Samples (N = 108) were successfully sequenced and generated all downstream data. Tables 1a and b present the demographic summary of the patient population for MRSA and VRE studies, respectively. Controls were recruited by matching on the demographic and comorbidities of cases (sex, age, race, ethnicity, unit of admission, and diagnostic-related group, when available). As indicated in Tables 1a and b, no statistically significant differences (t- and chi-squared tests) were observed in age, race, sex, and Elixhauser Comorbidity Index between the cases and controls for the MRSA and VRE studies.
Table 1
Comparison of demographic characteristics of enrolled samples from subjects colonized with (a) MRSA and (b) VRE and their respective uncolonized matched subject-pairs (controls). All subjects identified as non-Hispanic/Latino. No statistical significance (at a .05 level) between cases and controls across demographics of the recruited samples were observed.
|
a. MRSA Study
|
b. VRE Study
|
|
MRSA Cases (N = 28)
|
Controls (N = 27)
|
p-value*
|
VRE Cases (N = 27)
|
Controls (N = 26)
|
p-value*
|
Sex
|
|
|
|
|
|
|
Male
|
13 (46.4%)
|
13 (48.1%)
|
1
|
17 (63.0%)
|
18 (69.2%)
|
0.848
|
Female
|
15 (53.6%)
|
14 (51.9%)
|
|
10 (37.0%)
|
8 (30.8%)
|
|
Age (years)
|
|
|
|
|
|
|
Mean (SD)
|
56.7 (16.3)
|
55.6 (16.3)
|
0.8
|
57.5 (13.5)
|
61.0 (11.4)
|
0.31
|
Median [Min, Max]
|
56.0 [25.0, 93.0]
|
55.0 [25.0, 93.0]
|
|
60.0 [20.0, 79.0]
|
64.0 [38.0, 81.0]
|
|
Race
|
|
|
|
|
|
|
White
|
20 (71.4%)
|
20 (74.1%)
|
1
|
21 (77.8%)
|
20 (76.9%)
|
1
|
Black
|
8 (28.6%)
|
7 (25.9%)
|
|
6 (22.2%)
|
6 (23.1%)
|
|
Elixhauser Comorbidity Index
|
|
|
|
|
|
Mean (SD)
|
5.43 (2.50)
|
5.70 (3.99)
|
0.762
|
8.27 (4.13)
|
7.62 (3.77)
|
0.554
|
Median [Min, Max]
|
6.00 [0, 11.0]
|
6.00 [0, 13.0]
|
|
8.50 [1.00, 16.0]
|
8.00 [1.00, 14.0]
|
|
Missing
|
|
|
|
1 (3.7%)
|
0 (0%)
|
|
*Significance values were calculated using chi-square tests for categorical variables (sex and race) and two-sample t-test for numerical variables (age and Elixhauser Comorbidity Index).
For the MRSA study, 55 samples generated a total of 1,704,344 non-chimeric reads (mean [SD] = 30,988 [12,438.9], median [min, max] = 31,966 [2,298, 72,070]). These comprised 740 ASVs, five (5) of which were eukaryotic in origin and were only observed across four samples. For the VRE study, 53 samples generated a total of 1,648,085 non-chimeric reads (mean [SD] = 31,096 [12,157.35], median [min, max] = 27,840 [8,337, 68,097]). These reads comprised 962 ASVs, only one (1) of which was eukaryotic in origin and was observed across four samples. The eukaryotic ASVs were removed prior to analysis. Sequencing depth between MRSA/VRE colonized and uncolonized samples were assessed to ensure no significant read quality differences were present in any of the covariates. Case-control matched visualization of the top-10 most abundant genus-level communities revealed an interesting trend (Figs. 1a and b); uncolonized samples (top facets) are observed to have a relatively larger portion of their abundance comprise their non-top-10 genera (combined and colored in black), indicating a greater within sample diversity. This trend was confirmed and visualized in Supplementary Figs. 2a and b. As observed in Fig. 1a, the majority of ASVs in both colonized and uncolonized samples were identified within the Staphylococcus genus. This same trend was not observed in the VRE study; although in the cases we observe the Enterococcusgenus with the majority abundance, this is not the case in the matched negative controls (in 1b top facet).
