Molecular Detection of Salmonella typhi Isolated from Patients Undergoing Gallbladder Cholecystectomy in Baghdad.

BACKGROUND: Salmonella typhi is a specific strain of the Salmonella bacterium, responsible for triggering typhoid fever; a significant public health concern in developing nations. OBJECTIVE: The current study aimed to identify the bacteria from the gallbladder, taken during cholecystectomies of patients, by isolating Salmonella typhi and by using microscopic characteristics, biochemical and polymerase chain reaction (PCR) tests. METHODS: A total of 120 specimens were collected from the Baghdad Teaching Hospital, Iraq. A cross-sectional descriptive study was carried out from October, 2021, to July, 2022. During that study, 26 (54.2%) male patient tested positive for Salmonella typhias well as 22 (45.8%) female patients. The age of the patients varied from < 30 to > 60 years. p-value > 0.05 was considered significant to confirm a relationship between age and Salmonella typhi effect for patients. RESULTS: Out of the 120 blood samples taken for this study, 48 (40%) tested positive by use of PCR test, 40 (33.3%) tested positive by use of the Widal test, 35 (29.1%) were positive for biopsy culture, and 35 (29.1%) were positive for blood culture. All Salmonella typhi isolates were found to be sensitive to the imipenem, cefepime, and ceftriaxone, but were resistant to gentamycin, ciprofloxacin, amikacin, erythromycin, and tetracycline (72%, 29%, 43%, 100%, 100%, respectively). CONCLUSIONS: The real time polymerase chain reaction (RT-PCR) tests and the Vitek 2 compact system showed a high level of accuracy in the detection of Salmonella typhi. Multidrug resistance was observed, which should be a signal to reduce antibiotic consumption.

Helicobacter pylori CagA Promotes the Formation of Gallstones by Increasing the Permeability of Gallbladder Epithelial Cells.

BACKGROUND: The formation of gallstones is often accompanied by chronic inflammation, and the mechanisms underlying inflammation and stone formation are not fully understood. Our aim is to utilize single-cell transcriptomics, bulk transcriptomics, and microbiome data to explore key pathogenic bacteria that may contribute to chronic inflammation and gallstone formation, as well as their associated mechanisms. METHODS: scRNA-seq data from a gallstone mouse model were extracted from the Gene Expression Omnibus (GEO) database and analyzed using the FindCluster() package for cell clustering analysis. Bulk transcriptomics data from patients with gallstone were also extracted from the GEO database, and intergroup functional differences were assessed using GO and KEGG enrichment analysis. Additionally, 16S rRNA sequencing was performed on gallbladder mucosal samples from asymptomatic patients with gallstone (n = 6) and liver transplant donor gallbladder mucosal samples (n = 6) to identify key bacteria associated with stone formation and chronic inflammation. Animal models were constructed to investigate the mechanisms by which these key pathogenic bacterial genera promote gallstone formation. RESULTS: Analysis of scRNA-seq data from the gallstone mouse model (GSE179524) revealed seven distinct cell clusters, with a significant increase in neutrophil numbers in the gallstone group. Analysis of bulk transcriptomics data from patients with gallstone (GSE202479) identified chronic inflammation in the gallbladder, potentially associated with dysbiosis of the gallbladder microbiota. 16S rRNA sequencing identified Helicobacter pylori as a key bacterium associated with gallbladder chronic inflammation and stone formation. CONCLUSIONS: Dysbiosis of the gallbladder mucosal microbiota is implicated in gallstone disease and leads to chronic inflammation. This study identified H. pylori as a potential key mucosal resident bacterium contributing to gallstone formation and discovered its key pathogenic factor CagA, which causes damage to the gallbladder mucosal barrier. These findings provide important clues for the prevention and treatment of gallstones.

The frequency and associated factors of typhoid carriage in patients undergoing cholecystectomy for gallbladder disease in Pakistan: A cross-sectional study.

