Vijay Iyer PhD1
1 Independent Patient-Researcher
Email: viyer05@gmail.com
Cite as: Iyer, V. (2024). Hypothesis: Elevated abundance of the Streptococcaceae family within the intestinal microbiome is a consistent and relevant finding for ME/CFS. Patient-Generated Hypotheses Journal for Long COVID & Associated Conditions, Vol. 2, 4-9
Abstract
Alterations of the fecal microbiome have been reported in numerous studies of ME/CFS patients. However, only two sizable studies to date include quantifications of less abundant bacterial families which favor oxygenated environments, particularly Enterococcaceae and Streptococcaceae. Both studies contain data showing significant elevations of Streptococcaceae in ME/CFS patients compared to controls, although the more recent study does not remark upon it. This previously unnoticed replication suggests Streptococcaceae abundance may be useful as an ME/CFS biomarker, and may point towards a pathological role for Streptococcaceae in ME/CFS.
Hypothesis
The gut microbiome has gained growing attention in the last decades for its roles in human health and chronic disease.1,2,3 Such attention has not escaped the field of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) research. Recently, multiple groups have published reviews of several studies, finding altered gut microbiome composition in ME/CFS, as assessed via fecal samples.4,5,6
The present hypothesis draws attention to a potentially important common finding between an early study by Sheedy et al.7 and a recent study by Lupo et al.8 – elevated abundance of gut microbiota from the Streptococcaceae family in ME/CFS patients versus healthy controls. This finding appears to have been incidental, as Sheedy et al. is not cited in the study by Lupo et al.
This unintended replication gives rise to the present hypothesis: elevated abundance of Streptococcaceae in the gut microbiota is a potential marker and possible driver of pathophysiology for some cases of ME/CFS.
Sheedy et al. is an earlier study of the fecal microbiome which reports culturing ~1000x more Streptococcaceae colony-forming units (CFUs) in their ME/CFS patient cohort (n=108) versus healthy controls (n=177).7 The recent study by Lupo et al. looks at the gut microbiome of ME/CFS patients using 16S rRNA next-generation sequencing (NGS) technology.8 This study reports predominantly on the altered composition of the most abundant genera of gut microbiota, e.g., noting reduced and increased abundances of the Lachnospiraceae family and Bacteroides genera, respectively. Although the authors focus on the most abundant families and their genera, the study’s technology has sensitivity to less abundant families, including Streptococcaceae. Close examination of family data in Supplementary Figure 3 reveals that Streptococcaceae is clearly elevated in ME/CFS patients, in comparison both to healthy relatives and unrelated healthy controls.
The following sections address three important questions: How strongly does the data from these two studies support the hypothesis? What are the mechanisms by which Streptococcaceae might be associated with ME/CFS? What further studies should be done to test this hypothesis?
How strongly does the data support the hypothesis?
Of the numerous fecal microbiome studies, Sheedy et al. and Lupo et al. are the only two known to date which speak to Streptococcaceae abundance in ME/CFS cases vs controls. Both studies have sizable cohorts and exhibit methodological strengths, making the replication of elevated Streptococcaceae noteworthy.
The 2018 review by Du Preez et al. rates Sheedy et al. highly for study methodology, standing out as the largest study by a significant margin (over 2x).6,7 Among the culture-based studies, it also stands out as the only one to specifically utilize aerobic culture methods required to quantify the abundance of more aerophilic families, including both Enterococcaceae and Streptococcaceae. These reside at low levels in the oxygen-poor intestinal environment.
More recently, studies are gravitating towards NGS approaches, including metataxonomic studies (based on 16S rRNA sequencing) surveying genera and later metagenomic studies (based on whole-genome sequencing) capable of drilling down to species and even strains.9,10 This focus on quantifying the numerous comparisons among genera and species from highly speciated families often obscures less abundant families. Even when highly elevated, relatively aerophilic bacteria such as Streptococcaceae remain far less abundant in the intestinal environment. One should note, however, that the raw data of all NGS studies completed to date may yet contain unreported information about less abundant families such as Streptococcaceae. So far, Lupo et al. stands out as the one known NGS study which reports family-level data, albeit in the supplementary data.8 While not very large, it is sizable and carefully designed, including three cohorts of n=35, cases, close relatives, and unrelated controls.
