Frequent Standardized Oral Care Using Human Milk to Prevent Oral Dysbiosis and Improve Health Outcomes in Premature Infants in the Neonatal Intensive Care Unit
Phase: NA
Conditions: Ventilator Associated Pneumonia, Bronchopulmonary Dysplasia, Respiratory Disease
Sponsor: University of Florida

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Frequent Standardized Oral Care to Improve 
Health Outcomes in Premature Infants in the 
Neonatal Intensive Care Unit 
 
 
NCT # 06000761 
 
8/23/2023 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Protocol 
 
 
1. Project Title: Frequent Standardized Oral Care to Improve Health Outcomes in Premature Infants in the                       
Neonatal Intensive Care Unit 
 
2. Principal Investigators:  Leslie Parker PhD 
                  
3. Abstract:  
Premature very low birth weight (VLBW) infants are susceptible to complications related to infrequent and 
non-standardized oral care. Although the benefits of frequent standardized oral care are known to reduce oral 
dysbiosis (increased level of potentially pathogenic bacteria) and its associated complications in critically ill 
adults leading to established evidence-based guidelines, no such information exists for VLBW infants. 
Premature VLBW infants are highly susceptible to costly, life threatening and potentially preventable 
morbidities, such as ventilator associated pneumonia (VAP), bronchopulmonary dysplasia (BPD; oxygen 
requirement at 28 days of life), and need for prolonged respiratory support which require additional 
treatments, increase cost of care, and can lead to chronic illness, re-hospitalization, and developmental 
delay.1,2 A dearth of information exists regarding oral care in VLBW infants and no such guidelines exist for infants 
admitted to the neonatal intensive care unit (NICU) which may negatively affect their health. Thus, research 
regarding the effect of frequent, standardized oral care on the health of VLBW infants is essential to develop 
guidelines thus potentially improving the health of this vulnerable population. If successful, this research could 
change practice in NICUs across the nation.   
 
4.     Background: 
VLBW infants are at significant risk of adverse health outcomes including ventilator associated pneumonia, 
bronchopulmonary dysplasia, and prolonged respiratory support which increase costs and are associated with 
chronic illness, re-hospitalization, and developmental delay. The oral cavity of patients admitted to intensive care 
units contains high levels of pathogenic bacteria which can be aspirated, inhaled and invade the systemic, 
respiratory and gastrointestinal systems increasing the risk of adverse health outcomes. Extensive research 
regarding the benefits of oral care in critically ill adults has led to nationally recognized evidence-based guidelines.   
In critically ill adults, evidence based guidelines for oral care are well established, known to reduce 
complications, and are included in the Institute for Healthcare Improvement’s Five Million Lives Campaign.3 
However, in critically ill infants admitted to the neonatal intensive care unit (NICU), no such guidelines exist 
and there is a dearth of information regarding oral care for preterm VLBW infants who due to their suppressed 
immunity and physiologic immaturity are at increased risk of morbidity due to infrequent and non-
standardized oral care. However, in critically ill infants admitted to the neonatal intensive care unit (NICU), no 
such guidelines exist and there is a dearth of information regarding oral care for preterm VLBW infants who 
due to their suppressed immunity and physiologic immaturity are at increased risk of morbidity due to 
infrequent and non-standardized oral care.  
Lack of frequent, standardized oral care can result in abnormal levels of potentially pathogenic bacteria in the 
oral cavity including the cheeks, tongue, lips, and palate. When admitted to the intensive care unit, the oral 
cavity of adults become colonized with pathogenic bacteria within 24 hours4 and oral flora changes from gram 
positive (normal) to gram negative (potentially pathogenic) within 48 hours of ventilation.5 In premature 
infants admitted to the NICU, pathogenic gram-negative bacteria increase significantly in the oral cavity from 
day 1 to 10.6 Premature VLBW infants may be particularly susceptible to abnormal oral cavity colonization due 
to the routine placement of oral devices such as feeding and endotracheal tubes which are known to acquire 
biofilm and to alter the oral flora.7 In addition, the NICU environment contains abnormal and pathogenic 

