Dysbiosis of the Urinary Microbiota Associated With Urine Levels of Proinflammatory Chemokine Interleukin-8 in Female Type 2 Diabetic Patients

Abstract

Evidence has shown that dysbiosis of the urinary microbiota existed in female type 2 diabetes mellitus (T2DM) patients. Perturbations of intestinal microbiota are linked to proinflammatory chemokine interleukin-8 (IL-8); however, the correlations between urinary microbiota and IL-8 are not well studied. Here, we investigated the associations between the altered urinary microbiota and urinary IL-8 in female T2DM patients. A modified four-tube midstream urine technique was used to collect urine specimens from 70 female T2DM patients and 70 matched healthy controls (HCs). Bacterial genomic DNA from urine specimens was isolated using magnetic beads and the urinary microbiota was assessed using Illumina MiSeq platform targeting on the 16S rRNA gene V3-V4 region. Urinary IL-8 was determined by enzyme linked immunosorbent assay. Subsequently, the T2DM patients were separated into urine IL-8 detectable (WIL8) and undetectable (NIL8) groups, and the composition of urinary microbiota between the two groups was compared. Meanwhile, the levels of IL-8 between the "≥HCs" group (those specific bacterial genera were more than or equal to the HCs) and the "<HCs" group (those specific bacterial genera were less than the HCs) was also compared. Of 70 urine samples from T2DM patients without urinary tract infections, 46 patients had detectable IL-8 in their urine (64.31 ± 70.43 pg/mL), while 24 patients had undetectable IL-8. Compared to the NIL8 group, 11 bacterial genera increased in the WIL8 group, including Corynebacterium, Akkermansia, Enterococcus, etc., whereas 10 genera, such as Faecalibacterium, Bacteroides, and Pseudomonas decreased. One species of Lactobacillus, Lactobacillus iners, increased obviously in the WIL8 group. The "≥HCs" group showed 17 genera increased and 16 genera decreased. In addition, 18 genera contributed to the presence of urinary IL-8 in T2DM patients, which explained 95.60% of the total variance of urinary microbiota. Our study demonstrated that dysbiosis of the urinary microbiota with several key bacteria was associated with urinary IL-8 in female T2DM patients, which might be useful to explore the interactions between urinary microbiota and inflammatory responses and shed light on novel diagnosis and therapy for urinary microbiota associated with infections in T2DM patients.

Keywords: Akkermansia; Lactobacillus; interleukin-8; type 2 diabetes mellitus; urinary microbiota.

Figure 1

Principal coordinate analysis (PCoA) plot. PCoA plot of the urinary microbiota based on the unweighted UniFrac metric. Blue and yellow dots represent NIL8 and WIL8 specimens, respectively.

Figure 2

Cladogram showing differentially abundant taxa of urinary microbiota in type 2 diabetes mellitus patients. (A) LEfSe cladogram showed the most differentially abundant taxa between WIL8 and NIL8 groups. Taxonomic cladogram obtained from LEfSe analysis of 16S rDNA sequences. Taxa enriched for WIL8 in yellow; NIL8 enriched taxa in blue. The size of each dot is proportional to its effect size. (B) Only taxa meeting an LDA threshold > 2.0 are shown.

Figure 3

Phylum-level operational taxonomic units different between NIL8 and WIL8 groups. STAMP software was used to calculate the bacterial phylum proportions in the two groups: Proteobacteria (A) and Bacteroidetes (B). Welch’s t-test was used to compare abundance at the bacterial phylum level for NIL8 urine samples and WIL8 samples. The different phyla were assigned only to those presenting a minimum variation at a significant level [p (corrected) < 0.05].

Figure 4

Genus-level operational taxonomic units different between NIL8 and WIL8 groups. STAMP software was used to calculate the genus proportions in the two groups. Welch’s t-test was used to compare abundance at the genus level for NIL8 and WIL8 specimens. The different genera were assigned only to those presenting a minimum variation at a significant level [p (corrected) < 0.05].

Figure 5

Comparison of interleukin-8 (IL-8) levels between genus “≥HCs” group and “p < 0.05).