To prevent post cholecystectomy infection, the most common microorganisms causing it and their antibacterial susceptibility pattern should be determined. Therefore, the aim of the present study was to determine the exact incidence and nature of the microbial flora in the bile of the patients with cholelithiasis and chronic cholecystitis as well as their antibiotic sensitivity pattern. In this study, a total of 132 samples from the patients were tested for bacterial strains using the appropriate methods for testing them. The isolated bacteria were subsequently subjected to antibacterial susceptibility test using Kirby-Bauer method. The data were analyzed using Frequency, Chi-square and t-test. Fifty of 132 (37.87) studied patients were positive for bacteria. The most common isolated organisms were Escherichia coli (13; 26), Enterobacteriaceae (9; 18), and Salmonella typhi (7; 14). The most effective antibiotics were sequentially Amikacin, Ceftriaxone, and Clindamycin. Isolating bacteria and determining their sensitivity to different antibiotics may be help physicians take prophylactic measures against postoperative infection of cholelithiasis and chronic cholecystitis. © 2013 Tehran University of Medical Sciences. All rights reserved

Imani, Reza.

Moazeni-Bistgani, Mohammad.

To prevent post cholecystectomy infection, the most common microorganisms causing it and their antibacterial susceptibility pattern should be determined. Therefore, the aim of the present study was to determine the exact incidence and nature of the microbial flora in the bile of the patients with cholelithiasis and chronic cholecystitis as well as their antibiotic sensitivity pattern. In this study, a total of 132 samples from the patients were tested for bacterial strains using the appropriate methods for testing them. The isolated bacteria were subsequently subjected to antibacterial susceptibility test using Kirby-Bauer method. The data were analyzed using Frequency, Chi-square and t-test. Fifty of 132 (37.87%) studied patients were positive for bacteria. The most common isolated organisms were Escherichia coli (13; 26%), Enterobacteriaceae (9; 18%), and Salmonella typhi (7; 14%). The most effective antibiotics were sequentially Amikacin, Ceftriaxone, and Clindamycin. Isolating bacteria and determining their sensitivity to different antibiotics may be help physicians take prophylactic measures against postoperative infection of cholelithiasis and chronic cholecystitis

