Non-protein coding RNA (npcRNA) is a functional RNA molecule that is not translated into a protein. Bacterial npcRNAs are structurally diversified molecules, typically 50-200 nucleotides in length. They play a crucial physiological role in cellular networking, including stress responses, replication and bacterial virulence. In this study, by using an identified npcRNA gene (Sau-02) in Methicillin-resistant Staphylococcus aureus (MRSA), we identified the Gram-positive bacteria S. aureus. A Sau-02-mediated monoplex Polymerase Chain Reaction (PCR) assay was designed that displayed high sensitivity and specificity. Fourteen different bacteria and 18 S. aureus strains were tested, and the results showed that the Sau-02 gene is specific to S. aureus. The detection limit was tested against genomic DNA from MRSA and was found to be ~10 genome copies. Further, the detection was extended to whole-cell MRSA detection, and we reached the detection limit with two bacteria. The monoplex PCR assay demonstrated in this study is a novel detection method that can replicate other npcRNA-mediated detection assays.
Staphylococci are facultative anaerobes, perfectly spherical un-encapsulated cocci, with a diameter not exceeding 1 micrometer in diameter. Staphylococcus aureus are generally harmless and remain confined to the skin unless they burrow deep into the body, causing life-threatening infections in bones, joints, bloodstream, heart valves and lungs. Among the 20 medically important staphylococci species, Staphylococcus aureus is one of the emerging human pathogens. Streptomycin had its highest potency against Staphylococcus infections despite the likelihood of getting a resistant type of staphylococcus strains. Methicillin-resistant S. aureus (MRSA) is the persister type of Staphylococcus aureus and was evolved after decades of antibiotic misuse. Inadequate penetration of the antibiotic is one of the principal factors related to success/failure of the therapy. The active drug needs to reach the bacteria at concentrations necessary to kill or suppress the pathogen's growth. In turn the effectiveness of the treatment relied on the physical properties of Staphylococcus aureus. Thus understanding the cell integrity, shape and roughness is crucial to the overall influence of the therapeutic agent on S. aureus of different origins. Hence our experiments were designed to clarify ultrastructural changes of S. aureus treated with streptomycin (synthetic compound) in comparison to artonin E (natural compound). In addition to the standard in vitro microbial techniques, we used transmission electron microscopy to study the disrupted cell architecture under antibacterial regimen and we correlate this with scanning electron microscopy (SEM) to compare results of both techniques.
28 new pyrrolidine types of compounds as analogues for natural polyhydroxy alkaloids of codonopsinine were evaluated for their anti-MRSA activity using MIC and MBC value determination assay against a panel of S. aureus isolates. One pyrrolidine compound, MFM 501, exhibited good inhibitory activity with MIC value of 15.6 to 31.3 μg/mL against 55 S. aureus isolates (43 MRSA and 12 MSSA isolates). The active compound also displayed MBC values between 250 and 500 μg/mL against 58 S. aureus isolates (45 MRSA and 13 MSSA isolates) implying that MFM 501 has a bacteriostatic rather than bactericidal effect against both MRSA and MSSA isolates. In addition, MFM 501 showed no apparent cytotoxicity activity towards three normal cell lines (WRL-68, Vero, and 3T3) with IC50 values of >625 µg/mL. Selectivity index (SI) of MFM 501 gave a value of >10 suggesting that MFM 501 is significant and suitable for further in vivo investigations. These results suggested that synthetically derived intermediate compounds based on natural products may play an important role in the discovery of new anti-infective agents against MRSA.