Layout 1 The microbiota-gut-brain axis in myalgic encephalomyelitis/chronic fatigue syndrome Eur J Transl Myol 35 (1) 13690, 2025 doi: 10.4081/ejtm.2025.13690 The intricate relationship between gut microbiota and the brain has emerged as a pivotal area of research in recent years, particularly in understanding Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).1 This complex condition is characterized by debilitating fatigue, cognitive dysfunction, and a wide array of systemic manifestations, all of which present significant challenges not only for accurate diagnosis but also for the development of effective treatment strategies.2 Recent studies have illuminated the importance of the microbiota-gut-brain axis as a crucial pathway in the pathophysiology of ME/CFS, raising important questions about the causal mechanisms that may be at play.1-8 These studies suggest that alterations in gut microbial composition could significantly impact immune responses, neurochemical signaling, and neuronal health, thereby contributing to the diverse and often debilitating symptomatology associated with ME/CFS.1-8 However, it is essential to critically evaluate whether these Abstract The intricate relationship between gut microbiota and the brain has emerged as a pivotal area of research, particularly in understanding Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). This complex condition is characterized by debilitating fatigue, cognitive dysfunction, and a wide array of systemic manifestations, posing significant challenges for diagnosis and treatment. Recent studies highlight the microbiota-gut-brain axis as a crucial pathway in ME/CFS pathophysiology, suggesting that alterations in gut microbial composition may impact immune responses, neurochemical signaling, and neuronal health. This narrative review systematically explores English-language scholarly articles from January 1995 to January 2025, utilizing databases such as PubMed, Scopus, and Web of Science. The findings underscore the potential for targeted therapeutic interventions aimed at correcting gut dysbiosis. As research progresses, a deeper understanding of the microbiota-gut-brain connection could lead to innovative approaches for managing ME/CFS, ultimately enhancing the quality of life for affected individuals. Key Words: microbiota-gut-brain axis, myalgic encephalomyelitis, chronic fatigue syndrome. Eur J Transl Myol 35 (1) 13690, 2025 doi: 10.4081/ejtm.2025.13690 The microbiota-gut-brain axis in myalgic encephalomyelitis/chronic fatigue syndrome: a narrative review of an emerging field Amr Ali Mohamed Abdelgawwad El-Sehrawy,1 Ibtihal Ibrahim Ayoub,2 Subasini Uthirapathy,3 Suhas Ballal,4 Baneen C. Gabble,5,6,7 Abhayveer Singh,8 Kavitha V,9 Rajashree Panigrahi,10 Mostafa Kamali,11 Mohsen Khosravi11,12,13 1Department of Internal medicine, Diabetes, Endocrinology and Metabolism, Mansoura University, Mansoura, Egypt; 2Department of Internal Medicine, Batterjee Medical College, Jeddah, Saudi Arabia; 3Faculty of Pharmacy, Department of Pharmacology, Tishk International University, Erbil, Kurdistan Region, Iraq; 4Department of Chemistry and Biochemistry, School of Sciences, JAIN (Deemed to be University), Bangalore, Karnataka, India; 5Department of Medical Analysis, Medical Laboratory Technique College, The Islamic University, Najaf, Iraq; 6Department of Medical Analysis, Medical Laboratory Technique College, The Islamic University of Al Diwaniyah, Al Diwaniyah, Iraq; 7Department of Medical Analysis, Medical Laboratory Technique College, The Islamic University of Babylon, Babylon, Iraq; 8Centre for Research Impact & Outcome, Chitkara University Institute of Engineering and Technology, Chitkara University, Rajpura, Punjab, India; 9Department of Chemistry, Sathyabama Institute of Science and Technology, Chennai, Tamil Nadu, India; 10Department of Microbiology, IMS and SUM Hospital, Siksha ‘O’ Anusandhan (Deemed to be University), Bhubaneswar, India; 11Department of Psychiatry, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran; 12Health Promotion Research Center, Zahedan University of Medical Sciences, Zahedan, Iran; 13Community Nursing Research Center, Zahedan University of Medical Sciences, Zahedan, Iran. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. - 63 - The microbiota-gut-brain axis in myalgic encephalomyelitis/chronic fatigue syndrome Eur J Transl Myol 35 (1) 13690, 2025 doi: 10.4081/ejtm.2025.13690 associations consistently translate into causal relationships or if they merely reflect correlated phenomena without direct influence.8 This intricate interplay between microbiota and neurological function transcends traditional views of gut health, emphasizing the potential for targeted therapeutic interventions aimed at correcting gut dysbiosis.3 As the field continues to evolve, a thorough and critical overview of existing research will not only facilitate a deeper understanding of the complexities surrounding ME/CFS but also encourage the exploration of innovative approaches to both management and treatment.5-7 This reflective analysis marks a significant and necessary step toward addressing this multifaceted disorder, as we must remain open to questioning and potentially reshaping existing paradigms in the relentless quest for effective solutions.3 Ultimately, advancing our understanding of the gut-brain connection may lead to more effective interventions that can alleviate the burdens faced by those suffering from ME/CFS, paving the way for enhanced quality of life and well-being for affected individuals.3-8 Materials and Methods In this narrative review, the methodology for gathering data involved a systematic exploration of English-lan- guage scholarly articles published from January 1995 to January 2025. This approach was chosen to comprehen- sively cover research related to the microbiome and ME/CFS. Key databases such as PubMed, Scopus, and Web of Science were searched using specific terms includ- ing “microbiota-gut-brain axis,” “ME/CFS,” “microbiome dysbiosis,” “gut health,” “pathophysiology,” and “thera- peutic approach.” The objective was to collate both qual- itative and quantitative insights concerning the composition of gut microbiota, the evaluation of inflam- matory markers, and the effectiveness of microbiome-tar- geted therapeutic interventions. Where possible, additional information from clinical trials and observa- tional studies was incorporated to enhance the review and support the comprehensive narrative being constructed. The review adopted a narrative format, aiming to integrate current research findings while emphasizing crucial mech- anistic understandings and clinical ramifications. Results Definition, prevalence, and diagnosis of ME/CFS ME/CFS is a profound multisystem disorder marked by sig- nificant physical impairment, potentially confining individ- uals to complete bed rest with extensive care needs.1 The condition manifests as severe fatigue that does not improve with rest, cognitive dysfunctions, and symptoms akin to in- fluenza including muscle aches, pain, headaches, sore