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RESEARCH

esearch and Professional Briefs

entralized Infant Formula Preparation Room inhe Neonatal Intensive Care Unit Reducesncidence of Microbial Contamination

AROLINE STEELE, MS, RD, CSP; ROBERT SHORT, PhD

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BSTRACTncreasing concern regarding potential pathogens in in-ant formulas led to this evaluation of the influence ofrocedural and environmental factors on infant formulaontamination. In two phases of study, a total of 526ormula samples were analyzed for contamination. Inhase 1, conducted from October 2001 through May 2002,ursing staff prepared formulas in the neonatal intensiveare unit at bedside; in Phase 2, conducted from February006 through June 2006, dietetic technicians preparedormulas in a centralized feeding preparation room. Twoypes of formula, ready-to-feed and powder, were sam-led. Each sample was divided into two portions; the firstas cultured immediately, and the second after 8 hourst room temperature. Multivariate binary logistic re-ression models were tested to identify the major factorsontributing to contamination. Results showed formulasrepared at bedside were 24 times more likely to showontamination than those prepared in a central locationP�0.001) and that powdered formulas were 14 times

ore likely to be contaminated than ready-to-feed formu-as (P�0.001). In addition, samples that had beenarmed (P�0.050) and those that were either milk-basedr casein hydrolysate (P�0.001) were more likely to beontaminated. This study suggests that centralized feed-ng preparation results in a significant decrease in prev-lence of microbial growth. Because contamination riskncreases significantly with the use of powdered formu-as, sterile liquid formulas should be considered when

. Steele is a neonatal academic specialist, Mead John-on Nutritionals, Spokane, WA; at the time of the study,he was a neonatal intensive care dietitian, Sacredeart Children’s Hospital, Spokane, WA. R. Short isssistant director, Washington Institute for Mentalesearch and Training, Washington State University,pokane.Address correspondence to: Caroline Steele, MS, RD,SP, 1314 S. Grand Blvd #2 PMB 123, Spokane, WA9202. E-mail: caroline.steele@bms.comManuscript accepted: April 11, 2008.Copyright © 2008 by the American Dietetic

ssociation.0002-8223/08/10810-0012$34.00/0

wdoi: 10.1016/j.jada.2008.07.009

700 Journal of the AMERICAN DIETETIC ASSOCIATION

ossible to minimize risk of microbial exposure in theeonatal intensive care unit population. Am Diet Assoc. 2008;108:1700-1703.

n 2000, the Joint Commission started suggesting theuse of the Hazard Analysis Critical Control Pointguidelines regarding enteral feedings (1), and many

nstitutions explored using these guidelines specificallyor the preparation of infant and pediatric feedings (2,3).ecause the Food and Drug Administration and the Foodnd Agriculture Organization/World Health Organiza-ion recognize that powdered infant formulas are notterile (4), they currently recommend that powdered in-ant formulas not be used in neonatal intensive care unitNICU) settings unless no other nutritionally acceptablelternative is available (5-7). Ready-to-feed (RTF) andiquid concentrate versions of infant formulas are sterile.remature infants or ill, full-term infants in the NICU,owever, often have specialized nutritional needs thatay necessitate the use of powdered human milk fortifi-

rs or powdered formulas.According to the Infant Formula Act of 1980, bacterial

ounts of up to 10,000 colony-forming units (cfu) per gramf powder at the time of manufacture are consideredcceptable (8). Although current standards likely reducehe number of infections, they do not eliminate the risk,s shown by outbreaks caused by powdered formulas thateet current guidelines (4). No specific guidelines with

egard to acceptable bacterial counts in infant formulaseyond the manufacturing guidelines exist; however, pro-osed standards have suggested goals for no more than00 cfu/mL before feeding administration and no morehan 1,000 cfu/mL at the end of an enteral infusion (9).umerous studies have shown levels of enteral formula

ontamination that exceed these levels (9-13).One method suggested to reduce contamination rates is

he use of a centralized infant feeding preparation room14). The primary objective of this study was to comparehe incidence of infant formula contamination in theormer NICU location, in which feedings were preparedy nursing staff at bedside, with the incidence of contam-nation when feedings were prepared by nutrition staff in centralized feeding preparation area. The secondarybjective was to evaluate whether product form (RTF vsowdered formula) had a significant impact on con-amination. Also evaluated were additional factors thatould affect microbial growth, such as type of formula,

hether a formula was freshly prepared vs previously

© 2008 by the American Dietetic Association

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repared and refrigerated, hang times for continuouseedings, and the warming of infant formulas.

