Gallbladder microbiota variability in Colombian gallstones patients

Introduction: Gallbladder stones are a very frequently occurring condition. Despite bile bactericidal activity, many bacteria have been detected inside the gallbladder, and gallstones facilitate their presence. Between 3% and 5% of the patients with Salmonella spp. infection develop the carrier stage, with the bacteria persisting inside the gallbladder, shedding bacteria in their feces without signs of infection. The aim of this study was to isolate bacteria from Colombian patients with gallstones, using standard culturing methods, and to identify Salmonella spp. carriers by molecular techniques. Methodology: A total of 149 patients (120 female and 29 male) diagnosed with gallstones who underwent cholecystectomy and who did not have symptoms of acute inflammation were included. Gallbladder tissue and bile were cultured and used for DNA extraction and Salmonella spp. hilA gene detection. Results: Of the 149 patients 28 (19%) had positive cultures. Twenty-one (75%) patients with positive cultures were from Medellin’s metropolitan area. In this geographical location, the most frequent isolations were Pseudomonas spp. (38%), Klebsiella spp. (23%), and Proteus spp. (9%) in addition to unique cases of other bacteria. In Apartado, the isolates found were Enterobacter cloacae (50%), Raoultella terrigena (32%), and both Enterobacter cloacae and Raoultella terrigena were isolated in one (18%) male patient. Five (3.3%) of the 149 patients had positive polymerase chain reaction (PCR) results for the hilA gene of Salmonella spp., all of whom were female and residents of the Medellín metropolitan area. Conclusions: The gallbladder microbiota variability found could be related to geographical, ethnic, and environmental conditions.


Introduction
Gallbladder stones occur very frequently, with prevalence rates between 10% and 30% in adult populations of developed countries [1].The pathogenesis of gallstones is a multifactorial phenomenon related to age, ethnicity, diet, gender, body mass index, family history, and metabolic diseases, among other factors [2].
Bacteria have been implicated in the pathogenesis of gallstones, producing high levels of betaglucuronidase, biliary mud, and phospholipases A2 [3], not only in non-pigmented gallstones where oversaturation of cholesterol prevails, but also in pigmented gallstones where there are high proportions of unconjugated bilirubin [4].Additionally, gallstones facilitate the presence of bacteria inside the gallbladder, adhering to the stones and making a bacterial community inside a polymeric extracellular matrix called biofilm [5].
Bile is produced by hepatocytes and is stored and concentrated in the gallbladder [6].Its function is to facilitate the absorption of lipophilic substances such as fats and vitamins [7], to excrete cholesterol, and to regulate the intestinal microbiota through its bactericidal activity [8,9].
Despite bile bactericidal activity, effected by damage to the cellular membrane and to DNA, many bacteria are able to survive inside the gallbladder [10].This organ is reached by ascending from the duodenum, via the hematogenous route or via enterohepatic circulation [11].
Many bacteria have been detected from the bile using conventional methods (cultures) or molecular techniques, such as the amplification and sequencing of the 16S rRNA gene [12].Using molecular techniques, a study of Chinese patients with gallstones revealed Firmicutes and Bacteriodetes as the first and second most abundant phyla, mirroring the major distribution of bacteria in the intestine.Their bile also show increased relative abundance of phyla such as Proteobacteria, Actinobacteria, Thermi, Cyanobacteria, and Tenericutes when compared to normal feces [13].Groups such as Enterobacteriaceae, Ruminococcaceae, Bacteroidales, Lactococcus, Enterococcus, and Clostridiales predominated in the bile of patients with gallstones.Escherichia coli, Klebsiella, Enterobacter, Acinetobacter, Pseudomonas, and Citrobacter were the most frequently occurring species [13,14].
Between 3% and 5% of the patients with Salmonella spp.infection develop the carrier stage, in which the bacteria persists inside the gallbladder [15].Patients who develop the carrier stage can shed bacteria in their feces for months, years, or even the rest of their lives without signs of infection [16].These individuals are of utmost importance from the perspective of public health organizations as they become reservoirs, and 25% of them do not realize they are infected [17,18].
The isolation of Salmonella spp. is a difficult task due to scarce bacteria in the different samples.Due to the low sensitivity of the cultures and the high rate of false-positive results in the serologic test, an alternative approach for the diagnosis, using techniques based on the amplification and detection of nucleic acids, has been suggested [19].
Bacterial communities from different anatomic locations, principally in the gastrointestinal tract, vary in presence and relative abundance of phyla, genera, and species, depending on factors related to geography, population, ethnicity, diet, environment, and sanitation [20,21].Colombia is an endemic country for Salmonella spp.infections [22,23], yet there have been no local surveys of the gallbladder microbiota.The aim of this study was to isolate bacteria from the bile of Colombian patients' gallbladders using standard culturing methods, and to identify Salmonella spp.carriers by molecular techniques.