throats, and sensitive lymph nodes.2 A notable worsening of these symptoms occurs after minimal physical, orthos- tatic, or mental exertion, a phenomenon known as Post-Ex- ertional Malaise (PEM). PEM typically manifests with a delay, approximately 24 hours post-exertion, and results in a drastic decrease in energy and activity levels by at least 50%. Recovery from such episodes extends beyond 24 hours and may last several weeks. The progression of ME/CFS is variable, with symptom severity shifting dra- matically within days. Patients frequently report sensitiv- ities and allergic reactions to various stimuli including extreme temperatures, light, noise, certain smells or chem- icals, and even engaging in simple conversations.1 Gastro- intestinal issues like constipation, diarrhea, and abdominal pain are common, and there is a notable overlap with irri- table bowel syndrome, affecting between 38% and 92% of patients.9 Research involving 48 participants revealed that over 70% experience gastrointestinal complaints, and more than 35% use medication for these issues.10,11 A substantial portion of patients, at least 25%, are confined to their homes or beds, indicative of the moderate to severe spectrum of ME/CFS.12 Epidemiological data suggests nearly half of those affected are unable to maintain employment, with a previous study indicating an unemployment rate of 87% among patients.11,12 During their most severe episodes, 48% of individuals are completely unproductive, with those se- verely impacted often limiting communication and spend- ing extended periods in dark, quiet rooms, unable to partake in regular showers or engage in activities like listening to music or watching television.11 Although ME/CFS can be as incapacitating as diseases like multiple sclerosis or sys- temic lupus erythematous, it is classified by the ICD-11 as a disease of the nervous system under the code 8E49, post- viral fatigue syndrome.1,13 Nonetheless, ongoing debates re- garding its pathogenesis and etiology raise questions about the accuracy of this classification.14 The worldwide incidence of ME/CFS varies significantly depending on the definition of the disease used and is also influenced by the absence of objective diagnostic tests.9 A recent meta-analysis has shown that the prevalence can be as high as 0.89% according to the most commonly used case definition, CDC-1994, and 1.14% when based on clin- ical interviews.16 ME/CFS is more commonly diagnosed in females than males, with a ratio ranging from 1.5 to 2.15 Typically manifesting in mid-life, ME/CFS has two primary patterns of onset: a sudden onset that patients clearly recall and a gradual onset marked by progressive deterioration.16 The severity and symptoms of ME/CFS can fluctuate over time. The progression of the disease varies among individ- uals; however, a significant majority reports no improve- ment over time, with only about 4% experiencing a complete recovery.11 Despite the existence of multiple consensus criteria doc- umented in scholarly articles, such as the Canadian Con- sensus Criteria, Fukuda, Oxford, and International Criteria, there is currently no commercially available blood test or diagnostic tool for ME/CFS.9 The absence of a universally accepted set of consensus criteria could potentially lead to diagnostic inaccuracies in up to 80% of ME/CFS cases.1,9 This represents a significant challenge not only for affected individuals who encounter inexperienced healthcare pro- viders but also poses broader societal and public health challenges.1 However, recent scientific publications have suggested the potential development of diagnostic tools for ME/CFS in the foreseeable future. Innovations such as nano-electronic blood-based assays, CD8+ T cell analysis, - 64 - The microbiota-gut-brain axis in myalgic encephalomyelitis/chronic fatigue syndrome Eur J Transl Myol 35 (1) 13690, 2025 doi: 10.4081/ejtm.2025.13690 fecal metagenomic profiling, and metabolite analysis have shown promise in reliably distinguishing ME/CFS patients from healthy controls in certain studies.17-20 Overview of the microbiota-gut-brain axis Since the mid-19th century, it has been recognized that there is a bidirectional communication between the gut and the brain. More recently, this interaction has been conceptual- ized as the “gut-brain axis.” The interplay between the gut microbiota and various physiological and pathological mechanisms is crucial.21 For instance, one study highlighted the role of the gut microbiome in shaping the development of the hypothalamus-pituitary-adrenal axis and influencing stress responses in germ-free animals.22 Similarly, another study found that fecal microbiota transplantation in germ- free mice from their own strain preserved typical behaviors, whereas transplantation from a different strain altered their behavior to match the donor strain.23 While the precise physiological pathways involved remain under discussion, the significance of gut-brain communication in patients with ME/CFS is supported by multiple studies.24-27 Ho- wever, the specific pathophysiological mechanisms through which the gut-brain axis affects neuropsychiatric symptoms are still not fully understood.5 The potential pathways for this communication may involve changes in the immune system, such as alterations in regulatory T-cells, NK-cells, or CD8+ T-cells, as well as variations in cytokine produc- tion including increased TGF-ß and immunoglobulins.28-33 These findings are subject to debate since chronic inflam- mation might also stem from other underlying con- ditions.1,34 In ME/CFS, altered levels of tryptophan, influenced by the microbiome, have been associated with the condition.35 Additionally, the gut microbiome has been shown to directly affect neural stimulation of the vagal nerve, which also innervates the colon, suggesting a pos- sible bidirectional influence.36 Despite uncertainties about direct mechanisms, several in- direct indicators underscore the importance of the gut-brain axis in neuropsychiatric symptoms generally and in ME/CFS specifically.1-8 A study reported alleviation of ME/CFS symptoms following rectal bacterial infusion, with a significant proportion of participants also suffering from Irritable Bowel Syndrome (IBS), indicating that while the treatment targeted IBS, it also pointed to significant gut- brain communication in ME/CFS.37 Moreover, modulation of the intestinal microbiome through antibiotics or pro- biotics has shown improvements in various neuropsychia- tric symptoms, further supporting the relevance of the gut-brain axis.24-27 Nevertheless, a recent pilot study has re- ported faecal microbiota transplantation as safe but ineffec- tive on fatigue severity and health-related quality of life in patients with ME/CFS.38 This contradiction highlights the importance of further research in this regard and clarifying the potential role of the microbiota-gut-brain axis in the pathophysiology of ME/CFS. Metagenomic analysis has also identified distinct clusters of fecal bacteria that are characteristic of