Home Medicine Uncovering the hidden villain within the human respiratory microbiome
Article Open Access

Uncovering the hidden villain within the human respiratory microbiome

  • Chun Kiat Lee and Stephen James Bent EMAIL logo
Published/Copyright: August 5, 2014
Download Article (PDF)
Diagnosis
From the journal Diagnosis Volume 1 Issue 3

Abstract

Respiratory tract infection increases the risk of secondary bacterial infection and causes mortality. Despite advances in the field of targeted molecular diagnostics, there are still failed attempts in identifying a valid causative etiological agent in a large proportion of respiratory tract infections. To date, a comprehensive list of human respiratory infection-associated eukaryotic viruses has been identified. However, there has been little progress towards the characterisation of the viruses that infect bacteria (phages), which are capable of mediating the transfer of virulence genes into non-pathogenic bacterial species to cause respiratory tract infections. With the advent of next-generation-sequencing, the application of an unbiased comparative metagenomic survey on the viral communities within the human respiratory tract may reveal to us how the phage virome changes between healthy individuals and respiratory tract infection patients. With this useful information, it will be feasible to develop an alternative phage-based diagnostic panel for respiratory tract infections. The review herein presents the current status of human airway microbiome research and highlights potential gaps which can be translated into research possibilities for future work on respiratory tract infection diagnosis.

Keywords: bacteriophage; metagenome; microbiome; respiratory tract infection; virome

Introduction

Respiratory tract infection (RTI) and its complications (secondary bacterial infections and illnesses such as pneumonia) constitute a significant global health problem [1]. Viruses that infect eukaryotes have been identified as the leading cause of RTIs worldwide. Therefore, a number of eukaryote-infecting viral pathogens have been incorporated into routine diagnostic assays for RTI screening [2, 3]. Despite this, failure to isolate a likely etiological agent is common for many suspected RTI cases [4]. This could be explained by the lack of a reliable broad spectrum diagnostic assay which can target all known or recently emerged respiratory viruses or viruses with hitherto unreported mutations [5]. Hence, a rare or novel viral etiological agent can go undetected and undiagnosed.

Another possibility may be the causative agent responsible for the disease onset is a prokaryote-infecting virus which can exert an indirect pathogenetic effect through its bacterial host. Studies have shown that the bacterial viruses, called bacteriophages or phages, can serve as vehicles to mediate the transfer of virulence genes into non-pathogenic bacterial species during lysogenic conversion [6, 7] or enable the virulence genes to travel between bacterial species in the event of prophage induction [8]. The bacteriophages and their hosts can be shed into the saliva and aspirated into the lower respiratory system to cause lower RTI. Dental caries studies have also shown that periodontal disease patients suffered from frequent RTI episodes due to virulent dental plaque bacteria that may have obtained their virulence genes from phages [9, 10].

Currently, little has been done to learn about the prokaryote-infecting viral biota residing in the human respiratory system and their role in RTI pathogenesis. It is therefore crucial to encourage more virome studies to explore these research gaps in order to broaden our understanding on the possible health impact of bacteriophages in the human airway tract.

In this review, existing data on the human respiratory microbiome have been studied to provide helpful insights regarding future RTI diagnostic work. In addition, a novel diagnostic framework to enhance clinical assessment by physicians on RTI patients is proposed. This could potentially relieve the substantial socioeconomic and medical burden of RTIs.

Human respiratory tract

The human RT is constantly exposed to a host of viral and bacterial pathogens via the nasal and oral cavities through inhalation of airborne particles, ingestion of food and beverages, and routine activities such as talking and yawning. The human respiratory system is divided into the upper (oral cavity, nasal passage, pharynx and larynx) and lower (trachea, bronchi and lungs) airway tracts.

Upper respiratory tract

The nasopharynx and oropharynx are frequently sampled with pernasal flocked swabs to serve as non-invasive proxies for microbiota profiling and RTI diagnostic testing [11, 12]. Commensal inhabitants such as Streptococcus, Staphylococcus aureus and Neisseria species were reported to inhabit in these regions among healthy individuals [13, 14]. The human oral cavity harbours a plethora of microbes. Distinct sites such as the tongue dorsum, palatine tonsils, buccal mucosa, keratinised gingival, hard palate, subgingival, supragingival plaque and saliva, are commonly sampled for periodontal disease pathogens [10, 15]. Microbial studies have demonstrated the presence of common respiratory pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, Epstein–Barr virus and human cytomegalovirus within the oral cavity [16–18]. Consequently, experimental studies on dental care have shown that periodontal disease is associated with an increased risk of pneumonia [19–21]. With the timely arrival of the next-generation-sequencing deep sequencing 16S ribosomal RNA (16S rRNA) gene survey technology, scientists are actively probing into the microbiota of the oral cavity to look for unique bacterial signatures for human diseases [22–24].

Lower respiratory tract

Previously, the lower airway tract was believed to be sterile [25]. However, with the development of culture-independent molecular screening technique, it is now evident that microbes can also be found residing within the lower respiratory tract, albeit at a lower biomass when compared to the upper respiratory tract [14, 26]. There are studies that have demonstrated that the lower airway tract microbiome could be misrepresented due to upper respiratory tract microbial flora contamination during the sample collection process [27, 28]. Therefore, microbial study on the lower airway tract is a fledging area of interest with many challenges. The study experimental design and the sampling techniques used will have to be addressed properly before further studies can be performed on the lower respiratory tract to understand the microbial communities and their relationships.

Respiratory tract infection

Prevalence

Respiratory tract infection is among the top 10 leading causes of death in children, teenagers and the elderly in the United States (US) [29]. Moreover, the high health care costs make RTI among the most costly health diseases, adding to the socioeconomic burden of the world [29]. A study has shown that the health-care expenditure was significantly reduced if the causative pathogen responsible for RTI was detected early [30].

