Clinical and In Vitro Safety of Heyndrickxia coagulans AO 1167B: A Double-Blind, Placebo-Controlled Trial
Abstract
:1. Introduction
2. Materials and Methods
2.1. Provenance of H. coagulans AO1167B
2.2. Culture Conditions of Bacterial Strain
2.3. Phenotypic Characterization of the Organism
2.4. Genotypic Characterization of the Organism
2.5. Hemolytic Activity
2.6. Cytotoxicity in Vero Cells
2.7. Human Safety Assessment
2.7.1. Study Design
2.7.2. Study Outcomes
2.7.3. Safety Assessment
2.7.4. Sample Collection, Processing, and Data Management
2.7.5. Clinical Determinations
2.7.6. Occurrence of Adverse Events Determination
2.7.7. Statistical Analyses
3. Results
3.1. Species and Strain Identification
3.2. Traits Associated with Probiotic Properties
3.2.1. Phenotypic Traits
3.2.2. Genotypic Properties
3.3. Safety Assessment of H. coagulans AO1167B
3.3.1. Antimicrobial Resistance and Virulence Factors
3.3.2. Hemolytic Activity and Cytotoxicity
3.3.3. Clinical and Hematological Determinations
3.3.4. Adverse Events
3.3.5. Health Questionnaire Analysis
4. Discussion
4.1. Species and Strain Identification
4.2. Traits of H. coagulans AO1167B Associated with Probiotic Properties
4.2.1. Phenotypic Traits
4.2.2. Genotypic Traits
4.3. Antimicrobial Resistance
4.4. Virulence Genes
4.5. Bioactive Amines
4.6. Clinical Study
4.6.1. Demographic and Safety Overview
4.6.2. Limitations of the Study
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Enzyme | Product |
---|---|---|
hdcA | Histidine decarboxylase | Histamine |
tdcA | Tyrosine decarboxylase | Tyramine |
speC | Ornithine decarboxylase | Putrescine |
ldc | Lysine decarboxylase | Cadaverine |
aguA | Agmatine ureohydrolase | Agmatine |
aguD | Agmatine deiminase antiporter | Agmatine |
speE | Spermidine synthetase | Spermidine |
speF | Spermine synthetase | Spermine |
speG | Spermidine synthetase | Spermidine |
tyrDC | Tyrosine decarboxylase | Phenylethylamine |
tph | Tryptophan hydroxylase | Serotonin |
aadc | Aromatic amino acid decarboxylase | Serotonin |
tbh | Tyramine β-hydroxylase | Octopamine |
tam | Tryptamine synthase | Tryptamine |
sufI | Copper-containing nitrite reductase | Nitric oxide |
nasD | Nitrite reductase | Nitric oxide |
Demographic Variables | H. coagulans AO1167B Cohort (n = 50) | Placebo Cohort (n = 49) | p Value | |||
---|---|---|---|---|---|---|
Number | % | Number | % | |||
Sex | Female | 31 | 62 | 31 | 63.3 | 0.896 a |
Male | 19 | 38 | 18 | 36.7 | ||
Age (years) Median ± SD | 42.82 ± 16.3 | 44.43 ± 14.74 | 1.0 b | |||
BMI Median ± SD | 23.20 ± 3.93 | 23.63 ± 4.17 | 0.764 a |
Gene | Encoded Protein |
---|---|
Acid tolerance | |
