Biliary Microflora in Patients Undergoing Cholecystectomy

Abstract

Background:

The management of acute cholecystitis requires a sound knowledge of the biliary microflora.

Methods:

Bile samples were taken for culture according to a standard routine during all cholecystectomies performed from April 2007 to February 2009 in the Department of Surgery at Enköping Hospital. The use of antibiotics within the 3-mo period before surgery, indication for surgery, prophylactic antibiotics, and post-operative complications were recorded prospectively.

Results:

Altogether, 246 procedures were performed during the study period, of which 149 (62%) were done on women. The mean (±SD) age of the study subjects was 49±16 y. Bacterial growth was seen in cultures from 34 (14%) of the subjects. The mean age of subjects with positive cultures was 64 y and that of subjects with negative cultures was 47 y (p<0.001). Positive culture was seen in 16 (31%) of the 51 patients who underwent operations for acute cholecystitis, whereas positive cultures were obtained in 18 of 195 patients without acute cholecystitis (9%) (p<0.001). Resistance to ampicillin was recorded in three of 34 (9%) of the cultures with bacterial growth, to co-trimoxazole in one of the 34 (3%) cultures, to fluoroquinolones in one of the 34 (3%) cultures, and to cephalosporins in one of the 34 (3%) cultures. Resistance to piperacillin–tazobactam was not observed in any of the cultures. In multivariable logistic regression analysis, a positive culture was the only factor significantly associated with risk for post-operative infectious complications (p<0.05).

Discussion:

Bacterial growth in the bile is observed more often in patients undergoing surgery for acute cholecystitis. The microflora of the bile is probably important for the outcome of surgery, but further studies are required for assessing the effectiveness of measures for preventing infectious post-operative complications.

The timing and extent of bacterial colonization as part of the pathogenesis of acute cholecystitis is not understood completely. Bile duct obstruction is probably crucial for the development of cholecystitis [1], but how bacterial contamination affects its course is not known fully [2]. Bacterial colonization has been suggested to play both a primary and a secondary role [1]. To some extent, bacteria in the bile may increase the risk of an adverse post-operative outcome after laparoscopic cholecystectomy [3]. As in most visceral inflammatory processes, acute cholecystitis begins as sterile inflammation. The way in which mechanical, biochemical, and bacteriologic factors interact in this process has been described only partly [4], and it is not known when, if at all, this process becomes infected.

The role of antibiotics as prophylaxis during cholecystectomy, and as treatment in acute cholecystitis, whether or not surgery is performed subsequently, depends ultimately on the extent of bacterial growth in the bile. An association between the organisms in the bile and the risk and clinical manifestation of a post-operative infection may be assumed, but has not been shown unequivocally. The choice of prophylactic antibiotic, if any, for cholecystectomy is therefore a controversial issue [5,6].

To survey the bacterial flora in patients with uncomplicated gallstone disease and acute cholecystitis, we took bile samples for bacterial culture from all patients undergoing cholecystectomy at a single institution during a 2-y period.

Patients and Methods

Study population

All patients undergoing cholecystectomy between April 2007 and February 2009 in the Department of Surgery at Enköping Hospital in Enköping, Sweden, were included in the study. A standard protocol was used for collecting patient data. Included variables were indication for surgery, surgical approach, use of prophylactic antibiotics, and use of antibiotics within 3 mo before the procedure. All post-operative infectious complications were extracted through a review of the patients' records performed in 2009.

Bacteriology

During the study period, at least 10 mL of bile was aspirated under sterile conditions from the fundus of the gallbladder with a long needle before dissection of the peritoneum was begun. The bile was sent to the Department of Clinical Microbiology at Uppsala University Hospital in Uppsala, Sweden for culture. Upon arrival, the bile was plated onto chocolate agar, aerobic and anaerobic blood agar (Acumedia Manufacturers, Lansing, MI), and cysteine–lactose–electrolyte-deficient agar (Becton, Dickinson; Sparks, MD). The plates were incubated for 48–96 h in appropriate atmospheres (ambient air, 5% CO₂, or anaerobically) at 35°–37°C. Bacteria were identified with standard laboratory procedures or a Vitek 2 instrument (bioMérieux, Lyon, France). Aerobic growth was recorded after 18–24 h and 48 h, and anaerobic growth after 48 h and 96 h. Susceptibility testing was done according to the recommendations of the Swedish Reference Group for Antibiotics (SRGA) (www.srga.org). Species-related breakpoints defined by the SRGA were used to categorize isolates as susceptible, indeterminate, or resistant to a particular antibiotic.