Causative agents

The human airway tract is susceptible to infection by a range of pathogens. The eukaryotic viruses are commonly diagnosed as the first-line pathogens which are responsible for respiratory morbidities and mortalities worldwide [31]. Highly pathogenic virus strains such as the pandemic 1981 H1N1 influenza, H5N1 avian influenza, severe acute respiratory syndrome and the more recent H7N9 avian influenza and Middle East respiratory syndrome are known to cause mortality directly [32–35]. On the other hand, the mild pathogenic virus strains are less likely to cause direct death unless significant pre-existing comorbidities within the patient are triggered or secondary bacterial infections are promoted. To date, there are over 200 known RTI-linked eukaryotic viruses. Influenza viruses, parainfluenzavirus, respiratory syncytial virus, metapneumovirus, rhinovirus, enterovirus, adenovirus, coronavirus and the recently discovered bocavirus have been incorporated into singleplex or multiplex diagnostic assays for RTI diagnosis in the clinical and public health laboratories [2, 3]. Currently, most studies tend to associate eukaryotic virus as the causative agent for RTI [36]. As a result, no prokaryotic virus has been identified as a disease biomarker for RTI diagnosis thus far. Studies have shown that phage-encoded virulence factors can boost the virulence of the non-pathogenic bacteria strain through lysogenic conversion or phage induction [37]. In addition, phage can also infect and kill commensal bacteria, allowing the pathogenic bacteria to thrive. Recently, Duerkop et al. reported the discovery of a composite phage produced by Enterococcus faecalis strain V538 (vancomycin-resistant) that can kill off other commensal strains of E. faecalis (competitors for space and nutrients) within the human gut [38].

Diagnosis

A sensitive and specific diagnostic assay is crucial for accurate RTI diagnosis. A misdiagnosis can lead to a poor prognosis (likelihood of recovery) due to ineffective therapy or lack of prompt treatment. Furthermore, a plethora of viruses are known to cause RTI and each individual virus can have several subtypes, eliciting diverse symptoms and different prognoses. Therefore, a rapid and accurate diagnosis of the causative pathogen is essential in ensuring effective treatment. Conventionally, virus detection is based on immunofluorescence testing or virus culture. These techniques are generally slow with low throughput capability, allowing only one or two viruses to be detected in a single run. In most cases, immunofluorescence testing may have poor sensitivity and exhibit cross-reactivity. Similarly, virus culture is ineffective for rapid diagnosis as some viruses may be slow-growing or uncultivable [39]. To overcome these limitations, rapid multiplexed reverse-transcription polymerase chain reaction (RT-PCR) assays with high sensitivity and specificity have been developed [2, 3]. Commercial assays such as the US Food and Drug Administration approved xTAG® Respiratory Viral Panel (Luminex, TX, USA), Seeplex™ Respiratory Virus Detection system (Seegene, Seoul, South Korea), ResPlex II Panel v2.0 (Qiagen, CA, USA) and FilmArray Respiratory Panel (BioFire Diagnostics, Inc., UT, USA) are available in the market and used by diagnostic laboratories for RTI testing and epidemiology studies. With the arrival of next-generation-sequencing, several studies have begun to make use of this technology for broad spectrum pathogen detection and to discover novel emerging pathogens through established whole-genome-shotgun protocols and metagenomic pipelines [40–43]. These protocols and pipelines can also be modified to sequence and characterise the composition of the prokaryotic viral fraction.

Overview of human airway tract microbiome studies

Existing studies which have performed metagenomic surveillance on the human respiratory system are summarised in Table 1. A majority of these studies are focused exclusively on characterisation of the bacterial communities. The prokaryotic 16S rRNA gene is by far the most common target for bacterial phylogeny and taxonomic analysis in all studies of bacteria and archaea. Also, a number of these investigations collected several proxies from distinct human body sites to obtain reliable information on the respiratory bacterial biota composition. In particular, two whole-genome-shotgun metagenomic studies have conducted niche specialisation survey on multiple distinct respiratory sites (buccal mucosa, hard palate, keratinised gingival, palatine tonsils, saliva, subgingival plaque, supragingival plaque, oropharynx, tongue dorsum and the anterior nares) of 239 healthy individuals [44, 45]. Both studies complemented each other in findings that the distribution of the microbial communities was different even among closely related body habitats.

Table 1

Summary of existing human respiratory microbiome studies.

Human respiratory microbiome studies
Study, yearSubjects, nStudy typeSampling site(s)/Specimen(s)Platform(s)Metagenomic dataMicrobiome
Bogaert et al., 2011 [13]96Body site-specificNasopharynxRoche 45416S rRNABacterial
Charlson et al., 2011 [14]6Body site-specificNasopharynx, oropharynx and bronchusRoche 45416S rRNABacterial
Faust et al., 2012 [44]239Body site-specific1 nasal cavity site and 9 oral cavity sitesRoche 45416S rRNABacterial
Segata et al., 2012 [45]239Body site-specific9 oral cavity sitesRoche 45416S rRNABacterial
Sibley et al., 2011 [46]6Disease-specificSputumRoche 454 and ABI GA16S rRNABacterial
Rudkjobing et al., 2011 [28]5Disease-specificLungABI GA16S rRNABacterial
Cardenas et al., 2012 [47]48Disease-specificOropharynxRoche 45416S rRNABacterial
Delhaes et al., 2012 [48]4Disease-specificSputumRoche 45416S rRNABacterial
Fodor et al., 2012 [49]23Disease-specificSputumRoche 45416S rRNABacterial
Goddard et al., 2012 [27]10Disease-specificLungRoche 45416S rRNABacterial
Iwai et al., 2012 [50]20Disease-specificTongue and oropharynxAffymetrix PhyloChip16S rRNABacterial
Madan et al., 2012 [51]7Disease-specificOropharynxRoche 45416S rRNABacterial
Stressmann et al., 2012 [52]14Disease-specificSputumLI-COR16S rRNABacterial
Cabrera-Rubio et al., 2012 [53]8Disease-specificBronchus and sputumRoche 45416S rRNABacterial
Pragman et al., 2012 [54]32Disease-specificBronchusRoche 45416S rRNABacterial
Charlson et al., 2010 [55]62SmokingNasopharynx and oropharynxRoche 45416S rRNABacterial
Sapkota et al., 2010 [56]20SmokingCigaretteIn-house array and ABI GA16S rRNABacterial
Erb-Downward et al., 2011 [57]14SmokingBronchus and lungRoche 45416S rRNABacterial
Morris et al., 2013 [58]64SmokingMouth and bronchusRoche 45416S rRNABacterial
Willner et al., 2011 [59]19Body site-specificSalivaRoche 454Whole-genomeViral
Pride et al., 2012 [60]5Body site-specificSalivaRoche 454Whole-genome and 16S rRNAViral and bacterial
Pride et al., 2012 [61]4Body site-specificSalivaRoche 454 and ABI GAWhole-genome and CRISPR spacerViral
Robles-Sikisaka et al., 2013 [62]21Body site-specificSalivaIon TorrentWhole-genome and CRISPR spacerViral
Nakamura et al., 2009 [40]3Disease-specificNasopharynxRoche 454Whole-genomeViral
Willner et al., 2009 [11]10Disease-specificSputumRoche 454Whole-genomeViral
Greninger et al., 2010 [41]17Disease-specificNasopharynxIllumina GAWhole-genomeViral and bacterial
Wylie et al., 2012 [63]131Disease-specificNasopharynxIllumina GA and Roche 454Whole-genomeViral
Lysholm et al., 2012 [64]210Disease-specificNasopharynxRoche 454Whole-genomeViral and bacterial

