Sero-bacteriological detection of bacteria in the gall bladder , blood and stool of patients having cholecystectomy

Objectives: 1To identify the bacteria from the gall bladder and stool samples taken during cholecystectomy of patients with acute, and chronic cholecystitis of calculus and acalculus types. 2To use serological agglutination tests for the diagnosis of typhoid and brucellosis cholecystitis.3To find out the antibiogram of the isolated bacteria from the different cases. Methods: A prospective case series study was conducted at Al-Jumhori Teaching Hospital, and Ninevah and Al-Zahrawi Private Hospitals during the period from the 1 of October 2003 to the 30 of June 2004. A total of 150 patients undergoing cholecystectomy (134 females and 16 males)were studied. From each patient samples were collected from the gall bladder (bile, tissue biopsy and stone if present),blood for serological tests and stool (from 69/150 patients). Results: Among the gall bladder samples, 101/150 (67.3%) cases showed bacterial growth, the remaining 49 (32.7%) cases had no growth. The bacterial growth was seen among 83.3% and 64.3% of acute and chronic cholecystitis respectively. A single agent per sample was detected in 62.4% of the cases, whereas mixed growth was identified in the remaining 37.6%. The gram-negative bacteria were predominant and represented 86.7% of the isolates,while gram-positive ones were 13.3%. The most common aerobic bacteria isolated were E. coli (19.3%), while anaerobes were detected in only 5.3% of the isolates. The stool cultures showed similar microorganisms to those identified in the gall bladder samples in 74% of cases. In acute cholecyctitis all the gram-negative bacteria were sensitive to ciprofloxacin followed by ceftriaxone (96.5%) and gentamycin (96.5%). In chronic cholecystitis the most effective drug was ciprofloxacin (93.7%) followed by amikacin (82.2%). In both acute and chronic cholecystitis the least effective antibiotic was ampicillin.Typhoid cholecystitis was detected among four cases only, while brucellosis cholecystitis was not observed. Conclusions: The bacteria causing acute and chronic types of cholecystitis are similar and predominantly are gram-negative bacilli. The intestinal origin of these microorganisms suggests ascending infection from the intestine to the biliary tract. Ciprofloxacin is the most effective drug against the isolated bacteria.

holecystitis and cholelithiasis are fairly common diseases that are associated with serious complications.In 85-95% of the cases, inflammation of the gall bladder is associated with calculi (1) .Acute cholecystitis is mostly associated with an obstruction of the neck of the gall bladder or cystic duct due to stone impacted in Hartmann's pouch.The disease is more common in females than males (1) .Enteric bacteria can be cultured from gall bladder bile in approximately half of patients with acute cholecystitis, but bacteria are not thought to contribute to the actual onset of acute cholecystitis.Emphysematous cholecystitis is a rare form of acute cholecystitis.It is usually of gangrenous type and caused by gas-forming microorganisms (2) .Emphysematous cholecystitis is characterized by the presence of gas in the gall bladder, and is more common in males than females (3) .The pathogenesis is related to acute inflammation of the gall bladder, which often begins aseptically and then complicated by a secondary infection with gas forming bacilli.These may reach the gall bladder by the bile ducts, blood stream or lymphatic channels and grow in an anaerobic environment.
Chronic cholecystitis is a chronic inflammation of the gall bladder and is generally associated with cholelithiasis with fibrosis of its wall.The bile is initially sterile, but may be secondarily infected with coliform bacilli, Streptococcus and occasionally Clostridia or Salmonella.Cholecystitis is the most common intraabdominal localized infection due to Salmonella (4) .
Acalculus cholecystitis is an acute or chronic inflammatory disease of the gall bladder, which can occur without stones.Acute acalculus cholecystitis is frequently a complication of sepsis, multiple organ failure or major operation.The incidence of chronic acalculus cholecystitis is less than 5%, and the possible causes are thrombosis of major blood vessels, systemic disease as diabetes, specific infections as typhoid fever, actinomycosis and parasitic infestations (3) .

Materials and methods
One hundred fifty patients undergoing cholecystectomy for acute or chronic cholecystitis, with or without cholelithiasis, were studied.The patients were 134 (89.3%) females and 16 (10.7%)males.They were of age 5-75 years.
Thirty cases of cholecystitis and 120 had cholecystectomy by open and laparoscopic surgery respectively.At operation, bile from the gall bladder, tissue biopsy from the wall and stone (if present) were taken for bacterial culture.All specimens were collected aseptically in sterile sealed containers, and were cultured within 1-2 hours after sampling.Stool samples or rectal swabs were also taken from 69/150 enrolled in the present study.The stool samples were cultured within few minutes to 1 hour of the time of sampling.About 3 milliliters of venous blood were taken from all patients in plain tubes and centrifuged for 10 minutes at 2500 RPM.The sera were separated and used for serological tests either immediately or kept frozen at -20C° until use.

