Aim: To isolate and characterize bacteriocin, licheniocin 50.2, from soil bacteria identified as Bacillus licheniformis. Methods and Results: The strain B. licheniformis VPS50.2 was identified as bacteriocin producer, effective against Gram-positive bacteria, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and b-haemolytic streptococci. The start of bacteriocin production coincides with the beginning of sporulation. Ammonium sulfate precipitation, chloroform extraction and ultrafiltration were used for bacteriocin purification. MALDI TOF/TOF mass spectrometry of purified sample detected the protein with molecular mass of 3253Á209 Da. N-terminal sequencing recognized first 15 amino acids with the sequence: W E E Y N I I X Q L G N K G Q. We named the newly characterized bacteriocin as subclass II.3 bacteriocin, licheniocin 50Á2. The bacteriocin activity was insensitive to lysozyme and proteinase K, heat stable after incubation at 100°C for 30 min and over wide range of pH (2-12). MICs of crude bacteriocin extract were determined for L. monocytogenes and MRSA. Time-kill study showed that licheniocin had bactericidal effect to L. monocytogenes. Conclusion: A novel, thermostable, pH-tolerant bacteriocin active against Gram-positive bacteria was isolated. Significance and Impact of the Study: Attributes of new, stable licheniocin 50.2 make it a promising agent for application as biopreservative in food industry.

BackgroundCoagulase negative staphylococci (CoNS) and Listeria monocytogenes have important roles in pathogenesis of various genital tract infections and fatal foetomaternal infections, respectively. The aim of our study was to investigate the inhibitory effects of two novel bacteriocins on biofilms of CoNS and L. monocytogenes genital isolates.MethodsThe effects of licheniocin 50.2 from Bacillus licheniformis VPS50.2 and crude extract of bacteriocins produced by Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 (BGBU1-4 crude extract) were evaluated on biofilm formation and formed biofilms of eight CoNS (four S. epidermidis, two S. hominis, one S. lugdunensis and one S. haemolyticus) and 12 L. monocytogenes genital isolates.ResultsLicheniocin 50.2 and BGBU1-4 crude extract inhibited the growth of both CoNS and L. monocytogenes isolates, with MIC values in the range between 200–400 AU/ml for licheniocin 50.2 and 400–3200 AU/ml for BGBU1-4 crude extract. Subinhibitory concentrations (1/2 × and 1/4 × MIC) of licheniocin 50.2 inhibited biofilm formation by all CoNS isolates (p < 0.05, respectively), while BGBU1-4 crude extract inhibited biofilm formation by all L. monocytogenes isolates (p < 0.01 and p < 0.05, respectively). Both bacteriocins in concentrations of 100 AU/mL and 200 AU/mL reduced the amount of 24 h old CoNS and L. monocytogenes biofilms (p < 0.05, p < 0.01, p < 0.001).ConclusionsThis study suggests that novel bacteriocins have potential to be used for genital application, to prevent biofilm formation and/or to eradicate formed biofilms, and consequently reduce genital and neonatal infections by CoNS and L. monocytogenes.

A collection of 205 natural isolates of Bacillus was tested for the presence of genes for biosynthesis of antimicrobial lipopeptides, iturin, surfactin, fengycin and bacillomycin D. For the detection of iturin producers by PCR screening, we used forward ITUP1-F and reverse ITUP2-R primers which are capable of detecting a 2-kb region that includes the intergenic sequence between the ituA and ituB genes. A 675-bp fragment from the gene sfp from B. subtilis encoding 4’-phosphopantetheinyl transferase involved in the biosynthesis of surfactin was targeted for amplification by using primers P17 and P18. Other two pairs of primers were BACC1F and BACC1R for bacillomycin D and FEND1F and FEND1R for potential fengycin producers, respectively. The results of the screening showed that the majority of tested strains had more than one biosynthetic operon, since 81% possessed the genes for bacillomycin D production, 54% for surfactin, 38% for iturin and 25% for fengycin production. [Projekat Ministarstva nauke Republike Srbije, br. 173026

The aim of this study was genotyping and identification of collection of 164 Bacillus spp. isolates, from samples of soil, manure, and straw gathered from across Serbia, using Pulse field gel electrophoresis (PFGE) combined with sequencing of tuf gene, one of the housekeeping genes. The PFGE analysis with NotI enzyme was used to determine phylogenetic relationships of isolates and referent strains. Four large groups of Bacillus spp. were distinguishable: cereus, subtilis, pumilus and megaterium and within enormous genetic diversity. Bacillus subtilis Marburg referent strain did not group with rest of the strains from the subtilis group (Bacillus subtilis ATCC6633 and Bacillus atrophaeus ATCC9372). Strains from the cereus group were distinguished and closely grouped together. One representative isolate from each of 21 distinct PFGE groups was identified by sequencing of tuf gene. Eight different species were identified among chosen isolates: B. amyloliquefaciens, B. subtilis, B. pumilus, B. safensis, B. megaterium, B. cereus, B. anthracis and B. thuringiensis. Our results showed that PFGE analysis combined with sequencing of one of the housekeeping genes could be used for characterization of large collections of Bacillus isolates. The determination of tuf gene recommended itself to be an adequate and sufficient analysis for obtaining very clear and unambiguous results, with high resolution of separation of Bacillus species. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173026]

Background/Aim. Most cases of herpetic keratitis present a recurrent disease, as a result of herpes simplex virus type 1 reactivation from latency the nearest sensory ganglia. Therefore, understanding the mechanisms of latency and reactivation of latent virus is an important link in understanding the onset of recurrent eye disease itself. Epigenetic regulation of virus reactivation as a result of the presence of transcriptionally active LAT region (Latency-Associated Transcript-LAT) in the latent viral genome has already been demonstrated in several studies. The activity of the LAT region is directed to the chromatin arrangement. Epigenetic modulation of DNA methylation is associated with folat and vitamin B12 intake or their serum concentartions. The aim of this study was to analyze the potential role of folic acid and vitamin B12 in the control of ocular herpes simplex keratitis reactivation. Methods. The study included 50 patients older than 18 years of age with recurrent herpes simplex virus eye disease. Levels of vitamin B12 and folic acid were measured in the acute phase of the disease. All patients were followed up for at least one year and episodes of recurrent herpetic eye diseases were recorded. Results. Recurrence rate of herpetic keratitis is lower in patients with a higher blood level of vitamin B12. There is statistical significance between these two variables. In addition, recurrence rate of herpetic keratitis is lower in patients with higher blood level of folic acid. However, statistical significance is lower in comparison with vitamin B12. Conclusion.Vitamin B12 and folic acid might have important role in herpes simplex keratitis reactivation. To our knowledge there is no report on potential role of vitamin B12 and folic acid in HSV keratitis reactivation.