like in very low birth weight infants (born weighing less than 1500 g) or very premature infants (infants born at less than 32 weeks of gestational age) and those who are unable to maintain adequate blood sugar (premature, very sick infants those whose mothers are diabetic). Maternal conditions that may justify temporary avoidance of breastfeeding include HIV infection, severe illnesses such as sepsis, and the use of certain drugs and chemotherapy (WHO-UNICEF, 2009).In a setting like the Philippines where hygienic preparation of infant formula is highly questionable, along with higher temperature and humidity, there is high risk of bacterial contamination of powdered infant formulas. In reality, mothers are not educated as to their responsibilities in case they decide not to breastfeed. Anecdotal experiences and testimonies point to the lack of knowledge regarding the proper use of bottles, preparation of formulas and use of leftover milk. The latter often creates a problem as most mothers think that giving of leftover milk that has stood for four hours is still safe. There are various recommendations as to the suitability of giving milk that has been reconstituted for several hours but there is no one consensus. The WHO (2007) recommends that milks that have not been consumed in two hours or have not been refrigerated ay 5 degrees Celsius and below should be discarded, and that powdered milk should be reconstituted in pre-boiled water at 70 degrees Celsius after which it has to be cooled before feeding to an infant. The Centre for Health Promotion in Australia (2012) also recommends that the water has to be cooled down to room temperature before milk can be reconstituted. On the other hand the American Dietetic Association (2011) recommends that hang time of infant formula or expressed breast milk for tube feeding of infants should not exceed four hours. Because of these conflicting recommendations, this study was envisioned to determine that presence or absence of bacterial contamination of reconstituted powdered infant formula prepared according to standard guidelines that has stood for several hours at different temperatures compared with expressed breast milk.

STATEMENT OF THE PROBLEMAmong samples of newly reconstituted or left-over reconstituted powdered infant formula milk and expressed human milk exposed at different temperatures and time intervals, which will show the most bacterial colonization?OBJECTIVESGeneral ObjectivesTo compare the occurrence and rate of bacterial contamination in reconstituted powdered infant formula milk with expressed human breast milk at different temperatures and time intervals.Specific Objectives1. To determine the presence of bacterial colonies in reconstituted powdered infant formula milk left standing under different temperatures and time intervals.2. To determine the rate of bacterial colonization in standing infant formula milk and expressed human breast milk under different temperatures and time intervals.3. To compare the presence and rate of bacterial colonization in standing infant formula milk and expressed human milk under different temperatures and time intervals.4. To characterize the different bacterial colonies obtained from the leftover, newly reconstituted, and expressed human milk samples.

Hypotheses1. There is a significant difference in bacterial colonization in standing reconstituted powdered infant formula milk and expressed human breast milk.2. The degree of bacterial colonization is as follows: leftover reconstituted powdered infant formula milk > newly reconstituted > expressed human milk; whether exposed at room temperature or refrigerated.

Significance of the StudyThis study aims to determine if bacterial contamination is present and significant in reconstituted powdered infant formula. Once proven this will send a message that breastfeeding should be advocated at all costs. Aside from the nutritional, emotional and economic benefits, breastfeeding is the strategy that can promote child survival by prevention of illnesses like gastrointestinal diseases, which are the foremost cause of infant deaths particularly in developing countries. The presence of bacterial contamination due to improper use of bottle-feeding negates this benefit that can be achieved by breastfeeding. As such, this will be another evidence to promote and advocate exclusive breastfeeding during early infancy. All nutrients as well as antibody presence that are needed by an infant for the first six months are provided by breast milk, without the necessity of other food or liquids.In the event that breastfeeding is not possible, this study will promote the reduction of diseases due to food-borne illnesses (in this case from bottle feeding) such as diarrhea and gastrointestinal tract infections. It will create heightened awareness regarding the observance of strict hygienic practices in proper handling, preparation and storage of breast milk substitute. In situations when a mother is unable to breastfeed, maternal education in proper infant feeding practices will be emphasized. Good hygienic practices will also be reinforced in the preparation of powdered infant formulas. This strategy can reduce the occurrence of diseases. Prevention of bacterial contamination of infant formulas will lead to prevention of common childhood illnesses like diarrheas and respiratory infections that can reduce expenses for medical treatment, medicines, and hospitalization. Indirect benefits include avoidance of absences of mothers who will bring their infants for medical consultation or hospitalization due to these illnesses. Expenses that could have been used for these situations can be converted to family savings.