ME/CFS, which vary between patients with and without concurrent IBS.19 Specifically, in those suffering from IBS, there was a no- table increase in unclassified Alistipes and a reduction in Faecalibacterium. Conversely, in ME/CFS patients without IBS, there was a rise in unclassified Bacteroides and a de- crease in Bacteroides vulgatus. Furthermore, the study highlighted variations in metabolic pathways including the synthesis of unsaturated fatty acids, degradation of atrazine, production of vitamin B6, and breakdown of pyrimidine ri- bonucleosides.39 Considering that the gut microbiome in- cludes both bacterial and viral components, and acknowledging that ME/CFS is often reported as a post- viral condition such as following SARS-CoV-2 infection, another research effort examined viral taxa in feces, blood, and saliva. However, this investigation found no significant differences in viral populations between ME/CFS patients and healthy controls.40 The role of gut dysbiosis in CFS/ME Alterations in the intestinal microbiota and dysbiosis have been observed in numerous studies on ME/CFS, though a distinct microbial pattern has yet to be consistently identi- fied.10,19,41-44 Reviews of microbiome research in ME/CFS patients indicate variable outcomes, making it difficult to definitively link changes in gut bacteria to the pathophysi- ology of the disease.6 Newberry and colleagues, through a systematic review, noted eight concordant and seven dis- cordant findings across different studies, yet still supported the presence of dysbiosis.45 In a particular study, researchers were able to accurately identify 83% of subjects as either ME/CFS patients or healthy controls by assessing dysbiosis in the gut microbiome and noting elevated inflammatory markers in the blood due to microbial translocation. Ho- wever, this study had a small sample size (N=48) and re- quires further verification. Analysis of 16S rRNA stool samples indicated a decrease in bacterial diversity and rich- ness, reduced carriage of anti-inflammatory species, and an increase in pro-inflammatory bacterial species such as En- terobacteriaceae.10 Typically, greater microbial diversity is linked to a healthier host. The cardiorespiratory fitness, measured by peak oxygen consumption (VO2 peak), ac- counts for approximately 20% of the variation in gut diver- sity, suggesting that the reduced microbial diversity in ME/CFS patients might correlate with altered physical fit- ness levels. ME/CFS patients are known to exhibit signifi- cantly lower VO2 peaks.46,47 While Armstrong and colleagues reported significant reductions in intestinal an- aerobic bacteria, other studies have found increases in these microorganisms in ME/CFS patients.42,43 Elevated levels of Enterococcus spp., Streptococcus spp., and increased En- terobacteriaceae have been documented, alongside lower levels of beneficial Bifidobacteria and decreased anti-in- flammatory Firmicutes.10,25,33,41,43,48 Metagenomic analyses by Nagy-Szakal and colleagues identified specific bacterial taxa such as the Firmicutes phylum, and genera Faecal- ibacterium, Roseburia, and Clostridium as linked to ME/CFS. Notably, increases in Alistipes and decreases in butyrate-producing Faecalibacterium were highlighted as potential diagnostic biomarkers. Their observations of higher Clostridium abundance contrast with findings from Giloteaux and others. Replications also confirmed lower Faecalibacterium levels.10,19 A case study comparing ME/CFS-discordant monozygotic twins revealed reduced - 65 - The microbiota-gut-brain axis in myalgic encephalomyelitis/chronic fatigue syndrome Eur J Transl Myol 35 (1) 13690, 2025 doi: 10.4081/ejtm.2025.13690 abundance of Bifidobacterium and Faecalibacterium in the affected twin, along with lower gut alpha diversity.49 Inter- estingly, reductions in Faecalibacterium have also been ob- served in patients with fatigue associated with inflammatory bowel disease, cancer-related fatigue, and other autoim- mune conditions such as multiple sclerosis and type 1 dia- betes mellitus.50-53 Numerous investigations have indicated a decrease in a butyrate-producing Bacteroides species in individuals with ME/CFS.45 Advanced 16S rRNA sequen- cing of fecal samples has shown changes in the gut micro- biome associated with heightened gut inflammation, specifically notable increases in Lactonifactor and Alistipes.41 A particular study involving 48 patients and 52 controls pinpointed 26 markers, including bacterial taxa that distinctly characterized ME/CFS compared to healthy in- dividuals.54 The most significant differences between pa- tients and controls were observed in the levels of Coprobacillus, Eggerthella, and Blautia. There were reduc- tions in Faecalibacterium and rises in Coprobacillus, cor- roborating findings from earlier research.10,19 To assess the diagnostic capabilities of these markers, researchers cate- gorized patients based on the duration of ME/CFS into short-term and long-term groups. They noted that micro- biome alterations were substantially more common in cases with shorter disease duration (≤3 years) than those with longer disease spans (˃3 years). The researchers proposed that these microbial markers reflect transient shifts at the initial stages of the disease, presenting potential for their use as future biomarkers.54 Moreover, research comparing the microbiome profiles of patients with ME/CFS to those with acute Q-fever fatigue and healthy individuals revealed similar microbial patterns between the two patient groups, both of which were distinctly different from that of healthy controls. Both ME/CFS and Q-fever patients exhibited sim- ilar proinflammatory markers, allowing for their differen- tiation from healthy individuals. Increased levels of Firmicutes and Actinobacteria were observed in 50 ME/CFS patients compared to 72 healthy controls; while Bacteroidetes levels were lower.55 A subsequent study in- volving 35 patients and 70 controls also identified a unique microbial composition characterized by reduced anti-in- flammatory Firmicutes, supporting earlier findings.8,10,41 This research compared patients with internal (relatives) versus external controls, identifying several commonalities between patients and their relatives, but not with external controls, highlighting the importance of selecting appropri- ate controls that account for lifestyle and genetic factors.8 These findings, though somewhat inconsistent, clearly in- dicate dysbiosis in ME/CFS as noted in prior reviews; ho- wever, the precise role of these microbial discrepancies in the pathogenesis of the disease remains to be elucidated. The referenced studies vary significantly in terms of sample size, participant recruitment criteria including disease sev- erity at the time of sampling, and microbiota analysis tech- niques (predominantly culturing or 16S amplicon sequencing, with only one employing whole genome shot- gun sequencing).6,45 It is crucial to recognize that ME/CFS patients often use various medications regularly to manage their symptoms,11,56 and it is