Association between rhinovirus species and nasopharyngeal microbiota in infants with severe bronchiolitis.

To the Editor:

Bronchiolitis is the leading cause of hospitalization in US infants. Rhinovirus (RV) is the second most common cause of severe bronchiolitis (ie, bronchiolitis requiring hospitalization) following respiratory syncytial virus (RSV). RVs are RNA viruses consisting of more than 160 genotypes that are classified into 3 species (RV-A, RV-B, and RV-C). RV-A and RV-C are more frequently found than RV-B in children with acute respiratory infections (ARIs) and wheezing illnesses.3, 4 Emerging evidence suggests a complex interplay between viral infection, airway microbes, and host immune response in the pathobiology of ARI. Studies have shown that RV infection in children is associated with increased detection of pathogenic bacteria in the airways.5, 6 Furthermore, detection of RV together with specific airway pathogens (eg, Moraxella catarrhalis) is associated with increased ARI and asthma symptoms. Recently, RV-A and RV-C were reported to differentially associate with detection of pathogenic bacteria in school-age children. However, no study has investigated the relationships between rhinovirus species and airway microbiota in infants, let alone infants with bronchiolitis. To address the knowledge gap, we examined the association between rhinovirus species and the nasopharyngeal airway microbiota determined by 16S rRNA gene sequencing in 774 infants with severe bronchiolitis.

This was a post hoc analysis of data from the 35th Multicenter Airway Research Collaboration (MARC-35) cohort study—a multicenter prospective cohort study of infants hospitalized for bronchiolitis. The details of the study design, setting, virus and microbiota measurements, and analysis are described in this article's Online Repository at www.jacionline.org. Briefly, 1016 infants (age <1 year) hospitalized for bronchiolitis were enrolled in 17 sites across 14 US states (see Table E1 in this article's Online Repository at www.jacionline.org). Bronchiolitis was defined according to the American Academy of Pediatrics guidelines. The institutional review boards at participating sites approved the study. Informed consent was obtained from the infants' parent or legal guardian. Nasopharyngeal samples were collected within 24 hours of hospitalization and stored at −80°C locally. These samples were processed and tested for 17 respiratory pathogens by real-time PCR and for microbiota using 16S rRNA gene sequencing at Baylor College of Medicine (Houston, Tex). Singleplex real-time PCR was used to detect RV, and positive specimens were further genotyped by using molecular typing assay at the University of Wisconsin (Madison, Wis). By using partitioning around medoids unsupervised clustering with the use of weighted UniFrac distance, 4 distinct nasopharyngeal microbiota profiles were derived as previously described. In the current analysis, we grouped infants into 4 mutually exclusive virus categories: solo RSV (reference), RV-A, RV-B, and RV-C. We tested the association between these virus categories and nasopharyngeal microbiota profiles by constructing multinomial logistic regression model adjusting for 8 covariates. Data were analyzed using R version 3.4.4.

Table E1

Principal investigators at the 17 participating sites in MARC-35

Amy D. Thompson, MD	Alfred I. duPont Hospital for Children, Wilmington, Del
Federico R. Laham, MD, MS	Arnold Palmer Hospital for Children, Orlando, Fla
Jonathan M. Mansbach, MD, MPH	Boston Children's Hospital, Boston, Mass
Vincent J. Wang, MD, MHA	Children's Hospital of Los Angeles, Los Angeles, Calif
Michelle B. Dunn, MD	Children's Hospital of Philadelphia, Philadelphia, Pa
Juan C. Celedon, MD, DrPH	Children's Hospital of Pittsburgh, Pittsburgh, Pa
Michael Gomez, MD, MS-HCA, and Nancy Inhofe, MD	The Children's Hospital at St Francis, Tulsa, Okla
Brian M. Pate, MD, and Henry T. Puls, MD	The Children's Mercy Hospital & Clinics, Kansas City, Mo
Stephen J. Teach, MD, MPH	Children's National Medical Center, Washington, DC
Richard T. Strait, MD	Cincinnati Children's Hospital and Medical Center, Cincinnati, Ohio
Ilana Waynik, MD	Connecticut Children's Medical Center, Hartford, Conn
Sujit Iyer, MD	Dell Children's Medical Center of Central Texas, Austin, Tex
Michelle D. Stevenson, MD, MS	Kosair Children's Hospital, Louisville, Ky
Wayne G. Schreffler, MD, PhD, and Ari R. Cohen, MD	Massachusetts General Hospital, Boston, Mass
Anne K Beasley, MD	Phoenix Children's Hospital, Phoenix, Ariz
Thida Ong, MD	Seattle Children's Hospital, Seattle, Wash
Charles G. Macias, MD, MPH	Texas Children's Hospital, Houston, Tex

