Control of Salmonella Newport on cherry tomato using a cocktail of lytic bacteriophages.

Bacteriophages have been envisioned as tools to control a variety of foodborne pathogenic bacteria. Salmonella is a foodborne pathogen that is a threat to public health around the world. Contaminated tomatoes have been associated with several Salmonella outbreaks. Hence, the objective of this work was to identify and characterize different lytic bacteriophages against Salmonella Newport, as one of top ten Salmonella serovars associated with human salmonellosis in North America, and then apply these phages to enhance the safety of cherry tomatoes. Four lytic phages against Salmonella Newport were selected based on their ability to lyse a majority of the 26 screened Salmonella serovars. The selected phages belong to Myoviridae (vB_SnwM_CGG4-1, vB_SnwM_CGG4-2) and Siphoviridae (vB_SnwM_CGG3-1, vB_SnwM_CGG3-2) families. They were found to be stable at different temperatures and pH, have latent periods ranging from 53 to 65 min and burst sizes from 92 to 177. In addition, the two Myoviridae phages have a lower frequency of developing bacteriophage insensitive mutants when compared with the Siphoviridae phages. No significant change in virulence gene expression was observed in the developed bacteriophage insensitive mutants when compared to the parental phage sensitive strain. Furthermore, the vB_SnwM_CGG4-1 genome revealed no homology to virulence or lysogenic genes. A phage cocktail was used to control the growth of S. Newport in broth medium and on contaminated cherry tomato. Complete inhibition of bacterial growth in broth medium was observed at 25 °C for 24 h. In addition, a 4.5 log10 unit reduction in the bacterial count was observed when applying the phage cocktail onto contaminated tomatoes stored at 22 °C for 3 days. These findings suggest that the isolated phages can be used for biocontrol of S. Newport to improve the safety of ready-to-eat (RTE) produce.

Antimicrobial light-activated materials: towards application for food and environmental safety.

AIMS: To produce light-activated antimicrobial materials composed of the photodynamic dye phloxine B incorporated into paper or cellulose membranes and to investigate ability of these materials to decrease bacterial loads on their surfaces as well as on food surfaces that were in contact with these materials under illumination with regular white light. METHODS AND RESULTS: Antimicrobial cellulose-based materials with incorporated phloxine B were produced using a layer-by-layer deposition method. Antimicrobial properties of the materials were tested in model systems as well as for decontamination of food and food contact surfaces. Pseudomonas aeruginosa, Listeria monocytogenes and Bacillus anthracis were efficiently killed by exposure of the bacterial suspension to the dye-containing material under illumination with white light, but Salmonella Typhimurium and Escherichia coli O157:H7 were only partially affected. Application of the materials for decontamination of food surfaces artificially contaminated with L. monocytogenes was shown to be ineffective, while the self-decontamination of the material surface by exposure to white light resulted in eradication of L. monocytogenes cells from the material surface. CONCLUSIONS: The developed materials showed significant self-decontaminating ability when under illumination; however, decontamination of food surfaces in contact with the developed materials was not achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the antibacterial activity of materials with incorporated photodynamic dyes when under illumination with regular-intensity white light. Possible uses of the light-activated antimicrobial materials for food processing, as food contact surfaces, and surfaces in public areas to prevent cross-contamination are discussed.

[Bilateral massive pulmonary embolism: clinical observation and literature review].

The description of successful surgical treatment of a patient with bilateral massive pulmonary embolism (with the defeat of the equity and segmental branches), thrombosis of the right atrium and patent foramen ovale is given. The authors emphasize that determining predictors of successful surgical treatment of bilateral pulmonary embolism in a patient with high risk of death are the following: a) operational emergency diagnosis of disease; b) time from the beginning of clinical manifestations till embolectomy (within 1 hour); c) the maximum total removal of blood clots from the pulmonary artery and its branches. Dynamic 12 months observation showed a significant decrease of pulmonary perfusion deficiency, improvement of functional parameters of right heart chambers, absence of thromboembolism relapses.

