The Sinonasal Cavity as a Reservoir for Upper Airway Bacterial Development

The study of microbial community dynamics is critically important to human health, including
how to maintain or restore a healthy microbiome. Metagenomic studies have revolutionized
microbiology by addressing these issues in a culture-independent manner, and have defined
essential roles of the microbiota in host development. The initial development of gut
microbiota begins in utero and is strongly influenced by exposures at birth (e.g. vaginal vs
caesarean birth). The initial seeding by the mother's microflora and its ensuing bacterial
succession is thought to have critical long-term impacts on human health. Recent longitudinal
studies have documented a gradual increased in bacterial diversity, non-random community
assembly, the effects of breast milk and introduction of table foods, and large taxonomic
shifts as a result of antibiotics and environmental stresses that occur during infancy. Since
perturbations of these early events have been linked to diabetes, cancer, mental health and a
range of other maladies, efforts are underway to learn how manipulation of the infant
microbiome to a "healthy" state translates into long-term clinical outcomes.

By contrast, little attention has been paid to factors that govern the early-life development
of respiratory tract microbiota, including the oral cavity. At birth, the oral and
nasopharyngeal flora resemble those of the maternal vaginal tract or skin (depending on mode
of delivery). Preliminary datasets suggest that during the first year of life, the airway
microbiome evolves into a rich, adult-like microbial ecosystem. The keystone colonizers of
the airways are thought to condition the subsequent colonization by over 600 species, some of
which serve to establish robust bacterial communities characteristic of human health. Other
secondary colonizers are frequently implicated in oral and respiratory infections, including
chronic sinusitis, pneumonia, and periodontal diseases. As an example, infant colonization by
Streptococcus pneumoniae is an important risk factor for childhood middle ear disease. Early
microbial communities, therefore, represent major factors that govern the colonization of the
airways by both pathogenic and protective microbes during infant microbiome development.
Understanding the acquisition of infant airway microbial communities and the factors that
alter their composition is critical to the promotion of human health and the prevention of
airway diseases that represent an annual multi-billion dollar burden on the US healthcare
system.

While the maternal-newborn exchange of airway microbiota is well-documented, no studies have
examined within-subject relationships among the mouth, sinuses, nasopharynx and lungs and the
relative abundance of bacterial taxa at those sites. Recent evidence suggests the oral cavity
may serve as a reservoir for pathogens that trans-locate to non-oral locations;
oral-associated microbes infect most other body sites as evidence by 16S sequencing. However,
two caveats make this bacterial metastasis controversial. First, that oral bacteria are not
located solely in the mouth but are also found elsewhere in the airways makes their origin
difficult to establish. Thus, the directionality of microbial exchange between airway niche
spaces is not known. Secondly, most 16S ribosomal RNA (rRNA) surveys only reveal bacterial
composition at the genus or phylum level, providing little information about strain lineages,
and whether individual strains can migrate between sites. These data are critical to promote
development of protective microbiota while restricting growth of recalcitrant pathogens.

A more complete understanding of this trafficking begins with in-depth, strain-level surveys
of the bacterial communities present in each airway niche shortly after birth, and their
development over time. As a step in this direction, this Academic Health Center-sponsored
study will use metagenomic sequencing to assess the exchange of specific bacterial strains
throughout the upper airways. Our mouse studies (explained elsewhere) will be complemented by
a study of cystic fibrosis (CF) infants, who represent a unique population that facilitates
the capture of oral, sinonasal, and lung microbiota over the first two years of life. By
using a combination of oral and throat swabs, together with nasal suction of mucus samples, a
metagenomic sequencing approach will be used to characterize the composition of bacterial
communities at each anatomical site. Beginning at birth, the investigators will collect a
time-series of swabs from each subject, and monitor changes in the development of microbiota
over time. By doing so, our studies will illuminate airway trafficking of both beneficial and
pathogenic microbes and may represent an essential pathophysiological step towards shifting
the balance between airway health and disease.

EXPERIMENTAL PLAN The development of airway bacterial communities will be monitored in a
small CF patient cohort. Since CF infants are monitored from birth and are routinely sampled
for airway microbiota during routine outpatient visits as part of their standard-of-care,
they represent a unique population to monitor bacterial trafficking between the oral, nasal
and lung cavities. All newborns at the University of Minnesota (UMN) are genetically
screened. Those testing positive for CF will be confirmed using a pilocarpine sweat test.
Positive subjects (with 2 Cystic Fibrosis Transmembrane Conductance Regulator [CFTR]
mutations) will then be sampled during the participants' visits to the CF treatment center;
on average, infants will be seen every month for the first 6 months, every other month until
1 year of age, followed by quarterly visits to the UMN CF Center (in accordance with Cystic
Fibrosis Foundation guidelines). As part of standard of care, nasal samples are collected by
suction at each visit. Nylon swabs will also be used to sample each infant's buccal mucosa.
Oropharyngeal swabs will also be obtained, which are considered to be an accurate proxy of
lower airway (lung) microbiomes. Based on the number of CF newborns expected over the study
period, the investigators plan to recruit up to 10 subjects. From each infant, up to 10
temporal swabs will be collected from each airway site (10 infants x 10 swabs x 3 sites = 300
samples). Swabs will be stored in saline, frozen at -80C and processed in parallel.

DNA will be extracted from each swab sample, and sequencing performed at the University of
Minnesota Genomics Center.

Inclusion Criteria:

- Diagnosis of CF by sweat chloride test >60 mEq/L or by presence of two known CF
genetic mutations

- Age 0-3 years

- Willingness to comply with study procedures

- Willingness of parent/guardian to provide written consent.

Exclusion Criteria:

• Presence of vasculitis or rheumatologic disorder