In Vitro Evaluation of the Therapeutic Potential of Phage VA7 against Enterotoxigenic Bacteroides fragilis Infection

Since the beginning of the 20th century, bacteriophages (phages), i.e., viruses that infect bacteria, have been used as antimicrobial agents for treating various infections. Phage preparations targeting a number of bacterial pathogens are still in use in the post-Soviet states and are experiencing a revival in the Western world. However, phages have never been used to treat diseases caused by Bacteroides fragilis, the leading agent cultured in anaerobic abscesses and postoperative peritonitis. Enterotoxin-producing strains of B. fragilis have been associated with the development of inflammatory diarrhea and colorectal carcinoma. In this study, we evaluated the molecular biosafety and antimicrobial properties of novel phage species vB_BfrS_VA7 (VA7) lysate, as well as its impact on cytokine IL-8 production in an enterotoxigenic B. fragilis (ETBF)-infected colonic epithelial cell (CEC) culture model. Compared to untreated infected cells, the addition of phage VA7 to ETBF-infected CECs led to significantly reduced bacterial counts and IL-8 levels. This in vitro study confirms the potential of phage VA7 as an antibacterial agent for use in prophylaxis or in the treatment of B. fragilis infections and associated colorectal carcinoma.

Celiac Disease Prevalence in Patients with Chronic Noninflammatory Diarrhea in 2017

Introduction: Celiac disease is an autoimmune disorder. Eating gluten, which is part of the cereals, can lead to intestinal mucosal injury and diarrhea. Although celiac disease is more common today with non-digestive symptoms, we sometimes encounter cases of chronic non-inflammatory diarrhea, which is one of the clinical manifestations of celiac disease. This study was performed to evaluate the prevalence of celiac disease in patients with chronic non-inflammatory diarrhea. Because diarrhea in celiac disease has distinct and efficient treatment we decided to do this study to evaluate the prevalence of celiac disease in patients with chronic non-inflammatory diarrhea. Methods: This was a descriptive cross sectional study on 200 patients with chronic non-inflammatory diarrhea in Yazd in 2017. Stool exam and tissue transglutaminase Antibody (TTG Ab) was done for the patients; upper endoscopy and duodenum biopsy was done for the patients with high serum TTG Ab. Findings analyzed in SPSS.ver.17 statistical software. Chi-Square, t-test and Fisher Exact tests were used for statistical analysis. P-value less than 0.05 was considered significant. Results: Two hundred patients, including 93 (46.5%) men and 107 women (53.5%) participated in this study. The average age was 34.32 ± 17.84 years. Among these cases, 31 patients (15.5%) had celiac disease. Among cases of celiac (16.1%), 15 (16.1%) were male and 16 (15%) were female. The relative frequency of celiac disease was the same in both sexes and in all age groups. Conclusion: Significant portion of patients (15.5%) with chronic non-inflammatory diarrhea has celiac disease.

The sRNA MicC downregulates hilD translation to control the SPI1 T3SS in Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium invades the intestinal epithelium and induces inflammatory diarrhea using the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS). Expression of the SPI1 T3SS is controlled by three AraC-like regulators, HilD, HilC and RtsA, which form a feed-forward regulatory loop that leads to activation of hilA, encoding the main transcriptional regulator of the T3SS structural genes. This complex system is affected by numerous regulatory proteins and environmental signals, many of which act at the level of hilD mRNA translation or HilD protein function. Here, we show that the sRNA MicC blocks translation of the hilD mRNA by base pairing near the ribosome binding site. This binding blocks translation but does not induce degradation of the hilD message. Our data indicate that micC is transcriptionally activated by SlyA, and SlyA feeds into the SPI1 regulatory network solely through MicC. Transcription of micC is negatively regulated by the OmpR/EnvZ two-component system, but this regulation is dependent on SlyA. OmpR/EnvZ control SPI1 expression partially through MicC, but also affect expression through other mechanisms. MicC-mediated regulation plays a role during infection, as evidenced by an increase in Salmonella fitness in the intestine in the micC deletion mutant that is dependent on the SPI1 T3SS. These results further elucidate the complex regulatory network controlling SPI1 expression and add to the list of sRNAs that control this primary virulence factor.

