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2.  Bacteriophages and Phage-Derived Proteins – Application Approaches 
Current Medicinal Chemistry  2015;22(14):1757-1773.
Currently, the bacterial resistance, especially to most commonly used antibiotics has proved to be a severe therapeutic problem. Nosocomial and community-acquired infections are usually caused by multidrug resistant strains. Therefore, we are forced to develop an alternative or supportive treatment for successful cure of life-threatening infections. The idea of using natural bacterial pathogens such as bacteriophages is already well known. Many papers have been published proving the high antibacterial efficacy of lytic phages tested in animal models as well as in the clinic. Researchers have also investigated the application of non-lytic phages and temperate phages, with promising results. Moreover, the development of molecular biology and novel generation methods of sequencing has opened up new possibilities in the design of engineered phages and recombinant phage-derived proteins. Encouraging performances were noted especially for phage enzymes involved in the first step of viral infection responsible for bacterial envelope degradation, named depolymerases. There are at least five major groups of such enzymes – peptidoglycan hydrolases, endosialidases, endorhamnosidases, alginate lyases and hyaluronate lyases – that have application potential. There is also much interest in proteins encoded by lysis cassette genes (holins, endolysins, spanins) responsible for progeny release during the phage lytic cycle. In this review, we discuss several issues of phage and phage-derived protein application approaches in therapy, diagnostics and biotechnology in general.
PMCID: PMC4468916  PMID: 25666799
Bacteriophage application; endolysins; holins; phage-encoded proteins; polysaccharide depolymerases; spanins.
3.  Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm 
PLoS ONE  2015;10(5):e0127603.
We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.
PMCID: PMC4440721  PMID: 25996839
4.  A Suggested New Bacteriophage Genus, “Kp34likevirus”, within the Autographivirinae Subfamily of Podoviridae 
Viruses  2015;7(4):1804-1822.
Klebsiella pneumoniae phages vB_KpnP_SU503 (SU503) and vB_KpnP_SU552A (SU552A) are virulent viruses belonging to the Autographivirinae subfamily of Podoviridae that infect and kill multi-resistant K. pneumoniae isolates. Phages SU503 and SU552A show high pairwise nucleotide identity to Klebsiella phages KP34 (NC_013649), F19 (NC_023567) and NTUH-K2044-K1-1 (NC_025418). Bioinformatic analysis of these phage genomes show high conservation of gene arrangement and gene content, conserved catalytically active residues of their RNA polymerase, a common and specific lysis cassette, and form a joint cluster in phylogenetic analysis of their conserved genes. Also, we have performed biological characterization of the burst size, latent period, host specificity (together with KP34 and NTUH-K2044-K1-1), morphology, and structural genes as well as sensitivity testing to various conditions. Based on the analyses of these phages, the creation of a new phage genus is suggested within the Autographivirinae, called “Kp34likevirus” after their type phage, KP34. This genus should encompass the recently genome sequenced Klebsiella phages KP34, SU503, SU552A, F19 and NTUH-K2044-K1-1.
PMCID: PMC4411677  PMID: 25853484
“Kp34likevirus”; phikmvlikevirus; Autographivirinae; Klebsiella pneumoniae; bacteriophage; phage; Podoviridae
5.  In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas aeruginosa strains from cystic fibrosis patients 
Applied Microbiology and Biotechnology  2015;99(14):6021-6033.
The goal of the study was to determine the relationship between in vitro/in vivo efficacy of environmental Pseudomonas phages and certain phenotypical properties of Pseudomonas aeruginosa (PA) strains. We studied the diversity between particular isolates and determined phage sensitivity in vitro and in vivo in the Galleria mellonella insect model. Twenty-eight lytic bacteriophages specific for PA were tested against 121 CF PA isolates including 29 mucoid PA strains. Most strains from cystic fibrosis (CF) patients were lysed by at least three phages (93.6 %), but completely insensitive strains were also present (6.4 %). Two phages PA5oct and KT28 exhibited high rates of lytic potency on 55–68 % of PA strains (72–86 % of mucoid isolates). We further explored phage activity against six PA strains (CF and non-CF) in vitro, comparing clonal differences in phage susceptibility with bacterial properties such as the ability to form biofilms, mucosity, twitching motility, and biochemical profiles. We observed the relationship between variation in phage susceptibility and Fourier transform infrared spectroscopy (FTIR) analysis in the spectra window of carbohydrates. The protective efficacy of two selected phages against PA PAO1 and 0038 infection was confirmed in vivo in G. mellonella larvae. Generally, the wax moth model results confirmed the data from in vitro assays, but in massive infection of CF isolates, the application of lytic phages probably led to the release of toxic compound causing an increase in larvae mortality. We assumed that apart of in vitro phage activity testing, a simple and convenient wax moth larvae model should be applied for the evaluation of in vivo effectiveness of particular phage preparations.
