A Pseudomonas aeruginosa small RNA regulates chronic and acute infection.

The ability to switch between different lifestyles allows bacterial pathogens to thrive in diverse ecological niches1,2. However, a molecular understanding of their lifestyle changes within the human host is lacking. Here, by directly examining bacterial gene expression in human-derived samples, we discover a gene that orchestrates the transition between chronic and acute infection in the opportunistic pathogen Pseudomonas aeruginosa. The expression level of this gene, here named sicX, is the highest of the P. aeruginosa genes expressed in human chronic wound and cystic fibrosis infections, but it is expressed at extremely low levels during standard laboratory growth. We show that sicX encodes a small RNA that is strongly induced by low-oxygen conditions and post-transcriptionally regulates anaerobic ubiquinone biosynthesis. Deletion of sicX causes P. aeruginosa to switch from a chronic to an acute lifestyle in multiple mammalian models of infection. Notably, sicX is also a biomarker for this chronic-to-acute transition, as it is the most downregulated gene when a chronic infection is dispersed to cause acute septicaemia. This work solves a decades-old question regarding the molecular basis underlying the chronic-to-acute switch in P. aeruginosa and suggests oxygen as a primary environmental driver of acute lethality.

Contribution of Pseudomonas aeruginosa Exopolysaccharides Pel and Psl to Wound Infections.

Biofilms are the cause of most chronic bacterial infections. Living within the biofilm matrix, which is made of extracellular substances, including polysaccharides, proteins, eDNA, lipids and other molecules, provides microorganisms protection from antimicrobials and the host immune response. Exopolysaccharides are major structural components of bacterial biofilms and are thought to be vital to numerous aspects of biofilm formation and persistence, including adherence to surfaces, coherence with other biofilm-associated cells, mechanical stability, protection against desiccation, binding of enzymes, and nutrient acquisition and storage, as well as protection against antimicrobials, host immune cells and molecules, and environmental stressors. However, the contribution of specific exopolysaccharide types to the pathogenesis of biofilm infection is not well understood. In this study we examined whether the absence of the two main exopolysaccharides produced by the biofilm former Pseudomonas aeruginosa would affect wound infection in a mouse model. Using P. aeruginosa mutants that do not produce the exopolysaccharides Pel and/or Psl we observed that the severity of wound infections was not grossly affected; both the bacterial load in the wounds and the wound closure rates were unchanged. However, the size and spatial distribution of biofilm aggregates in the wound tissue were significantly different when Pel and Psl were not produced, and the ability of the mutants to survive antibiotic treatment was also impaired. Taken together, our data suggest that while the production of Pel and Psl do not appear to affect P. aeruginosa pathogenesis in mouse wound infections, they may have an important implication for bacterial persistence in vivo.

Assessing Biofilm Dispersal in Murine Wounds.

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring otitis media, and many more. Microbial cells within these infections are protected by an extracellular polymeric substance (EPS), which can prevent antibiotics and host immune cells from clearing the infection. To overcome this obstacle, investigators have begun developing dispersal agents as potential therapeutics. These agents target various components within the biofilm EPS, weakening the structure, and initiating dispersal of the bacteria, which can theoretically improve antibiotic potency and immune clearance. To determine the efficacy of dispersal agents for wound infections, we have developed protocols that measure biofilm dispersal both ex vivo and in vivo. We use a mouse surgical excision model that has been well-described to create biofilm-associated chronic wound infections. To monitor dispersal in vivo, we infect the wounds with bacterial strains that express luciferase. Once mature infections have established, we irrigate the wounds with a solution containing enzymes that degrade components of the biofilm EPS. We then monitor the location and intensity of the luminescent signal in the wound and filtering organs to provide information about the level of dispersal achieved. For ex vivo analysis of biofilm dispersal, infected wound tissue is submerged in biofilm-degrading enzyme solution, after which the bacterial load remaining in the tissue, versus the bacterial load in solution, is assessed. Both protocols have strengths and weaknesses and can be optimized to help accurately determine the efficacy of dispersal treatments.

Efficacy and safety of biofilm dispersal by glycoside hydrolases in wounds.

Novel anti-biofilm and dispersal agents are currently being investigated in an attempt to combat biofilm-associated wound infections. Glycoside hydrolases (GHs) are enzymes that hydrolyze the glycosidic bonds between sugars, such as those found within the exopolysaccharides of the biofilm matrix. Previous studies have shown that GHs can weaken the matrix, inducing bacterial dispersal, and improving antibiotic clearance. Yet, the number of GH enzymes that have been examined for potential therapeutic effects is limited. In this study, we screened sixteen GHs for their ability to disperse mono-microbial and polymicrobial biofilms grown in different environments. Six GHs, α-amylase (source: A. oryzae), alginate lyase (source: various algae), pectinase (source: Rhizopus sp.), amyloglucosidase (source: A. niger), inulinase (source: A. niger), and xylanase (source: A. oryzae), exhibited the highest dispersal efficacy in vitro. Two GHs, α-amylase (source: Bacillus sp.) and cellulase (source: A. niger), used in conjunction with meropenem demonstrated infection clearing ability in a mouse wound model. GHs were also effective in improving antibiotic clearance in diabetic mice. To examine their safety, we screened the GHs for toxicity in cell culture. Overall, there was an inverse relationship between enzyme exposure time and cellular toxicity, with twelve out of sixteen GHs demonstrating some level of toxicity in cell culture. However, only one GH exhibited harmful effects in mice. These results further support the ability of GHs to improve antibiotic clearance of biofilm-associated infections and help lay a foundation for establishing GHs as therapeutic agents for chronic wound infections.