Illumina GA, Illumina genome analyser; ABI GA, ABI genetic analyser; Roche 454, Roche 454 pyrosequencer; LI-COR, LI-COR DNA analyser.

Aside from studies on healthy respiratory system, there are disease-specific studies on obstructive lung diseases such as asthma [47] and cystic fibrosis, chronic obstructive pulmonary disease (COPD) [53, 54] and RTI [50]. A significant number of the disease-specific studies are concentrated on cystic fibrosis [27, 28, 46, 48, 49, 51, 52], signifying the need to redirect research efforts on the less well-studied areas such as asthma, COPD and RTI.

Metagenomic investigations on smokers is a growing field as there has been increasing recognition that tobacco smoking is one of the leading causes of COPD in the developed nations [65]. Current lines of research have shown that tobacco smoking facilitated bacterial acquisition and pathogenic bacteria colonisation [66, 67], along with an increased risk of pulmonary infections [68]. Interestingly, Sapkota et al. have isolated a broad diversity of viable pathogenic bacteria from multiple brands of cigarettes [56]. Furthermore, other studies have also observed significant differences in the airway bacterial communities between smokers and non-smokers [55, 57, 58]. Surprisingly, there has been no attempt to perform similar study on the airway virome. If a positive association between cigarette smoking and viral-induced respiratory infections is established, our scientific understanding on the interplay between viruses (either eukaryotic or prokaryotic) and bacteria on COPD progression could be potentially improved.

Most microbiome studies mentioned thus far have only explored the bacterial taxonomic compositions within the human respiratory airway. Notably, there has been little interest in exploring the viral biota within the respiratory tract. Moreover, the experimental designs of the virome-based studies were less comprehensive when compared to the bacterial studies. All available virome studies (Table 1) collected a single proxy for determining the respiratory virome composition which may not be representative enough for robust and accurate data analysis and inference. Moreover, Faust et al. and Segata et al. findings indicate that distinct microbial communities are found even between body sites with very similar conditions [44, 45]. This observed trend is very likely to be true for the respiratory viral communities as well, especially for the bacteriophages. Therefore, similar multi-niche surveillance studies should be designed for the human respiratory virome to confirm this speculation.

Virome studies on human airway tract and RTI

Healthy individuals

A detailed overview of the virome studies is shown in Table 2. One research group has performed four separate respiratory virome studies on healthy individuals [59–62]. Their primary aim was to examine the role of the prokaryotic viruses in shaping bacterial diversity within a healthy human oral ecosystem [59–61]. The initial investigations found many viral virulence factors and integrase homologs within the human salivary virome [59, 60]. The presence of integrase homologs illustrated that lysogenic phages were predominant in the saliva. These lysogenic phages could serve as potential virulence gene reservoirs, contributing to the development of pathogenic bacteria during lysogenisation. The discovery of the platelet-binding factors (pblA and pblB) within the human oral cavity was another key research finding [59]. These phage-encoded genes can improve Streptococcus miti adherence to the human platelets, thus playing a key role in the pathogenesis of infective endocarditis [69]. Building on the findings, they further isolated a significant proportion of other phage-encoded virulence factors such as the pneumococcal surface proteins (pspA, pspC) and choline-binding proteins (cbpD and cbpE) within the salivary virome [60]. Pneumococcal surface protein A is putatively involved in immune system evasion by inhibiting complement-mediated opsonisation [70, 71]. Defective pspC (also known as cbpA), cbpD and cbpE have also been shown to reduce nasopharyngeal colonisation of the Streptoccous Pneumoniae due to decreased adherence [71, 72]. The group went on to include CRISPR-virus analysis to interrogate the interactions between the phages and their respective bacterial hosts, revealing the availability of a repertoire of CRISPR spacer sequences in bacteria to defend themselves against the mutable prokaryotic viruses [61]. In a more recent study, they compared the virome compositions and CRISPR spacer sequences between healthy volunteers in shared and different households [62]. Their data showed that individuals from the same household shared similar virome composition and CRISPR spacer sequences, suggesting that the environment may have played a role in determining the virome composition within the respiratory tract. Taken together, their findings displayed a complex interplay between the phages and their hosts within the healthy human oral ecosystem. There is likely to be even more complexity in the patterns exhibited by prokaryotic viral communities when factors such as different airway niches and disease states are brought into consideration. Continual research efforts in this emerging field of research should eventually shed light on the influence of phages in respiratory disease progression.

Table 2

Detailed overview of the human respiratory virome studies.

