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\begin{document}

\title{Respiratory syncytial virus and airway microbiota -- A complex interplay
and its reflection on morbidity}



\author[1]{Giovanni Rossi}%
\author[2]{Stefania Ballarini}%
\author[3]{Michela Silvestri}%
\author[3]{Oliviero Sacco}%
\author[4]{Andrew Colin}%
\affil[1]{G. Gaslini Institute}%
\affil[2]{University of Perugia}%
\affil[3]{Istituto Giannina Gaslini}%
\affil[4]{Director, Division of Pediatric Pulmonology}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The immunopathology of respiratory syncytial virus (RSV) infection, the
most common cause of lower respiratory tract infections (LRTI) in the
pediatric population, with severe disease being the exception. The
variability of the clinical presentation is incompletely explained by
host, viral and environmental factors but, in infants and young
children, disease severity is certainly linked to the physiological
immune immaturity. There is evidence that the maturation of the host
immune response is, at least in part, promoted by the composition of the
nasopharyngeal microbiome that, modulating excessive inflammation, can
counteract the predisposition to develop viral respiratory infections
and lower the risk of disease severity. However, interaction between the
nasopharyngeal microbiota and respiratory viruses can be bidirectional.
Microbial dysbiosis can drive disease pathogenesis but may also
represents a reflection of the disease-induced alterations of the local
milieu. Moreover, viruses like RSV, can also increase the virulence of
potential pathogens in nasopharynx, which is a main reservoir of
bacteria, and therefore promote their spread to the lower airways
causing superinfection. Negative changes in microbial community
composition in early life may constitute a heightened risk towards
severe RSV respiratory infection and bacterial superinfection, whilst
specific groups of microorganisms can be associated with protection. A
better understanding into the potential negative and positive role of
the different nasopharyngeal bacterial species in disease prevention as
well as into the possible benefits of microbiome therapeutic
manipulation, may improve patient outcomes.%
\end{abstract}%



\sloppy


Respiratory syncytial virus and airway microbiota -- A complex interplay
and its reflection on morbidity

Giovanni A. Rossi\textsuperscript{1}, Stefania
Ballarini\textsuperscript{2}, Michela Silvestri\textsuperscript{1},
Oliviero Sacco\textsuperscript{1} and Andrew A. Colin\textsuperscript{3}

\textsuperscript{1} Department of Pediatrics, Pulmonary and Allergy
Disease Unit, G. Gaslini University Hospital, Genoa,
Italy,\textsuperscript{2}Department of Experimental Medicine, Section of
Immunometabolism, Immunogenetics and Translational Immunology,
University of Perugia, Italy, and \textsuperscript{3}Division of
Pediatric Pulmonology, Miller School of Medicine, University of Miami,
Miami, FL, USA.

\textbf{E-mail addresses:} giovannirossi@gaslini.org

Correspondence to. Giovanni A. Rossi, Department of Pediatrics,
Pulmonary and Allergy Disease Pediatric Unit and Cystic Fibrosis Center,
Istituto Giannina Gaslini, Genoa, Italy;- giovannirossi@gaslini.com

\subsection*{}

{\label{section}}

Abstract: 233 words; text: 4.382 words; reference: 55; figures 3

\textbf{Abstract}

The immunopathology of respiratory syncytial virus (RSV) infection, the
most common cause of lower respiratory tract infections (LRTI) in the
pediatric population, with severe disease being the exception. The
variability of the clinical presentation is incompletely explained by
host, viral and environmental factors but, in infants and young
children, disease severity is certainly linked to the physiological
immune immaturity. There is evidence that the maturation of the host
immune response is, at least in part, promoted by the composition of the
nasopharyngeal microbiome that, modulating excessive inflammation, can
counteract the predisposition to develop viral respiratory infections
and lower the risk of disease severity. However, interaction between the
nasopharyngeal microbiota and respiratory viruses can be bidirectional.
Microbial dysbiosis can drive disease pathogenesis but may also
represents a reflection of the disease-induced alterations of the local
milieu. Moreover, viruses like RSV, can also increase the virulence of
potential pathogens in nasopharynx, which is a main reservoir of
bacteria, and therefore promote their spread to the lower airways
causing superinfection. Negative changes in microbial community
composition in early life may constitute a heightened risk towards
severe RSV respiratory infection and bacterial superinfection, whilst
specific groups of microorganisms can be associated with protection. A
better understanding into the potential negative and positive role of
the different nasopharyngeal bacterial species in disease prevention as
well as into the possible benefits of microbiome therapeutic
manipulation, may improve patient outcomes.

