Dynamic Changes in the Nasal Microbiome Associated With Disease Activity in Patients With Granulomatosis With Polyangiitis

Abstract

Objective: Little is known about temporal changes in nasal bacteria in granulomatosis with polyangiitis (GPA). This study was undertaken to examine longitudinal changes in the nasal microbiome in association with relapse in GPA patients.

Methods: Bacterial 16S ribosomal RNA gene sequencing was performed on nasal swabs from 19 patients with GPA who were followed up longitudinally for a total of 78 visits, including 9 patients who experienced a relapse and 10 patients who remained in remission. Relative abundance of bacteria and ratios between bacteria were examined. Generalized estimating equation models were used to evaluate the association between bacterial composition and 1) disease activity and 2) levels of antineutrophil cytoplasmic antibody (ANCA) with specificity for proteinase 3 (PR3), adjusted for medication.

Results: Corynebacterium and Staphylococcus were the most abundant bacterial genera across all nasal samples. Patients with quiescent disease maintained a stable ratio of Corynebacterium to Staphylococcus across visits. In contrast, in patients who experienced a relapse, a significantly lower ratio was observed at the visit prior to relapse, followed by a higher ratio at the time of relapse (adjusted P < 0.01). Species-level analysis identified an association between a higher abundance of nasal Corynebacterium tuberculostearicum and 1) relapse (adjusted P = 0.04) and 2) higher PR3-ANCA levels (adjusted P = 0.02).

Conclusion: In GPA, significant changes occur in the nasal microbiome over time and are associated with disease activity. The occurrence of these changes months prior to the onset of relapse supports a pathogenic role of nasal bacteria in GPA. Our results uphold existing hypotheses implicating Staphylococcus as an instigator of disease and have generated a novel finding involving Corynebacterium as a potential mediator of disease in GPA.

Figure 1.. Bacterial composition of patients with GPA

(A) A heatmap illustrating the relative abundance of top 20 most abundant bacterial genera across all samples, grouped by outcome (relapsing vs non-relapsing GPA) and disease status (remission, pre-relapse, relapse, and post-relapse) among all samples (n = 78). The samples were primarily comprised of 5 bacteria (in descending order of abundance): Corynebacterium, Staphylococcus, Propionibacterium, Alloiococcus, and Streptococcus. (B) Bar plot showing mean relative abundance of 5 most abundant bacteria vs all other remaining bacteria. Over 80% of bacteria in samples were represented by the 5 most abundant bacteria. (C) Comparison of alpha diversity measured by Shannon diversity index among all visits grouped by relapsing (left) and non-relapsing (right) GPA (P-value = 0.78).

Figure 2.. Relative abundance of 5 most abundant bacteria grouped by disease status.

Boxplot demonstrates differences in relative abundance associated with disease status with most pronounced variations seen in the abundance of Corynebacterium (blue) and Staphylococcus (orange). Each box spans the first to the third quartile and the middle line indicates the median abundance; the whiskers below and above the box demonstrate the minimum and maximum abundance in the group with the dots representing outliers.

Figure 3.. Dynamic changes in ratio of Corynebacterium to Staphylococcus occur across visits of relapsing GPA.

Median relative abundance of Corynebacterium (black) and Staphylococcus (white) in non-relapsing (left) and relapsing (right) GPA. Consecutive visits are shown separately to display temporal shifts. Patients with non-relapsing GPA (n=10) had a relatively stable ratio of Corynebacterium to Staphylococcus across 3 consecutive visits (all P > 0.05); in contrast, patients with relapsing GPA (n=9) had significantly lower ratio at pre-relapse visit (adjusted P < 0.01) followed by an increase in ratio at relapse (adjusted P < 0.01), even after adjusting for antibiotics, immunosuppressive medications, and nasal irrigation.

Figure 4.. Increasing abundance of nasal Corynebacterium tuberculostearicum is associated with relapse in GPA.

Box plot depicts median relative abundance of C. tuberculostearicum by disease status: stable remission, pre- pre-relapse, pre-relapse, relapse, and post-relapse. Test for trend detected a significant linear increase in nasal C. tuberculostearicum abundance across visits even after adjusting for antibiotics, immunosuppressives, nasal irrigation, and presence of Staphylococcus aureus (adjusted P = 0.04).

Figure 5.. Relative abundance of nasal Corynebacterium tuberculostearicum is associated with PR3-ANCA level in GPA.

Scatterplot demonstrates the association between abundance of nasal C. tuberculostearicum and serum PR3-ANCA levels in 11 patients with a total of 29 visits (unadjusted P < 0.01). The association remained significant even after adjusting for use of antibiotics, immunosuppressives, and nasal irrigation (adjusted P = 0.02). Reference range for normal PR3-ANCA < 20 units.