Resensitisation of Methicillin-Resistant Staphylococcus aureus to Conventional Antibiotics in the Presence of an Engineered Enzybiotic.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most dreadful pathogens relevant in community and nosocomial-related infections around the world. Resensitising MRSA to antibiotics, once it became resistant, was a tough choice due to the high adaptability of this bacteria to savage conditions. This study aimed to create a chimeric enzybiotic against MRSA and test its efficiency, either individually or in combination with antibiotics. The novel enzybiotic BAC100 was constructed by fusing the catalytic domain from the bacteriocin BacL1 from Enterococcus faecalis with the cell-wall-binding domain from protein P17 of Staphylococcus aureus bacteriophage ϕ44AHJD. Apart from its partial lone activity, BAC100 was found to resensitise the MRSA strain to traditional antibiotics, including ampicillin and tetracycline. Both drugs were able to reduce live MRSA cells by 85 and 90%, respectively, within 60 min of treatment together with BAC100. However, no significant activity was observed against MRSA when these drugs were tested independently, pointing to the inherent resistance of MRSA against these conventional antibiotics. To our knowledge, this is one of the first instances where an engineered enzybiotic was found to resensitise MRSA to conventional antibiotics. This study will pave the way for the development of similar peptides that can be used together with antibiotics against gruesome pathogens of clinical importance.

An Enzybiotic Cocktail Effectively Disrupts Preformed Dual Biofilm of Staphylococcus aureus and Enterococcus faecalis.

Multidrug-resistant bacterial infections are on the rise around the world. Chronic infections caused by these pathogens through biofilm mediation often complicate the situation. In natural settings, biofilms are often formed with different species of bacteria existing synergistically or antagonistically. Biofilms on diabetic foot ulcers are formed predominantly by two opportunistic pathogens, Staphylococcus aureus and Enterococcus faecalis. Bacteriophages and phage-based proteins, including endolysins, have been found to be active against biofilms. In this study, we evaluated the activity of two engineered enzybiotics either by themselves or as a combination against a dual biofilm formed by S. aureus and E. faecalis in an inert glass surface. An additive effect in rapidly disrupting the preformed dual biofilm was observed with the cocktail of proteins, in comparison with mono treatment. The cocktail-treated biofilms were dispersed by more than 90% within 3 h of treatment. Apart from biofilm disruption, bacterial cells embedded in the biofilm matrix were also effectively reduced by more than 90% within 3 h of treatment. This is the first instance where a cocktail of engineered enzybiotics has been effectively used to impede the structural integrity of a dual biofilm.

Construction and Activity Testing of a Modular Fusion Peptide against Enterococcus faecalis.

The emergence of antibiotic resistance in enterococci is a great concern encountered worldwide. Almost all enterococci exhibit significant levels of resistance to penicillin, ampicillin, semi-synthetic penicillin and most cephalosporins, primarily due to the expression of low-affinity penicillin-binding proteins. The development of new and novel antibacterial agents against enterococci is a significant need of the hour. In this research, we have constructed a modular peptide against Enterococcus faecalis. The enzymatic domain of the constructed peptide BP404 is from the bacteriocin BacL1 and the cell wall binding domain from endolysin PlyV12 of phage ϕ1. The protein BP404 was found to be active against two tested strains of Enterococcus faecalis, with a reduction in cell density amounting to 85% and 65%. The cell wall binding assay confirms the binding of the protein to Enterococcus faecalis, which was not seen towards the control strain Escherichia coli, invariably pointing to the specificity of BP404. To the best of our knowledge, this is one of the first instances of the development of a chimeric peptide against Enterococcus faecalis. This study points out that novel proteins can be genetically engineered against clinically relevant enterococci.

An Engineered Multimodular Enzybiotic against Methicillin-Resistant Staphylococcus aureus.

Development of multidrug antibiotic resistance in bacteria is a predicament encountered worldwide. Researchers are in a constant hunt to develop effective antimicrobial agents to counter these dreadful pathogenic bacteria. Here we describe a chimerically engineered multimodular enzybiotic to treat a clinical isolate of methicillin-resistant Staphylococcus aureus (S. aureus). The cell wall binding domain of phage ϕ11 endolysin was replaced with a truncated and more potent cell wall binding domain from a completely unrelated protein from a different phage. The engineered enzybiotic showed strong activity against clinically relevant methicillin-resistant Staphylococcus aureus. In spite of a multimodular peptidoglycan cleaving catalytic domain, the engineered enzybiotic could not exhibit its activity against a veterinary isolate of S. aureus. Our studies point out that novel antimicrobial proteins can be genetically engineered. Moreover, the cell wall binding domain of the engineered protein is indispensable for a strong binding and stability of the proteins.

Biofabricated silver nanoparticles exhibit broad-spectrum antibiofilm and antiquorum sensing activity against Gram-negative bacteria.

The emergence and spread of antimicrobial resistance (AMR) among bacterial pathogens have created a global threat to human health and the environment. Targeting the quorum sensing (QS) linked virulent traits of bacteria is considered to be a novel approach for addressing the problem of AMR. In this study, green synthesized silver nanoparticles (AgNPs-MK) were evaluated for the inhibition of the formation of biofilms and quorum sensing controlled virulence factors against three Gram negative bacteria. Remarkable inhibition (>80%) of QS-mediated violacein production was recorded in C. violaceum 12472. Up to 90% inhibition of the QS-mediated virulent traits of S. marcescens MTCC 97 was observed. The virulence factors of P. aeruginosa PAO1 also decreased in a dose dependent manner in the presence of AgNPs-MK. Moreover, the development of biofilms of C. violaceum 12472, S. marcescens MTCC 97, and P. aeruginosa PAO1 was reduced by 87.39, 81.54, and 71.34%, respectively. Biofilms on glass surfaces were remarkably reduced, with less aggregation of bacterial cells and the reduced formation of extra polymeric substances. The findings clearly show the efficacy of AgNPs-MK against the development of biofilms and the QS mediated virulent traits of Gram negative bacterial pathogens. AgNPs-MK may be further exploited for the development of alternative antimicrobial agents after careful scrutiny in animal models for the management of bacterial infections, especially for topical applications.