Electrochemical, optical and mass-based immunosensors: A comprehensive review of Bacillus anthracis detection methods.

A biosensor is an analytical device whose main components include transducer and bioreceptor segments. The combination of biological recognition with the ligand is followed by transformation into physical or chemical signals. Many publications describe biological sensors as user-friendly, easy, portable, and less time-consuming than conventional methods. Among major categories of methods for the detection of Bacillus anthracis, such as culture-based microbiological method, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), microarray-based techniques sensors with bioreceptors have been highlighted which particular emphasis is placed on herein. There are several types of biosensors based on various chemical or physical transducers (e.g., electrochemical, optical, piezoelectric, thermal or magnetic electrodes) and the type of biological materials used (e.g., enzymes, nucleic acids, antibodies, cells, phages or tissues). In recent decades, antibody-based sensors have increasingly gained popularity due to their reliability, sensitivity and rapidness. The fundamental principle of antibody-based sensors is mainly based on the molecular recognition between antigens and antibodies. Therefore, immunosensors that detect B. anthracis surface antigens can provide a rapid tool for detecting anthrax bacilli and spores, especially in situ. This review provides a comprehensive summary of immunosensor-based methods using electrochemical, optical, and mass-based transducers to detect B. anthracis.

Three Novel Bacteriophages, J5a, F16Ba, and z1a, Specific for Bacillus anthracis, Define a New Clade of Historical Wbeta Phage Relatives.

Bacillus anthracis is a potent biowarfare agent, able to be highly lethal. The bacteria dwell in the soil of certain regions, as natural flora. Bacteriophages or their lytic enzymes, endolysins, may be an alternative for antibiotics and other antibacterials to fight this pathogen in infections and to minimize environmental contamination with anthrax endospores. Upon screening environmental samples from various regions in Poland, we isolated three new siphophages, J5a, F16Ba, and z1a, specific for B. anthracis. They represent new species related to historical anthrax phages Gamma, Cherry, and Fah, and to phage Wbeta of Wbetavirus genus. We show that the new phages and their closest relatives, phages Tavor_SA, Negev_SA, and Carmel_SA, form a separate clade of the Wbetavirus genus, designated as J5a clade. The most distinctive feature of J5a clade phages is their cell lysis module. While in the historical phages it encodes a canonical endolysin and a class III holin, in J5a clade phages it encodes an endolysin with a signal peptide and two putative holins. We present the basic characteristic of the isolated phages. Their comparative genomic analysis indicates that they encode two receptor-binding proteins, of which one may bind a sugar moiety of B. anthracis cell surface.

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.

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.

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.

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.

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.

[Sporicidal activity of hydrogen peroxide and peracetic acid against Bacillus anthracis spores]. / Sporobójcza aktywnosc nadtlenku wodoru i kwasu nadoctowego wobec przetrwalników Bacillus anthracis.

The aim of the presented study was determined the effectiveness of sporicidal activity the peracetic acid and the hydrogen peroxide against B. anthracis spores. In the investigations was used B. anthracis stain "Sterne" 34F2. As inactivators were applied 0,5 % natriumthiosulphate and catalase. The obtained results show that the sporicidal effect of studied substances depends from their concentration and operates time. 5% water solution of peracetic acid shows the full sporicidal activity after outflow 120 minutes and the hydrogen peroxide about concentration 30% after outflow 180 minutes. However the hydrogen peroxide.

[Anthrax--continuous threat to humans and animals]. / Waglik--stale aktualne zagrozenie dla ludzi i zwierzat.

Gram-positive, spore-forming, aerobic bacterium Bacillus anthracis is an etiological agent of anthrax a disease very dangerous to humans and all warm-blooded animals. The spore forms are markedly resistant to unfavourable environmental extremes of heat, cold, desiccation, chemicals, irradiation etc. The vegetative forms characterised virulence factors: the antiphagocytic poly-gamma-D-polipeptide capsule and three proteins, edema factor (EF), lethal factor (LF) and protective antigen (PA). Anthrax is mainly transmitted from animals to man through food of animal origin, animal products and contamination of the environment with B. anthracis and its spores. There are three types of this disease: cutaneous, intestinal and inhalation anthrax. Research on anthrax as a biological weapon began more then 80 years ago. Depending on the target chosen and the scale of the attack the anthrax spores may by used to contaminate of foodstuffs or liquids and water. The aerosolised release of anthrax spore can cause illness with a high fatality rate.

[Gamma radiation resistance of Bacillus anthracis spores]. / Opornosc przetrwalników Bacillus anthracis na promieniowanie gamma.

The aim of the presented study was determined the effectiveness of action the gamma radiation on water suspension B. anthracis spores. The irradiation was performed using a Cobalt 60 (Co 60) source, by using single and fractionary irradiation doses. In the investigations was used B. anthracis stain "Sterne" 34F2. The obtained results show, that gamma radiation effectively inactivates B. anthracis spores. On the efficiency of sterilization process influence the irradiation's method and the number of spores in 1 ml suspension. In the suspension 1.5 x 10(9) spore in 1 ml, sporicidal doses gamma radiation amount to 25.0 kGy (single dose) or 41.5 kGy (fractionary dose). The volume suspension about definite inoculum of spores, subjected working the gamma rays has not influence on sporicidal effectiveness of radiation sterilization.