well-established that not only antibiotics but also other medications can substantially alter the commensal microbiota.57-59 Future studies should con- sider these factors. Additionally, diet can quickly affect the microbiome composition in the short term, and long-term dietary patterns have been linked to specific fecal micro- biome genera; for instance, diets rich in protein/animal fats tend to favor Bacteroides, while carbohydrate-rich diets are associated with a higher prevalence of Prevotella.60,61 Therefore, dietary intake within at least 48-24 hours before stool sample collection should be taken into account. Fur- thermore, larger studies with well-defined clinical inclusion criteria are necessary to address the various subgroups within the ME/CFS patient population and to explore both viral and eukaryotic components of the gut micro- biome.6,44,45,62 Mechanisms linking gut microbiota to CFS/ME symptoms Under normal composition of the gut, bacterial transloca- tion through the gut barrier should not be possible.63 Ho- wever, numerous studies have found that patients with ME/CFS exhibit greater intestinal permeability, lending support to the theory that bacteria may translocate into the systemic circulation.64-67 This is used to account for the anomalously high concentrations of IgA and IgM antibodies against lipopolysaccharide observed in the peripheral blood of these patients, with 67% and 40% of ME/CFS patients showing elevated levels of IgA and IgM, respectively, in contrast to none in the control group. Moreover, it has been noted that IgA levels correlate with the severity of the dis- ease.33 Additionally, increased bacterial components in the plasma of ME/CFS patients suggest heightened gut permea- bility. Giloteaux and colleagues propose that elevated en- dotoxin levels might compromise the epithelial barrier, allowing entry into the bloodstream, triggering an immune response and leading to systemic inflammation. The pres- ence of higher levels of lipopolysaccharide in the blood- stream could be due to a greater abundance of Gram-negative bacteria in the intestine, as their outer mem- brane contains this bacterial endotoxin, often linked with gut dysbiosis.10,63 Nagy-Szakal and associates also observed raised plasma ceramines levels, suggesting these might re- sult from increased lipopolysaccharide hydrolysis. They hy- pothesized that elevated ceramines could induce toxic responses in various cell types including gut epithelial cells; potentially impairing the epithelial barrier and further in- creasing gut permeability.68 Notably, higher ceramines levels have been identified in other chronic conditions such as IBS, diabetes, cardiomyopathy, and atherosclerosis.68-70 An additional factor contributing to heightened intestinal permeability could be the elevated by-products resulting from the fermentation of amino acids to generate Short- Chain Fatty Acids (SCFAs).42 Furthermore, there has been a noted decrease in the presence of bacteria that produce SCFAs, particularly those that generate butyrate, such as Faecalibacterium, Roseburia (belonging to the Firmicutes phylum), and certain Bacteroides species.10,19,42 This obser- vation has been consistently made across various studies involving patients with ME/CFS. SCFAs, including buty- rate, are byproducts of bacterial fermentation and play cru- cial roles in the communication between the gut and brain. - 66 - The microbiota-gut-brain axis in myalgic encephalomyelitis/chronic fatigue syndrome Eur J Transl Myol 35 (1) 13690, 2025 doi: 10.4081/ejtm.2025.13690 Butyrate serves as the primary energy source for colon- ocytes and is thought to be a vital component in managing the neuroimmunoendocrine system. These fatty acids con- tribute to gut health by exerting anti-inflammatory proper- ties and improving the function of the intestinal epithelial barrier.71 A study utilizing high-throughput sequencing demonstrated that well-conditioned individuals exhibit not only elevated fecal butyrate concentrations but also a cor- relation with enhanced physical fitness levels, notably higher VO2 max values, and increased diversity in gut mi- crobiota. Particularly, the presence of butyrate-producing microorganisms was linked to superior fitness levels and a corresponding decrease in lipopolysaccharide biosynthesis in these fit subjects. Typically, elevated blood lipopolysac- charide triggers a significant inflammatory response; ho- wever, reduced lipopolysaccharide levels due to regular physical activity might contribute to diminished inflamma- tion in physically active individuals.25,43 When relating these findings from a healthy cohort to the clinical manifestations of ME/CFS, it becomes plausible that the observed scarcity of butyrate-producing bacteria in ME/CFS patients could stem from a decrease in SCFAs, potentially leading to PEM which then perpetuates a cycle of inactivity. This inactivity may further exacerbate the increase of lipopolysaccharide levels in ME/CFS patients. Conversely, it is conceivable that the chronic inactivity seen in ME/CFS could be a direct consequence of the illness itself, which over time might re- sult in decreased SCFAs production. Another perspective might consider the depletion of SCFAs as an indirect out- come of the disease, where SCFAs-producing bacteria are suppressed by an overgrowth of D-lactate producing bac- teria, which have been noted at elevated levels in ME/CFS patients and have been subject to prior investigations.46 Contrary to the butyrate deficiency theory, a particular study identified a notable rise in fecal concentrations of butyrate, isovalerate, and valerate in ME/CFS specimens. This in- crease in fecal SCFAs levels was associated with height- ened bacterial fermentation. The researchers proposed that these metabolic changes could be due to an increased gut pH or from the gut dysbiosis.42 Additionally, it’s worth not- ing that SCFAs can sometimes exert neurotoxic effects.72 Another theory for the elevated fecal SCFAs levels could involve a malabsorption issue. It is important to recognize that fecal SCFAs excretion does not fully represent the ac- tual SCFAs concentration and production within the gut; thus, analysis should also include serum levels.73,74 To date, no studies have measured butyrate levels in serum. Inter- preting these findings requires caution due to the potential for multiple direct and indirect causes. For instance, ME/CFS patients often undergo medication treatments and are limited in their physical activities, which could indi- rectly influence their gut microbiota composition. To estab- lish causality, longitudinal studies spanning extended periods, ideally starting before the disease onset, are crucial. These studies should consider factors like medication use and physical activity levels as they might confound the re- sults.11,75 As another possible mechanism, some researchers have suggested that an increase in D-lactic acid might stem from enhanced intestinal colonization by Gram-positive bacteria, such as Enterococci and Streptococci, which