Human respiratory virome studies
Study, yearRecruited subjectsCase, nControl, nData analysisMicrobiomeInsight
Willner et al., 2011 [59]Healthy subjects–19PooledDNA eukaryotic viruses

DNA prokaryotic viruses		The oral cavity is a reservoir of phage SM1-encoded platelet-binding factors. These factors are the virulence determinants for the Streptococcus miti.
Pride et al., 2012 [60]Healthy subjects–5IndividualDNA eukaryotic viruses

DNA prokaryotic viruses

Bacteria		Viral virulence factor and integrase homologs found within the salivary virome.
Pride et al., 2012 [61]Healthy subjects–4IndividualDNA prokaryotic viruses

Bacteria – CRISPR spacers		Newly identified Streptococcus Group II CRISPR spacers suggesting adaptation of the streptococcus to defend against local virulent phage infections.
Robles-Sikisaka et al., 2013 [62]Healthy subjects–21IndividualDNA eukaryotic viruses

DNA prokaryotic viruses

Bacteria – CRISPR spacers		Living environment determines the type of viruses we are exposed to and shaped the viral membership within the oral ecosystem. As with earlier CRISPR study, oral spacer repertoires were specifically adapted to oral viromes.
Nakamura et al., 2009 [40]Seasonal influenza A virus infected children3–IndividualRNA eukaryotic viruses–
Willner et al., 2009 [11]Healthy subjects and cystic fibrosis patients55IndividualDNA eukaryotic viruses

DNA prokaryotic viruses

Bacteria		Separate core sets of phage genomes were found in the airways of cystic fibrosis and healthy subjects.
Greninger et al., 2010 [41]Pandemic influenza A (2009 H1N1) infected patients17–IndividualsDNA and RNA eukaryotic viruses

DNA and RNA prokaryotic viruses

Bacteria		–
Wylie et al., 2012 [63]Febrile children with unexplained fever5081IndividualDNA and RNA eukaryotic virusesData showed that there were more viral sequences found within febrile children. However, presence of viral pathogens within the respiratory tract may not represent disease outcome.
Lysholm et al., 2012 [64]Inpatients (children and adults) with severe lower respiratory tract infections210–PooledDNA and RNA eukaryotic viruses

Bacteria		Viral families such as Paramyxoviridae, Orthomyxoviridae and Picornaviridae constituted 90% of the viral sequences in the pooled severe lower respiratory tract infection samples.

Individuals with a disease state

Most existing disease-specific studies are concentrated on the characterisation of the eukaryotic viral communities. Two studies have attempted to characterise the prokaryotic viral communities to a certain degree although their main research focus were on the eukaryotic viruses [11, 41]. Among the disease-specific studies, there were two comparative studies which differentiate the respiratory virome compositions between the healthy and disease states to identify distinctive virome patterns [11, 63]. Willner et al. have found separate core sets of phage genomes between cystic fibrosis patients and healthy individuals while Wylie et al. have reported that more eukaryotic viral reads can be found in febrile children when compared to afebrile children [11, 63].

The way forward

Deciphering the role of phages in RTI

Evidently, a majority of the metagenome studies have largely focused on bacteria due to the availability of a standardised operating protocol for isolating bacteria and characterising the bacterial 16S rRNA gene. In contrast, virome research on the human airway tract has lagged behind. Virulent eukaryotic viruses are capable of infecting human to cause morbidity and mortality. On the other hand, the prokaryotic virus can infect the bacteria, thus allowing a phage to regulate its bacterial host’s function and survival within the human body. Given the polymicrobial nature of pulmonary infection, it will be advantageous to extend our knowledge to the neglected members of the human airway tract microbiome. Scientists will then have a clearer picture of the multifaceted polymicrobial interactions in human respiratory diseases, and this insight could potentially enhance patient management.

Most virulence factors which enhance fitness, adhesion, colonisation, toxins and invasion are phage-encoded [37, 73]. From previous studies, group A Streptococcus (a bacterium responsible for pharyngitis) was able to acquire toxin genes from phages through lysogenisation [8, 74]. A more recent study has described a pathogenic bacteria strain which made use of phage-encoded antimicrobial-resistant genes to inhibit the growth of others, thus enhancing its own survival in cystic fibrosis patients [75]. Earlier, Willner et al. have shown that distinguishable phage virome compositions were observed between healthy individuals and cystic fibrosis patients [11]. From the salivary virome studies, a variety of phage-encoded virulence factors can be found within the human oral cavity [59–61]. These factors have been demonstrated to be not only important for the survival of the bacteria but also contribute to the pathogenicity of the bacteria which in turn lead to disease progression [70–72]. Therefore, a closer examination of this prokaryotic virus-bacteria mutual beneficial relationship, where phages seem to reinforce bacteria survival and disease progression, is a worthwhile alternative angle on which to focus in the never-ending search for means to improve the clinical management of RTI patients. Presently, no comprehensive viral metagenomic surveillance study has been conducted across healthy populations to establish a reference phage virome profile which is unique to a healthy respiratory tract. Furthermore, we have not yet obtained a complete picture of the disease state phage virome profile among the RTI patients due to the lack of research interest in this relatively new area. More studies on the role of phages in RTI pathogenesis are warranted to fill this dearth of information.

A proposed novel diagnostic panel

We propose a blueprint for the development and eventual application of a prokaryotic virus profiling system to advance RTI diagnosis. First, adequate knowledge on how the phage virome changes between healthy subjects and RTI patients is required. With the advent of whole-genome-shotgun metagenomic technology and the availability of robust bioinformatics tools for data comparison and analysis, a reference baseline phage virome profile can be extracted from a representative healthy population. A multi-site surveillance study approach should be undertaken to ensure the reliability and robustness of the metagenomic data. The baseline healthy airway phage virome profile should then be then compared against the diseased phage virome profiles from the RTI patients (same study protocol as described for the healthy population) to isolate RTI-specific profiles. These diseased profiles could potentially be incorporated into diagnostic assays as unique viral biomarkers for RTI screening.

Discussion

The number of RTI cases around the world is steadily increasing [1]. Studies have shown that the ability to rapidly identify the causative agent for RTI episodes will significantly decrease the length of hospitalisation, mortality rate and antibiotic dispensation [30, 76], significantly reducing hospital expenditures. In view of these social and economical implications, a more immediate goal will be to improve RTI diagnosis by discovering alternative etiological agents. However, our current understanding of the human respiratory virome is still nascent due to the lack of comprehensive viral metagenomic studies on the human respiratory tract.

This review has addressed the potential health implications of prokaryote-infecting viruses on the human respiratory system. We have also specifically highlighted the importance of characterising the phage communities within the human body, which should be considered as a potential area for future research. With the persistent reports of failed attempts in identifying a valid causative etiological agent in RTIs, there is a need to improve on the current RTI diagnostic assays. A blueprint for the eventual application of phage virome profiling in routine RTI diagnosis is advocated.

