Uncovering the hidden villain within the human ...

Diagnosis 2014; 1(3): 203–212

Review

Open Access

Chun Kiat Lee and Stephen James Bent*

Uncovering the hidden villain within the human respiratory microbiome 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. DOI 10.1515/dx-2014-0039 Received June 17, 2014; accepted July 6, 2014; previously published online August 5, 2014

*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: [email protected] Chun Kiat Lee: School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, SA, Australia; and Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital, Singapore

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

©2014, Stephen James Bent et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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204 Lee and Bent: Hidden villain within the human respiratory microbiome 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.

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

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

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


Lee and Bent: Hidden villain within the human respiratory microbiome 205

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 (vancomycinresistant) 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-genomeshotgun 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. 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



Body site-specific Body site-specific Body site-specific Body site-specific Disease-specific Disease-specific Disease-specific Disease-specific Disease-specific Disease-specific Disease-specific Disease-specific Disease-specific Disease-specific Disease-specific Smoking Smoking Smoking Smoking Body site-specific Body site-specific Body site-specific Body site-specific Disease-specific Disease-specific Disease-specific Disease-specific Disease-specific

16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA Whole-genome Whole-genome and 16S rRNA Whole-genome and CRISPR spacer Whole-genome and CRISPR spacer Whole-genome Whole-genome Whole-genome Whole-genome Whole-genome

Metagenomic data

Roche 454 Roche 454 Roche 454 Roche 454 Roche 454 and ABI GA ABI GA Roche 454 Roche 454 Roche 454 Roche 454 Affymetrix PhyloChip Roche 454 LI-COR Roche 454 Roche 454 Roche 454 In-house array and ABI GA Roche 454 Roche 454 Roche 454 Roche 454 Roche 454 and ABI GA Ion Torrent Roche 454 Roche 454 Illumina GA Illumina GA and Roche 454 Roche 454

Platform(s)

Nasopharynx Nasopharynx, oropharynx and bronchus 1 nasal cavity site and 9 oral cavity sites 9 oral cavity sites Sputum Lung Oropharynx Sputum Sputum Lung Tongue and oropharynx Oropharynx Sputum Bronchus and sputum Bronchus Nasopharynx and oropharynx Cigarette Bronchus and lung Mouth and bronchus Saliva Saliva Saliva Saliva Nasopharynx Sputum Nasopharynx Nasopharynx Nasopharynx

Sampling site(s)/Specimen(s)

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

96 6 239 239 6 5 48 4 23 10 20 7 14 8 32 62 20 14 64 19 5 4 21 3 10 17 131 210

Subjects, Study type n

Bogaert et al., 2011 [13] Charlson et al., 2011 [14] Faust et al., 2012 [44] Segata et al., 2012 [45] Sibley et al., 2011 [46] Rudkjobing et al., 2011 [28] Cardenas et al., 2012 [47] Delhaes et al., 2012 [48] Fodor et al., 2012 [49] Goddard et al., 2012 [27] Iwai et al., 2012 [50] Madan et al., 2012 [51] Stressmann et al., 2012 [52] Cabrera-Rubio et al., 2012 [53] Pragman et al., 2012 [54] Charlson et al., 2010 [55] Sapkota et al., 2010 [56] Erb-Downward et al., 2011 [57] Morris et al., 2013 [58] Willner et al., 2011 [59] Pride et al., 2012 [60] Pride et al., 2012 [61] Robles-Sikisaka et al., 2013 [62] Nakamura et al., 2009 [40] Willner et al., 2009 [11] Greninger et al., 2010 [41] Wylie et al., 2012 [63] Lysholm et al., 2012 [64]

Study, year

Human respiratory microbiome studies

Table 1 Summary of existing human respiratory microbiome studies.

Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Viral Viral and bacterial Viral Viral Viral Viral Viral and bacterial Viral Viral and bacterial

Microbiome

206 Lee and Bent: Hidden villain within the human respiratory microbiome


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.

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].


208 Lee and Bent: Hidden villain within the human respiratory microbiome Table 2 Detailed overview of the human respiratory virome studies. Human respiratory virome studies Study, year

Recruited subjects

Case, n Control, Data n analysis

Microbiome

Willner et al., 2011 [59]

Healthy subjects

–

Pride et al., 2012 [60]

Healthy subjects

–

5 Individual

Pride et al., 2012 [61]

Healthy subjects

–

4 Individual

Robles-Sikisaka Healthy subjects et al., 2013 [62]

–

21 Individual

Seasonal influenza A virus infected children Healthy subjects and cystic fibrosis patients

3

– Individual

DNA eukaryotic viruses The oral cavity is a reservoir of DNA prokaryotic viruses phage SM1-encoded plateletbinding factors. These factors are the virulence determinants for the Streptococcus miti. DNA eukaryotic viruses Viral virulence factor and DNA prokaryotic viruses integrase homologs found within Bacteria the salivary virome. DNA prokaryotic viruses Newly identified Streptococcus Bacteria – CRISPR Group II CRISPR spacers spacers suggesting adaptation of the streptococcus to defend against local virulent phage infections. DNA eukaryotic viruses Living environment determines the DNA prokaryotic viruses type of viruses we are exposed to Bacteria – CRISPR and shaped the viral membership spacers within the oral ecosystem. As with earlier CRISPR study, oral spacer repertoires were specifically adapted to oral viromes. RNA eukaryotic viruses –

Nakamura et al., 2009 [40] Willner et al., 2009 [11]

5

Greninger et al., Pandemic influenza A 2010 [41] (2009 H1N1) infected patients

17

Wylie et al., 2012 [63]

50

210

Febrile children with unexplained fever

Lysholm et al., Inpatients (children 2012 [64] and adults) with severe lower respiratory tract infections

19 Pooled

Insight

5 Individual DNA eukaryotic viruses Separate core sets of phage DNA prokaryotic viruses genomes were found in the Bacteria airways of cystic fibrosis and healthy subjects. – Individuals DNA and RNA eukaryotic – viruses DNA and RNA prokaryotic viruses Bacteria 81 Individual DNA and RNA eukaryotic Data showed that there were viruses more viral sequences found within febrile children. However, presence of viral pathogens within the respiratory tract may not represent disease outcome. – Pooled DNA and RNA eukaryotic Viral families such viruses as Paramyxoviridae, Bacteria Orthomyxoviridae and Picornaviridae constituted 90% of the viral sequences in the pooled severe lower respiratory tract infection samples.

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.

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.

A proposed novel diagnostic panel

Conflict of interest statement

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

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.

Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared.


210 Lee and Bent: Hidden villain within the human respiratory microbiome

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