A Rapid Lysostaphin Production Approach and a Convenient Novel Lysostaphin Loaded Nano-emulgel; As a Sustainable Low-Cost Methicillin-Resistant Staphylococcus aureus Combating Platform.

Staphylococcus aureus is a Gram-positive pathogen that is capable of infecting almost every organ in the human body. Alarmingly, the rapid emergence of methicillin-resistant S.aureus strains (MRSA) jeopardizes the available treatment options. Herein, we propose sustainable, low-cost production of recombinant lysostaphin (rLST), which is a native bacteriocin destroying the staphylococcal cell wall through its endopeptidase activity. We combined the use of E. coli BL21(DE3)/pET15b, factorial design, and simple Ni-NTA affinity chromatography to optimize rLST production. The enzyme yield was up to 50 mg/L culture, surpassing reported systems. Our rLST demonstrated superlative biofilm combating ability by inhibiting staphylococcal biofilms formation and detachment of already formed biofilms, compared to vancomycin and linezolid. Furthermore, we aimed at developing a novel rLST topical formula targeting staphylococcal skin infections. The phase inversion composition (PIC) method fulfilled this aim with its simple preparatory steps and affordable components. LST nano-emulgel (LNEG) was able to extend active LST release up to 8 h and cure skin infections in a murine skin model. We are introducing a rapid, convenient rLST production platform with an outcome of pure, active rLST incorporated into an effective LNEG formula with scaling-up potential to satisfy the needs of both research and therapeutic purposes.

Isogenic mutations in the Moraxella catarrhalis CydDC system display pleiotropic phenotypes and reveal the role of a palindrome sequence in its transcriptional regulation.

Moraxella catarrhalis is becoming an important human respiratory tract pathogen affecting significant proportions from the population. However, still little is known about its physiology and molecular regulation. To this end, the CydDC, which is a heterodimeric ATP binding cassette transporter that has been shown to contribute to the maintenance of the redox homeostasis across the periplasm in other Gram-negative bacteria, is studied here. Amino acids multiple sequence alignments indicated that M. catarrhalis CydC is different from the CydC proteins of the bacterial species in which this system has been previously studied. These findings prompted further interest in studying this system in M. catarrhalis. Isogenic mutant in the CydDC system showed suppression in growth rate, hypersensitivity to oxidative and reductive stress and increased accumulation of intracellular cysteine levels. In addition, the growth of cydC- mutant exhibited hypersensitivity to exogenous cysteine; however, it did not display a significant difference from its wild-type counterpart in the murine pulmonary clearance model. Moreover, a palindrome was detected 94bp upstream of the cydD ORF suggesting it might act as a potential regulatory element. Real-time reverse transcription-PCR analysis showed that deletion/change in the palindrome resulted into alterations in the transcription levels of cydC. A better understanding of such system and its regulation helps in developing better ways to combat M. catarrhalis infections.

New 3-pyridinecarbonitrile derivatives and their antimicrobial properties.

2-Alkoxy-4,6-diaryl-3-pyridinecarbonitriles 2a-f were prepared through the reaction of 1,3-diaryl-2-propen-1-ones 1a-c with malononitrile in the appropriate alcohol in the presence of sodium. The reaction was assumed to take place through Michael addition followed by cyclization due to the alkoxide nucleophilic attack at one of the nitrile groups. This assumption was substantiated by isolation of the open-chain Michael adduct 4, followed by independent cyclization to the corresponding 2-alkoxy-3-pyridinecarbonitriles 2 upon treatment with the appropriate alcohol in the presence of sodium. Bromination of 4a,b with bromine in glacial acetic acid, afforded directly the corresponding 2-bromo-3-pyridinecarbonitriles 6a,b. The latters readily underwent nucleophilic substitution with different amines. The antimicrobial properties of the prepared compounds against Gram positive, Gram-negative, acid-fast bacteria and yeast were screened. Many of the prepared compounds show remarkable antimicrobial activity.