Scope and LimitationThe study is limited to the use of powdered infant formula in young infants aged less than six months of age as this is the age most at risk to development of food-borne illnesses particularly with regards bottle feeding. Only samples of milk previously consumed by one infant will be used in the study. Only powdered infant formula will be used instead of ready-to-use liquid formula which is not readily available in the Philippine setting but is the type of infant formula that is recommended which is not commonly susceptible to bacterial contamination. Powdered infant formulas require reconstitution first prior to use. Only samples from reconstituted powdered infant formula will be examined for bacterial contamination. No samples will be taken from the raw milk prior to reconstitution, the feeding bottle and its accessories like cap and nipples, the water to be used in reconstitution, the hands of the person preparing the formula and the setting where the formula will be prepared like tabletop. Although the other variables like the air inside the laboratory where the cultures will be taken as well as the unopened can of the formula used.Reconstitution of powdered infant formula shall follow the WHO recommendations (Bachrach et. al., 2003), which will take into account the sterilization of feeding bottles, the use of pre-boiled water, and the cooling of reconstituted milk to room temperature. This will ensure uniformity in the procedures. As to expressed breast milk, this will be done by manual or hand expression and not the use of breast pump as the latter may introduce another variable in the study. Breast pump has a potential for being contaminated also even prior to use.

Definition of TermsBacterial contamination: the initial presence of any form and quantity of any species of bacteria, especially those of pathogenic and infectious strains.Infant formula: preparation in which cows milk or soya milk is used to substitute, modify and or strengthen/fortify human milk. This can come as powdered or liquid ready-to-use forms. Only the powdered form will be used in the study. Standard or starter infant formula, recommended for infants 0-6 months of age will be used.Reconstituted powdered infant formula: Powdered infant formula that is mixed with previously boiled water following the recommended dilution by the brand used.Newly reconstituted powdered infant formula: Reconstituted powdered infant formula that is cultured within five minutes from reconstitution.Leftover reconstituted powdered infant formula: Previously reconstituted powdered infant formula that has been partially consumed by the infant. The remainder of the milk will be used as samples for bacterial contamination at different temperatures and time intervals.Standing infant formula: Reconstituted powdered infant formula that is made to stand under specific condition at different time intervals.REVIEW OF RELATED LITERATUREBreastfeeding represents the best option for infant feeding due to its countless benefits and advantages to the infant, mother and society in general, yet even if this is highly recommended, there are mothers who are unable to comply with the recommendations due to varied reasons. As such they resort to the giving of breast milk substitutes or infant formulas, which are designed to approximate but not duplicate the nutritional contents of human milk. Additional properties of human milk such as presence of antibodies and other bioactive factors are difficult to simulate. Thus infant formulas still remain as inferior alternative. The main issue against the use of infant formula, particularly the powdered form is that it is not sterile and may be contaminated with bacterial organisms either before or after reconstitution. Every process in the production and manufacture of infant formula carries the potential for contamination starting from getting raw milks from cows to the process of converting liquid milk to powdered milk, to packaging, transport, storage and actual use by consumers who need to open the containers, prepare the milk for use like adequate cleaning of utensils used for milk feeding, to actual reconstitution. The time elapsed from reconstitution to actual consumption is crucial as well as use of left over milks. Several reports in the 1990s have documented the presence of bacterial contamination either in the powdered state or reconstituted state. Majority belonged to Salmonella species and other