BACKGROUND: Enteric fever is caused by Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A, B, and C. It continues to be a significant cause of morbidity and mortality worldwide. In highly endemic areas, children are disproportionately affected, and antimicrobial resistance reduces therapeutic options. It is estimated that 2-5% of enteric fever patients develop chronic asymptomatic infection. These carriers may act as reservoirs of infection; therefore, the prospective identification and treatment of carriers are critical for long-term disease control. We aimed to find the frequency of Salmonella Typhi carriers in patients undergoing cholecystectomy. We also compared the detection limit of culturing versus qPCR in detecting S. Typhi, performed a geospatial analysis of the carriers identified using this study, and evaluated the accuracy of anti-Vi and anti-YncE in identifying chronic typhoid carriage. METHODS: We performed a cross-sectional study in two centers in Pakistan. Gallbladder specimens were subjected to quantitative PCR (qPCR) and serum samples were analyzed for IgG against YncE and Vi by ELISA. We also mapped the residential location of those with a positive qPCR result. FINDINGS: Out of 988 participants, 3.4% had qPCR-positive gallbladder samples (23 S. Typhi and 11 S. Paratyphi). Gallstones were more likely to be qPCR positive than bile and gallbladder tissue. Anti-Vi and YncE were significantly correlated (r = 0.78 p<0.0001) and elevated among carriers as compared to qPCR negative controls, except for anti-Vi response in Paratyphi A. But the discriminatory values of these antigens in identifying carriers from qPCR negative controls were low. CONCLUSION: The high prevalence of typhoid carriers observed in this study suggests that further studies are required to gain information that will help in controlling future typhoid outbreaks in a superior manner than they are currently being managed.

Invasive Non-Typhoidal Salmonella Lineage Biofilm Formation and Gallbladder Colonization Vary But Do Not Correlate Directly with Known Biofilm-Related Mutations.

Non-typhoidal Salmonella (NTS) serovars have a broad host range and cause gastroenteritis in humans. However, invasive NTS (iNTS) bloodstream infections have increased in the last decade, causing 60,000 deaths annually. Human-specific typhoidal Salmonella colonizes and forms biofilms on gallstones, resulting in chronic, asymptomatic infection. iNTS lineages are undergoing genomic reduction and may have adapted to person-to-person transmission via mutations in virulence, bile resistance, and biofilm formation. As such, we sought to determine the capacity of iNTS lineages for biofilm formation and the development of chronic infections in the gallbladder in our mouse model. Of the lineages tested (L1, L2, L3 and UK), only L2 and UK were defective for the rough, dry and red (RDAR) morphotype, correlating with the known bcsG (cellulose) mutation but not with csgD (curli) gene mutations. Biofilm-forming ability was assessed in vitro, which revealed a biofilm formation hierarchy of L3 > ST19 > UK > L1 = L2, which did not correlate directly with either the bcsG or the csgD mutation. By confocal microscopy, biofilms of L2 and UK had significantly less curli and cellulose, while L1 biofilms had significantly lower cellulose. All iNTS strains were able to colonize the mouse gallbladder, liver, and spleen in a similar manner, while L3 had a significantly higher bacterial load in the gallbladder and increased lethality. While there was iNTS lineage variability in biofilm formation, gallbladder colonization, and virulence in a chronic mouse model, all tested lineages were capable of colonization despite possessing biofilm-related mutations. Thus, iNTS strains may be unrecognized chronic pathogens in endemic settings.

Genomic characterization of Salmonella enterica serovar Choleraesuis from Brazil reveals a swine gallbladder isolate harboring colistin resistance gene mcr-1.1.