Corresponding author: Stephen James Bent, PhD, The Robinson Research Institute, School of Paediatrics and Reproductive Health, The University of Adelaide, Adelaide 5005, Australia, e-mail:

  1. Conflict of interest statement

  2. Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article.

  3. Research funding: None declared.

  4. Employment or leadership: None declared.

  5. Employment or leadership: None declared.

  6. Honorarium: None declared.

References

1. The World Health Report 2008. Primary care – now more than ever. Available at: http://www.who.int/whr/2008/en/index.html. Accessed on June 1, 2014.Search in Google Scholar

2. Dabisch-Ruthe M, Vollmer T, Adams O, Knabbe C, Dreier J. Comparison of three multiplex PCR assays for the detection of respiratory viral infections: evaluation of xTAG respiratory virus panel fast assay, RespiFinder 19 assay and RespiFinder SMART 22 assay. BMC Infect Dis 2012;12:163.10.1186/1471-2334-12-163Search in Google Scholar

3. Kim HK, Oh SH, Yun KA, Sung H, Kim MN. Comparison of Anyplex II RV16 with the xTAG respiratory viral panel and Seeplex RV15 for detection of respiratory viruses. J Clin Microbiol 2013;51:1137–41.10.1128/JCM.02958-12Search in Google Scholar

4. Heikkinen T, Jarvinen A. The common cold. Lancet 2003;361:51–9.10.1016/S0140-6736(03)12162-9Search in Google Scholar

5. Lee HK, Lee CK, Loh TP, Chiang D, Koay ES, Tang JW. Missed diagnosis of influenza B virus due to nucleoprotein sequence mutations, Singapore, April 2011. Euro Surveill 2011;16.10.2807/ese.16.33.19943-enSearch in Google Scholar

6. Fortier LC, Sekulovic O. Importance of prophages to evolution and virulence of bacterial pathogens. Virulence 2013;4:354–65.10.4161/viru.24498Search in Google Scholar PubMed PubMed Central

7. Rolain JM, Fancello L, Desnues C, Raoult D. Bacteriophages as vehicles of the resistome in cystic fibrosis. J Antimicrob Chemother 2011;66:2444–7.10.1093/jac/dkr318Search in Google Scholar PubMed

8. Broudy TB, Pancholi V, Fischetti VA. Induction of lysogenic bacteriophage and phage-associated toxin from group a streptococci during coculture with human pharyngeal cells. Infect Immun 2001;69:1440–3.10.1128/IAI.69.3.1440-1443.2001Search in Google Scholar PubMed PubMed Central

9. Adachi M, Ishihara K, Abe S, Okuda K. Professional oral health care by dental hygienists reduced respiratory infections in elderly persons requiring nursing care. Int J Dent Hyg 2007;5:69–74.10.1111/j.1601-5037.2007.00233.xSearch in Google Scholar PubMed

10. Azarpazhooh A, Leake JL. Systematic review of the association between respiratory diseases and oral health. J Periodontol 2006;77:1465–82.10.1902/jop.2006.060010Search in Google Scholar PubMed

11. Willner D, Furlan M, Haynes M, Schmieder R, Angly FE, Silva J, et al. Metagenomic analysis of respiratory tract DNA viral communities in cystic fibrosis and non-cystic fibrosis individuals. PLoS One 2009;4:e7370.10.1371/journal.pone.0007370Search in Google Scholar PubMed PubMed Central

12. Cho CH, Chulten B, Lee CK, Nam MH, Yoon SY, Lim CS, et al. Evaluation of a novel real-time RT-PCR using TOCE technology compared with culture and Seeplex RV15 for simultaneous detection of respiratory viruses. J Clin Virol 2013;57:338–42.10.1016/j.jcv.2013.04.014Search in Google Scholar PubMed PubMed Central

13. Bogaert D, Keijser B, Huse S, Rossen J, Veenhoven R, van Gils E, et al. Variability and diversity of nasopharyngeal microbiota in children: a metagenomic analysis. PLoS One 2011;6:e17035.10.1371/journal.pone.0017035Search in Google Scholar PubMed PubMed Central

14. Charlson ES, Bittinger K, Haas AR, Fitzgerald AS, Frank I, Yadav A, et al. Topographical continuity of bacterial populations in the healthy human respiratory tract. Am J Respir Crit Care Med 2011;184:957–63.10.1164/rccm.201104-0655OCSearch in Google Scholar PubMed PubMed Central

15. Fourrier F, Duvivier B, Boutigny H, Roussel-Delvallez M, Chopin C. Colonization of dental plaque: a source of nosocomial infections in intensive care unit patients. Crit Care Med 1998;26:301–8.10.1097/00003246-199802000-00032Search in Google Scholar PubMed

16. Yildirim S, Yildiz E, Kubar A. TaqMan real-time quantification of Epstein-Barr virus in severe early childhood caries. Eur J Dent 2010;4:28–33.10.1055/s-0039-1697805Search in Google Scholar

17. Kubar A, Saygun I, Ozdemir A, Yapar M, Slots J. Real-time polymerase chain reaction quantification of human cytomegalovirus and Epstein-Barr virus in periodontal pockets and the adjacent gingiva of periodontitis lesions. J Periodontal Res 2005;40:97–104.10.1111/j.1600-0765.2005.00770.xSearch in Google Scholar PubMed

18. Bousbia S, Papazian L, Saux P, Forel JM, Auffray JP, Martin C, et al. Repertoire of intensive care unit pneumonia microbiota. PLoS One 2012;7:e32486.10.1371/journal.pone.0032486Search in Google Scholar PubMed PubMed Central

19. Adachi M, Ishihara K, Abe S, Okuda K, Ishikawa T. Effect of professional oral health care on the elderly living in nursing homes. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:191–5.10.1067/moe.2002.123493Search in Google Scholar PubMed

20. Bergmans DC, Bonten MJ, Gaillard CA, Paling JC, van der Geest S, van Tiel FH, et al. Prevention of ventilator-associated pneumonia by oral decontamination: a prospective, randomized, double-blind, placebo-controlled study. Am J Respir Crit Care Med 2001;164:382–8.10.1164/ajrccm.164.3.2005003Search in Google Scholar PubMed

