Antibiofilm activity and synergistic effects of DNase I and lysostaphinaggainst Staphylococcus aureus biofilms

Background:In recent years,food safety has become a global public health concern.Microbial contamination is one of the most common food safety issues.Staphylococcus aureus is a common foodborne pathogen that can form biofilms on the surface of food processing equipment,leading to greater resistance to antimicrobial agents than occurs with planktonic bacteria.Materials and methods:In this work,recombinant Escherichia coli BL21(DE3)cells expressing optimized lysostaphin(Lst)were constructed,recombinant Lst was produced and purified,and Lst enzymatic assays were performed,followed by antimicrobial testing of Lst.Finally,a mixtureof Lst and DNase I was tested for antibiofilmactivity.Results:The protein content of purified Lst was 0.6 mg/mL and the enzyme activity was 240 U/mL.The minimum inhibitory concentration(MiC)of Lst against S.aureus was O.1μg/mL.At 1 MiC,Lst exerted an effect on the growth,cell wall integrity and cell membrane permeability of S.aureus.Conclusions:Although Lst alone also showed good inhibition and disruption of S.aureus biofilms,the inhibition and disruption of S.aureus biofilms were significantly greater when Lst was mixed with DNase l.This is probably because DNase I removes extracellular DNA,affecting biofilm formation and dispersing mature biofilms,and thereby facilitating the penetration of Lst.

Characterization and complete genome sequence of vB_EcoP-Bp4,a novel polyvalent N4-like bacteriophage that infects chicken pathogenic Escherichia coli

Pathogenic Escherichia coli cause chicken colibacillosis, which is economically devastating to the poultry in- dustry worldwide （Bagheri et al., 2014）. Owing to in- creasing antibiotic resistance, phage therapy reagents have been developed to treat bacterial infections （Xu et al., 2015）.

Multiple-site mutations of phage Bp7 endolysin improves its activities against target bacteria

The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7(Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a p ET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated(L99A, M102E) to construct p ET28a-Bp7Δe, with p ET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.