Alpha diversity of samples was assessed (Table 2) prior to any abundance/prevalence filtering under the three scenarios described in the methods. For the MRSA study, a total of 735 ASVs across 55 subjects were assessed using t-test of observed, Shannon, and Simpson diversity measures, all three of which were found to be significantly associated with subjects’ colonization status (p = .026, .003, and .004 respectively). ASVs identified as Staphylococcus were removed and processed for the same analysis. This time only the observed alpha diversity count was seen to be significant (p = .02). Subsequently, Staphylococcus ASVs were isolated and processed using the same assessment of alpha diversity and no significance was observed on a 0.05-level. Similar trends were observed in the VRE study in which observed, Shannon, and Simpson diversity measures were found to be significantly associated with subjects’ colonization status (p = .003, .001, and .012 respectively) when all ASVs were included. ASVs identified as Enterococcus were removed and processed for the same analysis leading to significantly different alpha diversity measures again (p = .001, .001, and .003 for observed, Shannon, and Simpson respectively). Subsequently, Enterococcus ASVs were isolated and processed using the same assessment of alpha diversity and no significance was observed on a 0.05-level. We have included visualized alpha diversity measures as Supplementary Figs. 3a and b.
Table 2. Assessment of alpha diversity of observed, Shannon, and Simpson indices are assessed using paired t-test (within match analysis) under three scenarios:
-
in which all ASVs present (at least 1% relative abundance and in more than 1 sample);
- in which all ASVs identified as genus Staphylococcus (a) or Enterococcus (b) have been removed; and
- in which all ASVs except those classified as genus Staphylococcus (a) or Enterococcus (b) have been removed.
In both (a) and (b), within the first scenario we can see a significant difference (at a 0.05 level) in alpha diversity of observed counts, Shannon and Simpson indices. However, removal of the ASVs of the genus Staphylococcus (a) results in the disappearance of this significant difference of the Shannon and Simpson indices previously observed (observed counts remain significant). Likewise, when ASVs of Staphylococcus are isolated (a) in assessment, we observe no statistical significance. Removal of the ASVs of the genus Enterococcus (b) results in the increase in magnitude of the effect. However, when ASVs of Enterococcusare isolated (b) in assessment, we observe a drop in magnitude with no statistical significance.
a. MRSA Study: Assessment of Alpha-Diversity Measures
|
Measure
|
Mean
|
Conf Interval
|
df
|
t-statistic
|
p-value
|
MRSA-
|
MRSA+
|
Delta
|
Low
|
High
|
All ASVs
|
Observed
|
38.07
|
27.22
|
10.85
|
1.37
|
20.33
|
26
|
2.35
|
0.026
|
Shannon
|
2.12
|
1.63
|
0.49
|
0.19
|
0.80
|
26
|
3.31
|
0.003
|
Simpson
|
0.79
|
0.70
|
0.08
|
0.03
|
0.14
|
26
|
3.17
|
0.004
|
'Staphylococcus' ASVs removed
|
Observed
|
26.96
|
17.40
|
10.80
|
1.82
|
19.78
|
24
|
2.48
|
0.02
|
Shannon
|
1.97
|
1.88
|
0.16
|
-0.18
|
0.51
|
24
|
0.98
|
0.34
|
Simpson
|
0.74
|
0.76
|
0.00
|
-0.07
|
0.07
|
24
|
0.08
|
0.94
|
'Staphylococcus' ASVs only
|
Observed
|
11.54
|
11.11
|
0.46
|
-2.86
|
3.78
|
25
|
0.29
|
0.78
|
Shannon
|
1.48
|
1.31
|
0.18
|
-0.08
|
0.43
|
25
|
1.43
|
0.16
|
Simpson
|
0.69
|
0.65
|
0.04
|
-0.02
|
0.10
|
25
|
1.31
|
0.20
|
b. VRE Study: Assessment of Alpha-Diversity Measures
|
Measure
|
Mean
|
Conf Interval
|
df
|
t-statistic
|
p-value
|
MRSA-
|
MRSA+
|
Delta
|
Low
|
High
|
All ASVs
|
Observed
|
52.72
|
28.27
|
25.62
|
9.98
|
41.27
|
23
|
3.39
|
0.003
|
Shannon
|
2.29
|
1.58
|
0.76
|
0.34
|
1.18
