Biliary infection in patients is common and has been associated with invasive clinical procedures, septicemia, intestinal barrier dysfunction, and gut bacteria translocation (Chi et al., 2023; Ekpanyapong and Reddy, 2023). It continues to be a significant cause of severe complications and morbidity in patients, particularly in elderly patients with underlying comorbidities. The heightened mortality rate among patients with infected biliary secretions underscores the critical need for timely diagnosis and treatment of biliary tract infections. Therefore, for patients with bile infectious symptoms, bile samples are frequently used for clinical microbiological culture tests. However, the bile bacterial spectrum and antibiotic resistance characteristics are constantly changing. Based on previous literature reports, the widespread use of antibiotics globally has increased the likelihood of bacterial species alterations. Data indicates that pathogenic bacteria such as Escherichia coli, Enterococcus faecium, Klebsiella pneumoniae, and Serratia, particularly Enterococcus faecium, have become predominant in recent cases of biliary tract infections. It is noteworthy that the trend of drug resistance is worsening (Ruan et al., 2019).

Gram-negative bacilli, which are some of the most common opportunistic pathogens in hospitals, have shown increasing trends toward drug resistance in recent years. Gram-negative bacilli mainly include those in the Enterobacteriaceae family and nonfermentative bacteria, and easily evolve into multidrug-resistant (Chen S. et al., 2021) organisms (MDROs) that are resistant to three or more types of clinical antibiotics used at the same time. In recent years, with the extensive use of antibiotics for the treatment of bacterial infections, MDROs have become common and important pathogens responsible for clinical infections, resulting in an intractable challenge for clinical diagnosis and treatment (Kawanishi et al., 2017); therefore, it is necessary to clarify the risk factors for MDRO infection in patients.

We conducted this study to understand the characteristics of pathogens causing biliary infections and related risk factors in patients, guide clinical detection, promote the rational use of antibiotics, control multidrug-resistant bacterial infections, and improve the cure rate in biliary tract infectious patients. We analyzed the in-hospital data of patients from January 2019 to December 2021 to retrospectively identify the biliary pathogenic spectrum and drug resistance profiles, Gram-negative multidrug-resistant bacteria distributions and resistance rates, and associations of risk factors in the clinical characteristics of the patients. This analysis aimed to provide valuable insights into the changing trends of biliary tract infections and drug resistance, ultimately informing more effective clinical management, improved detection capability, and prevention strategies in the medical future. The study results are as follows.

Bile is a vital bodily fluid that plays a crucial role in digestion and metabolism within the human body. It typically maintains sterility, and samples are frequently collected during routine in-hospital examinations when infected by pathogens (Jang et al., 2023). Unfortunately, its infection is increasing worldwide. When the normal excretion of bile is blocked by tumors, stones, or worm infections, there is increased pressure in the biliary tract, allowing the invasion of intestinal bacteria through the intestine or portal vein. This can promote the development of acute or chronic cholecystitis, as well as abdominal infection (Zhang et al., 2023).

It is well-known that the rates of resistance to antibiotics vary by bacterial type and time (Shen et al., 2023; Yin et al., 2023). The authors of this paper have extensive experience in clinical microbiological detection within hospital settings. For them, gaining a comprehensive understanding of the distribution and drug resistance patterns of common biliary tract pathogens in China is crucial. This knowledge is essential for enhancing detection capabilities and reducing mortality rates among patients with biliary infections.

To address these issues, the intraoperative collection of bile and bacterial culture samples can greatly improve the accuracy of diagnosis and treatment by providing a more comprehensive understanding of the patient’s condition (Asukai et al., 2023; Papazyan et al., 2023). Moreover, although there are many methods to treat corresponding infectious complications, the effect is not satisfactory (Chi et al., 2023). Therefore, it is necessary to actively prevent pathogen infection in patients, and important to understand the characteristics of pathogen infections and their associated risk factors.

At present, there are scientific studies on clinical biliary infection in humans. The 2000 British study and the 2023 India paper reported ambiguity resulting from limited sample size and a lack of detailed pathogen classification (Kar et al., 2023). In contrast, the 2017 report from Japan, as well as the 2006 report of clinical infections in China, identified Pseudomonas aeruginosa, Enterococcus, Klebsiella pneumoniae, and Escherichia coli as the most prevalent bacteria in biliary tract infection. This aligns with our findings regarding species composition but in an entirely reversed sequence (Chen et al., 2006). Interestingly, the pathogenic dominant strains in our study were similar to those in the Iranian study (Shafagh et al., 2021). There were relatively few controversial studies on pathogen infections in China with the comparison that the dominant strains of acute pancreatic infection were different except for Escherichia coli (Chen H. L. et al., 2021). Hence, it is necessary to conduct in-depth research on infectious pathogens in the bile of biliary infection patients.

In this study, the bile samples were incubated and separated by a BacT/Alert3D automated blood culture instrument by international standard operation procedures for bacterial detection (Chen S. et al., 2021); this method can guarantee the growth and accurate identification of specific organisms, even if the samples contained multiple species.

