Escherichia coli virulence factors iutA, an iron acquisition factor, and ibeA, an invasion factor, are related to severity of bacteremic acute biliary tract infections


 BackgroundAlthough Escherichia coli is the most frequently isolated organism in acute biliary tract infections with bacteremia, data regarding its virulence are limited.ResultsBacteremic acute biliary tract infection cases in a retrospective study were collected from 2013 to 2015 at a tertiary care hospital in Japan. Factors related to the severity of infection were investigated, including patient background, phylogenetic typing, and virulence factors of E. coli, such as adhesion, invasion, toxins, and iron acquisition. In total, 72 E. coli strains were identified in 71 cases, most of which primarily belonged to the phylogenetic B2 group (68%). The presence of IutA (77.3% in the non-severe group, 46.4% in the severe group, P = 0.011) and ibeA (9.1% in the non-severe group, and 35.7% in the severe group, P = 0.012) were significantly associated with severity of infection, whereas patient characteristics did not relate to the severity of infection.ConclusionsWe showed that bacteremic E. coli strains from acute biliary tract infections belonged to a virulent (B2) group. The severity of biliary tract infection depended on iutA and ibeA.

Pathogenic E. coli express many common virulence factors even at different sites of infection, but an organ-speci c strategy is needed to identify speci c virulence factors. These common and/or different traits are now studied rigorously to understand pathogenesis and to cope with infection at different foci.
Our study aimed to analyze the relationship between the severity of biliary tract infections and virulence of BEC strains.

Results
A total of 71 cases of bacteremic acute biliary tract infections (72 BEC isolates) were identi ed. We compared the severe (Pitt's score 2 or greater) group to the non-severe (score less than 2) group. Patient background, such as age, gender, and comorbidity were the same in the two groups (Table 1). More than half of the patients in both groups had abnormalities of the biliary tract. Approximately half of the patients in both groups had experienced biliary tract infections in the past.  Table 2 shows the comparison of laboratory data by severity. Liver enzymes and biliary markers were elevated to the same degree except for alkaline phosphatase. Median white blood cell counts were higher in the non-severe group than in the severe group. detected frequently between four STs by multi-locus sequencing typing (MLST), but more than half belonged to other STs. The most detected phylogenetic group was B2 in both groups (70.5% in nonsevere, 64.3% in severe), but the proportion of each phylogenetic group was similar. The presence of iutA (77.3% in the non-severe group, 46.4% in the severe group, P = 0.011) and ibeA (9.1% in non-severe group, and 35.7% in severe group, P = 0.012) were signi cantly associated with severity, whereas patient characteristics were not related to severity.

Discussion
In this study, BEC mainly belonged to group B2, which is known as a virulent group, and ST131. Although acute biliary tract infections are caused by obstruction/stasis of biliary ow and in ux of commensal bacteria into the biliary tract, BEC detected from blood cultures belonged to a pathogenic group.
The human intestinal tract has been recognized as a reservoir of extraintestinal pathogenic E. coli strains, such as uropathogenic E. coli (10). BEC may also hide in the gut. Once BEC translocates into the biliary tract due to stasis/obstruction, acute biliary tract infections can occur and develop into bacteremia.
Commensal E. coli strains, once considered as avirulent, are now known to express many of the same virulence factors, such as adhesion factors, as pathogenic E. coli strains. This is because these factors are needed for persistent colonization in the gut. E. coli has resistance against the effects of bile (11). In response to bile stress, both commensal and pathogenic E. coli strains, and especially enteropathogenic E. coli strains, activate stress response pathways (12,13), e ux pumps (14), and production of toxins (15) in the gut. As there are high concentrations of bile acid in the biliary tract, resistance against bile might play an important role in pathogenicity.
In our study, other virulence factors which are needed during infection, such as iron acquisition, adhesion, and invasion, were analyzed. Factors needed for colonization of the gut as compared to those needed during biliary tract infection must be analyzed to clarify speci c virulence factors of the biliary tractpathogenic E. coli strains. The differences between commensal and enteropathogenic E. coli strains in the gut have yet to be clari ed.
In the present study, iutA was found at a lower level in the severe group than in the non-severe group. IutA codes for the aerobactin siderophore ferric receptor protein, which has a role in facilitating iron acquisition by mediating the uptake of siderophores (16). In a chicken infection model, iutA expression in extraintestinal pathogenic E. coli strains was at least 50-fold higher in organ tissues compared to in vitro grown bacteria during infection (17). In mammalian hosts, iron is tightly bound to various proteins, such as hemoproteins and ferritin (18), making free iron for use by pathogenic bacteria scarce. In biliary tract infections, bile is an iron-limiting environment (19). Bile stress also causes increased mRNA levels for virulence genes associated with iron scavenging in E. coli (20). Therefore, E. coli strains harboring the iutA gene may become competitive in bile, but those without the iutA gene might nd it di cult to proliferate in bile and escape into the blood. An iutA vaccine protected mice in a sepsis challenge model (21) and UTI model (22). Because iutA as an antigen might be easily recognized by host immune systems, E. coli strains harboring the iutA gene may be easy to eliminate in bile.
In contrast, E. coli strains containing the ibeA gene were found in a higher proportion in the severe group. IbeA codes for a 50 kDa protein used for penetration of human brain microvascular endothelial cells to invade through the blood brain barrier, and is thought to have an important role in neonatal meningitis (23,24). IbeA may also be essential for invasion into intestinal epithelial cells and macrophages (25). No information regarding the relationship between ibeA and biliary tract epithelium cells has been reported; therefore, the mechanism of ibeA on the severity of acute biliary infection is unknown. ibeA inhibitors have been discovered to prevent invasion of human brain microvascular endothelial cells in vitro (26). It would be worthwhile to investigate whether E. coli strains harboring ibeA can perform internalization, and whether ibeA is essential to invade from bile duct to vessels through in vitro experiments using ibeA inhibitors.
In a rat model with common bile duct ligation, clearance of E. coli from blood and trapping rates in the liver were decreased compared to controls, and also showed decreased phagocytic activity and superoxide production of Kupffer cells (27). In our results, no signi cant relation was observed between severity of infection and patient background, such as abnormalities in the biliary tract and laboratory data of jaundice markers, except ALP.
This study has several limitations. First, although cholangitis is a polymicrobial infection (3), we investigated only one E. coli strain from each patient without analyzing other E. coli strains found in the bile. This comparison might reveal whether ibeA-positive strains can more easily escape or invade into the blood compared to strains that remained in the bile. Second, this study was conducted at a single institution. A larger multi-center study is needed to assess potential bias in the epidemiology of phylogenetic patterns and virulence factors.