Of 1016 enrolled infants, 774 were in 1 of the 4 prespecified virus categories (580 RSV-only, 91 RV-A, 12 RV-B, and 91 RV-C) and had high-quality microbiota data; they comprised the analytic sample. Overall, the median age was 2.9 months (interquartile range, 1.6-5.3), 60% were male, and 16% infants received intensive care therapy. Compared with infants with RSV-only, those with RV-A or RV-C were older and more likely to have previous breathing problems (P < .001; see Table E2 in this article's Online Repository at www.jacionline.org).

Table E2

Characteristics of 774 infants hospitalized for bronchiolitis by RV category

Characteristic	RV				P value
	RSV-only (n = 580)	RV-A (n = 91)	RV-B (n = 12)	RV-C (n = 91)
Age (mo), median (IQR)	2.7 (1.5-4.8)	3.1 (1.9-5.7)	3.0 (2.1-4.7)	4.4 (2.3-7.4)	<.001
Female sex	249 (42.9)	30 (33.0)	6 (50.0)	27 (29.7)	.04
Race/ethnicity					.38
Non-Hispanic white	267 (46.0)	33 (36.3)	5 (41.7)	36 (39.6)
Non-Hispanic black	126 (21.7)	20 (22.0)	4 (33.3)	26 (28.6)
Hispanic	162 (27.9)	36 (39.6)	3 (25.0)	25 (27.5)
Other	25 (4.3)	2 (2.2)	0 (0)	4 (4.4)
Parental history of asthma	190 (32.8)	35 (38.5)	4 (33.3)	35 (38.5)	.54
Maternal smoking during pregnancy	84 (14.5)	14 (15.4)	1 (8.3)	13 (14.3)	.48
C-section delivery	214 (36.9)	25 (27.5)	3 (25.0)	34 (37.4)	.07
Prematurity (32-37 wk)	104 (17.9)	18 (19.8)	1 (8.3)	21 (23.1)	.51
Low birth weight (<2.3 kg)	33 (5.7)	8 (8.8)	0 (0)	8 (8.8)	.58
Sibling in the household	457 (78.8)	80 (87.9)	12 (100)	67 (73.6)	.03
Mostly breast-fed during the first 3 mo of life	248 (42.8)	37 (40.7)	7 (58.3)	33 (36.3)	.57
History of a breathing problem	76 (13.1)	21 (23.1)	2 (16.7)	31 (34.1)	<.001
Lifetime history of systemic antibiotic use	153 (26.4)	27 (29.7)	3 (25.0)	32 (35.2)	.36
Lifetime history of corticosteroid use	62 (10.7)	11 (12.1)	3 (25.0)	21 (23.1)	.005
Detected pathogens
RSV	580 (100)	52 (57.1)	10 (83.3)	47 (51.6)	<.001
RV	0 (0)	91 (100)	12 (100.0)	91 (100)	<.001
Other pathogen∗	0 (0)	22 (24.2)	2 (16.7)	24 (26.4)	<.001
Clinical outcomes
Intensive care therapy†	93 (16.0)	13 (14.3)	3 (25.0)	13 (14.3)	.78
Hospital length of stay (d), median (IQR)	2.0 (1.0-3.0)	2.0 (1.0-3.0)	1.5 (1.0-3.3)	2.0 (1.0-2.5)	.09

IQR, Interquartile range.