According to the data obtained from bile specimens from 1,556 patients in hospitals. The bile samples in this study had a positive pathogen detection rate of 72.30% during 2019–2021, supporting prior studies conducted in China (Mavri and Smole Mozina, 2013; Wajima et al., 2022). A total of 3,490 pathogenic bacteria were identified, primarily involving Gram-negative bacilli (67.05%), followed by Gram-positive cocci (29.48%) and fungi (3.12%).

During the 3 years, the predominant Gram-negative bacilli included Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae subspecies seweris, and Pseudomonas aeruginosa. The distribution pattern bears a certain degree of resemblance to the microecological arrangement observed in the gastrointestinal tract in SAP (Chen S. et al., 2021). It is noteworthy that Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae subspecies seweris exhibited a similar pattern of initial decline followed by an increase; however, their data in 2021 did not surpass that of 2019. In contrast, Pseudomonas aeruginosa displayed a distinct pattern of continuous growth. Among the Gram-positive cocci species identified mainly were Enterococcus faecium and Enterococcus faecalis; however, their trends differed. The former showed a slight decline followed by an increase while the latter demonstrated a decreasing trend. Candida albicans was identified as the major fungal species. Regarding obligate anaerobic bacteria, Clostridium perfringens remained the primary species but with a declining trend. The presence of fungi and obligate anaerobes detected, however, is relatively limited in quantity. Therefore, there may be some potential deviation, thus it is not necessary to draw excessive inferences.

These findings are consistent with those described in the National Bacterial Resistance Monitoring Network’s monitoring report (Kawecki et al., 2007; Oldereid et al., 2023). Escherichia coli, Klebsiella pneumoniae, Enterococcus, and other intestinal flora were the primary pathogenic Gram-negative bacilli identified by the current study. Biliary tract infection is often caused by opportunistic intestinal pathogens entering retrograde (Babekir et al., 2023), suggesting that they may be related to the placement of drainage tubes, biliary stents, and other interventional treatments (Quinn et al., 2020; Suh et al., 2021; Behroozian et al., 2022).

Most of the bile pathogens in this study were detected in hepatobiliary and pancreatic surgery and tumor surgery departments by the laboratory clinicians. The majority of patients were experiencing inflammation or tumors of the gallbladder, bile duct, liver, or pancreas, and most underwent surgery or interventional treatments, such as ERCP (Mistry and Zeng, 2022). Older age, a higher number of gallstones, hypoproteinemia, recurrent gallstones, relapsed biliary tract surgery, malignant biliary tract infection, and previous antibiotic exposure are all risk factors for biliary tract infection (Gouveia et al., 2017; Kawanishi et al., 2017; Xing et al., 2022). Thus, more attention should be given to these patient populations, including the implementation of intervention measures to prevent biliary tract infection.

Analysis of the Gram-negative bacteria confirmed that Escherichia coli and Klebsiella pneumoniae Extended-spectrum 𝛽-lactamase (ESBL) detection rates were 37.21 and 17.56%, respectively. While the rate of Escherichia coli antibiotic resistance remained stable during 2019–2021, the rate of resistance to ciprofloxacin increased significantly in 2021 (p < 0.05). This may be due to the high concentration of this drug in bile. Since ciprofloxacin is a common antibiotic used to treat biliary tract infections, more vigilance is required for the use of this drug. While Klebsiella pneumoniae was more susceptible to most antibiotics than Escherichia coli, its resistance to tigecycline and carbapenem increased significantly in 2021 (p < 0.01).

Klebsiella pneumoniae resistance to carbapenem antibiotics was mediated by several different resistance mechanisms, including the production of carbapenemase, increases in effervescent pump activity, and changes in outer membrane protein quantity and function (Yan et al., 2021; Wang et al., 2022; Jing et al., 2023). Tigecycline, one of the newest antibiotic drugs used to treat carbapenem-resistant strains, has been in clinical use in China since 2012 (Fergadaki et al., 2021); however, drug-resistant strains developed quickly and are increasing in number every year. Several mechanisms of resistance, mediated by both chromosomes and plasmids, have been identified (Chen H. L. et al., 2021; Yang J. et al., 2023; Yang Y. et al., 2023). The current study also showed an increase in the resistance of Klebsiella pneumoniae to cephalosporins, carbapenems, lactamase inhibitors, cephalases, sminoglycosides, and quinolones (p < 0.05). The increase in Klebsiella pneumoniae drug resistance indicates a need to intensify the prevention and control of these bacteria in clinical settings.

Pseudomonas aeruginosa was one of the primary causes of infection in bile. The rates of resistance to piperacillin, piperacillin/tazobactam, ticacillin/clavulanate, and amikacin decreased significantly over time (p < 0.05). After the decline in resistance to quinolones in 2020, however, Pseudomonas aeruginosa had varying degrees of resistance in 2021 (p < 0.05). This is an important opportunistic pathogen and a causative agent of clinical infections. Patients who are immunocompromised, have complicated underlying diseases, or have received long-term use of antibiotics or interventional therapy are at higher risk of infection and should be monitored more closely to prevent and effectively treat infection (Araya et al., 2023).