may lead to reduced intestinal pH, increased gut permeability, systemic inflammation, immune activation, and oxidative stress.25,43 Despite this theory, investigations targeting the bacterial overgrowth with antibiotics and probiotics have not demonstrated improvements in fatigue symptoms, cast- ing doubt on the role of D-lactate. Furthermore, discrep- ancies between cerebrospinal fluid lactate levels and fecal lactate levels highlight an incomplete understanding of in- testinal lactate metabolism’s impact on ME/CFS pa- tients.25,42,76 Additionally, recent research on the etiology of ME/CFS suggests an abnormal metabolism of tryptophan, which is converted into kynurenine via the enzyme Indo- leamine-2,3-Dioxygenase (IDO).77-83 This enzyme, ex- pressed in antigen-presenting cells, is pivotal as it metabolizes about 95% of tryptophan, with the remaining 5% converted into serotonin and melatonin. The kynurenine pathway, regulated by IDO, plays critical roles in the im- mune system by acting as an immunoregulator and im- munosuppressor. IDO is enhanced by proinflammatory cytokines, such as IFN-γ, as well as IFN-α, TNF-α, and li- popolysaccharide during infections and inflammations. Al- though high IDO levels seem to relate to disease severity by activating regulatory T-cells and suppressing effector T- cell function through tryptophan degradation to kynurenine, the implications for meaningfully treating or diagnosing diseases such as ME/CFS are still under investigation.84- 87 Robert Phair’s team has put forth a “metabolic trap hy- pothesis” suggesting that ME/CFS patients experience insufficient kynurenine production due to gene mutations in IDO isoforms, especially IDO-2.78 This results in el- evated tryptophan levels and impaired functions of the CNS, gastrointestinal, immune systems, and energy metab- olism. In their study with ME/CFS patients, they found nu- merous gene mutations in IDO-2, and these correlated with the severity of ME/CFS symptoms. The lack of IDO-2 enzyme activity hinders tryptophan’s conversion to kynure- nine, aligning with typical pathological states of ME/CFS. Elevated tryptophan and reduced kynurenine were con- firmed in these patients. Disruption in serotonin and mela- tonin pathways due to this blockage is linked to ME/CFS symptomatology. Additionally, insufficient Nicotinamide Adenine Dinucleotide (NAD+) synthesis from this meta- bolic disruption might explain the condition’s hypometa- bolic phenotype. Elevated tryptophan levels fail to protect intestinal integrity against lipopolysaccharide-induced dam- age, unlike lower kynurenine levels.78,88 The “metabolic trap hypothesis” suggests potential immunological and meta- bolic mechanisms involved in ME/CFS, emphasising the role of serotonin and tryptophan metabolism, with impacts on patients’ microbiomes. Serotonin acts as a neurotrans- mitter in the central nervous system and a hormone in gut- brain communication, while tryptophan undergoes metabolism via serotonin, kynurenine, melatonin pathways, and through gut microbiota into indole derivatives. The function of the enzyme IDO is crucial here; decreased IDO activity can disrupt tryptophan fermentation and influence the gut microbiome. This disruption can lead to an impaired intestinal mucosal barrier, increase endotoxin translocation, and cause chronic inflammation—all factors associated with ME/CFS. Changes in these biochemical pathways - 67 - The microbiota-gut-brain axis in myalgic encephalomyelitis/chronic fatigue syndrome Eur J Transl Myol 35 (1) 13690, 2025 doi: 10.4081/ejtm.2025.13690 highlight their importance in understanding disease mech- anisms and potential therapeutic targets for ME/CFS pa- tients.89-91 Recent studies in ME/CFS explore the potential increase in kynurenine production as opposed to the meta- bolic trap hypothesis suggesting decreased levels. The im- balance between tryptophan depletion and kynurenine generation, due to elevated IDO activity triggered by an im- mune response, is marked by the Kynurenine and Trypto- phan (KYN/TRP) ratio. This ratio serves as an indicator of IDO activity and cellular immune response. High IDO levels are linked with chronic inflammation, supporting in- creased kynurenine production theories. Elevated KYN/TRP ratios and neuroactive metabolites like quino- linic acid, observed in multiple disorders, parallel symp- toms found in ME/CFS such as heightened sensitivity and central sensitization. Additionally, viral infections like Ep- stein-Barr virus activating IDO to convert tryptophan to ky- nurenine support this hypothesis.92-95 Altogether, these results indicate that tryptophan and its derivatives could play a significant role in influencing the gut microbiome, as well as the activation of the gut mucosal immune system and its interactions with the host, including the immune re- sponse.90,96-98 However, recent clinical investigations indi- cate that altered immunity and kynurenine metabolism likely initiate fatigue in ME/CFS rather than sustaining it over time.77 Recent evidence has also explored the potential link between antibiotic use and the development of ME/CFS. It has been noted that altered microbiota in pa- tients with ME/CFS can be affected by antibiotics, yet no studies have specifically examined antibiotic intake as a trigger for ME/CFS. Many ME/CFS patients have a history of frequent infections, often treated with antibiotics, which might alter the microbiome and influence susceptibility to the syndrome.11,99-102 This suggests that frequent antibiotic use could change intestinal microbiota, potentially impair- ing the production of anti-inflammatory metabolites or fos- tering conditions like D-lactic acidosis due to an increase in D-lactate-producing bacteria.42,43,76,103,104 Women are more frequently diagnosed with ME/CFS, which may be linked to their higher antibiotic exposure compared to men.105,106 Additionally, antibiotics might contribute to oxidative stress through the production of Reactive Oxygen Species (ROS), aligning with findings of elevated ROS levels in ME/CFS patients.107-109 In contrast to some theories, antibiotics have been considered as a potential treatment for ME/CFS to ad- dress the overgrowth of certain bacterial species. A pilot study by Jackson and colleagues found that antibiotic treat- ment improved sleep, likely due to decreased levels of lactic acid-producing bacteria. They suggested that better sleep quality might result from a more balanced microbiome with reduced Gram-positive bacteria, consequently lowering proinflammatory cytokines.24 Additionally, Wallis and col- leagues observed improvements in neurological symptoms, including sleep quality, following a four-week combined antibiotic and probiotic treatment. However, this treatment did not alleviate fatigue symptoms associated with ME/CFS.25 These findings indicate a possible microbiome- related pathway to enhance certain symptoms of the con- dition while highlighting the complexity of treating fatigue specifically.24,25 Potential therapeutic approaches targeting gut health in CFS/ME