Data are n (%) of infants unless otherwise indicated.

Adenovirus, bocavirus, Bordetella pertussis, enterovirus, human coronavirus NL63, OC43, 229E, or HKU1, human metapneumovirus, influenza A or B virus, Mycoplasma pneumoniae, parainfluenza virus 3.

Defined as admission to intensive care unit or use of mechanical ventilation (continuous positive airway pressure or intubation).

Across the virus categories, there was a significant difference in the likelihood of nasopharyngeal microbiota profiles (P < .001; see Table E3 in this article's Online Repository at www.jacionline.org). For example, while infants with RSV had the highest likelihood of Streptococcus-dominant profile (the reference virus and microbiota profile), those with RV-A had the highest likelihood of Haemophilus-dominant profile (Fig 1 , A), corresponding to an adjusted relative rate ratio of 5.67 (95% CI, 2.76-11.67; P < .001; Table I ). In contrast, infants with RV-C were more likely to have Moraxella-dominant profile than Streptococcus-dominant profile (adjusted relative rate ratio, 2.69; 95% CI, 1.39-5.20; P = .003). Similarly, at the genus-level (Fig 1, B; see Table E3 in this article's Online Repository at www.jacionline.org), compared with infants with RSV-only, those with any RV species had lower relative abundance of Streptococcus (P = .002) and those with RV-A had a higher abundance of Haemophilus (P = .002).

Table E3

Nasopharyngeal microbiota of infants hospitalized for bronchiolitis by respiratory virus category

Characteristic	RV				P value
	RSV-only (n = 580)	RV-A (n = 91)	RV-B (n = 12)	RV-C (n = 91)
Richness
No. of genera, median (IQR)	17 (10-25)	13 (7-23)	14 (7-24)	15 (8-22)	.10
Alpha-diversity
Shannon index, median (IQR)	0.96 (0.58-1.49)	0.94 (0.41-1.39)	0.67 (0.14-1.34)	0.91 (0.57-1.28)	.20
Microbiota profiles					<.001
Haemophilus-dominant profile	83 (14.3)	30 (33.0)	3 (25.0)	17 (18.7)
Moraxella-dominant profile	119 (20.5)	17 (18.7)	4 (33.3)	27 (29.7)
Mixed profile	176 (30.3)	31 (34.1)	4 (33.3)	28 (30.8)
Streptococcus-dominant profile	202 (34.8)	13 (14.3)	1 (8.3)	19 (20.9)
Relative abundance of 10 most abundant genera, mean ± SD
Streptococcus	0.35 ± 0.31	0.23 ± 0.26	0.17 ± 0.25	0.29 ± 0.30	.002∗
Moraxella	0.27 ± 0.33	0.31 ± 0.33	0.42 ± 0.42	0.36 ± 0.36	.10∗
Haemophilus	0.16 ± 0.28	0.30 ± 0.36	0.29 ± 0.39	0.19 ± 0.29	.002∗
Prevotella	0.03 ± 0.07	0.02 ± 0.06	0.01 ± 0.01	0.02 ± 0.06	.77∗
Neisseria	0.02 ± 0.07	0.02 ± 0.07	0.02 ± 0.04	0.02 ± 0.10	.99∗
Staphylococcus	0.03 ± 0.10	0.01 ± 0.07	0.01 ± 0.02	0.02 ± 0.08	.72∗
Corynebacterium	0.02 ± 0.08	0.00 ± 0.01	0.00 ± 0.01	0.00 ± 0.02	.10∗
Alloprevotella	0.01 ± 0.05	0.02 ± 0.06	0.00 ± 0.01	0.01 ± 0.06	.75∗
Veillonella	0.01 ± 0.03	0.01 ± 0.02	0.00 ± 0.00	0.01 ± 0.02	.17∗
Gemella	0.01 ± 0.04	0.01 ± 0.02	0.00 ± 0.00	0.01 ± 0.02	.61∗

IQR, Interquartile range.

