Acute pancreatitis is one of the most serious gastrointestinal disorder necessitating hospital admissions . The clinical course of the disease is highly variable ranging from mild cases to multi-organ failure, and death. The mortality rate can rise up to 30% when necrosis develops within the pancreatic or the surrounding tissues . Furthermore, infection of the necrotic tissue is an even more feared complication that further complicates the clinical course leading to sepsis, multi-organ failure and death in many. Endoscopic and minimally invasive surgical techniques to remove the necrotic debris combined with the appropriate antibiotherapy and supportive measures are the pillars of management .
However, when it comes to the microbial spectrum of the infected pancreatic necrosis, the data are scarce and controversial. Generally, gram-negative bacteria are held responsible for infections which are most likely due to the disruption of the normal gut barrier and ascension through the gastrointestinal tract . However, this premise has not been thoroughly investigated in large-scale studies. Moreover, some studies reported a shift from a gram-negative to gram-positive microbial flora possibly due to the overuse of antibiotics [5,6]. It is also likely that hospital-specific microbial flora may affect the microbial spectrum which tends to be polymicrobial in 50% of the cases. Therefore, in this study, we assessed the microbial spectrum in patients with necrotizing acute pancreatitis (NAP) and signs and symptoms of infection. In addition, we also evaluated the impact of microbial findings on in-hospital and 1-year all-cause mortality.
Materials and methods
The data of patients admitted to our gastroenterology department from January 2013 to May 2018 due to acute pancreatitis were retrospectively analyzed and those with necrotizing pancreatitis were included in the study. Patients with chronic pancreatitis, incomplete data and those who were lost to 1-year follow-up were excluded from the study. Local ethical board approval was provided.
The definition of necrotizing pancreatitis was based on the revised Atlanta criteria . According to the culture results, the study patients were divided into two groups: (1) negative culture NAP which included patients with sterile cultures and without any sign of infection; (2) positive culture NAP. This latter group consisted of patients with signs and symptoms of infection (such as fever, elevated serum procalcitonin levels, gas within the pancreas) with either only positive blood cultures (suspected pancreatic necrosis infection) or with proven pancreatic infected necrosis based on pancreatic and peri-pancreatic material cultures. The degree of pancreatic necrosis and the computed tomography severity index scores were evaluated by a radiologist who had experience with pancreatic disorders and was blinded to the study results. Microbiological analysis regarding all culture results was reviewed by an infectious disease specialist and categorized accordingly.
Identification of agents isolated from the cultures and the impact of underlying microorganisms on in-hospital and 1-year all-cause mortality as compared to patients with sterile cultures were the main outcomes of this study.
The data for continuous variables were reported as median and interquartile ranges (IQRs). Categorical variables were reported as numbers and percentages. Continuous variables were compared between groups using independent sample T-test or Mann–Whitney as appropriate. Categorical data were compared using the chi-square or Fisher exact test. Survival curves for 1-year all-cause mortality were generated for patients with culture-negative and culture-positive (monomicrobial and polymicrobial) pancreatic necrosis using the Kaplan–Meier method. Differences among the groups were assessed using the log-rank test. ‘Number at risk’ tables were generated using the MedCalc statistical software (free trial version). A two-tailed P-value <0.05 was considered statistically significant. SPSS 22 software was used for statistical analysis (SPSS Inc., Chicago, Illinois, USA).
Overall 109 patients were included in the final analysis (median age: 53, IQR: 44–61, males: 70, 64.2%). Blood culture analysis was performed in all patients. In addition to that, in 49 patients pancreatic/peri-pancreatic cultures were evaluated which were obtained through aspiration of the pancreatic material. In detail, these cultures were obtained from the endoscopic necrosectomy materials in 14 (12.8%), from endoscopic drainage in 31 (28.4%) and from direct percutaneous drainage in four (3.7%) patients. Of all patients, 76 (69.7%) had sterile pancreatic necrosis. The remaining 33 patients (30.3%) had either positive blood or aspirate cultures of whom 22 had polymicrobial, 11 monomicrobial results. Baseline clinical characteristics of the study patients are displayed in Table 1. The most common etiologies were biliary and idiopathic (n = 48, 44.0% and n = 30, 27.5%, respectively). Treatment modalities included, ‘only medical treatment’ (n = 60, 55.0%), endoscopic drainage (n = 31, 28.4%), endoscopic necrosectomy (n = 14, 12.8%) and percutaneous drainage (n = 4, 3.7%). The patients with an identified microorganism had higher computed tomography severity index scores. Length of hospital stay and ICU admission rates were also higher in these patients (P < 0.01). The majority of the patients with sterile NAP were managed by medical treatment alone (n = 54, 71.1% vs. n = 6, 18.2%, P < 0.01). In terms of laboratory values, white blood cell count and 48-hour C-reactive protein values were significantly higher in-culture positive patients, however, there was no statistical difference in serum amylase, lipase, and hematocrit values.
