Distribution analysis of positive and negative pathogenic bacteria in patients with acute pancreatitis and the clinical characteristics and model prediction analysis of positive infection bacteria

Introduction

Acute pancreatitis (AP) is a common digestive system disease. About 80% of AP cases are mild and self-limiting. If severe AP (SAP) is infected, the mortality rate can be as high as 29.5–32% (1). Infection is an important factor leading to the death of patients with AP (2), and therefore, anti-infective therapy is crucial. Bacterial infection of pancreatic necrosis occurs in the early stage of the disease (3,4) but since pancreatic and extra-pancreatic infections often occur 2 weeks after the onset of pancreatitis, the targeted use of antibiotics in the early stage of the disease lacks pathogenic evidence and preventive drugs cannot reduce the mortality. Furthermore, the risk of multidrug-resistant bacteria and fungal infection is also increased (5). There are challenges to the use of antibiotics in the treatment of AP (6). AP is one of the most common acute abdominal diseases in clinical practice. In recent years, through the joint efforts of scholars at home and abroad, the mortality and complication rate of AP have decreased significantly, but the mortality rate of SAP is still high. Early clinical identification of SAP is essential to improve the prognosis of patients. Some scholars have proposed the model analysis of AP severity and bacterial culture, Clinical judgment of the severity of the disease will have more reference indicators. Evidence of infection obtained in the early stage is highly significant for the anti-infective therapy of AP. We found pathogens in the pancreatic juice of patients with early AP. The present study aims to provide a reference for the anti-infective therapy of AP by analyzing the distribution and clinical characteristics of pathogens in the pancreatic juice of patients with early AP. We present the following article in accordance with the STARD reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-22-6337/rc).

Methods

General information

Patients with acute biliary pancreatitis who underwent pancreatic stent implantation to prevent post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis and pancreatic juice bacterial culturing in the Department of Hepatobiliary surgery of the General Hospital of Ningxia Medical University from January 1, 2019, to June 30, 2020, were reviewed. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by the ethics committee of the General Hospital of Ningxia Medical University (No. 2019-467). All patients voluntarily participated in this study and signed informed consent and ERCP surgery consent.

Patients and inclusion criteria

All acute biliary pancreatitis and complications in our hospital were diagnosed according to the Atlanta pancreatitis classification (as revised in 2012). The presence of gallstones with or without choledocholithiasis was confirmed by imaging examination. All patients underwent ERCP lithotomy and preventive pancreatic stent placement, and pancreatic juice was obtained for bacterial culturing and drug sensitivity testing. The included patients were older than 18 years old. We included patients with a first-time AP infection and those with AP who could be followed-up for 6 months.

Exclusion criteria

Patients who were younger than 18 years old or had suspected specimen contamination were excluded. Furthermore, pancreatic juice culture specimens that were collected more than 1 week from the time of onset were excluded. Moreover, we also excluded patients with anesthesia or surgical contraindications, those with mental illnesses, patients with primary organ failure before onset, and those who could not cooperate with treatment, gave up treatment, or were transferred to another hospital.

Treatment plan

All patients were treated according to the Chinese guidelines for the diagnosis and treatment of AP: after admission, the patients were fasted, rehydrated, and given analgesia, enzyme inhibition, and other treatments. In addition to the above conservative treatment measures, patients with ERCP indications underwent ERCP examination and treatment within 72 hours after admission. Pancreatic stents were placed in high-risk groups to prevent post-ERCP pancreatitis, and pancreatic juice was collected for bacterial culturing.

Collection and culture of pancreatic juice

All endoscopic operations were completed by doctors with more than 10 years of surgical experience. The surgical procedure was as follows. Firstly, the endoscope reached the descending segment of the duodenum through the gastric cavity. Next, the surgeon looked for the great duodenal papilla on the inner side of the duodenum, rinsed the duodenum with normal saline, cut the knife before the sphincterotomy of the duodenal papilla, and then intubated the pancreatic duct through the guide wire. After successful intubation, an X-ray was used to confirm that the guide wire was placed along the direction of the pancreatic duct, and followed-up the incision knife to suck pancreatic juice. During the whole process, the lumen was filled with normal saline to avoid intestinal fluid pollution.

