Associations between self-reported psychological symptom severity and gut microbiota: further support for the microgenderome.

BACKGROUND: Research into the brain-gut-microbiota axis (BGMA) continues to reveal associations between gut microbiota (GM) and psychological symptom expression, inspiring new ways of conceptualising psychological disorders. However, before GM modulation can be touted as a possible auxiliary treatment option, more research is needed as inconsistencies in previous findings regarding these associations are prevalent. Additionally, the concept of the microgenderome, which proposes that GM may interact with sex hormones, has received limited attention in studies using human samples to date. However, such research has demonstrated sex specific associations between GM and psychological symptom expression. METHOD: This cross-sectional retrospective study explores associations between GM species (identified through faecal microbial analysis) and symptom severity across four psychological domains (Depressive, Neurocognitive, Stress and Anxiety, and Sleep and Fatigue) for males (N = 1143) and females (N = 3467) separately. RESULTS: GM species from several genera including Bifidobacterium, Clostridium, Enterococcus, and Leuconostoc were found to be differentially associated with psychological symptom severity for males and females. As such, the findings of the current study provide support for the concept of the microgenderome. CONCLUSION: While further research is needed before their implementation in psychological treatment plans, the current findings suggest that modulation of GM at the species level may hold promise as auxiliary diagnostic or treatment options. These findings may give further insight into a client's presenting problem from a more holistic, multidisciplinary perspective. The clear sex divergence in associations between GM and symptoms give insight into sex discrepancies in susceptibility to psychological disorders.

The role of the brain-gut-microbiota axis in psychology: The importance of considering gut microbiota in the development, perpetuation, and treatment of psychological disorders.

INTRODUCTION: The prevalence of psychological disorders remains stable despite steady increases in pharmacological treatments suggesting the need for auxiliary treatment options. Consideration of the brain-gut-microbiota axis (BGMA) has made inroads into reconceptualizing psychological illness from a more holistic perspective. While our understanding of the precise role of gut microbiota (GM) in psychological illness is in its infancy, it represents an attractive target for novel interventions. METHOD: An extensive review of relevant literature was undertaken. RESULTS: Gut microbiota are proposed to directly and indirectly influence mood, cognition, and behavior which are key components of mental health. This paper outlines how GM may be implicated in psychological disorders from etiology through to treatment and prevention using the Four P model of case formulation. CONCLUSION: Moving forward, integration of GM into the conceptualization and treatment of psychological illness will require the discipline of psychology to undergo a significant paradigm shift. While the importance of the GM in psychological well-being must be respected, it is not proposed to be a panacea, but instead, an additional arm to a multidisciplinary approach to treatment and prevention.

Correction to: Open-label pilot for treatment targeting gut dysbiosis in myalgic encephalomyelitis/chronic fatigue syndrome: neuropsychological symptoms and sex comparisons.

The original version of this article [1], published on 6 February 2018, contains a mistake in the 'Conclusions' section. The corrected version of the affected sentence is given below and the corrected part is marked in bold.

Open-label pilot for treatment targeting gut dysbiosis in myalgic encephalomyelitis/chronic fatigue syndrome: neuropsychological symptoms and sex comparisons.