members of the family Enterobacteriaceae.A study done by Muytiens, et. al. (1988) identified other types of bacterial species in addition to Salmonella. After analyzing 141 powdered infant formulas from 35 countries, 52% was positive for bacterial isolates which included Enterobacter agglomerans, Enetrobacter cloacae, Enterobacter sakazakii and Klebsiella pneumoniae as the predominant species. However, none of the sample formulas had bacterial concentrates greater than 3 cfu (colony forming units) per gram, which was the cut off used by the FAO as ceiling for coliform content of infant formulas.Leuscher and Bew (2004) isolated E. sakazakii in 13.8% of powdered infant formula from 11 countries. Iversen and Stephan (2004) isolated seven types of Enterobacteriacea from powdered infant formula milk and related products consisting of two isolates of Enterobacter sakazakii and Panoea spp. and one isolate each of Enterobacter cloacae, Klebsiella pneumoniae subsp. ozaenae, Serratia ficaria, Rahnella aquatilis and Citrobacter freundii. Chap, et. al. (2009) surveyed seven countries and analyzed 290 products from them in follow-up formulas and infant foods. They found that Cronobacter (previously Enterobacter) sakazakii was isolated from 27 products, 3 out of 91 (3%) from follow-up formulas and 24 out of 199 (12%) from infant foods and drinks. Other isolates included Acinetobacter baumanii, Enterobacter cloacae, Klebsiella pneumoniae, Citrobacter freundii and Serratia ficaria.A more recent study done in Japan by Oonaka and associates (2010) revealed that 36 out of 149 or 24.2% of powdered infant formulas produced domestically in Japan or obtained from foreign sources were positive for Enterobacteriaceae. Out of 61 domestic samples, 12 were positive (19.7%). Out of the 88 foreign samples, 24 were positive. These included 26 samples from the Philippines, of which seven were positive (26.9%). Only one or 3.8% from the Philippine samples was positive for E. sakazakii. In total E. sakazakii was isolated in nine out of 149 samples or 6.6%.The implications of bacterial contamination of powdered infant formula are very significant because most of the bacteria isolated are pathogenic or can cause diseases which often times can cause long-term consequences and even deaths among infants. These are documented in terms of the presence of Salmonella and Enterobacter sakazakii.In 1993 there was a reported outbreak of infection caused by Salmonella serotype Tenessee among infants in Canada and the U.S. involving use of powdered infant milk (CDC, 1993). These formulas were recalled after these cases were identified. In 1994, 48 cases of Salmonellosis were reported in Spain among infants less than one year of age. Salmonella virchow was isolated (Usera, et. al., 1998).In the succeeding years the focus was the presence of Enterobacter sakazakii in powdered infant formula as this bacteria became associated with severe infections and deaths of young infants. Enterobacter sakazakii was first implicated in a case of meningitis in a newborn in 1958 and since then there has been more than 70 reported cases of E. sakazakii infection as of 2006 (Drudy, et. al., 2006). Since then there has been sporadic reports of cases of infections due to this bacteria : a case of urinary tract infection in India (Bhat, et. al., 2009), meningitis in Spain (Simon, et. al., 2010), multiple brain abscess in Japan (Oonaka, et. al., 2010). Infants are at greater risk for infection. The range of diseases it can cause is often serious and even fatal (Simmons, et. al., 1989): meningitis, septicemia (blood infection), urinary tract infection (Bar-oz, et.al., 2001), necrotizing enterocolitis (infection then eventual gangrene of the intestines) and brain abscess that may cause paralysis (quadriplegia) (Norberg, et.al., 2012). One of the characteristics of this species is heat-resistance leading to its survival even under extreme physical conditions. In a study by Jacobs, et. al. (2011) on the reservoir and routes of transmission of E. sakazakii in a milk powder producing plant the organism was isolated from two areas: the spray drying area and the roller dryer area, proving that milk contamination can occur even during the manufacture of milk products and that the organism may withstand the conditions of the milk factory.Enterobacter sakazakii is a Gram-negative bacillus, facultative anaerobic, yellow pigmented, mesophilic and generally motile. It grows at a maximum