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) is a swine-adapted serovar associated to invasive infections in humans. In Brazil, data of strains of this serovar are scarce. In the present study, six S. Choleraesuis strains of animal (n = 5) and human (n = 1) origin from Brazil were screened for phenotypic antimicrobial resistance using disk-diffusion assay and using whole-genome sequencing data to search for antimicrobial resistance genes, plasmids, prophages, and Salmonella pathogenicity islands (SPIs). Its genetic relatedness was evaluated by MLST and SNP analysis. A single isolate from swine gallbladder harbored the colistin resistance gene mcr-1.1 into a IncX4 plasmid. In the six strains analyzed, resistance was found to tetracycline, nalidixic acid, ciprofloxacin, ampicillin, piperacillin, streptomycin, cefazoline, gentamycin, sulfamethoxazole-trimethoprim, and choloramphenicol, along with resistance genes aac(6')-Iaa, aac(3)-IV, aph(3'')-Ib, aph(6)-Id, aph(4)-Ia, aadA1, aph(3')-IIa, blaTEM-1A, floR, sul1, sul2, tet(B), drfA1, erm(B), mph(B), lnu(G), qacE, and gyrA point mutation Serine83 → Tyrosine and parC Threonine57 → Serine. Furthermore, IncF and IncH plasmids, ten SPIs, and seven prophage types were detected. All strains were assigned to ST145 and five belonged to a common SNP cluster of S. Choleraesuis strains from Brazil. The presence of S. Choleraesuis isolated from animals harboring relevant antimicrobial resistance profiles and virulence determinants reinforced the urge for enhanced surveillance to avoid its transmission to humans through food items.

A large panel of chicken cells are invaded in vivo by Salmonella Typhimurium even when depleted of all known invasion factors.

Poultry are the main source of human infection by Salmonella. As infected poultry are asymptomatic, identifying infected poultry farms is difficult, thus controlling animal infections is of primary importance. As cell tropism is known to govern disease, our aim was therefore to identify infected host-cell types in the organs of chicks known to be involved in Salmonella infection and investigate the role of the three known invasion factors in this process (T3SS-1, Rck and PagN). Chicks were inoculated with wild-type or isogenic fluorescent Salmonella Typhimurium mutants via the intracoelomic route. Our results show that liver, spleen, gall bladder and aortic vessels could be foci of infection, and that phagocytic and non-phagocytic cells, including immune, epithelial and endothelial cells, are invaded in vivo in each organ. Moreover, a mutant defective for the T3SS-1, Rck and PagN remained able to colonize organs like the wild-type strain and invaded non-phagocytic cells in each organ studied. As the infection of the gall bladder had not previously been described in chicks, invasion of gall bladder cells was confirmed by immunohistochemistry and infection was shown to last several weeks after inoculation. Altogether, for the first time these findings provide insights into cell tropism of Salmonella in relevant organs involved in Salmonella infection in chicks and also demonstrate that the known invasion factors are not required for entry into these cell types.

Association of Microbial Dysbiosis with Gallbladder Diseases Identified by Bile Microbiome Profiling.

BACKGROUND: Cholecystitis is an important risk factor for gallbladder cancer, but the bile microbiome and its association with gallbladder disease has not been investigated fully. We aimed to analyze the bile microbiome in normal conditions, chronic cholecystitis, and gallbladder cancer, and to identify candidate bacteria that play an important role in gallbladder carcinogenesis. METHODS: We performed metagenome sequencing on bile samples of 10 healthy individuals, 10 patients with chronic cholecystitis, and 5 patients with gallbladder cancer, and compared the clinical, radiological, and pathological characteristics of the participants. RESULTS: No significant bacterial signal was identified in the normal bile. The predominant dysbiotic bacteria in both chronic cholecystitis and gallbladder cancer were those belonging to the Enterobacteriaceae family. Klebsiella increased significantly in the order of normal, chronic cholecystitis, and gallbladder cancer. Patients with chronic cholecystitis and dysbiotic microbiome patterns had larger gallstones and showed marked epithelial atypia, which are considered as precancerous conditions. CONCLUSION: We investigated the bile microbiome in normal, chronic cholecystitis, and gallbladder cancer. We suggest possible roles of Enterobacteriaceae, including Klebsiella, in gallbladder carcinogenesis. Our findings reveal a possible link between a dysbiotic bile microbiome and the development of chronic calculous cholecystitis and gallbladder cancer.