Agents identified in cultures are presented in Table 2. In total, 20 different microorganisms were isolated from 74 positive cultures in 33 patients. Only six patients’ culture results were just based on blood samples alone. Although two patients had both positive blood and aspirate cultures, the rest 25 had positive cultures that are specifically obtained from the aspirates of the pancreatic collection either through percutaneous or endoscopic drainage. There was a high frequency of polymicrobial etiology with an overall dominance of gram-negative bacteria in particular Klebsiella spp. (18 cultures, 24%). Carbapenem and multi-drug resistant Klebsiella spp. were identified in five and two cultures, respectively. Candida spp. were the only fungi identified with a 10% (n = 8) frequency and all of them were polymicrobial. Among gram-positive bacteria, Staphylococcus spp. were the most common two of which were methicillin-resistant Staphylococcus aureus. Culture results of patients who died during the hospital admission are presented in Table 3. Again marked gram-negative dominance is seen along with fungi in three cases. There was a significant association between endoscopic interventions and the possibility of polymicrobial culture results (P < 0.001, odds ratio = 20, 95% confidence interval 4.3–91.4).
The most commonly used antimicrobial agents included carbapenems (n = 49, 45.0%), antipseudomonal penicillins (n = 8, 7.3%), third generation cephalosporins (n = 14, 12.8%), fluoroquinolones (n = 11, 10.1%) and antifungals (n = 10, 9.2%), respectively.
In-hospital, 30-day, and 1-year mortality rates were also higher in patients with positive culture NAP compared to patients with sterile culture results (n = 9, 27.3% vs. n = 4, 5.3%, P < 0.01 for in-hospital mortality; n = 11, 33.3% vs. n = 5, 6.6%, P < 0.01 for 30-day mortality; and n = 14, 42.4% vs. n = 10, 13.2%, P < 0.01 for 1-year mortality). Kaplan–Meier survival curves generated according to culture results for 1-year all-cause mortality are presented in Figs. 1 and 2, respectively. Figure 1 compares patients with proven infection (based on positive cultures directly obtained from the pancreatic materials) to patients with negative cultures (P [log-rank] <0.001). Figure 2 displays a more in-depth survival analysis according to culture results in which patients who had polymicrobial positive cultures had worse prognosis compared to the patients with monomicrobial and negative culture results (P [log-rank] <0.001).
When 33 patients with positive cultures were further stratified according to the development of persistent organ failure, those who had polymicrobial cultures had more organ failure compared to patients with monomicrobial culture results although it did not reach statistical significance (77.3% vs. 54.5%, P = 0.18).
In this study, it appeared that pancreatic necrosis with polymicrobial bacteremia has the worst prognosis with a 1-year all-cause mortality rate reaching to 42.4%.
The culture results clearly showed a gram-negative bacterial dominance, Klebsiella spp. being the most common. The high prevalence of gram-negative bacteria has been observed in numerous previous studies and thought to be secondary to the disruption of the normal gut barrier and permeation of bacteria through the gastrointestinal tract [8,9]. Other proposed mechanisms involved hematogenic or lymphatic spread from the adjacent tissues . However, recently investigators also proposed enterococcus as the most prevalent organism in some studies and therefore pointed a shift from gram-negative to gram-positive etiology . Up to now, the most common bacteria isolated in these studies were Streptococcus, Enterococcus and Staphylococci spp. This shift in infecting organisms was attributed to the extensive use of prophylactic antibiotics for the treatment of acute pancreatitis (AP) . Likewise, the study led by Sahar et al.  found a predominance of gram-positive bacteria in initial cultures drawn from walled-of-necrosis, especially if a single microorganism was isolated.
