L.Y. Brovko

Oriented Immobilization of Bacteriophages for Biosensor Applications

ABSTRACT A method was developed for oriented immobilization of bacteriophage T4 through introduction of specific binding ligands into the phage head using a phage display technique. Fusion of the biotin carboxyl carrier protein gene (bccp) or the cellulose binding module gene (cbm) with the small outer capsid protein gene (soc) of T4 resulted in expression of the respective ligand on the phage head. Recombinant bacteriophages were characterized in terms of infectivity. It was shown that both recombinant phages retain their lytic activity and host range. However, phage head modification resulted in a decreased burst size and an increased latent period. The efficiency of bacteriophage immobilization with streptavidin-coated magnetic beads and cellulose-based materials was investigated. It was shown that recombinant bacteriophages form specific and strong bonds with their respective solid support and are able to specifically capture and infect the host bacterium. Thus, the use of immobilized BCCP-T4 bacteriophage for an Escherichia coli B assay using a phage multiplication approach and real-time PCR allowed detection of as few as 800 cells within 2 h.

Role of efflux pumps in adaptation and resistance of Listeria monocytogenes to benzalkonium chloride

ABSTRACT In this study, potential mechanisms underlying resistance and adaptation to benzalkonium chloride (BC) in Listeria monocytogenes were investigated. Two groups of strains were studied. The first group consisted of strains naturally sensitive to BC which could be adapted to BC. The second group consisted of naturally resistant strains. For all adapted isolates, there was a correlation between the resistance to BC and ethidium bromide, but this was not the case for the naturally resistant isolates. To investigate the role of efflux pumps in adaptation or resistance, reserpine, an efflux pump inhibitor, was added to the strains. Addition of reserpine to the sensitive and adapted strains resulted in a decrease in the MIC for BC, whereas no such decrease was observed for the resistant strains, indicating that efflux pumps played no role in the innate resistance of certain strains of L. monocytogenes to this compound. Two efflux pumps (MdrL and Lde) have been described in L. monocytogenes. Studies showed low and intermediate levels of expression of the genes encoding the efflux pumps for two selected resistant strains, H7764 and H7962, respectively. Adaptation to BC of sensitive isolates of L. monocytogenes resulted in significant increases in expression of mdrl (P < 0.05), but no such increase was observed for lde for two adapted strains of L. monocytogenes, LJH 381 (P = 0.91) and C719 (P = 0.11). This indicates that the efflux pump Mdrl is at least partly responsible for the adaptation to BC.

Assessment of Photodynamic Destruction of Escherichia coli O157:H7 and Listeria monocytogenes by Using ATP Bioluminescence

ABSTRACT Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes. To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes. The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction and intracellular ATP leakage were different for E. coli and L. monocytogenes. These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.

Use of bioluminescent Salmonella for assessing the efficiency of constructed phage-based biosorbent

A bacteriophage-based biosorbent for Salmonella enteritidis was constructed, and bacterial bioluminescence was used for assessment of the efficiency of cell capture. A strain of S. enteritidis with bioluminescent phenotype was constructed by transformation with plasmid pT7 carrying the entire lux operon from Photorhabdus luminescens. The relation between relative light output (RLU) and colony-forming units (CFU/ml) of the bioluminescent strain was established. The bacteriophage specific to S. enteritidis was biotinylated, and the biotinylation procedure was optimized based on the maximum retention of phage infectivity. The biotinylated phages were then coated onto streptavidin-labeled magnetic beads, and were used to capture the bioluminescent S. enteritidis cells. Our preliminary results showed that the number of cells captured by constructed biosorbent was five times higher than that of the control, magnetic beads coated with nonbiotinylated phage, indicating the capture is specific. Journal of Industrial Microbiology & Biotechnology (2000) 25, 273–275.

Antibacterial Photosensitization-Based Treatment for Food Safety

Development of novel methods for decontamination of food and food-processing, food-handling environment, which are compatible with the consumer demand for minimally processed safe foods, remains one of the urgent topics of food science. One of these methods—antibacterial photosensitization-based treatment—is gaining more attention recently due to its unique properties. The method is based on combined action of nontoxic dye (called photosensitizer), visible light, and oxygen-producing cytotoxic effect (photodynamic effect). Presently, this method is widely used as anticancer and antiinfections treatments under the name photodynamic therapy. Photosensitization-based treatment was shown to be effective against a wide range of microorganisms (Gram-positive and Gram-negative bacteria in both vegetative form and spores, as well as in biofilms, mammalian viruses and bacteriophages, fungi and yeasts, and parasitic protozoa); no existing or emerging resistance against this treatment was observed even after multiple applications and the possibility of that is considered very unlikely; possibility to initiate the process on-spot and on-demand by targeted delivery of illumination provides multiple treatment options tailored to each particular case. Recent advances in the area of antibacterial photosensitization-based treatment applications in food and food-processing environment are presented in this review. Advantages and shortcomings of the method are discussed.