ETHODStudy Designn two phases of the study, 526 formula samples were an-lyzed for contamination. To determine presence of anyorm of microbial contamination, the NICU registered die-itian (RD) collected 5-mL formula samples via sterile vialnd syringe and delivered these samples within 5 minuteso the Sacred Heart Medical Center Microbiology Labora-ory. Microbiology staff then plated 0.01 mL of each formulaample within 10 minutes of delivery onto Trypticase Soygar (BD Biosciences, Sparks, MD) with sheep blood and

ncubated it in a 4% carbon dioxide atmosphere for 48 hourst 35°C (95°F). Colony forming units per milliliter werealculated based on the volume of formula plated. Becausef the concern that any level of microbial contaminationoses a risk for the neonate, contamination was defined ashe presence of any microbes.

hase 1—Bedside Preparationuring the first phase of the study, formula samples were

ollected between October 2001 and May 2002 by theICU RD. All formulas were prepared by nursing staff atedside. Staff were unaware of the study so that prepa-ation technique would not be influenced. Formula sam-les were designated as RTF or powdered formula, and asilk-based (including full-term standard and full-term

actose-free, preterm, and transitional formulas), soy-ased, or casein hydrolysate formulas. The formulas sam-led were randomly selected based on what was beingrepared on the day of collection; the goal was to obtain ainimum of 500 samples with a distribution consistentith the typical formula usage in the unit. Formula mix-

ures containing modular components were excluded.Three additional variables were studied. The first washether the sample was freshly opened (RTF) or freshlyrepared (powdered formula) vs previously opened/pre-ared and refrigerated at 2°C to 4°C (35°F to 40°F) foress than 24 hours.

Freshly opened/prepared formulas were obtained im-ediately after preparation on day-shift due to timeeeded for the microbiology lab to culture the sample.ormulas were opened/prepared within the previous 24ours per unit protocol; such formulas may have beenpened/prepared by either day-shift or night-shift nurses.The second variable was hang-time at room tempera-

ure. Each sample was divided into two smaller samples.he first sample was taken to the microbiology laboratory

or immediate inoculation into culture media. The secondas left at room temperature for 8 hours then taken to

he microbiology laboratory to be cultured.The final factor evaluated was whether the sample wasarmed. Approximately 40% of the formula samples werearmed per unit practice by placing the bottle of formula

nto a cup of 82.2°C (180°F) hot water for fewer than 5inutes.

hase 2—Centralized Preparation Roomuring the second phase of the study, formula samples

ere collected between February 2006 and June 2006 by t

he NICU RD. All formulas were prepared by associate oraccalaureate degree–prepared dietetic technicians perutrition center protocols, based on American Dieteticssociation guidelines (14). All of the formula samplesere matched to the samples in Phase 1 with regard to

ormula form and type, freshly vs previously prepared,nd warmed or not . As with Phase 1, each sample wasivided into two, with one sample cultured immediatelynd the other cultured after 8 hours at room temperature.

hase 2 Subseturing the second phase of the study, the NICU imple-ented additional policies and procedures to reduce the

isk of contamination. New formula recipes were devel-ped to maximize the use of sterile RTF and sterile liquidoncentrate and reduce the use of powdered formula. AllTF, liquid concentrate, and sterile water used to pre-are formulas were refrigerated at 2°C to 4°C (35°F to0°F) before use to reduce the length of time to chill anished product to proper storage temperatures. To eval-ate the effectiveness of these measures, an additionalubset of 40 samples, including milk-based, soy-based,nd casein hydrolysate formulas, was obtained and cul-ured. Each sample was divided into two portions, withne sample cultured immediately and the other after 8ours at room temperature. These samples were not in-luded in the statistical analysis due to insufficient powero detect statistical differences in bacterial counts.

tatistical Methodsmultivariate binary logistic regression was used to iden-

ify variables with a significant impact on microbial growth,hile controlling for other variables. �2 tests were usedhen contrasting two conditions. A P value �0.05 was con-

idered statistically significant. A sample size of 526 wasufficient to detect a 7% difference in bacterial counts withpower of 80%. All statistical tests were computed using

PSS version 14.0 (2005, SPSS, Chicago, IL).