The interrupted pancreatic juice was extracted, placed into a sterile vacuum specimen collection tube, and then stored in an incubator. All specimens were sent to the laboratory for examination within 8 hours. The BacT/Alert3D blood culture and vitek-2compact bacterial identification and drug sensitivity instruments (Biomerie, France) were utilized for bacterial culturing of the pancreatic juice. The reagents applied were specifically for the instrument.

Monitoring indicators

The data were extracted by two researchers according to the pre-designed data extraction table. The extracted data contents mainly included the patient’s age, gender, etiology, APACHE II score, cultured bacteria, systemic complications, local complications, Balthazar computed tomography (CT) score, inflammatory factors, the use of antibiotics, the drug sensitivity analysis results, and the patient’s co-infection.

Statistical analysis

Statistical analyses were performed using the Statistical Package for the Social Sciences version 20.0 (SPSS Inc., Chicago, IL, USA). Non-normally distributed metric variables were analyzed by the Kruskal-Wallis test and Mann-Whitney U-test. Logistic regression analysis was used to draw the subject-working curve [receiver operating characteristic (ROC) curve] to analyze the value of related factors. Values were expressed as the mean ± standard deviation (SD) unless stated otherwise. The measurement data of normal distribution are expressed by mean ± SD with sample t-test, and the measurement data of non-normal distribution were expressed by median [interquartile range (IQR)]. P value ≤0.05 was considered statistically significant.

Results

Basic characteristics

A total of 186 patients with pancreatic juice bacterial cultures were retrieved, of which 156 patients met the inclusion criteria, including 83 males and 73 females. The average age of the included patients was 50±17 years old, and the median time from admission to onset was 48 h (24–96 h) (Table 1).

Results of pancreatic juice culture and bacterial distribution

All 156 pancreatic juice samples were sent for examination, of which 64 (41.03%) were cultured with pathogens. A total of 94 strains were isolated from 64 positive samples, including 55 (58.51%) strains of Gram-negative bacteria, 36 (38.30%) strains of Gram-positive bacteria, and three (3.19%) strains of fungus (Table 2).

The pathogen susceptibility analysis results showed that all of the Gram-negative bacteria had the highest sensitivity to amikacin, among which Escherichia coli and Citrobacter freundii also had relatively high sensitivity to imipenem, cefotetan, piperacillin/tazobactam, Klebsiella pneumoniae, Enterobacter cloacae, imipenem, and cefepime. Moreover, Acinetobacter baumannii and Morganella morganii were generally resistant, and Morganella morganii only had a high sensitivity rate to amikacin (Table 3).

The drug sensitivity results of Gram-positive bacteria demonstrated that Enterococcus faecium had the highest sensitivity to tigecycline, vancomycin, and quinupristin/dalfopristin. Enterococcus faecalis had the highest sensitivity to linezolid, tigecycline, and vancomycin. Except for erythromycin and vancomycin, Enterococcus aureus generally exhibited high drug sensitivity to other antibiotics. Streptococcus has the highest drug sensitivity to linezolid and vancomycin (Table 4).

The drug sensitivity results of the main fungi showed that Candida albicans and Candida tropicalis had high sensitivity to fluconazole, 5-fluorocytosine, itraconazole, and voriconazole (Table 5).

We then compared the clinical data between the two groups with positive and negative culture results. Patients with positive and negative culture results were divided into a positive culture group and a negative culture group accordingly, and the clinical data of the two groups were compared. The results indicated that there was no significant difference in age distribution, sex ratio, body mass index (BMI) value, and time from onset to admission between the two groups. However, the C-reactive protein (CRP), D-dimer, the ratio of neutrophils to lymphocytes, APACHE II score, and Balthazar CT score in the positive culture group were higher than those in the negative culture group (P<0.05). The incidence of complications and duration of fever were also markedly higher in the positive culture group (P<0.05) (Table 6).

