Dynamics of the respiratory tract flora in the first years of life: a comparative analysis between infants / children with cystic fibrosis and healthy controls.

Nasopharyngeal colonization establishes soon after birth. By 6 months of age,
frequent encountered potential pathogens in the nasopharynx are S. pneumoniae,
H. influenza, M. catharralis and S. aureus. Upper airway tract colonization is
dynamic and often balanced; dysbalance is associated with diseases such as
acute otitis media, sepsis and meningitis.Competitive pathogen interactions
(e.g. between S. pneumoniae and S. aureus) are present in the upper airways but
are still hardly understood.Host- and environmental factors such as day care
attendance, age, immunity, viral infections, 7-valent pneumococcal conjugate
vaccine and antimicrobial agent use have been shown to modify colonization.
Children with cystic fibrosis (CF) are known to acquire respiratory tract
infections very early in life, in particular with S. aureus, non-typable H.
influenzae and P. aeruginosa. P. aeruginosa infection is a major determinant
for morbidity in CF. Pulmonary inflammation is observed already in the first
months of life; it is still debated whether inflammation occurs independently
before pathogen colonisation. In patients diagnosed by newborn screening,
broncho-alveolar specimens from the first months of life discovered pathogens
in 20 % of patients, the majority being asymptomatic. Pulmonary infection in CF
patients follows upper respiratory tract colonization, among others in the
sinuses.Unlike in healthy children, only scarce data are available for
nasopharyngeal colonisation since majority of CF studies focus on the lower
airways. Nasopharyngeal specimens from CF patients detect most common pathogens
concomitantly present in the lung; there is an extra diagnostic yield with
respect to upper respiratory tract colonizing pathogens.
In CF, competitive interactions within the microbial flora seem to be an
important determinant for respiratory exacerbations. In healthy children, P.
aeruginosa colonization occurs infrequently, and is increased in presence of
respiratory viruses. In contrast, in CF patients viral upper respiratory tract
infections are clearly associated with lower respiratory tract morbidity and
increased risk for initial P. aeruginosa infection. Synergisms within the
microbial flora may cause renewed virulence of P. aeruginosa in a chronic
infection condition, leading to increased inflammation and respiratory
exacerbations.
The quality and quantity of the complete respiratory flora in healthy children
and children with CF is largely unknown because of limitations in current
microbiological methods, leaving possible windows of treatment opportunities
unnoticed. Molecular targeting of pulmonary specimens manifested much more
colonizing species in CF patients than conventional cultures; not taking into
account colonization rates in the upper airways.
To gain more understanding about microbial colonization profiles, interactions,
the impact of use of antibiotics and respiratory viral infections and risks for
subsequent pulmonary disease in children with CF, it is necessary to begin at
the start and to unravel the microbial colonization profiles from a very early
age. Modern high-throughput molecular techniques can facilitate detailed
analysis of microbial profiles and -shifts, which was impossible until now
using conventional culturing techniques.
Since antibiotic treatment in CF patients may affect the microbiome and
resistance, we also will collect faeces for evaluation of flora and resistome.
Amendement 2:
The pneumococcal conjugate vaccine is recommended in infants with CF and
currently included in the national infant immunization schedule for all
newborns, though the immunogenicity and protectivity of this vaccine hasn*t
been tested in this patient group. Recently we showed that saliva IgG levels
correlate well with systemic IgG antibodies (Rodenburg et al., under review) .
CF patients may have an altered attenuated antibody response to pneumococcus
for several reasons; due to a lot of antibiotic use, organisms are eradicated
before a natural antibody response can be generated or vaccine responses can be
boosted. The abnormal airway secretions in CF and overgrowth of the respiratory
epithelium by other organisms may also impair the ability to recognize and
mount an appropriate antibody response to pneumococcus and CF patients may have
an attenuated IgG2 response to encapsulated organisms.
We will collect saliva samples to investigate the mucosal immune response in
relation to natural boosting by pneumococcal colonization and childhood
vaccinations and in relation to vaccination in children with CF compared to
controls (saliva IgA and IgG).
Amendement 3:
Amendement 3: (see separate reference list 3)
Since January 2011, a persistent large number of different types of
micro-organisms has been discovered in the respiratory samples of both the
control and CF patients, aged under 18 months. In literature, correlations have
been found between bacterial community profiles and clinical disease markers
(2,3,5). Bacterial community complexity was inversely correlated with patient
age, presence of P. aeruginosa and antibiotic exposure, and was related to CF
genotype. In the current study, untill the age of 18 months, we observed no
substantial differences in diversity between control and CF patients. This
might be due to the fact that 1) the microbiome is still establishing at this
very young age and 2) the antibiotic use is still low in both groups,
antibiotics which are essential in the decrease of diversity. Therefore , we
believe we need to follow these children for a longer period of time to gain
more understanding about microbiome diversity in relation to age and
CFTR-genotype. Consequently,
we want to extend the duration of the follow-up part of this case-control
study, investigating the dynamics and diversity of the airway-microbiome in
children.
In the present study we observe events of nasopharyngeal recolonization with
the same pathogen, therefore we intend to investigate the correlation between
(re)colonization and species-specific local antibody responses in saliva (local
IgA-production)(1). It is interesting to know if nasopharyngeal colonization
elecits protective antibody levels against recolonization.

