healthmedicinet

It is generally believed that exposure to microorganism compromises health. Reduced
exposure to microbiota results in decrease of incidence of infectious diseases but
may adversely increase the incidence of allergic disorders 1]-3]. Recent developments of culture-independent tools make it possible to identify microbial
species not previously detected by conventional methods. Unbeknownst to us, man had
been living with these microoraganisms since the dawn of time.

The human body harbors from 10 to 100 trillion microbes which greatly outnumber our
own human cells 4]. This bacterial assemblage has been coined, “the human microbiota” 4]. Subsequently, a project called “Human Microbiome” was established to investigate
the flora in healthy volunteers and their relationship to human health and disease
5]. The study of the host-microbe relationship has shown that microbes play a major
role in our well-being 4],6]. Alterations of microbial composition have been linked to several human diseases
4]. There is also evidence showing that, in the respiratory system, composition of airway
microbiota varies between healthy people and people with diseases such as asthma 6]-8] and cystic fibrosis (CF) 8],9]. Unfortunately, with limited studies currently available, it cannot be concluded
with the same degree for chronic rhinosinusitis (CRS) 10].

Research on microbiome in CRS is therefore needed to elucidate pathophysiology of
this disease such as; 1) the relationship between the microbiome and inflammatory
patterns, 2) possible causal relationships between microbe and CRS, 3) investigation
of the microbiome regarding possible therapeutic properties. Dysregulation of the
interactions between the immune system and commensal bacteria is a contributing factor
to the development and chronicity of a number of inflammatory diseases 11]. Microorganisms in the gut may play a significant role in regulating T helper cells
(Th cells), regulatory T cells (Tregs) and dendritic cells as well as Toll-like receptor
expression in sentinel cell (macrophage and dendritic cells) which are relevant to
airway illnesses such as asthma and allergic diseases 10].

Techniques in microbiota study

Principal approaches to analyze human microbiota are: culture-dependent and culture-independent
techniques. Culture-dependent methods involve isolation and culturing of microorganisms
prior to their identification according to morphological, biochemical or genetic characteristics.
These methods are time-consuming, due to culture and bias, as certain media and growth
conditions favor the growth of some bacteria over others 12]. In addition, this approach may not provide a true reflection of the diversity of
microbes in a sample. A “no growth” result does not necessarily imply that a sample
is sterile. It is estimated that up to 99% of microorganisms observable in nature
typically cannot be cultured by standard techniques 13]. Un-cultivability is a widespread condition that includes: (i) organisms for which
the specific growth requirements (nutritional, temperature, aeration, etc.) are not
fulfilled; (ii) slow-growing organisms are out-competed in the presence of fast-growing
microorganisms and (iii) disfavored organisms, which cannot stand the stressful conditions
imposed by cultivation 13],14]. This approach camouflages the true bacterial community. There needed to be a better
approach to analyze these microorganisms.

Since the 1980s, the application of molecular detection methods has allowed culture-independent
investigations of the microbial communities 15]. Molecular techniques have proven effective in characterizing complex microbial assemblages
in environmental samples 16]. However, an important usefulness of molecular techniques is the ability to detect
genetic materials of non-viable microorganisms 17],18]. Culture-independent methods are based on the direct analysis of bacterial DNA (or
RNA) without culturing. Due to the sensitivity of these techniques, special care and
attention is required for procedures that include sample collection and handling,
DNA extraction, amplification of gene fragments, distinction of different fragments,
identification of microorganism and analysis of the microbial community 15].

