Some research studies have revealed that abnormalities in
Risk factors for T1DM and T2DM
Interestingly, a number of studies have demonstrated that the microbiota of the gastrointestinal tract may be one of these key contributors [8,9,10]. Due to the beneficial role of
The study included 64 patients admitted to the Department of Metabolic Diseases, University Hospital, Krakow, Poland (33 with T1DM, 31 with T2DM), according to the inclusion criteria: age 30–65 years, clinical diagnosis of T1DM or T2DM, and disease duration of at least 2 years. The exclusion criteria included: antibiotic therapy up to 30 days prior to entering the study, and the use of probiotic preparations up to 30 days before entering the study. The control group consisted of 33 healthy individuals, who had not been treated with antibiotics in the period of at least 30 days before collecting the fecal sample, had not taken probiotic preparations, and did not have reported or clinically confirmed gastrointestinal symptoms. All study participants gave written consent to use the data obtained from the biological materials.
The tested materials were fecal samples. Additionally, blood samples were collected in order to evaluate the following parameters: glycated hemoglobin (HbA1c), serum fasting glucose levels, lipid profile (HDL, LDL, total cholesterol, triglycerides (TGs)), and alanine transaminase (ALT). All analysis from blood samples was performed in specialized laboratories of the University Children’s Hospital in Krakow according to routine laboratory procedures designed for medical diagnosis in accordance with GPL and IVD/FDA standards. Fecal samples were transported under deep-freeze conditions to the laboratory at the Department of Microbiology, Jagiellonian University Medical College. Bacterial DNA was extracted using a QIAamp DNA Stool Mini Kit (QIAGEN, GmbH, Germany) with our modified preliminary procedure [11, 12], involving mechanical lysis using the FastPrep homogenizer (MP Biomedicals, California, USA) and enzymatic lysis with mutanolysin (A&A Biotechnology, Gdansk, Poland), lysozyme (Sigma, Saint Louis, USA), and lysostaphin (A&A Biotechnology, Gdansk, Poland) Obtained DNA isolates were used for quantitative real-time PCR amplification (qPCR) to estimate the number of bifidobacteria and the total number of bacterial cells in 1 gram of the fecal samples.
Specific primers targeting
Sequences of primers, reaction mixture and amplification program used in the study
Oligonucleotide sequence (5′−> 3′) | F: CTCCTGGAAACGGGTGG |
F: CCTACGGGNGGCWGCAG |
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Reaction mixture | Water | 2.8 |
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SYBR Green PCR Master Mix | 5 |
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Primer F (20mM) | 0.1 |
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Primer R (20mM) | 0.1 |
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DNA | 2 |
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Amplification program | 95°C – 5 min | 95°C – 5 min | ||
95°C – 30 sec | 40 cycles | 95°C – 30 sec | 40 cycles | |
53°C – 30 sec | 55°C – 30 sec | |||
72°C – 30 sec | 72°C – 30 sec |
The reactions were conducted with a negative control containing water instead of DNA and a positive control containing the DNA of the appropriate reference strain of the species. Each sample was tested in duplicate.
Statistical analyses were performed using IBM SPSS Statistics 28. Differences between the studied groups were analyzed with a one-way ANOVA test. In the case of the ANOVA test, the assumption regarding the homogeneity of variance was not met; therefore, Welch’s F correction was applied. Correlations between biochemical parameters in diabetic patients and the logarithm of number of bacteria in each group was calculated with a Spearman correlation test. In every case, p-values < 0.05 were considered statistically significant. All data shown are mean ± standard deviation.
Anthropometric and clinical characterizations of the examined study groups, and also the mean number of bacteria belonging to
Characteristics of the studied patient groups
Sex: male: female (n) | 9 : 24 | 13 : 18 | 10 : 23 | 0.424 |
Age (yrs) | 39.36 (± 13.27) | 62.68 (± 8.87) | 41.21 (± 13.96) | <0.001 |
BMI [kg/m2] | 24.27 (± 4.80) | 29.28 (± 4.95) | 23.62 (± 1.74) | <0.001 |
Glucose [mmol/l] | 7.26 (± 1.63) | 7.85 (± 2.62) | 4.67 (± 0.56) | <0.001* |
Total cholesterol [mmol/l] | 4.66 (± 0.89) | 4.71 (± 1.19) | 5.17 (± 0.70) | 0.025 |
HDL [mmol/l] | 1.68 (± 0.40) | 1.12 (± 0.37) | 1.65 (± 0.33) | <0.001 |
LDL [mmol/l] | 2.60 (± 0.75) | 2.70 (± 1.06) | 3.09 (± 0.54) | 0.009 |
Triglycerides (TG) [mmol/l] | 0.99 (± 0.57) | 2.29 (± 1.83) | 0.98 (± 0.43) | <0.001* |
ALT [U/l] | 17.73 (± 8.57) | 28.58 (± 16.46) | 18.92 (± 6.36) | <0.001* |
HbA1c [%] | 8.37 (± 2.72) | 8.11 (± 2.72) | 5.38 (± 0.24) | <0.001 |
7.08 × 106 (± 2.14 × 107) |
6.38 × 106 (± 9.59 × 106) |
1.41 × 107 (± 1.76 × 107) |
<0.001* | |
All bacteria |
7.27 × 1011 (± 9.04 × 1011) |
7.29 × 1011 (± 7.57 × 1011) |
1.48 × 1012 (± 4.09 × 1012) |
0.397 |
The number of
In the T1DM group, a positive correlation was found between the number of
In this study, we carried out an assessment of the number of bacteria belonging to the genus
In our study, the numbers of bacteria belonging to
When comparing total levels of bacteria, our work and earlier studies showed no significant differences between the T2DM and control groups [17]. It is possible that the total content of bacteria in the colon, even if its qualitative composition was changed, was similar in both groups. This conclusion has also been drawn by other research [21, 22].
There were no such quantitative sets of published data for adults for us to draw comparisons with the T1DM patients from our study. Most studies are based on comparisons of microbiota between healthy children and those with T1DM [23, 24]. However, it is difficult to compare these results with our data, since young individuals have a physiologically larger number of these bacteria.
We also compared the number of
In their meta-analysis, Akbari et al. concluded that supplementation of probiotics (including, among others,
Another relationship observed in the T1DM group was a positive correlation between age and the number of
Interestingly, in patients with T2DM, a significant negative correlation between the triglyceride levels (TGs) and the number of bifidobacteria was observed. In turn, in patients with T1DM, we documented a negative correlation between the alanine aminotransferease (ALAT) level and
In conclusion, the number of