21. Fourrier F, Cau-Pottier E, Boutigny H, Roussel-Delvallez M, Jourdain M, Chopin C. Effects of dental plaque antiseptic decontamination on bacterial colonization and nosocomial infections in critically ill patients. Intensive Care Med 2000;26:1239–47.10.1007/s001340000585Search in Google Scholar PubMed

22. Chen T, Yu WH, Izard J, Baranova OV, Lakshmanan A, Dewhirst FE. The Human Oral Microbiome Database: a web accessible resource for investigating oral microbe taxonomic and genomic information. Database (Oxford) 2010;2010:baq013.10.1093/database/baq013Search in Google Scholar PubMed PubMed Central

23. Lazarevic V, Whiteson K, Huse S, Hernandez D, Farinelli L, Osteras M, et al. Metagenomic study of the oral microbiota by Illumina high-throughput sequencing. J Microbiol Methods 2009;79:266–71.10.1016/j.mimet.2009.09.012Search in Google Scholar PubMed PubMed Central

24. Ahn J, Yang L, Paster BJ, Ganly I, Morris L, Pei Z, et al. Oral microbiome profiles: 16S rRNA pyrosequencing and microarray assay comparison. PLoS One 2011;6:e22788.10.1371/journal.pone.0022788Search in Google Scholar PubMed PubMed Central

25. Robinson J. Colonization and infection of the respiratory tract: What do we know? Paediatr Child Health 2004;9:21–4.10.1093/pch/9.1.21Search in Google Scholar PubMed PubMed Central

26. Beck JM, Young VB, Huffnagle GB. The microbiome of the lung. Transl Res 2012;160:258–66.10.1016/j.trsl.2012.02.005Search in Google Scholar PubMed PubMed Central

27. Goddard AF, Staudinger BJ, Dowd SE, Joshi-Datar A, Wolcott RD, Aitken ML, et al. Direct sampling of cystic fibrosis lungs indicates that DNA-based analyses of upper-airway specimens can misrepresent lung microbiota. Proc Natl Acad Sci USA 2012;109:13769–74.10.1073/pnas.1107435109Search in Google Scholar PubMed PubMed Central

28. Rudkjobing VB, Thomsen TR, Alhede M, Kragh KN, Nielsen PH, Johansen UR, et al. True microbiota involved in chronic lung infection of cystic fibrosis patients found by culturing and 16S rRNA gene analysis. J Clin Microbiol 2011;49:4352–5.10.1128/JCM.06092-11Search in Google Scholar PubMed PubMed Central

29. National Center for Health Statistics. Health, United States, 2012: With special feature on emergency care. Hyattsville, MD, 2013.Search in Google Scholar

30. Barenfanger J, Drake C, Leon N, Mueller T, Troutt T. Clinical and financial benefits of rapid detection of respiratory viruses: an outcomes study. J Clin Microbiol 2000;38:2824–8.10.1128/JCM.38.8.2824-2828.2000Search in Google Scholar PubMed PubMed Central

31. Shay DK, Holman RC, Newman RD, Liu LL, Stout JW, Anderson LJ. Bronchiolitis-associated hospitalizations among US children, 1980–1996. J Am Med Assoc 1999;282:1440–6.10.1001/jama.282.15.1440Search in Google Scholar PubMed

32. Beigel JH, Farrar J, Han AM, Hayden FG, Hyer R, de Jong MD, et al. Avian influenza A (H5N1) infection in humans. N Engl J Med 2005;353:1374–85.10.1056/NEJMra052211Search in Google Scholar PubMed

33. Ke Y, Wang Y, Zhang W, Huang L, Chen Z. Deaths associated with avian influenza A(H7N9) virus in China. Ann Intern Med 2013;159:159–60.10.7326/0003-4819-159-2-201307160-00669Search in Google Scholar PubMed

34. Booth CM, Matukas LM, Tomlinson GA, Rachlis AR, Rose DB, Dwosh HA, et al. Clinical features and short-term outcomes of 144 patients with SARS in the greater Toronto area. J Am Med Assoc 2003;289:2801–9.10.1001/jama.289.21.JOC30885Search in Google Scholar PubMed

35. Assiri A, McGeer A, Perl TM, Price CS, Al Rabeeah AA, Cummings DA, et al. Hospital outbreak of Middle East respiratory syndrome coronavirus. N Engl J Med 2013;369:407–16.10.1056/NEJMoa1306742Search in Google Scholar PubMed PubMed Central

36. Pavia AT. Viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis. Clin Infect Dis 2011;52(Suppl 4):S284–9.10.1093/cid/cir043Search in Google Scholar PubMed PubMed Central

37. Brussow H, Canchaya C, Hardt WD. Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiol Mol Biol Rev 2004;68:560–602, table of contents.10.1128/MMBR.68.3.560-602.2004Search in Google Scholar PubMed PubMed Central

38. Duerkop BA, Clements CV, Rollins D, Rodrigues JL, Hooper LV. A composite bacteriophage alters colonization by an intestinal commensal bacterium. Proc Natl Acad Sci USA 2012;109: 17621–6.10.1073/pnas.1206136109Search in Google Scholar PubMed PubMed Central

39. Rodriguez RA, Pepper IL, Gerba CP. Application of PCR-based methods to assess the infectivity of enteric viruses in environmental samples. Appl Environ Microbiol 2009;75: 297–307.10.1128/AEM.01150-08Search in Google Scholar PubMed PubMed Central

40. Nakamura S, Yang C-S, Sakon N, Ueda M, Tougan T, Yamashita A, et al. Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach. PLoS One 2009;4:e4219.10.1371/journal.pone.0004219Search in Google Scholar PubMed PubMed Central

41. Greninger AL, Chen EC, Sittler T, Scheinerman A, Roubinian N, Yu G, et al. A metagenomic analysis of pandemic influenza A (2009 H1N1) infection in patients from North America. PLoS One 2010;5:e13381.10.1371/journal.pone.0013381Search in Google Scholar PubMed PubMed Central

42. Mokili JL, Dutilh BE, Lim YW, Schneider BS, Taylor T, Haynes MR, et al. Identification of a novel human papillomavirus by metagenomic analysis of samples from patients with febrile respiratory illness. PLoS One 2013;8:e58404.10.1371/journal.pone.0058404Search in Google Scholar PubMed PubMed Central

