Background

Staphylococcus aureus remains the predominant pathogen in diabetic foot infections and prevalence of methicillin resistant S.aureus (MRSA) strains further complicates the situation. The incidence of MRSA in infected foot ulcers is 15–30% and there is an alarming trend for its increase in many countries. Diabetes acts as an immunosuppressive state decreasing the overall immune functioning of body and to worsen the situation, wounds inflicted with drug resistant strains represent a morbid combination in diabetic patients. Foot infections caused by MRSA are associated with an increased risk of amputations, increased hospital stay, increased expenses and higher infection-related mortality. Hence, newer, safer and effective treatment strategies are required for treating MRSA mediated diabetic foot infections. The present study focuses on the use of lytic bacteriophage in combination with linezolid as an effective treatment strategy against foot infection in diabetic population.

Methodology

Acute hindpaw infection with S.aureus ATCC 43300 was established in alloxan induced diabetic BALB/c mice. Therapeutic efficacy of a well characterized broad host range lytic bacteriophage, MR-10 was evaluated alone as well as in combination with linezolid in resolving the course of hindpaw foot infection in diabetic mice. The process of wound healing was also investigated.

Results and Conclusions

A single administration of phage exhibited efficacy similar to linezolid in resolving the course of hindpaw infection in diabetic animals. However, combination therapy using both the agents was much more effective in arresting the entire infection process (bacterial load, lesion score, foot myeloperoxidase activity and histopathological analysis). The entire process of tissue healing was also hastened. Use of combined agents has been known to decrease the frequency of emergence of resistant mutants, hence this approach can serve as an effective strategy in treating MRSA mediated foot infections in diabetic individuals who do not respond to conventional antibiotic therapy.

Background & objectives:

Catheter associated urinary tract infections are the second most common nosocomial infections and Pseudomonas aeruginosa is the third most common organism responsible for these infections. In this study P. aeruginosa isolates from catheterized urinary tract infection patients were screened and profiled for the presence of different type of quorum sensing (QS) signal molecules.

Methods:

Screening and quantitation of AHLs was done by using cross feeding assay and by determining β-galactosidase activity respectively using Escherichia coli MG4 as reporter strain. Further, AHL profiles were determined by separating AHLs on TLC coupled with their detection using Chromobacterium violaceum CV026 and Agrobacterium tumifaciens A136 biosensor strains.

Results:

All uroisolates from catheterized patients having urinary tract infections were found to be producers of QS signal molecules. There were differences in amounts and type of AHL produced amongst uroisolates of P. aeruginosa. Several AHLs belonging to C4-HSL, C6-HSL, oxo-C6-HSL, C8-HSL, C10-HSL and C12-HSL were determined in these strains.

Interpretation & conclusions:

Simultaneous use of more than one reporter strain and assay method proved useful in determining the AHLs profile in uroisolates of P. aeruginosa. Observed differences in the amounts and types of AHLs may reflect differences in virulence potential of P. aeruginosa to cause UTIs which can be further confirmed by employing animal model system. The present study speculates that production of QS signal molecules may act as a new virulence marker of P. aeruginosa responsible for causing catheter associated UTIs and can be considered as futuristic potential drug targets towards treatment of UTIs.

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.

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.

This study investigated the effect of copper as an antibacterial agent on the infectivity of Salmonella enterica serovar Typhimurium. Mice were infected orally with a standardized dose of unstressed Salmonella Typhimurium and copper-stressed cells of Salmonella Typhimurium. Bacterial counts in ileum, blood, liver and spleen were observed up to 168 h under normal aerobic conditions. Serum sensitivity, phagocytosis, malondialdehyde levels and histopathology were studied for both set of animals. A decreased bacterial count in the organs with mild symptoms of infection and a complete recovery by 48 h was observed in mice infected with copper-stressed bacteria. Histopathological examination of ileum tissue demonstrated regeneration of damaged tissue post-infection with copper-stressed bacteria and no malondialdehyde levels were detected after 24 h in ileum, spleen and liver. Exposure to copper sensitized Salmonella Typhimurium to the lytic action of serum and intracellular killing by peritoneal macrophages. It can be concluded that copper stress confers a decrease in the infectivity of healthy Salmonella Typhimurium in normal mice. This study highlights the significance of use of copper as an antibacterial agent against Salmonella Typhimurium in reducing the risk of incidence of Salmonella infections from contaminated water.

Urinary tract infections may induce severe inflammation, transient impairment in renal function and scar formation, ranging in severity from acute symptomatic pyelonephritis to chronic pyelonephritis, and have the potential to lead to renal failure and death. In the present study, the relationship between production of tumour necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), neutrophil recruitment, bacterial colonization and tissue damage was investigated using a mouse model of acute ascending pyelonephritis induced with planktonic and biofilm cells of Pseudomonas aeruginosa. Neutrophil influx correlated with rise in TNF-α and IL-1β, indicating an association between these cytokines and neutrophil infiltration. However, biofilm cells of P aeruginosa induced higher levels of TNF-α and IL-1β leading to higher neutrophil infiltration causing tissue damage, assessed in terms of malondialdehyde, lactate dehydrogenase and glutathione content, which may have contributed to bacterial persistence compared with their planktonic counterparts. The results of the present investigation suggest that exaggerated cytokine production during P aeruginosa-induced pyelonephritis causes tissue damage operative through neutrophil recruitment leading to bacterial persistence in host tissues. The findings of the present study may be relevant for the better understanding of disease pathophysiology and for the future developments of preventive strategies against pyelonephritis based on anti-inflammatory intervention.