Detection of Acinetobacter Baumannii and Staphylococcus Capitis in Bile from Two Patients with Chronic Xanthogranulomatous Cholecystitis: The Impact of Metagenomic Analysis

Background Escherichia coli is thought to cause xanthogranulomatous cholecystitis (XGC). However, it is unclear whether other pathogens are associated with the cause and progression of XGC. Case presentation Patient 1 was a 55-year-old man with a previous surgical history of right lung cancer. He presented with abdominal pain and was diagnosed with acute cholecystitis. He underwent endoscopic nasogallbladder drainage (ENGBD), antimicrobial therapy, and endoscopic sphincterotomy (EST). He underwent cholecystectomy on day 59. The patient was pathologically diagnosed with chronic phase XGC. Acinetobacter baumannii was isolated from the bile sample during the operation. Patient 2 was a 58-year-old man with no previous medical history. He presented with abdominal pain and was diagnosed with acute cholecystitis. He underwent endoscopic retrograde biliary drainage (ERGBD) and antimicrobial therapy. His symptoms improved, but acute cholecystitis became exacerbated on day 53. The patient was treated with antimicrobial therapy. He underwent cholecystectomy on day 88. The patient was pathologically diagnosed with focal acute inflammatory phase XGC. Staphylococcus capitis was isolated from the bile during the operation. This study describes two patients with XGC, one infected with A. baumannii and the other with S. capitis, in their gallbladders, which was identified by bacterial culture. Metagenomic analysis revealed that the genera Acinetobacter and Staphylococcus predominated and that other genera, including Delftia and Anaerobacillus, were also present, suggesting that these bacteria play a significant role in the pathological changes associated with XGC Conclusions This is the first report of A. baumannii and S. capitis infections in patients with XGC.


Introduction
Xanthogranulomatous cholecystitis (XGC) is characterized histologically by the accumulation of numerous foamy macrophages in the gallbladder, resulting in thickening of the gallbladder wall 1) .Moreover, XGC is frequently diagnosed as gallbladder carcinoma.Escherichia coli antigens have been detected immunohistologically in XGC lesions 2) , and E. coli was reported to be involved in the pathogenesis of XGC, with scavenger receptor class A and CXCL16-CXCR interactions 3) .These results suggested that E. coli infections of the gallbladder play an important role in the onset and/or early stages of XGC.However, it is unclear whether other pathogens are associated with the cause and progression of XGC.
This study describes the isolation of two other species of bacteria, Acinetobacter baumannii and Staphylococcus capitis, from the bile samples of two patients with XGC.Metagenomic analysis of the microbiota in bile samples confirmed that these pathogens, as well as other microorganisms, were present in bile.