Bioluminescent high-throughput assay for the bacteria adherence to the tissue culture cells.

The goal of this study was develop a rapid high-throughput method for the assessment of the bacterial adhesion to tissue culture cells and test this method by investigation of the adhesion and growth of pathogenic and non-pathogenic Escherichia coli strains in the presence of HeLa human epithelial cells. Fifteen strains of E. coli were transformed with a plasmid carrying the entire lux operon of Photorhabdus luminescens to make them bioluminescent. By using the Time-to-Detection approach and bioluminescence imaging in microplate format, the adherence and growth of bacteria in tissue culture medium in the presence of HeLa cells was monitored. It was observed that Eagle's minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) significantly inhibited growth of E. coli. However, in the presence of HeLa cells the detected growth of E. coli was similar to the growth observed in LB medium. It was established that the initial number of E. coli cells present in the microplate directly correlated with the time necessary for the bioluminescence signal to reach the threshold level, hence allowing the accurate assessment of the adhered cells within 8-10 h. Neither bacterial adherence nor growth kinetics correlated with the pathogenicity of the strain though they were strain-specific. The developed approach provided new information on the interaction of E. coli with epithelial cells and could be used for both pathogenicity research and for the screening of potential therapeutic agents for the ability to minimize pathogen colonization of human tissues.

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.

Oriented immobilization of bacteriophages for biosensor applications.

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.

Immobilization of bacteriophages on gold surfaces for the specific capture of pathogens.

Techniques for the chemical attachment of wild-type bacteriophages onto gold surfaces and the subsequent capture of their host bacteria have been developed. The surfaces were modified with sugars (dextrose and sucrose) as well as amino acids (histidine and cysteine) to facilitate such attachment. Non-specific attachment was prevented by using bovine serum albumin as blocking layer. Surfaces modified with cysteine (and cysteamine) followed by activation using 2% gluteraldehyde resulted in an attachment density of 18+/-0.15 phages/microm(2). This represented a 37-fold improvement compared to simply applying physisorption. Subsequently, the phage immobilized surfaces were exposed to the host E. coli EC12 bacteria and capture was confirmed by fluorescence microscopy. We obtained a bacterial capture density of 11.9+/-0.2/100 microm(2), a 9-fold improvement when compared to those on physically adsorbed phages. The specificity of recognition was confirmed by exposing similar surfaces to three strains of non-host bacteria. These negative control experiments do not show any bacterial capture. In addition, no capture of the host was observed in the absence of the phages.

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

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.

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.

Protein structure and bioluminescent spectra for firefly bioluminescence.

Modern theory on general and specific effects of microenvironment on emission spectra was used for explanation of spectral differences for both natural and mutant forms of beetle luciferases, as well as for bioluminescence emitter oxyluciferin in model systems. For the analysis, both authors' and other published data were used. It was shown that active site mutations that resulted in spectral shifts of bioluminescence as a rule caused substantial decrease in the catalytic activity of the enzyme. At the same time, mutations in the conservative regions of the protein amino acid sequence that were in the periphery of the protein globe resulted in red shift of the bioluminescence spectra without affecting catalytic activity. Correlation was observed between the value of spectral shift and polarizability of the introduced amino acid residue: the higher the polarizability, the larger was the red shift of bioluminescence.

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). METHODS AND RESULTS: The capturing efficiency of biosorbents for Escherichia coli O157:H7, constructed using streptavidin-coated magnetic beads and biotinylated antibodies, was tested using both in vivo and ATP bioluminescence. Both methods were suitable for the enumeration of bacteria captured by the biosorbents. The level of both ATP and in vivo bioluminescence depended on the media used, but was unaffected by the magnetic beads. The capture efficiency depended on time and sample volume, but did not depend on the length of spacer arm of the biotinylation agent. For cell concentrations of

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.