Emerging therapeutic approaches targeting gut health in ME/CFS underscore the importance of the microbiota-gut- brain axis in managing this complex condition. Research indicates that dysbiosis in individuals with ME/CFS is as- sociated with heightened fatigue and cognitive difficulties, prompting interest in interventions such as probiotics, pre- biotics, dietary adjustments, and Fecal Microbiota Trans- plantation (FMT) to restore microbiome balance.2 The normalization of gut microbiota through microbial treat- ments could potentially alleviate symptoms by enhancing immune function and reducing inflammation.2 Additionally, the exploration of synbiotics highlights the need for com- prehensive strategies that address both digestive health and neuropsychiatric symptoms.4 Current evidence suggests that modifications in the gut microbiome, particularly de- creases in beneficial butyrate-producing bacteria, may con- tribute to cognitive impairments commonly reported in ME/CFS patients.7 As research progresses, it becomes in- creasingly clear that a nuanced understanding of gut health is vital for developing effective, multidisciplinary treatment modalities for ME/CFS (see Figure 1). Probiotics and their potential benefits The emerging role of probiotics in the management of ME/CFS holds significant promise for addressing the com- plex interplay between gut health and neurocognitive symptoms. Probiotics, which are live microorganisms that confer health benefits when consumed, may help restore gut microbiome balance, potentially alleviating symptoms associated with dysbiosis often observed in ME/CFS pa- tients.4 Two systematic reviews assessed the efficacy of probiotics in treating ME/CFS, but found limited and in- consistent data, including only two studies each due to vari- able outcomes and poor quality research.56,111 A small cohort demonstrated significant increases in Bifidobacteria and Lactobacillus levels after probiotic intake compared to a placebo.27 Groeger’s rare RCT indicated decreased inflam- matory markers, such as CRP and TNF-a, in ME/CFS pa- tients following Bifidobacterium infantis consumption.112 Another study by Sullivan suggested an improvement in anxiety through probiotics containing Lactobacillus and Bi- fidobacterium, although anxiety is not a core ME/CFS symptom, and the study had only 15 participants.26 Ad- ditionally, a non-controlled pilot study with 13 ME/CFS patients reported enhanced well-being and reduced oxi- dative and inflammatory parameters after probiotic con- sumption.113 Overall, while some positive outcomes were noted, the research is limited by small sample sizes and methodological inconsistencies. All in all, research on the microbiome’s potential as a treatment target for ME/CFS shows promise, but no direct, long-term improvements in PEM or physical activity levels have been confirmed.1 Cor- bitt and colleagues’ systematic review highlights the current lack of evidence supporting probiotics for gastrointestinal symptoms in ME/CFS patients, mainly due to poor study quality and conflicting outcomes.56 This aligns with a Na- ture evaluation indicating insufficient impact assessments - 68 - The microbiota-gut-brain axis in myalgic encephalomyelitis/chronic fatigue syndrome Eur J Transl Myol 35 (1) 13690, 2025 doi: 10.4081/ejtm.2025.13690 and approved therapy recommendations for probiotics, par- ticularly as biased industry-driven studies yield inconsistent results.114 Recent suggestions propose using biopsies in- stead of stool samples to better understand gut health, con- sidering individual variability and gut microbiota resilience. In other neuroimmunological diseases like mul- tiple sclerosis, probiotics seem to modulate immune re- sponses by increasing anti-inflammatory mediators. Additionally, SCFA supplementation emerges as a novel area of interest; depleted SCFA-producing bacteria in pa- tients may lead to inflammatory states relevant in such dis- eases. Clinical studies with multiple sclerosis patients show propionic acid supplementation boosts Treg cells and in- terleukin-10 levels, potentially relevant findings for ME/CFS treatment strategies as well.115-123 Prebiotics and their potential benefits Prebiotics are indigestible carbohydrates that nourish gut microbiota, primarily including fructo-oligosaccharides and galacto-oligosaccharides. They are broken down by bacteria into SCFAs, which benefit both gastrointestinal and systemic health.1 By selectively enhancing beneficial bacteria and altering gut microbiota composition and func- tion, prebiotics show promise as supportive treatments for disorders like IBS, Crohn’s disease, autism, obesity, and colorectal cancer.3-7 Research demonstrates that various oligosaccharides can correct microbiota imbalances by fostering Lactobacilli growth, decreasing Proteobacteria, and reducing the Firmicutes/Bacteroidetes ratio in obese rodents.3 These studies also show improvements in gut barrier integrity and reduced systemic inflammation. Ro- dents consuming prebiotics such as bovine milk oligosac- charides and oligofructose-enriched inulin experienced lowered plasma lipopolysaccharides, serum pro-inflam- matory cytokines, intestinal inflammation, and enhanced tight-junction integrity.1-8 Collectively, these findings sug- gest that prebiotics could help manage conditions like ME/CFS involving dysbiosis and chronic inflammation. - 69 - Figure 1. Mechanisms linking chronic inflammation, dysbiosis, and therapeutic options for cognitive symptoms in ME/CFS (Adapted from Varesi et al. 20213). The microbiota-gut-brain axis in myalgic encephalomyelitis/chronic fatigue syndrome Eur J Transl Myol 35 (1) 13690, 2025 doi: 10.4081/ejtm.2025.13690 However, more clinical studies are required to confirm these potential benefits. Dietary interventions and their impact on gut microbiota Fasting and fasting mimicking diets may positively impact the immune system, enhancing chronic inflammation reg- ulation without compromising defense against infections. They could inhibit hyperinflammation by targeting the NLRP3 inflammasome, modulating type 2 immune re- sponses, decreasing pro-inflammatory T helper-17 cells in the gut, and exerting anti-inflammatory effects through various pathways.124-129 These diets could also interact with circadian rhythms and mitochondrial function, which are implicated in ME/CFS pathophysiology.130-131 As mi- tochondrial dysfunction is crucial in ME/CFS, interven- tions like caloric restriction, fasting, and ketogenic diets aim to protect mitochondria and potentially alleviate symptoms.132,133 Ketogenic diets may bypass glycolytic blockages, providing an alternative energy substrate for cells and showing promise in managing fatigue across conditions like multiple sclerosis, Parkinson’s disease, and cancer-related fatigue. However, due to potential risk of bias and adverse effects during initial weeks, these reg- imens should be undertaken cautiously under medical guidance. Their mechanisms are partly mediated by the gut microbiome but remain incompletely understood.134- 145 Altogether, A