Mohammad Moazeni-Bistgani

Reza Imani

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ACTA MEDICA IRANICA

Bile Bacteria of Patients with Cholelithiasis and Theirs Antibiogram

shahrekord university of medical scinces

 ORIGINAL REPORT   Corresponding Author: Mohammad Moazeni- Bistgani Department of Surgery, Faculty of Medicine, Shahrekord University of Medical Sciences, shahrekord, Iran Tel: +98 913 1812974 .Fax: +98 381 2224492, E-mail: dr_m_moazeni@yahoo.com  Bile Bacteria of Patients with Cholelithiasis and Theirs Antibiogram Mohammad Moazeni-Bistgani1 and Reza Imani2 1 Department of Surgery, Faculty of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran 2 Department of Infectious Diseases, Faculty of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran  Received: 7 Nov. 2012; Accepted: 26 Feb. 2013  Abstract- To prevent post cholecystectomy infection, the most common microorganisms causing it and their antibacterial susceptibility pattern should be determined. Therefore, the aim of the present study was to determine the exact incidence and nature of the microbial flora in the bile of the patients with cholelithiasis and chronic cholecystitis as well as their antibiotic sensitivity pattern. In this study, a total of 132 samples from the patients were tested for bacterial strains using the appropriate methods for testing them. The isolated bacteria were subsequently subjected to antibacterial susceptibility test using Kirby-Bauer method. The data were analyzed using Frequency, Chi-square and t-test. Fifty of 132 (37.87%) studied patients were positive for bacteria. The most common isolated organisms were Escherichia coli (13; 26%), Enterobacteriaceae (9; 18%), and Salmonella typhi (7; 14%). The most effective antibiotics were sequentially Amikacin, Ceftriaxone, and Clindamycin. Isolating bacteria and determining their sensitivity to different antibiotics may be help physicians take prophylactic measures against postoperative infection of cholelithiasis and chronic cholecystitis. © 2013 Tehran University of Medical Sciences. All rights reserved.  Acta Medica Iranica, 2013; 51(11): 779-783.   Keywords: Antibacterial susceptibility pattern; Bacteria; Cholelithiasis  Introduction  As one of the most common diseases of gastrointestinal tract, cholelithiasis occurs in 10% of the western country's population and 17% of the Asian country's population, with an overall prevalence of 11-36%. In Iran, there are no reliable data about the overall prevalence of cholelithiasis and chronic cholecystitis. Symptomatic cholelithiasis may lead to serious complications or mortality. The most prevalent complication of cholelithiasis is chronic cholecystitis that usually requires cholecystectomy (1, 2). The biliary tract is usually sterile; however, if cholelithiasis occurs, different microbes might be identified in and/or cultured from the bile or gallbladder wall (3,4). Microscopic examinations indicated that 20-50% of the patients with chronic cholecystitis have a positive bile culture (5,6). Different reasons for biliary tract infection have been presented, e.g. ascending infection due to reflux of duodenal contents, blood-borne infection and infection spread through the portal-venous channels. Ascending infection from the duodenum is thought to be the primary mechanism by which bacteria enter the bile (7). Reviewing the literature, there are conflicting reports regarding the significance of bacterial infection in patients with chronic cholecystitis and cholelithiasis (8). As prophylaxis, a number of antibiotics could be prescribed to prevent potential post-cholecystectomy infections. Thus, understanding the most common organisms causing them and their antibacterial susceptibility pattern would be useful in prevention of these infections. The present study was carried out to achieve this aim.  Materials and Methods  In this cross sectional study, 132 patients with cholelithiasis and chronic cholecystitis without risk factors for postoperative sepsis (older than 70 years old, previous biliary tract operation, jaundice, chills and fever within 1 month of cholecystitis acute attack) were subjected to elective laparoscopic cholecystectomy from September 2009 to September 2010. Using a questionnaire, patients' age, sex, clinical features of the patients, the isolated bacteria, and antibiogram were recorded. Since inadvertent entry into the gallbladder during laparoscopic cholecystectomy is not uncommon and could lead to spillage of bile or stone into the Bile bacteria of patients with cholelithiasis and theirs antibiogram 780    Acta Medica Iranica, Vol. 51, No. 11 (2013)   peritoneal cavity, all  our patients received antimicrobial prophylaxis with 1g Keflin preoperatively and patients who had diabetes mellitus or spillage of bile or stone into the peritoneal cavity continued receiving 1g Keflin every 6 hours postoperatively. During surgery 5-10 ml of the bile samples was aspirated with a sterile syringe from gallbladder immediately after cholecystectomy; 3-5 ml was placed in a sterile container; another 3-5 ml was inoculated directly into an aerobic and anaerobic BacT/Alert blood culture bottle (Organon Teknika, Durham, NC, USA). The