Linearized Siderophore Products Secreted via MacAB Efflux Pump Protect Salmonella enterica Serovar Typhimurium from Oxidative Stress

ABSTRACT Nontyphoidal salmonellae (NTS) are exposed to reactive oxygen species (ROS) during their residency in the gut. To survive oxidative stress encountered during infection, salmonellae employ several mechanisms. One of these mechanisms involves the multidrug efflux pump MacAB, although the natural substrate of this pump has not been identified. MacAB homologs in pseudomonads secrete products of nonribosomal peptide synthesis (NRPS). In Salmonella enterica serovar Typhimurium, the siderophore enterobactin is produced by NRPS in response to iron starvation and this molecule can be processed into salmochelin and several linear metabolites. We found that Salmonella mutants lacking the key NRPS enzyme EntF are sensitive to peroxide mediated killing and cannot detoxify extracellular H2O2. Moreover, EntF and MacAB function in a common pathway to promote survival of Salmonella during oxidative stress. We further demonstrated that S. Typhimurium secretes siderophores in iron-rich media when peroxide is present and that these MacAB-secreted metabolites participate in protection of bacteria against H2O2. We showed that secretion of anti-H2O2 molecules is independent of the presence of the known siderophore efflux pumps EntS and IroC, well-described efflux systems involved in secretion of enterobactin and salmochelin. Both salmochelin and enterobactin are dispensable for S. Typhimurium protection against ROS; however, linear metabolites of enterobactin produced by esterases IroE and Fes are needed for bacterial survival in peroxide-containing media. We determined that linearized enterobactin trimer protects S. Typhimurium against peroxide-mediated killing in a MacAB-dependent fashion. Thus, we suggest that linearized enterobactin trimer is a natural substrate of MacAB and that its purpose is to detoxify extracellular reactive oxygen species. IMPORTANCE Nontyphoidal Salmonella bacteria induce a classic inflammatory diarrhea by eliciting a large influx of neutrophils, producing a robust oxidative burst. Despite substantial progress understanding the benefits to the host of the inflammatory response to Salmonella, little is known regarding how Salmonella can simultaneously resist the damaging effects of the oxidative burst. The multidrug efflux pump MacAB is important for survival of oxidative stress both in vitro and during infection. We describe a new pathway used by Salmonella Typhimurium to detoxify extracellular reactive oxygen species using a multidrug efflux pump (MacAB) to secrete a linear siderophore, a metabolite of enterobactin. The natural substrates of many multidrug efflux pumps are unknown, and functional roles of the linear metabolites of enterobactin are unknown. We bring two novel discoveries together to highlight an important mechanism used by Salmonella to survive under the oxidative stress conditions that this organism encounters during the classic inflammatory diarrhea that it also induces.

A new role for heat shock factor 27 in the pathophysiology of Clostridium difficile toxin B