temperature of 41-45 degrees Celsius while the minimum temperature for growth is 5.5 -8.0 degrees Celsius. Growth is inhibited at 4 degrees Celsius. The generation times are as follows: 40 minutes at 23 degrees Celsius; 4.18-5.52 hours at 10 degrees Celsius and 75 minutes at 25 degrees Celsius in reconstituted powdered infant formula. Virulence is conferred through the production of enterotoxin (Fiore, et. al., 2008). In 2007, the genus was deemed as separate from Enetrobacteriaceae and was renamed Cronobacter sakazakii.Several outbreaks of E. sakazakii have been reported in the literature leading to recalls of infant formulas. In 2004, France and New Zealand reported outbreaks of infection (WHO-FAO, 2006). Nine cases were reported in France from five hospitals, which led to two deaths. Eight cases were premature infants. Upon investigation one hospital was not following standard practices for the preaparation, handling nd storage of infant formulas and four were storing reconstituted formula for more than 24 hours in a refrigerator without temperature control. In the United States, the Centers for Disease Control (CDC) (2001) reported an outbreak of E. sakazakii associated with the use of infant formula in Tennessee. During this outbreak, a total of 10 colonization or infection events were identified among 49 screened infants. The organism was identified among samples of powdered formula used in the neonatal ICU where this outbreak was reported. In addition, pulsed-field electrophoresis revealed that isolates ofE. sakazakiifrom the cerebrospinal fluid of a neonate with meningitis and isolates from the cultures of powdered formula were similar.Studies on bacterial contamination of powdered infant formula subjected to different physical conditions are lacking. The WHO recommendations on the safe preparation, handling and storage of infant formula comes from guidelines on best practices from manufacturing of these milks to the observance of strict hygienic practices in the preparation of these products. Temperature during storage is a major concern. A study done by Marino, et. al. (2007) on the prevalence of bacterial contamination of powdered infant feeds in a hospital environment showed that formulas incubated and stored at different temperatures and time intervals are at risk for contamination. They obtained aliquots of milk and incubated onto agar. Pre-incubation samples were cultured. Ten samples were incubated at 25 degrees Celsius overnight. Forty-eight samples were incubated at 30 degrees overnight. Thirty-four samples were incubated at 30 degrees Celsius for six hours. Post-incubation samples were cultured again. Contamination was defined as any positive culture before incubation or > 102 cfu/ml post incubation. Results showed that 50 out of 82 were contaminated pre-incubation with 25 of them (30.4%) considered as heavily contaminated. Post incubation, 43 (46.7%) was contaminated.Because of the concern that temperature of water to be used for reconstitution may play a role in the inactivation of pathogens, particularly E. sakazakii, WHO recommends that the water to be used should be put int a rolling boil then cooled to 70 degrees Celsius , not at room temperature, before the milk can be reconstituted. At this temperature this pathogen can be eradicated. In a study done by Osaili, et. al. (2008) on the effect of extended storage and use of hot water, they concluded that, reconstitution of contaminated powder with water at 70 degrees Celsius after one month of dry storage reduced E. sakazakii viability slightly and after powder was stored for 1-2 months, all E. sakazakii strains were eliminated.The infant formula industry is universally one of the most regulated industries, mainly because the consumers are infants with specific nutritional needs and requirements and whose physical and mental developments have to be ensured. Standards as to nutritional adequacy, minimum and maximum levels of nutrients are strongly adhered as stipulated by the Codex Alimentarius. The Codex Alimentarius is a list of internationally recognized standards, codes of practice, guidelines and other recommendations relating tofoods, food production andfood safety maintained by a Commission established by the Food and Agriculture Organization (FAO) of the United Nations established in 1961. The primary aim is to ensure safe good food for everyone, everywhere (International Food Standards, n.d.).In addition to nutrient