BACKGROUND: Preliminary evidence suggests that the enteric microbiota may play a role in the expression of neurological symptoms in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Overlapping symptoms with the acute presentation of D-lactic acidosis has prompted the use of antibiotic treatment to target the overgrowth of species within the Streptococcus genus found in commensal enteric microbiota as a possible treatment for neurological symptoms in ME/CFS. METHODS: An open-label, repeated measures design was used to examine treatment efficacy and enable sex comparisons. Participants included 44 adult ME/CFS patients (27 females) from one specialist medical clinic with Streptococcus viable counts above 3.00 × 105 cfu/g (wet weight of faeces) and with a count greater than 5% of the total count of aerobic microorganisms. The 4-week treatment protocol included alternate weeks of Erythromycin (400 mg of erythromycin as ethyl succinate salt) twice daily and probiotic (D-lactate free multistrain probiotic, 5 × 1010 cfu twice daily). 2 × 2 repeated measures ANOVAs were used to assess sex-time interactions and effects across pre- and post-intervention for microbial, lactate and clinical outcomes. Ancillary non-parametric correlations were conducted to examine interactions between change in microbiota and clinical outcomes. RESULTS: Large treatment effects were observed for the intention-to-treat sample with a reduction in Streptococcus viable count and improvement on several clinical outcomes including total symptoms, some sleep (less awakenings, greater efficiency and quality) and cognitive symptoms (attention, processing speed, cognitive flexibility, story memory and verbal fluency). Mood, fatigue and urine D:L lactate ratio remained similar across time. Ancillary results infer that shifts in microbiota were associated with more of the variance in clinical changes for males compared with females. CONCLUSIONS: Results support the notion that specific microorganisms interact with some ME/CFS symptoms and offer promise for the therapeutic potential of targeting gut dysbiosis in this population. Streptococcus spp. are not the primary or sole producers of D-lactate. Further investigation of lactate concentrations are needed to elucidate any role of D-lactate in this population. Concurrent microbial shifts that may be associated with clinical improvement (i.e., increased Bacteroides and Bifidobacterium or decreased Clostridium in males) invite enquiry into alternative strategies for individualised treatment. Trial Registration Australian and New Zealand Clinical Trial Registry (ACTRN12614001077651) 9th October 2014. https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=366933&isReview=true.

Examining clinical similarities between myalgic encephalomyelitis/chronic fatigue syndrome and D-lactic acidosis: a systematic review.

BACKGROUND: The pursuit for clarity in diagnostic and treatment pathways for the complex, chronic condition of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) continues. This systematic review raises a novel question to explore possible overlapping aetiology in two distinct conditions. Similar neurocognitive symptoms and evidence of D-lactate producing bacteria in ME/CFS raise questions about shared mechanisms with the acute condition of D-lactic acidosis (D-la). METHODS: D-la case reports published between 1965 and March 2016 were reviewed for episodes describing both neurological symptoms and high D-lactate levels. Fifty-nine D-la episodes were included in the qualitative synthesis comparing D-la symptoms with ME/CFS diagnostic criteria. A narrative review of D-la mechanisms and relevance for ME/CFS was provided. RESULTS: The majority of neurological disturbances reported in D-la episodes overlapped with ME/CFS symptoms. Of these, the most frequently reported D-la symptoms were motor disturbances that appear more prominent during severe presentations of ME/CFS. Both patient groups shared a history of gastrointestinal abnormalities and evidence of bacterial dysbiosis, although only preliminary evidence supported the role of lactate-producing bacteria in ME/CFS. LIMITATIONS: Interpretation of results are constrained by both the breadth of symptoms included in ME/CFS diagnostic criteria and the conservative methodology used for D-la symptom classification. Several pathophysiological mechanisms in ME/CFS were not examined. CONCLUSIONS: Shared symptomatology and underlying microbiota-gut-brain interactions raise the possibility of a continuum of acute (D-la) versus chronic (ME/CFS) presentations related to D-lactate absorption. Measurement of D-lactate in ME/CFS is needed to effectively evaluate whether subclinical D-lactate levels affect neurological symptoms in this clinical population.

Support for the microgenderome invites enquiry into sex differences.

The microgenderome defines the interaction between microbiota, sex hormones and the immune system. Our recent research inferred support for the microgenderome by showing sex differences in microbiota-symptom associations in a clinical sample of patients with myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS). This addendum expands upon the sex-specific pattern of associations that were observed. Interpretations are hypothesized in relation to genera versus species-level analyses and D-lactate theory. Evidence of sex-differences invites future research to consider sex comparisons in microbial function even when microbial abundance is statistically similar. Pairing assessment of clinical symptoms with microbial culture, DNA sequencing and metabolomics methods will help advance our current understandings of the role of the microbiome in health and disease.

Association of clinical parameters with periodontal bacterial haemolytic activity.

