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Introduction to Newborn Colonization
Newborn colonization, as it is discussed here, focuses primarily upon the sequence of the
varieties of microbial growth in the intestinal tract. On the periphery of this review exist other
varieties of human microflora which include, but are not limited to, skin, inner and outer ear,
upper GI tract and vaginal bacterial symbionts.1 Because these microbes exist in places where
the benefit is necessarily limited in comparison to the multifunctional inhabitants of the lower GI
tract, most research is adjusted accordingly.2
A mature human microflora population includes more individual bacterium, tenfold more
on average, than a human has cells. At maturity, the makeup of the population is composed of
strict and facultative anaerobes with a transient, minor component of aerobic genera.3 The
function of the microflora can be categorized into three main functionalities. Protection from
potential pathogens is done via production of anti-microbial elements and by maintaining a
healthy biofilm on the intestinal epithelium which disallows adherence and growth of pathogenic
bacteria. Structural benefits include antibody response from stimuli from the microflora and
changes in the tight junctions in the epithelium which cause the “squeezing together” of the skin
cells to protect from potential invading pathogenic bacteria. Metabolic benefits vary from the
consumption of toxic compounds that arise from direct ingestion or secretion from pathogens to
synthesizing vitamins for nutritional uptake by the host human. By individualized metabolic
functionality, bacteria are also able to change non-digestible elements to bio-available
compounds and also aid in the maintenance of a healthy pH. How this equilibrium is attained
from an initial sterile environment that is the newborn gut is the whole of this topic.4
During a vaginal birth, a baby is inoculated with bacteria found in the vaginal canal,
surface skin and feces of the mother. Initial levels of transmitted microbes are quite low, but due
to the sterile, yet hospitable environment of the newborn allows for rapid growth. At first,
aerobic and facultative anaerobic microbes (which makes sense when considering the source of
these pioneer colonizers) are the only species found in the baby. However, within one week the
environment within the gut has become drastically reduced so that strict anaerobes are the
primary member of the growing population. At two years of age the gut microbiota is in essence,
what it will be throughout an individual’s life, barring any major disruption in health or
location.5
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Discussion Concerning: Dynamics of Gut Colonization and Source of Intestinal Flora in
Healthy Newborn Infants6
I. Abstract
The evaluation of the dynamics of gut colonization, so that the primary source of
bacterial flora during the first period of life are identified, via qualitative techniques using
computer aided analysis of cellular fatty acid (CFA) profiles is the aim of this study. Stool was
collected from10 healthy babies during the first 2-7 days after birth, with another sample gained
at 6 months of age. Also, stool samples were collected from the mothers and the nurses involved
in initial care. Gas-liquid chromatography was the method used to produce CFA profiles, which
for the infants fluctuated on an hour to hour basis during the first days of life, resembling the
mother’s and the nurse’s at different times. From these findings, it may be ascertained that the
maternal flora effect on the initial gut colonization is lesser in magnitude than previously
believed.
In adults, the total mass of the intestinal bacterial flora can be up to 1.5 kg and contain
more than 400 species. The colonization of the intestine begins during birth when exposure to
vaginal, fecal and environmental microflora occurs and continues up to 24 months, at which time
a unique and stable microbiota is established. Possible long term repercussions concerning the
colonization by fecal bacteria have been identified. For instance, individuals delivered via
caesarean section have a different gut flora at 6 months of age and subsequent different
immunological responses which place them at a higher predisposition to atopic diseases when
compared to vaginally born infants.
A pathogen acquired at birth or during the neonatal birth, even a single one can increase
the risk factor for future infections. An example which demonstrates this clearly comes from a
survey in which a nosocomial spread of an Escherichia coli strain found to be uropathogenic
increases later susceptibility to UTI through, at least, the first two years of life.
Previous attempts to evaluate gut colonization were done via platelet count analysis of
fecal samples. However, this is inefficient, time consuming and only detects cultivatable
bacteria, which estimates place, at best, 50% of all fecal bacteria. From these analyses the
4. Pamer, 4
approximate rate of time for the colonization of the neonatal gut has been ascertained, but the
limited data available from these techniques have left much of the overall dynamics in the dark.
By determining the bacterial CFA profiles from the infant stool and comparing it with the
potential sources of the bacteria, a complete picture of all microbiota in the gut can be identified,
and from whence they came. CFAs are structural components of all bacteria, and changes or
differences, even slight ones can indicate and identify the composition and diversity of the
microflora over time. A computer program which was previously developed was used for this
study.
II. Methods
Stool Samples
At the Oulu University Hospital in Finland, 10 healthy neonates, on two consecutive days
participated in this study. All were born vaginally and stayed, during the night, in the hospital’s
maternity ward with the assistance of nurses. A total of 159 stool samples were obtained; of
these all 10 infants were represented, 9 of the 10 mothers and 13 of the 19 nurses submitted
samples. In the 6 month follow up only 5 of the 10 infants were available for samples. The
infants stay in the maternity ward ranged from 2-7 days and stool samples were continuously
taken during this time; a single sample was taken from the mothers and the nurses during this
same period. The time of each feeding and each stool sample was recorded.
