Characterization of Modular Bacteriophage Endolysins from Myoviridae Phages OBP, 201ϕ2-1 and PVP-SE1

Peptidoglycan lytic enzymes (endolysins) induce bacterial host cell lysis in the late phase of the lytic bacteriophage replication cycle. Endolysins OBPgp279 (from Pseudomonas fluorescens phage OBP), PVP-SE1gp146 (Salmonella enterica serovar Enteritidis phage PVP-SE1) and 201ϕ2-1gp229 (Pseudomonas chlororaphis phage 201ϕ2-1) all possess a modular structure with an N-terminal cell wall binding domain and a C-terminal catalytic domain, a unique property for endolysins with a Gram-negative background. All three modular endolysins showed strong muralytic activity on the peptidoglycan of a broad range of Gram-negative bacteria, partly due to the presence of the cell wall binding domain. In the case of PVP-SE1gp146, this domain shows a binding affinity for Salmonella peptidoglycan that falls within the range of typical cell adhesion molecules (Kaff = 1.26×106 M−1). Remarkably, PVP-SE1gp146 turns out to be thermoresistant up to temperatures of 90°C, making it a potential candidate as antibacterial component in hurdle technology for food preservation. OBPgp279, on the other hand, is suggested to intrinsically destabilize the outer membrane of Pseudomonas species, thereby gaining access to their peptidoglycan and exerts an antibacterial activity of 1 logarithmic unit reduction. Addition of 0.5 mM EDTA significantly increases the antibacterial activity of the three modular endolysins up to 2–3 logarithmic units reduction. This research work offers perspectives towards elucidation of the structural differences explaining the unique biochemical and antibacterial properties of OBPgp279, PVP-SE1gp146 and 201ϕ2-1gp229. Furthermore, these endolysins extensively enlarge the pool of potential antibacterial compounds used against multi-drug resistant Gram-negative bacterial infections

A thermostable salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids

Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.This work was supported by the projects FCOMP-01-0124-FEDER-019446, FCOMP-01-0124-FEDER-027462 and PEst-OE/EQB/LA0023/2013 from "Fundacao para a Ciencia e Tecnologia" (FCT), Portugal. The authors thank the Project "BioHealth - Biotechnology and Bioengineering approaches to improve health quality", Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON. 2 - O Novo Norte), QREN, FEDER. Hugo Oliveira acknowledges the FCT grant SFRH/BD/63734/2009. Maarten Walmagh held a PhD scholarship of the IWT Vlaanderen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Review of laser speckle contrast techniques for visualizing tissue perfusion

When a diffuse object is illuminated with coherent laser light, the backscattered light will form an interference pattern on the detector. This pattern of bright and dark areas is called a speckle pattern. When there is movement in the object, the speckle pattern will change over time. Laser speckle contrast techniques use this change in speckle pattern to visualize tissue perfusion. We present and review the contribution of laser speckle contrast techniques to the field of perfusion visualization and discuss the development of the techniques

Time domain algorithm for accelerated determination of the first order moment of photo current fluctuations in high speed laser Doppler perfusion imaging

Advances in optical array sensor technology allow for the real time acquisition of dynamic laser speckle patterns generated by tissue perfusion, which, in principle, allows for real time laser Doppler perfusion imaging (LDPI). Exploitation of these developments is enhanced with the introduction of faster algorithms to transform photo currents into perfusion estimates using the first moment of the power spectrum. A time domain (TD) algorithm is presented for determining the first-order spectral moment. Experiments are performed to compare this algorithm with the widely used Fast Fourier Transform (FFT). This study shows that the TD-algorithm is twice as fast as the FFT-algorithm without loss of accuracy. Compared to FFT, the TD-algorithm is efficient in terms of processor time, memory usage and data transport

Characterization and genome sequencing of a Citrobacter freundii phage CfP1 harboring a lysin active against multidrug-resistant isolates