For bacterial identification, the predominant gene target for amplification has been
the 16S ribosomal RNA gene (or 16S rRNA) 19],20], which is a component of the 30S small subunit of prokaryotic ribosomes 21]. It has been widely targeted because of (i) its presence in almost all bacteria,
often existing as a multigene family, or as operon; (ii) the conservation of the 16S
rRNA gene, suggesting that random sequence changes are a measure of time (evolution)
rather than a reflection of different bacteria; and (iii) the size of 16S rRNA genes
(1,500 bp) being large enough for informatic purposes 22]. Moreover, there are several available reference sequences and taxonomies databases
such as Greengenes, SILVA and the Ribosomal database project 23]. However, amplification of target genes using polymerase chain reaction (PCR) has
made it impossible to completely avoid PCR-based biases and chimera production. It
thus may distort the level of diversity and bacterial composition in a sample because
of the amplification of pseudogenes 24]. Therefore, other technologies are often used as complementary approaches to 16S
rRNA gene sequencing for reducing distortion of bacterial diversity and composition.
They are DNA microarray, fluorescence in situ hybridization (FISH), and quantitative
PCR (qPCR), and are based on oligonucleotide probes and primers that target the ribosomal
RNA sequences or other genes in different hybridization procedures. Thus these techniques
require a prior knowledge of the microbial DNA sequence. A DNA microarray (also commonly
known as DNA chip or biochip) is a collection of microscopic DNA spots (oligonucleotide
probes) attached to a solid surface. It is usually used for gene expression analysis
or screening of single nucleotide polymorphisms. The FISH technique uses fluorescent
probes that bind to only those parts of the chromosome with which they show a high
degree of sequence complementarity. It detects and localizes the presence of specific
DNA sequences on chromosomes. qPCR or real-time PCR follows the general principle
of PCR with the advantage of detecting the amount of initial DNA in samples using
either fluorescent DNA-binding dyes or fluorescence-tagged oligonucleotide probes
15].

The introduction of next generation sequencing changed the history of genomic research
as it increased sequencing throughput, and did not require prior cloning steps 25]. These technologies are not only changing our genome sequencing approaches and the
associated timelines and costs 26], but also developing many exciting fields such as metagenomics, metatranscriptomics
and single-cell genomics 15]. Three platforms for high throughput parallel DNA sequencing are in reasonably widespread
use at present: the Roche/454 FLX, the Illumina (MiSeq, HiSeq, and NextSeq), and the
Ion Torrent.

At presence, researchers have a large choice in formulating methodological strategies:
depending on the access to the technology, budget, and objectives of research. Each
culture-independent methodology has its own limitations and biases, investigators
must take additional measures; for example one may use more than one molecular technique
or a culture-dependent approach in parallel to provide additional validation of results
and reduce the possibility of false findings due to methodological errors and biases.
Although the culture-independent techniques have the ability to detect more microbes
than culture technique, the culture-dependent methods so far remain a better means
of obtaining individual isolates contributing and obtaining isolates for further assays.

Microbiota in allergic rhinitis

As the gate into our body, the respiratory tract itself harbors a heterogeneous microbiota
that decreases in biomass from upper to lower tract 27]. Even in health, recent findings indicated that direct exposure to bacterial communities
in the airways may provide an explanation for how commensal bacteria can regulate
chronic airway inflammation 11]. Since the observation that infections within households in early childhood have
a role in preventing allergic rhinitis 3], numerous epidemiologic and experimental studies have sought to clarify and extend
the so-called hygiene hypothesis with respect to asthma and other allergic and autoimmune
disorders. The evidence supporting the hygiene hypothesis established the “microbiota
(microflora) hypothesis”. This concept proposes that perturbations in gastrointestinal
bacteria have disrupted the mechanisms of mucosal immunologic tolerance, which has
led to an increase in the incidence of allergic airway disease 28]. Independent studies found that a reduced diversity of the gut microbiota in infancy
is associated with an increased risk of allergic manifestation at school age 29]-31]. The association between the composition of microbiota in the intestine, asthma and
allergic disease is nowadays of high interest 32],33], although the exact mechanism of the interaction of intestinal-systemic immunity
is still not defined 34],35]. There are several publications reviewing the relationships between intestinal microbes
and asthma 7],10],36]. Suggesting that the intestinal microbiome contributes to the regulation of local
and systemic inflammatory responses via short-chain fatty acids, a product of fermentation
of dietary fibers by intestinal bacteria 11]. Following this model, it is likely that the respiratory microbiota may also have
an impact on airway inflammation in allergic responses 1],11], however, this needs further investigation.