\textbf{INTRODUCTION}

Respiratory syncytial virus (RSV) is the main cause of acute viral
respiratory infection leading to hospitalization in infants and young
children worldwide {[}1{]}. The spectrum of its clinical manifestations
ranges from mild upper tract illness to severe LRTI. The latter,
requiring hospitalization in approximately 20\% of the cases and of
these \textasciitilde{}15\% require intensive care management {[}2{]}.
RSV virulence and a variety of environmental factors contribute to
increased RSV susceptibility, as well, specific host related conditions
have been identified which increase the risk for severe disease
{[}1-3{]}, yet these are not present in most infants hospitalized with
RSV LRTI. The mechanisms accounting for the wide variability in the
clinical presentation of RSV infection are only partly understood, but
recent insights suggest that modifications of the nasopharyngeal
microbiota composition established or induced by a previous infection
might be involved. In early life, the baseline system structure and
function of the airway immunity is regulated by the local microbial
milieu, and distinct microbiota have been associated with sequelae of
specific viral and bacterial LRTI {[}4-6{]}. Recent insights suggest
that modifications of the nasopharyngeal microbiota composition,
established by a preceding infection, might be involved. Whether
imbalance in the microbial community can drive disease pathogenesis or,
conversely, reflects disease-induced alterations of the local milieu
following respiratory infections is still to be elucidated {[}5,6{]}. A
paradigmatic example is represented by the bidirectional interplay
between resident nasopharyngeal bacterial communities and RSV in
bronchiolitis. Modifications of the upper airway microbiota composition
appears to be associated with RSV disease severity, at the same time
there is evidence that RSV can increase the virulence of potential
nasopharyngeal pathogens, a large bacterial reservoir, that can
consequently spreads to the lower airways causing superinfection
{[}7,8{]}. The current evidences on the epidemiologic link between RSV
and nasopharyngeal microbiome, the mechanisms involved in these
interactions, and the possible clinical consequences will be reviewed,
summarized and discussed.