AIMS: To determine whether haemolytic activity of subgingival bacteria is associated with periodontitis clinical parameters and to identify which bacteria produce the haemolysins. MATERIALS AND METHODS: Subgingival plaque samples from 22 untreated chronic periodontitis patients were investigated by culture and identified with matrix assisted laser desorption/ionisation time-of-flight mass spectrometry. RESULTS: Total aerobic and anaerobic bacterial viable counts, percentage distribution of α- and ß-haemolytic bacteria were significantly elevated in diseased sites in relation to healthy sites (p < 0.001). Periodontal pathogens were more frequently detected at diseased sites: Porphyromonas gingivalis, Tannerella forsythia, Treponema sp., Prevotella sp., Parvimonas micra, Fusobacterium sp., Campylobacter sp., Capnocytophaga sp., and Selenomonas sp. Haemolytic unidentifiable species and Gram-positive anaerobes such as Slackia exigua, Solobacterium moorei, and Bulledia extructa were also more frequently detected at diseased sites. In diseased sites, the presence of different haemolytic characteristics was more strongly correlated with clinical measures of disease than the mere absence or presence of specific species. The strongest correlation with probing pocket depth was observed for overall ß-haemolytic toxicity (r = 0.73, p < 0.001). CONCLUSION: A strong association was observed between subgingival bacterial haemolytic activity and clinical parameters in patients with chronic periodontitis. Further investigations are warranted to delineate the role of haemolysins in the pathogenesis of periodontitis.

Support for the Microgenderome: Associations in a Human Clinical Population.

The 'microgenderome' provides a paradigm shift that highlights the role of sex differences in the host-microbiota interaction relevant for autoimmune and neuro-immune conditions. Analysis of cross-sectional self-report and faecal microbial data from 274 patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) suggests that commensal gut microorganisms may play both protective and deleterious roles in symptom expression. Results revealed significant sex-specific interactions between Firmicutes (Clostridium, Streptococcus, Lactobacillus and Enterococcus) and ME/CFS symptoms (including neurological, immune and mood symptoms), regardless of compositional similarity in microbial levels across the sexes. Extending animal studies, we provide support for the microgenderome in a human clinical population. Applied and mechanistic research needs to consider sex-interactions when examining the composition and function of human microbiota.

Sleep quality and the treatment of intestinal microbiota imbalance in Chronic Fatigue Syndrome: A pilot study.

Chronic Fatigue Syndrome (CFS) is a multisystem illness, which may be associated with imbalances in gut microbiota. This study builds on recent evidence that sleep may be influenced by gut microbiota, by assessing whether changes to microbiota in a clinical population known to have both poor sleep and high rates of colonization with gram-positive faecal Streptococcus, can improve sleep. Twenty-one CFS participants completed a 22- day open label trial. Faecal microbiota analysis was performed at baseline and at the end of the trial. Participants were administered erythromycin 400 mg b.d. for 6 days. Actigraphy and questionnaires were used to monitor sleep, symptoms and mood. Changes in patients who showed a clinically significant change in faecal Streptococcus after treatment (responders; defined as post-therapy distribution<6%) were compared to participants who did not respond to treatment. In the seven responders, there was a significant increase in actigraphic total sleep time (p=0.028) from baseline to follow up, compared with non-responders. Improved vigour scores were associated with a lower Streptococcus count (ρ=-0.90, p=0.037). For both the responders and the whole group, poorer mood was associated with higher Lactobacillus. Short term antibiotic treatment appears to be insufficient to effect sustainable changes in the gut ecosystem in most CFS participants. Some improvement in objective sleep parameters and mood were found in participants with reduced levels of gram-positive gut microbiota after antibiotic treatment, which is encouraging. Further study of possible links between gut microorganisms and sleep and mood disturbances is warranted.

Metabolism in chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a poorly understood condition that presents as long-term physical and mental fatigue with associated symptoms of pain and sensitivity across a broad range of systems in the body. The poor understanding of the disorder comes from the varying clinical diagnostic definitions as well as the broad array of body systems from which its symptoms present. Studies on metabolism and CFS suggest irregularities in energy metabolism, amino acid metabolism, nucleotide metabolism, nitrogen metabolism, hormone metabolism, and oxidative stress metabolism. The overwhelming body of evidence suggests an oxidative environment with the minimal utilization of mitochondria for efficient energy production. This is coupled with a reduced excretion of amino acids and nitrogen in general. Metabolomics is a developing field that studies metabolism within a living system under varying conditions of stimuli. Through its development, there has been the optimisation of techniques to do large-scale hypothesis-generating untargeted studies as well as hypothesis-testing targeted studies. These techniques are introduced and show an important future direction for research into complex illnesses such as CFS.

Updating the taxonomic toolbox: classification of Alteromonas spp. using multilocus phylogenetic analysis and MALDI-TOF mass spectrometry.