ESULTS AND DISCUSSIONo assess the influence of procedural and environmen-al factors on formula contamination, this study eval-ated infant formulas prepared at bedside vs thoserepared in a centralized feeding preparation area. Of26 samples, 79 were contaminated (Table 1). Thereatest risk for contamination of infant formulas wasedside preparation. Formulas prepared at bedsideere 24 times more likely to be contaminated than

hose prepared in the centralized feeding preparationoom (Table 2; P�0.001; odds ratio [OR]�24; 95% con-dence interval [CI]�10, 58). Among the powdered for-ula samples mixed at bedside, 43.7% (n�66) showed

ontamination, whereas only 4% (n�6) of samplesixed in the centralized preparation room showed con-

amination (Table 1). All contaminated samples con-ained less than 1,000 cfu/mL.

The second most prominent risk factor for contamina-ion was the use of powdered formula. Powdered formulasere 14 times more likely to exhibit contamination thanTF formulas (Table 2; P�0.001; OR�14; 95% CI�6

o 33). RTF formulas had minimal levels of contamina-

October 2008 ● Journal of the AMERICAN DIETETIC ASSOCIATION 1701

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ion, with 93.8% of 112 samples in Phase 1 having norowth and 100% of 112 samples in Phase 2 having norowth (�2�7.23, P�0.007).Our data did not support the concept that freshly pre-

ared formulas would have lower levels of contaminationhan those prepared in advance and refrigerated. No de-ectable differences were found between freshly prepared

Table 1. Number of samples and the frequency of contamination foof preparation (zero hour) and after 8 hours at room temperature

Phase 1: PreparN�112a;

Zero Hour

n c

Ready-to-Feed FormulasMilk-based, freshly opened 23 1Milk-based, opened/refrigerated 17 1Soy-based, freshly opened 6 1Soy-based, opened/refrigerated 1 0Casein hydrolysate, freshly opened 7 1Casein hydrolysate, opened/refrigerated 2 0Total 56 4

Phase 1: PrepaN�151

Zero Hour

n c

Powdered FormulasMilk-based, freshly prepared 24 13Milk-based, prepared/refrigerated 28 15Soy-based, freshly prepared 6 0Soy-based, prepared/refrigerated 7 0Casein hydrolysate, freshly prepared 8 1Casein hydrolysate, prepared/refrigerated 3 0Total 76 29

an�total number of samples.bc�number of contaminated samples.

Table 2. Results of multivariate binary logistic regression analysis othe incidence of microbial growth, expressed as odds ratios with th

Phase 1 (n�263) vs Phase 2 (n�263)Powder (n�302) vs RTFb (n�224)Combined milk-based/casein hydrolysate (n�446) vs soy-based (n�

Milk-based (n�366) vs soy-based (n�80)Casein hydrolysate (n�80) vs soy-based (n�80)

Fresh (n�296) vs previously prepared (n�230)Zero (n�264) vs 8 hours at room temperature (n�262)Warmed (n�202) vs not warmed (n�324)

aCI�confidence interval.bRTF�ready-to-feed.

r previously prepared formulas (Table 2; P�0.869). w

702 October 2008 Volume 108 Number 10

ang-time at room temperature was not a factor in in-uencing incidence of contamination (P�0.159). For pow-ered formulas prepared at bedside, warming the for-ula contributed to the incidence of contamination; thosearmed were more likely to be contaminated (P�0.050;R�1.8; CI 95%�1.0-3.3). However, the difference wasot dramatic, with 35 of the 66 contaminated samples

y-to-feed and powdered formulas under various conditions at time

Bedside (totall c�7b)