\textbf{INFANT NASOPHARYNGEAL MICROBIOME IN HEALTH AND DISEASE}

The airways represent a large surface area that interacts with the
surrounding environment. They exhibit a gradient of bacterial load that
is relatively high in the nasopharynx and substantially diminished in
the lower respiratory tract {[}1{]}. A growing body of literature has
demonstrated that the nasopharyngeal microbiome, with its mixed
microbial communities, plays an important role in maturation and
homeostasis of the host immune response in the upper and lower airways
{[}1,4,5{]}. The balanced competitive/synergistic interplay between
commensal and potentially pathogenic taxa, that is the hallmark of a
healthy status, can be altered by acquisition of new pathogenic
bacteria, increased virulence or prevalence of potential pathogens,
and/or decreased efficiency of host defenses, the latter being the cause
but also the result of dysbiosis {[}1,4,6{]}. In newborns and infants,
obligatory nasal breathers, nasopharyngeal airways are both the first
line of defense but also the main port of entry for bacteria and
viruses. Overgrowth of virulent taxa will lead to symptomatic infection,
with surrounding tissue invasion and inflammation. {[}9{]}. Therefore,
in early life, microbiota composition of nasopharyngeal airways reflects
exposures to the surrounding atmospheric environment which, shaping the
host immune responses, will favor or inhibit the predisposition to
respiratory disorders {[}1,4,10-12{]}. Nasopharyngeal colonization by
environmental potential respiratory pathogens is established early in
childhood {[}2{]}. In a study on 234 infants, it was shown that
nasopharyngeal microbiome has a relatively simple structure, dominated
by six clusters: \emph{Moraxella} ,\emph{Haemophilus} (Gram negative
bacteria), \emph{Staphylococcus} ,\emph{Corynebacterium} ,
\emph{Streptococcus} and \emph{Alloiococcus}(Gram positive bacteria)
(figure 1) {[}13{]}. Healthy nasopharyngeal samples collected around 2
months of age were dominated by\emph{Staphylococcus} and
\emph{Corynebacterium,} but the frequency of these microbiome profile
groups declined with age and, at 12 months of age, an increased
prevalence of \emph{Alloiococcus} and \emph{Moraxella}species (spp) was
detected (figure 1A) {[}13{]}. Transient incursions
of\emph{Streptococcus} , \emph{Moraxella} and/or
\emph{Haemophilus}occurred during acute viral respiratory tract
infections,\emph{Streptococcus} and \emph{Moraxella} colonizing,
respectively, 54\% and 72\% of the children by 1 year of age {[}14{]}
and a progressive decline in \emph{Staphylococcus}
and\emph{Corynebacterium} spp (figure 1B) {[}13{]}. Antibiotic
treatments were associated with higher abundances of the potential
respiratory pathogens \emph{Haemophilus} , \emph{Streptococcus}
and\emph{Moraxella} , with lower abundances of \emph{Alloiococcus}
and\emph{Corynebacterium} (figure 1C) {[}13{]}. This microbial dysbiosis
may predict subsequent respiratory infections {[}2,15-17{]}.
Colonization with \emph{Streptococcus} , \emph{Moraxella}
or\emph{Haemophilus} spp was linked to an increased risk for LRTI,
whilst nasopharyngeal microbiota dominated by \emph{Corynebacterium}
or\emph{Alloiococcus} clusters was associated with less respiratory
morbidity (figure 1D) {[}11-13{]}. When related to early exposure to
noxious environmental factors negative changes in microbial community
composition may constitute a heightened risk towards severe viral
respiratory infections and bacterial superinfections {[}11,17{]}. This
can be observed in severely premature infants admitted to neonatal
intensive care units (NICU). These infants face the combined effects of
deprivation of the normal intrauterine physical environment,
supplemental oxygen with relative hyperoxia, barotrauma from mechanical
respiratory support, exposure to nosocomial pathogens, and
broad-spectrum antimicrobials {[}16{]}. They show abnormal nasal airway
immune responses against respiratory viruses, persisting beyond the PICU
period and, in comparison with full term infants, a higher
nasopharyngeal microbial heterogeneity and within-group dissimilarity
{[}18{]}. Emergence of a mixed flora, including \emph{Moraxella} and
other Gram-negative bacteria (\emph{Burkholderia} , \emph{Neisseria} and
\emph{Janthinobacterium} ), was detected. At phylum level, an increase
in \emph{Proteobacteria} (a major group of Gram-negative bacteria) was
associated with a decrease in\emph{Firmicute} (Gram-positive bacteria
such as \emph{Lactobacillus} ) (figure 1E) {[}14{]}. This airway
microbiome signature has been recently described also in preterm
newborns at increased risk of bronchopulmonary dysplasia {[}16,19{]}.
The presence of within-group dissimilarity, identified also in the
nasopharyngeal microbiota of otherwise healthy premature infants,
suggests that this group does not develop a stable microbiome in early
life. Longitudinal data showed that these prematurity-related microbiota
features persisted during viral infection and played important negative
role in modulating airway inflammatory and immune responses in this
vulnerable group {[}16,19{]}. Upper airways colonization by potential
pathogens in neonates, also in term infants, may be associated with
increased susceptibility to LRTI during the first 3 years of life. A
prospective birth cohort study of 411 children born to mothers with
asthma, showed that neonatal hypopharyngeal colonization with
\emph{Streptococcus pneumoniae} , \emph{Haemophilus influenzae} ,
or\emph{Moraxella catarrhalis} , at age 4 weeks, was associated with
increased risk of bronchiolitis, recurrent wheeze, asthma and pneumonia,
during the first 3 years of life {[}20,21{]}. The viral etiology of
bronchiolitis, i.e. whether it was RSV or human rhinovirus (HRV)
related, was not reported in this study.