Among Gram-positive bacteria, Enterococcus faecium was the most likely to develop drug resistance, followed by Enterococcus faecalis. The resistance rate of Enterococcus faecium to tetracycline increased over time (p < 0.05), and strains that were resistant to tigecycline and vancomycin also emerged. Low hemoglobin is significantly associated with the production of vancomycin-resistant enterococci, indicating that drug-resistant bacteria may acquire iron to evolve (Brunson et al., 2023). These findings indicate a need to pay more attention to biliary enterococcal infection in patients with anemia.

While Enterococcus faecalis had higher drug sensitivity than Enterococcus faecium, the rate of resistance to erythromycin and high-concentration gentamicin decreased over time (p < 0.05). Meanwhile, Enterococcus faecalis showed no resistance to tigecycline, linezolid, or vancomycin, indicating that these should be the first choice for treatment in severely infected patients. Aminoglycosides can be combined as needed to achieve synergistic effects (Anjum et al., 2022).

Candida albicans and Candida tropicalis were the top fungal strains isolated from bile, and most were sensitive to common antifungal drugs (Jabeen et al., 2023). These findings indicate that the use of conventional antifungal drugs can have strong therapeutic effects. However, clinicians should make sure to use standardized operating and drug application procedures and strive for aseptic infection.

It is important to note that 12 anaerobic bacteria (0.34%) were also isolated from the bile samples. This finding highlights a need to test bile samples for both aerobic and anaerobic bacteria to avoid missing anaerobic infections. In addition, treatment directed at both aerobic and anaerobic bacteria should be considered for patients with a history of bilio-entero-anastomosis (Ligero-Lopez et al., 2023).

With the extensive use of antimicrobial agents at present, MDROs have become important pathogens of clinical infections. MDROs are resistant to ampicillin, first-generation cephalosporins, and second-generation cephalosporins. Moreover, the sensitivity trends of ceftazidime, piperacillin/tazobactam, cefoperazone/sulbactam, gentamicin, and tobramycin were similar. However, resistance to both ceftriaxone and ciprofloxacin was higher than that to ciprofloxacin. In contrast, resistance to aminoglycosides, cefepime, and carbapenems was higher (Xiao et al., 2023). It was concluded that the drug resistance patterns of MDROs were very different, the patterns of MDROs differ slightly between the northern and southern regions in China, even within the same country (Ruan et al., 2019). This variability is less conducive to clinical anti-infection treatment in patients. Merely, analysis of resistance of MDROs and risk factors for Gram-negative MDRO infection can help identify relevant risk factors and prevent infection (Chen S. et al., 2021). Our results showed that six factors, such as the length of hospital stay, time, antibacterial drug use, antimicrobial type, use of third- or fourth-generation cephalosporins, use of penicillium carbon alkene antimicrobial agents, and invasive operations, were risk factors for Gram-negative MDRO infection in biliary infectious patients. Multivariable logistic regression analysis of these factors indicated that the use of third- or fourth-generation cephalosporins in biliary infectious patients with the presence of biliary Gram-negative MDROs was an independent risk factor for infection and high drug resistance. According to a report by Skorochod et al. (2022), independent risk factors for Israeli patients with Staphylococcus aureus hepatobiliary infection included hypertension, bedridden status, and nursing home residence. This suggests that different countries may have their own unique set of independent risk factors for this condition. The rational use of third- or fourth-generation cephalosporins in China was an important measure in preventing MDRO infection in this type of patient.

In conclusion, this study has determined that Gram-negative bacteria were the predominant pathogens isolated from patients with biliary infections, particularly Escherichia coli. The rates and patterns of drug resistance were found to be high and constantly changing. In clinical practice, it is crucial to judiciously use third- or fourth-generation cephalosporins as a key measure to prevent multidrug-resistant organism (MDRO) infections in patients with biliary infections. While our study did not investigate resistance mechanisms, the findings have the potential to inform decision-making in the healthcare sector. Furthermore, this information can be utilized to educate both healthcare professionals and the general public about the importance of appropriate antibiotic use. Additionally, the results of this study could lay a foundation for future research efforts, enabling scientists to explore new detection, therapeutic options, and strategies for biliary infections.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The studies involving humans were approved by Ethical Committee of Nankai Clinical College, Tianjin Medical University. The studies were conducted in accordance with the local legislation and institutional requirements. The ethics committee/institutional review board waived the requirement of written informed consent for participation from the participants or the participants' legal guardians/next of kin because Due to its retrospective design, a waiver of participant informed consent was granted by the Ethical Committee of Nankai Clinical College, Tianjin Medical University.

SC: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing. WL: Data curation, Writing – original draft, Writing – review & editing. XS: Data curation, Funding acquisition, Investigation, Methodology, Writing – original draft, Writing – review & editing. JLu: Data curation, Funding acquisition, Investigation, Methodology, Writing – original draft, Writing – review & editing. JLi: Data curation, Funding acquisition, Investigation, Methodology, Writing – original draft, Writing – review & editing. HO: Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. WZ: Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. JC: Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. ZY: Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. HL: Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. YZ: Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing.