Comparison of bacterial profile of gallbladder with gallstones from patients undergoing cholecystectomy due to complicated and uncomplicated cholelithiasis: changes in the epidemiological scenario

ABSTRACT Introduction: cholelithiasis is a highly prevalent disease of the digestive system in the world. In Brazil, it is a routine condition, whose studies suggest a prevalence of around 10% of adults. Colonization of bile and gallstone pathogens can occur when there is bacterial stasis and proliferation. This proliferation is facilitated by the adhesion and biofilm formation capacity of some bacteria. There are also lithogenic processes that involve bacterial participation. Studies have shown changes in the microbiota of the gallbladder of patients undergoing cholecystectomy, which may impact empirical treatment with antibiotics. Methodology: microbiological analyzes of the sonication fluid of the gallstones and of two samples with bile were performed. Identification and antimicrobial susceptibility testing were performed according to a standard routine. Results: of the 34 patients, 76.4% were female. The age group was 48 years +/- 16.61. Acute cholecystitis occurred in 50% of cases. Bactobilia was evidenced in 32.1% of the cases. Klebisiella pneumoniae was noted as the most prevalent pathogen in acute cholecystitis; and Enterobacter sp, in cases of uncomplicated cholelithiasis. Greater sensitivity was obtained in the search for microorganisms in the sonication fluid samples of the stones in relation to the bile samples (p=0.0058). Conclusion: there was a higher prevalence of bactobilia in patients with acute cholecystitis compared to those with uncomplicated cholelithiasis. The use of sonication in bacterial investigation proved to be superior to the conventional method and can be considered.


INTRODUCTION
C holelithiasis is a highly prevalent disease 1,2 .
Bile acquires lithogenic capacity when there is a change in its content.The majority (>80%) of gallstones are of cholesterol, yellow in color 1,7 .The formation of pigmented calculi is the result of cholestasis associated with bacterial colonization in the biliary tract 5,6 and/ or biliary parasites [7][8] .Although cholelithiasis is a predominantly asymptomatic disease, gallstones are associated with important complications, such as acute cholecystitis (AC) 9 .
Foreign bodies in the biliary tract can facilitate bacterobilia 13 .Yu, J.-L et al. implanted solid objects into the temporarily occluded biliary tract of rats.Electron microscopy showed bacterial colonization and biofilm formation on the surface of the implanted material and on the surface of the biliary tract mucosa 11 .This finding showed that physiological bacterial elimination loses its value when microorganisms attach to a foreign body, such as gallstones.Biofilm formation confers resistance to antimicrobial agents and is responsible for the low positivity of culture tests.Sonication is a method that, through ultrasound waves, promotes the rupture of the polymeric matrix of the biofilm, detaching the sessile bacteria from the surfaces that had adhered without damaging them.The fluid culture resulting from this process seems to be more positive when compared with the conventional culture 12 .
In the present study, we performed analysis of bile and sonication fluid from gallstones taken from patients undergoing cholecystectomy for uncomplicated cholelithiasis and for AC, and analyzed the epidemiological profile resulting from the findings of the gallbladder microbiota and gallstones.

Study Population
The population studied consisted of patients treated at Hospital São Luiz Gonzaga and Hospital Central da Irmandade da Santa Casa de Misericórdia de São Paulo, over 14 years of age with cholelithiasis, who underwent cholecystectomy for uncomplicated cholelithiasis or for AC, submitted to a standard surgical procedure, and who agreed to participate in the study by signing the Informed Consent Form.We excluded those with fistulas (Mirizzi syndrome or fistulas to the digestive tract), chronic inflammatory diseases of the biliary tract, biliary malformations, those whose anatomopathological examination of the specimen indicated neoplastic disease, and patients who refused to participate in the study.In addition, we excluded samples in which there was obvious contamination, doubts in identification, insufficient material, or in which the interval between collection and processing in the microbiology laboratory was greater than six hours.We considered a valid case one with successful examination of both the calculus and the bile.

Study Variables
Regarding the patients and the surgical procedure, we studied age, sex, height, weight, Body Mass Index (BMI), comorbidities (systemic arterial hypertension, diabetes mellitus, chronic renal failure, smoking, chronic obstructive pulmonary disease, alcoholism, chronic hepatitis, cirrhosis, sickle cell anemia, use of corticosteroids, positive serology for HIV), reason for surgery (uncomplicated cholelithiasis or AC), antibiotic therapy in the last two weeks, previous endoscopic manipulation of the biliary tract, access route for removal of the gallbladder (laparotomic, laparoscopic, or laparoscopic converted into laparotomy), type of stone (yellow, brown, or black), and, in cases of AC, classification of severity, C-Reactive Protein (PCR), Alkaline Phosphatase (AP), and Gamma Glutamyl Transferase (GGT).

Source of Information and Data Collection
All patients underwent cholecystectomy.Bile samples were collected in 0.9% saline solution, bile in thioglycolate and gallstone sonication fluid in 0.9% saline solution.The 102 samples were seeded on blood agar and chocolate agar media in aerobic and anaerobic environments.Bile samples were sent directly to the central laboratory, where they were seeded on blood agar and chocolate agar.The Falcon tubes containing the calculi were sent to the microbiology laboratory of the Faculty of Medical Sciences of Santa Casa de São Paulo, where they were subjected to the following processes: 1. vortexing for 30 seconds using the Vortex-Genie 2 (Scientific Industries, Inc., Bohemia, NY, USA).At the end of this process, the resulting fluid was sent to the central laboratory for seeding on blood agar and chocolate agar media (the same ones in which the bile sample was seeded).Bile and sonication fluid samples were stored and frozen at the end of these procedures.
The aerobic plates (blood agar and chocolate agar) were incubated at 35-37°C with 5-7% CO 2 for 7 days; the anaerobic plates, at 37°C for 14 days.All plates were inspected daily to monitor the growth of microorganisms.
Finally, microbial susceptibility tests followed the standards established by the CLSI (Clinical Laboratory Standards Institute) and were performed in all identified colonies.
The CLSI standardization for disc diffusion was used across all strains to identify the susceptibility profile.The quality control of the susceptibility test was performed with the standard strains (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 25923).The reference standard adopted for these tests is the one used in the laboratory and follows the international criteria of the CLSI.

Diagnostic Criteria
Uncomplicated cholelithiasis: detection of stones in the gallbladder by any imaging exam (most commonly by abdominal ultrasonography, but those detected by computed axial tomography, magnetic nuclear resonance, or echo-endoscopy would also be accepted) and confirmed after cholecystectomy (anatomopathological exam without acute inflammatory signs).
Acute cholecystitis: detection of stones in the gallbladder by any imaging exam (most commonly by abdominal ultrasonography, but those detected by computed axial tomography, magnetic nuclear resonance, or echo-endoscopy would also be accepted) and confirmed after cholecystectomy (anatomopathological exam with signs acute inflammation).
To classify the severity of acute cholecystitis, we used the Tokyo Guidelines 2018 14 : • Mild: No complications.
• Severe: systemic complications (Glasgow coma scale less than 15, PaO 2 / FiO 2 ratio less than 300mmHg, need to use any dose of intravenous noradrenaline to maintain blood pressure, less than 100,000 platelets / µL, INR greater than 1.5, and / or serum creatinine greater than 2mg/dL).

Surgical Technique
Cholecystectomies were performed either openly or laparoscopically.In the former, the gallbladder was immediately taken to the instrument technician's table and punctured to extract the bile in a syringe.
In the latter, the bile was aspirated through a syringe during the removal of the organ from the abdominal cavity.Patients with an amount of obtained bile less than 5 mL were excluded from the study.
After collection, two bile samples were separated.One of them was placed in a Falcon tube containing 0.9% saline and the other in Thioglycolate.
All containers were duly labeled with the patient's record, date of surgery, and collection time.

Statistical Analysis
For the sample description, we used percentile and mean for qualitative variables and median and standard deviation for quantitative ones.To compare the results between the different samples (bile and calculus), we used the chi-square, the Fisher, and the Mann-Whitney tests according to the analyzed variables.We considered the value of p<0.05 as statistically relevant.
We used the REDCap software to analyze the studied variables.

Ethical Aspects
This project was initially registered on

Study Population
During the study period, 34 patients were included.There were no exclusions.Of these, 26 were female (76.4%), 11 were older than 60 years (average 48 +/-16.61years, range 23-76), only nine patients were eutrophic (26.4%), while the others were above the ideal Body Mass Index Kg/m 2 (average 27.64 +/-4.27,range 15.97-36.44).without the clinical need for the laboratory analysis described in Table 3.

Surgical Procedure
With the exception of one patient diagnosed with TG III acute cholecystitis, who underwent surgery via laparotomy (right subcostal incision), all patients were operated via laparoscopy (Table 2).Furthermore, there was predominance of yellow-colored calculi (Figure 1).

Microbiological Findings
There was growth of microorganisms in 32.4% (11/34) of the patients studied (Table 3).In     Table 4 shows the comparative analysis between bacterial growth and the stratification of the clinical picture according to the Tokyo Guidelines 2018 (TG18).We fond no statistical differences between disease severity and pathogen identification.
There were no statistical differences between the comorbidities studied and bactobilia, as described in Table 6.Table 7 shows the analysis between the quantitative variables in the study (BMI, C-reactive protein, leukocyte count, neutrophils, lymphocytes, Alkaline Phosphatase, and Gamma-glutamyl transferase) and the presence of bacterial growth, using the Mann-Whitney test.
We observed the highest positivity of microbiological tests in patients with AC (Figure 2).In general, there was an unfavorable sensitivity pattern for the pathogens found, mainly Gram-negative bacteria (Klebsiella pneumoniae and Enterobacter spp.), showing a profile of resistance to first-generation cephalosporins (cefazolin and cephalothin) and to ampicillin.Importantly, these patients did not present a description of antibiotics use before surgery.The isolated Gram-positive bacteria were sensitive to penicillins.Figure 3 shows the relative frequency of the isoated microorganisms, and Figue 4 shows the microorganisms found in each diagnosis.
Using the Kappa coefficient, we noticed an important association (Kappa between 0.61 and 0.8) between the methods using bile in saline solution 0.9% and bile in thioglycolate, and between bile in saline solution 0.9% and sonication fluid from the gallstones (Table 7).Furthermore, we observed an almost perfect association (Kappa between 0.81 -0.99) between bile samples in thioglycolate and gallstone sonication fluid (0.929).

Comparison Of Microbiological Findings Between Sonication Fluid From Gallstones And Bile Samples
The sensitivity of identification of pathogens by the sonication test of the calculi occurred in 32.3% (11/34 stones).The sensitivity of identifying bile pathogens was 10.2% (7/68 bile samples) (p=0.0058).Among the cases in which there was bacterial growth, the sample with sonication fluid from the gallstone was positive in all cultures, while in the samples of bile in saline solution 0.9% and in thioglycolate, seven and ten of the cases, respectively (Figure 5).The bile sample in 0.     sonication fluid from gallstones were used for bacterial investigation.By amplifying the sampling of microbiological cellular material, this method improves the sensitivity of cultures, in addition to providing sensitivity tests to antibiotics 13 .Such a finding may help us in understanding bacterial colonization in gallstones.In addition to sonication, we also noticed that bile in thioglycolate increased sensitivity when compared with the conventional method.
This work found the presence of bacteria in samples from 41.1% of patients with AC.Other studies suggest the incidence of bactobilia between 35% to 65% of cases of AC and around 10% in those with uncomplicated cholelithiasis 10,15 .Despite the statistical limitation, we observed that the prevalence of bacterial growth was higher the more sevre was the patient.It is likely that the bile microflora contained in the gallbladder is important for the clinical-surgical outcome of patients undergoing cholecystectomy 14 .
hospital course 19 .We did not find statistical relevance in the use of antibiotics for prophylactic or therapeutic use in eradicating bactobilia and its clinical outcome in both groups of patients.There is controversy about the use of antibiotics for prophylaxis in cholecystectomy for uncomplicated cholelithiasis 19 .It is postulated that the lack of antimicrobial effect is associated with poor diffusion of drugs in the biliary tract 14 .However, adequate bacterial identification can contribute to decision-making in case of complications, such as intra or postoperative bile leakage 14 .

CONCLUSION
The prevalence of bacteria found in the bile of patients with acute cholecystitis was higher than that of those with uncomplicated cholelithiasis.However, we found no relevant statistical association between the evaluated clinical and laboratory variables and bacterial growth in the samples.The use of sonication as a method of bacterial investigation proved to be superior to the conventional method of bile culture, and can be considered in the medical routine.

R E S U M O R E S U M O
unicentric, cross-sectional study comparing the results of microbial identification and sensitivity tests of microorganisms identified in bile and sonication fluid from gallstones in patients with cholelithiasis who underwent cholecystectomy for uncomplicated cholelithiasis or for AC in a secondary hospital in the city of São Paulo.Study Area During the period from January 2021 to June 2022, we studied all patients over 14 years of age with cholelithiasis who underwent cholecystectomy for uncomplicated cholelithiasis or for AC at the Hospital São Luiz Gonzaga and Hospital Central da Irmandade da Santa Casa de São Paulo.Gallstones and bile samples were sent to the central hospital of the Irmandade da Santa Casa de Misericórdia de São Paulo.The study was approved by the Ethics in Research Committee (CEP) of the Irmandade da Santa Casa de São Paulo, under number CAAE: 40713420.0.0000.5479.

2 .
sonication for 5 minutes at a frequency of 40±2 kHz and density power of 0.22±0.04W/cm 2 using the ultrasonic washer model BactoSonic 14.2 (BANDELIN Electronic GmbH & Co. KG, Germany).3. eddy for an additional 30 seconds. 4. concentration of the sonication fluid through centrifugation performed in 50mL aliquots at 2,500rpm for 5 minutes.

Plataforma
Brasil and forwarded to the Ethics in Research Committee of the Faculty of Medical Sciences of Santa Casa de São Paulo, where it was approved (CAAE: 40713420.0.0000.5479).Only patients who were informed about the content of the study and who agreed with the terms contained in the Informed Consent Form were accepted in the study.

(
GGT) tests and their respective classification by the Tokyo Guidelines 2018 (TG).The others underwent elective surgeries for uncomplicated cholelithiasis,

Figure 1 .
Figure 1.Gallstones present in a patient with Acute Cholecystitis who underwent laparoscopic cholecystectomy.

Figure 2 .
Figure 2. Bacterial growth in acute cholecystitis and uncomplicated cholelithiasis and their respective findings.

Figure 3 .
Figure 3. General frequency of microorganisms found.

*
Chi-square testDISCUSSIONThe main finding of this study was the greater sensitivity of pathogen identification when samples of Hirata Comparison of bacterial profile of gallbladder with gallstones from patients undergoing cholecystectomy due to complicated and uncomplicated cholelithiasis: changes in the epidemiological scenario However, further studies are needed to evaluate techniques to prevent postoperative infections and assess their effectiveness.Despite the lithogenic theory of pigmented calculi, which consists of cholestasis associated with bacterial colonization (Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis)12 , there was a lower prevalence of bactobilia in individuals with brown calculi compared with those with yellow ones.However, such findings were inconclusive and were not statistically relevant in the association between the presence of bacteria in bile or gallstones and the incidence of complications in cholelithiasis.As in previous studies, the enteric bacteria Klebsiella pneumoniae and Enterobacter spp.predominated among positive cultures10,12,15,16 .It is postulated that such microorganisms reach the gallbladder lumen by ascending the duodenum.Gram positive bacteria such as Streptococcus spp.were also isolated in the study, however it is likely that they also have an enteric origin14 .Of the 19 antibiotics used in the sensitivity profiles, ampicillin proved to be inappropriate for the therapeutic management of bactobilia.Furthermore, only 50% (5/10) of the microorganisms were sensitive to cefazolin.R. Reiss et.al. investigated the presence of bactobilia in 800 patients undergoing cholecystectomy.Of these, 27% (217/800) of the cases had bacterial growth and 12% (27/217) were strains of Klebsiella pneumoniae, whose sensitivities to first-generation cephalosporin and ampicillin were 56% and 10%, respectively19 .In our study, the strains of Klebsiella pneumoniae (5/11) showed 100% resistance to ampicillin and only 20% resistance to cefazolin.The use of antibiotics in medical practice for the management of AC is widely accepted19 .Mazeh et al. performed a comparative study between patients with mild AC (TG-I) who used antibiotic therapy and those who did not.They observed that the use of intravenous antibiotics did not affect the length of hospital stay, the number of admissions, or the surgical outcome.Their study suggests that the genesis of AC in these cases is more associated with the inflammatory process than with the infectious process, and that antibiotic therapy should be individualized based on its

Figure 4 .
Figure 4. Microorganisms found in Uncomplicated Cholelithiasis and in Acute Cholecystitis.

Figure 5 .
Figure 5.Comparison between samples with bacterial growth.

Table 1 -
Demographic analysis of the studied patients and Body Mass Index.

Table 2 -
Diagnosis and treatment of operated patients.

Table 3 -
Bacterial growth according to diagnosis.

Table 4 -
Bacterial growth in the acute cholecystitis caes, stratified by the Tokyo Guidelines 2018 sevetiry classification.

Table 6 -
Quantitative variables and the presence of bacterial growth..

Table 7 -
Agreement index (Kappa) obtained between the collected samples regarding bacterial growth.

Table 5 -
Comorbidities and the presence of bactobilia.