Samples were sent immediately to the Microbiology Laboratory of Medical Faculty and incubated at 37°C for 24 h. In the laboratory, the samples were cultured in thioglycolate medium to detect anaerobic and other bacteria and were cultured on blood agar and MacKoncky agar incubated in 37°C for 24 hours. Differential tests such as IMVIC (Indole, Methyl Red, Voges presquare, and Citrate), motility, urease, and lysine decarboxylase were performed after staining and microscopic observation. After the bacteria were isolated, we performed antibiotic sensitivity tests by the isolates carried out using Kirby-Bauer method with a colony in Mueller-Hinton agar medium (pH: 7.2-7.4) (Merck, Germany). The agar was prepared and sterilized according to the manufacturer's instructions.     The results were reported as susceptible, resistant and intermediate based on the diameter of the clear zone around disks with reference to the antibiotic standard table.  The applied antibiotic disks were Penicillin, Ampicillin, Amikacin, Gentamicin, Cefotaxime, Cefepime, Erythromycin, Clindamycin, Vancomycin, Tetracycline, Novobiocin, Ceftriaxone, Imipenem, Co-trimoxazole and Ciprofloxacin.  Results  In this study, 132 patients hospitalized due to cholelithiasis and biliary colic were included. All of the patients had cholelithiasis and 74 patients had chronic cholecystitis. The study population included 88 (66.7%) female and 44 (33.3%) male with a mean age of 55.6±14.3 years (range; 18 and 63 yr). Among the total 132 bile samples, bacteria were isolated in 50 samples (37.87%). Mean age of patients with positive and negative cultures were 56.9±14 and 52.7±14.7 years respectively, having significant difference based on Student’s t-test (P<0.05). Frequency of isolated bacteria in females and males had no significant difference (P>0.05). Monomicrobial and polymicrobial infection were observed in 47 (94%) and 3 (6%) respectively. Anaerobic bacteria were detected in 8 (16%) patients; Bacteroides fragilis (2; 4%) and Bacteroides perfringens (6; 12%). Escherichia coli was the most common isolate (13; 26%). Enterobacter was isolated from 9 samples (18%) followed by Salmonella typhi (7; 14%), Coagulase-negative Staphylococcus (6; 12%), Klebsiella pneumoniae (2; 4%), Proteus (2; 4%). Salmonella typhi was isolated only from samples of female patients (Table 1).  Antibiotic susceptibility tests were performed for organisms that were isolated from the bile samples and the following data were obtained. E.coli was 92.3% susceptible to Amikacin, while it was nearly resistant to Erythromycin (90%). Enterococcus was 69.23% susceptible to Ceftriaxone, while it was 46.12% resistant to Gentamicin, Penicillin, and Cefotaxime.  Table 1. The distribution of various microorganisms. Microorganism  Total number Percentage Monomicrobial    E.coli 13 26    Enterobacter 9 18    Salmonella typhi 7 14    Coagulase-negative Staphylococcus 6 12    Klebsiella pneumoniae 2 4    Proteus 2 4    Anaerobic bacteria 8 16    Total 47 94 Polymicrobial    Proteus with Klebsiella 1 2    E. coli with anaerobic bacteria 2 4    Total 50 100 M. Moazeni Bistgani and R. Imani     Acta Medica Iranica, Vol. 51, No. 11 (2013)    781 Table 2. Antimicrobial* susceptibility of isolates in the study. Isolate(N) P Amp Ctz Ci Cp Im Ak G Ery Cli Van Cf Sxt Tet NovE. coli 6 6 3 7 6 9 12 7 1 8 1 9 7 4 3 Coagulase-negative Staphylococcus 1 1 5 1 2 2 1 7 3 1 2 2 2 2 1 Klebsiella pneumoniae 1 1 2 2 2 2 3 1 2 3 2 2 3 1 1 Salmonella typhi 2 1 6 3 4 1 3 2 4 8 7 0 5 7 8 Enterobacter 3 1 6 9 5 8 4 4 6 0 1 6 0 1 0 Proteus 0 0 2 2 2 3 3 0 1 3 2 2 0 3 3 Total 13 10 24 24 21 25 26 21 17 23 15 21 17 18 16 *P=Penicillin; Amp=Ampicilin; Ctz=Cefotaxime; Ci=Ceftriaxone; Cp=Cefepime; Im=Imipenem; Ak=Amikacin; G=Gentamycin; Ery=Erythromycin; Cli=Clindamicin; Van=Vancomycin; Cf=Ciprofloxacin; Sxt=Co-trimoxazole; Tet=Tetracyclin; Nov=Novobiocine.   The most susceptible antibiotic for microorganisms on the whole was Amikacin (52%). The antibiogram of the isolates are also summarized (Table 2). We did not have any infective complications.  Discussion  In our study the mean age of patients was 55.6±14.3 years (range; 18-63 yr), nearly in line with 46 and 51.7 years in two other studies (1, 9). From the total 132 bile samples, bacteria were isolated in 50 samples (37.87%). Some other studies have shown that 20-50% of the patients with chronic cholecystitis have a positive bile culture (5, 6). Mean age of patients with positive and negative cultures was 56.9±14 and 52.7±14.7 years respectively, having significant difference based on t student test (P<0.05). A study by Al Harbi et al. has shown being older than 50 years was the only factor significant in view of preoperative positive bile culture (8), which is in agreement with our study. Anaerobic bacteria were detected in 8 (16%), monomicrobial infection in 47 (94%), and polymicrobial infection in 3 (6%) patients. In a study by Ballal et al. in India, bile cultures for aerobic and anaerobic bacteria were carried out on 125 samples from patients with chronic cholecystitis with cholelithiasis; 71 (56.8%) aerobic and 17 (13.6%) anaerobic bacteria were detected. Among the mixed flora, 2 had only aerobes and the remaining had both aerobes and anaerobes (10). Al Harbi et al.'s study also showed polymicrobial infection in 4 (3.57%) and anaerobic bacteria in none of the cases (8). Anaerobic bacteria may grow when the bile duct is seriously infected by anaerobic and aerobic bacteria and hence body's immunity becomes low. Lou et al. suggested the paucity of anaerobes in the human biliary system (11). The effect of anaerobic bacteria on bile pigment stone has been reported widely (12, 13), according to which the anaerobes such as B. fragilis and B. fusiformis may produce an E.coli substance called β-Lactamase which resolves bilirubin. The bilirubin when integrated with calcium ion forms calcium bilirubinate.  