Next, we compared the clinical data between patients with and without pancreatic infection in the later stage. The patients were divided into a pancreatic infection group and a non-pancreatic infection group according to whether a pancreatic infection occurred. The clinical data of the two groups were compared and the results showed that the age distribution, sex ratio, BMI, time from onset to admission, CRP, neutrophils/lymphocytes, and APACHE II score were not significantly different between the two groups. However, the Balthazar CT score, D-dimer, fever duration, hospital stay, and fasting time in the pancreatic infection group were higher than those in the non-pancreatic infection group (P<0.05). The incidence of systemic inflammatory response syndrome (SIRS), multiple organ failure (MOF), pancreatic necrosis, and positive rate of pancreatic juice culture were also considerably higher in patients with pancreatic infection (P<0.05) (Table 7).

The multivariate regression analysis of pancreatic infection and the ROC curve included positive pancreatic juice culture, diabetes, fasting time, hospitalization time, continuous fever time, SIRS, MOF, D-dimer, and Balthazar CT score ≥7. A multivariate logistic regression equation was constructed. The results demonstrated that the effect of positive pancreatic juice culture on pancreatic infection was statistically significant [odds ratio (OR) =8.406, 95% confidence interval (CI): 1.490–47.426]. Furthermore, a Balthazar CT score ≥7 at admission had a statistically significant effect on later pancreatic infection (OR =14.632, 95% CI: 2.240–95.579) (Table 8). The area under the ROC curve (AUC) of these two indicators was 0.856 (95% CI: 0.758–0.954), the specificity was 90.00%, the sensitivity was 65.30%, and the Youden index was 0.553 (Figure 1).

Figure 1 ROC curve of the regression model of pancreatic infection. ROC, receiver operation characteristic.

Discussion

For the vast majority of SAP patients, infection is still an unavoidable problem that seriously affects the prognosis of the disease. Among them, pancreatic necrosis infection is closely related to mortality in SAP (7). In necrotizing pancreatitis, if the necrotic pancreas is secondary to an infection, the risk of death will more than double (8). The occurrence of infection in SAP is not a small probability event. In relevant reports, the overall incidence of secondary infection in SAP is 73.0–82.1%, the incidence of pancreatic infection is 39–57%, and the incidence of extra-pancreatic infection is 43.0–79.5% (1,9,10). Some scholars believe that AP is mainly caused by damage to the pancreatic barrier, which has little to do with pancreatic bacterial infection (11). Therefore, it is particularly important to clarify the relevant factors causing pancreatic infection in SAP patients, promptly grasp the distribution of pathogens caused by infection, and administer corresponding antibiotic treatment according to the distribution of pathogens.

At present, there are relatively few studies on the pathogenic bacteria of pancreatic infection. Current research shows that pancreatic and extra-pancreatic infections often occur 2 weeks after onset. At this stage, antibiotic intervention can prevent the deterioration of the disease but it cannot prevent the occurrence of infection. Thus, determining methods of identifying pathogenic bacteria in the early stage of the disease is very important (12). The discharge of pancreatic juice and bile has a similar anatomical structure. Although bile is sterile under normal conditions, some studies have found pathogenic bacteria in the bile of patients with biliary tract infections (13). Based on this, our study boldly cultured the pancreatic juice of AP patients, seeking to clarify the distribution of pathogenic bacteria in pancreatitis and provide a reference for clinical treatment (14).

In our study, the positive rate of pancreatic juice bacterial cultures was 41.03%, and 94 strains were cultured, including 55 (58.51%) Gram-negative bacteria, 36 (38.30%) Gram-positive bacteria, and three fungi (3.19%) strains. A study has shown that regardless of whether the infection is pancreatic or extra-pancreatic, Gram-negative bacteria are the main pathogenic bacteria, and Escherichia coli is the most common (15). Also, the pathogenic bacteria found in pancreatic juice may be related to secondary infection in patients with late pancreatitis, especially pancreatic infection.

