Isolation and characterization of a protective bacterial culture isolated from honey active against American Foulbrood disease

Bacterial strains isolated from US domestic honey were screened for antibacterial activity against Paenibacillus larvae ssp. larvae, the causative agent of American Foulbrood (AFB) in apiaries. A bacterial isolate (TH13) showing a high level of antimicrobial activity against P. larvae ssp. larvae ATCC 9545 was selected and identified as Paenibacillus polymyxa by 16S rRNA gene sequencing. The antimicrobial compound was purified by 80% saturated ammonium sulfate precipitation followed by CM-sepharose chromatography and reverse-phase HPLC. The molecular mass of the compound was determined to be 1168.78 Da by ESI-qTOF MS, matching that of polymyxin E1. The producer strain showed a broad range of antibacterial activity against Gram-positive and -negative bacteria including P. larvae ssp. larvae ATCC 25747 and foodborne pathogens such as Bacillus cereus F4552 and Escherichia coli O157:H7 ATCC 43895. The selection of antibiotic-producing bacterial strains indigenous to honey as protective cultures against AFB may lessen the use of antibiotics in apiarie

Biosynthesis and transcriptional analysis of thurincin H, a tandem repeated bacteriocin genetic locus, produced by Bacillus thuringiensis SF361

Thurincin H, a bacteriocin produced by Bacillus thuringiensis SF361 isolated from honey, strongly inhibited the growth of Bacillus cereus F4552. The bacteriocin was purified by 65% ammonium sulfate precipitation of the culture supernatant, followed by octyl-sepharose CL-4B and reverse-phase HPLC. The molecular mass of the bacteriocin was determined to be 3139.51 Da and the 14 amino acids of the bacteriocin at the N-terminus were identified. The complete amino acid sequence of mature thurincin H was deduced from three structural genes, thnA1, thnA2, and thnA3 found in tandem repeats on the chromosome, all of which encode for the same bacteriocin, thurincin H. The genetic determinants for thurincin H biosynthesis consist of 10 ORFs, including three thurincin H structural genes. Northern hybridization elucidated that the transcription of all three bacteriocin structural genes was regulated by a putative promoter located upstream of thnA

Isolation of Bacteriocin-producing Staphylococcus spp. Strains from Human Skin Wounds, Soft Tissue Infections and Bovine Mastitis

A collection of 206 Staphylococcus spp. isolates was investigated for their ability to produce compounds exhibiting antistaphylococcal activity. This group included Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus xylosus strains recovered from bovine mastitis (n = 158) and human skin wounds and soft tissues infections (n = 48). Production of substances with antimicrobial activity was observed in six strains. Five of them were recovered from bovine mastitis, and one was isolated from the infected human skin wound. Three of the six antimicrobials produced by the different strains showed substantial loss of antimicrobial activity upon treatment with proteolytic enzymes, which suggests their peptidic structure. Additional studies have shown that one of the putative bacteriocins was efficiently secreted to the liquid medium, facilitating its large-scale production and isolation. The peptide produced by the M2B strain exhibited promising activity; however, against narrow spectrum of Staphylococcus spp. clinical and animal isolates. Growth inhibition was observed only in the case of 13 (including nine S. aureus, three S. xylosus and one S. epidermidis strains) out of 206 strains tested. Important advantage of the produced agent was its high thermal stability. Fifteen minutes of incubation at 90°C did not affect its antimicrobial potential. The highest efficiency of production of the agent was demonstrated in TSB medium after 24 hours at 37°C. The researches revealed that ability to production of bacteriocin among staphylococci is not very common. Only one (S. xylosus strain assigned as M2B) out of 206 strains tested produced satisfactory amounts of antistaphylococcal bacteriocin. In spite of that, we would encourage other researchers for investigation of their collections of Staphylococcus spp. isolates towards selection strains producing antimicrobial agents

Characterization of Mundticin L, a Class IIa Anti-Listeria Bacteriocin from Enterococcus mundtii CUGF08▿

Enterococcus mundtii CUGF08, a lactic acid bacterium isolated from alfalfa sprouts, was found to produce mundticin L, a new class IIa bacteriocin that has a high level of inhibitory activity against the genus Listeria. The plasmid-associated operons containing genes for the mundticin L precursor, the ATP binding cassette (ABC) transporter, and immunity were cloned and sequenced. The fifth residue of the conservative consensus sequence YGNGX in the mature bacteriocin is leucine instead of valine in the sequences of the homologous molecules mundticin KS (ATO6) and enterocin CRL35. The primary structures of the ABC transporter and the immunity protein are homologous but unique

