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

An in vitro and in vivo evaluation of peroxyacetic acid as an alternative sanitizer for wine barrels

Peroxyacetic acid is a common sanitizer used in the food and wine industry, but its use as a sanitizer for wine barrels has not been reported. We are reporting the findings for in vitro studies using three different concentrations of peroxyacetic acid (0, 60, and 120 mg/L) as sanitization challenges against seven strains of wine spoilage yeast representing three different species: Dekkera /Brettanomyces bruxellensis (three strains), Saccharomyces cerevisiae (three strains) and Zygosaccharomyces bailii (one strain). In vitro sensitivity to peroxyacetic acid concentration varied within and between species. A post hoc study (in vivo ) using the highest concentration from the in vitro studies (120 mg/L) as well as 200 mg/L was performed to validate a sanitization method for wine barrels. Exposure of barrels to 200 mg/L of peroxyacetic acid for one week resulted in no detectable levels of wine spoilage microorganisms after treatment. These findings are crucial for establishing protocols to assure the maximum reduction of microbial contaminants

Diversity, antimicrobial production, and seasonal variation of honey bee microbiota isolated from the honey stomachs of the domestic honey bee, Apis mellifera

The antimicrobial nature of honey and its related apiological origins typically focus on basic chemical analysis without attempting to understand the diversity of the microbial component. The antibacterial activity, chemical characterization, and diversity of bacteria isolated from Apis mellifera honey stomachs and hive honey collected throughout the honey production season are presented. After screening >2,000 isolates, 50 isolates were selected and characterized by 16S rRNA gene homology, Gram stain, catalase and protease tests, as well as for antibacterial activity against select indicators. Antibacterial-producing isolates were predominantly from the Pseudomonas, Paenibacillus, Lonsdalea, Serratia, and Bacillus genera. Isolates collected from honey stomachs in April displayed the highest level of activity (27%). While April isolates did not demonstrate activity against the Gram-negative bacteria tested. Whereas 59% of July isolates, 33% of September isolates, and 100% of the honey isolates did. The predominant honey stomach isolates were Pseudomonas spp. (April), Paenibacillus polymyxa (July, Sept.), and Lonsdalea iberica (Sept.). Chemical characterizations of the antimicrobial compounds show most to be antibiotic in nature with the minority being potential bacteriocins. This study offers the first glimpse into the variability and diversity of the bacteria/host interactions found within the honey stomach of the domestic honey bee while revealing a novel source of potentially beneficial antimicrobial compounds

Non-chlorine Sanitizer Options for the Wineries

Discusses non-chlorine sanitizer options for wineries

Microbial safety and quality evaluation of UV-Treated, cold-pressed colored and turbid juices and beverages

The growing demand for fruit and vegetable juice blends, with improved nutritional and sensory attributes, has prompted the industrial adoption of nonthermal processing technologies, including UV light. Limited studies have explored conditions to overcome the well-known limitations of UV when treating liquid foods with a high content of particles that absorb or scatter UV light. This study addressed the effectiveness of the application of UV light, using a commercial processing unit, to inactivate pathogenic Escherichia coli O157:H7, Salmonella enterica (hereafter Salmonella), and Listeria monocytogenes, as well as spoilage microorganisms, in colored and turbid juices and beverages. The inactivation of cocktails of five strains (or serotypes) of E. coli O157:H7, Salmonella, and L. monocytogenes isolated from fruit- and vegetable-derived products linked to outbreaks was determined in seven colored and turbid cold-pressed juices and beverages. Juices and beverages were UV treated at a constant flow rate of 150 L/h through multiple consecutive passes. The inactivation of aerobic mesophilic bacteria, molds and yeasts, and lactic acid bacteria was also assessed at the cumulative dose that guaranteed a 5-log reduction of the most UV-tolerant pathogen for each product. A 5-log reduction of the three pathogens was achieved in all juices and beverages at a maximum cumulative UV dose of 12.0 ± 0.6 mJ/cm2. The dose required to ensure the targeted reduction varied depending on the tested product and the inoculated pathogen. The reduction of aerobic mesophiles, molds and yeasts, and lactic acid bacteria varied from 0.5 to 3.6, from 0.2 to 2.0, and from 0.5 to 3.6 log CFU/mL, respectively. Thus, the proposed treatment represents a suitable processing alternative to ensure the safety and extend the shelf life of colored and turbid cold-pressed juices and beverages.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Centro Nacional de Ciencia y Tecnología de Alimentos (CITA

The Antimicrobial Potential of Bacteria Isolated from Honey Samples Produced in the Apiaries Located in Pomeranian Voivodeship in Northern Poland