atpA | ATP synthase subunit alpha |
atpB | ATP synthase subunit beta |
ldh1 | L-lactate dehydrogenase 1 |
ldhD | D-lactate dehydrogenase |
pgi | Glucose-6-phosphate isomerase |
groL | 60 kDa chaperonin |
cspB | Cold shock protein |
teaD | TRAP-T-associated universal stress protein |
ald | Alanine dehydrogenase |
gabD | Succinate-semialdehyde dehydrogenase |
fdhD | formate dehydrogenase |
pgi | Glucose-6-phosphate isomerase |
atpB | ATP synthase subunit B |
Acid/bile tolerance | |
arcD1 | Arginine/ornithine antiporter ArcD1 |
iPGM | bisphosphoglycerate-independent phosphoglycerate mutase |
argF | Ornithine carbamoyltransferase |
gpmI | 2,3-bisphosphoglycerate-independent phosphoglycerate mutase |
argR | Arginine repressor |
Adhesion and aggregation | |
eno | Enolase |
mntH | Manganese transferase/Divalent metal cation transporter |
ywgD | Tyrosine-protein kinase |
tpiA | Triosephosphate isomerase |
ugtP | Glucosyltransferase |
tuf | Elongation factor Tu |
spaCBA | Pilin |
Antioxidant defense | |
trxA | Thioredoxin |
katE | Catalase |
Detoxification | |
arsC | Arsenate reductase |
bsh | Bile salt hydrolase |
cadA | Cadmium-transporting ATPase |
hsp18 | 18 kDa heat shock protein |
Bile tolerance | |
nagB | Glucosamine-6-phosphate deaminase |
pyrG | CTP (cytidine triphosphate synthetase) synthase |
nagB | Glucosamine-6-phosphate deaminase |
bshB | Bile salt hydrolase |
Biofilm formation/Adhesion/Chemotaxis | |
luxS | Lyase |
cheA | Chemotaxis protein CheA |
srfA | Surfactin synthetase |
hag | Flagellin |
motB | Motility protein |
manX | mannose-specific EIIAB component |
Carbohydrate metabolism | |
galT | UDP-glucose--hexose-1-phosphate uridylyl transferase |
nagA | Glucosamine-6-phosphate deaminase |
bgaB | Beta-galactosidase |
agaA | alpha galactosidase |
bbmA | intracellular maltogenic amylase |
gntR | Xylose utilization operon |
xylA | Xylose isomerase |
galE | UDP-glucose 4-epimerase |
Cell wall remodeling | |
pbp | Penicillin-binding protein |
murA | UDP-N-acetylglucosamine enol pyruvyl transferase |
Immune modulation | |
hemA | Glutamyl-tRNA reductase |
groEL | Chaperonin GroEL |
magl | Monoacylglycerol lipase |
ctkA | Serine/threonine protein kinase |
Metabolism | |
feoB | Ferrous ion transporter |
ldh | L-lactate dehydrogenase |
lacA | Beta-galactosidase |
glnA | Glutamine synthetase |
UreA | Urease |
methH | Methionine synthase |
phoB | Phosphate regulon protein |
pstS | Phosphate-binding protein |
clpP | Protease ClpP |
purA | Adenylosuccinate synthetase |
SCFA (acetate) production | |
ackA | Acetate kinase |
pta | Phosphotransacetylase |
Stress tolerance/Response | |
