Purification and synergistic antibacterial activity of arginine derived cyclic dipeptides, from Achromobacter sp. associated with a rhabditid entomopathogenic nematode against major clinically relevant biofilm forming wound bacteria.

Skin and chronic wound infections caused by various pathogenic bacteria are an increasing and urgent health problem worldwide. In the present investigation ethyl acetate extract of an Achromobacter sp. associated with a Rhabditis entomopathogenic nematode (EPN), displayed promising antibacterial property and was further purified by silica gel column chromatography to get three different cyclic dipeptides (CDPs). Based on the spectral data and Marfey's analyses, the CDPs were identified as cyclo(D-Leu-D-Arg) (1), cyclo(L-Trp-L-Arg) (2), and cyclo(D-Trp-D-Arg) (3), respectively. Three CDPs were active against all the 10 wound associated bacteria tested. The significant antibacterial activity was recorded by CDP 3, and highest activity of 0.5 µg/ml was recorded against Staphylococcus aureus and Pseudomonas aeruginosa. The synergistic antibacterial activities of CDPs and ampicillin were assessed using the checkerboard microdilution method. The results of the current study recorded that the combined effects of CDPs and ampicillin principally recorded synergistic activity. Interestingly, the combination of CDPs and ampicillin also recorded enhanced inhibition of biofilm formation by bacteria. Moreover, CDPs significantly stimulate the production of IL-10 and IL-4 (anti-inflammatory cytokines) by human peripheral blood mononuclear cells. CDPs do not make any significant effect on the production of pro-inflammatory cytokines like TNF-α. The three CDPs have been studied for their effect on intracellular S. aureus in murine macrophages (J774) using 24 h exposure to 0.5X, 1X, and 2X MIC concentrations. Significant decrease in intracellular S. aureus burden was recorded by CDPs. CDPs also recorded no cytotoxicity toward FS normal fibroblast, VERO, and L231 normal lung epithelial cell lines. Antimicrobial activity of the arginine containing CDPs against the wound associated bacteria is reported here for the first. Moreover, this is also the first report on the production of CDPs by Achromobacter sp. Finally, we conclude that the Achromobacter sp. is an incredibly promising source of natural bioactive secondary metabolites especially against wound pathogenic bacteria that may receive significant benefit in the field of human medicine in near future as topical agents.

Rapid and sensitive detection of Phytophthora colocasiae responsible for the taro leaf blight using conventional and real-time PCR assay.

Conventional and real-time PCR assays were developed for sensitive and specific detection of Phytophthora colocasiae, an oomycete pathogen that causes leaf blight and corm rot of taro. A set of three primer pairs was designed from regions of the RAS-related protein (Ypt1), G protein alpha-subunit (GPA1) and phospho-ribosylanthranilate isomerase (TRP1) genes. In conventional PCR, the lower limit of detection was 50 pg DNA, whereas in real-time PCR, the detection limit was 12.5 fg for the primer based on Ypt1 gene. The cycle threshold values were linearly correlated with the concentration of the target DNA (range of R(2) = 0.911-0.999). All the primer sets were successful in detecting P. colocasie from naturally infected leaves and tubers of taro. Phytophthora colocasiae was detected from artificially infested samples after 18 and 15 h of postinoculation in conventional and real-time PCR assay, respectively. The developed PCR assay proved to be a robust and reliable technique to detect P. colocasiae in taro planting material and for assessing the distribution of pathogen within fields, thus aid in mitigating taro leaf blight.