Bacteria of the genus Alteromonas are Gram-negative, strictly aerobic, motile, heterotrophic marine bacteria known for their versatile metabolic activities. Identification and classification of novel species belonging to the genus Alteromonas generally involves DNA-DNA hybridization (DDH) as distinct species often fail to be resolved at the 97 % threshold value of the 16S rRNA gene sequence similarity. In this study, the applicability of Multilocus Phylogenetic Analysis (MLPA) and Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for the differentiation of Alteromonas species has been evaluated. Phylogenetic analysis incorporating five house-keeping genes (dnaK, sucC, rpoB, gyrB, and rpoD) revealed a threshold value of 98.9 % that could be considered as the species cut-off value for the delineation of Alteromonas spp. MALDI-TOF MS data analysis reconfirmed the Alteromonas species clustering. MLPA and MALDI-TOF MS both generated data that were comparable to that of the 16S rRNA gene sequence analysis and may be considered as useful complementary techniques for the description of new Alteromonas species.

Green-lipped mussel extract (Perna canaliculus) and glucosamine sulphate in patients with knee osteoarthritis: therapeutic efficacy and effects on gastrointestinal microbiota profiles.

OBJECTIVE: To investigate how changes in the gastrointestinal tract (GIT) microbiota profile may influence nutraceutical efficacy in osteoarthritis (OA) and allow the formulation of a hypothesis that explains in part the inconsistent and contentious findings from OA clinical studies with green-lipped mussel (GLM) and glucosamine. METHODS: A non-blinded randomised clinical trial was conducted with 38 subjects diagnosed with knee OA. Each participant received either 3,000 mg/day of a whole GLM extract or 3,000 mg/day of glucosamine sulphate (GS), p.o. for 12 weeks. Faecal microbial analyses were carried out after collecting stools at T (0) and T (12) weeks. Additional pharmacometric measures were obtained from changes in arthritic scores in the Western Ontario McMaster Universities Arthritis Index (WOMAC) and the Lequesne algofunctional indices and the Gastrointestinal Symptom Rating Scale (GSRS). An intention-to-treat analysis was employed and participant data collected at T (0), T (6) and T (12) weeks. RESULTS: There were no statistically significant changes in bacterial growth patterns determined by the Wilcoxon test. In both groups there was a trend towards a decrease in Clostridium and Staphylococcus species and increase in Lactobacillus, Streptococcus and Eubacterium species. In the GLM group Bifidobacterium tended to increase and Enterococcus and yeast species to decrease. The GS-treated group demonstrated a trend towards a decrease in Bacteroides and an increase in yeasts and Coliforms species, most notably Escherichia coli. We further confirm significant improvement (p < 0.05) in all OA outcome measures from T (0) to T (12) weeks for both the GLM and GS groups. The GSRS scores indicated that GIT function significantly improved over the 12 weeks duration with GLM and GS supplementation. CONCLUSION: Both GLM and GS reduced OA symptoms and non-significantly altered the gut microbiota profile from baseline. Changes in the microbiota profiles occurred in both treatment groups; the most notable being a reduction in the Clostridia sp. This study suggests that nutritional supplements such as GLM and GS may regulate some of the metabolic and immunological activities of the GIT microbiota. The decrease in Clostridia, a potent modulator of colonic Th17 and CD4+ regulatory T cells, was consistent with a decrease in inflammation; improved GSRS scores and OA symptoms for these OA participants. The GIT microbiota may be important factor in the first-pass metabolism of these nutraceuticals.

The gastrointestinal microbiome and musculoskeletal diseases: a beneficial role for probiotics and prebiotics.