|
23
|
3.70
|
0.001
|
Simpson
|
0.76
|
0.62
|
0.15
|
0.04
|
0.26
|
23
|
2.72
|
0.012
|
‘Enterococcus’ ASVs removed
|
Observed
|
50.92
|
24.85
|
27.17
|
11.73
|
42.61
|
23
|
3.64
|
0.001
|
Shannon
|
2.42
|
1.55
|
0.91
|
0.44
|
1.38
|
23
|
4.00
|
0.001
|
Simpson
|
0.79
|
0.60
|
0.20
|
0.07
|
0.32
|
23
|
3.32
|
0.003
|
‘Enterococcus’ ASVs only
|
Observed
|
2.81
|
3.42
|
-0.94
|
-2.50
|
0.62
|
15
|
-1.28
|
0.22
|
Shannon
|
0.31
|
0.42
|
-0.15
|
-0.48
|
0.17
|
15
|
-1.02
|
0.32
|
Simpson
|
0.18
|
0.22
|
-0.07
|
-0.25
|
0.12
|
15
|
-0.78
|
0.45
|
A filtering criterion of at least 1% relative abundance and observed in more than one sample was applied. In the MRSA study, this eliminated 566 low abundance and 523 low prevalence ASVs (total of 631), resulting in 104 ASVs that were used for univariate and multivariate assessments. CLR-transformed data was assessed in ASV-wise manner across colonization status (Table 3a). This assessment revealed ASVs 1 and 2 (both identified in the Staphylococcus genus) as statistically significant (\(\varvec{q}=4.98\times {10}^{-5}\) and \(6.32\times {10}^{-5}\), respectively). These two ASVs are also the most abundant overall. When corrected for multiple comparisons using FDR approach, q-values were observed to be non-significant (on a .05 level). A table containing all 104 ASVs is included as Supplementary Table 1a. A similar comparison was performed on genus agglomerated CLR data (Table 4a). We do not see any differences in q-values following corrections for multiple testing. A table containing all 30 genera of the MRSA study is included as Supplementary Table 2a.
Table 3
ASV-level univariate analysis of centered log-ratio (CLR) abundances on MRSA (a) and VRE (b) colonized and uncolonized respective pairs using paired t-test. Only the top 10 most significant ASVs are displayed here and larger tables including all 104 ASVs (for a) and 176 ASVs (for b) are included as Supplementary Table 1. P-values were corrected for multiple testing using FDR methods (q-values). In (a) we see ASVs 1 and 2 showing a significant q-value (on .05 level) even after these corrections. Both of these ASVs have been identified within the Staphylococcus genus and no species level information is available. In (b) we see no individual ASV as statistically significant (on .05 level) after multiple testing corrections.
a. MRSA Study: ASV-level univariate analysis using paired t-test on CLR-transformed relative abundance values between MRSA colonized and non-colonized matches.
|
ASV
|
Genus
|
Species
|
Mean
|
Conf Interval
|
t-statistic
|
p-value
|
q-value
|
MRSA-
|
MRSA+
|
Delta
|
Low
|
High
|
ASV1
|
Staphylococcus
|
NA
|
0.03
|
0.23
|
-0.21
|
-0.27
|
-0.14
|
-6.64
|
4.79E-07
|
4.98E-05
|
ASV2
|
Staphylococcus
|
NA
|
0.03
|
0.21
|
-0.18
|
-0.24
|
-0.12
|
-6.27
|
1.22E-06
|
6.32E-05
|
ASV11
|
Staphylococcus
|
NA
|
0.01
|
3.05E-03
|
0.01
|
2.93E-03
|
0.02
|
2.78
|
9.88E-03
|
0.26
|
ASV4
|
Staphylococcus
|
NA
|
0.14
|
0.07
|
0.08
|
0.02
|
0.14
|
2.61
|
0.01
|
0.26
|
ASV9
|
Staphylococcus
|
epidermidis
|
0.02
|
4.12E-03
|
0.01
|
2.73E-03
|
0.02
|
2.61
|
0.01
|
0.26
|
ASV3
|
Staphylococcus
|
NA
|
0.17
|
0.08
|
0.09
|
0.02
|
0.17
|
2.61
|
0.01
|
0.26
|
ASV8
|
Corynebacterium
|
NA
|
0.03
|
2.81E-03
|
0.03
|
7.15E-04
|
0.06
|
2.11
|
0.05
|
0.5
|
ASV7
|
Corynebacterium
|
accolens
|
0.04
|
3.98E-03
|
0.04
|
-1.01E-03
|
0.07
|
2
|
0.06
|
0.5
|
ASV35
|
Cutibacterium
|
NA
|
4.84E-03
|
-3.39E-04
|
5.25E-03
|
-1.77E-04
|
0.01
|
1.99
|
0.06
|
0.5
|
ASV42
|
Cutibacterium
|
NA
|
3.46E-03
|
-3.59E-04
|
3.91E-03
|
-2.04E-04
|
8.02E-03
|
1.95
|
0.06
|
0.5
|
b. VRE Study: ASV-level univariate analysis using paired t-test on CLR-transformed relative abundance values between MRSA colonized and non-colonized matches.
