2012
DOI: 10.1089/mdr.2012.0024
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Phage Endolysins with Broad Antimicrobial Activity AgainstEnterococcus faecalisClinical Strains

Abstract: Increasing antibiotic resistance of bacterial pathogens has drawn the attention to the potential use of bacteriophage endolysins as alternative antibacterial agents. Here we have identified, characterized, and studied the lytic potential of two endolysins, Lys168 and Lys170, from phages infecting Enterococcus faecalis. Lys168 and Lys170 belong to the cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) and amidase-2 protein families, respectively. Lys168 is quite a unique enterococcal phage endolysi… Show more

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Cited by 31 publications
(41 citation statements)
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References 51 publications
(61 reference statements)
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“…Although, the protein remained active at various salt concentrations, it showed the highest activity in the absence of NaCl. The results of the current study also highlighted that our recombinant endolysin shows more potent activity (at 10 and 5 μg/mL concentration) against MRSA 252 compared with previously studied endolysin constructs (Becker et al, 2009; Fernandes et al, 2012; Proença et al, 2012). …”
Section: Discussionsupporting
confidence: 72%
See 1 more Smart Citation
“…Although, the protein remained active at various salt concentrations, it showed the highest activity in the absence of NaCl. The results of the current study also highlighted that our recombinant endolysin shows more potent activity (at 10 and 5 μg/mL concentration) against MRSA 252 compared with previously studied endolysin constructs (Becker et al, 2009; Fernandes et al, 2012; Proença et al, 2012). …”
Section: Discussionsupporting
confidence: 72%
“…The colony forming unit (CFU/mL) was calculated at the end of each assay. The buffer without endolysin was used as negative control (Becker et al, 2009; Fernandes et al, 2012; Proença et al, 2012). …”
Section: Methodsmentioning
confidence: 99%
“…The underlying idea is that these enzymes should be able to efficiently lyse viable bacteria when added exogenously in the form of purified proteins. In apparent conflict with the ideas conveyed here, a great number of in vitro studies (fewer in vivo ), including some emanating from our group (Fernandes et al ., ; Proença et al ., ) seem to support the lytic efficacy of endolysins added externally to Gram‐positive bacteria. However, a large number of these studies have been performed in conditions that do not support bacterial growth, i.e., before endolysin challenge cells are typically washed and suspended in nutrient‐depleted, buffered solutions which are unable to sustain normal pmf.…”
Section: Discussionmentioning
confidence: 79%
“…Induction of EC300 and mEC300 synthesis by E. coli CG61 carrying pDPEC300 or pDPmEC300, respectively, production of total protein extracts, and subsequent purification by metal chelate affinity chromatography (AFC) were carried out as described by Proença et al (2012), except for the following changes: induction of protein production by heat shock occurred in a shaking water bath set to 42°C (100 rpm, model BSH, Raypa-R. Espinar, SL) for 30 min, followed by a 3-h incubation at 37°C (200 rpm, model SM 30 control, Edmund Bühler GmbH), and removal of insoluble material from total protein extracts was by centrifugation at 35,000g, 25 min, 4°C in a High-Performance Centrifuge Avanti J-25I (rotor JA 25-50, Beckman Coulter). Fractions eluted from the AFC step (HisTrap HP columns, GE Healthcare) were analyzed by SDS-PAGE, and those containing the partially purified enzymes were subjected to size-exclusion chromatography (SEC) using a Hi-load 16/600 superdex 75 prep grade column (GE Healthcare), equilibrated and run in protein buffer (20 mM HEPES-Na, 500 mM NaCl, 1 % glycerol, and 1 mM DTT, pH8.0) at a flow rate of 1 ml/min.…”
Section: General Protein Techniquesmentioning
confidence: 99%
“…In the vast majority of the studies reporting the antibacterial activity of phage PG hydrolases, the lytic enzymes are tested in conditions that do not support robust bacterial growth; most commonly, in vitro experiments are performed with target cells washed and suspended in buffered solutions (Gu et al 2011;Proença et al 2012). Often, the high lytic activity observed in these conditions does not translate to the expected results when assays are transposed to animal infection models, and in some cases, satisfactory levels of animal survival are only obtained when lytic enzymes are administrated to animals soon after the injection of the deadly bacterial inoculum (Gu et al 2011;Loeffler et al 2003;Oechslin et al 2013).…”
Section: Introductionmentioning
confidence: 99%