systematic review found no evidence sup- porting the benefits of elimination or modified diets for patients with ME/CFS.146 This conclusion also applied to nutritional supplements, though they might help alleviate some specific symptoms experienced by ME/CFS pa- tients.146-148 FMT as a treatment option FMT has emerged as a compelling treatment option for ad- dressing gut dysbiosis associated with ME/CFS. As re- search increasingly highlights the critical interplay between gut health and neurological function, FMT offers a means to potentially restore microbial diversity and improve clin- ical outcomes. Studies indicate that patients with ME/CFS frequently experience gastrointestinal symptoms, and alter- ations in gut microbiota composition, specifically reduced diversity and dysbiosis, are common.5 By re-establishing a healthier gut microbiome, FMT may mitigate chronic in- flammation and enhance intestinal barrier integrity, thus ad- dressing both gastrointestinal and neurological symptoms.7 Additionally, the overlap between ME/CFS and Long co- ronavirus disease 2019 suggests that FMT might also alle- viate fatigue and cognitive impairments linked to these conditions.4 However, while preliminary findings are prom- ising, rigorous clinical trials are essential to ascertain the efficacy and safety of FMT for ME/CFS patients.1 Empha- sizing holistic approaches, the study of FMT underscores the necessity for integrative therapies in chronic diseases characterized by complex etiologies.3 Additional approaches mediated by the gut microbiome Thiamine, essential for the citrate cycle and glycolysis, is partially produced by gut bacteria like Bacteroides, re- duced in ME/CFS patients.149 Faecalibacterium, also re- duced in these patients, needs thiamine for growth.10,19,45,150 Though microbial thiamine production is minimal, it in- fluences the competitive microbial environment.151 With limited therapeutic options for ME/CFS beyond dietary and pharmaceutical interventions, non-pharmaceutical treatments have been explored. Notably in China, acu- puncture and moxibustion have been studied for their po- tential to alleviate fatigue symptoms.152 A recent randomized controlled trial suggested improvements in fatigue with these therapies linked to changes in intestinal microbiome; however, possible bias exists due to chal- lenges such as lack of blinding and subjective fatigue as- sessments.153 Another non-pharmacological option is ginseng administration, shown to reduce fatigue in ME/CFS and other conditions through potentially gut mi- crobiome-mediated pharmacokinetics.154 Although indi- rect and not conclusive, these findings indicate a possible connection between intestinal microbiome changes and fatigue reduction in ME/CFS.155,156 Discussion Challenges and limitations of current therapeutic strategies The current therapeutic strategies for ME/CFS face numer- ous challenges and limitations, particularly as the field grap- ples with a complex interplay of biological and psychological factors. The absence of definitive biomarkers complicates diagnosis and has led to inconsistencies in treatment approaches, leaving many patients dissatisfied with conventional therapies like cognitive-behavioral ther- apy and graded exercise, which have mixed results in effi- cacy.5 Furthermore, emerging evidence linking gut dysbiosis to symptom severity highlights potential thera- peutic avenues involving probiotics and dietary interven- tions; however, substantial variability in study designs and outcomes restricts the ability to draw robust conclusions.3,4 Moreover, the multifactorial nature of ME/CFS necessitates a more nuanced understanding of its pathogenesis, includ- ing interactions between genetic, environmental, and mi- crobial factors.2 Thus, ongoing research is critical to refine these strategies while addressing methodological limitations inherent in the current studies.1,7 Future directions in research Future directions in research on the microbiota-gut-brain axis in ME/CFS hold great promise for elucidating the un- derlying mechanisms of this complex condition. Investigat- ing the gut microbiomes alterations, including the notable dysbiosis observed in ME/CFS patients, may uncover po- tential therapeutic targets, such as probiotics and dietary in- terventions that could ameliorate symptoms like fatigue and cognitive dysfunction.4 Furthermore, as emerging evidence links gut health with neuropsychiatric symptoms, exploring the effects of psychobiotic treatments may provide new avenues for intervention.3 Additionally, addressing the methodological inconsistencies in previous studies is vital, suggesting a need for standardized diagnostic criteria and - 70 - The microbiota-gut-brain axis in myalgic encephalomyelitis/chronic fatigue syndrome Eur J Transl Myol 35 (1) 13690, 2025 doi: 10.4081/ejtm.2025.13690 larger, longitudinal studies that encompass diverse patient populations.1,6 Understanding the intersection of immune function, gut microbiota, and ME/CFS pathogenesis will be crucial for developing effective personalized treatment strategies and enhancing overall patient outcomes.2,7,8 Need for standardized diagnostic criteria in studies The disparate findings across studies investigating the mi- crobiota-gut-brain axis in ME/CFS highlight the urgent need for standardized diagnostic criteria. Current research often employs varying definitions of ME/CFS, which complicates comparisons and may obscure true relation- ships between gut dysbiosis and symptomatology. For in- stance, the lack of consistency in diagnostic protocols has resulted in differential reporting of comorbid conditions and symptom severity among participants, undermining the validity of conclusions drawn from these studies.6 Es- tablishing uniform criteria would not only facilitate reli- ably replicating results but also enhance our understanding of shared pathways in related conditions such as Long co- ronavirus disease 2019.4 The identification of specific mi- crobial profiles and metabolic disturbances in ME/CFS patients necessitates rigorous methodologies to inform treatment strategies and improve outcomes, making stan- dardized diagnostic criteria critical for future advance- ments in this emerging field.1,2 Importance of longitudinal studies to establish causality Longitudinal studies play a crucial role in establishing caus- ality within the emerging field of the microbiota-gut-brain axis as it relates to ME/CFS. These studies allow research- ers to observe changes over time, which can help identify whether alterations in gut microbiota precede the onset of ME/CFS symptoms or result from the condition itself. This distinction is essential, as highlighted by the correlation of gut dysbiosis with increased intestinal permeability and in- flammation noted in previous research.7 Moreover, longitu- dinal designs facilitate the tracking of various confounding factors, such as infections and stressors, that may contribute to symptom development.1 The discrepancies in current studies regarding microbiome composition