Benjamini-Hochberg false-discovery rate–adjusted P value accounting for multiple comparisons.

Fig 1

Between-virus difference in nasopharyngeal microbiota in infants hospitalized for bronchiolitis. A, Between the 4 virus categories, the proportion of nasopharyngeal microbiota profiles differed. For example, compared with infants with RSV-only bronchiolitis, those with RV-A infection were more likely to have a Haemophilus-dominant, mixed, or Moraxella-dominant profile than a Streptococcus-dominant profile. Infants with RV-C infection were more likely to have a Moraxella-dominant profile. P values were derived from adjusted multinomial logistic regression model. Corresponding relative rate ratios are presented in Table I. *P < .05. B, Between the 4 virus categories, the distribution of relative abundance of 3 most common genera in the nasopharyngeal microbiota differed. Data are presented using violin plots, which are boxplots with a rotated kernel density plot on each side. P values adjusted for multiple comparisons are presented in Table E3.

Table I

Unadjusted and adjusted associations of respiratory viruses (exposure) with nasopharyngeal microbiota profiles (outcome) in infants hospitalized for bronchiolitis

Model and virus category	Microbiota profile
	Haemophilus-dominant (n = 133)	P value	Moraxella-dominant (n = 167)	P value	Mixed profile (n = 239)	P value	Streptococcus-dominant (n = 235)
	RRR (95% CI)		RRR (95% CI)		RRR (95% CI)		RRR (95% CI)
Unadjusted model
RSV-only (n = 580)	Reference		Reference		Reference		Reference
RV-A (n = 91)	5.62 (2.79-11.30)	<.001	2.22 (1.04-4.73)	.04	2.74 (1.39-5.39)	.004	Reference
RV-B (n = 12)	7.30 (0.75-71.21)	.09	6.79 (0.75-61.46)	.09	4.59 (0.51-41.50)	.17	Reference
RV-C (n = 91)	2.18 (1.08-4.40)	.03	2.41 (1.29-4.53)	.006	1.69 (0.91-3.13)	.09	Reference
Adjusted model∗
RSV-only (n = 580)	Reference		Reference		Reference		Reference
RV-A (n = 91)	5.67 (2.76-11.67)	<.001	2.26 (1.05-4.89)	.04	2.74 (1.38-5.44)	.004	Reference
RV-B (n = 12)	7.50 (0.74-76.08)	.09	5.72 (0.62-52.71)	.12	4.73 (0.52-43.04)	.17	Reference
RV-C (n = 91)	1.81 (0.86-3.81)	.12	2.69 (1.39-5.20)	.003	1.57 (0.83-2.96)	.17	Reference

RRR, Relative rate ratio.

Multinomial logistic regression model adjusting for 8 patient-level covariates (age, sex, race/ethnicity, gestational age, siblings in the household, breast-feeding, history of breathing problems, and lifetime history of systemic antibiotic use). RSV-only infection was used as the reference of exposure (virus category), and Streptococcus-dominant microbiota profile was used as the reference for the outcome (nasopharyngeal microbiota profile).

Earlier studies reported that RV-C infection is associated with higher risks of subsequent ARI in young children and that enrichment of Moraxella abundance in the upper airways is related to higher frequency of ARIs. Furthermore, a recent analysis from RhinoGen study (310 children [aged 4-12 years] with or without asthma, using quantitative PCR for 3 bacteria) reported that RV-A and RV-C are differentially associated with increased quantity of H influenzae, M catarrhalis, and S pneumoniae. Our observations—for example, the association between RV-C and higher likelihood of Moraxella-dominant microbiota— corroborate these earlier findings, and extend them by applying 16S rRNA gene sequencing to the airway samples of a large multicenter prospective cohort of infants with severe bronchiolitis.

