Evaluation of interleukin-10 production in Pseudomonas aeruginosa induced acute pyelonephritis

SummaryPseudomonas aeruginosa is an opportunistic pathogen of immunocompromised hosts. This pathogen has a tendency to form biofilms on the surface of indwelling catheters leading to acute and chronic urinary tract infections that result in significant morbidity and mortality. In the present study, kinetics of interleukin-10 (IL-10) production in mouse renal tissue was studied employing experimental mouse model of acute pyelonephritis induced with planktonic and biofilm cells of P. aeruginosa. IL-10 production was found to be significantly lower in biofilm cell instilled mice compared to planktonic cell infected animals, which corroborated with higher bacterial load and tissue damage. The data suggests that downregulation of IL-10 production may be novel strategy employed by biofilm cells to cause tissue damage and hence bacterial persistence. The results of the present study may open up avenues of research that will ultimately provide the foundation for the development of preventative measures and therapeutic strategies to successfully treat P. aeruginosa biofilm infections based on the administration of anti-inflammatory agents

OPTIMIZATION OF GROWTH CONDITIONS AND PURIFICATION OF QUORUM SENSING SIGNAL MOLECULES PRODUCED BY

Expression of virulence factors and biofilm formation in P.aeruginosa is associated with production of quorum sensing signal molecules (QSSMs) belonging to the class of acyl homoserine lactones (AHLs). Besides regulating virulence factors, these molecules also interact with eukaryotic cells and can modulate immune response. In most of the studies, synthetic QSSMs have been employed as therapeutic agents. Although 98-99 % homology exist between synthetic and natural AHLs but the biological response against either may in fact be different in natural host. In the present study, under optimized growth conditions there is increase in the production of natural AHLs. Extracted AHLs were detected using C18 reverse phase analytical thin layer chromatography (RP TLC) by employing Agrobacterium tumefaciens as biosensor strain. Preparative TLC assay was successfully performed to purify the 3oxo-C12-HSL and 3-oxo-C10-HSL. This study provides easy and simple method for purification of natural AHLs under optimized conditions, hence these molecules can be employed for future research involving control of infections associated with P.aeruginosa

Acyl Homoserine Lactones from Culture Supernatants of Pseudomonas aeruginosa Accelerate Host Immunomodulation

The virulence of Pseudomonas aeruginosa is multifactorial and under the control of quorum sensing signals, such as acyl homoserine lactones (AHLs). The importance of these molecules in the establishment of infection has been previously reported. These molecules either improve the virulence potential of P. aeruginosa or modulate the host immune response. To establish the immune modulating potential of quorum sensing signal molecules, previous studies have only used synthetic AHLs. However, there can be differences in the biological properties of synthetic and natural AHLs. The use of naturally extracted AHLs from the culture supernatant of P. aeruginosa is likely to simulate natural conditions more than the use of synthetic AHLs. Therefore, in the present study, the immune modulating potential of synthetic and naturally extracted AHLs was compared using a thymidine uptake assay, immunophenotyping and sandwich ELISA in order to assess mouse T-cell proliferation and production of Th1 and Th2 cytokines. Natural AHLs were able to suppress T-cell proliferation, even at low concentrations, compared to synthetic AHLs. The majority of cells undergoing proliferation were CD4+, as revealed by immunophenotyping. The inhibition of T-cells was stronger with natural AHLs compared to synthetic AHLs. Moreover, the natural AHLs were also able to shift immune responses away from host protective Th1 responses to pathogen protective Th2 responses

Inactivation and sub-lethal injury of salmonella typhi, salmonella typhimurium and vibrio cholerae in copper water storage vessels

Background: This study provides information on the antibacterial effect of copper against the water-borne pathogens Salmonella Typhi, Salmonella Typhimurium and Vibrio cholerae. Methods: Suspensions of each pathogen were kept in water within a traditional copper vessel at 30°C for 24 h. Samples were withdrawn, diluted and plated onto suitable growth media. Conventional enumeration of healthy (uninjured) bacteria was carried out using standard aerobic incubation conditions. Additionally, reactive oxygen species-neutralised (ROS-n) conditions were achieved by adding the peroxide scavenger sodium pyruvate to the medium with anaerobic incubation, to enumerate uninjured (ROS-insensitive) and injured (ROS-sensitive) bacteria. Differences between log-transformed means of conventional (aerobic) and ROS-n counts were statistically evaluated using t tests. Results: Overall, all three pathogens were inactivated by storage in copper vessels for 24 h. However, for shorter-term incubation (4-12 h), higher counts were observed under ROS-n conditions than under aerobic conditions, which demonstrate the presence of substantial numbers of sub-lethally injured cells prior to their complete inactivation. Conclusions: The present study has for the first time confirmed that these bacterial pathogens are inactivated by storage in a copper vessel within 24 h. However, it has also demonstrated that it is necessary to account for short-term sub-lethal injury, manifest as ROS-sensitivity, in order to more fully understand the process. This has important practical implications in terms of the time required to store water within a copper vessel to completely inactivate these bacteria and thereby remove the risk of water-borne disease transmission by this route

Acute lung inflammation in Klebsiella pneumoniae B5055-induced pneumonia and sepsis in BALB/c mice: a comparative study

Lungs play an important role in the body's defense against a variety of pathogens, but this network of immune system-mediated defense can be deregulated during acute pulmonary infections. The present study compares acute lung inflammation occurring during Klebsiella pneumoniae B5055-induced pneumonia and sepsis in BALB/c mice. Pneumonia was induced by intranasal instillation of bacteria (10 4 cfu), while sepsis was developed by placing the fibrin-thrombin clot containing known amount of bacteria (10 2 cfu) into the peritoneal cavity of animals. Mice with sepsis showed 100% mortality within five postinfection days, whereas all the animals with pneumonia survived. In animals suffering from K. pneumoniae B5055-induced pneumonia, all the inflammatory parameters (TNF-α, IL-1α, MPO, MDA, and NO) were found to be maximum till third post-infection day, after that, a decline was observed, whereas in septic animals, all the above-mentioned markers of inflammation kept on increasing. Histopathological study showed presence of alternatively activated alveolar macrophages (or foam cells) in lungs of mice with pneumonia after third post-infection day, which might have contributed to the induction of resolution of inflammation, but no such observation was made in lungs of septic mice. Hence, during pneumonia, controlled activation of macrophages may lead to resolution of inflammation