Bacteria inside gallstones have long been thought to be dead, as is explicit in the well known aphorism of Lord Moynihan 1: ‘Tomb stone erected to the memory of organisms which lie dead with in them’. Yet calculi spilled at laparoscopic cholecystectomy cause a variety of complications (Table 1) 2,3,4,5,6,7,8,9,10,11,12. Peritoneal lavage and retrieval of spilled stones prevent intraperitoneal abscess formation, thus directly incriminating gallstones in the infective complications 13,14,15. Experimental studies have also confirmed that implantation of human gallstones in the peritoneal cavity of rat, rabbit and dog produces intraperitoneal abscesses, sinuses and adhesions 2,16,17,18.

Table 1.Complications caused by retained stones following laparoscopic cholecystectomy

The infective process originating from gallstones is independent of contamination of the peritoneal cavity with infected bile. Gurleyik and colleagues 19 have shown that the chemical composition of stones also influences septic complications observed after laparoscopic cholecystectomy, which are common when brown pigment stones are retained in the abdominal cavity. The cholesterol and black pigment in stones is thought to be unrelated to the presence of bacteria. Bacterial DNA is present in all brown pigment stones but only occasionally in black pigment stones or stones with a high cholesterol content (70–95%) 20,21,22. There are no reports of bacteria being isolated from stones with cholesterol content >95%. The presence of bacteria or bacterial DNA as shown by scanning electron microscopy and molecular genetic techniques does not prove that bacteria found in the cores of gallstones are live and capable of multiplying, and thus a potential cause of infective complication.

Gallstone culture is a suitable alternative for demonstrating bacteria walled up within the calculus and also reflects their infective potential. In this study we set out to culture bacteria from the core of gallstones in patients undergoing cholecystectomy for various reasons, irrespective of stone type and size. The bacterial species isolated have been correlated with age, sex and diagnosis.

The percentage of cases in each group was calculated and was related to age, sex and diagnosis. Statistical analysis was carried out using the unpaired Student's t-test and the χ² test.

Enteric organisms were grown more frequently than non-enteric organisms (46 versus 16 cases) (Table 2). Age and sex had no significant effect. As for the underlying diagnosis, only enteric organisms were retrieved from the 11 patients with carcinoma. The mean age of patients with cholelithiasis was lower than that of patients with carcinoma of the gallbladder (43.48±14–07 versus 57.45±12.51, p≤0.005). The mean age of patients harbouring enteric bacteria in their calculi was greater than that of patients harbouring non-enteric bacteria (47.52±12.86 versus 36.57±12.63, p=0.0069). The percentages of different bacteria isolated are shown in Table 3. In three patients calculi grew more than one bacterium, including Pseudomonas spp. in two of these cases. Table 4 shows the results of bacterial culture of bile and stones as reported in the literature 22,23,24,25,26,27.

Table 2.Culture results according to diagnosis, age and sex

Table 3.Bacteria isolated and relative percentage of cases

Table 4.Culture positivity rates as observed by bile culture and stone culture in various reported studies

The association between cholelithiasis and bacteria has been confirmed by bile culture, scanning electron microscopy and molecular genetic techniques 20,22,23,28,29. Although bile culture can also demonstrate the infective potential of bacteria colonising the gallbladder, it yields lower positive culture rates than stone culture in patients with gall stones 22,23,24,25,26,27. Moreover, bile culture does not prove whether bacter-obilia is the cause or effect of cholelithiasis. Thus methods other than stone culture cannot conclusively determine whether bacteria inside gallstones are alive and capable of multiplying so as to cause infection.

Besides the threat of sepsis and cholangitis, the presence of live bacteria in the core of gallstones underlines the potential hazards of leaving stones in the peritoneal cavity during laparoscopic cholecystect-omy. The chance of infection has been seen to be greater in such patients, other than those with stones having a cholesterol content >95% or pure black pigment stones 29,30. Our study has shown live bacteria in the core of gallstones in 81% of cases and thus substantiates the need for complete retrieval of all stones and debris dropped in the peritoneal cavity during cholecystectomy.

The incidence of bile and gallstone infection varies considerably from area to area in the world and also among different countries in a given area. Thus β-glucuronidase-producing Escherichia coli was more frequent in bile from Chinese than from Japanese patients with gallstones (51% vs 22%), in line with the incidence of calcium bilirubinate stones in the two countries (49% versus 17%) 25. In Ghana 26, only 21% of patients with gallstones containing <75% cholesterol had positive bile cultures, despite the fact that the most non-cholesterol stones and mixed cholesterol stones have been shown to harbour bacteria as seen by scanning electron microscopy and molecular genetic methods. However, gallstone culture should provide a better profile of bacterial species causing cholelithiasis in a given area in view of the higher positive culture rates obtained by this method.

Enteric organisms have often been suspected of causing cholelithiasis, and intestinal flora have frequently been recovered following interventions on the biliary tree 31,32. The acquisition of certain outer membrane characteristics can distinguish these bacteria from normal gut flora and may explain their selective colonisation of the biliary tract 22. In our study non-enteric pathogens were isolated in 24% of cases while enteric pathogens were isolated in 54%.

The present study shows Klebsiella spp. to be the commonest organisms isolated (18%) followed by E. coli (15%), Enterococcus spp. (7.5%) and Enterobacter spp. in 7.5% cases, whereas Salmonella sp. was observed in only 1.5% of cases. Psendomonas spp. (8.8%) was the commonest non-enteric pathogen followed by Acineto-bacter spp. (7.5%) and Staphylococcus aureus (3.8%). These proportions coincide with the bacterial flora recovered from the cultures in cases of cholelithiasis and hepatolithiasis in South-East Asia 33.

Carcinoma of the gallbladder is highly prevalent in South Asia and it is here that non-cholesterol stones (stones due to infection) are also common. Increasing age is an important risk factor for gallbladder carcinoma. Higher rates of positive bile cultures 23 and of calcified pigment stones 34 have been found in the elderly. Csendes and colleagues 23 observed that age >60 years has a significant influence on the percentage of positive biliary cultures. Whereas 81% of cases of bile cultures showed organisms in patients with gallbladder carcinoma, only 41% were positive in those with cholelithiasis. The equivalent figures in our scores for positive stone culture were 77% for carcinoma and 81% for cholelithiasis. Infection has been considered a minor risk factor for carcinoma of the gallbadder, but data are sparse.

It is of interest that only enteric pathogens were recovered from the stones of patients with carcinoma of the gallbladder; no Salmonella was grown, although this has long been considered a causative agent. The mean age of patients harbouring enteric bacteria was higher than those harbouring non-enteric organisms and gallbladder cancer is known to occur in older age groups (mean age 57.45±12.51). It is possible that chronic colonisation of the gallbladder with gallstones harbouring enteric bacteria may predispose to gallbladder carcinoma in the elderly, but further studies are required before definitive conclusions can be drawn in this regard.

In summary, the gallstone is a viable dynamic home or fortress for bacteria rather than a tomb.

We are grateful to Mr Girija Dwevedi and Mr Ramji for collection of the stones and also transportation to the Microbiology Department.