Influence of phage population on the phage-mediated bioluminescent adenylate kinase (AK) assay for detection of bacteria.

AIMS: The effect of phage concentration on the activity of adenylate kinase (AK) released from the cells lysed during infection was investigated in order to optimize a bioluminescent phage-mediated method for bacterial enumeration. METHODS AND RESULTS: The number of bacteria lysed by phages specific to Salmonella enteritidis and E. coli was determined using a bioluminescent method for the detection of AK released. In order to optimize the assay, the effect of phage concentration and time of infection on the amount of AK released was investigated. The release of AK was greatest at a multiplicity of infection (moi) of 10-100. CONCLUSION: The amount of AK released from Salmonella enteritidis and E. coli G2-2 cells by specific phages, SJ2 and AT20, respectively, depended on the type of bacteria, the stage of growth, the nature of phage, moi and time. SIGNIFICANCE AND IMPACT OF THE STUDY: An assay is described which allows detection of E. coli and Salmonella Enteritidis within 2 h at levels of 103 cfu ml-1.

Fluorescent properties of firefly luciferases and their complexes with luciferin.

Fluorescence of luciferases from Luciola mingrelica (single tryptophan residue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417, Trp-426) was studied. Analysis of quenching of tryptophan fluorescence showed that the tryptophan residue conserved in all luciferases is not accessible for charged quenchers, which is explained by the presence of positively and negatively charged amino acid residues in the close vicinity to it. An effective energy transfer from tryptophan to luciferin was observed during quenching of tryptophan fluorescence of both luciferases with luciferin. From the data on the energy transfer, the distance between the luciferin molecule and Trp-417 (419) in the luciferin luciferase complex was calculated: 11-15 A for P. pyralis and 12-17 A for L. mingrelica luciferases. The role of the conserved Trp residue in the catalysis is discussed.

Fluorescence of tryptophan residues in firefly luciferases and enzyme--substrate complexes.

Quenching of tryptophan fluorescence of Luciola mingrelica (single tryptophan residue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417 and Trp-426) luciferases with different quenchers (I-, Cs+, acrylamide) was studied. The conserved Trp-417(419) residue was shown to be not accessible to charged particles, and positively and negatively charged amino acid residues are located in close vicinity to it. We found previously unreported effective energy transfer from this tryptophan to luciferin during the quenching of the tryptophan fluorescence. The distance between the luciferin molecule and Trp-417(419) was calculated: 11-15 and 12-17 A for P. pyralis and L. mingrelica luciferases, respectively. The role of the conserved Trp residue in the catalysis is discussed. ATP and AMP are also quenchers of the tryptophan fluorescence of the luciferases. In this case, an allosteric mechanism of the interaction of Trp-417(419) with an excess of ATP (AMP) is proposed.

[Bioluminescent assay of total bacterial contamination of raw milk]. / Bioliuminestsentnyi metod opredeleniia obshcheo bakterial'noi obsemenennosti syrogo moloka.

A rapid (30-35 min) bioluminescence assay of total bacterial contamination (TBC) of raw milk was optimized. This method includes incubation of milk samples in the presence of Neonol-10 and medical purity grade pancreatin with further removal of nonbacterial ATP by filtration through a membrane filter, cell disruption by treatment with dimethyl sulfoxide, and measurement of ATP concentration in a reaction with the bioluminescent reagent Immolum. The TBC detection threshold is 0.5 x 10(5) colony-forming units (CFU) per ml milk. Coefficients of correlation between the standard plate count method and bioluminescence assay (R) and residual standard deviations (Sxy) in raw milk samples (n = 140) were 0.83 and 0.54, respectively. In sterilized milk samples artificially contaminated with pure cultures of the main representatives of milk microflora (coli-forms, Staphylococcus aureus, Streptococcus thermophilus, and Streptococcus group D), these values were 0.89-0.99 and 0.09-0.29, respectively. The specific content of ATP was found to be (0.8 +/- 0.1) x 10(-18) mol/CFU in coli-forms; (12.0 +/- 8.1) x 10(-18) mol/CFU in S. aureus; (35.2 +/- 16.9) x 10(-18) mol/CFU in S. thermophilus; and (42.5 +/- 1.3) x 10(-18) in Streptococcus group D.