Evaluation of high pressure processing (HPP) inactivation of Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes in acid and acidified juices and beverages

Increasing consumer demand for high-quality foods has driven adoption by the food industry of non-thermal technologies such as high pressure processing (HPP). The technology is employed as a post-packaging treatment step for inactivation of vegetative microorganisms. In order to evaluate HPP inactivation of Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes in acid and acidified juices and beverages, pressure tolerance parameters were determined using log-linear and Weibull models in pH-adjusted apple juice (pH 4.5) at 5 °C. A commercial processing HPP unit was used. The Weibull model better described the inactivation kinetics of the three tested pathogens. According to estimates from the Weibull model, 1.5, 0.9, and 1.5 min are required at 600 MPa to produce 5-log reductions of E. coli, Salmonella, and L. monocytogenes, respectively, whereas according to the log-linear model, 3.2, 1.8, and 2.1 min are required. The effects of process conditions were verified using commercial products (pH between 3.02 and 4.21). In all tested commercial juices or beverages, greater than 5-log reductions were achieved for all tested pathogens using HPP process conditions of 550 MPa for 1 min. These findings demonstrate that the HPP conditions of 600 MPa for 3 min, typically used by the food industry provide an adequate safety margin for control of relevant vegetative pathogens in acid and acidified juices and beverages (pH < 4.5). Results from this study can be used by food processors to support validation studies and may be useful for the future establishment of safe harbors for the HPP industry.Universidad de Costa Rica/[735-B6-777]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Centro Nacional de Ciencia y Tecnología de Alimentos (CITA

The effectiveness of treating irrigation water using ultraviolet radiation or sulphuric acid fertilizer for reducing generic Escherichia coli on fresh produce—a controlled intervention trial

Aims The aims of this study were to: (i) estimate the effectiveness of ultraviolet radiation (UV) and sulphuric acid-based fertilizer (SA), at reducing levels of generic Escherichia coli in surface irrigation water and on produce and surface soil in open produce fields; and (ii) describe the population dynamics of generic E. coli in produce fields. Methods and Results Spinach and cantaloupe plots were randomly assigned to control, UV or SA treatment groups. Irrigation water was inoculated with Rifampicin-resistant E. coli prior to treatment. More than 75% of UV- and SA-treated tank water samples had counts below the detection limit, compared to a mean count of 3·3 Log10 CFU per ml before treatment. Levels of Rifampicin-resistant E. coli in soil and produce both increased and decreased over 10–15 days after irrigation, depending on the plot and time-period. Conclusions UV and SA treatments effectively reduce the levels of E. coli in surface irrigation water. Their effectiveness at reducing contamination on produce was dependent on environmental conditions. Applying wait-times after irrigation and prior to harvest is not a reliable means of mitigating against contaminated produce. Significance and Impact of the Study The results are of timely importance for the agricultural industry as new FSMA guidelines require producers to demonstrate a low microbial load in irrigation water or allow producers to apply a wait-time to mitigate the risk of contaminated produce.Universidad de Costa Rica/[735-B6-777]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Centro Nacional de Ciencia y Tecnología de Alimentos (CITA

Inactivation of Salmonella and Escherichia coli in surface agricultural water using a commercial UV processing unit

Treatment of agricultural water aids in the prevention of foodborne disease outbreaks linked to contaminated fresh produce. UV light is a suitable alternative for treating drinking water but is not always effective for surface irrigation water due to interference caused by turbidity and high microbial loads. The effectiveness of UV treatment for reducing Escherichia coli and Salmonella in surface water used in agriculture was evaluated. Six pond water samples were collected on each of 16 sampling dates over a 3-year period. On each corresponding testing date, three samples were inoculated with Salmonella enterica serovars Hartford, Montevideo, and Gaminara and the other three samples were inoculated with E. coli ATCC 25922, targeting a concentration of 7 log CFU/mL. Inoculated water was UV treated with a commercially available juice processing UV device at a constant UV dose of 14.2 mJ/cm2 and a turbulent flow regime. The effects of date, initial bacterial counts, and water pH and turbidity on log reductions of both microorganisms were determined. Initial bacterial counts and test date significantly predicted microbial reduction (multivariate P 0.05). UV treatment reduced both Salmonella and E. coli by a mean of >6 log CFU/mL.Universidad de Costa Rica/[735-B6-777]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Centro Nacional de Ciencia y Tecnología de Alimentos (CITA