phoPR | Two-component response regulator |
sigH | Sigma factor H |
sigB | Sigma factor B |
clpL | Stress response protein |
msrA | Methionine sulfoxide reductase |
acoA | Oxidoreductase |
yvbW | Amino acid permease |
emrY | Multidrug resistance protein |
TeaD | Universal stress protein |
Vitamin biosynthesis | |
cobA | Adenosylcobalamin synthase |
fadA | 3-ketoacyl-CoA thiolase @ Acetyl-CoA acetyltransferase |
fadD_1 | Long-chain-fatty-acid-CoA ligase |
bioB | Biotin synthase |
hpt | Hypoxanthine-guanine phosphoribosyltransferase |
dfrA | Dihydrofolate reductase |
thyA2 | Thymidylate synthase |
serA | D-3-phosphoglycerate dehydrogenase |
dagK | Diacylglycerol kinase |
thieE | Thiamine phosphate synthase |
Antimicrobial | M100 Susceptible Breakpoint for Staphylococcus aureus | Staphylococcus aureus ATCC 29213 | H. coagulans AO1167B | ||
---|---|---|---|---|---|
Zone Diameter (mm) 1 | Interpretation | Zone Diameter (mm) | Interpretation | ||
Ampicillin | ≥29 | 41.8 | S 2 | 45.3 | S |
Chloramphenicol | ≥19 | 24.5 | S | 33.2 | S |
Clindamycin | NA | 29 | S | 44.1 | S |
Erythromycin | ≥15 | 28.4 | S | 41.5 | S |
Gentamicin | ≥18 | 24.2 | S | 36.2 | S |
Kanamycin | NA | 24 | S | 36.2 | S |
Streptomycin | ≥18 | 17.1 | S | 25.4 | NA 3 |
Tetracycline | ≥23 | 31.9 | S | 45.8 | S |
Vancomycin | ≥21 | 18.7 | S | 36.3 | NA |
Gene | Length (bp) | Resistance Mechanism | AMR Gene Function | % Identity of Matching Region |
---|---|---|---|---|
vanH gene in vanO cluster | 990 | glycopeptide | glycopeptide resistance gene cluster | 31.78 |
vanY gene in vanM cluster | 795 | glycopeptide | glycopeptide resistance gene cluster | 35.63 |
vanT gene in vanG cluster | 1152 | glycopeptide | glycopeptide resistance gene cluster | 34.38 |
vanW gene in vanI cluster | 1824 | glycopeptide | glycopeptide resistance gene cluster | 32.00 |
qacG | 744 | efflux | small multidrug resistance protein that facilitates the efflux of QACs 1 | 44.4 |
qacJ | 744 | efflux | small multidrug resistance protein that facilitates the efflux of QACs | 46.46 |
acrB | 3165 | multidrug | resistance-nodulation-cell division (RND) efflux pump | 80.41 |
Clinical Chemistry and Haematology | Normal Range | H. coagulans Cohort (n = 50) | Placebo Cohort (n = 49) | p Value | |||||
---|---|---|---|---|---|---|---|---|---|
Baseline | Day 60 | Baseline | Day 60 | p1 | p2 | p3 | p4 | ||
Creatinine | 47.6–113.4 μmol/L | 78.23 ± 18.09 | 75.53 ± 17.22 | 73.10 ± 21.59 | 76.167 ± 17.84 | NS | NS | 0.032 | NS |
Urea | <8.3 mmol/L | 4.27 ± 1.05 | 4.28 ± 1.01 | 4.02 ± 1.16 | 4.47 ± 1.12 | NS | NS | NS | 0.012 |
ALAT | <45 U/L | 14.98 ± 6.55 | 13.73 ± 5.78 | 19.44 ± 9.45 | 16.26 ± 6.71 | 0.008 | 0.047 | NS | 0.002 |