Differential Efficacy of Glycoside Hydrolases to Disperse Biofilms.

Chronic wounds will impact 2% of the United States population at some point in their life. These wounds are often associated with a reoccurring, chronic infection caused by a community of microorganisms encased in an extracellular polymeric substance (EPS), or a biofilm. Biofilm-associated microbes can exhibit tolerance to antibiotics, which has prompted researchers to investigate therapeutics that improve antibiotic efficacy. Glycoside hydrolases (GHs), enzymes that target the polysaccharide linkages within the EPS, are one potential adjunctive therapy. In order to develop GH-based therapeutics, it is imperative that we understand whether the composition of biofilm EPS changes based on the environment and/or presence of other microbes. Here, we utilized α-amylase and cellulase to target the polysaccharides within the EPS of mono- and dual-species Pseudomonas aeruginosa and Staphylococcus aureus biofilms in three different models that vary in clinical relevancy. We show that biofilms established in an in vitro well-plate model are not strongly adhered to the polystyrene surface and do not accurately reflect the GH efficacy seen with biofilms grown in vivo. However, dispersal efficacy in an in vitro wound microcosm model was more reflective of that seen in a murine wound model. We also saw a striking loss of efficacy for cellulase to disperse S. aureus in both mono- and dual species biofilms grown in the wound models, suggesting that EPS constituents may be altered depending on the environment.

Utilizing glycoside hydrolases to improve the quantitation and visualization of biofilm bacteria.

The complexity of microbial biofilms offers several challenges to the use of traditional means of microbial research. In particular, it can be difficult to calculate accurate numbers of biofilm bacteria, because even after thorough homogenization or sonication, small pieces of the biofilm remain, which contain numerous bacterial cells and result in inaccurately low colony forming units (CFU). In addition, imaging of infected tissue ex vivo often results in a disparity between the CFU and the number of bacterial cells observed under the microscope. We hypothesized that this phenomenon is due to the biofilm extracellular polymeric substance decreasing the accessibility of stains and antibodies to the embedded bacterial cells. In this study, we describe incorporating EPS-degrading glycoside hydrolases for CFU determination to obtain a more accurate estimation of the viable cells and for immunohistochemistry to disrupt the biofilm matrix and increase primary antibody binding to the bacterial cells.

Immune responses in liver and spleen against Plasmodium yoelii pre-erythrocytic stages in Swiss mice model.

Though the immunity to malaria has been associated with cellular immune responses, the exact function of the phenotypic cell population is still unclear. This study investigated the host immune responses elicited during the pre-erythrocytic stage, post-Plasmodium yoelii sporozoite infection in Swiss mice model. For this purpose, we analyzed the dynamics of different subsets of immune cells population and cytokine levels in the hepatic mononuclear and splenic cells population during pre-erythrocytic liver-stage infection. We observed a significant reduction in the effectors immune cells population including CD8+ T cell, F4/80+ macrophage and in plasmacytoid dendritic cells (CD11c+ B220+). Interestingly, substantial down-regulation was also noted in pro-inflammatory cytokines (i.e. IFN-γ, TNF-α, IL-12, IL-2, IL-17 and iNOS), while, up-regulation of anti-inflammatory cytokines (i.e. IL-10, IL-4 and TGF-ß) during asymptomatic pre-erythrocytic liver-stage infection. Collectively, this study demonstrated that during pre-erythrocytic development, Plasmodium yoelii sporozoite impaired the host activators of innate and adaptive immune responses by regulating the immune effector cells, gene expression and cytokines levels for the establishment of infection and subsequent development in the liver and spleen. The results in this study provided a better understanding of the events leading to malarial infection and will be helpful in supportive treatment and vaccine development strategy.

Neurological disorder and psychosocial aspects of cerebral malaria: what is new on its pathogenesis and complications? A minireview.

Recently, malaria is remain considered as the most prevalent infectious disease, affecting the human health globally. High morbidity and mortality worldwide is often allied with cerebral malaria (CM) based disorders of the central nervous system, especially across many tropical and sub-tropical regions. These disorders are characterised by the infection of Plasmodium species, which leads to acute or chronic neurological disorders, even after having active/effective antimalarial drugs. Furthermore, even during the treatment, individual remain sensitive for neurological impairments in the form of decrease blood flow and vascular obstruction in brain including many more other changes. This review briefly explains and update on the epidemiology, burden of disease, pathogenesis and role of CM in neurological disorders with behaviour and function in mouse and human models. Moreover, the social stigma, which plays an important role in neurological disorders and a factor for assessing CM, is also discussed in this review.