However, the proportion of fungi in our study is compared with previous studies, which is much smaller. After consulting the relevant literature for analysis, we believe that this difference is primarily due to our early pancreatic juice bacterial culture, which avoids the bacterial spectrum changes caused by the long-term preventive use of antibiotics, and the trend of this change is the transformation from Gram-negative to Gram-positive bacteria and fungus (16-19). Therefore, the proportion of fungus in pancreatic juice in the early stage of pancreatitis is low. If antibiotics are used blindly, the probability of fungal infection increases, which elevates the risk of death (5). The blind prophylactic use of antibiotics may not reduce the risk of infection and death (20); only by selecting sensitive and effective antibiotics according to the results of pathogen drug sensitivity analysis can the production of drug-resistant bacteria be reduced. In this study, pathogenic bacteria were found in pancreatic juice in the early stage of AP. Although a positive bacterial culture does not necessarily lead to infection, the results can still provide a theoretical basis for empirical antibiotic selection (21).

The pathogens of SAP secondary infection exhibit the characteristics of multi-drug resistance (22), and the selection of antibiotics is more difficult. The drug sensitivity test results in this study indicated that common gram-negative bacteria had a high sensitivity to amikacin and imipenem, but Acinetobacter baumannii and Morganella morganii were generally resistant, among which Morganella morganii only had high sensitivity to amikacin. Common gram-positive bacteria showed a high drug sensitivity to vancomycin, linezolid, and tigecycline, which can provide a reference basis for the selection of clinical antibiotics (23,24). Therefore, amikacin and imipenem can be selected for gram-negative bacterial infection in SAP, and vancomycin and linezolid can be selected for suspected Gram-positive bacterial infection (25-27).

By comparing the clinical data of the two groups of patients with positive and negative cultures, we found that there were no significant differences in age distribution, sex ratio, BMI, and the time from onset to admission between the two groups. However, the indicators reflecting the severity of the disease (APACHE II score, Balthazar CT score, complication rate, etc.) were higher in positive patients than in negative patients (P<0.05). Some indicators that can reflect the level of inflammation and predict later pancreatic infection (e.g., CRP, D-dimer, neutrophils/lymphocytes), were also significantly higher in patients with positive cultures compared to those in negative patients (P<0.05). Therefore, the condition of positive patients is more serious, and the risk of pancreatic infection in the later stage is also higher.

The pathogenic bacteria in the early pancreatic juice were related to the condition and later complications of patients. On the other hand, it also showed that we cultivated pathogenic bacteria rather than polluting bacteria. There was no significant difference in leukocyte values between the two groups at admission but CRP, PCT, and neutrophils/leukomonocyte were significantly different in leukocytes, and thus, leukocytes were less sensitive than CRP and the other inflammatory factors in the early evaluation of AP and the prediction of infection. Therefore, we should pay more attention to these inflammatory indicators in anti-infective therapy and pancreatic juice was sterile under normal conditions (28).

We conducted a multivariate regression analysis of pancreatic infection to explore the influence of pathogenic bacteria in pancreatic juice on pancreatic infection in patients at a later stage. The results showed that positive pancreatic juice culture and Balthazar CT score ≥7 were independent risk factors for pancreatic infection. The ROC curve combining the two risk factors showed that a positive pancreatic juice culture and a Balthazar CT score ≥7 had a certain predictive value for pancreatic infection. Imaging plays an important role in the evaluation of the AP condition. CT examination can not only accurately display the anatomical morphology of SAP lesions and evaluate the degree of pancreatic necrosis, as a non-invasive examination method, it can also dynamically monitor the changes in patients’ conditions and provide guidance for clinical treatment and prognostic evaluation. The Balthazar CT score is an evaluation method established according to the degree of involvement of the peripancreatic and retroperitoneal space. Some scholars believe that this method should be the first choice to evaluate the severity of AP patients in the early stage; however, the Balthazar CT score results are affected by the subjective factors of the operator. Therefore, under the premise of allowable conditions, routine pancreatic juice bacterial culture should be carried out. Then, the occurrence of pancreatic infection in the later stage of AP can be predicted according to the culture results combined with the Balthazar CT score, which has a positive significance for the anti-infection treatment of AP.