Baseline Characteristics of the Newborn
The means by which the mothers and infants were selected to enter this study came by a
questionnaire and from the delivery records. No antimicrobials or antibiotics were administered
during labor. All of the newborns were kept with the mother, in the same room during the
follow-up. None of the mothers used probiotics on a regular basis prior to their delivery. All the
mothers, with the exception of one, were fed with a mixture of mother’s milk supplemented with
formula and bovine milk, the one was fed exclusively with mother’s milk.
Analysis of Cellular Fatty Acid Profiles
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Immediately after collection the stool samples were frozen at -24 °C and purified samples
were analyzed by gas-liquid chromatography (GLC) which produced the CFA profiles. This
method and repeatability of this method has been verified previously. The fatty acid profiles
show the composition and diversity of a sample of bacteria, but it does not demonstrate the
amount of bacteria present, only the differences in the sample.
Fig 1. Similarity indices for the CFA profiles for each of the 10 infants of each
of their subsequent stoolsamples are shown. Each dot is a stool sample and each
curve represents one infant. (A) represents a rapid colonization, (B)
demonstrates a slow and fluctuating colonization and (C) is erratic with an
infirmity introduced.
Using the prior mentioned computer aided data analysis program, the stool samples after
comparison were assigned a similarity index between 0-100%, as can be seen in Figure 1. A
100% similarity index would denote complete similarity.
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Statistical Analyses
A regression line was developed for the similarity indices for the infants and their
mothers and was compared with a regression line procured from the data correlating the infants
and their respective nurses. The slope was tested statistically with a null hypothesis that the true
slope is equal to zero. Other statistical tests, all available with the software used for this study,
were used to demonstrate statistical fidelity.
III. Results
During the first days of life, the CFA profiles dramatically fluctuated from hour to hour.
For five of the infants, the greatest differences between samples occurred between 10 – 20 hours
after birth, and in three of the newborns the biggest differences were found to happen 50 – 60
hours after birth and in the
remaining two children, the
fluctuation was highly irregular.
This can be seen in graphical
form in figure 1.
The CFA profiles began
to resemble those of the
mothers and the nurses in the
first week of life. Surprisingly
there was no statistical
difference between the trends of
the mother – infant and nurse –
infant pairs; this can be seen
clearly in Figure 2.
At 6 months, however,
the CFA profiles more closely
resembled those of their
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mother’s by a large margin compared to those of the nurses; this data is presented in Figure 3.
During the time between
the infants stay in the
maternity ward and the
follow up sample taken at
6 months of life, the mean
similarity index had
increased from 51 to 67,
showing that the
resemblance to the
mother’s intestinal
microflora had increased
16 percentage points.
However, the similarity
mean index for the nurses demonstrated an increase from 51 to 62, clearly demonstrating that the
transference of bacteria from the nurse during the first days of life have a lasting impact.
Discussion
The manner in which the study was performed, with the computerized analysis of CFA,
was of unique design. The data gathered from the CFA profiles obtained from the stool samples
allowed determination and identification of all bacterial species (not in name but in relevance to
source of colonizing bacteria) involved in the gut colonization from the infant’s mothers and
nurses. This was the first known study to incorporate the already established methods of GLC to
produce CFA profiles in the assessment of initial gut colonization and the sources of the
colonizers. There are many benefits to using this method instead of the conventional techniques,
the most important being that is a cheap, rapid and culture-independent allowing multiple
comparisons with multiple individuals. The main drawback of this technology comes with the
inability of the CFA to positively identify specific species of bacteria nor the quantity of specific
bacteria; only the diversity. This means that the specific bacterial species that are causative for
the changes in microflora profiles cannot be identified taxonomically.
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IV. Unanswered Questions
Authorial Interrogative
The significant differences between a number of the infants and their succession of
colonization, specifically the timing, in that some of the infants the rapid changes in CFA
profiles occurred markedly later than a majority of the others remains a mystery. There seems to
be, from the data gathered, no differences which would indicate why the timing differs so
dramatically.
Student Interrogative
The mechanisms of how the microbiota gained by the infants from the nurses is clear, in
that, in the maternity ward the nurses handle the newborns frequently and skin on skin contact
along with items that may end up in the infant’s mouth occur with abundance. However, the fact
that the similarity indices increase for the mother and the nurse with the infants at 6 months,
even though the change is larger for the mother, is peculiar to me. The reason for this is not
specifically discussed in this paper.
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TvetenY, KristiansenBE,AskE,JenkinsA,&Hofstad T. (1991). DNA fingerprintingof isolatesof
Staphylococcusaureusfromnewbornsandtheircontacts.Journal of Clinical Microbiology.29
(6),1100-5.
3
BezirtzoglouE.(1997). The intestinalmicrofloraduringthe firstweeksof life.Anaerobe.3(2-3).
4
Morelli L. (2008). Postnatal developmentof intestinal microfloraasinfluencedbyinfantnutrition.The
Journal of Nutrition.138 (9).
5
Are A,AronssonL,Wang S, GreiciusG, Lee YK, GustafssonJA,etal.(2008). Enterococcusfaecalisfrom
newbornbabiesregulateendogenousPPARgammaactivityandIL-10levelsincolonicepithelial
cells.Proceedingsof the National Academyof Sciencesof the UnitedStatesof America.105 (6),
1943-8.
6
Tapiainen,T.,Ylitalo,S.,Eerola,E.,& Uhari,M. (2006). Dynamicsof gut colonizationandsource of
intestinal florainhealthynewborninfants.APMIS.114 (11), 812-817.