Clinical implication of bactibilia in moderate to severe acute cholecystitis undergone cholecystostomy following cholecystectomy.

There is little evidence of clinical outcome in using antibiotics during the perioperative phase of acute cholecystitis with bactibilia. The aim of current study is to examine the effect of bactibilia on patients with acute cholecystitis and their perioperative clinical outcome. We performed a retrospective cohort analysis of 128 patients who underwent cholecystectomy for acute cholecystitis with moderate and severe grade. Patients who were positive for bactibilia were compared to bactibilia-negative group in following categories: morbidity, duration of antimicrobial agent use, in-hospital course, and readmission rate. There was no difference in morbidity when patients with bactibilia (n = 70) were compared to those without (n = 58) after cholecystectomy. The duration of antibiotics use and clinical course were also similar in both groups. In severe grade AC group (n = 18), patients used antibiotics and were hospitalized for a significantly longer period of time than those in the moderate grade AC group. The morbidity including surgical site infection, and readmission rates were not significantly different in moderate and severe grade AC groups. In moderate and severe AC groups, bactibilia itself did not predict more complication and worse clinical course. Antibiotics may be safely discontinued within few days after cholecystectomy irrespective of bactibilia when cholecystectomy is successful.

Antibiotic selection based on microbiology and resistance profiles of bile from gallbladder of patients with acute cholecystitis.

With the progression of acute cholecystitis, antimicrobial therapy becomes important for infection control. Current antibiotic recommendations were mostly based on reports of patients with acute cholangitis whose bile specimens were sampled from the biliary tract. However, as most infections of acute cholecystitis are limited to the gallbladder, direct sampling from the site increases the probability of identifying the causative pathogen. We investigated 321 positive bile cultures from 931 patients with acute cholecystitis who underwent laparoscopic cholecystectomy between January 2003 and December 2017. The frequency of enterococci declined (P = 0.041), whereas that of Enterobacteriales (P = 0.005), particularly Escherichia (P = 0.008), increased over time. The incidence of ciprofloxacin-resistant Enterobacteriales showed a significant increasing trend (P = 0.031). Vancomycin-resistant E.faecium, carbapenem-resistant Enterobacteriales, and extended-spectrum beta-lactamase-producing Enterobacteriales were recently observed. In grade I and II acute cholecystitis, there were no significant differences in perioperative outcomes in patients with and without early appropriate antimicrobial therapy. In conclusion, the changing incidence of frequently isolated microorganisms and their antibiotic resistance over time would be considered before selecting antibiotics for the treatment of acute cholecystitis. Surgery might be a crucial component of infection control in grade I and II acute cholecystitis.

Biliary microbiota and mucin 4 impact the calcification of cholesterol gallstones.

BACKGROUND: Cholesterol gallstones account for over 80% of gallstones, and the pathogenesis of gallstone formation involves genetic and environmental factors. However, data on the evolution of cholesterol gallstones with various densities are limited. This study aimed to determine the roles of microbiota and mucins on the formation of calcified cholesterol gallstones in patients with cholelithiasis. METHODS: Paired gallbladder tissues and bile specimens were obtained from cholelithiasis patients who were categorized into the isodense group and calcified group according to the density of gallstones. The relative abundance of microbiota in gallbladder tissues was detected. Immunohistochemistry and enzyme-linked immunosorbent assay were performed to detect the expression levels of MUC1, MUC2, MUC3a, MUC3b, MUC4, MUC5ac and MUC5b in gallbladder tissues and bile. The correlation of microbiota abundance with MUC4 expression was evaluated by linear regression. RESULTS: A total of 23 patients with gallbladder stones were included. The density of gallstones in the isodense group was significantly lower than that of the calcified group (34.20 ± 1.50 vs. 109.40 ± 3.84 HU, P < 0.0001). Compared to the isodense group, the calcified group showed a higher abundance of gram-positive bacteria at the fundus, in the body and neck of gallbladder tissues. The concentrations of MUC1, MUC2, MUC3a, MUC3b, MUC5ac and MUC5b in the epithelial cells of gallbladder tissues showed no difference between the two groups, while the concentrations of MUC4 were significantly higher in the calcified group than that in the isodense group at the fundus (15.49 ± 0.69 vs. 10.23 ± 0.54 ng/mL, P < 0.05), in the body (14.54 ± 0.94 vs. 11.87 ± 0.85 ng/mL, P < 0.05) as well as in the neck (14.77 ± 1.04 vs. 10.85 ± 0.72 ng/mL, P < 0.05) of gallbladder tissues. Moreover, the abundance of bacteria was positively correlated with the expression of MUC4 (r = 0.569, P < 0.05) in the calcified group. CONCLUSIONS: This study showed the potential clinical relevance among biliary microbiota, mucins and calcified gallstones in patients with gallstones. Gram-positive microbiota and MUC4 may be positively associated with the calcification of cholesterol gallstones.