In conclusion, we found pathogens in the pancreatic juice of 41.03% of patients with AP, among which Gram-negative bacteria were the most prevalent, followed by Gram-positive bacteria and fungi. The presence of pathogens in pancreatic juice is closely related to the patient’s condition and prognosis, and the presence of pathogens in pancreatic juice is an independent risk factor for pancreatic infection in the later stage of the disease. If antibiotics can be used in the early stage of pancreatitis according to the results of pancreatic juice culture and drug sensitivity testing, this may provide better outcomes for patients with AP (29).

Conclusions

Bacterial culture of pancreatic juice can obtain evidence of infection in the early stage of AP, which has certain reference significance for anti-infection treatment of AP. Our study first reported the distribution and clinical characteristics of pancreatic juice bacteria in AP. Pancreatic juice bacterial cultures can provide evidence of infection in the early stage of AP, which provides a reference for the anti-infective therapy of AP.

Acknowledgments

Funding: This research was supported by the Key Projects of Ningxia Autonomous Region’s Key R&D Program (Nos. 2020BEG02002 and 2021BEG03042), and the Ningxia Autonomous Region Science and Technology for the People Project (No. 2021CMG03013).

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-22-6337/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-22-6337/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-6337/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by the ethics committee of the General Hospital of Ningxia Medical University (No. 2019-467). All patients voluntarily participated in this study and signed informed consent and ERCP surgery consent.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Pintado MC, Trascasa M, Arenillas C, et al. New Atlanta Classification of acute pancreatitis in intensive care unit: Complications and prognosis. Eur J Intern Med 2016;30:82-7. [Crossref] [PubMed]
2. Tenner S, Baillie J, DeWitt J, et al. American College of Gastroenterology guideline: management of acute pancreatitis. Am J Gastroenterol 2013;108:1400-15; 1416.
3. Beger HG, Bittner R, Block S, et al. Bacterial contamination of pancreatic necrosis. A prospective clinical study. Gastroenterology 1986;91:433-8. [Crossref] [PubMed]
4. Baron TH, DiMaio CJ, Wang AY, et al. American Gastroenterological Association Clinical Practice Update: Management of Pancreatic Necrosis. Gastroenterology 2020;158:67-75.e1. [Crossref] [PubMed]
5. Bai Y, Gao J, Zou DW, et al. Prophylactic antibiotics cannot reduce infected pancreatic necrosis and mortality in acute necrotizing pancreatitis: evidence from a meta-analysis of randomized controlled trials. Am J Gastroenterol 2008;103:104-10. [Crossref] [PubMed]
6. Besselink MG, van Santvoort HC, Witteman BJ, et al. Management of severe acute pancreatitis: it's all about timing. Curr Opin Crit Care 2007;13:200-6. [Crossref] [PubMed]
7. Petrov MS, Shanbhag S, Chakraborty M, et al. Organ failure and infection of pancreatic necrosis as determinants of mortality in patients with acute pancreatitis. Gastroenterology 2010;139:813-20. [Crossref] [PubMed]
8. Werge M, Novovic S, Schmidt PN, et al. Infection increases mortality in necrotizing pancreatitis: A systematic review and meta-analysis. Pancreatology 2016;16:698-707. [Crossref] [PubMed]
9. De Waele JJ, Rello J, Anzueto A, et al. Infections and use of antibiotics in patients admitted for severe acute pancreatitis: data from the EPIC II study. Surg Infect (Larchmt) 2014;15:394-8. [Crossref] [PubMed]