ASAT | 40 U/L | 13.74 ± 4.76 | 22.83 ± 6.18 | 18.13 ± 4.78 | 13.35 ± 5.01 | <0.01 | <0.01 | <0.01 | <0.01 |
GGT | <50 U/L | 21.70 ± 16.82 | 19.64 ± 14.51 | 22.57± 16.62 | 24.80 ± 20.21 | NS | NS | NS | 0.026 |
Total Protein | 60–80 g/L | 70.84 ± 4.03 | 71.07 ± 3.53 | 70.59 ± 6.22 | 70.77 ± 4.00 | NS | NS | NS | NS |
Albumin | 35–52 g/L | 44.45 ± 3.40 | 39.92 ± 2.19 | 44.78 ± 3.38 | 44.65 ± 2.89 | NS | <0.01 | <0.01 | NS |
Glycemia | 4.2–6.1 μmol/L | 4.70 ± 0.46 | 4.61 ± 0.60 | 4.74 ± 0.55 | 4.69 ± 0.46 | NS | NS | NS | NS |
Cholesterol | 2.81–5.2 mmol/L | 4.38 ± 1.15 | 4.47 ± 0.92 | 4.95 ± 1.24 | 4.49 ± 1.11 | 0.020 | NS | NS | <0.01 |
Triglycerides | 0.46–1.8mmol/L | 1.131 ± 0.74 | 1.60 ± 0.52 | 1.29 ±0.87 | 1.33 ± 0.69 | NS | 0.031 | 0.001 | NS |
Total bilirubin | <17 mmol/L | 8.05 ± 4.47 | 9.37 ± 3.21 | 9.32 ± 5.33 | 7.62 ± 4.49 | NS | 0.028 | 0.01 | 0.002 |
Direct bilirubin | <5.1 mmol/L | 3.02 ± 1.23 | 2.95 ± 1.13 | 4.03 ± 4.59 | 2.95 ± 1.24 | NS | NS | NS | 0.001 |
WBC | (4.5–11) × 109/μL | 6.37± 2.00 | 6.31± 2.35 | 6.26 ± 1.62 | 6.52 ± 1.96 | NS | NS | NS | NS |
RBC | (F = 4.2–5.4/M = 4.7–6.1) cels/μL | 4.56 ± 0.38 | 4.55 ± 0.38 | 4.51 ± 0.40 | 4.56 ± 0.39 | NS | NS | NS | NS |
HBG | (F = 12.3–15.3/M = 14.0–17.5) g/dL | 132.74 ± 13.65 | 132.88 ± 13.31 | 131.53 ± 15.45 | 132.51 ± 13.71 | NS | NS | NS | NS |
HTC | (F = 36–45/M = 42–50) % | 0.42 ± 0.03 | 0.42 ± 0.03 | 0.40 ± 0.04 | 0.42 ± 0.03 | NS | NS | NS | 0.001 |
MVC | 80–96.1% | 92.57± 5.24 | 91.49 ± 5.31 | 90.79 ± 6.15 | 92.30 ± 5.53 | NS | NS | 0.003 | <0.01 |
MCH | 33.4–35.5 g/dL | 29.11 ± 2.05 | 29.24 ± 1.96 | 29.15 ± 2.36 | 29.02 ± 2.13 | 0.046 | 0.013 | <0.01 | NS |
PLT | (172–450) × 103/mL | 257.70 ± 70.08 | 267.06 ± 66.51 | 261.88 ± 52.73 | 259.67 ± 62.52 | NS | NS | NS | NS |
RDWCV | (11–14) % | 1.05 ± 1.22 | 12.96 ± 1.08 | 12.82 ± 1.90 | 13.12 ± 1.22 | NS | NS | 0.017 | 0.014 |
MPV | (F: 12–16/M: 14–17.4) g/dL | 10.45 ± 0.90 | 10.66 ± 0.88 | 10.34 ± 0.90 | 10.35 ± 0.80 | NS | NS | 0.040 | NS |
Neutrophil | 1.42–6.34 × 109/L | 3.60± 1.55 | 3.65 ± 1.90 | 3.36 ± 1.16 | 3.66 ± 1.56 | NS | NS | NS | NS |
lymphocytes | 0.71–4.53 × 109/L | 2.02 ± 0.64 | 1.91 ± 0.63 | 2.12 ± 0.63 | 2.10 ± 0.66 | NS | NS | NS | NS |
Monocytes | 0.14–0.72 × 109/L | 0.52 ± 0.17 | 0.55 ± 0.23 | 0.52 ± 0.13 | 0.52 ± 0.16 | NS | NS | NS | NS |
Eosinophils | 0–0.54 × 109/L | 0.19 ± 0.16 | 0.19 ± 0.19 | 0.20 ± 0.17 | 0.19 ± 0.13 | NS | NS | NS | NS |
Basophils | 0–0.18 × 109/L | 0.02 ± 0.01 | 0.02 ± 0.01 | 0.02 ± 0.01 | 0.02 ± 0.01 | NS | NS | NS | NS |
Bioimpedance Variables | Baseline | Day 60 | Baseline | Day 60 | p1 | p2 | p3 | p4 | |
