Aerococcus urinae and Globicatella sanguinis Persist in Polymicrobial Urethral Catheter Biofilms Examined in Longitudinal Profiles at the Proteomic Level

Abstract

Aerococcus urinae (Au) and Globicatella sanguinis (Gs) are gram-positive bacteria belonging to the family Aerococcaceae and colonize the human immunocompromised and catheterized urinary tract. We identified both pathogens in polymicrobial urethral catheter biofilms (CBs) with a combination of 16S rDNA sequencing, proteomic analyses, and microbial cultures. Longitudinal sampling of biofilms from serially replaced catheters revealed that each species persisted in the urinary tract of a patient in cohabitation with 1 or more gram-negative uropathogens. The Gs and Au proteomes revealed active glycolytic, heterolactic fermentation, and peptide catabolic energy metabolism pathways in an anaerobic milieu. A few phosphotransferase system (PTS)-based sugar uptake and oligopeptide ABC transport systems were highly expressed, indicating adaptations to the supply of nutrients in urine and from exfoliating squamous epithelial and urothelial cells. Differences in the Au vs Gs metabolisms pertained to citrate lyase and utilization and storage of glycogen (evident only in Gs proteomes) and to the enzyme Xfp that degrades d-xylulose-5'-phosphate and the biosynthetic pathways for 2 protein cofactors, pyridoxal 6'-phosphate and 4'-phosphopantothenate (expressed only in Au proteomes). A predicted ZnuA-like transition metal ion uptake system was identified for Gs while Au expressed 2 LPXTG-anchored surface proteins, one of which had a predicted pilin D adhesion motif. While these proteins may contribute to fitness and virulence in the human host, it cannot be ruled out that Au and Gs fill a niche in polymicrobial biofilms without being the direct cause of injury in urothelial tissues.

Keywords: Aerococcus; Globicatella; catheter biofilm; host-pathogen interaction; infection; proteomics; urinary tract.

Figure 1.

Relative quantities of polymicrobial proteomes in CB and UP samples from patients (A) P5 and (B) P6. The segmented bars are ordered from left to right according to the sequence of catheter collection time points. The time points of P5 and P6 were 2 and 3 weeks apart, respectively, thus indicating that the CBs persisted over several months. The colored segments of a bar represent the relative contribution of each microbial proteome to the entire sample’s proteome (including human proteins). Contributions of the latter (represented by the difference of 1 and the bar height) varied from 15% in #37_CB to 96% in #156_UP_s. We estimate that the protein quantity is roughly equivalent to biomass contribution. Using color coding, the text on the right of the bar diagrams denotes the species represented by colored bar segments. A matching sample number for CB and UP samples in the plot (B) indicates specimen collection at the same timepoint. For comparative purposes, the graphic in (A) shows proteomic data from 3 unrelated cases of UTI with or without short-term catheterization (Au as one of the identified species; UPs: “s” for short term-catheterized; UPn: “n” for not catheterized). Au and Gs proteomes that were functionally interrogated in the following paragraphs are #27_CB, #37_CB, #168_UPs, #156_UPs, #53_UP, #53_CB, and #54_CB. CB indicates catheter biofilm; UP, urinary pellet; UTI, urinary tract infection.

Figure 2.

Anaerobically cultured bacteria from catheter biofilm extracts of P5 and P6. (A) Small grayish-white colonies on 5% sheep blood agar were identified as Aerococcus urinae. Eight other bacterial species were also identified from distinct agar colonies growing on the plate. (B) A Globicatella sanguinis strain from P6 (extract #53_CB) was grown aerobically on 5% sheep blood agar over 48 hours. The pinpoint-sized Gs colonies were α-hemolytic.

Figure 3.

Peptide/amino acid transport and metabolism and cofactor synthesis in Gs and Au cells. The schematic representation contains protein names and gene loci (if protein short names were not provided for ORFs based on conserved sequences) in red. Each gene locus contains only the last 4 and 5 numbers of the Au and Gs gene accession terms, respectively. Details on the proteins (quantities and descriptions) are provided in datasets of the Supplemental File S2. Metabolite names are depicted in black. Blue arrows illustrate an enzymatic activity or pathway step, while black arrows indicate a transport process. The darker the color of the circle behind each protein name, the higher its average abundance in CB datasets. Blue: Gs proteins; green: Au proteins; gray: protein not detected in the proteomes. Cofactors, in light green script, are depicted next to enzymes where applicable: Me²⁺ (metal ion), Zn²⁺ (zinc), py (pyridoxal-5′-phosphate). DHA indicates dihydroxyacetone; “P,” phosphate; PPP, pentose-phosphate pathway; “?” indicates that a gene is predicted to catalyze an enzymatic step based on a domain with a predicted function or its gene neighborhood.

Figure 4.

Inference of carbohydrate uptake and metabolism pathways used by Aerococcus urinae and Globicatella sanguinis in urethral catheter biofilms. The legend of Figure 3 already described most acronyms, symbols, and colors of circles that follow protein names/gene identifiers as well as the pathway connecting arrows used here. IIA, IIB, and IIC are terms generally used to define the subunits of PTS for ATP-dependent sugar import. ATP indicates adenosine tri-phosphate; PTS, phosphotransferase systems.

Figure 5.

Active anaerobic energy metabolism pathways used by Aerococcus urinae and Globicatella sanguinis in urethral catheter biofilms. (A) Heterolactic fermentation pathways active in Gs and Au. (B) Citrate lyase pathway active in Gs only. The legend of Figure 3 describes most of the acronyms, symbols, and colors of circles that follow protein names/gene identifiers as well as the connecting arrows used here. The individual early glycolytic pathway steps catalyzed by Pgi, PfkA, and Fba (class II) are not shown in the schematic representation. These enzymes were highly abundant in the Au and Gs proteomes. MAF indicates mixed acid fermentation.