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bacteria that can enter the infant’s oral cavity during routine care. Finally, due to prematurity and physiologic 
instability, VLBW infants often have limited maternal contact and are unable to orally feed which interferes 
with healthy oral colonization that normally occurs following birth. 
If potentially pathogenic bacteria are not removed via frequent standardized oral care, it is possible that VLBW 
infants can aspirate or inhale saliva containing high levels of bacteria. In addition, these bacteria could invade 
their systemic circulation, respiratory and gastrointestinal tract through the fragile mucosal barrier thus 
increasing the risk of health problems such as VAP, BPD, and need for prolonged respiratory support. VAP is 
defined as bacterial lung invasion developing after > 48 hours of invasive ventilation.8 VAP accounts for up to 
32.2% of hospital acquired infections in neonates9 and has been shown to prolong length of stay and increase 
cost of care.10 Because the endotracheal tube bypasses the natural upper and lower respiratory track defense 
mechanisms against harmful microorganisms and bacteria laden oral secretions can leak around the tube into 
the airway, the risk of VAP is high. This is especially important in VLBW infants who use un-cuffed 
endotracheal tubes which are more likely to allow entry of oral secretions into the lungs. In adults, the same 
bacteria found in the oral cavity was present in the lungs of ~75% of VAP cases and VAP was preceded by an 
oral culture of the same bacterial species.11 While minimal information exists regarding VAP and the oral 
microbiome in VLBW infants, VAP has been shown to be poly-microbial from primarily gram-negative 
organisms which significantly increase in the oral cavity over the first days after NICU admission.9 Even when 
endotracheal intubation and invasive ventilation are not required, infants likely have micro-aspiration of oral 
secretions. When these secretions contain high levels of potentially pathogenic bacteria, prolonged 
respiratory support may be required. This is important because both invasive and non-invasive respiratory 
support can damage the VLBW infant’s fragile lungs increasing the risk of BPD.12  
To date, researchers do not know whether frequent standardized oral care can decrease the number of 
potentially pathogenic bacteria in the oral cavity of premature VLBW infants. Because frequent standardized 
oral care is known to reduce the amount of oral pathogenic bacteria in adult patients which are known to 
increase the risk of VAP, this is a vital piece of information that may allow NICU clinicians to: (a) reduce the 
amount of oral pathogenic bacteria, (b) reduce VAP, (c) decrease the incidence of BPD, and (d) decrease the 
need for respiratory support.   
In a national survey of 129 neonatal nurses from 35 states, we found that while over 98% stated oral care was 
important for the health of VLBW infants, less than 37% felt their NICU adequately addressed oral care. In 
addition, tremendous variation existed in practices regarding oral care including the method, timing, 
equipment and whether or not oral care was even performed. 
 
5. Specific Aims:  
The long-term goal of this research is to improve short and long-term health outcomes for premature very low 
birth weight (VLBW; <1500 grams) infants by decreasing complications caused by lack of frequent standardized 
oral care. 
Specific aims are as follows: 
Aim 1: To compare the oral microbiome between groups including bacterial pathogenicity determinants. 
Aim 2: To compare respiratory outcomes including the incidence of ventilator associated pneumonia, 
bronchopulmonary dysplasia and need for respiratory support between groups.   
 
 
 

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6. Research Plan:  
 
Sample and Setting:  
The proposed study will prospectively follow 217 VLBW infants and 217 mothers (dyad) for 4 weeks following 
birth (includes 69 previously enrolled mother/infant dyads). Infants will be randomized into 1 of 3 groups. 
Standardized oral care will be performed every 3-4 hours (Group 1) and every 12 hours (Group 2) using sterile 
water. Group 3 will have standardized oral care performed every 3-4 hours using human milk (mother’s breast 
milk or donor breast milk). Mothers of multiples will also be potential subjects. UF NICU nursing staff will receive 
UF Qualtrics survey links to their UF email yearly. Survey responses are voluntary and anonymous and will 
include up to 225 RN responses.   
Infants will be sampled by convenience from the NICU at UF Health Shands Children’s Hospital. Approximately 
150 premature VLBW infants are admitted to this NICU per year. Our consent rate in similar studies is > 60% 
which strongly suggests we will be able to recruit 217 infants and 217 mothers (dyad) over a 12-month period.  
 
