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Surgery Today Jpn. J. Surg. (1992) 22:504-507
© Springer-Verlag 1992
Original Articles Bacterial Adherence to Human Gallbladder Epithelium SATOSHI SAKURAI, NAGAO SHINAGAWA,TAKUJI FUKUI, and JIRO YURA The First Department of Surgery, Nagoya City University Medical School, 1 Kawasumi, Mizuho, Nagoya, 467 Japan
Abstract: The adherence of Escherichia coli and Pseudomonas aeruginosa to the epithelium of the gallbladders obtained from 32 patients with negative bile culture was quantified by a scanning electron microscope. Of the gallbladders, 5 were histologically normal (group A), 21 had chronic calculus cholecystitis (group B), and 6 had acute calculus cholecystitis (group C). The data were expressed as the mean + S.D. of the numbers of adherent bacteria to 1,000~m 2 of the gallbladder epithelium. The number of adherent E. coli were 0.1 + 0.2 in group A, 4.2 + 2.8 in group B, and 9.2 + 3.3 in group C. A similar result was also observed with P. aeruginasa. The number of adherent bacteria, both ofE. coli and P. aeruginosa were significantly higher in group C than in groups A and B, and were also significantly higher in group B compared to group A. The amount of bacterial adherence paralleled that of the degree of epithelial damage, and the normal epithelium proved to have an inhibiting ability. Thus, a secondary bacterial infection is more likely to happen in patients with contaminated bile, and therefore, the treatment for acute cholecystitis should be based either on the results of a bile culture or according to predictive factors for bactibilia. Key Words: bacterial adherence, cholecystitis, scanning electron microscopy
Introduction
More than 90% of the patients presenting with acute cholecystitis bare gallstones, and the results of bile cultures are reported to be positive in 40%-60% of them. 1'2 Acute cholecystitis is regarded to normally be initiated by cystic duct obstruction, mostly due to gallstones. It is widely accepted that the course of the disease differs according to the presence or absence of bacteria in the bile, and therefore, the type of treatment should be based on the results of a bile culture. Reprint requests to: N. Shinagawa (Received for publication on Mar. 19, 1991; accepted on Mar. 13, 1992)
Johanson et al. 3 have reported a correlation between the adherence and colonization of gram-negative bacilli to the upper respiratory tract epithelium in vitro, and they also stated that the in vivo colonization is also associated with bacterial adherence. It has also been reported that gram-negative bacilli adhered in greater numbers to buccal epithelial cells sampled from colonized patients compared to those sampled from healthy individuals, in vitro. 4 The ability of the E. coli, isolated from the urine of patients with urinary tract infections, to induce urinary tract infection symptoms paralleled closely that of its in vitro ability to attach itself to human urinary tract epithelial cells, s Our previous report 6 demonstrated that bacteria was easier to adhere to an injured epithelium when compared to a normal epithelium, according to our study using a rabbit gallbladder, and injuring it with either bile acid, HC1 or active carbon. However, little is known about bacterial adherence to the human gallbladder epithelium, which led us to conduct this study in order to evaluate the relationship between bacterial adherence and the degree of inflammation of the human gallbladder.
Materials and Methods
The gallbladders utilized in this study were obtained from 32 patients whose bile was negative both in aerobic and anaerobic bacterial cultures. They were admitted to the First Department of Surgery, Nagoya City University Hospital from January, 1989 to April, 1990. The clinical diagnoses of the patients were cholelithiasis in 27 patients, gallbladder polyps in 2 patients, periampullary carcinoma in 2 patients, and cancer of the pancreas in 1 patient. Immediately after cholecystectomy, a longitudinal section of the gallbladder was taken from the free surface for histological diagnosis and a small block
S, Sakurai et al.: Bacterial Adherence to Human Gallbladder
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Fig. 1. Scanning electron microscopy of group A (E. coli) was taken from the section for electron microscopy. They were divided into three groups according to the pathological findings; 5 patients showed no signs of inflammation (group A), 21 had chronic calculus cholecystitis (group B), and 6 had acute calculus cholecystitis (group C). The small block taken for electron microscopy was first washed in a phosphate buffer solution (PBS) and placed in a small plastic petri dish, to which 1 ml of the bacterial suspension was added while making sure that the block was always fully soaked. After a 1-h incubation at 37°C, the tissue block was rinsed gently in three changes of PBS, prefixed in a 5% glutalaldehyde solution, and postfixed in a 2% osmium tetraoxide solution. Then, it was dehydrated through an ethanol series, immersed in t-butyl alcohol, and a goldpalladium coating was applied as previously reported. 7'8 Adherence was quantified by counting the bacteria under a scanning electron microscope as previously reported. 9 Briefly, the size of the microscopi'c field was fixed by keeping the distance from the sample to the bottom pole of the objective lens to 10mm, and by keeping the viewing angle at 90 ° , and using the magnification power of ca. x3,600. Fifty randomly selected fields were counted on each sample, and the data were expressed as the mean + one standard deviation of the number of bacteria adherent to 1,000 gm 2 of the specimen. E. coli NIHJ-JC2 and P. aeruginosa LLO-1210 were employed in this study. The strains were first transferred from deep agar stab cultures to lactosebromothymol blue agar plates, and an inoculum was
taken from the confluent bacterial growth to avoid selection of an unrepresentative mutant colony. After incubation for 16h without shaking in 3 ml of liquid growth medium, the bacteria were collected by centrifugation and resuspended in PBS as to give a final concentration of 7 x 10 CFU/ml. Either Student's t-test or Welch's t-test were used for statistical analysis, and the difference was considered to be significant when the P value was less than 5%.