Clostridium difficile (CD) is a common pathogen that causes severe gastrointestinal inflammatory diarrhea in patients undergoing antibiotic therapy. Its virulence derives from two toxins, toxin CD, A and B (TcdA and TcdB) (Borriello et al. Rev Infect Dis 12, Suppl 2: S185-191, 1990). Among the prime candidates for CD colonization are patients with cystic fibrosis (CF), who are routinely treated with antibiotics and frequently hospitalized. Indeed, ~50% of patients with CF are colonized with virulent forms of CD but do not exhibit diarrhea (Bauer et al. Clin Microbiol Infect 20: O446–O449, 2014; Binkovitz et al. Am J Roentgenol 172: 517–521, 199; Zemljic et al. Anaerobe 16: 527–532, 2010). We found that TcdB has global effects on colonic cells, including reducing the steady-state levels of sodium-proton exchange regulatory factors, reducing the levels of heat shock protein (Hsp) 27, and increasing the fraction of total Hsp27 bound to the cystic fibrosis transmembrane conductance regulator (CFTR). Also, since some mutations in CFTR seem to be protective, we asked whether CFTR is a target of TcdB. We show here that TcdB increases the maturation of CFTR and transiently increases its function. These combined effects promote increased surface expression of CFTR, resulting in a transient increase in Cl− secretion. This increase is followed by a precipitous decline in both CFTR-dependent Cl− secretion and transepithelial resistance (TER), suggesting a breakdown in the epithelial cells’ tight junctions. We also found that overexpressing Hsp27 reverses some of the deleterious effects of TcdB, in particular preserving TER and therefore likely the maintenance of barrier function. Thus, our data suggest that Hsp27 plays a role in the diarrhea generated by CD infection and is a potential therapeutic target for treating this diarrhea. NEW & NOTEWORTHY Clostridium difficile (CD) is a common pathogen that causes severe gastrointestinal inflammatory diarrhea in patients undergoing antibiotic therapy. We provide new evidence that heat shock protein (Hsp) 27 is one of the key players in CD pathology and that increasing Hsp27 can prevent the decrease in transepithelial resistance induced by toxin CD B, pointing the way for pharmacologic therapies for patients with chronic CD infection that can increase Hsp27 as a means to mitigate the effects of CD on gastrointestinal pathology.

The Small RNA PinT Contributes to PhoP-Mediated Regulation of theSalmonellaPathogenicity Island 1 Type III Secretion System inSalmonella entericaSerovar Typhimurium

ABSTRACTSalmonella entericaserovar Typhimurium induces inflammatory diarrhea and bacterial uptake into intestinal epithelial cells using theSalmonellapathogenicity island 1 (SPI1) type III secretion system (T3SS). HilA activates transcription of the SPI1 structural components and effector proteins. Expression ofhilAis activated by HilD, HilC, and RtsA, which act in a complex feed-forward regulatory loop. Many environmental signals and other regulators are integrated into this regulatory loop, primarily via HilD. After the invasion ofSalmonellainto host intestinal epithelial cells or during systemic replication in macrophages, the SPI T3SS is no longer required or expressed. We have shown that the two-component regulatory system PhoPQ, required for intracellular survival, represses the SPI1 T3SS mostly by controlling the transcription ofhilAandhilD. Here we show that PinT, one of the PhoPQ-regulated small RNAs (sRNAs), contributes to this regulation by repressinghilAandrtsAtranslation. PinT base pairs with both thehilAandrtsAmRNAs, resulting in translational inhibition ofhilA, but also induces degradation of thertstranscript. PinT also indirectly represses expression of FliZ, a posttranslational regulator of HilD, and directly represses translation ofssrB, encoding the primary regulator of the SPI2 T3SS. Ourin vivomouse competition assays support the concept that PinT controls a series of virulence genes at the posttranscriptional level in order to adaptSalmonellafrom the invasion stage to intracellular survival.IMPORTANCESalmonellais one of the most important food-borne pathogens, infecting over one million people in the United States every year. These bacteria use a needle-like device to interact with intestinal epithelial cells, leading to invasion of the cells and induction of inflammatory diarrhea. A complex regulatory network controls expression of the invasion system in response to numerous environmental signals. Here we explore the molecular mechanisms by which the small RNA PinT contributes to this regulation, facilitating inactivation of the system after invasion. PinT controls several important virulence systems inSalmonella, tuning the transition between different stages of infection.

INTERACTION OF SALMONELLA WITH HOST ORGANISM

Diseases caused by Salmonella enterica species bacteria remain a healthcare challenge. Salmonella enterica species is divided into typhoid serovars that cause systemic infection and non-typhoid serovars that most frequently have a course of gastroenteritis with a development of inflammatory diarrhea. Both types of salmonella are opportunistic intracellular parasites able to invade and reproduce in both professional and non-professional phagocytes, e.g. M- cells and enterocytes. Invasion of cells and reproduction in them relates to functioning of salmonella pathogenicity island genes that determined synthesis of the third type of secretory system (T3SS). Contrary to the salmonella typhoid group serovars, non-typhoid serovars cause a development of inflammatory diarrhea, and effector molecules of T3SS as well as innate immunity components take part in it.