Subinhibitory concentration of ciprofloxacin targets quorum sensing system of Pseudomonas aeruginosa causing inhibition of biofilm formation & reduction of virulence.

Background & objectives: Biofilms formed by Pseudomonas aeruginosa lead to persistent infections. Use of antibiotics for the treatment of biofilm induced infection poses a threat towards development of resistance. Therefore, the research is directed towards exploring the property of antibiotics which may alter the virulence of an organism besides altering its growth. The aim of this study was to evaluate the role of subinhibitory concentration of ciprofloxacin (CIP) in inhibiting biofilm formation and virulence of P. aeruginosa. Methods: Antibiofilm potential of subinhibitory concentration of CIP was evaluated in terms of log reduction, biofilm forming capacity and coverslip assay. P. aeruginosa isolates (grown in the presence and absence of sub-MIC of CIP) were also evaluated for inhibition in motility, virulence factor production and quorum sensing (QS) signal production. Results: Sub-minimum inhibitory concentration (sub-MIC) of CIP significantly reduced the motility of P. aeruginosa stand and strain and clinical isolates and affected biofilm forming capacity. Production of protease, elastase, siderophore, alginate, and rhamnolipid was also significantly reduced by CIP. Interpretation & conclusions: Reduction in virulence factors and biofilm formation was due to inhibition of QS mechanism which was indicated by reduced production of QS signal molecules by P. aeruginosa in presence of subinhibitory concentration of CIP.

Inhibitory effect of polyunsaturated fatty acids on apoptosis induced by Streptococcus pneumoniae in alveolar macrophages.

Background & objectives: Apoptosis is considered as a major defense mechanism of the body. Multiple pathogens induce macrophage apoptosis as a mode of immune evasion. In earlier studies, n-3 polyunsaturated fatty acids (PUFA) have been reported to be protective against neuronal apoptosis and neuronal degeneration, seen after spinal cord injury. In this study, we tried to evaluate the role of n-3 polyunsaturated fatty acids on the process of macrophage phagocytic activity and apoptosis in mice. Methods: Mice were divided into three groups (n=60); Group I was fed on sea cod oil; Group II on flaxseed oil supplementation for 9 wk along with standard laboratory chow diet. Group III was fed on standard diet and served as control. After supplementation, phagocytic and apoptotic (morphological staining: acridine orange plus ethidium bromide; H-33342 plus propidium iodide staining and DNA ladder formation) activities of mouse alveolar macrophages were assessed. Results: Alveolar macrophages (obtained from sea cod oil and flaxseed oil fed group mice) showed significant increase in bacterial uptake as well as intracellular killing (P< 0.05) of Streptococcus pneumoniae. Significant decrease (P<0.05) in apoptotic cells was observed among alveolar macrophages from sea cod and flaxseed oil fed mice whereas maximum apoptosis was observed in control alveolar macrophages on interaction with bacteria in vitro which was confirmed by DNA laddering. Interpretation & conclusions: These findings suggest that dietary supplementation with n-3 polyunsaturated fatty acids to mice led to enhanced phagocytic capability of their alveolar macrophages as well as provided protection against apoptosis upon challenge with S. pneumoniae.

Screening & profiling of quorum sensing signal molecules in Pseudomonas aeruginosa isolates from catheterized urinary tract infection patients.

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.

Survival & detection of the faecal indicator bacterium Enterococcus faecalis in water stored in traditional vessels.

BACKGROUND & OBJECTIVE: Enterococci are used as indicators of faecal pollution and are typically detected using agar-based growth media incubated under standard aerobic conditions. However, such conditions may not be fully effective in enumerating injured bacteria, due to their sensitivity to reactive oxygen species (ROS) under aerobic conditions. Investigations were carried out to assess the extent of sub-lethal damage and ROS-sensitivity on different strains of Enterococcus faecalis in water stored in traditional brass and earthern vessels by enumerating the bacteria under standard aerobic conditions and under conditions designed to neutralize the effect of ROS. METHODS: Pure cultures of E. faecalis were maintained for up to 48 h in brass and earthern vessels and enumerated on various selective and non-selective media either under (i) standard aerobic conditions, (ii) aerobic conditions in a growth medium supplemented with the peroxide scavenger sodium pyruvate, (iii) anaerobic conditions using unsupplemented medium; and (iv) anaerobic conditions in a growth medium supplemented with sodium pyruvate, the latter being regarded as ROS-neutralized conditions. RESULTS: Counts of E. faecalis decreased substantially after storage for 12 h in water kept in the brass vessel but not in the earthern vessel. However, the decrease in counts depended upon the growth medium and the conditions used for enumeration, with a non-selective medium giving the highest count under ROS-neutralized conditions. While the counts obtained on various selective media were also enhanced under ROS-neutralized conditions, they remained lower than those of the non-selective medium. INTERPRETATION & CONCLUSION: Our study showed that growth conditions where reactive oxygen species are neutralized, were effective in enhancing the colony count of stressed E. faecalis, irrespective of the type of medium used for enumeration.

Induction & resolution of lobar pneumonia following intranasal instillation with Klebsiella pneumoniae in mice.

BACKGROUND & OBJECTIVES: Pneumonia caused by Klebsiella pneumoniae is important due to its high morbidity and mortality, especially in context of nosocomial infections. Many experimental studies have focused on the induction and progression of infection till it peaks, but the process of resolution has not been described. In the present study, we successfully attempted to establish an acute respiratory tract infection model in BALB/c strain of mice with K. pneumoniae employing a simple, reproducible intranasal instillation method. METHODS: Experimental pneumonia was induced by two strains of K. pneumoniae in BALB/c mice following intranasal instillation, and the course of pneumonia was studied by bacteriological and histopathological evaluation of the lung tissue. RESULTS: Both the strains were similar in their ability to induce infection which peaked on day 3, post infection. However, a strain dependent difference in relation to bacterial load and the process of resolution was observed. INTERPRETATION & CONCLUSION: The present study provides a model of lobar pneumonia produced by K. pneumoniae which can be useful for studying therapeutic and preventive interventions.