Microorganisms in the airways of cystic fibrosis patients

Cystic fibrosis (CF) is an autosomal recessive genetic disorder that affects among
other organs the lungs and sinuses. It is characterized by abnormal transport of chloride
and sodium across the epithelium, leading to thick, viscous secretions. This leads
to defective mucociliary clearance and chronic airway infection with a complex microbiota
37]. Lung disease in cystic fibrosis results from chronic airway infection and inflammation
leading to progressive bronchiectasis and respiratory failure 38]. Specimens for molecular microbial analysis in CF have for the most part all derived
from sputum 37],39]-42], while swabs from the middle meatus in patients with sinusitis were not used until
now. Thus, no conclusions can be made for the upper airways yet. Samples consisted
of serial collections of more than six patients in most of the studies 37],42]-44].

Previous studies indicated that exacerbations might be associated with changes in
microbial densities and the acquisition of new microbial species 37]. Bacterial pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia are known contributors to such exacerbations. Recent studies using the latest culture-dependent
techniques and culture-independent molecular techniques have broadened our view of
CF airway bacterial communities 38]. Each CF patient presented a unique microbiome 40]. The species present tended to vary more “between” than “within” subjects, suggesting
that each CF airway infection is unique, with relatively stable and resilient bacterial
communities 44]. The diversity and species richness of fungal and bacterial communities were significantly
lower in patients with decreased lung function and poor clinical status 39]. The authors observed a strong positive correlation between low species richness
and poor lung function 37]. These findings show the critical relationship between airway bacterial community
structure, disease stage, and clinical state at the time of sample collection 42].

The main microorganisms found in CF airways are the genera Haemophilus, Pseudomonas, Staphylococcus and Stenotrophomonas. Less common are gram-negatives, Streptococcus and Mycobacterium spp45]. Most bacteria of CF airways are difficult to culture using conventional clinical
methods; therefore, molecular approaches may confirm or reveal novel bacteria that
might be related to the pathogenesis of cystic fibrosis. Examples of interest are
the Streptococcus milleri group (Streptococcus anginosus, Streptococcus intermedius, Streptococcus constellatus) 46], Pseudomonas intermedia46], and Gemella species47] (Table 1). Further experiments suggested that these bacteria could act as co-pathogens or
enhance the virulence of conventional CF pathogens 48].

Table 1. Summary of cystic fibrosis microbiota studies; type of sample, technique used and
genus identified

The microbiome in chronic rhinosinusitis

Specimen collection is one of the most important steps in the analysis of remote areas
such as the sinuses. An appropriate collection of the samples is the first step to
perform a meaningful, high quality analysis. It must not be biased by interference
from the nares. Specimen can be tissue, nasal secretions 55] or material sampled by a swab. The use of an endoscope for the sampling during sinus
surgery is advisable 17],18],56], although simple swabs are often used 57] in both healthy and diseased patients 18]. Samples can be collected from various anatomical locations in the nose such as the
inferior turbinate 55], the middle-meatus 56], the ethmoidal sinuses, the sphenoid 18], and the anterior nasal cavity 58]. Mucosal surfaces of the lateral, central, and medial portions of the maxillary sinus
are also collected from locations in the nose 17]. The use of middle-meatus swabs for DNA-based bacterial assays is appropriate for
the detection of multiple bacterial species, including anaerobes, which may be undetected
when swabs are used solely for culture. Based on available cultivation-based studies,
the microbiology of the middle meatus correlates well with pathogenic organism of
CRS, whereas swabs of the nares would not be appropriate as a replacement of middle
meatus swabs in investigations of CRS pathogen 59]. Swabs should not be contaminated by the microorganism of the nares during insertion/retraction
from the middle meatus or sinuses 56]. To avoid contamination by the nasal vestibule, researchers often use appropriate
protective devices such as a sterilized Killian nasal speculum with long leaves 58].