While the pool of sizable studies speaking to Streptococcaceae abundance is currently limited to only two studies, it is noteworthy that a newly published but small metagenomic study (n=10) also reports higher Streptococcaceae in ME/CFS patients for 4 of 5 case-control matched pairs.11
The two larger studies also report on Enterococcaceae, another relatively aerobic family, describing either a much smaller elevation7 or no difference8 in the ME/CFS population compared to the healthy controls. Thus, Streptococcaceae appears to stand out among more aerophilic families of the human fecal microbiome.
How might Streptococcaceae be associated with ME/CFS?
If further replicated, elevated Streptococcaceae in ME/CFS patients could imply one of three scenarios:
- a pathogenic role for one or more species in the Streptococcaceae family;
- a pathway by which ME/CFS pathology drives elevated Streptococcaceae; or
- a common factor driving both Streptococcaceae elevation and ME/CFS pathology.
The latter two scenarios are made plausible by the strongly aerobic nature of Streptococcaceae relative to other gut-resident microbes, i.e., any pathological increase in gut oxygenation could drive higher Streptococcaceae abundance. However, the two studies anchoring this hypothesis hint at a more direct role for Streptococcaceae, as both show a much stronger elevation of Streptococcaceae compared to Enterococcaceae, which is another rather aerobic family in the human gut microbiome.
It is plausible that Streptococcaceae plays a pathogenic role based on several lines of evidence. First, several of its species are known pathobionts (adaptively pathogenic) and capable of intracellular persistence.12 Secondly, the largest genus of Streptococcaceae, Streptococcus, has been implicated as a trigger for autoimmune-like cardiovascular disease through molecular mimicry, including cross-reactivity of antibodies against Streptococcus with endothelial cell epitopes.13 Thirdly, Streptococcus molecular mimicry appears to directly contribute to a documented innate immune evasion pathway from platelet cell defense against Streptococcus..14 Finally, some Streptococcus immune mechanism findings potentially comport with recent reports of endothelial dysfunction and platelet hyperactivation in ME/CFS.15,16
Overall, there are several reasons to further investigate if Streptococcus is playing a driving role in the pathobiology of a subset of ME/CFS patients.
How to test this hypothesis?
Recent comparisons suggest NGS technology can accurately recapitulate culture-based assays.17 This bolsters the comparison of the two disparate studies which form the basis of the present hypothesis. Furthermore, it points to an immediate next step: re-analyzing raw data from the several existing NGS studies using different computational processing pipelines designed to consider relative abundance comparisons across a broad range of known gut microbiome families including facultative anaerobes, such as Streptococcaceae and Enterococcaceae. This screening of facultative anaerobes should ideally also include known opportunistic gut pathogens, such as Staphylococcaceae and Enterobacterioaceae, as another reference point to further determine how specific the sign or role of Streptococcaceae may be.
For new studies seeking to replicate the elevated Streptococcaceae findings with new data, quantitative PCR (qPCR) may be the most robust technique. It is increasingly validated against both older culture techniques and NGS for microbial quantification.20,21 As with metagenomic NGS sequencing, qPCR is typically based on amplifying and reading 16S rRNA sequences which contain both highly conserved and variable regions. However, it quantifies targeted species of interest instead of sequencing against a database of known taxa.
For further robustness, opportunities to collect cecal samples (vs fecal) should be sought, e.g., concurrent with colonoscopy examinations. The cecal region has the highest oxygen concentration in the colon, giving Streptococcaceae a relative advantage. Thus, the elevation effect, if the hypothesis holds, is likely strongest there.
Should any or all of these approaches (i.e., re-computation, qPCR, further NGS) confirm the elevation of Streptococcaceae in ME/CFS, then further new data/analyses should be sought that would drill deeper into specific Streptococcus species. While family or genus level studies are the fastest path to replication, any bacterially-driven pathology is apt to arise from specific species.
In tandem with these single time point population studies, clinical treatment studies that longitudinally track both specific symptoms and Streptococcaceae abundance can be considered utilizing antibiotics with efficacy against Streptococcaceae. To date, two open-label studies of ME/CFS patients with confirmed elevations in Streptococcus have reported reductions of both specific ME/CFS symptoms and Streptococcus abundance.22,23
Conclusion
The present hypothesis, that elevated Streptococcaceae is a promising marker and potential driving mechanism for ME/CFS, is supported by 1) two studies of patient and healthy cohorts with distinct microbiome quantification techniques, and 2) a range of mechanistic studies revealing Streptococcus-associated pathology potentially consistent with a key role in ME/CFS.
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