Weight | 68.49 ± 12.09 | 66.45 ± 12.60 | 66.64 ± 13.72 | 66.43 ± 13.57 | NS | ||||
BMI | 24.07 ± 3.65 | 23.13 ± 3.99 | 23.63 ± 4.17 | 23.57 ± 4.15 | NS |
Adverse Event | H. coagulans Cohort (n = 50) | Placebo Cohort (n = 49) | ||||
---|---|---|---|---|---|---|
Mild | Moderate | Severe | Mild | Moderate | Severe | |
Abdominal/GI discomfort | 2 (4.0) | 0 | 0 | 0 | ||
acne rosacea | 0 | 0 | 0 | 0 | 0 | 0 |
Anxiety Depression | 0 | 0 | 0 | 0 | 0 | 0 |
Joint pain | 0 | 0 | 0 | 0 | 0 | 0 |
Bronchitis | 0 | 0 | 0 | 0 | 0 | 0 |
Excision of birthmarks | 0 | 0 | 0 | 0 | 0 | 0 |
Bruises after a fall | 0 | 0 | 0 | 0 | 0 | 0 |
Crotid stenosis | 0 | 0 | 0 | 0 | 0 | 0 |
Cataract Surgery | 0 | 0 | 0 | 0 | 0 | 0 |
Chondrocalcinosis | 0 | 0 | 0 | 0 | 0 | 0 |
Colonoscopy and fibroscopy | 0 | 0 | 0 | 0 | 0 | 0 |
Cystitis | 0 | 0 | 0 | 0 | 0 | 0 |
Dental pain | 0 | 0 | 0 | 0 | 0 | 0 |
Diarrhoea | 3 (6.0) | 0 | 0 | 2 (2.0) | 0 | 0 |
Dizziness and nausea | 0 | 0 | 0 | 0 | 0 | 0 |
Oedema | 0 | 0 | 0 | 0 | 1 (1.0) | 0 |
Gases | 5 (10.0) | 0 | 0 | 0 | 0 | 0 |
General aches | 0 | 0 | 0 | 0 | 0 | 0 |
Genital herpes | 0 | 0 | 0 | 0 | 0 | 0 |
Headache | 1 (2.0) | 0 | 0 | 0 | 0 | 0 |
Haemorrhoids | 0 | 0 | 0 | 0 | 0 | 0 |
Infection | 0 | 0 | 0 | 0 | 0 | 0 |
Inflamed prostate | 0 | 0 | 0 | 0 | 0 | 0 |
Migraine | 0 | 0 | 0 | 0 | 0 | 0 |
Mouth ulcer | 0 | 0 | 0 | 0 | 0 | 0 |
Muscle discomfort | 0 | 0 | 0 | 0 | 0 | 0 |
Nasal obstruction | 0 | 0 | 0 | 0 | 0 | 0 |
Orthopaedic pain | 0 | 0 | 0 | 0 | 0 | 0 |
Pain following capsule consumption | 0 | 0 | 0 | 0 | 0 | 0 |
Palpitations | 0 | 0 | 0 | 0 | 0 | 0 |
Radio-infiltration (shoulder) | 0 | 0 | 0 | 0 | 0 | 0 |
Rhinitis | 0 | 0 | 0 | 0 | 0 | 0 |
Sore throat | 0 | 0 | 0 | 0 | 0 | 0 |
Tracheitis | 0 | 0 | 0 | 0 | 0 | 0 |
Trouble sleeping (insomnia) | 0 | 0 | 0 | 0 | 0 | 0 |
Vaginal dryness | 0 | 0 | 0 | 0 | 0 | 0 |
Vagal seizures during or after taking a blood sample. | 0 | 0 | 0 | 0 | 0 | 0 |
vitamin D deficiency | 0 | 0 | 0 | 0 | 0 | 0 |
Others (constipation) | 2 (4.0) | 0 | 0 | 0 | 0 | 0 |
Question | Associated Answer | Placebo (n = 49) | Cases (n = 50) | Kappa Test/p Value | |
---|---|---|---|---|---|
Before/After | Before/After | Placebo | Cases | ||
1-general, would you say your health is: | Excellent | 5/6 | 4/8 | 0.94 p = 0.000 | 0.78 p = 0.000 |
Very Good | 21/21 | 32/28 | |||
Good | 22/21 | 14/14 | |||
Fair | 2/2 | 0/0 | |||
6-During the past 4 weeks, to what extent has your physical health or emotional problems interfered with your normal social activities with family, friends, neighbors, or groups? | Not at all | 14/36 | 39/40 | 0.95 p = 0.000 | 0.82 p = 0.000 |