Contrary to the previous studies that pointed a shift towards gram-positive bacterial dominance, in our study there was a clear Klebsiella (gram-negative) dominance which may be due to a number of reasons. First, the baseline clinical characteristics of the study patients were different from the studies conducted in western countries where alcoholic AP was much more common. In our study, the most common etiology was biliary followed by idiopathic, whereas alcoholic pancreatitis constituted only 11% of the patients. Second, in our department the most common nosocomial infectious agent is Klebsiella. Third, 44 (40%) patients received antibiotics even if they had sterile culture results. The most likely reason for culture negativity may be related to the early empirical initiation of wide-spectrum antibiotics in cases where an infection is suspected. Although current guidelines recommend against the prophylactic use of antibiotics, real-world practice is controversial. There are studies showing that antibiotics are still administered empirically if there is enough suspicion for its justification. For instance, in the study led by Sahar et al. , empirical usage of antimicrobial agents reached 21% despite sterile cultures emphasizing the difficulty in the precise selection of infected patients. Moreover, there is also a discrepancy among guidelines. According to the Japanese guidelines, prophylactic administration of antibiotics in severe acute necrotizing pancreatitis may improve the prognosis, if initiated early, that directly opposes other international guidelines [13,14]. Besides that, there is also evidence showing that in the context of necrotizing pancreatitis, early initiation of antibiotics based on the clinical findings may decrease pancreatic infection and length of hospital stay . In a Cochrane review, it was demonstrated that prophylactic imipenem administration in cases of pancreatic necrosis decreased infection rates . Taken together these factors might have all influenced the culture results.
We did not identify any anaerobe microorganisms in cultures like most other previous studies. Beger et al.  found anaerobes as the only microbial finding in only 5% of patients with infected pancreatic necrosis. Although the precise mechanism is not clear, one proposed hypothesis why anaerobes rarely invade the infected pancreatic tissue is based on the fact that the translocation of bacteria occurs mainly from the small bowel in which anaerobes are rarely found .
One other important association was observed between invasive interventions and culture results. The risk of polymicrobial pancreatic necrosis increased 20 times in those undergoing percutaneous and endoscopic procedures. It is possible that the identification of microbes from the aspiration or necrosectomy materials might have been easier compared to peripheral blood cultures. In terms of fungal involvement, Candida spp. were the only identified fungi in cultures which were polymicrobial representing a more severe disease course and prolonged broad-spectrum antibiotic usage as pointed out as major risk factors in earlier studies . The rate of candida-positive culture results was approximately 10%. The majority of the previous studies reported Candida spp. as the only fungi with a frequency ranging from 20 to 60% [19,20]. This wide range of frequency has been attributed to the definition and culture techniques. One important consideration here may be related to the site where Candida is isolated. Culture positivity confined to a particular location may overestimate disseminated infection as it may represent colonization only .
To our knowledge, the association of culture results in pancreatic necrosis with 1-year all-cause mortality has not been adequately addressed in previous studies . Kaplan–Meier analysis showed that patients with identified microorganisms, particularly due to polymicrobial etiology, had the worst prognosis compared to those with sterile and monomicrobial culture-positive results. As can be appreciated from the survival curves, those with polymicrobial culture results had particularly worse outcomes both in the hospital and within the first months after discharge. This could be related to the higher persistent organ failure rate seen in these patients.
This was a single-center, retrospective and observational study. Also, the empirical antibiotic usage rate was high. Nevertheless, this represents a real-world practice. We did not specifically address antibiotic resistance patterns as the data was inadequate for complete analysis. We also did not report previous exposure to antibiotics which might have affected our culture results. Although only six out of 33 patients had positive cultures which are based on blood samples alone, we cannot prove infected pancreatic necrosis in these patients.
In conclusion, the patients who had necrotizing pancreatitis with polymicrobial culture results had the worst outcome both in terms of in-hospital and 1-year mortality. The identified bacteria were markedly gram negative, mostly Klebsiella species. When administering antibiotics to patients with suspected infected pancreatic necrosis, a polymicrobial etiology should be suspected and local hospital flora should be taken into consideration. Although the administration of antibiotics in pancreatic necrosis with suspected infection without positive cultures is still a controversial issue, any clinical or radiological evidence of infection should prompt the early administration of antibiotics due to the very poor prognosis of such patients.
We want to thank the statistics department for their contribution.
Conflicts of interest
There are no conflicts of interest.
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