Bacteriophages as an alternative strategy for fighting biofilm development.

The ability of microbes to form biofilms is an important element of their pathogenicity, and biofilm formation is a serious challenge for today's medicine. Fighting the clinical complications associated with biofilm formation is very difficult and linked to a high risk of failure, especially in a time of increasing bacterial resistance to antibiotics. Bacterial species most commonly isolated from biofilms include coagulase-negative staphylococci, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. The frequent failure of antibiotic therapy led researchers to look for alternative methods and experiment with the use of antibacterial factors with a mechanism of action different from that of antibiotics. Experimental studies with bacteriophages and mixtures thereof, expressing lytic properties against numerous biofilm-forming bacterial species showed that bacteriophages may both prevent biofilm formation and contribute to eradication of biofilm bacteria. A specific role is played here by phage depolymerases, which facilitate the degradation of extracellular polymeric substances (EPS) and thus the permeation of bacteriophages into deeper biofilm layers and lysis of the susceptible bacterial cells. Much hope is placed in genetic modifications of bacteriophages that would allow the equipping bacteriophages with the function of depolymerase synthesis. The use of phage cocktails prevents the development of phage-resistant bacteria.

Poly-γ-(D)-glutamic acid capsule interferes with lytic infection of Bacillus anthracis by B. anthracis-specific bacteriophages.

The poly-γ-d-glutamic acid capsule of Bacillus anthracis is a barrier to infection by B. anthracis-specific bacteriophages. Capsule expression was found to completely inhibit lytic infection by γ phage, an observation supported by the demonstration that this phage does not elaborate a hydrolase that would facilitate penetration through the protective capsule outer layer.

Characterization of a bacteriophage, isolated from a cow with mastitis, that is lytic against Staphylococcus aureus strains.

Methicillin-resistant strains of Staphylococcus aureus (MRSA) are now the most commonly reported antibiotic-resistant bacterium in clinical settings. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophage therapy is a potential alternative treatment for MRSA infections. The objective of this study was characterization of a novel virulent bacteriophage (MSA6) isolated from a cow with mastitis. Electron microscopy showed its resemblance to members of the family Myoviridae, with an isometric head (66 nm) and a long contractile tail (173 nm). The genome of phage MSA6 was tested by pulsed-field gel electrophoresis and estimated to be about 143 kb. It exhibited rapid adsorption (>82% in 5 min), a short latent period (15 min) and a relatively small burst size (23 PFU/cell). Isolated phage was capable of infecting a wide spectrum of staphylococcal strains of both human and bovine origin. The results of this investigation indicate that MSA6 is similar to other bacteriophages belonging to the family Myoviridae (Twort, K, G1, 812) that have been successfully used in bacteriophage therapy.

Isolation and characterization of a novel bacteriophage φ4D lytic against Enterococcus faecalis strains.

In recent years, Enterococcus faecalis has emerged as an important opportunistic nosocomial pathogen capable of causing dangerous infections. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophages have very effective bactericidal activity and several advantages over other antimicrobial agents and so far, no serious or irreversible side effects of phage therapy have been described. The objective of this study was to characterize a novel virulent bacteriophage φ4D isolated from sewage. Electron microscopy revealed its resemblance to Myoviridae, with an isometric head (74 ± 4 nm) and a long contractile tail (164 ± 4 nm). The φ4D phage genome was tested using pulsed-field gel electrophoresis and estimated to be 145 ± 2 kb. It exhibited short latent period (25 min) and a relatively small burst size (36 PFU/cell). Tests were conducted on the host range, multiplicities of infection (MOI), thermal stability, digestion of DNA by restriction enzymes, and proteomic analyses of this phage. The isolated phage was capable of infecting a wide spectrum of enterococcal strains. The results of these investigations indicate that φ4D is similar to other Myoviridae bacteriophages (for example φEF24C), which have been successfully used in phagotherapy.

[Peptides--a new strategy for combating viral infections]. / Peptydy--nowe mozliwosci zwalczania zakazen wirusowych.

The efficiency of peptides against many species of bacteria, fungi and parasites has been widely described. Recent studies on peptides have also demonstrated their antiviral activity. Some peptides exhibit direct virucidal activity, others disturb attachment of virus particles to the cell membrane surface or interfere with intracellular replication of virus. Due to limited effectiveness of commonly used drugs and emerging resistance of viruses, antiviral peptides may have the potential to be developed as putative therapeutic agents.

Bacteriophages, phage endolysins and antimicrobial peptides - the possibilities for their common use to combat infections and in the design of new drugs.