The underlying mechanisms of the virus-microbiota relationships are beyond the scope of our data. The observed associations may be causal—that is, specific respiratory virus species (eg, RV-C) alters the airway microbiota. Alternatively, unique microbiota profiles in conjunction with airway immune response might have contributed to susceptibility to specific virus infection. These potential mechanisms are not mutually exclusive. Despite this complexity, the identification of the association between specific virus species and airway microbiota in infants with bronchiolitis is important given their relation to subsequent respiratory health in children.

Our study has potential limitations. First, the study design precluded us from examining the relation between the temporal pattern of airway microbiota and respiratory health in children. To address this question, the cohort is currently being followed longitudinally for 6+ years with serial examinations of microbiota. Second, the current study did not have healthy controls. However, the study aim was to determine the association of virus species with microbiota among infants with bronchiolitis. Finally, although the study cohort comprised a racially/ethnically diverse US sample of infants, we must generalize the inferences cautiously beyond infants with severe bronchiolitis. Regardless, our data are highly relevant for 130,000 US children hospitalized with bronchiolitis each year.

In summary, on the basis of this multicenter prospective cohort study of infants with severe bronchiolitis, we observed that compared with infants with RSV-only infection, infants with RV-A or RV-C infection had distinct nasopharyngeal microbiota profiles—for example, those with RV-C infection had a higher likelihood of Moraxella-dominant microbiota profile, whereas those with RV-A infection had a higher likelihood of Haemophilus-dominant profile. Although causal inferences remain premature, our data should advance research into delineating the complex interrelations between respiratory viruses, airway microbiome, and respiratory outcomes in children.

Table E4

Characteristics of 774 infants hospitalized for bronchiolitis by nasopharyngeal microbiota profiles

Characteristic	Microbiota profile				P value
	Haemophilus-dominant (n = 133)	Moraxella-dominant (n = 167)	Mixed profile (n = 239)	Streptococcus-dominant (n = 235)
Age (mo), median (IQR)	3.9 (2.1-7.6)	2.9 (1.5-5.4)	2.9 (1.7-4.8)	2.5 (1.3-4.3)	<.001
Female sex	53 (39.8)	71 (42.5)	95 (39.7)	93 (39.6)	.93
Race/ethnicity
Non-Hispanic white	49 (36.8)	70 (41.9)	100 (41.8)	122 (51.9)	.20
Non-Hispanic black	29 (21.8)	42 (25.1)	60 (25.1)	45 (19.1)
Hispanic	49 (36.8)	48 (28.7)	68 (28.5)	61 (26.0)
Other	6 (4.5)	7 (4.2)	11 (4.6)	7 (3.0)
Parental history of asthma	44 (33.1)	49 (29.3)	80 (33.5)	91 (38.7)	.18
Maternal smoking during pregnancy	17 (12.8)	21 (12.6)	38 (15.9)	36 (15.3)	.43
C-section delivery	47 (35.3)	60 (35.9)	82 (34.3)	87 (37.0)	.60
Prematurity (32-37 wk)	26 (19.5)	27 (16.2)	48 (20.1)	43 (18.3)	.78
Low birth weight (<2.3 kg)	9 (6.8)	12 (7.2)	13 (5.4)	15 (6.4)	.94
Sibling in the household	98 (73.7)	142 (85.0)	188 (78.7)	188 (80.0)	.11
Mostly breast-fed during the first 3 mo of life	59 (44.4)	79 (47.3)	88 (36.8)	99 (42.1)	.43
History of a breathing problem	27 (20.3)	22 (13.2)	44 (18.4)	37 (15.7)	.34
Lifetime history of systemic antibiotic use	52 (39.1)	29 (17.4)	67 (28.0)	67 (28.5)	.001
Lifetime history of corticosteroid use	20 (15.0)	19 (11.4)	30 (12.6)	28 (11.9)	.79
Virus category∗
RSV-only	83 (62.4)	119 (71.3)	176 (73.6)	202 (86.0)	<.001
RV-A	30 (22.6)	17 (10.2)	31 (13.0)	13 (5.5)
RV-B	3 (2.3)	4 (2.4)	4 (1.7)	1 (0.4)
RV-C	17 (12.8)	27 (16.2)	28 (11.7)	19 (8.1)