The proposed randomized controlled pilot study will follow a prospective cohort (n=217) of racially and 
economically diverse premature VLBW male and female infants following birth. Infants will be randomly 
assigned to one of three groups using a randomized blocks approach as implemented in SAS PROC PLAN. A 
randomized blocks approach will ensure comparable treatment group sizes, and a priori assignment will 
reduce possibility of bias in treatment group assignment. All groups will receive standardized oral care for 4 
weeks. Infants in Group 1 will have standardized oral care performed every 3-4 hours with sterile water 
(dependent on scheduled care times), infants in Group 2 will have standardized oral care performed every 12 
hours with sterile water, and Group 3 will have standardized oral care performed every 3-4 hours (dependent 
on scheduled care times) with human milk. Groups were chosen based upon recommendations for adults 
(every 3-4 hours) and data regarding common practices of neonatal nurses obtained from our national survey 
(every 12 hours) and the benefits of placing drops of human milk into the oral cavity of preterm infants. 
 
Inclusion criteria: 1) mothers who are > 18 years of age, 2) infant (s), including multiples, born at < 30 weeks, 3) 
infant with birth weight at < 1500 grams 
 
Exclusion criteria: 1) infants with born congenital anomalies of the face, lungs, or gastrointestinal system 2) 
infant not expected to live > 7 days following delivery  
Informed consent will be will be obtained from the mother or father within 72 hours after birth at the infant’s 
bedside or in the mother’s hospital room.   
 
Procedure:  
Oral care will be performed in infants in Group 1 (every 3-4 hours) using sterile water, Group 2 (every 12 
hours) using sterile water and Group 3 (every 3-4 hours) using human milk (if mother’s breast milk, not 
available, donor breast milk will be used). The following procedure and a checklist to ensure treatment fidelity. 
Equipment will be easily accessible and provided in sterile pre-packaged kits at the infant’s bedside. Study 
personnel involved with subjects will be unable to be blinded since information regarding oral care is visually 
obvious and recorded in the chart by nursing staff. However, those performing analysis of saliva and collecting 
data on respiratory outcomes will be blinded to the infant’s designated group. Feeding tubes will be changed 
no less than every 7 days. 
 

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1. Using a sponge-tipped swab saturated with sterile water or human milk, the bedside nurse or research 
coordinator (with extensive NICU nursing experience) will clean all 4 quadrants of the gum surfaces 
and upper posterior part of the oropharynx for 15 seconds each.  
2. Using a new sterile water or human milk saturated swab, they will clean the ventral and posterior 
surfaces of the tongue for 15 seconds.  
3. Using a new sterile water or human milk saturated swab, they will gently clean the outer surface of the 
endotracheal tube (if present), feeding tube or other oral tubes.   
4. The lips will be cleaned with a sterile gauze saturated with sterile water or human milk. 
5. The oral cavity will be suctioned as needed with an oral suction devise to remove secretions (oral 
suction devises are changed every 24 hours per NICU protocol). 
6. After the above oral care is complete,  0.1 mL of mom’s breast milk will be placed into each buccal 
cavity (if available) until reaching day of life #7, if infant meets the NICU policy of a birth weight of < 
1250 grams. Orally administered breast milk is routine nursing care for premature infants in this NICU 
and may interact with the oral lymphoid tissue to help support immune system development.15  
 