The antibiotic resistance in many pathogenic bacteria has become a major clinical problem, therefore, the necessity arises to search for new therapeutic strategies. The most promising solution lies in bacteriophages, phage endolysins and antimicrobial peptides. The aim of this study is to review the possibilities for the common use of bacteriophages, phage endolysins and antimicrobial peptides, both in the form of combined therapies and new strategies for the production of peptide drugs. Bacteriophages are viruses that specifically infect and destroy pathogenic bacteria by penetration into bacterial cells, causing metabolism disorders and, consequently, cell lysis. Phage-encoded endolysins are bacteriolytic proteins produced at the end of the phage lytic cycle that destroy elements of bacterial cell wall and enable the release of phage progeny from host cells. Antimicrobial peptides (AMPs) constitute an element of the innate immunity of living organisms and are characterized by the activity against a broad spectrum of bacteria. In the literature, there are only a few reports on the direct interaction of bacteriophages, phage endolysins and antimicrobial peptides against pathogenic bacteria. In each of them, a synergistic effect was observed, and Phage-encoded antimicrobial peptides as a specific group of AMPs have were also discussed. Phage-display technique was also reviewed in terms of its applications to produce and deliver biologically active peptides. The literature data also suggest that bacteriophages, phage endolysins and antimicrobial peptides can be used in combined therapy, thus negating many of the limitations resulting from their specificity as a single antimicrobial agent.

[The structure and mechanism of action of clostridial neurotoxins]. / Struktura i mechanizm dzialania neurotoksyn bakterii rodzaju Clostridium.

Clostridium botulinum and Clostridium tetani produce highly potent neurotoxins, called botulinum toxins and tetanus toxin, respectively. The clostridial neurotoxins specifically bind to neuronal cells and disrupt neurotransmisser release by cleaving proteins involved in specific vesicle membrane fusion. Each toxin is synthesized as an inactive approximately 150 kDa single-chain protein. The protein is posttranslationally proteolyzed to form the active dichain molecule in which the chains approximately 50 and approximately 100 kDa, remain linked by a disulfide bond. The structural organization is funcionally related to the fact that CNTs intoxicate neurons via four-step mechanism consisting of 1. binding, 2. internalization, 3. membrane translocation, and 4. enzymatic target modification. The L chain is responsible for the intracellular catalitic activity. The NH2-terminal 50-kDa domain of the H chain (HN) is implicated in membrane translocation, whereas the COOH-terminal part (HC) is mainly responsible for neurospecific binding.

Utilisation of peptides against microbial infections - a review.

The emergence of resistance in microorganisms on a global scale has made it necessary to search for new antimicrobial factors. Antimicrobial peptides (AMPs) seem to meet these expectations. AMPs are produced by bacteria, viruses, plants, and animals, and may be considered as a new class of drugs intended for the prophylaxis and treatment of both systemic and topical infections. The aim of this study is to review the results of studies on the use of peptides to combat infections in vivo. Antimicrobial peptides may be applied topically and systemically. Among the peptides used topically, a very important area for their application is ophthalmology. AMPs in ophthalmology may be used mainly for the protection of contact lenses from ocular pathogens. Many AMPs are in clinical trials for application in the therapy of local infections. There may be mentioned such preparations as: pexiganan (magainin analogue), MX-226 (based on indolicidin), NEUPREX (isolated from human BPI (bactericidal/permeability-increasing) protein), IB-367 (variant of porcine protegrin), P113 (based on histatin), daptomycin, polymyxins, as well as peptidomimetics. In the combat against systemic infections are used such peptides as: P113D (modified P113 peptide containing D-amino acids), colistin, peptoids, and peptides containing non-typical amino acids or non-peptide elements. AMPs are also used as antiprotozoal, antifungal, antitoxic and immunostimulatory agents. The limitations in the use of peptides in the treatment of infections, such as susceptibility to proteolysis, and resistance of microorganisms to the peptides, are also discussed. AMPs are a promising strategy in the fight against microbial infections.

Isolation of bacteriophages and their application to control Pseudomonas aeruginosa in planktonic and biofilm models.

Pseudomonas aeruginosa is frequently identified as a cause of diverse infections and chronic diseases. It forms biofilms and has natural resistance to several antibiotics. Strains of this pathogen resistant to new-generation beta-lactams have emerged. Due to the difficulties associated with treating chronic P. aeruginosa infections, bacteriophages are amongst the alternative therapeutic options being actively researched. Two obligatorily lytic P. aeruginosa phages, vB_PaeM_MAG1 (MAG1) and vB_PaeP_MAG4 (MAG4), have been isolated and characterized. These phages belong to the PAK_P1likevirus genus of the Myoviridae family and the LIT1virus genus of the Podoviridae family, respectively. They adsorb quickly to their hosts (∼90% in 5 min), have a short latent period (15 min), and are stable during storage. Each individual phage propagated in approximately 50% of P. aeruginosa strains tested, which increased to 72.9% when phages were combined into a cocktail. While MAG4 reduced biofilm more effectively after a short time of treatment, MAG1 was more effective after a longer time and selected less for phage-resistant clones. A MAG1-encoded homolog of YefM antitoxin of the bacterial toxin-antitoxin system may contribute to the superiority of MAG1 over MAG4.