Bacteriophages for Detection and Control of Bacterial Pathogens in Food and Food-Processing Environment

This chapter presents recent advances in bacteriophage research and their application in the area of food safety. Section 1 describes general facts on phage biology that are relevant to their application for control and detection of bacterial pathogens in food and environmental samples. Section 2 summarizes the recently acquired data on application of bacteriophages to control growth of bacterial pathogens and spoilage organisms in food and food-processing environment. Section 3 deals with application of bacteriophages for detection and identification of bacterial pathogens. Advantages of bacteriophage-based methods are presented and their shortcomings are discussed. The chapter is intended for food scientist and food product developers, and people in food inspection and health agencies with the ultimate goal to attract their attention to the new developing technology that has a tremendous potential in providing means for producing wholesome and safe food.

Bacteriophage-based biosorbents coupled with bioluminescent ATP assay for rapid concentration and detection of Escherichia coli

Wild type T4 bacteriophage and recombinant T4 bacteriophages displaying biotin binding peptide (BCCP) and cellulose binding module (CBM) on their heads were immobilized on nano-aluminum fiber-based filter (Disruptor), streptavidin magnetic beads and microcrystalline cellulose, respectively. Infectivity of the immobilized phages was investigated by monitoring the phage-mediated growth inhibition of bioluminescent E. coli B and cell lysis using bioluminescent ATP assay. The results showed that phage immobilization resulted in a partial loss of infectivity as compared with the free phage. Nevertheless, the use of a biosorbent based on T4 bacteriophage immobilized on Disruptor filter coupled with a bioluminescent ATP assay allowed simultaneous concentration and detection of as low as 6 x 10(3)cfu/mL of E. coli in the sample within 2h with high accuracy (CV=1-5% in log scale). Excess of interfering microflora at levels 60-fold greater than the target organism did not affect the results when bacteriophage was immobilized on the filter prior to concentration of bacterial cells.

Characterization of immune-active peptides obtained from milk fermented by Lactobacillus helveticus.

The objectives of this research were to confirm the effect of compounds derived from milk fermented by Lactobacillus helveticus (LH-2) on the nonspecific host defence system, and isolate and characterize the active peptides that mediate the immune response. The cell-free supernatant obtained from the fermented milk and its fractions were tested in vitro for immuno-modulating activity using murine macrophages (RAW 264.7 cell line). Cytokine production (Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-alpha), and Interleukin-1beta (IL1-beta)), nitric oxide (NO) production and phagocytosis were used as biomarkers. Macrophages stimulated with cell-free supernatant of fermented milk showed higher production of cytokines and NO compared with macrophages stimulated with LPS (Lipopolysaccharide) and a commercial immunomodulator derived from beta-casein (f54-59). Phagocytosis was observed by macrophages stimulated with the supernatant. Two of nine fractions collected from the supernatant using size exclusion chromatography produced the highest response when used to stimulate macrophages. The results of the dose-response study of the effect of the fraction with the highest stimulation effect on the production of TNF-alpha showed a direct correlation between protein concentration and TNF-alpha release. The fraction contained four novel peptides, three derived from the hydrolysis of beta-casein and one from the hydrolysis of alpha-lactalbumin. These results confirm that fermentation of milk by Lactobacillus helveticus (LH-2) results in the production of specific peptides capable of modulating macrophage activity.

Comparison of ATP and in vivo bioluminescence for assessing the efficiency of immunomagnetic sorbents for live Escherichia coli O157:H7 cells

Aims: To develop methods to assess the efficiency of immunomagnetic separation (IMS).

Photodynamic treatment: a new efficient alternative for surface sanitation.

A novel and promising technology-photodynamic treatment (PDT), aimed for surface cleaning and sanitation in food industry-is presented. It is based on the treatment of surfaces with nontoxic dyes (photosensitizers), followed by illumination of the surface with regular white light. The method is currently used in the medical field and was proved to have wide specificity against a variety of bacterial and viral pathogens as well as against yeasts and protozoa. An additional advantage of this approach is that development of resistance of microorganisms to PDT was shown to be unlikely. The theoretical basis of light-induced antimicrobial treatment is described, followed by examples of its application for the cleaning and disinfection of surfaces. All available information supports the idea that PDT could offer a very efficient and cost-effective way to combat microbial contamination of foods. The advantages and pitfalls of the technique are discussed. Directions of future research needed for bringing the technology to commercial reality are identified.