\textbf{3. NASOPHARYNGEAL MICROBIOTA COMMUNITY COMPOSITION AND RSV
INFECTION}

\textbf{3.1 Modulation of RSV infection severity}

In infants, RSV bronchiolitis severity is linked to a dysregulation of
the immune response, related to a relative immaturity of the host
defenses {[}3{]}. Recent literature suggests that prior or concomitant
colonization of nasopharyngeal microbiota by potentially pathogenic
bacteria may enhance the severity of the RSV infection {[}7,22,23{]}. A
17-site, multicenter, prospective cohort study of 1,016 infants (age
\textless{}1 year) hospitalized with bronchiolitis, showed that during
RSV infection a greater abundance of \emph{Haemophilus influenzae}
and\emph{Streptococcus pneumoniae} in the nasal microbiota can be
associated with more severe disease (figure 2A) {[}22{]}. In a
prospective observational study, during four consecutive seasons, 106
young children with a first episode of RSV infection and 26 healthy
control subjects were enrolled {[}7{]}. Three groups of children less
than 2 years of age were evaluated: a) with mild RSV disease (RSV
infection), b) with severe RSV disease (RSV hospitalization) and c)
healthy controls. Five nasopharyngeal microbiota clusters were
identified, characterized by enrichment of either\emph{Haemophilus
influenzae} , \emph{Streptococcus} ,\emph{Corynebacterium} ,
\emph{Moraxella} , and \emph{Staphylococcus aureus} {[}7{]}. RSV
infection and hospitalization were positively associated with
\emph{Haemophilus influenzae} and \emph{Streptococcus} and negatively
associated with\emph{Staphylococcus aureus}abundance (figure 2A). Rates
of PICU admission and length of stay tended to be lower in children
within the \emph{Moraxella}cluster than in children included in the
other clusters and higher abundance of \emph{Moraxella} was observed
most often in outpatients {[}7{]}. Independently from microbiota
cluster, children with RSV infection showed overexpression of
IFN-related genes. However, in those with transcriptome profiles
dominated by \emph{Haemophilus influenzae}and \emph{Streptococcus} , an
upregulation of genes linked to toll-like receptor and
neutrophil-macrophage activation and signaling was also detected.
Overexpression of \emph{Haemophilus} and loss in commensals, like
\emph{Veillonella} , were described in nasopharyngeal aspirates of a
cohort of 54 infants, younger than 6 months of age, hospitalized with
RSV infection {[}24{]}. Transcriptome profiles dominated
by\emph{Haemophilus} was associated with increase IL-6 and CXCL-8
responses, cytokines that in respiratory virus infections are associated
with more severe disease (figure 2A). {[}24-26{]}. Moreover, in human
bronchial epithelial cell culture pretreated with \emph{Haemophilus
influenzae} , the release of IL-6 and CXCL-8 was synergistically
increased after RSV, but not after HRV infection, buttressing the notion
that this virus-bacterial interplay is specific for RSV {[}27{]}. The
clinical implication of these observation is reflected in a multicenter
prospective cohort study of 1,005 infants hospitalized for
bronchiolitis. In this study the odds ratio for PICU admission was
higher for subjects with RSV infection and \emph{Haemophilus} -dominant
profiles compared to RSV-infected infants with profiles dominated by
other bacteria, such as \emph{Moraxella}(figure 2A) {[}28{]}. Thus,
specific nasopharyngeal microbiota clusters, dominated
by\emph{Haemophilus influenzae} and \emph{Streptococcus pneumoniae} can
modulate the host immune response, potentially affecting clinical
disease severity. Further evidence for the ability of the nasopharyngeal
microbiota to positively modulate the host immune response is the
demonstration of the presence of specific metabolic profiles, linked to
the microbiome composition, in children with severe bronchiolitis. In a
multicenter prospective cohort study on infants \textless{}1 year of
age, hospitalized with bronchiolitis, Stewart et al. determined
microbiome profiles and metabolome in nasopharyngeal airway samples and
their association with disease severity {[}22{]}. Among 254 metabolites
identified, a panel of 25 showed high sensitivity (84\%) and specificity
(86\%) in predicting the use of positive pressure ventilation (PPV)
support. Metagenomic sequencing demonstrated that \emph{Haemophilus
influenzae} , \emph{Moraxella catarrhalis} , and \emph{Streptococcus
pneumoniae} dominated the nasopharyngeal airway. Of these, however, only
the relative abundance of \emph{Streptococcus}
\emph{pneumoniae}correlated with the intensity of these selected
metabolites to predict increased risk of PPV support (figure 2A). In the
pathway analysis, sphingolipid metabolism was the sub-pathway most
significantly enriched in infants with PPV use, and in turn, enrichment
of sphingolipid metabolites was positively correlated with the relative
abundance of\emph{Streptococcus} \emph{pneumoniae} {[}22{]}. In
contrast, the relative \emph{Moraxella} abundance was negatively
correlated with respective intensity of the same metabolites.
Sphingolipids~are linked to the inflammatory-mediated pathogenesis of
airway diseases and play an important role in multiple aspects
of~viral~replication, including the initial~infection~of mammalian
cells, depression of host immune response, and assembly and budding of
newly synthesized~viral~components {[}29{]}. Thus, curiously,
\emph{Moraxella} spp, thought to be associated with a general increase
in infant morbidity {[}11,13{]}, appears to have a ``protective effect''
on RSV bronchiolitis.