Before the era of culture-independent methods, conventional cultures have implicated
Staphylococcus aureus and coagulase-negative Staphylococcus as principal pathogens in chronic rhinosinusitis (CRS) 60]. The development of culture-independent molecular techniques allowed the detection
of more bacteria 60] and revealed greater biodiversity than conventional culture 56]. Thus, the etiology of CRS may be polymicrobial 55] and the role of anaerobe bacteria may be more prominent than presumed; however, it
is likely that the bacteria detected by culture-dependent techniques still are of
clinical relevance 60].

Using comparative microbiome profiling in a cohort of a small number of not further
defined CRS patients and healthy subjects, it was proposed that the sinus microbiota
of CRS patients exhibit significantly reduced bacterial diversity compared to those
of healthy controls. Abreu et al., found a depletion of multiple phylogenetically
distinct lactic acid bacteria coincident with an increase in the relative abundance
of a single species, Corynebacterium tuberculostearicum17]. These microbe caused goblet cell hyperplasia and mucin hypersecretion in a murine
model of sinus infection. In this model, Lactobacillus sakei represented a potentially protective species 17]. However, the finding of this single species has not been confirmed by others 55],56] (see Table 2). In a larger study, Staphylococcus aureus and Propionibacterium acnes were the most common organisms in CRS (mostly CRSwNP) and controls, respectively
18]. Recently, the investigators detected Staphylococcus aureus, Staphylococcus epidermidis and Propionibacterium acnes as the most prevalent and abundant microorganisms in healthy sinuses 61].

Table 2. Summary of chronic rhinosinusitis microbiota studies; type of sample, technique used
and genus identified

Using culture-independent (qPCR and 16S rRNA gene sequencing) methodologies for pathogen
identification in chronic rhinosinusitis patients, among 57,407 pyrosequences were
generated. The most prevalent ones were from coagulase-negative staphylococci (100%),
21/21 specimens, Corynebacterium spp (not specifically Corynebacterium tuberculostearicum) (85.7%) 18/21, P. acnes (76.2%), 16/21, and Staphylococcus aureus (66.7%) 14/21. Although these authors found significantly different distributions
of 16S rRNA sequences recovered from CRS vs. non-CRS cases, neither richness nor evenness
indices showed statistically significant differences 56]. In another approach using 16S rRNA gene clone sequencing in a terminal restriction
fragment length polymorphism (T-RFLP) analysis, the bacteria present in 70 clinical
samples from 43 CRS patients undergoing endoscopic sinus surgery were characterized;
a total of 48 separate bands were detected. Species belonging to 34 genera were identified
as present by clone sequence analysis. Of the species detected, those within the genera
Pseudomonas, Citrobacter, Haemophilus, Propionibacterium, Staphylococcus, and Streptococcus were found numerically dominant, with Pseudomonas aeruginosa being the most frequently detected species 55]. Another prospective study collected mucosal biopsies from 18 patients undergoing
endoscopic sinus surgery for CRS and 9 control patients with healthy sinuses (indication:
pituitary adenomas) compared swab culture with bTEFAP (bacterial tag-encoded FLX amplicon
pyrosequencing). Standard cultures mainly showed Staphylococcus aureus and Coagulase-negative Staphylococcus aureus, whereas the molecular analysis identified up to 20 predominant organisms per sample.
Staphylococcus aureus was nevertheless detected in about 50%; moreover, they disclosed anaerobic species
with so far unknown impact in CRS, Diaphorobacter and Peptoniphilus. Interestingly, Diaphorobacter is described as a strong biofilm creator 55],60].

Table 2 provides a summary of previous studies related to the microbiome in chronic rhinosinusitis,
including sample size, type of sample, technique used and genus found.