43. Alquezar-Planas DE, Mourier T, Bruhn CA, Hansen AJ, Vitcetz SN, Mork S, et al. Discovery of a divergent HPIV4 from respiratory secretions using second and third generation metagenomic sequencing. Sci Rep 2013;3:2468.10.1038/srep02468Search in Google Scholar PubMed PubMed Central

44. Faust K, Sathirapongsasuti JF, Izard J, Segata N, Gevers D, Raes J, et al. Microbial co-occurrence relationships in the human microbiome. PLoS Comput Biol 2012;8:e1002606.10.1371/journal.pcbi.1002606Search in Google Scholar PubMed PubMed Central

45. Segata N, Haake SK, Mannon P, Lemon KP, Waldron L, Gevers D, et al. Composition of the adult digestive tract bacterial microbiome based on seven mouth surfaces, tonsils, throat and stool samples. Genome Biol 2012;13:R42.10.1186/gb-2012-13-6-r42Search in Google Scholar PubMed PubMed Central

46. Sibley CD, Grinwis ME, Field TR, Eshaghurshan CS, Faria MM, Dowd SE, et al. Culture enriched molecular profiling of the cystic fibrosis airway microbiome. PLoS One 2011;6:e22702.10.1371/journal.pone.0022702Search in Google Scholar PubMed PubMed Central

47. Cardenas PA, Cooper PJ, Cox MJ, Chico M, Arias C, Moffatt MF, et al. Upper airways microbiota in antibiotic-naive wheezing and healthy infants from the tropics of rural Ecuador. PLoS One 2012;7:e46803.10.1371/journal.pone.0046803Search in Google Scholar PubMed PubMed Central

48. Delhaes L, Monchy S, Frealle E, Hubans C, Salleron J, Leroy S, et al. The airway microbiota in cystic fibrosis: a complex fungal and bacterial community–implications for therapeutic management. PLoS One 2012;7:e36313.10.1371/journal.pone.0036313Search in Google Scholar PubMed PubMed Central

49. Fodor AA, Klem ER, Gilpin DF, Elborn JS, Boucher RC, Tunney MM, et al. The adult cystic fibrosis airway microbiota is stable over time and infection type, and highly resilient to antibiotic treatment of exacerbations. PLoS One 2012;7:e45001.10.1371/journal.pone.0045001Search in Google Scholar PubMed PubMed Central

50. Iwai S, Fei M, Huang D, Fong S, Subramanian A, Grieco K, et al. Oral and airway microbiota in HIV-infected pneumonia patients. J Clin Microbiol 2012;50:2995–3002.10.1128/JCM.00278-12Search in Google Scholar PubMed PubMed Central

51. Madan JC, Koestler DC, Stanton BA, Davidson L, Moulton LA, Housman ML, et al. Serial analysis of the gut and respiratory microbiome in cystic fibrosis in infancy: interaction between intestinal and respiratory tracts and impact of nutritional exposures. MBio 2012;3.10.1128/mBio.00251-12Search in Google Scholar PubMed PubMed Central

52. Stressmann FA, Rogers GB, van der Gast CJ, Marsh P, Vermeer LS, Carroll MP, et al. Long-term cultivation-independent microbial diversity analysis demonstrates that bacterial communities infecting the adult cystic fibrosis lung show stability and resilience. Thorax 2012;67:867–73.10.1136/thoraxjnl-2011-200932Search in Google Scholar PubMed

53. Cabrera-Rubio R, Garcia-Nunez M, Seto L, Anto JM, Moya A, Monso E, et al. Microbiome diversity in the bronchial tracts of patients with chronic obstructive pulmonary disease. J Clin Microbiol 2012;50:3562–8.10.1128/JCM.00767-12Search in Google Scholar PubMed PubMed Central

54. Pragman AA, Kim HB, Reilly CS, Wendt C, Isaacson RE. The lung microbiome in moderate and severe chronic obstructive pulmonary disease. PLoS One 2012;7:e47305.10.1371/journal.pone.0047305Search in Google Scholar PubMed PubMed Central

55. Charlson ES, Chen J, Custers-Allen R, Bittinger K, Li H, Sinha R, et al. Disordered microbial communities in the upper respiratory tract of cigarette smokers. PLoS One 2010;5:e15216.10.1371/journal.pone.0015216Search in Google Scholar PubMed PubMed Central

56. Sapkota AR, Berger S, Vogel TM. Human pathogens abundant in the bacterial metagenome of cigarettes. Environ Health Perspect 2010;118:351–6.10.1289/ehp.0901201Search in Google Scholar PubMed PubMed Central

57. Erb-Downward JR, Thompson DL, Han MK, Freeman CM, McCloskey L, Schmidt LA, et al. Analysis of the lung microbiome in the “healthy” smoker and in COPD. PLoS One 2011;6:<softenter;e16384.10.1371/journal.pone.0016384Search in Google Scholar PubMed PubMed Central

58. Morris A, Beck JM, Schloss PD, Campbell TB, Crothers K, Curtis JL, et al. Comparison of the respiratory microbiome in healthy nonsmokers and smokers. Am J Respir Crit Care Med 2013;187:1067–75.10.1164/rccm.201210-1913OCSearch in Google Scholar PubMed PubMed Central

59. Willner D, Furlan M, Schmieder R, Grasis JA, Pride DT, Relman DA, et al. Metagenomic detection of phage-encoded platelet-binding factors in the human oral cavity. Proc Natl Acad Sci USA 2011;108 (Suppl 1):4547–53.10.1073/pnas.1000089107Search in Google Scholar PubMed PubMed Central

60. Pride DT, Salzman J, Haynes M, Rohwer F, Davis-Long C, White RA, 3rd, et al. Evidence of a robust resident bacteriophage population revealed through analysis of the human salivary virome. ISME J 2012;6:915–26.10.1038/ismej.2011.169Search in Google Scholar PubMed PubMed Central

61. Pride DT, Salzman J, Relman DA. Comparisons of clustered regularly interspaced short palindromic repeats and viromes in human saliva reveal bacterial adaptations to salivary viruses. Environ Microbiol 2012;14:2564–76.10.1111/j.1462-2920.2012.02775.xSearch in Google Scholar PubMed PubMed Central

