Consequences of early-life antibiotic exposure on antimicrobial gene selection: what regimen causes least harm?

Multidrug-resistant bacteria are becoming more prevalent and hamper antibiotic
treatment options. Antibiotic pressure has shown to induce selection of
antimicrobial resistance (AMR) genes within the human microbiome. Especially
the developing microbiome of neonates is prone to antibiotic perturbation.
Therefore, we hypothesize that antibiotic use in neonates will cause
significant selection of AMR genes and outgrowth of resistant microbes within
the human microbiome with long-term consequences for microbiome composition and
the reservoir of AMR genes within the child*s microbiome.

Since suspected neonatal infections requiring broad-spectrum antibiotic
treatment are very common (approx. 10% of all newborns), and do not make any
distinction between gender, race or socio-economic status, potential
side-effects on microbiome development and AMR gene selection and persistence
will have significant impact on society in general. Better insight into those
side effects will directly help us to tailor therapy to reduce those side
effects in early life and optimize chances for a healthy microbiome development
for those children over time. Because of the extend of antibiotic use in early
life, tailoring therapy to reduce AMR gene selection and persistence may have
major effects on community-wide spread of AMR genes as well. Therefore, the
potential impact of this study is expected to be high.

Primary Objective:
1) To assess the short- and long-term prevalence of AMR genes in the microbiome
of the gut in infants exposed to broad-spectrum antibiotics in the first week
of life compared to controls.
2) To investigate the short- and long-term effects of broad-spectrum
antibiotics administered in the first week of life on the composition of the
microbiome of the gut in the first 5 year of life compared to controls.

Secondary Objective(s):
To identify which of the recommended and generally used broad-spectrum
antibiotic regimens for treating neonatal infections induces least AMR gene
selection and disturbance of the microbial composition as compared to
non-treated control neonates.

We propose to execute a randomised study to guarantee comparability between
groups. In this study we wish to include 147 vaginally born infants or infants
born by emergency caesarean section with suspected early onset neonatal
infection, who need empirical antibiotic treatment in the first week of life.
Collaborating hospitals are the Spaarne Gasthuis in Hoofddorp and Haarlem Zuid,
Diakonessenhuis Utrecht and Tergooiziekenhuis Blaricum. Upon presentation of
the sick neonate in the hospital, parents will be addressed for participation
and receive study information by the responsible physician to explain the
objectives and impact of the study.

In their 5th year of life (before turning 5 years), two groups of ZEBRA study
participants will be approached to participate in a one-off intensified sample
and data collection moment at age 5 named the *ZEBRA-5 protocol*:
1. Participants (both active and drop out) of the follow up study.
2. ZEBRA participants who did not give informed consent for the follow up study
but have given permission to contact them.

A control group will be composed of 49 vaginally born infants or infants born
by emergency caesarean section who have been recruited prenatally and followed
in parallel through home-visits, receiving no antibiotics in the first week of
life. Ethical approval for the follow-up protocol and one-off intensified
sample and data collection moment at the age of 5 years has been obtained as
part of an observational microbiome development study (MUIS study, study
number: M012-015) which is currently operable and so far, highly successful.

Participation in this study holds no additional risks than negligible risk,
since we will be comparing the ecological side-effects of 3 routinely used
treatment regimens, which will be prescribed as standard medical care.
Participants will have no benefits. In case participants choose for the
optional spirometry and/or allow the optional venepuncture under the ZEBRA-5
protocol at the age of 5 years, this information will be shared with the
parents, and therefore can be seen as potentially beneficial for participants
and parents. Collection of faecal samples, saliva samples, rectal swabs, and at
the age of 5 years additional skin swabs are non-invasive, are generally
accepted as fully save and have a negligible burden. Collection of the
transnasal nasopharyngeal swab at the age of 5 years has a minimal burden and
can give a short (2-3 seconds) unpleasant tickly feeling, cough and watery
eyes, in very rare cases a minimal self-limiting nose bleed may occur (less
than 1:3000 in our experience). In case a nose bleed occurs the member of the
research team will give standard care according to the standard operating
procedures.

If parents agree with the optional venepuncture in the ZEBRA-5 protocol, there
is a small risk (2%) of developing a local extravasation (a haematoma). During
the procedure the participant can experience anxiety and some pain. With the
use of an EMLA patch we try to abate pain during the procedure. Further anxiety
(and therewith pain levels) will be reduced by distracting the child during the
procedure.

The optional spirometry and fractional exhaled nitrous oxide test are both save
and non-invasive. Children are playfully trained before doing the tests.
We will follow the code of conduct relating to expressions of objection by
minors participating in medical research, as stated by the CCMO.

Each sample moment including signing of the informed consent will take no more
than 15minutes of participant*s time. For the ZEBRA-5 protocol the participants
will have to visit the outpatient clinic instead of our study team executing a
home visit. This outpatient clinic visit will take about 1-1,5 extra hours and
the more extensive questionnaire will take parents approximately 20 minutes
prior to the outpatient clinic appointment. Parents receive a financial
compensation for travelling to and parking at the hospital. We try to plan the
visit on Wednesday or Friday afternoons as much as possible, or another moment
when the child has no school obligations.