|
ASV
|
Genus
|
Species
|
Mean
|
Conf Interval
|
t-statistic
|
p-value
|
q-value
|
VRE-
|
VRE+
|
Delta
|
Low
|
High
|
ASV1
|
Enterococcus
|
NA
|
0.07
|
0.22
|
-0.16
|
-0.28
|
-0.04
|
-2.73
|
0.01
|
0.54
|
ASV8
|
Enterococcus
|
faecium
|
-9.44e-04
|
0.02
|
-0.02
|
-0.04
|
-5.28e-03
|
-2.64
|
0.01
|
0.54
|
ASV20
|
Klebsiella
|
NA
|
-9.44e-04
|
8.91e-03
|
-1.67e-03
|
-3.30e-03
|
-4.41e-05
|
-2.12
|
0.04
|
0.54
|
ASV32
|
Anaerococcus
|
NA
|
4.83e-03
|
6.01e-04
|
4.89e-03
|
-1.76e-04
|
9.96e-03
|
1.99
|
0.06
|
0.54
|
ASV72
|
Anaerococcus
|
NA
|
2.29e-03
|
3.45e-04
|
6.78e-04
|
-3.69e-05
|
1.39e-03
|
1.96
|
0.06
|
0.54
|
ASV37
|
Eremococcus
|
NA
|
8.15e-03
|
-9.00e-04
|
4.43e-03
|
-4.64e-04
|
9.33e-03
|
1.87
|
0.07
|
0.54
|
ASV18
|
Peptoniphilus
|
NA
|
9.20e-03
|
9.68e-04
|
8.77e-03
|
-1.09e-03
|
0.02
|
1.84
|
0.08
|
0.54
|
ASV124
|
Staphylococcus
|
lugdunensis
|
1.49e-03
|
-7.55e-04
|
1.88e-03
|
-2.51e-04
|
4.01e-03
|
1.82
|
0.08
|
0.54
|
ASV52
|
Murdochiella
|
asaccharolytica
|
4.00e-03
|
-6.36e-04
|
4.82e-03
|
-7.11e-04
|
0.01
|
1.80
|
0.08
|
0.54
|
ASV165
|
Facklamia
|
languida
|
9.60e-04
|
-9.00e-04
|
1.93e-03
|
-2.88e-04
|
4.15e-03
|
1.80
|
0.09
|
0.54
|
Table 4Genus-level univariate analysis of centered log-ratio (CLR) abundances on MRSA (a) and VRE (b) colonized and uncolonized respective pairs using paired t-test. Only the top 10 most significant genera are displayed here and the larger tables including all 30 and 64 genera, for MRSA and VRE samples respectively are included as Supplementary Table 2. P-values were corrected for multiple testing using FDR methods (q-values). In (a) we observe genus Cutibacterium on the threshold of significance (on a 0.05 level). However, q-values after multiple testing corrections show no significance. In (b) we observe genera Enterococcus, Corynebacterium, Mobiluncus, and Facklamia as significance (on a 0.05 level). Likewise here, the q-values after multiple testing corrections show no significance for any genera.