Comparisons of molecular analyses suggest that the detection of microorganisms by
Fluorescence in-situ hybridization (FISH) and culture-dependent techniques is related to the abundance
of an organism, furthermore, cultivation tends to give advantage to rapidly growing
bacteria 18]. The investigators employed conventional cultivation, molecular diagnostics and FISH
to detect Staphylococcus aureus as a standard. They found that FISH analysis had a sensitivity of 78% with a specificity
of 93% compared to the molecular technique 18]. Evidence from high-sensitivity techniques demonstrates that the healthy sinus is
clearly not sterile 18], but shows high diversity of the resident microbiota 17]. The nasal microbiota of healthy subjects mainly consist of members of the phylum
Actinobacteria (e.g., Propionibacterium spp. and Corynebacterium spp.), whereas the phyla Firmicutes (e.g., Staphylococcus spp.) and Proteobacteria (e.g. Enterobacter spp) are less frequent 55],56],60],63]. It appears that the prevalence and abundance of organisms is critical in determining
healthy conditions 18].

Thus, similar to CF, findings in CRS have pointed out that the microbiome is unique
for each individual patient 42]-44] and the community of microbes is diversified 10]. As a general principle, a decreasing bacterial diversity is correlated with disease
severity in CF 37]-39],42],44], whereas CRS patients were characterized by an altered microbial composition and
greater abundance of Staphylococcus aureus56]. There was no single common microbiota profile among patients with similar clinical
conditions in the studies performed so far, although Staphylococcus aureus was prominent in most studies 10],11],68]. Thus, there is a clear need for larger series of well-defined patients sampled and
investigated in an optimal way, also avoiding the interference of recently applied
antibiotics, to establish the correlation between microbe and CRS disease.

Limitations of the current studies

Airway microbiome studies revealed several critical factors, which also may impact
CRS studies. First of all, the inclusion of well-defined patients, using pheno- and
potentially endotypes of upper airway disease 68]-71], and matched controls in meaningful numbers is necessary to draw supportable conclusions.
Furthermore, recent antibiotic treatment within 1 month 44] prior to collection could significantly reduce the diversity of the microbiome in
samples 42],43],56], and contamination by bacteria from other organs such as the skin should be taken
into account 9],27]. Factors which may perturb the collection or evaluation procedures are contaminating
host DNA 40] or RNA, the existence of viruses such as bacteriophages in the samples, which may
impact on the number and genes of microbes 72], and technical issues such as extraction methods (e.g. modified lysostaphin-lysozyme
method to enhance staphylococcus DNA extraction) 41].

Currently, most of the publications in human microbiome studies have spotlighted sequencing
of 16S rRNA in the identification of bacteria. Their results may misjudge the level
of diversity and microbial composition by amplification of chimera and pseudogenes
and/or inappropriate primer selection. Metagenomic shotgun sequencing may avoid these
problems by omitting amplification and allows to detect gene contents of complex microbiota
and to compare functional gene contents between samples, but still may have limitations
as discussed above and in low-microbial burden samples. However, researchers are now
increasingly employing novel techniques to study the human microbiome 25].

Conclusion and perspective of nasal microbiome studies

The new molecular techniques enhance our chance to identify new bacteria within the
nose and nasal cavities; as the pivotal host functions evolved under high microbial
pressure, they will show a very complex network of microbes and thus microbe-host
interactions 41]. On the host side, specific pheno- and endotypes of CRS have been described characterized
by an imbalance of Th1 and Th2 function 71]. In CRSwNP patients, Staphylococcus aureus has been identified to unfold impact on the mucosal immune functions 10],68],70]. The relationship between the microbiome and mucosal immunity may be bidirectional,
with pressure coming from the bacteria and inadequate defense from the host 70]. Research on how specific bacteria impact on the immune response of nasal and sinus
mucosa may shed new light on the pathophysiology of CRS and may result in new strategies
for its treatment.

The manipulation of microbiota or the introduction of specifically healthy microbiota
may prove to be useful for the treatment of inflammatory disease 73]. Staphylococcus aureus and Pseudomonas aeruginosa are principal offenders in the development of persistent severe airway disease in
CRS and CF patients. As bacterial resistance complicates the efficacy of antibiotics,
the use of probiotic bacteria as colonizers and antimicrobial agents that may inhibit
the growth of pathogenic bacteria awaits further development.