Natural medicines are an attractive option for patients diagnosed with common and debilitating musculoskeletal diseases such as Osteoarthritis (OA) or Rheumatoid Arthritis (RA). The high rate of self-medication with natural products is due to (1) lack of an available cure and (2) serious adverse events associated with chronic use of pharmaceutical medications in particular non-steroidal anti-inflammatory drugs (NSAIDs) and high dose paracetamol. Pharmaceuticals to treat pain may disrupt gastrointestinal (GIT) barrier integrity inducing GIT inflammation and a state of and hyper-permeability. Probiotics and prebiotics may comprise plausible therapeutic options that can restore GIT barrier functionality and down regulate pro-inflammatory mediators by modulating the activity of, for example, Clostridia species known to induce pro-inflammatory mediators. The effect may comprise the rescue of gut barrier physiological function. A postulated requirement has been the abrogation of free radical formation by numerous natural antioxidant molecules in order to improve musculoskeletal health outcomes, this notion in our view, is in error. The production of reactive oxygen species (ROS) in different anatomical environments including the GIT by the epithelial lining and the commensal microbe cohort is a regulated process, leading to the formation of hydrogen peroxide which is now well recognized as an essential second messenger required for normal cellular homeostasis and physiological function. The GIT commensal profile that tolerates the host does so by regulating pro-inflammatory and anti-inflammatory GIT mucosal actions through the activity of ROS signaling thereby controlling the activity of pathogenic bacterial species.

NMR metabolic profiling of serum identifies amino acid disturbances in chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a debilitating multisystem disorder characterised by long-term fatigue with a variety of other symptoms including cognitive dysfunction, unrefreshing sleep, muscle pain, and post-exertional malaise. It is a poorly understood condition that occurs in ~5 in every 1000 individuals. We present here a preliminary study on the analysis of blood samples from 11 CFS and 10 control subjects through NMR metabolic profiling. Identified metabolites that were found to be significantly altered between the groups were subjected to correlation analysis to potentially elucidate disturbed metabolic pathways. Our results showed a significant reduction of glutamine (P=0.002) and ornithine (P<0.05) in the blood of the CFS samples. Correlation analysis of glutamine and ornithine with other metabolites in the CFS sera showed relationships with glucogenic amino acids and metabolites that participate in the urea cycle. This indicates a possible disturbance to amino acid and nitrogen metabolism. It would be beneficial to identify any potential biomarkers of CFS for accurate diagnosis of the disorder.

Increased d-lactic Acid intestinal bacteria in patients with chronic fatigue syndrome.

Patients with chronic fatigue syndrome (CFS) are affected by symptoms of cognitive dysfunction and neurological impairment, the cause of which has yet to be elucidated. However, these symptoms are strikingly similar to those of patients presented with D-lactic acidosis. A significant increase of Gram positive facultative anaerobic faecal microorganisms in 108 CFS patients as compared to 177 control subjects (p<0.01) is presented in this report. The viable count of D-lactic acid producing Enterococcus and Streptococcus spp. in the faecal samples from the CFS group (3.5 x 10(7) cfu/L and 9.8 x 10(7) cfu/L respectively) were significantly higher than those for the control group (5.0 x 10(6) cfu/L and 8.9 x 10(4) cfu/L respectively). Analysis of exometabolic profiles of Enterococcus faecalis and Streptococcus sanguinis, representatives of Enterococcus and Streptococcus spp. respectively, by NMR and HPLC showed that these organisms produced significantly more lactic acid (p<0.01) from (13)C-labeled glucose, than the Gram negative Escherichia coli. Further, both E. faecalis and S. sanguinis secrete more D-lactic acid than E. coli. This study suggests a probable link between intestinal colonization of Gram positive facultative anaerobic D-lactic acid bacteria and symptom expressions in a subgroup of patients with CFS. Given the fact that this might explain not only neurocognitive dysfunction in CFS patients but also mitochondrial dysfunction, these findings may have important clinical implications.

Hematologic and urinary excretion anomalies in patients with chronic fatigue syndrome.

Patients with chronic fatigue syndrome (CFS) have a broad and variable spectrum of signs and symptoms with variable onsets. This report outlines the results of a single-blind, cross-sectional research project that extensively investigated a large cohort of 100 CFS patients and 82 non fatigued control subjects with the aim of performing a case-control evaluation of alterations in standard blood parameters and urinary amino and organic acid excretion profiles. Blood biochemistry and full blood counts were unremarkable and fell within normal laboratory ranges. However, the case-control comparison of the blood cell data revealed that CFS patients had a significant decrease in red cell distribution width and increases in mean platelet volume, neutrophil counts, and the neutrophil-lymphocyte ratio. Evaluation of the urine excretion parameters also revealed a number of anomalies. The overnight urine output and rate of amino acid excretion were both reduced in the CFS group (P < 0.01). Significant decreases in the urinary excretion of asparagine (P < 0.0001), phenylalanine (P < 0.003), the branch chain amino acids (P < 0.005), and succinic acid (P < 0.0001), as well as increases in 3-methylhistidine (P < 0.05) and tyrosine (P < 0.05) were observed. It was concluded that the urinary excretion and blood parameters data supported the hypothesis that alterations in physiologic homeostasis exist in CFS patients.

