Unusual Manifestation of Live Staphylococcus saprophyticus, Corynebacterium urinapleomorphum, and Helicobacter pylori in the Gallbladder with Cholecystitis

Abstract

Culture-independent studies have identified DNA of bacterial pathogens in the gallbladder under pathological conditions, yet reports on the isolation of corresponding live bacteria are rare. Thus, it is unclear which pathogens, or pathogen communities, can colonize the gallbladder and cause disease. Using light microscopy, scanning electron microscopy, culture techniques, phylogenetic analysis, urease assays and Western blotting, we investigated the presence of live bacterial communities in the gallbladder of a cholecystitis patient after cholecystectomy. 16S rRNA gene sequencing of isolated bacterial colonies revealed the presence of pathogens most closely resembling Corynebacterium urinapleomorphum nov. sp., Staphylococcus saprophyticus and Helicobacter pylori. The latter colonies were confirmed as H. pylori by immunohistochemistry and biochemical methods. H. pylori cultured from the gallbladder exhibited both the same DNA fingerprinting and Western cagA gene sequence with ABC-type EPIYA (Glu-Pro-Ile-Tyr-Ala) phosphorylation motifs as isolates recovered from the gastric mucus of the same patient, suggesting that gastric H. pylori can also colonize other organs in the human body. Taken together, here we report, for the first time, the identification and characterization of a community consisting of live S. saprophyticus; C. urinapleomorphum, and H. pylori in the gallbladder of a patient with acute cholecystitis. Their potential infection routes and roles in pathogenesis are discussed.

Keywords: 16S rRNA gene sequencing; CagA; Corynebacterium urinapleomorphum; EPIYA; GGT; Helicobacter pylori; HtrA; Staphylococcus saprophyticus; cholecystitis; electron microscopy; gallbladder; immunohistochemistry; protein profiling; urease test.

Figure 1

Three isolates of live bacterial species isolated from the gallbladder of a patient with cholecystitis were subjected to 16S rRNA gene sequencing and phylogenetic analysis. The DNA relatedness of sequences with known taxa is shown using the unweighted pair group method and arithmetic average method (UPGMA). The optimal tree with the sum of branch length was 1.04, 0.51, and 1.22 for panels (A–C), respectively. All positions containing gaps and missing data were eliminated, and the analyses were conducted in MEGA7.

Figure 2

Histological sections from the gallbladder of the patient. The magnification is 40× for the left panels, and a 200× magnification of the identified section (white box) is shown to the right. (A) Identification of collagen-rich fibrosis using hematoxylin–eosin staining and mucosal cholesteatosis. Very focal active inflammatory infiltrates are present; (B) Warthin–Starry silver staining of the section shown in panel A inside the white square; (C) Immunohistochemistry using a monoclonal antibody against Helicobacter pylori showing red, partly intracellular positive signals. In the right panel, H. pylori-like spiral shaped bacteria of approximately 2–3 μm in length are visible (blue arrows). Coccoid bacteria with up to 1 μm in diameter are also present (red arrows).

Figure 2

Histological sections from the gallbladder of the patient. The magnification is 40× for the left panels, and a 200× magnification of the identified section (white box) is shown to the right. (A) Identification of collagen-rich fibrosis using hematoxylin–eosin staining and mucosal cholesteatosis. Very focal active inflammatory infiltrates are present; (B) Warthin–Starry silver staining of the section shown in panel A inside the white square; (C) Immunohistochemistry using a monoclonal antibody against Helicobacter pylori showing red, partly intracellular positive signals. In the right panel, H. pylori-like spiral shaped bacteria of approximately 2–3 μm in length are visible (blue arrows). Coccoid bacteria with up to 1 μm in diameter are also present (red arrows).

Figure 3

Urease test and Western blotting analysis of H. pylori-specific pathogenicity factors from gallbladder strains. (A) Two H. pylori isolates (Gallbladder Hp-1 and Hp-2) were grown on acidified agar supplemented with urea (left samples). The observed color change from orange to red indicated that bacterial colonies were producing functional urease. The right samples represent positive controls. The color change occurred with the wild-type (wt) strain 26695 as expected, and was not observed with the negative control of an isogenic ΔureB deletion mutant, indicating that functional urease enzyme was not being produced; (B) Protein profiling using Coomassie staining. Asterisks label the following protein bands: CagA (*), Urease B (**), and Urease A (***); (C) Western blots of two reference strains (P12 and 26695) and the two gallbladder isolates that identifies presence of H. pylori proteins CagA, VacA, GGT and HtrA.

Figure 4

Scanning electron microscopic and genetic analyses of the stomach and gallbladder H. pylori isolates. High resolution scanning electron microscopy of the cultures obtained from the stomach (A) and gallbladder (B) of the same patient revealed spiral-shaped H. pylori bacteria. Arrows in the enlarged sections indicate typical monopolar flagella being present; (C) PCR-based randomly amplified polymorphic DNA (RAPD) produced identical fingerprints for the two H. pylori strains isolated from stomach and gallbladder. This method uses a set of single indicated primers (D1254 or D14307, top and bottom), which arbitrarily anneal and amplify genomic DNA resulting in strain-specific fingerprinting patterns. M = DNA size marker.