Integrated next-generation sequencing of 16S rDNA and metaproteomics differentiate the healthy urine microbiome from asymptomatic bacteriuria in neuropathic bladder associated with spinal cord injury

Abstract

Background: Clinical dogma is that healthy urine is sterile and the presence of bacteria with an inflammatory response is indicative of urinary tract infection (UTI). Asymptomatic bacteriuria (ABU) represents the state in which bacteria are present but the inflammatory response is negligible. Differentiating ABU from UTI is diagnostically challenging, but critical because overtreatment of ABU can perpetuate antimicrobial resistance while undertreatment of UTI can result in increased morbidity and mortality. In this study, we describe key characteristics of the healthy and ABU urine microbiomes utilizing 16S rRNA gene (16S rDNA) sequencing and metaproteomics, with the future goal of utilizing this information to personalize the treatment of UTI based on key individual characteristics.

Methods: A cross-sectional study of 26 healthy controls and 27 healthy subjects at risk for ABU due to spinal cord injury-related neuropathic bladder (NB) was conducted. Of the 27 subjects with NB, 8 voided normally, 8 utilized intermittent catheterization, and 11 utilized indwelling Foley urethral catheterization for bladder drainage. Urine was obtained by clean catch in voiders, or directly from the catheter in subjects utilizing catheters. Urinalysis, urine culture and 16S rDNA sequencing were performed on all samples, with metaproteomic analysis performed on a subsample.

Results: A total of 589454 quality-filtered 16S rDNA sequence reads were processed through a NextGen 16S rDNA analysis pipeline. Urine microbiomes differ by normal bladder function vs. NB, gender, type of bladder catheter utilized, and duration of NB. The top ten bacterial taxa showing the most relative abundance and change among samples were Lactobacillales, Enterobacteriales, Actinomycetales, Bacillales, Clostridiales, Bacteroidales, Burkholderiales, Pseudomonadales, Bifidobacteriales and Coriobacteriales. Metaproteomics confirmed the 16S rDNA results, and functional human protein-pathogen interactions were noted in subjects where host defenses were initiated.

Conclusions: Counter to clinical belief, healthy urine is not sterile. The healthy urine microbiome is characterized by a preponderance of Lactobacillales in women and Corynebacterium in men. The presence and duration of NB and method of urinary catheterization alter the healthy urine microbiome. An integrated approach of 16S rDNA sequencing with metaproteomics improves our understanding of healthy urine and facilitates a more personalized approach to prevention and treatment of infection.

Figures

Figure 1

Differences in relative bacterial OTU counts between neuropathic and healthy bladder in males and females. For every individual, the OTU counts were normalized to the individual's total OTU count. A heat map of the clustered distribution of OTU taxonomy at the level of bacterial order (A) and genus (B) was constructed using the Bray-Curtis index. For panels A and B, only the taxa with a standard deviation > 5% across all individuals are shown. Differences in the average OTU count between females and males are plotted in light and dark gray, respectively (C). In panel (C), the top 15 (8%) most abundant bacterial OTUs are represented. The X-axis indicates the difference in relative OTU counts per bacterial genus indicated on the Y-axis. Statistical significance was established using Kruskal-Wallis test. Significant differences (P < 0.05) between the relative OTU counts are indicated by an asterisk (*) for bladder function, and a plus sign (+) for gender.

Figure 2

OTU differences among individuals by duration of neuropathic bladder. A PCA analysis of the OTU counts of 52 individuals. The points are circled and colored based on the duration (in months) of neuropathic bladder (see key). The inset depicts a vector plot indicating the most influential principal component (bacterial genus).

Figure 3

Differences in the relative OTU counts between males and females stratified by bladder management. For every individual, the OTU counts were normalized to the individual’s total OTU count. Differences between relative OTU counts were calculated by subtracting the average OTU counts from females and males per bladder management category; healthy control, void (SCI patient with no catheter usage), indwelling catheter (IC), and Foley catheter (FC) (see key for color coding and sample sizes). The X-axis indicates the difference in relative OTU counts per bacterial genus indicated on the Y-axis. Significant differences (P < 0.05) between the relative OTU counts are indicated by plus sign (+) for gender, and an asterisk (*) for bladder management. The inset depicts the mean and standard deviation of OTU counts of the indicated genera for each catheter management group.

Figure 4

Phylogenetic diversity of Lactobacillales 16S rDNA sequences in human urine. NJ tree clustering of Lactobacillales OTU representatives labeled based on similarity to known RDP database sequences (gray), and OTU composition. Leaves are colored as follows: OTUs consisting of only healthy individuals (dark blue), mostly healthy (light blue), only NB (red), mostly NB (pink/salmon). Branches were highlighted and labeled by identifiable bacterial genera. Genus-level classification was based on the OTU representative RDP classification and the classification of nearest neighbors the RDP alignment. The nodes show SequenceID_#male/#female_#SCI/#healthy subjects.

Figure 5

Phylogenetic diversity of Enterobacteriales 16S rDNA sequences in human urine. NJ tree clustering of Enterobacteriales OTU representatives labeled based on similarity to known RDP database sequences (gray), and OTU composition. Leaves are colored as follows: OTUs consisting of only healthy individuals (dark blue), mostly healthy (light blue), only NB (red), mostly NB (pink/salmon). Branches were highlighted and labeled by identifiable bacterial genera. Genus-level classification was based on the OTU representative RDP classification and the classification of nearest neighbors the RDP alignment. The nodes show SequenceID_#male/#female_#SCI/#healthy subjects.