content, of utmost importance is the safety of these products in the form of absence of microbiological and environmental contamination. Infant formula, particularly the powdered form is not sterile (Bachrach et. al., 2003). Contamination can exist either intrinsically within the milk itself from processing and storage or extrinsically with the actual preparation and handling of milk because of environmental conditions and unhygienic practices. Safety of breast milk substitutes must be ensured in all the steps involved in the production, from getting raw milk from the source to processing, packaging and storage. Indeed several studies have documented presence of pathogenic bacteria in raw milk and powdered milk with several detrimental consequences to the infant. Only a few studies have documented presence of bacterial contamination of reconstituted infant formulas. It has always been assumed that bottle-feeding poses greater risk to infants. Bottlefed babies are more at risk to developing infections like respiratory, gastrointestinal infections. Bacharach et. al. (2003) in a meta-analysis of seven studies found that infants who were not breastfed were 3.6 times more at risk for hospitalization for lower respiratory infections in the first year of life compared to exclusively breastfed infants. Chien & Howie (2001) showed from a meta-analysis and 14 studies that babies given bottle-feeding or mixed feeding (bottle feeding and breastfeeding) were 2.8 times more likely to develop gastrointestinal illnesses like diarrheas.METHODOLOGYThis is an in vitro study that involves acquiring samples of infant formula milk and expressed human milk to be undertaken at the College of Public Health Microbiology Laboratory. Samples of leftover reconstituted powdered infant formula, newly reconstituted powdered infant milk and expressed human breast milk will be exposed at different temperatures and time intervals. Samples will be obtained from one volunteer mother and her infant aged 3-6 months accustomed to infant formula feeding and another volunteer mother who is currently breastfeeding. Room temperature will be measured as well as refrigerator temperature, which will be set between 2 to 5 C.I. Preparation and sterilization of powdered infant formula milk bottlesSix new feeding bottles will be cleaned, sterilized, and used prior to the study proper. This will follow the recommendations as promoted by the WHO (2007).1. Clean and disinfect the surface on which the feed will be prepared using detergent and bleach.2. Wash hands with soap and water, and dry using a clean cloth or a single-use napkin.3. Boil a sufficient volume of safe water. If using an automatic kettle, wait until the kettle switches off; otherwise make sure that the water comes to a rolling boil. Note: bottled water is not sterile and must be boiled before use. Microwaves should never be used in the preparation of PIF as uneven heating may result in 'hot spots' that can scald the infant's mouth.II. Elimination of confounding variables such as contamination from the can of milk used and the bacteria present in the air of the laboratorySwab technique for presence or absence of bacteria in infant formula milk can. Swab samples will also be collected from the can of milk to be used. The swab samples will be collected in sterile saline and then further analyzed. Both total plate and coliform counts of the swabs will be determined.Airborne bacterial counts for presence or absence of air-borne bacteria in the laboratory. A total of twenty (20) samples for bacterial count in air will be collected by exposing nutrient agar plates inside the laboratory for 10 minutes. The lid of the petri plates will be covered and incubated for 24 hours in an incubator at a temperature of 37C to study the bacterial count and airborne emission to the immediate environment.III. Reconstitution of powdered infant formula milkThis will follow the recommendations as promoted by the WHO (2007).1. Taking care to avoid scalds, pour the appropriate amount of boiled water that has been allowed to cool to no less than 70 C, into a cleaned and sterilized feeding cup or bottle. To achieve this temperature, the water should be left for no more than 30 minutes after boiling2. To the water, add the exact amount of formula as instructed on the label. Adding more or less powder than instructed could make infants ill.a. If using bottles: assemble the cleaned and sterilized parts of the bottle according to the manufacturer's instructions. Shake or swirl gently until the contents are mixed thoroughly, taking care to avoid scalds.b. If using feeding cups: mix thoroughly by stirring with a cleaned and sterilized spoon, taking care to avoid scalds.3. Immediately after preparation, quickly cool feeds to feeding temperature by holding the bottle or feeding cup under running tap water, or by placing in a container of cold or iced water. Ensure that the level of the cooling water is below the top of the feeding cup or the lid of the bottle.4. Dry the outside of the feeding cup or bottle with a clean or disposable cloth. 