a. MRSA Study: Genus-level univariate analysis using paired t-test on CLR-transformed relative abundance values between MRSA colonized and non-colonized matches.
|
Genus
|
Mean
|
Conf Interval
|
t-statistic
|
p-value
|
q-value
|
MRSA-
|
MRSA+
|
Delta
|
Low
|
High
|
Cutibacterium
|
7.37E-03
|
-4.83E-03
|
0.01
|
-4.57E-04
|
0.03
|
1.98
|
0.058
|
0.59
|
Lawsonella
|
-5.32E-03
|
-6.77E-03
|
1.42E-03
|
-3.91E-04
|
3.24E-03
|
1.61
|
0.12
|
0.59
|
Moraxella
|
-6.84E-03
|
3.24E-03
|
-0.01
|
-0.02
|
3.63E-03
|
-1.52
|
0.14
|
0.59
|
Enterococcus
|
0.03
|
0.01
|
0.02
|
-8.72E-03
|
0.05
|
1.4
|
0.17
|
0.59
|
Staphylococcus
|
0.46
|
0.54
|
-0.08
|
-0.2
|
0.05
|
-1.28
|
0.21
|
0.59
|
Enterobacter
|
0.02
|
-6.37E-03
|
0.03
|
-0.02
|
0.07
|
1.07
|
0.29
|
0.59
|
Neisseria
|
-3.05E-03
|
-6.68E-03
|
3.60E-03
|
-3.46E-03
|
0.01
|
1.05
|
0.3
|
0.59
|
Proteus
|
-6.90E-03
|
9.99E-03
|
-0.02
|
-0.05
|
0.02
|
-1.02
|
0.32
|
0.59
|
Providencia
|
-6.72E-03
|
0.01
|
-0.02
|
-0.06
|
0.02
|
-1
|
0.33
|
0.59
|
Bacillus
|
1.51E-04
|
-6.52E-03
|
6.64E-03
|
-7.76E-03
|
0.02
|
0.95
|
0.35
|
0.59
|
b. VRE Study: Genus-level univariate analysis using paired t-test on CLR-transformed relative abundance values between MRSA colonized and non-colonized matches.
|
Genus
|
Mean
|
Conf Interval
|
t-statistic
|
p-value
|
q-value
|
MRSA-
|
MRSA+
|
Delta
|
Low
|
High
|
Enterococcus
|
0.13
|
0.33
|
-0.2
|
-0.33
|
-0.07
|
-3.18
|
0.004
|
0.26
|
Corynebacterium
|
0.11
|
0.02
|
0.08
|
0.02
|
0.13
|
2.79
|
0.01
|
0.32
|
Mobiluncus
|
-4.78E-03
|
-2.97E-03
|
-2.30E-04
|
-4.25E-04
|
-3.63E-05
|
-2.45
|
0.022
|
0.46
|
Facklamia
|
3.94E-03
|
-3.84E-03
|
8.05E-03
|
9.07E-04
|
0.02
|
2.33
|
0.029
|
0.46
|
Klebsiella
|
-4.58E-03
|
0.03
|
-0.01
|
-0.03
|
9.64E-04
|
-1.93
|
0.065
|
0.53
|
Campylobacter
|
-2.67E-03
|
-3.82E-03
|
1.64E-03
|
-1.35E-04
|
3.41E-03
|
1.91
|
0.069
|
0.53
|
Prevotella
|
7.73E-03
|
-3.88E-03
|
0.01
|
-1.29E-03
|
0.03
|
1.86
|
0.075
|
0.53
|
Bacteroides
|
0.04
|
7.88E-03
|
0.03
|
-3.86E-03
|
0.07
|
1.86
|
0.076
|
0.53
|
Eremococcus
|
4.44E-03
|
-4.58E-03
|
4.38E-03
|
-6.53E-04
|
9.42E-03
|
1.8
|
0.085
|
0.53
|
Atopobium
|
-4.74E-03
|
-3.33E-03
|
-1.75E-04
|
-3.77E-04
|
2.78E-05
|
-1.78
|
0.088
|
0.53
|
We used an identical filtering criterion for the VRE study removing 670 low abundance and 659 low prevalence ASVs (total of 785), resulting in 176 ASVs that were used for our downstream analyses. CLR-transformed data was assessed in ASV-wise manner across VRE colonization status (Table 3b). Although three ASVs (1, 8, and 20) showed significant p-values, when corrected for multiple comparisons using FDR approach, q-values were observed to be non-significant (on a .05 level). ASVs 1 and 8 belong to the Enterococcus genus and ASV20 belongs to the Klebsiella genus. A table containing all 176 ASVs is included as Supplementary Table 1b. A similar comparison was performed on genus agglomerated CLR data (Table 4b) which showed no significant differences once corrected for multiple comparison. A table containing all 64 genera of the VRE study is included as Supplementary Table 2b.