Statistics

The association between the outcome of bacterial culture and age was tested with the Student t-test, and the association between culture results and dichotomous variables (presence of acute cholecystitis and gender) was tested with the χ² test. The risk of post-operative infection was tested through multi-variable logistic regression analysis, with bacterial growth in culture, gender, age, indication for surgery, and administration of antibiotics as co-variates. The multivariable model was constructed through stepwise selection, with entry testing of a variable based on the significance of the score statistic, and removal testing based on the probability of a likelihood-ratio statistic based on the maximum partial likelihood estimates. SPSS version 20 (IBM, Armonk, NY) was used for all statistical analyses.

Results

Altogether 246 cholecystectomies, of which 149 (62%) were done on women and 93 (38%) on men, were performed during the study period. The patients' mean (±SD) age was 49±16 y. Bacterial growth was seen in 34 (14%) of the patients' cultures. The findings on culture are shown in Table 1. The mean age of patients with positive cultures was 64 y and that of patients with negative cultures was 47 y (p<0.001). This age difference remained significant even when patients who had operations for acute cholecystitis were excluded (p<0.003). There was no significant difference between men and women in the frequency of positive culture. In 51 (21%) cases the indication for surgery was acute cholecystitis. Antibiotics were given to 14 (6%) of the patients. Culture was positive for 16 (31%) of the patients undergoing surgery for acute cholecystitis and for 18 (9%) of those undergoing cholecystectomy without macroscopic signs of acute cholecystitis (p<0.001). Post-operative infectious complications were seen in 14 (6%) of the patients, including seven (14%) of those with acute cholecystitis. By multivariable logistic regression analysis (Table 2 and 3) only a positive culture was significantly associated with the risk of post-operative infectious complications (p<0.05). Age, gender, ongoing or previous acute cholecystitis, or prophylactic administration of antibiotics had no significant effect on the risk of post-operative infection.

Table 1.

Bacterial Growth in Cultured Specimens of Bile

	Uncomplicated cholecystectomy (n=195)	Cholecystectomy for acute cholecystitis (n=51)	All procedures
Streptococcus spp.	5	5	10
Escherichia coli	5	3	8
Klebsiella spp.	1	2	3
Coagulase-negative Staphylococcus spp.	2	1	3
Clostridium perfringens		3	3
Enterococcus	1	1	2
Mixed flora	1	1	2
Enterobacter	1		1
Haemophilus	1		1
Propionibacterium	1		1

Table 2.

Multivariate Logistic Regression Analysis of Factors Predicting Post-Operative Infectious Complications in Patients Undergoing Cholescystectomy ^a

	Univariate models odds ratio (95% confidence interval)	AUC (c-statistic)	p value	Multivariable model odds ratio (95% confidence interval)	AUC (c-statistic)	p value
Gender
Women (vs. men)	1.13 (0.37–3.49)	0.514	0.83	1.13 (0.37–3.49)		0.51^*
Age
Greater than the median (vs. less than the median)	2.75 (0.84–9.03)	0.620	0.095	2.75 (0.84–9.03)		0.36^*
Antibiotic prophylaxis
Prophylaxis (vs. no prophylaxis)	1.30 (0.16–10.68)	0.509	0.81	1.30 (0.16–10.68)		0.94^*
Indication for surgery
Cholecystitis (vs. other indication)	4.27 (1.42–12.81)	0.654	0.010	4.27 (1.42–12.81)		0.070^*
Culture outcome
Bacterial growth (vs. no growth)	5.46 (1.76–16.91)	0.655	0.003	5.58 (1.80–17.32)	0.655	0.003^†
Constant				0.04		<0.001^†

The multivariable model was constructed through stepwise selection, with entry testing based on the significance of the score statistic, and removal testing based on the probability of a likelihood-ratio statistic obtained from maximum partial likelihood estimates. The correlation coefficient for the culture outcome and the constant in the multivariate model was −0.62.

Variable rejected in from the multivariate model

†

Variable selected in the multivariate model.

Table 3.

Contingency Table for Hosmer–Lemeshow Test of the Multivariable Model in Table 2

	No postoperative infection		Postoperative infection
Iteration	Observed	Expected	Observed	Expected	Total
1	201	201.00	8	8.00	209
2	27	27.00	6	6.00	33

Resistance to ampicillin was recorded in three of 34 (9%) of the positive cultures in the study, to co-trimoxazole in one of 34 (3%), to quinolones in one of 34 (3%), and to cephalosporins in one of 34 (3%). Resistance to piperacillin–tazobactam was not observed in any of the cultures.