PMCID: PMC4480334  PMID: 25758956
Myoviridae bacteriophages; Phage treatment; Pseudomonas aeruginosa; Cystic fibrosis; Galleria mellonella model
6.  Bactericidal activities of GM flax seedcake extract on pathogenic bacteria clinical strains 
BMC Biotechnology  2014;14:70.
The antibiotic resistance of pathogenic microorganisms is a worldwide problem. Each year several million people across the world acquire infections with bacteria that are antibiotic-resistant, which is costly in terms of human health. New antibiotics are extremely needed to overcome the current resistance problem.
Transgenic flax plants overproducing compounds from phenylpropanoid pathway accumulate phenolic derivatives of potential antioxidative, and thus, antimicrobial activity. Alkali hydrolyzed seedcake extract containing coumaric acid, ferulic acid, caffeic acid, and lignan in high quantities was used as an assayed against pathogenic bacteria (commonly used model organisms and clinical strains). It was shown that the extract components had antibacterial activity, which might be useful as a prophylactic against bacterial infection. Bacteria topoisomerase II (gyrase) inhibition and genomic DNA disintegration are suggested to be the main reason for rendering antibacterial action.
The data obtained strongly suggest that the seedcake extract preparation is a suitable candidate for antimicrobial action with a broad spectrum and partial selectivity. Such preparation can be applied in cases where there is a risk of multibacterial infection and excellent answer on global increase in multidrug resistance in pathogenic bacteria.
PMCID: PMC4134466  PMID: 25073883
Antimicrobial compound; Phenolic acid; Flax; Alternative antibiotic; Flax seedcake
7.  Developing an international Pseudomonas aeruginosa reference panel 
MicrobiologyOpen  2013;2(6):1010-1023.
Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis (CF) patients and causes a wide range of infections among other susceptible populations. Its inherent resistance to many antimicrobials also makes it difficult to treat infections with this pathogen. Recent evidence has highlighted the diversity of this species, yet despite this, the majority of studies on virulence and pathogenesis focus on a small number of strains. There is a pressing need for a P. aeruginosa reference panel to harmonize and coordinate the collective efforts of the P. aeruginosa research community. We have collated a panel of 43 P. aeruginosa strains that reflects the organism's diversity. In addition to the commonly studied clones, this panel includes transmissible strains, sequential CF isolates, strains with specific virulence characteristics, and strains that represent serotype, genotype or geographic diversity. This focussed panel of P. aeruginosa isolates will help accelerate and consolidate the discovery of virulence determinants, improve our understanding of the pathogenesis of infections caused by this pathogen, and provide the community with a valuable resource for the testing of novel therapeutic agents.
PMCID: PMC3892346  PMID: 24214409
Cystic fibrosis; genotype; pathogen; Pseudomonas aeruginosa
8.  A giant Pseudomonas phage from Poland 
Archives of Virology  2013;159(3):567-572.
A novel giant phage of the family Myoviridae is described. Pseudomonas phage PA5oct was isolated from a sewage sample from an irrigated field near Wroclaw, Poland. The virion morphology indicates that PA5oct differs from known giant phages. The phage has a head of about 131 nm in diameter and a tail of 136 × 19 nm. Phage PA5oct contains a genome of approximately 375 kbp and differs in size from any tailed phages known. PA5oct was further characterized by determination of its latent period and burst size and its sensitivity to heating, chloroform, and pH.
PMCID: PMC3936114  PMID: 24072472
Bacteriophage; DNA size; Giant; Myoviridae; Pseudomonas
9.  Laser interferometry analysis of ciprofloxacin and ampicillin diffusion from liposomal solutions to water phase 
European Biophysics Journal  2013;42(7):549-558.