The difference in anaerobic and polymicrobial positive culture between our study and others' could be attributed to the method of antibiotic therapy; here, we ordered antibiotic prophylaxis routinely while in Ballal et al. study and Al Harbi et al. study were selectively it , we had not known.  In our study E. coli was the most common isolate (13; 26%), as previously reported (14-17). Enterobacter was the second one (9; 18%) followed by Salmonella typhi (7; 14%), Coagulase-negative Staphylococcus (6; 12%), Klebsiella pneumoniae (2; 4%) and Proteus (2; 4%). The significance of E. coli dominance is also supported by previous reports indicating a potential role for E.coli's glucoronidase enzymatic activity in formation of calcium bilirubinate gall stone (18, 19). In our study, Salmonella typhi grew in 18% cases, sensitive to Clindamicin and Novobiocine in 88.8%. The prevalence of Salmonella typhi in bile of cholelithiasis patients varied widely from 1% to 34% (20, 22), perhaps due to typhoid fever which is, similar to some parts of our country, endemic in some regions. Analysis of our patients showed that Salmonella typhi was more common in females compared to males. Their high incidences in females have been attributed to hormonal effects related to menstrual cycle and pregnancy (10). Eslami et al. study conducted in Iran indicated E. coli (25%) as the most prevalent isolated bacterium, and the prevalence of Klebsiella, Aerobacter, Pseudomonas, Enterococci, and Proteus as 12%, 10%, 9%, 8%, and 3% respectively (23). In Ballal et al. study, E. coli and Klebsiella were predominant among the aerobes with 45.07% and 25.35% prevalence respectively and B. fragilis (58.82%) predominant among the anaerobes (10). According to Al Harbi et al., the most common organisms isolated were E. coli (26.1%), Enterococcus Bile bacteria of patients with cholelithiasis and theirs antibiogram 782    Acta Medica Iranica, Vol. 51, No. 11 (2013)   facials (15.6%), and Pseudomonas aeruginosa (9.6%) (8). We do not know why our findings were not completely similar to others' studies. However, we support taking cultures of the bile at cholecystectomy because appropriate antibiotics can be administered in case of cultures being positive, hence avoiding serious complications, e.g. gram negative sepsis. In our study E. coli was 92.3% susceptible to Amikacin and Enterococci 69.23% susceptible to Ceftriaxone. The most susceptible antibiotic for microorganisms on the whole was Amikacin. Ballal et al. have shown anaerobes were sensitive to Cefotaxime, Metronidazole, Chloramphenicol, Cefazolin, and Tetracycline, and aerobes isolated to Ampicillin, Chloramphenicol, Streptomycin, Tetracycline, Gentamicin, and second generation Fluoroquinolones such as Ciprofloxacin and Norfloxacin (10). In spite of the fact that we ordered antibiotic prophylaxis for all the patients, bacterial isolates and infectious complications showed no significant difference. Thus, we recommend starting antibiotics selectively, if supported by the clinical conditions and/or culture reports, in case of cholecystitis and cholelithiasis. However, routine culture of all bile samples is strongly advised. Given our and others' studies anaerobes are rare in the human biliary system; therefore, if antibiotic therapy is considered, aerobic coverage should be satisfactory. In addition because the most susceptible antibiotic for microorganisms on the whole was Amikacin, this antibiotic can be started selectively.  Acknowledgments  The authors would like to thank Research and Technology Department of Shahrekord University of Medical Sciences for cooperating in this study and Kashani University Hospital staff for assisting in data collection.   References  1. Csendes A, Burdiles P, Maluenda F, Diaz JC, Csendes P, Mitru N.Simultaneous bacteriologic assessment of bile from gallbladder and common bile duct in control subjects and patients with gallstones and common duct stones. Arch Surg 1996;131(4):389-94. 2.  Vitetta L, Best SP, Sali A. Single and multiple cholesterol gallstones and the influence of bacteria. Med Hypotheses 2000;55(6):502-6.Csendes  3. Wetter LA, Hamadeh RM, Griffiss JM, Oesterle A, Aagaard B, Way LW. Differences in outer membrane characteristics between gallstone-associated bacteria and normal bacterial flora. 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Bile bacteria of patients with cholelithiasis and theirs antibiogram

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