Acalculous cholecystitis with gallbladder necrosis in a patient presenting without abdominal pain.

An 83-year-old man with a history of chronic myelogenous leukaemia in remission maintained with bosutinib presented with new-onset fevers. He denied pain and had no other focal symptoms. Ultrasound imaging revealed mild gallbladder wall thickening. Non-contrasted CT revealed right upper quadrant inflammation of indeterminate source. The diagnosis of acalculous cholecystitis was made on the third day when a CT with oral contrast demonstrated a remarkably inflamed biliary tree. The gallbladder was surgically removed and found to be necrotic. The case highlights an unusual presentation for a well-known condition. Both ultrasound and CT have limited diagnostic sensitivity for acalculous cystitis. This case adds to existing literature to support development of acalculous cholecystitis in non-critically ill patients. Clinicians should maintain awareness of this condition among patients presenting to the hospital or clinic with abdominal pain. Careful discussion with radiology and surgery is indicated to guide diagnostic testing when initial imaging results are indeterminate.

A dual-therapy approach for the treatment of biofilm-mediated Salmonella gallbladder carriage.

Asymptomatic carriage of Salmonella Typhi continues to facilitate the transmission of typhoid fever, resulting in 14 million new infections and 136,000 fatalities each year. Asymptomatic chronic carriage of S. Typhi is facilitated by the formation of biofilms on gallstones that protect the bacteria from environmental insults and immune system clearance. Here, we identified two unique small molecules capable of both inhibiting Salmonella biofilm growth and disrupting pre-formed biofilm structures without affecting bacterial viability. In a mouse model of chronic gallbladder Salmonella carriage, treatment with either compound reduced bacterial burden in the gallbladder by 1-2 logs resulting in bacterial dissemination to peripheral organs that was associated with increased mortality. Co-administration of either compound with ciprofloxacin not only enhanced compound efficacy in the gallbladder by a further 1-1.5 logs for a total of 3-4.5 log reduction, but also prevented bacterial dissemination to peripheral organs. These data suggest a dual-therapy approach targeting both biofilm and planktonic populations can be further developed as a safe and efficient treatment of biofilm-mediated chronic S. Typhi infections.

[Prevalence and Characterization of Campylobacter in Bile from Bovine Gallbladders].