Patients
A total of 149 patients diagnosed with gallstones who underwent cholecystectomy were included in the study after informed consent was procured.Of the patients, 13 were from Apartadó (Antioquia), 2 were from Quibdó (Chocó), and 134 were from Medellin's metropolitan area.

Gallbladder and bile samples
Instructions were given to the surgeons in charge of the gallbladder resections in each city.The instructions involved sampling tissue of the gallbladder to be used in histopathology (10% tamponade formol) for DNA extraction (70% ethanol) and bile in lactose broth for cultures and DNA extraction.

Cultures
Bile samples were collected in lactose broth (BBL, Sparks, USA) for pre-enrichment, incubated at 35°C for 12 hours, and then replicated in blood agar, MacConkey and Salmonella-Shigella (SS) agars (all from BBL, Sparks, USA) and incubated again for 12 hours at 35°C.One milliliter of the previously enriched bile was cultured in selenite broth and incubated again for 12 hours at 35°C, and then replicated in MacConkey and SS agars.Biochemical identification of the isolated colonies was done using the API20E system (bioMérieux, Durham, USA).

DNA extraction
DNeasy Blood and Tissue Kit (QIAGEN, Duesseldorf, Germany) was used for DNA extraction from gallbladder tissue and bile.

Polymerase chain reaction (PCR)
Salmonella spp.hilA gene detection was performed as previously described [24,25].Briefly, PCR mix comprised 40 µL of SuperMix (Invitrogen, Carlsbad, USA), 0.5 µL of the forward primer at 10 µM, 0.5 µL of the reverse primer at 10 µM, and 9 µL of the DNA, for a total volume of 50 µL at 0.1 µM.The amplification protocols were as follows: initial denaturation at 94°C for 5 minutes, 30 cycles at 94°C for 1 minute, 65°C and 72°C for 1 minute each, and a final elongation phase of 10 minutes at 72°C.The visualization of the 854 base pairs was done using a 1% agarose gel stained with SYBR Safe (Invitrogen, Carlsbad, USA) and revealed in an EpiChemi Darkroom image analyzer (Upland, USA).
Pseudomonas fluorescens/putida was the sole isolate from the Quibdó region's positive patient.Pseudomonas spp.(38%), Klebsiella spp.(23%), and Proteus spp.(9%) were the most frequent species in the Medellín metropolitan area, in addition to unique cases of other bacteria (Table 1).

Salmonella spp. hilA PCR
Five (3.3%) of the 149 patients had positive PCR results for the hilA gene of Salmonella spp., all of whom were female and residents of the Medellín metropolitan area.Two (40%) had positive cultures (Escherichia coli and Pseudomonas fluorescens/putida) and three (60%) had negative cultures.Table 2 shows the characteristics of Salmonella spp.PCR-positive patients.