Pain intensity, illness duration, and protein catabolism in temporomandibular disorder patients with chronic muscle pain.

AIMS: To investigate whether the duration of chronic pain in temporomandibular disorder (TMD) patients is associated with a net depletion of amino acids, and a distinct process from pain intensity. METHODS: Twenty-nine patients defined by the research diagnostic criteria/TMD as having Type 1a muscle pain (TMD1A group), and 34 age- and sex-matched control subjects, were assessed for variation in urinary organic and amino acid excretion by gas chromatography-mass spectrometry. RESULTS: The TMD1A patients' mean pain intensity, assessed on a visual analog scale (VAS), was 5.4 (95% confidence limits: 4.5 to 6.3), TMD1A illness duration was 5.0 +/- 1.2 (SD) years, number of body areas with pain/subject was 6.3 +/- 2.4 (range 0 to 10), and symptom prevalence from the Symptom Check List-90-Revised (SCL-90-R) was 25.5 +/- 11.3 symptoms/subject, which was higher than the controls (5.2 +/- 5.0 symptoms/subject, P < .001). TMD1A patient illness duration was positively correlated with symptom prevalence and body pain distribution, and all were independent of pain intensity. The TMD1A patients had: (1) and increased tyrosine:leucine ratio; and (2) reduced leucine concentrations (both P < .001), which suggests deregulated catabolism. Pain intensity was associated with: (1) changes in the multivariate urinary metabolite excretion patterns (P < .001); (2) reduced leucine concentrations (P < .001); and (3) increases in total urinary metabolites (P < .04), and in 2 unidentified molecules, UM28 (P < .001) and CFSUM1 (P < .002). TMD1A illness duration was associated with lower (1) urinary metabolite concentrations and (2) succinic acid and combined glutamine + glutamic acid levels, suggesting a progressive depletion of metabolite reserves. CONCLUSION: In TMD1A patients, total amino acid excretion was positively correlated with pain intensity and negatively correlated with illness duration, which indicated that illness duration was associated with a different set of metabolic anomalies compared with those identified for pain intensity.

Coagulase-negative staphylococcal membrane-damaging toxins, pain intensity, and metabolic changes in temporomandibular disorder patients with chronic muscle pain.

AIMS: To investigate the association between toxin-producing staphylococci, symptom expression, and changes in urinary excretion of metabolites in temporomandibular disorder (TMD) patients and age- and sex-matched control subjects. METHODS: Twenty-nine patients defined by the research diagnostic criteria/TMD as having Type 1a muscle pain (TMD1A), and 34 age- and sex-matched control subjects were assessed for the carriage of staphylococcal species, staphylococcal toxin production, expression of symptoms, and changes in urinary excretion of amino and organic acids. RESULTS: TMD1A patients had an increased incidence of carriage of toxin-producing coagulase-negative staphylococcus (MDT-CoNS, P < .004), which produced increased levels of delta-like membrane-damaging toxins. The TMD1A patients also had a reduction in the incidence of carriage of Staphylococcus aureus (P < .02). Increased incidence of MDT-CoNS was positively associated with increased pain intensity as assessed by a visual analog scale (P < .001). Odds ratio analysis revealed a 9.2-fold increase in MDT-CoNS recovery from the nose of TMD1A patients compared with the control subjects (odds ratio = 9.2, > 95% confidence limits: 2.3 to 37.5, P < .001). Increases in the carriage incidence of MDT-CoNS were also associated with increases in the urinary tyrosine:leucine ratio (P < .004), which represents a change in the balance of proteolysis and protein synthesis. The toxin production by these CoNS species was also associated with an increased urinary excretion of glutamic acid (P < .03). CONCLUSION: These data suggest that an increased colonization of MDT-CoNS on skin and mucosal membranes was associated with changed proteolysis, increased pain intensity, and an increase in excitatory amino acids consistent with events associated with the development of chronic orofacial muscle pain in TMD patients.