The composition of the top 30 most abundant ASVs across samples is displayed using a heatmap in Fig. 2a and b. We see specific ASVs that are associated primarily or exclusively with each group (colonized and non-colonized). In 2a we see Staphylococcus ASVs 3 and 4 to be widely present in both cases and controls, whereas ASVs 1 and 2 (also Staphylococcus) are almost exclusively observed in the cases (MRSA colonized). On the other hand, Staphylococcus ASVs 9 and 11 are observed largely in the controls (non-colonized for MRSA). We have included a larger heatmap including all 104 AVSs within the MRSA study as Supplementary Fig. 4. Assessing these differences across colonization using Pearson’s correlation test shows significance (correlation coefficient = .287, p = .033). In 2b (VRE study) we see Enterococcus ASVs 1 and 3 to be primarily present in the cases (VRE colonized), whereas ASV 2 (Escherichia-Shigella), ASV 13 (Staphylococcus), and ASV 18 (Peptoniphilus) are primarily observed in the controls (non-colonized for VRE). We have included a larger heatmap including all 176 AVSs within the VRE study as Supplementary Fig. 5. Assessing these differences across colonization using Pearson’s correlation test shows no significance (correlation coefficient = .095, p = .49).
Visualized principal coordinates analysis (PCoA) of the Bray-transformed relative abundance data (\(\beta\)-diversity) revealed interesting interaction of ASVs (Figs. 3 and 4). Here, the assessments were made using the three scenarios (for MRSA and VRE studies) described within the methods section. Statistical assessment was made using \({W}_{d}^{*}\) test which has been developed in our group to account for heteroscedasticity and control for confounders and covariates [23]. Starting with the MRSA study, when all the ASVs are present (filtering at least 1% relative abundance and seen in more than one sample) (Scenario a, Fig. 3a), we see that the MRSA colonized cases are differentiated from the non-colonizing controls (\({W}_{d}^{*}\)=10.41, p=.001). Removal of the Staphylococcus genus (Scenario b, Fig. 3b) results in an overlap of group centroids meaning these differences are primarily driven by the Staphylococcus genus, which accounts for most observed microbiome shift (\({W}_{d}^{*}\)=.82, p=.581). Looking at the Staphylococcus genus in isolation (Scenario c, Fig. 3c) we see the first axis of PCoA accounting for a large 68.2% of the differences observed (\({W}_{d}^{*}\)=24.54, p = .001), further evidence that the signal results from within this genus. Performing a similar assessment for the VRE study following standard filtering criteria (filtering at least 1% relative abundance and seen in more than one sample), when all the ASVs are present (Scenario a, Fig. 4a), we see that the VRE colonized cases are relatively differentiated from the non-colonizing controls (\({W}_{d}^{*}\)=2.65, p=.009). Removal of the Enterococcus genus (Scenario b, Fig. 4b) results in an overlap of group centroids meaning these differences are primarily driven by the Enterococcus genus, which accounts for most observed microbiome shift (\({W}_{d}^{*}\)=.92, p=.461). Looking at the Enterococcus genus in isolation (Scenario c, Fig. 4c) we see the first axis of PCoA accounting for a large 80.1% of the differences observed (\({W}_{d}^{*}\)=3.86, p=.089). We have performed similar analyses using Jensen-Shannon divergence metric (on relative abundances) and Euclidean distances (on CLR-transformed abundances), along with respective \({W}_{d}^{*}\) test results for the three aforementioned scenarios. These are included as Supplementary Figs. 6—9 and show similar trends as described above.