The paper presents experimental investigations of diffusion of antibiotics (ciprofloxacin or ampicillin) into the water phase from mixtures of neutral or negatively charged liposomes, and antibiotic–liposome interactions. Using the laser interferometry technique, the amounts and fluxes of released antibiotics, concentration field evolution, and the velocity of the concentration boundary layer’s “growth” were determined. To avoid the limitations of membranes, a measurement system without the artificial boundary of phases with a free water–solution interface has been proposed. It was found that the diffusion of anionic and neutral liposomes into the water phase was insignificant and mainly the diffusion of antibiotics was measured. Differences in the diffusion kinetics of ciprofloxacin and ampicillin from liposomal solutions to the water phase were observed. Ampicillin diffused more efficiently than ciprofloxacin regardless of the liposomal solution type. Moreover, the amount of ampicillin and ciprofloxacin released from the anionic liposomal phase was higher than that from the neutral one. Our results confirm that ciprofloxacin at neutral pH shows little tendency to bind neutral liposomes. Additionally, it was also observed that ciprofloxacin disrupts negatively charged liposomes as a final effect of antibiotic–lipid interactions.
PMCID: PMC3674336  PMID: 23604440
Laser interferometry; Diffusion; Liposomes; Ciprofloxacin; Ampicillin
10.  Characterising the biology of novel lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae 
Virology Journal  2013;10:100.
Members of the genus Klebsiella are among the leading microbial pathogens associated with nosocomial infection. The increased incidence of antimicrobial resistance in these species has propelled the need for alternate/combination therapeutic regimens to aid clinical treatment. Bacteriophage therapy forms one of these alternate strategies.
Electron microscopy, burst size, host range, sensitivity of phage particles to temperature, chloroform, pH, and restriction digestion of phage DNA were used to characterize Klebsiella phages.
Results and conclusions
Of the 32 isolated phages eight belonged to the family Myoviridae, eight to the Siphoviridae whilst the remaining 16 belonged to the Podoviridae. The host range of these phages was characterised against 254 clinical Enterobacteriaceae strains including multidrug resistant Klebsiella isolates producing extended-spectrum beta-lactamases (ESBLs). Based on their lytic potential, six of the phages were further characterised for burst size, physicochemical properties and sensitivity to restriction endonuclease digestion. In addition, five were fully sequenced. Multiple phage-encoded host resistance mechanisms were identified. The Siphoviridae phage genomes (KP16 and KP36) contained low numbers of host restriction sites similar to the strategy found in T7-like phages (KP32). In addition, phage KP36 encoded its own DNA adenine methyltransferase. The φKMV-like KP34 phage was sensitive to all endonucleases used in this study. Dam methylation of KP34 DNA was detected although this was in the absence of an identifiable phage encoded methyltransferase. The Myoviridae phages KP15 and KP27 both carried Dam and Dcm methyltransferase genes and other anti-restriction mechanisms elucidated in previous studies. No other anti-restriction mechanisms were found, e.g. atypical nucleotides (hmC or glucosyl hmC), although Myoviridae phage KP27 encodes an unknown anti-restriction mechanism that needs further investigation.
PMCID: PMC3620542  PMID: 23537199
Bacteriophage; Klebsiella spp.; Multidrug resistance; Restriction endonuclease patterns; Myoviridae; Siphoviridae; Podoviridae
11.  Innate immune properties of selected human neuropeptides against Moraxella catarrhalis and nontypeable Haemophilus influenzae 
BMC Immunology  2012;13:24.
Considerable evidence supports the concept of active communication between the nervous and immune systems. One class of such communicators are the neuropeptides (NPs). Recent reports have highlighted the antimicrobial activity of neuropeptides, placing them among the integral components of innate immune defense. This study examined the action of four human neuropeptides: calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), substance P (SP) and somatostatin (SOM), which are accessible in the upper respiratory tract, against two human-specific respiratory pathogens. We studied: (i) neuropeptide-mediated direct antibacterial activity exerted against Moraxella catarrhalis and nontypeable Haemophilus influenzae, and (ii) indirect immunomodulatory role of these neuropeptides in the neutrophil-mediated phagocytosis of indicated pathogens.