Results
Scanning electron microscopy of group A gallbladder challenged with E. coil (Fig. 1) revealed that the topography of the luminal mucosa varies from a flat to a gently undulating surface, and the epithelium consisted of a homogeneous population of cells with hexagonalshaped apical surfaces. The numbers of adherent E. coli and P. aeruginosa in this group were 0.1 + 0.2 and 0.5 + 0.6 respectively (Figs. 2 and 3). In group B with histologically proven chronic cholecystitis, the following changes were observed in the mucosal surface: moderate dehiscence of the epithelial cells without detachment, and shortening and bounding of the microvilli with a small amount of detachment. The number of adherent bacteria was 4.2 + 2.8 for E. coli and 5.6 + 3.6 for P. aeruginosa. In group C with acute cholecystitis, the epithelial cells were dehiscent and detached, and marked changes of microvilli such as detachment, shortening and bounding were observed (Fig. 4), arid the number of
506
S. Sakurai et al.: Bacterial Adherence to Human Gallbladder
Cells/Field 14
p < 0.05
p < 0.05
p
Surgery Today Jpn. J. Surg. (1992) 22:504-507
© Springer-Verlag 1992
Original Articles Bacterial Adherence to Human Gallbladder Epithelium SATOSHI SAKURAI, NAGAO SHINAGAWA,TAKUJI FUKUI, and JIRO YURA The First Department of Surgery, Nagoya City University Medical School, 1 Kawasumi, Mizuho, Nagoya, 467 Japan
Abstract: The adherence of Escherichia coli and Pseudomonas aeruginosa to the epithelium of the gallbladders obtained from 32 patients with negative bile culture was quantified by a scanning electron microscope. Of the gallbladders, 5 were histologically normal (group A), 21 had chronic calculus cholecystitis (group B), and 6 had acute calculus cholecystitis (group C). The data were expressed as the mean + S.D. of the numbers of adherent bacteria to 1,000~m 2 of the gallbladder epithelium. The number of adherent E. coli were 0.1 + 0.2 in group A, 4.2 + 2.8 in group B, and 9.2 + 3.3 in group C. A similar result was also observed with P. aeruginasa. The number of adherent bacteria, both ofE. coli and P. aeruginosa were significantly higher in group C than in groups A and B, and were also significantly higher in group B compared to group A. The amount of bacterial adherence paralleled that of the degree of epithelial damage, and the normal epithelium proved to have an inhibiting ability. Thus, a secondary bacterial infection is more likely to happen in patients with contaminated bile, and therefore, the treatment for acute cholecystitis should be based either on the results of a bile culture or according to predictive factors for bactibilia. Key Words: bacterial adherence, cholecystitis, scanning electron microscopy
Introduction
More than 90% of the patients presenting with acute cholecystitis bare gallstones, and the results of bile cultures are reported to be positive in 40%-60% of them. 1'2 Acute cholecystitis is regarded to normally be initiated by cystic duct obstruction, mostly due to gallstones. It is widely accepted that the course of the disease differs according to the presence or absence of bacteria in the bile, and therefore, the type of treatment should be based on the results of a bile culture. Reprint requests to: N. Shinagawa (Received for publication on Mar. 19, 1991; accepted on Mar. 13, 1992)
Johanson et al. 3 have reported a correlation between the adherence and colonization of gram-negative bacilli to the upper respiratory tract epithelium in vitro, and they also stated that the in vivo colonization is also associated with bacterial adherence. It has also been reported that gram-negative bacilli adhered in greater numbers to buccal epithelial cells sampled from colonized patients compared to those sampled from healthy individuals, in vitro. 4 The ability of the E. coli, isolated from the urine of patients with urinary tract infections, to induce urinary tract infection symptoms paralleled closely that of its in vitro ability to attach itself to human urinary tract epithelial cells, s Our previous report 6 demonstrated that bacteria was easier to adhere to an injured epithelium when compared to a normal epithelium, according to our study using a rabbit gallbladder, and injuring it with either bile acid, HC1 or active carbon. However, little is known about bacterial adherence to the human gallbladder epithelium, which led us to conduct this study in order to evaluate the relationship between bacterial adherence and the degree of inflammation of the human gallbladder.