Bacterial culture
The bile and stool samples were processed for aerobic and anaerobic bacterial isolation.The samples were inoculated separately on MacConkey's (Difco,USA) and S-S (HiMedia,India) agars and incubated aerobically at 37C ° for 24 hours, extended to 48 hours incubation if there was no growth.The samples were also inoculated on duplicate of blood agar plates; one incubated anaerobically using Gas Pack Generating Kit (Oxoid,England) for 48 hours and the other plate incubated aerobically for 24 hours.The bile and the rectal swabs were also inoculated in tetrathionate broth at 37C ° for 24 hours and then subcultured on MacConkey's and S-S agars.If bacterial growth was detected in any of the culture medium, the result was reported as positive.Pure culture was obtained from the positive media for further identification by standard biochemical tests and antibiotic sensitivity testing (5) .

Serological tests
The following serological tests were done on the serum samples: a-Brucella agglutination test and 2-Mercaptoethanol test (2-ME) for brucellosis using rose bengal reagent (Merck-U.K.) by rapid slide titration method (6) .
b-Widal test using stained febrile antigens (Pasmatec Laboratory products-UK) applying rapid slide agglutination according to the manufacturers and 2ME test (7) .
Results of the tested microorganisms were compared with those of the control group and then reported.The isolates were tested against different antibiotics according to their Gram staining results.The Gram-negative bacteria were tested against ciprofloxacin, amikacin, norfloxacin, ceftriaxone, gentamicin, chloramphenicol, ceftazidime, tetracycline, cephalexin, pipericillin, cotrimoxazole, cefotaxim, ampicillin and cefoxitin.

Results:
The 150 patients studied were subdivided into two groups.The first one included 126 (84%) patients with chronic cholecystitis and the second group involved 24 (16%) patients with acute cholecystitis.
The presence of cholelithiasis was investigated by ultrasonography prior to the operation as well as by direct identification during surgery.One hundred twenty (80%) of the first group had gall bladder stones (calculus) and 6 (4%) were acalculus.Eighteen (12%) of the second group were calculus and 6 (4%) were acalculus.
Aerobic bacteria were detected in 94 (93.1%) cases, while mixed aerobes and anaerobes were identified only in 7 (6.9%)cases (Table 2).No anaerobic microorganism was identified alone.These anaerobic microorganisms were 6 (4%) Bacteroides fragilis and 2 (1.3%) Clostridium perfringens.The stool culture showed microorganisms similar to those identified in the gall bladder samples in 74% of cases.Culturing all samples collected via open and laparoscopic approaches showed similarity in the types of microorganisms isolated.
The antibiograms of the bacteria isolated from the gall bladder samples of acute and chronic cholecystitis were determined.The gramnegative isolates from these two conditions showed more comparable antimicrobial sensitivity pattern (< 5% difference) than the gram positive bacteria for most (12/14) of the antimicrobial agents used.
Brucella agglutination test was positive only among 9/150 cases of cholecystitis studied.All positive cases were of chronic calculus cholecystitis type.None of these cases showed the Brucella microorganism in cultures of their gall bladder samples.
Widal agglutination test was positive among 31/150 cases studied.The majority (25/31) of the Widal positive cases belong to chronic calculus cholecystitis.Moreover, the 2ME-Widal agglutination test was only positive among 4/31 cases.Three out of the four cases were chronic calculus cholecystitis type.All the four 2ME-Widal positive cases showed growth of microorganisms causing enteric fever in their gall bladder samples.Three of them were Salmonella typhi and only one was Salmonella paratyphi-A.Since the bile of normal subjects is sterile, the bacterial infection seems to play a role in the development of the gall bladder disorders (8) .
Out of the 150 cases studied, 101 (67.3%) cases yielded growth of many bacteria from the cultures of the gall bladder samples.Also, other studies had shown bacterial

Gram positive isolates
cases was also confirmed (besides the culture) by positive Widal and Widal-2ME tests.
The culture of the gall bladder wall and bile gave almost identical results, except in some cases (10/77) of chronic calculus cholecystitis and one case of acute calculus cholecystitis in which the bacteria were found more often in the gall bladder wall than in the bile.This may be due to sessile bacterial population associated with the mucosa.Some studies reported variations between the rates of bacteria detected in the bile (8%) from those isolated from the gall bladder wall (23%) specimens (12) .
According to the antibiogram detected, it was evident that ciprofloxacin constituted the first-choice drug for the treatment of biliary tract infection caused by gram negative and gram-positive bacteria.This result is in agreement with other studies (11,26,27) .The use of quinolones is recommended because these agents can effectively penetrate the obstructed biliary tree.
The bacterial sensitivity to most antimicrobial agents in acute cholecystitis was higher than that in chronic cholecystitis among the majority of cases.This may be attributed to the repeated uses of antibiotics in chronic cholecystitis which lead to the development of resistant microorganisms.

Figure( 1 )
Figure(1): The frequency of the gram-negative bacteria in cholecystitis

Table 1 :
The distribution of culture positive and stone formation according to the age group of patients.

Table 2 :
The types of bacteria in culture positive cases.