Campylobacter is one of the most important causes of food-borne infectious diseases. Antibiotics are rarely needed to treat campylobacteriosis, but occasionally used in severe or prolonged cases. Consumption of contaminated bovine liver is a source of campylobacteriosis. Bovine liver can be contaminated with Campylobacter on the surface and inside by the bile at slaughterhouses. Therefore, we investigated the current prevalence and characteristics of Campylobacter in bovine bile at a slaughterhouse. Campylobacter was isolated from 35.7% (55/154) of bile samples. C. jejuni and C. fetus were the two most frequent species. High antimicrobial resistant rates in C. jejuni were observed against tetracycline (63.0%) and ciprofloxacin (44.4%). Multi-locus sequence typing divided C. jejuni isolates (27 isolates) into 12 sequence types (STs) in which ST806 was the most frequent ST and accounted for 37.0%. All C. fetus were identified as C. fetus subsp. fetus which can cause systemic infections. High antimicrobial resistant rates in C. fetus were observed against ciprofloxacin (66.6%), streptomycin (58.3%) and tetracycline (33.3%). All the C. fetus isolates were divided into two STs, ST3 (16 isolates) and ST6 (8 isolates). Of the 16 ST3 isolates, 15 (93.8%) were resistant to both streptomycin and ciprofloxacin. Our data shows high prevalence of Campylobacter in bovine bile and their high rates of antimicrobial resistance. Preventing bile contamination of bovine liver at slaughterhouses is thus considered to be one of control measures to reduce the risk of Campylobacter infections.

Gallbladder carriage generates genetic variation and genome degradation in Salmonella Typhi.

Despite recent advances in typhoid fever control, asymptomatic carriage of Salmonella Typhi in the gallbladder remains poorly understood. Aiming to understand if S. Typhi becomes genetically adapted for long-term colonisation in the gallbladder, we performed whole genome sequencing on a collection of S. Typhi isolated from the gallbladders of typhoid carriers. These sequences were compared to contemporaneously sampled sequences from organisms isolated from the blood of acute patients within the same population. We found that S. Typhi carriage was not restricted to any particular genotype or conformation of antimicrobial resistance genes, but was largely reflective of S. Typhi circulating in the general population. However, gallbladder isolates showed a higher genetic variability than acute isolates, with median pairwise SNP distances of 21 and 13 SNPs (p = 2.8x10-9), respectively. Within gallbladder isolates of the predominant H58 genotype, variation was associated with a higher prevalence of nonsense mutations. Notably, gallbladder isolates displayed a higher frequency of non-synonymous mutations in genes encoding hypothetical proteins, membrane lipoproteins, transport/binding proteins, surface antigens, and carbohydrate degradation. Specifically, we identified several gallbladder-specific non-synonymous mutations involved in LPS synthesis and modification, with some isolates lacking the Vi capsular polysaccharide vaccine target due to the 134Kb deletion of SPI-7. S. Typhi is under strong selective pressure in the human gallbladder, which may be reflected phylogenetically by long terminal branches that may distinguish organisms from chronic and acute infections. Our work shows that selective pressures asserted by the hostile environment of the human gallbladder generate new antigenic variants and raises questions regarding the role of carriage in the epidemiology of typhoid fever.

Genotoxic Effect of Salmonella Paratyphi A Infection on Human Primary Gallbladder Cells.

Carcinoma of the gallbladder (GBC) is the most frequent tumor of the biliary tract. Despite epidemiological studies showing a correlation between chronic infection with Salmonella enterica Typhi/Paratyphi A and GBC, the underlying molecular mechanisms of this fatal connection are still uncertain. The murine serovar Salmonella Typhimurium has been shown to promote transformation of genetically predisposed cells by driving mitogenic signaling. However, insights from this strain remain limited as it lacks the typhoid toxin produced by the human serovars Typhi and Paratyphi A. In particular, the CdtB subunit of the typhoid toxin directly induces DNA breaks in host cells, likely promoting transformation. To assess the underlying principles of transformation, we used gallbladder organoids as an infection model for Salmonella Paratyphi A. In this model, bacteria can invade epithelial cells, and we observed host cell DNA damage. The induction of DNA double-strand breaks after infection depended on the typhoid toxin CdtB subunit and extended to neighboring, non-infected cells. By cultivating the organoid derived cells into polarized monolayers in air-liquid interphase, we could extend the duration of the infection, and we observed an initial arrest of the cell cycle that does not depend on the typhoid toxin. Non-infected intoxicated cells instead continued to proliferate despite the DNA damage. Our study highlights the importance of the typhoid toxin in causing genomic instability and corroborates the epidemiological link between Salmonella infection and GBC.IMPORTANCE Bacterial infections are increasingly being recognized as risk factors for the development of adenocarcinomas. The strong epidemiological evidence linking Helicobacter pylori infection to stomach cancer has paved the way to the demonstration that bacterial infections cause DNA damage in the host cells, initiating transformation. In this regard, the role of bacterial genotoxins has become more relevant. Salmonella enterica serovars Typhi and Paratyphi A have been clinically associated with gallbladder cancer. By harnessing the stem cell potential of cells from healthy human gallbladder explant, we regenerated and propagated the epithelium of this organ in vitro and used these cultures to model S. Paratyphi A infection. This study demonstrates the importance of the typhoid toxin, encoded only by these specific serovars, in causing genomic instability in healthy gallbladder cells, posing intoxicated cells at risk of malignant transformation.