We found that 100 micromolar concentrations of CGRP, NPY, SP, and SOM effectively permeabilized bacterial membranes and showed (except SOM) bactericidal activity against both pathogens. SOM acted only bacteriostatically. However the killing efficacy was dependent on the bactericidal assay used. The rank order of killing NP effect was: NPY ≥ CGRP > SP >> SOM and correlated with their potency to permeabilize bacterial membranes. The killing and permeabilization activity of the analyzed NPs showed significant correlation with several physicochemical properties and amino acid composition of the neuropeptides. M. catarrhalis was more sensitive to neuropeptides than nontypeable H. influenzae.
The immunomodulatory bimodal effect of physiological concentrations of CGRP, NPY, and SP on the phagocytic function of human neutrophils against M. catarrhalis and H. influenzae was observed both in the ingestion (pathogen uptake) and reactive oxygen species generation stages. This effect was also dependent on the distinct type of pathogen recognition (opsonic versus nonopsonic).
The present results indicate that neuropeptides such as CGRP, NPY, and SP can effectively participate in the direct and indirect elimination of human-specific respiratory pathogens. Because the studied NPs show both direct and indirect modulating antimicrobial potency, they seem to be important molecules involved in the innate host defense against M. catarrhalis and nontypeable H. influenzae.
PMCID: PMC3460729  PMID: 22551165
Neuropeptide Y; Substance P; CGRP; Somatostatin; Killing; Permeabilization; Phagocytosis; Immunomodulation; Moraxella catarrhalis; Haemophilus influenzae
12.  Isolation and characterisation of KP34—a novel φKMV-like bacteriophage for Klebsiella pneumoniae 
Bacteriophage KP34 is a novel virus belonging to the subfamily Autographivirinae lytic for extended-spectrum β-lactamase-producing Klebsiella pneumoniae strains. Its biological features, morphology, susceptibility to chemical and physical agents, burst size, host specificity and activity spectrum were determined. As a potential antibacterial agent used in therapy, KP34 molecular features including genome sequence and protein composition were examined. Phylogenetic analyses and clustering of KP34 phage genome sequences revealed its clear relationships with “phiKMV-like viruses”. Simultaneously, whole-genome analyses permitted clustering and classification of all phages, with completely sequenced genomes, belonging to the Podoviridae.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-011-3149-y) contains supplementary material, which is available to authorized users.
PMCID: PMC3082699  PMID: 21327407
φKMV-like bacteriophage; Genome; Klebsiella pneumoniae; Phage therapy; Podoviridae; phiKMV-like viruses
13.  Molecular Epidemiology of Acquired-Metallo-β-Lactamase-Producing Bacteria in Poland 
We have analyzed 40 metallo-β-lactamase (MBL)-producing isolates of Pseudomonas aeruginosa (n = 38), Pseudomonas putida (n = 1), and Acinetobacter genospecies 3 (n = 1) from 17 hospitals in 12 cities in Poland that were identified in 2000 to 2004. Pulsed field gel electrophoresis typing classified the P. aeruginosa isolates into eight types, with two types differentiated further into subtypes. Each of the types was specific either to a given center or to several hospitals of the same or neighboring geographic area. Almost all of the organisms produced β-lactamase VIM-2; the only exceptions were several P. aeruginosa isolates from two centers which expressed VIM-4. The blaVIM genes resided exclusively within class 1 integrons, and these were located in either chromosomal or plasmid DNA. PCR-restriction fragment length polymorphism study of the variable regions of the integrons, followed by DNA sequencing, revealed the presence of eight different, mostly novel gene cassette arrays, six of which contained blaVIM-2 and two of which contained blaVIM-4. The occurrence of the integron variants correlated well with the geographic distribution of the MBL-producing organisms, and this suggested that their emergence in particular parts of the country had been likely due to a number of independent events. The following regional dissemination of MBL producers could be attributed to various phenomena, including their clonal spread, horizontal transmission of resistance determinants, or both. All of the data collected in this study revealed that even at this early stage of detection, the epidemiological situation concerning MBL producers in Poland has already been complex and very dynamic.
PMCID: PMC1426447  PMID: 16495246
14.  Learning from Bacteriophages - Advantages and Limitations of Phage and Phage-Encoded Protein Applications 
Current Protein & Peptide Science  2012;13(8):699-722.
The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application.
PMCID: PMC3594737  PMID: 23305359
antibiotics; bacterial multidrug resistance; bacteriophage therapy; phage-encoded proteins application,

Results 1-14 (14)