62. Robles-Sikisaka R, Ly M, Boehm T, Naidu M, Salzman J, Pride DT. Association between living environment and human oral viral ecology. ISME J 2013;7:1710–24.10.1038/ismej.2013.63Search in Google Scholar PubMed PubMed Central

63. Wylie KM, Mihindukulasuriya KA, Sodergren E, Weinstock GM, Storch GA. Sequence analysis of the human virome in febrile and afebrile children. PLoS One 2012;7:e27735.10.1371/journal.pone.0027735Search in Google Scholar PubMed PubMed Central

64. Lysholm F, Wetterbom A, Lindau C, Darban H, Bjerkner A, Fahlander K, et al. Characterization of the viral microbiome in patients with severe lower respiratory tract infections, using metagenomic sequencing. PLoS One 2012;7:e30875.10.1371/journal.pone.0030875Search in Google Scholar PubMed PubMed Central

65. Rycroft CE, Heyes A, Lanza L, Becker K. Epidemiology of chronic obstructive pulmonary disease: a literature review. Int J Chron Obstruct Pulmon Dis 2012;7:457–94.10.2147/COPD.S32330Search in Google Scholar PubMed PubMed Central

66. Brook I. The impact of smoking on oral and nasopharyngeal bacterial flora. J Dent Res 2011;90:704–10.10.1177/0022034510391794Search in Google Scholar PubMed

67. Brook I. Effects of exposure to smoking on the microbial flora of children and their parents. Int J Pediatr Otorhinolaryngol 2010;74:447–50.10.1016/j.ijporl.2010.01.006Search in Google Scholar PubMed

68. Aronson MD, Weiss ST, Ben RL, Komaroff AL. Association between cigarette smoking and acute respiratory tract illness in young adults. J Am Med Assoc 1982;248:181–3.10.1001/jama.1982.03330020025023Search in Google Scholar

69. Mitchell J, Siboo IR, Takamatsu D, Chambers HF, Sullam PM. Mechanism of cell surface expression of the Streptococcus mitis platelet binding proteins PblA and PblB. Mol Microbiol 2007;64:844–57.10.1111/j.1365-2958.2007.05703.xSearch in Google Scholar PubMed

70. Kadioglu A, Weiser JN, Paton JC, Andrew PW. The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol 2008;6:288–301.10.1038/nrmicro1871Search in Google Scholar PubMed

71. Ogunniyi AD, LeMessurier KS, Graham RM, Watt JM, Briles DE, Stroeher UH, et al. Contributions of pneumolysin, pneumococcal surface protein A (PspA), and PspC to pathogenicity of Streptococcus pneumoniae D39 in a mouse model. Infect Immun 2007;75:1843–51.10.1128/IAI.01384-06Search in Google Scholar PubMed PubMed Central

72. Gosink KK, Mann ER, Guglielmo C, Tuomanen EI, Masure HR. Role of novel choline binding proteins in virulence of Streptococcus pneumoniae. Infect Immun 2000;68:5690–5.10.1128/IAI.68.10.5690-5695.2000Search in Google Scholar PubMed PubMed Central

73. Wagner PL, Waldor MK. Bacteriophage control of bacterial virulence. Infect Immun 2002;70:3985–93.10.1128/IAI.70.8.3985-3993.2002Search in Google Scholar PubMed PubMed Central

74. Banks DJ, Lei B, Musser JM. Prophage induction and expression of prophage-encoded virulence factors in group A Streptococcus serotype M3 strain MGAS315. Infect Immun 2003;71: 7079–86.10.1128/IAI.71.12.7079-7086.2003Search in Google Scholar PubMed PubMed Central

75. Fancello L, Desnues C, Raoult D, Rolain JM. Bacteriophages and diffusion of genes encoding antimicrobial resistance in cystic fibrosis sputum microbiota. J Antimicrob Chemother 2011;66:2448–54.10.1093/jac/dkr315Search in Google Scholar PubMed

76. Woo PC, Chiu SS, Seto WH, Peiris M. Cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients. J Clin Microbiol 1997;35:1579–81.10.1128/jcm.35.6.1579-1581.1997Search in Google Scholar PubMed PubMed Central

Received: 2014-6-17
Accepted: 2014-7-6
Published Online: 2014-8-5
Published in Print: 2014-9-1

©2014, Stephen James Bent et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Less is more, but do not throw out the baby with the bathwater either!
  4. Reviews
  5. Uncovering the hidden villain within the human respiratory microbiome
  6. The role of leukotriene receptor antagonists in exercise induced bronchoconstriction in children
  7. Original Articles
  8. Developing checklists to prevent diagnostic error in Emergency Room settings
  9. Framingham Risk Score for the prediction of coronary artery disease in patients with chronic rheumatic heart disease
  10. A survey of doctors reveals that few laboratory tests are of primary importance at the Emergency Department
  11. Case Report
  12. The bias of the question posed: a diagnostic “invisible gorilla”
  13. Letter to the Editor
  14. Thrombophilia testing and diagnostic dilemma – a tertiary centre experience
https://doi.org/10.1515/dx-2014-0039
Keywords for this article
bacteriophage; metagenome; microbiome; respiratory tract infection; virome
Creative Commons
BY-NC-ND 3.0

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Less is more, but do not throw out the baby with the bathwater either!
  4. Reviews
  5. Uncovering the hidden villain within the human respiratory microbiome
  6. The role of leukotriene receptor antagonists in exercise induced bronchoconstriction in children
  7. Original Articles
  8. Developing checklists to prevent diagnostic error in Emergency Room settings
  9. Framingham Risk Score for the prediction of coronary artery disease in patients with chronic rheumatic heart disease
  10. A survey of doctors reveals that few laboratory tests are of primary importance at the Emergency Department
  11. Case Report
  12. The bias of the question posed: a diagnostic “invisible gorilla”
  13. Letter to the Editor
  14. Thrombophilia testing and diagnostic dilemma – a tertiary centre experience
Sign up now to receive a 20% welcome discount
Institutional Access
How does access work?
Have an idea on how to improve our website?
Please write us.
© 2026 De Gruyter Brill
Downloaded on 27.1.2026 from https://www.degruyterbrill.com/document/doi/10.1515/dx-2014-0039/html
Scroll to top button