Aim 1 will determine the effect of standardized frequent oral care using human milk on the oral microbiome 
including bacterial pathogenicity determinants in premature VLBW infants. We hypothesize that standardized 
oral care performed every 3-4 hours using human milk will be associated with less dysbiosis than standardized 
oral care every 3-4 hours or 12 hours using sterile water. A sample of the infant’s saliva will be collected 
weekly for 4 weeks by the research coordinator (a registered nurse experienced in the care of critically ill 
infants) by gently inserting a sterile brush into the infant’s mouth and gently rotating the swab. Collection will 
occur immediately prior to the infant’s scheduled oral care. Our team is skilled in the collection, processing 
and analysis of infant saliva.  
DNA Extraction & 16S rRNA sequencing. DNA will be extracted from saliva using PowerFecal DNA isolation kit 
(MO BIO Laboratories) and quantified using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, 
DE). V4 universal primers will be used for 16S rRNA amplification and subsequent Illumina MiSeq sequencing. 
Samples will be amplified in 25 μl reactions containing 0.5 Units Phusion High-Fidelity Polymerase (New 
England Biolabs, Ipswich, MA), 1X Phusion HF Reaction Buffer, 0.75 μl DMSO, and 0.2 mM each dNTP. 
Triplicate PCR amplifications will be pooled for each sample, and cleaned with a QIAquick PCR Purification kit 
(Qiagen). 200 nanograms of each cleaned amplicon library will be submitted for sequencing at the ICBR core 
facility at UF. Sequencing will be performed on an Illumina MiSeq with a 300-bp paired-end protocol, using 
single indexing.  
Microbiome 16S rRNA Gene Analysis. Sequencing reads will be parsed by Illumina index at the sequencing 
center. Paired reads will be merged, and primers and adaptors will be removed using a combination of tools in 
cutadapt, Galaxy, and eautils. Sample names will be added to the definition lines of sequencing reads using 
sed and concatenated into one fasta file, to make them compatible for analysis in QIIME v1.9.16 Clustering of 
Operational Taxonomic Units (OTUs) at 97% similarity will be performed with the subsampled open-reference 
OTU picking method,17 with no removal of singletons. The Greengenes reference dataset version 13.8  will be 
used as the reference for OTU picking and for taxonomy assignment with uclust.18 Community structure will 
be analyzed in R with phyloseq and will be plotted with ggplot2. Analysis of similarities (ANOSIM) will be 
performed in R using VEGAN v2.0-8. Statistical significance of QIIME profiles will be assessed using STAMP for 
comparison of two independent groups. TIME will be utilized for analysis of the longitudinal microbiome data. 
These tests are appropriate for analysis between groups and over time.   
Stool samples will be collected at the end of week 1 and  4 by the research nurse. Following collection, all 
samples will be placed in a sterile specimen container and immediately frozen at -80 degrees Celsius for later 
analysis for inflammatory markers and 16S rRNA sequencing. 