further under- score the necessity of consistently applying longitudinal methodologies to clarify the biological mechanisms at play, thus solidifying the potential relationship between microbial health and ME/CFS symptomatology.5 Through rigorous evaluation over extended periods, these studies can contrib- ute significantly to refining treatment strategies that target gut health as part of a holistic approach to managing ME/CFS.4 Exploration of specific microbial metabolites in ME/CFS The exploration of specific microbial metabolites in ME/CFS reveals significant insights into the potential role of gut dysbiosis in the pathogenesis of this complex con- dition. Dysregulated microbial profiles, characterized by decreased diversity and altered metabolic pathways, may contribute to the debilitating symptoms experienced by individuals with ME/CFS. Research indicates that patients exhibit reduced levels of beneficial SCFA, particularly bu- tyrate, which are critical for maintaining gut barrier inte- grity and modulating inflammation.7 Additionally, metab- olites such as glutamic and argininosuccinic acids have been shown to be elevated in ME/CFS patients, raising concerns about their contribution to neuroinflammation and cognitive dysfunction.2 Evidence supports the idea that gut-brain communication may be disrupted due to these microbial imbalances, emphasizing the need for tar- geted interventions like probiotics and dietary modifica- tions to restore homeostasis and alleviate symptoms.4,5 Understanding these metabolic abnormalities opens path- ways for innovative treatment strategies aimed at improv- ing patient outcomes.8 Investigating the interplay between genetics and gut microbiota The investigation into the interplay between genetics and gut microbiota offers critical insights into the etiology of ME/CFS. Research indicates that genetic predispositions may influence the composition of gut microbiota, poten- tially affecting immune responses and symptom severity in ME/CFS patients. Alterations in microbial diversity have been consistently documented, with studies observing a de- cline in beneficial microbial populations like Bifidobacteria and Lactobacillus, which are linked to improved mental health outcomes.6 Moreover, associations between gut dys- biosis and various metabolic pathways suggest that genetic factors might modulate microbial responses to environmen- tal triggers, further complicating ME/CFS pathogenesis.4 Understanding this dynamic relationship could pave the way for targeted interventions, leveraging specific probiotic therapies to restore microbial balance and ameliorate symp- toms resulting from gut-brain axis disturbances.2 Thus, dis- entangling the genetic influences on gut microbiome composition represents a promising avenue for future re- search.1 Potential for personalized treatment approaches based on microbiome profiles The potential for personalized treatment approaches based on microbiome profiles in ME/CFS marks a significant ad- vancement in understanding and managing this complex condition. Growing evidence indicates that dysbiosis of the gut microbiome may play a critical role in ME/CFS pathol- ogy, with specific bacterial populations influencing symp- tom severity and metabolism.7 By analyzing the unique microbiome profiles of individuals, clinicians could tailor interventions such as probiotics, synbiotics, or dietary mod- ifications to restore microbial balance and alleviate symp- toms.4 Additionally, understanding the interactions between gut microbiota and neurological function could inform more comprehensive treatment options that address both physical and cognitive challenges.8 Despite the promising avenues for personalized therapies, further research is nec- essary to establish standardized protocols and clarify the mechanisms by which microbiome alterations impact the health of ME/CFS patients.1,5 Ultimately, this personalized approach toward treatment may enhance patient outcomes and improve quality of life. - 71 - The microbiota-gut-brain axis in myalgic encephalomyelitis/chronic fatigue syndrome Eur J Transl Myol 35 (1) 13690, 2025 doi: 10.4081/ejtm.2025.13690 Conclusions In conclusion, the exploration of the microbiota-gut-brain axis offers significant insights into the pathophysiology of ME/CFS, highlighting the intricate interplay between mi- crobial communities, immune responses, and neurological function. As research uncovers distinct dysbiotic profiles among ME/CFS patients, it becomes increasingly crucial to consider how alterations in gut microbiota may contrib- ute to the symptomatology of this complex condition. The evidence supporting the relationship between gut health and cognitive and physical fatigue underscores the need for tar- geted interventions, including dietary modifications and probiotic therapies, which could potentially alleviate symp- toms experienced by sufferers. However, the field remains in its infancy, necessitating further rigorous studies to es- tablish causative links and develop comprehensive treat- ment strategies. Ultimately, a deeper understanding of the microbiota-gut-brain axis could pave the way for innovative and effective therapeutic approaches in managing ME/CFS, transforming the landscape of care for affected individuals. Contributions AMAES, IIA, SU, SB, BCG, AS, KV, RP, MKa, and MKh: Conceptualization, data collection, writing-original draft preparation, and writing-review and editing. The authors have read and agreed to the published final version of the manuscript. Funding The authors received no specific funding for this work. Conflict of interest The authors declare that they have no conflict of interest. Ethical publication statement Not applicable. Informed consent Not applicable. Corresponding author Mostafa Kamali, Department of Psychiatry, School of Med- icine, Zahedan University of Medical Sciences, Zahedan, Iran. ORCID ID: 0009-0009-8606-3961 E-mail: mostafa78kamali@gmail.com Co authors Amr Ali Mohamed Abdelgawwad El-Sehrawy ORCID ID: 0000-0001-5481-5617 E-mail: sehrawyamr@gmail.com Ibtihal Ibrahim Ayoub ORCID ID: 0009-0008-6555-5943 E-mail: Ibtihalayoub5@gmail.com Subasini Uthirapathy ORCID ID: 0000-0003-1250-388X E-mail: subasini.uthirapathy@tiu.edu.iq Suhas Ballal ORCID ID: 0000-0002-6041-8332 E-mail: b.suhas@jainuniversity.ac.in Baneen C. Gabble ORCID ID: 0009-0001-5190-7185 E-mail: baneen.j.ja@iunajaf.edu.iq Abhayveer Singh ORCID ID: 0009-0002-1274-5117 E-mail: abhayveer_singh@outlook.com Kavitha V ORCID ID: 0000-0001-7281-5960 E-mail: kavitha.chemistry@sathyabama.ac.in Rajashree Panigrahi ORCID ID: 0009-0009-8606-3961 E-mail: rajashreepanigrahy@soa.ac.in Mostafa Kamali ORCID ID: 0009-0009-8606-3961 E-mail: mostafa78kamali@gmail.com Mohsen Khosravi ORCID ID: 0009-0009-8606-3961 E-mail: dr_khosravi2016@yahoo.com References 1. König RS, Albrich WC, Kahlert CR, et al. 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Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. Submitted: 2 February 2025. Accepted: 3 February 2025. Early access: 12 February 2025. - 77 -