Citrobacter spp., although frequently ignored, is emerging as an important nosocomial bacterium able to cause various superficial and systemic life-threatening infections. Considered to be hard-to-treat bacterium due to its pattern of high antibiotic resistance, it is important to develop effective measures for early and efficient therapy. In this study, the first myovirus (vB_CfrM_CfP1) lytic for Citrobacter freundii was microbiologically and genomically characterized. Its morphology, activity spectrum, burst size, and biophysical stability spectrum were determined. CfP1 specifically infects C. freundii, has broad host range (>85 %; 21 strains tested), a burst size of 45 PFU/cell, and is very stable under different temperatures (20 to 50 °C) and pH (3 to 11) values. CfP1 demonstrated to be highly virulent against multidrug-resistant clinical isolates up to 12 antibiotics, including penicillins, cephalosporins, carbapenems, and fluroquinoles. Genomically, CfP1 has a dsDNA molecule with 180,219 bp with average GC content of 43.1 % and codes for 273 CDSs. The genome architecture is organized into function-specific gene clusters typical for tailed phages, sharing 46 to 94 % nucleotide identity to other Citrobacter phages. The lysin gene encoding a predicted D-Ala-D-Ala carboxypeptidase was also cloned and expressed in Escherichia coli and its activity evaluated in terms of pH, ionic strength, and temperature. The lysine optimum activity was reached at 20 mM HEPES, pH 7 at 37 °C, and was able to significantly reduce all C. freundii (>2 logs) as well as Citrobacter koseri (>4 logs) strains tested. Interestingly, the antimicrobial activity of this enzyme was performed without the need of pretreatment with outer membrane-destabilizing agents. These results indicate that CfP1 lysin is a good candidate to control problematic Citrobacter infections, for which current antibiotics are no longer effective.This study was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER006684), and the PhD grants SFRH/BPD/111653/2015 and SFRH/BPD/69356/2010

Intracellular Vesicles as Reproduction Elements in Cell Wall-Deficient L-Form Bacteria

Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells

High-resolution reconstruction of a Jumbo-bacteriophage infecting capsulated bacteria using hyperbranched tail fibers

NWO184.034.014Computer Systems, Imagery and Medi

Time-reversed adapted-perturbation (TRAP) optical focusing onto dynamic objects inside scattering media

The ability to steer and focus light inside scattering media has long been sought for a multitude of applications. At present, the only feasible strategy to form optical foci inside scattering media is to guide photons by using either implanted or virtual guide stars, which can be inconvenient and limits the potential applications. Here we report a scheme for focusing light inside scattering media by employing intrinsic dynamics as guide stars. By adaptively time-reversing the perturbed component of the scattered light, we show that it is possible to focus light to the origin of the perturbation. Using this approach, we demonstrate non-invasive dynamic light focusing onto moving targets and imaging of a time-variant object obscured by highly scattering media. Anticipated applications include imaging and photoablation of angiogenic vessels in tumours, as well as other biomedical uses

Biophotonics methods for functional monitoring of complications of diabetes mellitus

The prevalence of diabetes complications is a significant public health problem with a considerable economic cost. Thus, the timely diagnosis of complications and prevention of their development will contribute to increasing the length and quality of patient life, and reducing the economic costs of their treatment. This article aims to review the current state-of-the-art biophotonics technologies used to identify the complications of diabetes mellitus and assess the quality of their treatment. Additionally, these technologies assess the structural and functional properties of biological tissues, and they include capillaroscopy, laser Doppler flowmetry and hyperspectral imaging, laser speckle contrast imaging, diffuse reflectance spectroscopy and imaging, fluorescence spectroscopy and imaging, optical coherence tomography, optoacoustic imaging and confocal microscopy. Recent advances in the field of optical noninvasive diagnosis suggest a wider introduction of biophotonics technologies into clinical practice and, in particular, in diabetes care units

Hemodynamic Responses Evoked by Neuronal Stimulation via Channelrhodopsin-2 Can Be Independent of Intracortical Glutamatergic Synaptic Transmission

Maintenance of neuronal function depends on the delivery of oxygen and glucose through changes in blood flow that are linked to the level of ongoing neuronal and glial activity, yet the underlying mechanisms remain unclear. Using transgenic mice expressing the light-activated cation channel channelrhodopsin-2 in deep layer pyramidal neurons, we report that changes in intrinsic optical signals and blood flow can be evoked by activation of a subset of channelrhodopsin-2-expressing neurons in the sensorimotor cortex. We have combined imaging and pharmacology to examine the importance of glutamatergic synaptic transmission in this form of neurovascular coupling. Blockade of ionotropic glutamate receptors with the antagonists CNQX and MK801 significantly reduced forepaw-evoked hemodynamic responses, yet resulted in no significant reduction of channelrhodopsin-evoked hemodynamic responses, suggesting that stimulus-dependent coupling of neuronal activity to blood flow can be independent of local excitatory synaptic transmission. Together, these results indicate that channelrhodopsin-2 activation of sensorimotor excitatory neurons produces changes in intrinsic optical signals and blood flow that can occur under conditions where synaptic activation of neurons or other cells through ionotropic glutamate receptors would be blocked