Our primary hypothesis is that the process of nasopharyngeal and fecal
colonization from birth to childhood differs in quality and quantity between
infants with CF and healthy controls and that these differences are related to
subsequent respiratory morbidity in patients with CF. The following specific
questions will be answered:
1. What is the general nasopharyngeal microbial colonization profile shortly
after birth in healthy newborns and infants with CF and how do these profiles
change over time during the first years of life?
2. Are clinical respiratory exacerbations preceded by changes in quality or
quantity of the nasopharyngeal microbial profile?
3. What are the colonization dynamics before and after acquisition of the most
important pathogen in CF, P. aeruginosa?
4a. What is characteristic of the resistome and how does the resistome change
over time in gut of CF infants (versus controls) during the first years of life?
4b. What is the effect of prophylactic antibiotics (macrolides) on the
respiratory and gut microbiota, in relation to protective flora and recovery
4c. What are the effects of therapeutic antibiotics on the selection and
development of resistance in respiratory pathogens in relation to the existing
(gut) resistome
4d. What is the role of respiratory viral infections on the respiratory
microbiota, in relation to protective flora and recovery
5. What is the mucosal immune response in relation to pneumococcal colonization
and vaccination.
6. Is bacterial community complexity inversely correlated with patient age and
related to CF genotype?
7. What is the impact of (re)colonization on species-specific local antibody
responses?

This explorative study aims to identify the sequential nasopharyngeal and gut
microbial colonization profile in the first years of life of 20 newborns with
CF (diagnosed with heelprick screening) and 45 age- and sex-matched healthy
controls. Saliva will be collected at the age of 3, 6 and 12 months till the
age of 18 months, thereafter annually in both groups. Nasopharyngeal and fecal
samples will be obtained regularly and during respiratoire complaints
(voluntarily one month after respiratory complaints) in all children. To
identify differences and shifts in microbial flora, high-throughput
pyrosequencing will first be performed on 3-monthly samples. Conventional
cultures and viral multiplex PCRs will be performed on the nasopharyngeal
samples. According to the pyrosequencing outcomes, representative
nasopharyngeal bacterial flora CHIPS and/or multiplex PCR will be developed to
analyze the remaining samples and to identify the colonization process in
relationship to disease on an individual base.

New knowledge about nasopharyngeal colonization dynamics and species
interaction in childrenwith CF leads to earlier recognition of pathogenic
microbial profiles leading to respiratory exacerbations. The final aim of the
study is to explore risk factors for respiratory exacerbations and to
facilitate monitoring and specific early interventions with respect to
colonization with pathogenic microorganisms (eg. P. aeruginosa and S. aureus)
in children with CF. In the future, rapid extensive evaluation of colonization
status by sensitive and innovative techniques might lead to immediate and
proper treatment and subsequent prevention of serious and chronic pulmonary
infections. In addition, we feel that detailed studies of the development of
resistance in pathogens in relation to the evolution of the human resistome
(resistance gene pool as present in the most important gene reservoir, e.g. the
gut) will help us in the future to design tailored antibiotic regimes to
prevent resistance development in the respiratory pathogens. Finally this study
might lead to data regarding vaccinations to common respiratory pathogens like
pneumococcus in infants with Cystic Fibrosis.