Phase 2: Centralized PreparationRoom (total N�112; total c�0)

8 Hours Zero Hour 8 Hours

n c n c n c

23 1 23 0 23 017 0 17 0 17 06 0 6 0 6 01 0 1 0 1 07 2 7 0 7 02 0 2 0 2 0

56 3 56 0 56 0

t Bedside (totall c�66)

Phase 2: Centralized PreparationRoom (total N�151; total c�6)

8 Hours Zero Hour 8 Hours

n c n c n c

24 15 24 1 24 127 16 28 1 27 16 0 6 1 6 07 1 7 0 7 08 3 8 0 8 13 2 3 0 3 0

75 37 76 3 75 3

samples assessing the independent influence of each condition on% confidence intervals and their associated probability values

Odds ratio

95% CIa

P valueLower Upper

23.8 9.8 58.1 �0.00114.2 6.1 32.9 �0.0019.6 2.7 32.5 �0.001

10.3 2.9 36.0 �0.0015.8 1.4 24.4 0.0171.1 0.6 1.9 0.8691.5 0.8 2.8 0.1591.8 1.0 3.3 0.050

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Milk-based and casein hydrolysate formulas combinedboth of which originate with a cow’s milk protein) were 9imes more likely to be contaminated than soy-basedormulas (P�0.001; OR�9.4; CI 95%�3.7�32.5). Phase 2emonstrated similar findings: five of six contaminatedamples were milk-based or casein hydrolysate (Table 1).A subset of an additional 40 samples in Phase 2 was

nalyzed to evaluate the effectiveness of preparing for-ulas using sterile liquids that had been refrigerated

head of time at 2°C to 4°C (35°F to 40° F). Under theseonditions, there was no incidence of contamination, sug-esting that these measures could help further reduceontamination risk, particularly if chilled sterile liquidoncentrates are substituted for nonsterile powdered for-ulas. Indeed, the fact that there was no contaminationhen RTF or other sterile liquids were used in Phase 2

compared with 4% when powders were used) suggestshat using chilled sterile liquids continues to be a reason-ble precaution whenever possible.The results of this study suggest that centralized feed-

ng preparation significantly decreases in the incidence oficrobial growth, as does the use of sterile liquid formu-

as. It is therefore prudent to minimize the use of pow-ered formulas in the NICU setting, particularly whenormulas are prepared at bedside.

Organisms cultured were either gram-positive cocci orram-positive rods with the majority (68 of 79 samples)eing gram-positive rods (bacillus). Although these or-anisms themselves are not pathogenic, the introductionf any microorganism to the neonate may be of concern.nfections and/or sepsis have been linked to several or-anisms, including gram-positive rods such as varieties ofacillus (15-18).One potential limitation of this study was that the dataere collected 5 years apart between Phase 1 and Phase 2.owever, the staff members directly involved in the

tudy remained consistent during the entire study time-rame, and the same nutritional products were used toinimize any potential impact on study results. Another

otential limitation was that many of the formulas pre-ared were not actually fed to infants; therefore, inci-ence of infections and other medical complications couldot be correlated with formula contamination rates. It isossible that the sample size may have limited the abilityo detect smaller differences between groups with regardo the three minor variables studied (fresh vs previouslyrepared, hang-time at room temperature, and warmingf feedings). However, the two primary objectives of thistudy were to compare the incidence of contaminationased on preparation location and product form; for bothf these variables, there was adequate power to detecttatistically significant differences between groups.The parameters used in this study were unique. Stud-

es investigating both preparation location and formulaype, as well as the influence of other environmentalactors, are lacking in the literature.