Association between cell-bound haemolysin and cell-detaching activity of Escherichia coli isolated from children.

Haemolysin alpha (HlyA) produced by cell-detaching Escherichia coli, a putative new class of enteric pathogen, is considered to be the main factor responsible for detachment of cells cultured in vitro. HlyA is one of the few E. coli proteins actively secreted into the medium during exponential growth. In the present study 27 HlyA-positive E. coli isolates, randomly selected from stool specimens, produced a cell-bound haemolysin that was detectable during the exponential and stationary growth phases. The influence of both cell-free and cell-bound haemolysins of the selected isolates on cell-detaching activity of E. coli in vitro was determined. The results suggest that cell-bound haemolysin rather than cell-free HlyA was responsible for the cell-detaching activity of E. coli strains tested.

Microarrays--new possibilities for detecting biological factors hazardous for humans and animals, and for use in environmental protection.

Both the known biological agents that cause infectious diseases, as well as modified (ABF-Advanced Biological Factors) or new, emerging agents pose a significant diagnostic problem using previously applied methods, both classical, as well as based on molecular biology methods. The latter, such as PCR and real-time PCR, have significant limitations, both quantitative (low capacity), and qualitative (limited number of targets). The article discusses the results of studies on using the microarray method for the identification of viruses (e.g. Orthopoxvirus group, noroviruses, influenza A and B viruses, rhino- and enteroviruses responsible for the FRI (Febrile Respiratory Illness), European bunyaviruses, and SARS-causing viruses), and bacteria (Mycobacterium spp., Yersinia spp., Campylobacter spp., Streptococcus pneumoniae, Salmonella typhi, Salmonella enterica, Staphylococcus aureus, Neisseria meningitidis, Clostridium difficile , Helicobacter pylori), including multiple antibiotic-resistant strains. The method allows for the serotyping and genotyping of bacteria, and is useful in the diagnosis of genetically modified agents. It allows the testing of thousands of genes in one experiment. In addition to diagnosis, it is applicable for gene expression studies, analysis of the function of genes, microorganisms virulence, and allows the detection of even single mutations. The possibility of its operational application in epidemiological surveillance, and in the detection of disease outbreak agents is demonstrated.

The presence of free shiga-like toxins in stool specimens of patients with diarrhea during one year study.

During one year study, 394 stool samples obtained from a random cases of diarrhea were examined for the presence of free shiga-like toxins Stx1 and Stx2 in Vero cells assay. Of 394 stool specimen supernatants tested, 2 (0.5%) gave positive results. The two stool supernatants positive on Vero cell line were wholly capable of being neutralized with the monoclonal antibody to Stx2. Broth cultures of strains isolated from the two positive stool samples were negative for Stxs-production in Vero cytotoxicity assay and by PCR.

[Isolation of shiga toxin-producing Escherichia coli strains from healthy cattle of the region of Lower Silesia]. / Izolacja shiga-toksycznych szczepów Escherichia coli od zdrowego bydla z regionu dolnego slaska.

The aim of the study was to determine if cattle from the region of Lower Silesia is the reservoir of shiga toxin-producing E. coli strains (STEC) and the analysis of virulence factors of isolated STEC strains. The ability of tested animal strains to shiga toxin synthesis was analysed in cytotoxicity assay in vitro on Vero cell line and then confirmed by detection of shiga toxin-encoding genes by PCR. STEC strains were isolated from 12 (15,2%) of animals examined, 21,4% of these strains were obtained from 9 of 42 calves, and 8,1% from 3 of 37 cows. Most of STEC isolated (75%) was enterohemolysin-producing. The cattle from the region of Lower Silesia is the reservoir of pathogenic for humans sorbitol-fermenting non-O157 STEC strains.

Characterization of five newly isolated bacteriophages active against Pseudomonas aeruginosa clinical strains.