Slightly | 14/11 | 11/10 | |||
Moderately | 18/1 | 0/0 | |||
Quite a bit | 1/0 | 0/0 | |||
Extremely | 1/0 | 0/0 | |||
7-How much bodily pain have you had during the past 4 weeks? | None | 14/18 | 19/22 | 0.82 p = 0.000 | 0.77 p = 0.000 |
Very mild | 14/14 | 15/16 | |||
mild | 18/14 | 13/9 | |||
moderate | 1/1 | 3/3 | |||
severe | 1/1 | 0/0 | |||
11a-I seem to get sick a little easier than other people | Definitely true | 0/0 | 0/0 | 0.96 p = 0.000 | 0.92 p = 0.000 |
Mostly true | 3/3 | 0/0 | |||
Don’t know | 2/2 | 5/4 | |||
Mostly False | 10/9 | 12/12 | |||
Definitely False | 33/34 | 32/33 | |||
11b-I am as healthy as anybody I know | Definitely true | 19/19 | 17/17 | 0.97 p = 0.000 | 0.97 p = 0.000 |
Mostly true | 16/15 | 25/26 | |||
Don’t know | 6/6 | 5/4 | |||
Mostly False | 4/4 | 1/1 | |||
Definitely False | 3/4 | 1/1 | |||
11c-I expect my health to get worse | Definitely true | 1/1 | 0/0 | 0.96 p = 0.000 | 0.91 p = 0.000 |
Mostly true | 0/0 | 0/1 | |||
Don’t know | 19/18 | 7/6 | |||
Mostly False | 5/5 | 6/7 | |||
Definitely False | 23/24 | 36/35 | |||
11d-My health is excellent | Definitely true | 9/10 | 9/8 | 0.93 p = 0.000 | 0.92 p = 0.000 |
Mostly true | 27/25 | 31/33 | |||
Don’t know | 6/6 | 8/7 | |||
Mostly False | 3/3 | 1/1 | |||
Definitely False | 3/4 | 0/0 |
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Share and Cite
García, G.; Soto, J.; Díaz, A.; Barreto, J.; Soto, C.; Pérez, A.B.; Boffill, S.; Gutiérrez, Á.; Cano, R.d.J. Clinical and In Vitro Safety of Heyndrickxia coagulans AO 1167B: A Double-Blind, Placebo-Controlled Trial. Microorganisms 2024, 12, 2584. https://doi.org/10.3390/microorganisms12122584
García G, Soto J, Díaz A, Barreto J, Soto C, Pérez AB, Boffill S, Gutiérrez Á, Cano RdJ. Clinical and In Vitro Safety of Heyndrickxia coagulans AO 1167B: A Double-Blind, Placebo-Controlled Trial. Microorganisms. 2024; 12(12):2584. https://doi.org/10.3390/microorganisms12122584
Chicago/Turabian StyleGarcía, Gissel, Josanne Soto, Antonio Díaz, Jesús Barreto, Carmen Soto, Ana Beatriz Pérez, Suselys Boffill, Ángela Gutiérrez, and Raúl de Jesús Cano. 2024. "Clinical and In Vitro Safety of Heyndrickxia coagulans AO 1167B: A Double-Blind, Placebo-Controlled Trial" Microorganisms 12, no. 12: 2584. https://doi.org/10.3390/microorganisms12122584
APA StyleGarcía, G., Soto, J., Díaz, A., Barreto, J., Soto, C., Pérez, A. B., Boffill, S., Gutiérrez, Á., & Cano, R. d. J. (2024). Clinical and In Vitro Safety of Heyndrickxia coagulans AO 1167B: A Double-Blind, Placebo-Controlled Trial. Microorganisms, 12(12), 2584. https://doi.org/10.3390/microorganisms12122584