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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.
Hypotheses
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.
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 Terms
Bacterial 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 LITERATURE
Breastfeeding 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
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
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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.
METHODOLOGY
This 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
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II.
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
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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.
IV.
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V.
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.
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VI.
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.
(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
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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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