\textbf{3.2 Delayed viral clearance, RSV and HRV infection and recurrent
wheezing}

Respiratory virus clearance requires a complex response initiated by
resident respiratory tract cells and innate immune cells and ultimately
resolved by adaptive immune cells {[}30{]}. This viral clearance that is
largely dependent on the interplay between infecting agents and host
immune response, may be influenced by nasopharyngeal microbiota
composition in infants with RSV bronchiolitis. A 17-center prospective
cohort study of infants aged less than 1 year, with RSV bronchiolitis,
found that those with a nasopharyngeal \emph{Haemophilus} -dominant
microbiota profile at time of hospitalization were more likely than
those with a mixed profile to have delayed viral clearance, also after
adjustment for 11 factors, including viral load (figure 2B) {[}23{]}.
Nasopharyngeal microbiota composition at the time of hospitalization was
also described as associated to an increased risk of recurrent wheezing.
In children \emph{\textless{}} 2 year of age, with severe bronchiolitis
due to RSV and/or HRV enrichment of \emph{Haemophilus influenzae} and
\emph{Moraxella catarrhalis} was found in their nasopharynx (figure 2B)
{[}31{]}. \emph{Haemophilus influenzae} was detected in most RSV-only
infected and co-infected samples, but not in HRV-only samples, whilst
\emph{Moraxella} spp were detected in co-infection and were more common
among children with wheezing upon admission {[}31{]}. No enrichment of
\emph{Haemophilus influenzae} in HRV-only and of \emph{Moraxella} spp in
RSV- or HRV-only was reported. It is therefore possible that specific
bacterial species increase the likelihood of one viral infection or
another, and play a role in the onset of wheezing (figure 2B). This may
be the case of the association of \emph{Moraxella} spp enrichment with
HRV infection. In 17 U.S.A. centers, nasal swabs were collected in
infants hospitalized for bronchiolitis and repeated 3 weeks and 1 year
after hospitalization {[}32{]}. The viral etiology of bronchiolitis was
not reported. An increase in relative abundance of \emph{Moraxella}
or\emph{Streptococcus} spp, at 3 weeks, and of \emph{Streptococcus} spp,
at 1-year follow-up was associated with increased risk of recurrent
wheezing in the study subjects {[}32{]}. In a similar study performed in
Beijing Children's Hospital; 74 infants aged 6 months or less,
hospitalized for an initial episode of severe RSV bronchiolitis, were
included and followed until age 3 years {[}33{]}. A higher relative
abundance of \emph{Haemophilus} , \emph{Moraxella} and
\emph{Klebsiella}was detected in nasotracheal aspiration in the 26
infants (35.1\%) who later developed recurrent wheezing. In these
infants, higher abundance of \emph{Haemophilus} or of \emph{Moraxella}
was respectively associated with elevated CXCL8 levels or of IL-6 and
IL-10 (figure 2B). Resolving the conundrum of the differential role of
\emph{Moraxella} , as having an apparent protective role on the severity
of RSV infection in the acute phase, yet being consistently identified
as a risk pathogen for recurrent wheezing, will advance our
comprehension on both its role and that of other nasopharyngeal
microbial species' in the course, severity and complications of
different respiratory conditions. A different nasopharyngeal microbiota
composition was observed as associated with a reduced risk of wheeze
after RSV infection. A population-based birth cohort of 118 previously
healthy term infants was evaluated during the first confirmed RSV acute
respiratory infection in a Tennessee-based study {[}34{]}. Of the 113
(95.8\%) children who had 2-year outcome data, 46 (40.7\%) had at least
one parental reported wheezing episode. There was no association between
the nasopharyngeal microbiome taxonomic composition, diversity and
richness assessed both during the first RSV infection and the subsequent
wheeze development. However, detection as well as relative abundance
of\emph{Lactobacillus} in nasopharyngeal aspirate was consistently
higher in infants who did not develop wheezing. \emph{Lactobacillus}
also ranked first among the different genera in a model distinguishing
infant with and without subsequent wheeze {[}34{]}. Increasing evidence
supports that dysbiosis, in the first years of life may also have a role
in the development of allergy and asthma because of the ``gut-lung
axis'' interaction. Therefore, probiotics were suggested as a possible
therapeutic approach in prevention of wheezing episodes {[}35{]}.
However, in a randomized double-blind study conducted in 131 young
children (6-24 months old) with at least two wheezing episodes and a
first-degree family history of atopic disease, 6 months treatment with
oral \emph{Lactobacillus rhamnosus} showed no clinical efficacy on
asthma-related events and only mild effects on allergic sensitization
which persisted 6 months after its cessation {[}36{]}. In a similar
study of 160 children and adolescents (6--18 years old), with asthma
diagnosis, 3 months of treatment with other species of
probiotics,\emph{Lactobacillus paracasei} , \emph{Lactobacillus
fermentum} , as well as their combination decreased IgE levels, and
improved asthma control based on the Childhood Asthma Control Test,
Pediatric Asthma Quality of Life Questionnaire scores, and improved peak
expiratory flow rates{[}37{]}. Further studies are needed to confirm
these inconsistent findings and, importantly, to investigate the
possible long-term benefits of some probiotics in infants and young
children at high risk for allergic sensitization, recurrent wheezing and
asthma.