The gallbladder and vermiform appendix influence the assemblage of intestinal microorganisms.

Surgical procedures for the symptomatic removal of the gallbladder and the vermiform appendix have been posited to adversely shift the assemblage of the intestinal microbiome increasing the risk of disease. The associated mechanisms have been linked with dysbiosis of the gut microbiota. Cholecystectomy causes changes of bile acid compositions and bile secretion patterns as bile acids interact with the intestinal microbiota in a bidirectional capacity. An appendectomy precludes the further recolonization of the proximal colon with a commensal biofilm that could maintain a stable intestinal microbiome. Epidemiological studies indicate that there is an increased risk of disease rather than causality following a cholecystectomy and appendectomy. This narrative review summarizes studies that report on the role that bile salts and the appendix, contribute to the assemblage of the intestinal microbiome in health and disease.

Salmonella Persistence and Host Immunity Are Dictated by the Anatomical Microenvironment.

The intracellular bacterial pathogen Salmonella is able to evade the immune system and persist within the host. In some cases, these persistent infections are asymptomatic for long periods and represent a significant public health hazard because the hosts are potential chronic carriers, yet the mechanisms that control persistence are incompletely understood. Using a mouse model of chronic typhoid fever combined with major histocompatibility complex (MHC) class II tetramers to interrogate endogenous, Salmonella-specific CD4+ helper T cells, we show that certain host microenvironments may favorably contribute to a pathogen's ability to persist in vivo We demonstrate that the environment in the hepatobiliary system may contribute to the persistence of Salmonella enterica subsp. enterica serovar Typhimurium through liver-resident immunoregulatory CD4+ helper T cells, alternatively activated macrophages, and impaired bactericidal activity. This contrasts with lymphoid organs, such as the spleen and mesenteric lymph nodes, where these same cells appear to have a greater capacity for bacterial killing, which may contribute to control of bacteria in these organs. We also found that, following an extended period of infection of more than 2 years, the liver appeared to be the only site that harbored Salmonella bacteria. This work establishes a potential role for nonlymphoid organ immunity in regulating chronic bacterial infections and provides further evidence for the hepatobiliary system as the site of chronic Salmonella infection.

Comparison of endoscopic naso-gallbladder drainage and percutaneous transhepatic gallbladder drainage in acute suppurative cholecystitis: Study Protocol Clinical Trial (SPIRIT Compliant).

INTRODUCTION: Transitional drainage, which is followed by cholecystectomy plays a key role in the management of acute cholecystitis, especially in high-risk surgical patients. Endoscopic naso-gallbladder drainage (ENGBD) is an alternative to percutaneous transhepatic gallbladder drainage (PTGBD) for patients who need temporary drainage. There is a lack of prospective comparison on the relevant outcomes of the two drainage methods during the period of drainage, especially the subsequent cholecystectomy. METHODS: This is a randomized controlled two-arm non-blind single center trial. Patients with acute cholecystitis undergo emergent or early cholecystectomy and need drainage will be randomly assigned to group PTGBD or ENGBD. Pain score is defined as the primary endpoint, whereas several secondary endpoints, such as the rates of technical success, clinical remission, open conversion of cholecystectomy will be determined to elucidate more detailed differences between two groups. The general feasibility, safety, and quality checks required for high-quality evidence will be adhered to. DISCUSSION: This study would provide the first type A evidence concerning the comparison of ENGBD versus PTGBD in surgically high-risk patients with acute cholecystitis, it will be the first trial designed to determine the impact of two drainage methods on not only peri-drainage but also peri-LC. TRIAL REGISTRATION: NCT03701464. Registered on October 10, 2018.