IQR, Interquartile range.

Data are n (%) of infants unless otherwise indicated.

Of these, 48 had coinfection with a non-RSV/non-RV, which did not have statistically significant association with the microbiota profiles (P = .06).

Table E5

Full results of the multivariable analysis on associations of RVs (exposure) with nasopharyngeal microbiota profiles (outcome) in infants hospitalized for bronchiolitis∗

Variable	Microbiota profile
	Haemophilus-dominant (n = 133)		Moraxella-dominant (n = 167)		Mixed profile (n = 239)		Streptococcus-dominant (n = 235)
	RRR (95% CI)	P value	RRR (95% CI)	P value	RRR (95% CI)	P value	RRR (95% CI)
Virus category
RSV-only (n = 580)	Reference		Reference		Reference		Reference
RV-A (n = 91)	5.67 (2.76-11.67)	<.001	2.26 (1.05-4.89)	.04	2.74 (1.38-5.44)	.004	Reference
RV-B (n = 12)	7.50 (0.74-76.08)	.09	5.72 (0.62-52.71)	.12	4.73 (0.52-43.04)	.17	Reference
RV-C (n = 91)	1.81 (0.86-3.81)	.12	2.69 (1.39-5.20)	.003	1.57 (0.83-2.96)	.17	Reference
Age ≥ 6 mo	2.82 (1.58-5.02)	<.001	2.09 (1.18-3.72)	.01	1.34 (0.78-2.29)	.29	Reference
Female (vs male) sex	1.08 (0.68-1.71)	.74	1.11 (0.73-1.69)	.63	1.01 (0.69-1.48)		Reference
Race/ethnicity						.95
Non-Hispanic white	Reference		Reference		Reference		Reference
Non-Hispanic black	1.75 (0.96-3.22)	.07	1.58 (0.92-2.69)	.10	1.56 (0.96-2.53)	.07	Reference
Hispanic	1.94 (1.14-3.30)	.01	1.41 (0.86-2.33)	.17	1.28 (0.82-2.00)	.28	Reference
Other	2.19 (0.67-7.11)	.19	1.74 (0.57-5.31)	.33	1.98 (0.73-5.34)	.18	Reference
Prematurity (32-37 wk)	1.04 (0.59-1.83)	.90	0.83 (0.48-1.43)	.49	1.06 (0.67-1.70)	.80	Reference
Sibling in the household	0.65 (0.38-1.11)	.11	1.49 (0.86-2.58)	.16	0.89 (0.56-1.40)	.61	Reference
Breast-feeding during the first 3 mo of life	1.13 (0.70-1.84)	.62	1.26 (0.81-1.97)	.31	0.80 (0.53-1.20)	.28	Reference
History of breathing problems before the index hospitalization	0.80 (0.43-1.49)	.49	0.65 (0.35-1.22)	.18	1.05 (0.62-1.76)	.86	Reference
Lifetime history of systemic antibiotic use	1.39 (0.86-2.27)	.18	0.49 (0.29-0.82)	.007	0.94 (0.62-1.44)	.79	Reference

RRR, Relative rate ratio.

Multinomial logistic regression model adjusting for 8 patient-level covariates. RSV-only infection was used as the reference of exposure (virus category), and Streptococcus-dominant microbiota profile was used as the reference for the outcome (nasopharyngeal microbiota profile).