Materials and Methods
The gallbladders utilized in this study were obtained from 32 patients whose bile was negative both in aerobic and anaerobic bacterial cultures. They were admitted to the First Department of Surgery, Nagoya City University Hospital from January, 1989 to April, 1990. The clinical diagnoses of the patients were cholelithiasis in 27 patients, gallbladder polyps in 2 patients, periampullary carcinoma in 2 patients, and cancer of the pancreas in 1 patient. Immediately after cholecystectomy, a longitudinal section of the gallbladder was taken from the free surface for histological diagnosis and a small block
S, Sakurai et al.: Bacterial Adherence to Human Gallbladder
505
Fig. 1. Scanning electron microscopy of group A (E. coli) was taken from the section for electron microscopy. They were divided into three groups according to the pathological findings; 5 patients showed no signs of inflammation (group A), 21 had chronic calculus cholecystitis (group B), and 6 had acute calculus cholecystitis (group C). The small block taken for electron microscopy was first washed in a phosphate buffer solution (PBS) and placed in a small plastic petri dish, to which 1 ml of the bacterial suspension was added while making sure that the block was always fully soaked. After a 1-h incubation at 37°C, the tissue block was rinsed gently in three changes of PBS, prefixed in a 5% glutalaldehyde solution, and postfixed in a 2% osmium tetraoxide solution. Then, it was dehydrated through an ethanol series, immersed in t-butyl alcohol, and a goldpalladium coating was applied as previously reported. 7'8 Adherence was quantified by counting the bacteria under a scanning electron microscope as previously reported. 9 Briefly, the size of the microscopi'c field was fixed by keeping the distance from the sample to the bottom pole of the objective lens to 10mm, and by keeping the viewing angle at 90 ° , and using the magnification power of ca. x3,600. Fifty randomly selected fields were counted on each sample, and the data were expressed as the mean + one standard deviation of the number of bacteria adherent to 1,000 gm 2 of the specimen. E. coli NIHJ-JC2 and P. aeruginosa LLO-1210 were employed in this study. The strains were first transferred from deep agar stab cultures to lactosebromothymol blue agar plates, and an inoculum was
taken from the confluent bacterial growth to avoid selection of an unrepresentative mutant colony. After incubation for 16h without shaking in 3 ml of liquid growth medium, the bacteria were collected by centrifugation and resuspended in PBS as to give a final concentration of 7 x 10 CFU/ml. Either Student's t-test or Welch's t-test were used for statistical analysis, and the difference was considered to be significant when the P value was less than 5%.
Results
Scanning electron microscopy of group A gallbladder challenged with E. coil (Fig. 1) revealed that the topography of the luminal mucosa varies from a flat to a gently undulating surface, and the epithelium consisted of a homogeneous population of cells with hexagonalshaped apical surfaces. The numbers of adherent E. coli and P. aeruginosa in this group were 0.1 + 0.2 and 0.5 + 0.6 respectively (Figs. 2 and 3). In group B with histologically proven chronic cholecystitis, the following changes were observed in the mucosal surface: moderate dehiscence of the epithelial cells without detachment, and shortening and bounding of the microvilli with a small amount of detachment. The number of adherent bacteria was 4.2 + 2.8 for E. coli and 5.6 + 3.6 for P. aeruginosa. In group C with acute cholecystitis, the epithelial cells were dehiscent and detached, and marked changes of microvilli such as detachment, shortening and bounding were observed (Fig. 4), arid the number of
506
S. Sakurai et al.: Bacterial Adherence to Human Gallbladder
Cells/Field 14
p < 0.05
p < 0.05
p