Sm-p80-based schistosomiasis vaccine: double-blind preclinical trial in baboons demonstrates comprehensive prophylactic and parasite transmission-blocking efficacy.

Schistosomiasis is of public health importance to an estimated one billion people in 79 countries. A vaccine is urgently needed. Here, we report the results of four independent, double-blind studies of an Sm-p80-based vaccine in baboons. The vaccine exhibited potent prophylactic efficacy against transmission of Schistosoma mansoni infection and was associated with significantly less egg-induced pathology, compared with unvaccinated control animals. Specifically, the vaccine resulted in a 93.45% reduction of pathology-producing female worms and significantly resolved the major clinical manifestations of hepatic/intestinal schistosomiasis by reducing the tissue egg-load by 89.95%. A 35-fold decrease in fecal egg excretion in vaccinated animals, combined with an 81.51% reduction in hatching of eggs into the snail-infective stage (miracidia), demonstrates the parasite transmission-blocking potential of the vaccine. Substantially higher Sm-p80 expression in female worms and Sm-p80-specific antibodies in vaccinated baboons appear to play an important role in vaccine-mediated protection. Preliminary analyses of RNA sequencing revealed distinct molecular signatures of vaccine-induced effects in baboon immune effector cells. This study provides comprehensive evidence for the effectiveness of an Sm-p80-based vaccine for schistosomiasis.

Sm-p80-based vaccine trial in baboons: efficacy when mimicking natural conditions of chronic disease, praziquantel therapy, immunization, and Schistosoma mansoni re-encounter.

Sm-p80-based vaccine efficacy for Schistosoma mansoni was evaluated in a baboon model of infection and disease. The study was designed to replicate a human vaccine implementation scenario for endemic regions in which vaccine would be administered following drug treatment of infected individuals. In our study, the Sm-p80-based vaccine reduced principal pathology producing hepatic egg burdens by 38.0% and egg load in small and large intestines by 72.2% and 49.4%, respectively, in baboons. Notably, hatching rates of eggs recovered from liver and small and large intestine of vaccinated animals were significantly reduced, by 60.4%, 48.6%, and 82.3%, respectively. Observed reduction in egg maturation/hatching rates was supported by immunofluorescence and confocal microscopy showing unique differences in Sm-p80 expression in worms of both sexes and matured eggs. Vaccinated baboons had a 64.5% reduction in urine schistosome circulating anodic antigen, a parameter that reflects worm numbers/health status in infected hosts. Preliminary analyses of RNA sequencing revealed unique genes and canonical pathways associated with establishment of chronic disease, praziquantel-mediated parasite killing, and Sm-p80-mediated protection in vaccinated baboons. Overall, our study demonstrated efficacy of the Sm-p80 vaccine and provides insight into some of the epistatic interactions associated with protection.

Gastrointestinal helminths of Coyotes (Canis latrans) from Southeast Nebraska and Shenandoah area of Iowa.

AIM: This survey was carried out on the carcasses of 29 coyotes from Southeastern Nebraska and Shenandoah area of Iowa to document the helminths present in the intestinal track of these carnivorous animals. MATERIALS AND METHODS: A total of 29 adult coyote carcasses were generously donated in the autumn and winter (November-February) of 2014-2015 by trappers, fur buyers and hunters of Southeast Nebraska and Shenandoah area of Iowa. The intestine of individual animals were examined for the recovery of helminth parasites as per the established procedures. RESULTS: We found that as many as 93.10% of the investigated coyotes were infected with one or more helminth infections. A total of 10 different species of helminth parasites were recovered from the intestines of coyotes under investigation. Among the 10 species of helminths, 5 were identified as cestodes while the remaining 5 were nematodes. A total of 82.75% of the animals were infected with one or more species of nematodes, while 75.86% of them were colonized with one or more species of cestode parasites. The most abundant species in coyotes were Toxascaris leonina (68.95%) closely followed by Taenia hydatigena (58.62%). The prevalence of Ancylostoma caninum and Taenia pisiformis were recorded at 31.03%, followed by those of Toxocara canis and Echinococcus spp. at 24.13%, respectively. Three animals were infected with Trichuris vulpis while three other coyotes each were found to be harboring Uncinaria stenocephala, Dipylidium caninum, or Hymenolepis diminuta. The presence of H. diminuta might have been the result of the ingestion of a rodent by the respective coyotes. CONCLUSION: From the overall analysis of the present data and comparing it with the previous reports of various scientists over several decades, we can conclude that intestinal helminths are still very much prevalent among the coyote population in the Southeast Nebraska and Iowa area. The relatively high prevalence of the zoonotic parasite species further warrants a more comprehensive investigation with larger numbers of wild predators from the region to ascertain the possible contribution of coyotes to the disease cycle as these animals are more frequently spotted in and around the densely populated urban areas.