Systemic infection facilitates transmission of Pseudomonas aeruginosa in mice.

Health care-associated infections such as Pseudomonas aeruginosa bacteremia pose a major clinical risk for hospitalized patients. However, these systemic infections are presumed to be a "dead-end" for P. aeruginosa and to have no impact on transmission. Here, we use a mouse infection model to show that P. aeruginosa can spread from the bloodstream to the gallbladder, where it replicates to extremely high numbers. Bacteria in the gallbladder can then seed the intestines and feces, leading to transmission to uninfected cage-mate mice. Our work shows that the gallbladder is crucial for spread of P. aeruginosa from the bloodstream to the feces during bacteremia, a process that promotes transmission in this experimental system. Further research is needed to test to what extent these findings are relevant to infections in patients.

Establishment of Chronic Typhoid Infection in a Mouse Carriage Model Involves a Type 2 Immune Shift and T and B Cell Recruitment to the Gallbladder.

Typhoid fever, caused primarily by Salmonella enterica serovar Typhi (S Typhi), is a life-threatening systemic disease responsible for significant morbidity and mortality worldwide. Three to 5% of individuals infected with S Typhi become chronic carriers due to bacterial persistence in the gallbladder. We have demonstrated that Salmonella forms biofilms on gallstones to establish gallbladder carriage. However, an in-depth molecular understanding of chronic carriage in the gallbladder, from the perspective of both the pathogen and host, is poorly defined. To examine the dynamics of the gallbladder in response to Salmonella infection, we performed transcriptional profiling in the mouse gallbladder at early (7 days) and chronic (21 days) time points. Transcriptome sequencing (RNA-Seq) revealed a shift from a Th1 proinflammatory response at 7 days postinfection (dpi) toward an anti-inflammatory Th2 response by 21 dpi, characterized by increased levels of immunoglobulins and the Th2 master transcriptional regulator, GATA3. Additionally, bioinformatic analysis predicted the upstream regulation of characteristic Th2 markers, including interleukin-4 (IL-4) and Stat6. Immunohistochemistry and fluorescence-activated cell sorter (FACS) analysis confirmed a significant increase in lymphocytes, including T and B cells, at 21 dpi in mice with gallstones. Interestingly, the levels of Salmonella-specific CD4 T cells were 10-fold higher in the gallbladder of mice with gallstones at 21 dpi. We speculate that the biofilm state allows Salmonella to resist the initial onslaught of the Th1 inflammatory response, while yet undefined events influence a switch in the host immunity toward a more permissive type 2 response, enabling the establishment of chronic infection.IMPORTANCE The existence of chronic typhoid carriers has been in the public eye for over 100 years in part because of the publicity around Typhoid Mary. Additionally, it has been known for decades that the gallbladder is the main site of persistence and recently that gallstones play a key role. Despite this, very little is known about the physiological conditions that allow Salmonella enterica serovar Typhi to persist in the gallbladder. In this study, we analyze the transcriptional profile of the gallbladder in a mouse model of chronic carriage. We found a shift from an early proinflammatory immune response toward a later anti-inflammatory response, which could explain the stalemate that allows Salmonella persistence. Interestingly, we found a 10-fold increase in the number of Salmonella-specific T cells in mice with gallstones. This work moves us closer to understanding the mechanistic basis of chronic carriage, with a goal toward eradication of the disease.