\par\null

\textbf{4. NASOPHARYNGEAL MICROBIOTA COMMUNITY COMPOSITION AND BACTERIAL
SUPER-INFECTION DURING RSV INFECTIONS}

The interaction between nasopharyngeal microbiota and RSV in children
can be bidirectional. As discussed above, specific nasopharyngeal
microbiota compositions may modulate RSV severity but, at the same time,
RSV-induced infections can stimulate proliferation of potentially
pathogenic microorganisms in the upper airways and thus favor bacterial
superinfections and morbidity {[}7,38,39{]}. RSV infection can promote
the invasion of the respiratory epithelium by non-typeable
\emph{Haemophilus influenzae} and\emph{Streptococcus pneumoniae} through
a variety of mechanisms, including upregulation of bacterial adhesion
protein expression, loss of epithelial barrier integrity and decrease of
bacterial clearance mechanisms {[}3,8,40,41{]}. Using an \emph{in vitro}
and a lung mouse model of infection, incubation with RSV or purified G
protein was shown to increase \emph{Streptococcus pneumoniae} adherence
to differentiated ciliated cells of the respiratory epithelium, thus
enhancing the severity of the airway infection and decreasing animal
survival {[}8,42{]}. Also detected in these studies were extensive
changes in the pneumococcal transcriptome, with upregulation in the
expression of key pneumococcal virulence genes, including those encoding
the pneumococcal toxin pneumolysin {[}42{]}. On the airway epithelium
end, RSV infection upregulated the cellular surface expression of other
known receptors for \emph{Streptococcus pneumoniae}and \emph{non-typable
Haemophilus influenzae} , notably the intercellular adhesion molecule-1
(ICAM-1) and platelet-activating factor receptor {[}8{]}. In clinical
practice, for infants with RSV bronchiolitis the risk of secondary
bacterial infections (SBI) is present, yet the severity of the resulting
bacterial infection appears to parallel that of the RSV infection
(figure 3A). The low risk of SBI in mild RSV is born out in a study on
1,248 febrile infants \emph{\textless{}} 60 days of age, from 88
pediatric Emergency Departments in New York. The rate of SBI was
significantly lower in those with RSV infection (7.0\%) than in those
without RSV infection (12.5\%) {[}43{]}. Additionally, as compared to
the RSV-negative infants, RSV-positive infants had a lower rate of
bacteremia (1.1\% vs 2.3) and of urinary tract infections (UTI) (5.4\%
vs 10.1\%). Similar results were reported in a retrospective cohort
study of \emph{\textless{}} 8-week-old infants, who presented with fever
and a positive RSV antigen test to the Emergency Department at an urban
child facility in Nashville, Tennessee {[}44{]}. Buttressing these
results is a systematic review of 11 studies on ``in- and
out-patients'', \emph{\textless{}} 60 to 90 day old, with bronchiolitis,
reporting no case of bacteremia, yet the rate of UTI resulting slightly
higher in the RSV-positive subgroup (5.1\% vs 2.0\%) {[}45{]}. These
data suggest that, in nontoxic-appearing infants with a positive RSV
test, full septic evaluations are not necessary, however, the finding of
a low, but clinically relevant rate of urinary tract infection, probably
justifies urine work up {[}44,45{]}. Conversely, the incidence of SBI
and concomitant bacterial pneumonia is more frequently observed (or
diagnosed) in infants with severe RSV bronchiolitis (figure 3B). Out of
127 infants (median age 1.7 months) admitted to the PICU of the Zurich
University Children's Hospital, 57 were mechanically ventilated
{[}46{]}. Tracheal cultures obtained from intubated children within the
first hour of intubation were positive in 25 infants, half of them for
community and half for nosocomial acquired bacteria. Microorganisms
associated with bacterial LRTI included \emph{Moraxella catarrhalis}
,\emph{Haemophilus influenzae} type b and \emph{Streptococcus
pneumoniae} . The incidence of sepsis was 3/127 patient. Despite only
documenting 25 bacterial concomitant infection, antibiotics were
administered to 73/127 infants over the first two days of hospital
admission, namely, to a high proportion of those with negative bacterial
cultures. The authors conclude that to avoid inappropriate antibiotic
prescription, tracheal aspirates should be investigated
microbiologically on a routine basis in this patient group {[}46{]}. The
high frequency of bacterial coinfection in young infants with severe RSV
bronchiolitis was confirmed in the study of the Royal Liverpool
Children's Hospital {[}47{]}. Out of the 165 children (median age 1.6
months) admitted to PICU with RSV bronchiolitis, 70 (42.4\%) lower
airway secretions tested positive for bacteria,
including\emph{Haemophilus influenzae} , \emph{Moraxella catarrhalis}
,\emph{Streptococcus pneumoniae} and \emph{Staphylococcus