Conclusion
We showed that many BEC belonged to a virulent group (B2) with a high prevalence of ST131. The severity of biliary tract infections depended on the presence of iutA and ibeA in BEC.

Patients
This retrospective study was conducted at the University of Tokyo Hospital, a 1217-bed tertiary-care teaching hospital in Tokyo, Japan. Patients with acute biliary tract infection who also had E. coli isolates detected in their blood were included in the study from April 2013 to February 2015. Each patient in this study was included only once, even if the patients repeatedly suffered E. coli bacteremia with acute biliary tract infection. Patient data, including clinical symptoms and microbiological data were collected from the medical records.

Data collection and de nitions
Patient data collected included age, sex, underlying disease (diabetes mellitus, malignancy with or without metastasis, lymphoma, and collagen disease), use of immunosuppressants, biliary tract abnormalities, such as insertion of an intrabiliary stent or surgery for biliary carcinoma, gastrointestinal tract abnormalities, and past history of acute biliary infection and bacteremia. History of residence in a nursing home and antibiotic use within three months before onset of bacteremia was also noted. Collected patient laboratory data included white blood cell counts, platelet counts, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyltransferase, and C-reactive protein. Cases in which E. coli was detected from blood cultures obtained within 48 h after admission were de ned as community acquired infection. Others were considered hospital acquired infections.
Biliary tract infection was de ned according to the Tokyo guideline (28). Cholangitis was de ned in cases where all the following sings were positive: 1) generalized in ammation sign such as fever (more than 38.0 °C), elevation of in ammation indicators in blood tests (white blood cell counts less than 4000/mL or more than 10,000/mL, and C-reactive protein 1 mg/dL or more), 2) signs of bile stasis such as jaundice (total bilirubin 2 mg/dL or more), elevation of liver function and biliary function tests (more than one-anda-half times of the upper limit for normal values for alkaline phosphatase(ALP), gammaglutamyltransferase(g-GTP), aspartate aminotransferase(AST), or alanine aminotransferase(ALT), normal range; ALP 106-322 U/L, g-GTP 13-64 U/L in men and 9-32 U/L in women, AST 13-30 U/L, ALT 10-42 U/L in men and 7-23 U/L in women, respectively), and 3) imaging of biliary tract abnormalities, such as dilatation of the biliary tract or the presence of a stent, and constriction. Cholecystitis was de ned as: 1) localized clinical signs such as Murphy's sign and pain in right upper abdomen, 2) generalized in ammation such as fever and elevation of in ammation indicators in blood tests, and 3) typical ndings, such as acute cholecystitis with echocardiography or CT scan. Severity was divided using the Pitt bacteremia score (29) as severe (score of 2 or more) and non-severe (score less than 2).

Microbiological procedures
All isolates were identi ed using the Walkaway system (Siemens, Berlin, Germany) or matrix-assisted laser desorption/ionization time-of-ight mass spectrometry (using the MALDI Biotyper; Bruker Daltonik, Germany).

Statistical analysis
The two-tailed Fisher's exact test was used for analysis of categorical data. Non-parametric data were analyzed using the Mann-Whitney U test. Values of P <0.05 were considered signi cant. All statistical analyses were performed using JMP Pro version 11 software (SAS Institute, Cary, NC, USA).

Ethical considerations
This study was approved by the research ethics committee at the University of Tokyo Hospital. Obtaining written informed consent from each patient was waived because it was an observational retrospective study. The data were analyzed anonymously.

Declarations
Ethics approval and consent to participate This study was approved by the research ethics committee at the University of Tokyo Hospital. Obtaining written informed consent from each patient was waived because it was an observational retrospective study. The data were analyzed anonymously.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This work was supported by JSPS KAKENHI Grant Number JP18K16171. The funding agencies did not have a role in the design of the study or the collection, analysis, and interpretation of data.
Authors' contributions MI designed this study, acquired and analyzed the patients' data, and was a major contributor in writing the manuscript. MI and TK performed PCR to analyze virulence of E. coli. FF and TH identi ed and collected the E. coli strains. KT made database of bacteremia, YO, SO and KM revised the manuscript. All authors read and approved the nal manuscript.