Encrustations on ureteral stents from patients without urinary tract infection reveal distinct urotypes and a low bacterial load

Abstract

Background: Current knowledge of the urinary tract microbiome is limited to urine analysis and analysis of biofilms formed on Foley catheters. Bacterial biofilms on ureteral stents have rarely been investigated, and no cultivation-independent data are available on the microbiome of the encrustations on the stents.

Results: The typical encrustations of organic and inorganic urine-derived material, including microbial biofilms formed during 3-6 weeks on ureteral stents in patients treated for kidney and ureteral stones, and without reported urinary tract infection at the time of stent insertion, were analysed. Next-generation sequencing of the 16S rRNA gene V3-V4 region revealed presence of different urotypes, distinct bacterial communities. Analysis of bacterial load was performed by combining quantification of 16S rRNA gene copy numbers by qPCR with microscopy and cultivation-dependent analysis methods, which revealed that ureteral stent biofilms mostly contain low numbers of bacteria. Fluorescence microscopy indicates the presence of extracellular DNA. Bacteria identified in biofilms by microscopy had mostly morphogenic similarities to gram-positive bacteria, in few cases to Lactobacillus and Corynebacterium, while sequencing showed many additional bacterial genera. Weddellite crystals were absent in biofilms of patients with Enterobacterales and Corynebacterium-dominated microbiomes.

Conclusions: This study provides novel insights into the bacterial burden in ureteral stent encrustations and the urinary tract microbiome. Short-term (3-6 weeks) ureteral stenting is associated with a low load of viable and visible bacteria in ureteral stent encrustations, which may be different from long-term stenting. Patients could be classified according to different urotypes, some of which were dominated by potentially pathogenic species. Facultative pathogens however appear to be a common feature in patients without clinically manifested urinary tract infection.

Trial registration: ClinicalTrials.gov, NCT02845726 . Registered on 30 June 2016-retrospectively registered.

Keywords: Biofilm; Cultivation-independent methods; Encrustation; Microbiome; Next-generation sequencing; Ureteral stent; Urinary tract microbiota; qPCR.

Fig. 1

a Representative scanning electron micrographs of ureteral stent encrustations and biofilms. Numbers refer to sample IDs. Based on NGS and cultivation, bacteria visible likely include L. jensenii (ST55), G. vaginalis (ST22), S. anginosus or A. tetradius (ST08), Staphylococcus (ST45), S. epidermidis (ST85) and Corynebacterium (ST30). Further samples included structures with size and morphology of fungal cells or blood cells (ST05, ST51 and ST80). Scale bars = 5 μm. b Confocal laser scanning micrographs of SYBR Green-stained ureteral stent encrustations, shown as maximum intensity projection. Fluorescence was associated with a layered structure. Colour allocation—grey, reflection; green, SYBR Green. Scale bars = 50 μm

Fig. 2

a–d Representative scanning electron micrographs of dominant crystalline phases in ureteral stent encrustations. Scale bars = 10 μm. a Dicalcium phosphate dihydrate (ST06). b Whewellite (ST16). c Weddellite (ST31). d Absence of crystals (no XRD signal), organic deposits (ST04). e Distribution of encrustation biomass. f Distribution of encrustation biomass among crystalline phases. ND absence of crystals, Whe whewellite, Wed weddellite, Cap dicalcium phosphate dihydrate

Fig. 3

Assessment of bacterial load by 16S qPCR. a Detection of bacterial DNA in water controls (NTC), blind samples (unused ureteral stents) and samples below and above the limit of detection (LOD). Each data point (open diamonds) represents a PCR reaction. b Samples (IDs indicated on y-axis) with evidence for biofilm bacteria. Grey bars indicate samples above the LOD, white bars those below. Error bars represent the standard deviation between three technical qPCR replicates. The table below illustrates indications for the presence of bacteria (black boxes) according to analyses by SEM, and bacterial cultivation from encrustation or urine samples

Fig. 4

Heat map representing absolute normalized abundance of bacterial nearest neighbour species (OTUs, y-axis), and of ureteral stent encrustation samples (x-axis) ordered according to inter-sample distance. Note, horizontal dense rows of points representing OTUs found in more than 50% of the samples, likely representing commensal bacteria. The normalized abundance represents bacteria equivalents per sequencing reaction

Fig. 5

Relative abundance of 28 most abundant bacterial taxa (very similar taxa summarized under one identifier, brackets indicate abundant species or genera) in the ureteral stent encrustation samples, sorted according to inter-sample distance (beta diversity). Similar samples formed 11 clusters, termed “urotypes” (UT1–UT6, indicated by purple dashed boxes), with 6 samples of a very different community structure not being part of the clusters

Fig. 6

Crystal phases identified in urotypes by XRD analysis. ND absence of crystals, Whe whewellite, Wed weddellite, CAP dicalcium phosphate dihydrate