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To determine bacteria in the infant’s mouth which may originate from the mother’s breast milk or donor milk, 
a small sample of breast milk (3-5 mL) will be obtained from the infant’s mother (after her consent) at the end 
of week 1 and 4 and 16S rRNA sequencing will be performed. Samples will be obtained from the NICU milk 
room. If mother is pumping < 5mL/, no sample will be obtained but no deviation reported. If donor breast milk 
is used for oral care (mother is not providing breast milk to her infant), 3-5 mL of donor breast milk will be 
obtained and 16s rRNA sequencing performed   
Aim 2: will compare respiratory outcomes including evidence of VAP (positive ETT culture, incidence of 
ventilator associated events), days on invasive and non-invasive respiratory support, and incidence of BPD 
between groups. We hypothesize that infants who undergo standardized oral care every 3-4 hours will have 
less VAP, require fewer days of respiratory support, and have less BPD than Group 2.  Descriptive statistics, 
including confidence intervals, will be generated to describe the distribution of days of respiratory support in 
each group, and measures of effect size (Cohen’s f) will be calculated from the between-subjects (Treatment 
Group, with 3 levels) general linear model (GLM). Incidence of occurrence for VAP and BPD will be described 
for each group using frequencies and percentages along with confidence intervals for the proportions. Effect 
sizes (Cohen’s w) for each of those two outcome variables will be calculated from a 3 (Treatment) by 2 
(Occurrence) contingency table.19  
Information concerning infant demographics and health outcomes will be collected from the infant’s 
electronic health records. Traditionally, VAP has been diagnosed using radiographic evidence of pneumonia. 
However, radiographs have not been shown to be a valid or reliable method of diagnosis especially in 
neonates who often have underlying lung disease making radiographic diagnosis even more difficult. 
Therefore, in accordance with the National Healthcare Safety Network 2021 recommendations for neonatal 
and pediatric patients, VAP will be defined as the incidence of ventilator associated events (VAE).1 VAEs are 
objectively defined events consisting of deterioration in respiratory status after a period of stability or 
improvement in ventilated infants. VAEs occur when a ventilated infant has either an increase in daily 
minimum oxygen requirement of > 0.25 or mean airway pressure of > 4 cm sustained for more than 2 days 
after more than 2 days of stable or decreasing daily minimum oxygen level or mean airway pressure.  
If the infant is ventilated with an endotracheal tube, a weekly (for 4-weeks) culture and gram stains will be 
obtained from the endotracheal tube and sent to the laboratory for analysis to assess for bacteria which may 
be in the infant’s airway. This ETT culture will be obtained with assistance by the infant’s nurse or respiratory 
therapist when suctioning was to occur as part of routine care. The culture and gram stain will be sent to UF 
Shands Laboratory. Furthermore, days on respiratory support (ventilation, continuous positive airway 
pressure, high flow nasal cannula or low flow nasal cannula), incidence of BPD (oxygen requirement at 28 days 
of life), and length of hospital stay will be collected during the infant’s NICU stay. Other clinical information 
which could potentially affect the risk of oral secretion aspiration will be collected including days requiring 
sedation, days of antibiotic therapy, and number of intubations.6  
All de-identified saliva, stool and breast milk samples will be conducted at a UF lab outside of Shands. No EPIC 
charges apply, as these are being directly paid to the lab by research funds. ETT culture samples will also be 
de-identified before sending to Shands CoreLab. There will be no EPIC orders and no results of culture will be 
placed in EPIC. Core lab services are being set up as a direct pay. 
 
A yearly anonymous and voluntary survey will be administered to the NICU nursing staff using a UF Qualtrics 
link sent to their email regarding staff concerns and questions. An IRB approved waiver of consent, will be sent 
with each survey, with an expected response of 225 staff. Yearly Q & A sessions will be scheduled as needed 
for staff questions and/or concerns.  To encourage protocol compliance, nurses will receive quarterly emails or 
flyers will be posted with study updates or information important to study compliance. New nurses will be 

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formally oriented to the study during their orientation period. We have previously successfully used these 
strategies.   
 
Data Entry and Management  
All data will be entered into REDCap by a research assistant and verified by a Research Coordinator. A unique 
identifier will be assigned to each enrolled participant that will serve as the unique subject ID. Personal Health 
Information will not be stored in the study database. The study case report forms (CRFs) will be maintained in 
study specific folders. The CRFs are considered the primary data collection instruments for this study. When 
CRFs are not actively being processed, they will be secured in locked file cabinets. In addition to the use of 
passwords and other security measures, all documents containing identifying information are considered 
confidential materials and are safeguarded to the greatest possible extent. No individually identifying 
information will be released or discussed with anyone other than study staff.   
Table 1. Instruments 
 