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections, especially in patients with immunodeficiency. It exhibits multiple mechanisms of resistance, including efflux pumps, antibiotic modifying enzymes and limited membrane permeability. The primary reason for the development of novel therapeutics for P. aeruginosa infections is the declining efficacy of conventional antibiotic therapy. These clinical problems caused a revitalization of interest in bacteriophages, which are highly specific and have very effective antibacterial activity as well as several other advantages over traditional antimicrobial agents. Above all, so far, no serious or irreversible side effects of phage therapy have been described. Five newly purified P. aeruginosa phages named vB_PaeM_WP1, vB_PaeM_WP2, vB_PaeM_WP3, vB_PaeM_WP4 and vB_PaeP_WP5 have been characterized as potential candidates for use in phage therapy. They are representatives of the Myoviridae and Podoviridae families. Their host range, genome size, structural proteins and stability in various physical and chemical conditions were tested. The results of these preliminary investigations indicate that the newly isolated bacteriophages may be considered for use in phagotherapy.

The carrier state of shiga-like toxin II (SLT II) and hemolysin-producing enteroaggregative Escherichia coli strain.

Shiga-like toxin-producing (SLTEC) Escherichia coli strains are one of the most important food borne emerging pathogens. One hundred and fifty-seven E. coli strains isolated from 39 children with diarrhea of unknown origin and one hundred and five E. coli strains from 20 healthy children were examined for Shiga-like toxin production in Vero cell line assay. The synthesis of Shiga-like toxin was observed on Vero cell line and confirmed by PCR for one of 262 E. coli strains tested. The shiga-like toxin II-positive E. coli strain was isolated from 2-years old healthy child with no symptoms of gastrointestinal tract infection.

Research on substances with activity against orthopoxviruses.

Although smallpox was eradicated over 30 years ago, the disease remains a major threat. High mortality, high infectivity and low resistance of the contemporary population make the smallpox virus very attractive to terrorists. The possible presence of illegal stocks of the virus or risk of deliberate genetic modifications cause serious concerns among experts. Hence, it is reasonable to seek effective drugs that could be used in case of smallpox outbreak. This paper reviews studies on compounds with proven in vitro or in vivo antipoxviruses potential, which show various mechanisms of action. Nucleoside analogues, such as cidofovir, can inhibit virus replication. Cidofovir derivatives are developed to improve the bioavailability of the drug. Among the nucleoside analogues under current investigation are: ANO (adenozine N1-oxide) and its derivatives, N-methanocarbothymidine [(N)-MCT], or derivatitives of aciklovir, peninclovir and brivudin. Recently, ST-246 - which effectively inhibits infection by limiting release of progeny virions - has become an object of attention. It has been also been demonstrated that compounds such as: nigericin, aptamers and peptides may have antiviral potential. An interesting strategy to fight infections was presented in experiments aimed at defining the role of individual genes (E3L, K3L or C6L) in the pathogenesis, and looking for their potential blockers. Additionally, among substances considered to be effective in the treatment of smallpox cases, there are factors that can block viral inhibitors of the human complement system, epidermal growth factor inhibitors or immunomodulators. Further studies on compounds with activity against poxviruses are necessary in order to broaden the pool of available means that could be used in the case of a new outbreak of smallpox.

Prevalence of non-O157 Escherichia coli strains among shiga-like toxin-producing (SLTEC) isolates in the region of Lower Silesia, Poland.

During 5 y (1997-2002) in the region of Lower Silesia, Poland, 55 shiga-like toxin-producing strains were isolated from children with diarrhoea, none of whom developed haemolytic uremic syndrome. Shiga-like toxin production was detected on Vero cells and confirmed by the detection of slt1 and slt2 gene sequences. Most of isolates examined possessed slt2 gene alone (41.8%), or in combination with slt1 gene (38.2%). None of the shiga-like toxin-producing strains was of serotype O157:H7. It appears that non-O157 shiga-like toxin-producing E. coil strains are prevalent as the causal agent of severe diarrhoea in the region.

Comparison of multiplex PCR, and an immunochromatographic method sensitivity for the detection of Escherichia coli O157:H7 in minced beef.

In this study, the sensitivities of multiplex PCR and an immuno-chromatographic methods to detect Escherichia coli O157:H7 in minced beef were compared. The detection of Escherichia coli O157:H7 in minced beef inoculated with 1-100 cells of this bacterium was possible after enrichment of culture and subsequent analysis by either of the two methods. Enrichment conditions were eight hours of incubation at 37 degrees C or 42 degrees C in a non-selective medium (Buffered Peptone Water). Multiplex PCR analysis was performed using three primer sets with analysis by gel electrophoresis. The Quix immuno-chromatographic assay which is a new kit being marketed by New Horizons Diagnostics, Columbia, MD, was used for immunological analysis of the enriched broths.The sensitivity of both tests was similar. The results depended on the concentration of the specific bacterium in the culture since the influence of the proportion of other bacteria to the E. coli O157:H7 was not observed. The data suggests that either method or used together, when coupled with an enrichment technique, could provide a rapid mean to detect the presence of this pathogen in minced meat samples.