ONCLUSIONShe findings of this study suggest that the primary factor

eading to contamination of infant feedings is the locationf the initial preparation. The use of a centralized for-

ula preparation room in this study decreased the inci-

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ence of contamination. Because contamination may re-ult in illness or be life-threatening for the neonate, it isrudent to enact measures to reduce this risk. The use ofhilled sterile liquids instead of formula powders is an-ther such measure to reduce risk. Because of the higherncidence of contamination for powdered formulas com-ared with sterile liquids, particularly when prepared atedside, limiting the use of powdered formulas amongulnerable populations seems to be a reasonable precau-ion. According to Robbins and Becker, “The objectivesre to limit the entry of bacteria and microorganisms interile feedings and to limit microorganism growth fromon-sterile PF [powdered formula] during the assemblynd delivery of enteral feedings” (14). Health care provid-rs need to take every precaution to protect this vulner-ble population against exposure to pathogens in infanteedings that could result in more serious health problems.

his research was supported in part by a research grantrom Mead Johnson Nutritionals, Evansville, IN.

eferences1. Becker DS. Interdisciplinary HACCP in acute care. Management in

Food and Nutrition Systems. 2000:4.2. Henry BW, Nelms J, Foley S. Implementing a HACCP plan with

pediatric formula preparation. PNPG Post. 2000;Fall:4-5.3. Hunter PR. Application of Hazard Analysis Critical Control Point

(HACCP) to the handling of expressed breast milk on a neonatal unit.J Hosp Infect. 1991;17:139-146.

4. World Health Organization. Enterobacter sakazakii and other micro-organisms in powdered infant formula. World Health OrganizationWeb site. http://www.who.int/entity/foodsafety/publications. AccessedJune 7, 2007.

5. Mohan Nair MK, Venkitanarayanan KS. Cloning and sequencing ofthe ompA gene of Enterobacter sakazakii and development of anompA-targeted PCR for rapid detection of Enterobacter sakazakii ininfant formula. Appl Environ Microbiol. 2006;72:2539-2546.

6. Seo KH, Brackett RE. Rapid, specific detection of Enterobacter saka-zakii in infant formula using a real-time PCR assay. J Food Prot.2005;68:59-63.

7. Iversen C, Lane M, Forsythe SJ. The growth profile, thermotoleranceand biofilm formation of Enterobacter sakazakii grown in infant for-mula milk. Lett Appl Microbiol. 2004;38:378-382.

8. Baker RD. Infant formula safety. Pediatrics. 2002;110:833-835.9. Campbell SM. Preventing Microbial Contamination of Enteral Formu-

las and Delivery Systems. Columbus, OH: Ross Products; 2000.0. Schreiner RL, Eitzen H, Gfell MA, Kress S, Gresham EL, French M,

Myoe L. Environmental contamination of continuous drip feedings.Pediatrics. 1979;63:232-237.

1. Anderson KR, Norris DJ, Godfrey LB, Avent CK, Butterworth CE Jr.Bacterial contamination of tube-feeding formulas. JPEN J ParenterEnteral Nutr. 1984;8:673-678.

2. Patchell CJ, Anderton A, MacDonald A, George RH, Booth IW. Bac-terial contamination of enteral feeds. Arch Dis Child. 1994;70:327-330.

3. Freedland CP, Roller RD, Wolfe BM, Flynn NM. Microbial contami-nation of continuous drip feedings. JPEN J Parenter Enteral Nutr.1989;13:18-22.

4. Robbins ST, Becker LT. Infant Feedings: Guidelines for Preparation ofFormula and Breastmilk in Health Care Facilities. Chicago, IL: Amer-ican Dietetic Association, 2003.

5. Hilliard NJ, Schelonka RL, Waites KB. Bacillus cereus bacteremia ina preterm neonate. J Clin Microbiol. 2003;41:3441-3444.

6. Adler A, Gottesman G, Dolfin T, Arnon S, Regev R, Bauer S, Litmano-vitz I. Bacillus species sepsis in the neonatal intensive care unit.J Infect. 2005;51:390-395.

7. Ko KS, Oh WS, Lee MY, Lee JH, Lee H, Peck KR, Lee NY, Song JH.Bacillus infantis sp. Nov. and Bacillus indriensis sp. Nov., isolatedfrom a patient with neonatal sepsis. Int J Syst Evol Microbiol. 2006;56:2541-2544.

8. Boyle RJ, Robins-Browne RM, Tang MLK. Probiotic use in clinicalpractice: What are the risks? Am J Clin Nutr. 2006;83:1256-1264.

October 2008 ● Journal of the AMERICAN DIETETIC ASSOCIATION 1703