Discussion
Gallbladder microbiota is a dynamic and complex bacterial community with variability between populations related to geographical, ethnic, and environmental conditions, which additionally could have important clinical implications in the setting of gallstones patients with acute inflammatory complications.
An important finding of our study is the detection of Salmonella spp. in 3.3% of the study population, or in all patients from the Medellin metropolitan area.This reveals that the chronic carriage rates in our population are similar to those reported in the literature [15], which further highlights the complexity of Salmonella spp. as an infectious agent.There are no a highly efficient, costeffective, and non-invasive methods for the identification of Salmonella spp.chronic carriage.High antibody titer against the Vi antigen is the surrogate  marker for chronic carriage state, but serology is not a reliable technique [26].Gallbladder resection and antibiotics can reduce the rates of chronic carriage, but none of these interventions can guarantee a complete cure [27].
It is important to point out the differences between the results of the current and previous studies concerning the frequency of bacterial species, geographical origin of patients, and the Afro-Latin American proportion of the population.In the Medellín metropolitan area, located 1,475 meters above sea level, with an average temperature of 22°C, well-developed public services and sanitation facilities, and a population comprising 19% Afro-Latin Americans [28,29], the most frequent bacteria were Pseudomonas spp., Klebsiella spp., and Proteus spp.In contrast, in Apartadó, located 25 meters above sea level, with an average temperature of 28ºC [30], under-developed public services and sanitation facilities, and an Afro-Latin American population of 69% [28,31], the most frequent bacteria were Enterobacter cloacae and Raoultella terrigena, suggesting differences in the microbiota between populations.
Differences found in our two Colombian populations contrast with the results found in other similar studies from other geographical origins.The relative frequencies of the isolated bacteria in the different studies has shown some consistency within the United States population (E.coli, Enterococcus, Klebsiella, and Enterobacter), as was reported by Stewart et al. [32,33] and Brook et al. [34], and similarities between American and Chilean patients (E.coli, Streptococcus spp., Klebsiella spp., Enterobacter spp.) [10].
The results are less homogeneous in the Asiatic surveys from India (E.coli, Klebsiella spp., Citrobacter freundii, and Salmonella Typhi) [35] and Sri Lanka (E.coli, Pseudomonas spp., Streptococcus spp., and Klebsiella spp.) [36], with important differences when compared to our study.Differences can be explained by the low frequency of E. coli and Enterococcus and the predominance of species such as Pseudomonas and Klebsiella found in the studied population.
The variability in the bacterial isolations is a phenomenon related to, in a simplistic manner, differences in the kind of samples used, culture methodologies [37], the anatomical site where the sample was taken, and associated inflammatory conditions [38].There are variations between the cited studies explained by the previously considered variables.In our study, however, the isolations are different and may be related to the environmental conditions and the ethnicity of the population factors that produce variations in the microbiome of many anatomical locations [20,21].
As in our study, most of the bacteria isolated in the cited studies belong to the Enterobacteriaceae family and are Gram-negative bacteria, some of which are considered fecal coliforms (E.coli, Klebsiella, Enterobacter, Raoultella); these bacteria are ubiquitous in soils, water fonts, vegetables, and contaminated meat products [39].This kind of bacteria colonize the gastrointestinal tract very early in life, becoming part of the microbiome [40].
High numbers of coliforms in water destined for human consumption correlates to poor water treatment, a condition present among in the Apartadó population and, in much lower proportions, the Medellín metropolitan area.Therefore, to aid in the explanation of the differences among all cited studies and between them and our results, it would be useful to conduct an eco-epidemiological study.This study should use molecular methods to analyze the bacterial communities of the food in a regular diet and the water destined for human consumption in these cities, as well as inside the gastrointestinal tract and gallbladder.The intent would be to establish more objective differences or associations, which would allow for expansion of our knowledge of the microbiome dynamics in our population and the related conditions.
Knowing the core gallbladder microbiota of a population in non-acute inflammatory conditions is an important issue, because some of this bacteria can become pathogenic and there could be no other opportunity to isolate them because microbiota may change, as in the gut, by the inflammatory microenvironment and previous antibiotic use [41].
Acute cholecystitis is a complication of patients with gallstones, and although it is considered a non-lifethreatening condition, it may evolve to more serious complaints like empyema, gangrene, and perforation [42].In cases of acute complications, gallbladder tissue or bile for culture are not available and management options agree on supportive care and early antibiotic use, but delayed cholecystectomy and percutaneous drainage cholecystostomy remain controversial [43][44][45][46].
Without results from gallbladder tissue or bile cultures, antibiotic treatment is empiric, relying on the assumption of the probable local microbiota.There have been many studies trying to support the use of antibiotics, with difficulties for comparisons in relation to timing, choice, and duration [47], and disappointing results with respect to benefits (symptoms severity, perioperative course, post-operative complications, hospital stays) [48,49].These surveys have not considered the gallbladder microbiota and its variability as a possible source for fluctuations in the proposed outcomes; these fluctuations could be explained by the antibiotic selection and the response for each specific type of microbiota in different populations.Being aware of the probable gallbladder microbiota of the population in the city of medical practice may help optimize the antibiotic selection in acute complications, increasing the benefits of this therapy and even leading to the possibility of avoiding surgery [50].
The low rate of isolations and the lack of anaerobic cultures are weaknesses in our study.Future studies may ameliorate these limitations by including molecular techniques to increase knowledge of the gallbladder microbiota in patients with pathologies such as acute cholecystitis and biliary tract carcinomas in Colombia.

Conclusions
The gallbladder microbiota variability found in the current population belongs to the Enterobacteriaceae family and comprises Gram-negative bacteria, some of which are considered fecal coliforms (E.coli, Klebsiella, Enterobacter, Raoultella).
Gallstones, sclerosing cholangitis, and gallbladder carcinoma are pathologies with an unraveled unequal distribution around the world.The gallbladder microbiota and its variability, as in the different Colombian regions studied, could be a key factor in such uneven distribution.A core gallbladder microbiota should be defined, and this definition must be related to geographical, ethnic, and environmental conditions, to support the idea that the microbiota is really associated with gallbladder pathologies.Future studies in other regions of Colombia could exhibit more variability in the microbiota since diet and ethnic origin are quite different.This is the first study of gallbladder microbiota in Colombian patients.

Table 2 .
Positive Salmonella hilA gene PCR, sex, age, and culture results.