Discussion

A positive bile culture was obtained from 31% of patients undergoing surgery for acute cholecystitis and in 9% of patients with no signs of acute cholecystitis. The only factor that, along with the presence of acute cholecystitis, was associated with an increased prevalence of positive cultures was high age; treatment with antibiotics was not so associated. Positive culture was the only factor that had an effect on the risk of post-operative infection.

The crucial etiologic factor in acute cholecystitis, in the vast majority of cases, is the impaction of gallstones in the cystic duct [1]. Bile becomes congested in the gallbladder, leading to a cascade of inflammation, ischemic changes, and finally necrosis. At some stage during this process, bacteria proliferate within the gallbladder, although it is difficult to determine whether this is a secondary event during the development of acute cholecystitis or an early causal factor [7]. In the present study we could not show any association between the duration of acute cholecystitis and the presence of a positive culture. This indicates that bacterial colonization may occur at any stage of the disease and does not seem to follow a predictable course.

As in previous studies [4,7 –9], Escherichia coli, Enterococcus, and Klebsiella predominated among the pathogens in positive cultures. These are all bacteria present typically in the intestinal flora, and probably colonize the gallbladder by ascension from the duodenum. In a few cases bacteria not usually found in the enteric flora were isolated, including Clostridium perfringens and Staphylococcus species. However, even these bacteria probably have an enteric origin. Their hematogenous spread to the gallbladder seems less likely, although Staphylococcus aureus has occasionally been recovered from the gallbladder in patients with endocarditis [10]. Anaerobic organisms were found only in mixed growth together with aerobes.

Drug resistance to the antibiotics used most commonly for pre-operative prophylaxis and treatment in acute cholecystitis (ampicillin, co-trimoxazole, quinolones, cephalosporins, and piperacillin-tazobactam) was not observed. These drugs have been recommended by the Surgical Infection Society (SIS) [11,12] and the Infectious Diseases Society of America (IDSA)[13] as treatment agents for intraabdominal infections. However, the efficacy of these antibiotics is affected not only by drug resistance but also by the extent of their penetration into the infected tissue and colonized bile. Whereas in our study a positive culture was associated with an increased risk for subsequent infectious complications, neither prophylactic antibiotics nor any other factor predicted the patients' post-operative course. Although we cannot rule out the possibility of an association having been found had the patient sample been larger than it was, the absence of any relationship reflects the multi-factorial origin of infectious complications. Other risk factors that may be of equal importance in the development of a local post-operative infection, such as impaired immune defense mechanisms, post-operative bleeding, contamination of the abdominal cavity, and subcutaneous tissue, and operating time may have blurred the association between the prophylactic use of antibiotics and the risk of post-operative infection.

Previous studies have not shown any benefit of antibiotic prophylaxis in planned laparoscopic cholecystectomy [6,7,14]. To our knowledge, only one randomized controlled study has assessed the benefit of antibiotic prophylaxis in patients undergoing surgery for mild acute cholecystitis [15]. In that study intravenous antibiotic treatment did not improve the hospital course or early outcome in most of the patients. In the present study we could not find any association between the risk of post-operative infectious complications and the presence of acute cholecystitis or administration of antibiotic prophylaxis. The lack of effect of antibiotic prophylaxis may be explained by poor diffusion of the drugs used into the contaminated bile. However, the study was not designed primarily to test the effectiveness of antibiotic prophylaxis. Reasons exist to believe that the predominant risk factor for infectious complications of cholecystectomy is surgical complications, and particularly post-operative bile leakage. In this context, antibiotic prophylaxis has only a minor effect that is difficult to detect in the presence of factors with much greater effects. Nevertheless, a good knowledge of the biliary microflora may aid in making treatment decisions in case infectious complications do occur.

In many cases the presence of E. coli in the bile precedes rather than follows pigment stone formation. Whereas cholesterol stones usually develop as the result of bile stasis, bactibilia is crucial for the formation of brown pigment stones [16,17]. Pigment stones, and particularly brown stones, may develop as a result of the deconjugation of bilirubin by bacterial beta-glucuronidase, which results in the precipitation of calcium bilirubinate [18]. Bacterial slime production also facilitates bacterial adherence and stone formation. This slime may form a biofilm around stones and could result in false-negative bile cultures [17]. It is also possible that Helicobacter spp. play an active role in the development of cholelithiasis [19]. However, cultures for Helicobacter were not done in the present study.

In conclusion, a positive culture was the only factor associated with an increased risk for postoperative infection in cholecystectomy in the present study. Bacterial content may be crucial in the development of local infection following this procedure.

Footnotes

Author Disclosure Statement

The authors do not have any financial or other interest in the preparation of this work.

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