aureus}{[}47{]}. All those with bacterial co-infection were mechanically
ventilated and required ventilatory support for longer than those with
only RSV. Gender, co-morbidity, origin, prior antibiotics, time on
preceding antibiotics, admission oxygen, and ventilation index were not
predictive of positive bacterial cultures {[}47{]}. The risk of
prolonged hospitalization and PICU admission may be even higher in
preterm born infants, hospitalized with RSV disease, if concurrent
bacterial infections are present. Out of a total of 464 infants, median
age 2.75 months (range 0.25--96), hospitalized due RSV infection to the
Graz Medical University, 42 (9.1\%) were born\textless{}37 weeks of
gestational age {[}48{]}. Concurrent bacterial infections were detected
in 3.1\% of term and 9.5\% of preterm infants and the percentage of
those referred to PICU was 5.2\% and 23.8\% in term and preterm infants
respectively. In all these preterm a diagnosis of pneumonia was made,
and tracheal aspirates were positive for \emph{Streptococcus
pneumoniae}and \emph{Haemophilus influenzae} . Mean hospital stay was
longer (22.3 days) in preterm infants with bacterial co-infection than
in the ones without it (10.3 days) {[}48{]}. Concomitant bacterial
infection can occur also in older children hospitalized with RSV
bronchopulmonary infections as shown in a study performed of 188
pediatric inpatients, aged up to 5 years, admitted to 2 hospitals in
Chiba Prefecture, Japan {[}49{]}. Pathogenic bacteria were isolated from
washed sputum bacterial culture in 43.6\% of the children, the three
most frequently isolated being \emph{Haemophilus influenzae} (43.9\%),
\emph{Streptococcus pneumoniae} (36.6\%) and \emph{Moraxella
catarrhalis} (29.3\%). Before admission, 28.7\% of the patients received
oral antibiotics, most often macrolides and amoxicillin. Of the
\emph{Haemophilus influenzae} strain isolated 38.9\% were
ampicillin-resistant, whilst all\emph{Streptococcus pneumoniae} strains
were penicillin G sensitive, minimum inhibitory concentration values
were 2 \selectlanguage{greek}µ\selectlanguage{english}g/ml {[}49{]}. Thus, RSV bronchopulmonary infection in
hospitalized children can often be associated with super-infection by
antimicrobial-resistant bacterial. Prior antibiotic treatment and
Gram-negative pathogen prevalence at nasopharyngeal level can be
associated with more disease severity. A prospective, observational
cohort study, of 136 infants with RSV bronchiolitis enrolled at 24 h of
hospitalization; 104 on the ward and 32 in the PICU {[}50{]}. As
compared to an age-matched group of healthy controls, RSV bronchiolitis
infants had more frequent nasopharyngeal colonization
with\emph{non-typable Haemophilus influenzae} or \emph{Moraxella
catarrhalis} (Gram-negative bacteria) and\emph{Staphylococcus aureus}
and\emph{Streptococcus pneumoniae} (Gram-positive bacteria). Higher
proportion of Gram-negative pathogens was associated with higher blood
neutrophil percentages, greater number of nasal wash leukocytes,
increased concentrations of plasma IL-8 and IL-6, a trend towards
increased disease severity and longer need for oxygen.
Thus,\emph{Staphylococcus aureus} , which seems to have a protective
role? on RSV infection, may be detected in the lower airways of young
infants with severe RSV bronchiolitis but its presence does not seem to
be associated with increase disease severity. In this study, 47\%
(64/136) of all RSV infants and 75\% of those in PICU received
antibiotics prior to sample collection. This common use of antibiotic
was associated with a decreased likelihood of recovering potentially
pathogenic bacteria by culture, but also with enhanced disease severity
as defined by clinical parameters and radiologic findings. Infants
treated with antibiotics prior to hospitalization required oxygen for a
longer time and radiographic lobar consolidation was more frequently
identified compared to those with no prior antibiotics. Atelectasis or
lobar consolidation was associated with higher clinical disease severity
scores {[}50{]}. The elevated antimicrobial-resistance in bacterial
superinfection and the higher risks of enhanced disease severity
highlight the need for microbiological investigation of airway secretion
in this patient population. Finally, the incidence of
\emph{Streptococcus pneumoniae}coinfection could be modified following
the introduction of seven-valent pneumococcal conjugate vaccine (PCV7).
Weinberger DM et al, evaluating hospitalization data from community
hospitals in 18 U.S.A. states and comparing the 1992/1993 and 2008/2009
seasons, demonstrated a significant decline in RSV-coded
hospitalizations in children aged \textless{}1 y and 1--2-year-old
following PCV7 introduction in 2000 {[}51{]}. We could find no data on
the effect of the effect of the subsequent introduction of PCV13.