The principal objective of this study was to determine whether the honeys produced in apiaries located in Pomeranian Voivodeship (Northern Poland) contain bacteria producing metabolites with growth inhibition potential against important human and animal pathogens. The pathogens included Staphylococcus aurues, Staphyloccocus epidermidis, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Candida albicans. From 12 samples of honey, 163 strains of bacteria were isolated. Activity against reference staphylococci: S. aurues ATCC 25923; S. aureus ATCC 29213; S. epidermidis 12228 was observed in 33 (20.3%), 38 (23.3%), and 41 (25.1%) isolates, respectively. High inhibitory activity was also found against Listeria monocytogenes ATCC 7644 in 34 strains (20.9%). Activity against Candida albicans ATCC 10231 and especially Gram-negative bacteria: Pseudomonas aeruginosa ATCC 27857 and Escherichia coli ATCC 25922 was rarely observed. Production of metabolites exhibiting activity against the three pathogens mentioned above was confirmed for 13 (7.8%), 3 (1.8%), and 2 (1.2%) isolates, respectively. Forty-six isolates were selected for further analysis. Within this group, metabolites synthesized by 18 producing strains (39.13%) inhibited growth of only one of the reference strains of pathogenic microorganisms. However, 14 (30.44%), 8 (17.39%), and 6 (13.04%) strains produced agents active against three, two, and four pathogens, respectively. Sequencing of the 16S rRNA gene revealed that 80.4% of these 46 producing strains belong to the genus Bacillus. However, some producing strains belonging to the genus of Peanibacillus, Lysinibacillus, Microbacterium, and Staphylococcus were also identified. Furthermore, the analysis of the sequences of 16S rRNA, as well as RAPD-PCR, exhibited a significant diversity in the strains tested, even in the case of bacteria isolated from the same honey (and classified to the same genus, usually Bacillus spp.). This observation suggests environmental origin (nectar, water, or pollen) of the producing strains. The research carried out confirmed that honey produced in Northern Poland is a promising source of strains of bacteria producing metabolites with antimicrobial activity

Bee Pollen and Bee Bread as a Source of Bacteria Producing Antimicrobials

The principal objective of the study was the isolation and identification of bacteria that are present in mature bee bread (BB) and dried (ready for selling and consumption) bee pollen (BP). Obtained isolates were screened for their potential to inhibit select human pathogenic bacteria and their ability to produce enzymes of particular industrial importance. Four and five samples of BP and BB, respectively, were used for the study. In total, 81 strains of bacteria were isolated, and 34 (42%) of them exhibited antagonistic interactions with at least one reference strain of pathogenic bacteria, namely Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis 12228, Pseudomonas aeruginosa ATCC 27857, and Escherichia coli ATCC 25922. The sequencing of the 16S rRNA gene revealed that all strains producing antimicrobials belong to the genus Bacillus spp., and among them, five species were identified: B. pumilus (n = 17), B. altitudinis (n = 9), B. licheniformis (n = 4), B. subtilis (n = 2), and B. safensis (n = 1). Furthermore, 69, 54, 39, and 29 of the strains exhibited lipolytic, proteolytic, cellulolytic, and esterolytic activity, respectively. Alpha amylase and beta galactosidase activity were rarely observed, and none of the strains produced laccase. The outcomes of the study revealed that BP and BB can be considered potential sources of bacteria producing antimicrobial agents and/or enzymes of particular industrial importance. Of course, additional research is required to verify this hypothesis, but the results of preliminary studies are promising

Paenibacillus alvei MP1 as a Producer of the Proteinaceous Compound with Activity against Important Human Pathogens, Including Staphylococcus aureus and Listeria monocytogenes

An emerging need for new classes of antibiotics is, on the one hand, evident as antimicrobial resistance continues to rise. On the other hand, the awareness of the pros and cons of chemically synthesized compounds’ extensive use leads to a search for new metabolites in already known reservoirs. Previous research showed that Paenibacillus strain (P. alvei MP1) recovered from a buckwheat honey sample presented a wide spectrum of antimicrobial activity against both Gram-positive and Gram-negative pathogens. Recent investigation has confirmed that P. alvei MP1 (deposited at DDBJ/ENA/GenBank under the accession WSQB00000000) produces a proteinaceous, heat-stable compound(s) with the maximum antimicrobial production obtained after 18 h of P. alvei MP1 growth in LB medium at 37 °C with continuous shaking at 200 RPM. The highest activity was found in the 40% ammonium sulfate precipitate, with high activity also remaining in the 50% and 60% ammonium sulfate precipitates. Moderate to high antimicrobial activity that is insensitive to proteases or heat treatment, was confirmed against pathogenic bacteria that included L. monocytogenes FSL – X1-0001 (strain 10403S), S. aureus L1 – 0030 and E. coli O157: H7. Further studies, including de novo sequencing of peptides by mass spectrometry, are in progress