Case report
Two patients (P1 and P2) underwent cholecystectomy for suspected chronic cholecystitis in July 2018 at the Department of Hepato-biliary Pancreatic Surgery, Juntendo University Hospital.Written informed consent was obtained from these patients before surgery.This study was conducted according to the principles of the Declaration of Helsinki, and approved by the Juntendo University ethics committee (JHS 18-060 Juntendo University Hospital Independent Ethics Committee).The clinical characteristics of these two patients are summarized in Table 1.Chronic cholecystitis in these patients was diagnosed as XGC by histopathological examination of the gallbladder samples.
Patient 1 was a 55-year-old Japanese man with a previous surgical history of right lung cancer seven years earlier, and was admitted to an intensive care unit (ICU) with a mechanical ventilator.He presented with abdominal pain and was diagnosed with acute cholecystitis.He underwent endo- scopic nasogallbladder drainage (ENGBD) on the first day and was treated with 3 g/day cefmetazole (CMZ) for three days.The bile sample obtained during ENGBD was negative for bacterial culture, but his AST/ALT was elevated.Endoscopic sphincterotomy (EST) was performed on the third day, and he was treated with 1.5 g/day meropenem (MEPM) for three days, followed by 3 g/day CMZ for three days.His symptoms disappeared and the patient was not administered any antimicrobial agents after day 10.He underwent cholecystectomy on day 59.Gross examination of the resected gallbladder showed the hemorrhagic mucosa with marked wall thickness (Figure 1A).Microscopic examination showed diffuse infiltration of foam cells along with multinucleated giant cells, lymphocytes, and cholesterol deposit (Figure 1B, C).No bacterial colonies or neutrophilic reactions were evident histologically.The patient was pathologically diagnosed with chronic phase XGC. A. baumannii was isolated from the bile sample obtained from Patient 1 during the operation.This isolate was susceptible to all drugs tested (Table 2).Metagenomic analysis of the bile sample from Patient 1 showed bacterial DNA derived from seven genera, with the genus Acinetobacter being predominant.
Filtering of the data sets to include OTUs present in > 0.5% of the samples revealed bacterial DNA from four phyla, Actinobacteria (0.8%), Bacteroidetes (1%), Firmicutes (10%) and Proteobacteria (88%) (Figure 2).Patient 2 was a 58-year-old Japanese man with no previous medical history.He presented with abdominal pain and was diagnosed with acute cholecystitis.He underwent endoscopic retrograde biliary drainage (ERGBD) on the first day, and was treated with 6 g/day CMZ for two days, followed by 18 g/day piperacillin-tazobactam (PIPC/TAZ) for five days.His symptoms improved, but acute cholecystitis became exacerbated on day 53.The patient was treated with 1 g/day ceftriaxone (CTRX) for one day, followed by 0.5 g/day levofloxacin (LVFX) for seven days.His symptoms disappeared and the patient was not administered any antimicrobial agents after day 60.We adjusted the waiting period for a month to improve the inflammation, and he underwent cholecystectomy on day 88.Gross examination of the resected gall- bladder showed that rough and coarse mucosa at the fundus with marked wall thickness and microscopic examination showed infiltration of bile containing foam cells along with lymphocytes (Figure 1D-F).Gram staining showed no evident bacterial colonies, although some neutrophilic reactions were observed, histologically.The patient was pathologically diagnosed with focal acute inflammatory phase XGC. S. capitis was isolated from the bile sample obtained from Patient 2 during the operation, with this isolate being resistant to ABPC, CAZ, EM, GM, LVFX, OXA, and PCG, but susceptible to ABK, TEIC, and VCM (Table 2).Metagenomic analysis of the bile sample from Patient 2 showed bacterial DNA derived from four genera, with the genus Staphylococcus being predominant.Filtering of the data sets revealed bacterial DNA from two phyla, Firmicutes (97%)  and Proteobacteria (3%) (Figure 2).

Discussion
Because E. coli is the organism most frequently isolated from bile samples of patients with cholecystitis, it has been regarded as a cause of this condition.Other microorganisms isolated from the bile samples of patients with cholecystitis include Enterobacter, Enterococcus, Klebsiella, Streptococcus, and Pseudomonas spp. 4).These pathogens are thought to enter the gallbladder from the duodenum in a retrograde manner, although there is other possibility that they enter the gall bladder via the portal vein through the hepatic sinusoids and space of Disse 5) .Gallbladder stones play an important role in the pathological conditions observed in patients with cholecystitis.
Pathologically, XGC is characterized by thickening of the gallbladder wall, mimicking advanced gallbladder carcinoma 1) .Although these pathological changes are thought to be due to intense acute or chronic inflammation, the pathogenesis of XGC remains unclear.XGC is often associated with gallstones.Gallstones cause ulceration of the gallbladder mucosa, rupture of Rokintansky-Aschoff sinuses, and eventually xanthogranulomatous changes 6) .
Next-generation DNA sequencing has enabled analysis of the microbiota in the biliary tracts of patients with various diseases, including bacterial

Figure 1
Figure 1 Pathological view of the 2 cases of gallbladder with xanthogranulomatous cholecystitis 1A-C: Patient 1. A: Grossly, a thickened black gallbladder is seen.B and C: Microscopically, diffuse infiltration of foam cells and lymphocytes are observed along with bile and cholesterol deposits.1D-F.Patient 2. D. Grossly, a thickened gallbladder with coarse mucosa at the fundus (Arrows) is seen.E and F: Microscopically, diffuse infiltration of foam cells with bile pigment is seen.

Figure 2
Figure 2 Relative abundance of major bacteria in bile