\textbf{5. NASOPHARYNGEAL BACTERIAL COMMUNITY AS A SHIELD AGAINST
RESPIRATORY INFECTIONS}

Nasopharyngeal bacteria may counteract bacterial or viral infection. In
infant mice, nasal priming with viable \emph{Corynebacterium
pseudodiphtheriticum} , a Gram-positive bacterium member of the normal
microbiota of the upper respiratory tract, improved their resistance to
primary RSV infection and secondary pneumococcal pneumonia {[}52{]}. The
protection against RSV infection and pneumococcal superinfection was
related to modulation of Toll-like receptor 3 activation in the
respiratory tract leading to enhanced production of the protective
cytokines TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-6, IFN-\selectlanguage{greek}γ \selectlanguage{english}and IFN-\selectlanguage{greek}β, \selectlanguage{english}but also of IL-10 that could
contribute to restrict the inflammatory reaction and ultimately to limit
tissue injury. IFN-\selectlanguage{greek}γ \selectlanguage{english}producing CD3+CD4+ cells were also detected in the
lungs of treated infant mice, viral load was significantly lower and
lung tissue damage markers (LDH and albumin concentrations) in
bronchoalveolar lavage were decrease in RSV-infected mice. Priming with
viable \emph{Corynebacterium pseudodiphtheriticum}also reduced lung
bacterial cell counts and prevented the dissemination of \emph{S.
pneumoniae} into the blood {[}52{]}. Thus, a possible perspective use of
these results could be a seasonal application of a nasal probiotic spray
to boost respiratory innate immunity in immunocompetent subjects.
Bearing in mind the ``gut-lung axis'' interaction, one could consider
orally given probiotics or bacterial derived products as alternative
options; a fascinating proposal whose theoretical efficacy needs
eventually to be demonstrated by further studies {[}53{]}. A possible
approach can be to assess bacterial lysate in germ free mice models to
determine the effect on immune maturation and the capacity to mimic the
microbiota effect in respiratory virus infection.

\textbf{6. CONCLUSIONS}

The nasopharyngeal microbiome plays an important role in modulating the
susceptibility to respiratory infections in infants and young children.
However, due to the complexity of the pathogenetic mechanisms involved,
the role of dysbiosis at the nasopharyngeal levels on the pathogenesis
of viral infections is still not completely understood. Recent insights
have shown that early negative changes in microbial community
composition, due to exposure to noxious environmental factors, may
constitute a heightened risk for severe RSV respiratory infection as
well as bacterial superinfection, in both term and preterm infants
{[}2,10,15,16{]}. An exaggerated and ineffective inflammatory response
to infection, associated with delayed viral clearance and increased risk
of recurrent wheezing in later life are also observed {[}22{]}. The risk
of serious bacterial superinfection, low in infants with mild RSV
bronchiolitis, becomes higher when hospital admission with intensive
care is required {[}46-50{]}. The elevated antimicrobial-resistance
observed during bacterial super infections mandates careful stewardship
and avoidance of inappropriate antibiotic prescription. Antimicrobial
treatment does not eradicate bacteria from the nasopharynx. The
reduction in carriage is temporary, followed by a rapid replacement,
with overgrowth of more resistant strains, newly acquired or previously
masked by other organisms {[}2,8,54{]}. The finding that specific groups
of microorganisms might be associated with protection against RSV
infection and less disease severity {[}28{]} suggests that studies
should be done to understand the mechanistic insight into the potential
positive role of these species in disease prevention. Further studies
are also needed to investigate the possible short and long-term benefits
of microbiome therapeutic manipulation and immune training with the goal
of improving patient outcomes.

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\textbf{Figure legends}

Figure 1. Nasopharyngeal microbiome clusters in healthy infants and
changes associated with age, acute respiratory infections (ARI),
antibiotic treatment, increase morbidity and severe prematurity.

Figure 2. Nasopharyngeal microbiome modifications increasing the risk of
RSV disease severity, delayed viral clearance and increased risk of
recurrent wheezing.

Figure 3. Nasopharyngeal microbiome modifications induced or associated
with mild or severe RSV infection promoting bacterial superinfections.\selectlanguage{english}
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