Bioluminescence of free and poly(vinyl alcohol) cryogel-entrapped recombinant Escherichia coli cells expressing firefly luciferase.

Bioluminescence of free and poly(vinyl alcohol) cryogel-entrapped recombinant E. coli cells expressing firefly luciferase was investigated. It was shown that bioluminescence intensity and time-course of the bioluminescent signal changed upon immobilization and depended on intracellular ATP concentration and permeability of the cell membrane.

[A bioluminescent method of determining antibiotic sensitivity of microbial cells in septic blood]. / Bioliuminestsentnyi metod opredeleniia antibiotikochuvstvitel'nosti mikrobnykh kletok v septicheskoi krovi.

A bioluminescence assay for rapid (5-7 h) determination of susceptibility to antibiotics was applied to samples of septic blood and optimized. The method comprises hemolysis of blood, reduction of osmolarity by adding concentrated nutritive media, and further incubation of the samples in the presence or absence of therapeutic doses of the antibiotic examined. Growth of bacteria is estimated by the level of bacterial ATP in the sample, which is determined by a bioluminescence assay. Hemolysis and further incubation of samples in nutrition media reduced the concentration of nonbacterial ATP to a level that did not interfere with the determination of bacterial ATP. There was a positive correlation between the levels of resistance to antibiotics determined by the bioluminescence assay and standard plate counts.

[Comparative evaluation of methods of intracellular ATP extraction from different types of microorganisms for bioluminescent determinationof microbial cells]. / Sravnenie metodov ekstraktsii vnutrikletochnogo ATF mikroorganizmov razlichnogo tipa dlia bioliuminestsentnogo opredeleniia kletok mikro organizma.

The efficiency of dimethyl sulfoxide (DMSO), trichloroacetic acid (TCA), and cetyltrimethylammonium bromide (CTAB) as extractants of intracellular ATP from various microorganisms was compared in bioluminescent measurements of microbial cell concentrations. Extraction with CTAB was found to provide an approximately ten times higher sensitivity of the bioluninescent assay of microbial cells than extraction with DMSO or TCA. In Gram-positive bacteria and yeasts, the ATP concentration in the extract was a linear function of the microbial suspension density only within a cell concentration range of D600 0.02-3.5 in the three types of tested extracts. In Gram-negative bacteria, a significant deviation from the linear dependence between ATP concentration and microbial suspension density was observed in CTAB extracts at D600 > 1.

[The ATP content of the neutrophils and whole blood in the inhabitants of regions in the Altai Territory subjected to nuclear testing exposure]. / Soderzhanie ATF v neitrofilakh i tsel'noi krovi u zhitelei raionov Altaiskogo kraia, podvergshikhsia vozdeistviiu iadernykh ispytanii.

The bioluminescent method was used in the studies of the influence of ionizing irradiation and/or xenobiotics on the content of ATP in RBC and neutrophils of rats, and in whole blood and neutrophils of 80 examined women of Altai Region exposed to ionizing radiation during a series of nuclear tests in Semipalatinsk in 1949-1965. Deviations from the normal ATP content were measured with due to account of the natural variability of a given metabolite. For rats, deviations from the content of ATP in erythrocytes were short-term, those in neutrophils were long-term. For people, a statistically significant increase in the content of ATP in neutrophils as compared to the control was observed. A non-linear correlation between the content of ATP in neutrophils and the calculated dose of radiation was observed. An increase in the ATP content in whole blood, with regard to the control, was not statistically significant for all groups of examined persons.