Variable 
Measurement 
Timing 
Descriptive (1-2) below 
 
 
1. Maternal, prenatal and 
perinatal demographics 
Data extracted from medical record: Maternal 
history, prenatal and perinatal complications and 
medications, mode of delivery, whether mother 
received antibiotics and/or antenatal steroids  
Collected upon entry into study. 
2. Infant demographics 
Data extracted from medical record: 
Race/ethnicity, sex, gestational age, birth weight, 
Apgar scores, resuscitation at birth and neonatal 
acuity (SNAP II) score 
Collected upon entry into study.  
3. Infant data 
Type and amount of feeding, whether or not the 
infant is NPO, number of days and type of 
antibiotics, whether the infant was diagnosed with 
oral thrush and type of treatment. Length of skin-
to-skin care 
Collect upon entry into study and 
until infant discharge 
Respiratory Factors (1-2)  
 
 
1. Presence of bacteria in 
the infant’s breathing 
tube 
Presence of positive tracheal aspirate on ventilated 
infants 
Weekly for 4 weeks 
2. Respiratory Support 
Days of invasive and non-invasive respiratory 
support. 
Day of birth until NICU discharge 
3. Evidence of pneumonia 
Episodes of radiologic evidence of pneumonia 
Day of birth until NICU discharge 
Analysis of stool (1-2) 
 
 
1. Microbial analysis 
16S rRNA and metagenomics sequencing 
End of week 1 & 4 
2. Inflammatory markers 
Analysis of inflammatory markers 
End of week 1 & 4 
Nutritional outcomes (1-6) below 
 
 
1. Enteral intake  
24-hour enteral feeding intake in mL/kg and 
calories, episodes of emesis, # of oral colostrum 
buccal swabs received 
Day of birth until discharge from 
the NICU.   
2. Time to full feeds 
First day infant received 120 mL/kg/d and 150 
ml/kg/d of feedings 
Recorded daily until 150 mL/kg/d 
reached 
3. Hours of parenteral 
nutrition 
Number of hours infant received some parenteral 
nutrition 
Daily until NICU discharge  

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     4.  Parenteral nutrition 
associated liver disease 
Liver function tests (level of direct bilirubin, 
alkaline phosphatase, AST and ALT) 
Weekly or biweekly until NICU 
discharge (with clinically sent labs) 
 
     5.  Hours of central line access 
Number of hours infant has a central line 
Daily until NICU discharge  
     6.  Growth indices 
Weight, length, and head circumference 
Daily (weight) and weekly (head 
circumference and length) until 
NICU discharge.  
     7.  Length of time feeding and 
endotracheal tubes were in place 
Hours tubes remained in place.  
Daily for 5 weeks.  
Other health outcomes (1-3) below 
 
 
     1.  Episodes of late onset sepsis 
CBC lab results, episodes of culture proven or 
presumed sepsis (treated with >5 days of antibiotics 
but with negative cultures) occurring >3 days after 
birth 
Daily from birth until NICU 
discharge 
     2.  Episodes of necrotizing 
enterocolitis 
Episodes of radiologic or surgical necrotizing 
enterocolitis 
All incidents until NICU discharge  
     3. Discharge summary 
Days infant remains in hospital, hospital course, 
subject diagnosis 
From birth until discharge 
Covariates 
 
 
1. Analysis of maternal breast 
milk (if mom pumping) 
and donor breast milk 
Microbiota and metagenomic sequencing (3-5mls) 
At the end of week 1 and 4  
Oral Conditions 
 
 
1. Bacteria in saliva 
Amount and type of bacteria in the mouth 
Sputum samples weekly for 4 weeks 
2. Examination of mouth  
Assessment of skin integrity and presence of 
erythema, exudate or thrush.  
3x a week for 5 weeks.  
3. Oral diseases and treatment  
Oral medications and disease processes 
From birth to NICU discharge.   
 
7. Possible Discomforts and Risks:  
There may be some mild discomfort for the infant when cleaning the mouth and lips.   
8. Possible Benefits: 
Frequent and standardized oral care may improve the health of VLBW infants by reducing oral dysbiosis and its 
associated complications in critically ill infants.  
9. Conflict of Interest: 
There are no conflicts of interest. 
 
 
 
 

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