10. Cacopardo B, Pinzone M, Berretta S, et al. Localized and systemic bacterial infections in necrotizing pancreatitis submitted to surgical necrosectomy or percutaneous drainage of necrotic secretions. BMC Surg 2013;13:S50. [Crossref] [PubMed]
11. Koutroumpakis E, Slivka A, Furlan A, et al. Management and outcomes of acute pancreatitis patients over the last decade: A US tertiary-center experience. Pancreatology 2017;17:32-40. [Crossref] [PubMed]
12. Diaconu CC, Gheorghe G, Hortopan A, et al. Pancreatic Tuberculosis-A Condition That Mimics Pancreatic Cancer. Medicina (Kaunas) 2022;58:1165. [Crossref] [PubMed]
13. Gu XX, Zhang MP, Zhao YF, et al. Clinical and microbiological characteristics of patients with biliary disease. World J Gastroenterol 2020;26:1638-46. [Crossref] [PubMed]
14. Balthazar EJ. Acute pancreatitis: assessment of severity with clinical and CT evaluation. Radiology 2002;223:603-13. [Crossref] [PubMed]
15. Sugiyama M, Atomi Y. Does endoscopic sphincterotomy cause prolonged pancreatobiliary reflux? Am J Gastroenterol 1999;94:795-8. [Crossref] [PubMed]
16. Luiten EJ, Hop WC, Lange JF, et al. Controlled clinical trial of selective decontamination for the treatment of severe acute pancreatitis. Ann Surg 1995;222:57-65. [Crossref] [PubMed]
17. Howard TJ, Temple MB. Prophylactic antibiotics alter the bacteriology of infected necrosis in severe acute pancreatitis. J Am Coll Surg 2002;195:759-67. [Crossref] [PubMed]
18. Isenmann R, Schwarz M, Rau B, et al. Characteristics of infection with Candida species in patients with necrotizing pancreatitis. World J Surg 2002;26:372-6. [Crossref] [PubMed]
19. Hoerauf A, Hammer S, Müller-Myhsok B, et al. Intra-abdominal Candida infection during acute necrotizing pancreatitis has a high prevalence and is associated with increased mortality. Crit Care Med 1998;26:2010-5. [Crossref] [PubMed]
20. Lee HS, Lee SK, Park DH, et al. Emergence of multidrug resistant infection in patients with severe acute pancreatitis. Pancreatology 2014;14:450-3. [Crossref] [PubMed]
21. Fishman JE, Levy G, Alli V, et al. The intestinal mucus layer is a critical component of the gut barrier that is damaged during acute pancreatitis. Shock 2014;42:264-70. [Crossref] [PubMed]
22. Papachristou GI, Muddana V, Yadav D, et al. Comparison of BISAP, Ranson's, APACHE-II, and CTSI scores in predicting organ failure, complications, and mortality in acute pancreatitis. Am J Gastroenterol 2010;105:435-41; quiz 442. [Crossref] [PubMed]
23. Ke L, Ni HB, Tong ZH, et al. D-dimer as a marker of severity in patients with severe acute pancreatitis. J Hepatobiliary Pancreat Sci 2012;19:259-65. [Crossref] [PubMed]
24. Oppenlander KE, Chadwick C, Carman K. Acute Pancreatitis: Rapid Evidence Review. Am Fam Physician 2022;106:44-50. [PubMed]
25. Schütte K, Malfertheiner P. Markers for predicting severity and progression of acute pancreatitis. Best Pract Res Clin Gastroenterol 2008;22:75-90. [Crossref] [PubMed]
26. Liu H, Li W, Wang X, et al. Early gut mucosal dysfunction in patients with acute pancreatitis. Pancreas 2008;36:192-6. [Crossref] [PubMed]
27. Szatmary P, Grammatikopoulos T, Cai W, et al. Acute Pancreatitis: Diagnosis and Treatment. Drugs 2022;82:1251-76. [Crossref] [PubMed]
28. Flint RS, Windsor JA. The role of the intestine in the pathophysiology and management of severe acute pancreatitis. HPB (Oxford) 2003;5:69-85. [Crossref] [PubMed]
29. Wang PF, Zou H, Zhu JH, et al. Acute pancreatitis caused by tigecycline: A case report and literature review. Medicine (Baltimore) 2021;100:e28245. [Crossref] [PubMed]

(English Language Editor: A. Kassem)