Fig 1.
The oropharyngeal microbial diversity between CCO and HC groups in the discovery cohort.
A) Rarefaction analysis showed as the number of samples raised, the number of OTUs approached saturation in CCO (n = 198) and HC (n = 133). The number of OTU in CCO group was significantly lower than that in HC group. B) Shannon index showed that the oropharyngeal microbial α-diversity in CCO group was significantly lower than that in HC group (p<0.01). C) A Venn diagram displaying the overlaps between groups showed that 760 of 1072 OTUs were shared in CCO and HC groups, while 30 of 1072 OTUs were unique to the CCO group. D) and E) The PCoA and NMDS based on OTU distribution showed the oropharyngeal taxonomic composition was significantly different between the two groups. F) The ANOSIM analysis in the form of boxplot shows that the difference between CCO and HC groups is greater than that within groups (R = 0.373, P = 0.001). CCO, confirmed cases of COVID-19 Omicron variant; HC, healthy controls; OTUs, operational taxonomy units; PCoA, principal coordinate analysis; NMDS, nonmetric multidimensional scaling; centerline, median; box limits, upper and lower quartiles; error bars, 95% CI.
Fig 2.
Comparison of the composition and abundance of oropharyngeal microbiome between CCO (n = 198) and HC (n = 133) groups.
A) Average compositions and relative abundance of the bacterial community at the phylum level between CCO and HC groups. B) Compared with HC group, 5 phyla increased significantly and 11 phyla decreased significantly in CCO group (Only part of the bacteria is displayed. Please see the S8 Table for details). C) Average compositions and relative abundance of the bacterial community at the genus level between CCO and HC groups. D) Compared with HC group, 10 genera increased significantly and 53 genera decreased significantly in CCO group (Only part of the bacteria is displayed. Please see the S9 Table for details). E) Heatmap showed the relative abundances of differential OTUs for each sample between CCO and HC groups. *p<0.05, **p<0.01, ***p<0.001. CCO, confirmed cases of COVID-19 Omicron variant; HC, healthy controls; OTUs, operational taxonomy units; centerline, median; box limits, upper and lower quartiles; circle or square symbol, mean; error bars, 95% CI.
Fig 3.
Diagnostic efficacy of oropharyngeal microbial classifier on Omicron variant.
A) Six microbial markers were selected as the best markers set by random forest model. B) Importance distribution map of the selected microbial makers in the model. C) The POD value was significantly higher in CCO group (n = 198) compared with that in HC group (n = 133) in the discovery cohort. D) The POD value achieved an AUC of 98.52% (95% CI 97.09% to 99.94%, p<0.0001) between CCO group (n = 198) versus HC group (n = 133) in the discovery cohort. E) The POD value was significantly higher in CCO group (n = 99) compared with that in HC group (n = 66) in the verification cohort. F) The POD value achieved an AUC of 98.42% (95% CI 96.95% to 99.88%, p<0.0001) between CCO group (n = 99) versus HC group (n = 66) in the verification cohort. CCO, confirmed cases of COVID-19 Omicron variant; HC, healthy controls; OTUs, operational taxonomy units; POD, probability of disease; AUC, area under the curve; centerline, median; box limits, upper and lower quartiles; error bars, 95% CI.
Fig 4.
The oropharyngeal microbial diversity among CCO, CCOR and HC groups.
A) Rarefaction analysis showed as the number of samples raised, the number of OTUs approached saturation in CCO (n = 297), CCOR (n = 222) and HC (n = 199) group. The number of OTUs in CCOR group and CCO group was lower than that in HC group, and the number of OTUs in CCOR group was higher than that in CCO group. Shannon index showed that the oropharyngeal microbial α-diversity in CCO and CCOR group was significantly lower than that in HC group (p<0.05). B) and C) The PCoA based on OTU distribution showed the oropharyngeal taxonomic composition was significantly different among the three groups. D) A Venn diagram displaying the overlaps among groups showed that 954 of 1441 OTUs were shared in CCO, CCOR and HC groups, while 233 of 1441 OTUs were unique to the HC group. CCO, confirmed cases of COVID-19 Omicron variant; CCOR, confirmed cases of omicron who recovered; HC, healthy controls; OTUs, operational taxonomy units; PCoA, principal coordinate analysis; NMDS, nonmetric multidimensional scaling; centerline, median; box limits, upper and lower quartiles; error bars, 95% CI.
Fig 5.
Comparison of the composition and abundance of oropharyngeal microbiome among CCO (n = 297), CCOR (n = 222) and HC (n = 133) groups.
A) In the process of infection recovery, 1 phylum gradually increased and 1 phylum decreased gradually. There was no difference between CCO group and CCOR group, but there was a significant decrease in 7 phyla compared with HC group. There was no difference between CCO group and CCOR group, but it was significantly higher in 2 phyla than that in HC group (Only part of the bacteria is displayed. Please see the S23 Table for details). B) 4 genus increased gradually during recovery of infection, while 7 genera decreased gradually. There was no difference between CCO group and CCOR group, but there was a significant decrease in 31 genera compared with HC group. There was no difference between CCO group and CCOR group, but it was significantly higher in 10 genera than that in HC group (Only part of the bacteria is displayed. Please see the S24 Table for details). C) The cladogram, representing oropharyngeal microbial structure and their predominant bacteria, revealed the greatest differences in different taxa among CCO group (n = 297), CCOR group (222) and HC group (n = 199). D) Based on the LDA selection, 37 gene functions were significantly enhanced in CCO group, 11 gene functions in CCOR group, and 41 gene functions in HC group (p<0.05, LDA>3) (Only part of the bacteria is displayed. Please see the S27 Table for details). *p<0.05, **p<0.01, ***p<0.001. CCO, confirmed cases of COVID-19 Omicron variant; CCOR, confirmed cases of omicron who recovered; HC, healthy controls; LEfSe, linear discriminant analysis (LDA) effect size. OTUs, operational taxonomy units; centerline, median; box limits, upper and lower quartiles; error bars, 95% CI.
Fig 6.
The oropharyngeal microbial diversity between CCO (n = 134) and CCOS (n = 73) groups.
A) Rarefaction analysis showed as the number of samples raised, the number of OTUs approached saturation in CCO (n = 134) and CCOS (n = 73). B) Shannon index showed that the oropharyngeal microbial α-diversity in CCOS group was significantly lower than that in CCO group (p<0.001). C) A Venn diagram displaying the overlaps between groups showed that 813 of 1210 OTUs were shared in CCO and CCOS groups, while 211 of 1210 OTUs were unique to the CCOS group. D) and F) The PCoA and E) NMDS based on OTU distribution showed the oropharyngeal taxonomic composition was significantly different between the two groups. CCO, confirmed cases of COVID-19 Omicron variant; CCOS, confirmed cases of original strain; OTUs, operational taxonomy units; PCoA, principal coordinate analysis; NMDS, nonmetric multidimensional scaling; centerline, median; box limits, upper and lower quartiles; error bars, 95% CI.
Fig 7.
Comparison of the composition and abundance of oropharyngeal microbiome between CCO (n = 134) and CCOS (n = 73) groups.
A) Average compositions and relative abundance of the bacterial community at the phylum level between CCO and CCOS groups. B) Compared with CCOS group, 6 phyla increased significantly and 4 phyla decreased significantly in CCO group (Only part of the bacteria is displayed. Please see the S33 Table for details). C) Average compositions and relative abundance of the bacterial community at the genus level between CCO and CCOS groups. D) Compared with CCOS group, 23 genera increased significantly and 19 genera decreased significantly in CCO group (Only part of the bacteria is displayed. Please see the S34 Table for details). E) Heatmap showed the relative abundances of differential OTUs for each sample between CCO and CCOS groups. *p < 0.05, **p < 0.01, ***p < 0.001. CCO, confirmed cases of COVID-19 Omicron variant; CCOS, confirmed cases of original strain; OTUs, operational taxonomy units; centerline, median; box limits, upper and lower quartiles; error bars, 95% CI.
Fig 8.
Associations between the oropharyngeal microbiomes and clinical indices of Omicron variant.
The Heatmap shows the Spearman’s correlation coefficients between 27 oropharyngeal OTUs and 6 clinical indicators in CCO (n = 297) and HC (n = 199) groups. The red line indicates a positive correlation, and the blue line indicates a negative correlation. OTUs, operational taxonomic units; CCO, confirmed cases of COVID-19 Omicron variant; HC, healthy controls; WBC, white blood cell; Neut, neutrophils; Eos, eosinophils; PLT, platelets; ALB, albumin; CREA, creatinine.