8. Because very hot water has been used to prepare the feed, it is essential that the feeding temperature is checked before feeding in order to avoid scalding the infant's mouth. If necessary, continue cooling as outlined in step 3.IV. Leftover infant formula milk sampleTwo feeding bottles previously sterilized in accordance with the recommendation of WHO will be used. Powdered infant formula from a newly opened can of milk will be used. The infant formula will be prepared based on the standard dilution set forth in the label. Eight (8) ounces of infant formula will be reconstituted and will be given to the baby at room temperature. Once the baby consumes four (4) ounces of infant formula, the sample of the left-over formula will be retrieved. From the 4 ounces obtained, two (2) ounces will be left standing at room temperature in the laboratory while 2 ounces will be refrigerated at 2 to 5 C. Standard refrigerator temperature will be measured using a refrigerator thermometer. Samples under both temperatures will be taken and plated on a nutrient medium at baseline (0) hour, 1 hour, 2 hours, 3 hours and 4 hours.

V. Newly-reconstituted infant formula milk sampleTwo empty feeding bottles previously sterilized in accordance with the recommendation of WHO will be used. Powdered infant formula from the same can of newly opened milk will be used. The researcher will prepare the infant formula based on the standard dilution set forth in the label. Four (4) ounces of infant formula will be reconstituted. Two (2) ounces will be left standing at room temperature in the laboratory while 2 ounces will be refrigerated at 2 to 5 C. Standard refrigerator temperature will be measured using a refrigerator thermometer. Samples under both temperatures will be taken and plated on a nutrient medium at baseline (0) hour, 1 hour, 2 hours, 3 hours and 4 hours.VI. Expressed human breast milk sampleTwo empty cups of previously sterilized in accordance with the recommendation of WHO will be used as containers of expressed human milk. Another volunteer mother will express her breast milk using manual expression inside our just outside the laboratory. It is important to first clean the breast area around the nipple using sterile water prior to expression. Four (4) ounces of expressed human milk will be obtained. Two (2) ounces will be left standing at room temperature in the laboratory while 2 ounces will be refrigerated at 2 to 5 C. Standard refrigerator temperature will be measured using a refrigerator thermometer. Samples under both temperatures will be taken and plated on a nutrient medium at baseline (0) hour, 1 hour, 2 hours, 3 hours and 4 hours.

VII. Microbiological analysis of all samplesMethylene Blue Reduction Test (MBRT) for presence or absence of bacteria. One (1) mL of methylene blue will be added to ten (10) mL of each milk sample in a twenty 20 mL test tube, shaken. The test tubes will then be incubated at 37 C in a hot water bath for 30 minutes and the change in color will be carefully observed.Total plate count for calculation of aerobic mesophilic counts. For enumerations as per ICMSF (1986) will be used. Samples will be serially diluted in peptone water and the appropriate dilutions will be plated on plate count or nutrient agar using the spread plate method. The plates will then be incubated at 37 C for 24 hours for aerobic mesophilic counts.Coliform counts for calculation of coliform colony counts. For enumeration of coliforms procedure described in the Bureau of Indian Standards: 5401Part I (2002) will be used. The formula milk samples will be serially diluted in peptone water and the appropriate dilutions will be plated on MacConkeys agar using the spread plate method. The plates will then be incubated at 37 C for 24 hours for coliform counts.

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