METHOD: Endolysin and VAPGH genes were cloned and expressed in L. lactis NZ9000 after fusion with the SPK1 signal peptide for secretion. The recombinant proteins were expressed and purified, then analyzed for antimicrobial activity using plate assay and turbidity reduction assay. In addition, the spent media of the recombinant lactococcal culture was analyzed for its ability to inhibit the growth of the S. aureus PS 88.
RESULTS: Extracellular recombinant endolysin (Endo88) and VAPGH (VAH88) was successfully expressed and secreted from L. lactis which was able to inhibit S. aureus PS 88, as shown by halozone formation on plate assays as well as inhibition of growth in the turbidity reduction assay. Moreover, it was observed that the spent media from L. lactis NZ9000 expressing Endo88 and VAH88 reduced the viability of PS 88 by up to 3.5-log reduction with Endo88 being more efficacious than VAH88. In addition, Endo88 was able to lyse all MRSA strains tested and Staphylococcus epidermidis but not the other bacteria while VAH88 could only lyse S. aureus PS 88.
CONCLUSION: Recombinant L. lactisNZ9000 expressing phage 88 endolysin may be potentially developed into a new antimicrobial agent for the treatment of MDRSA infection.
AIM: The objective of this study was to determine the pathogenicity of Salmonella enterica subspecies enterica serovar Enteritidis (SE) phage type (PT) 1 in one-day-old specific pathogen-free (SPF) chicks.
METHODS: Seventy, one-day-old SPF chicks, were divided into SE group (30 chicks), mortality group (10 chicks), both orally inoculated (1.0 ml) with SE PT1 (1 × 108 colony-forming unit per 1.0 ml), and one control group (30 chicks). The chicks were sacrificed at 6 and 12 hours, and days 1, 2, 3, 5, 7, 10, 14, and 21 post-inoculation (pi). Samples were collected for bacterial isolation, histological examination, and ultrastructural examination.
RESULTS: Starting from day 2 pi, the body weight in the SE group significantly (p < 0.05) decreased. The SE isolation percentages from the liver, spleen, mid-intestinal content, cecal content, cecal tonsil, blood, and cloacal swab were 0.73, 0.77, 0.33, 0.33, 0.36, 0.40, and 0.30, respectively. The isolation percentage in the liver was significantly (p < 0.05) higher than the blood and cloacal swab. The villi heights and crypt depths in the SE group were significantly (p < 0.05) greater and smaller, respectively. Ultrastructurally, erosion and necrosis were observed in the microvilli of the cecal tonsil. The bacteria were engulfed by macrophages at the interepithelial clefts of the M-like M cells.
CONCLUSION: It was concluded that the inoculation of SE PT 1 in one-day-old chicks caused a systemic infection with diarrhea, a decrease in the body weight and villi height in the duodenum, jejunum, and ileum, and high bacterial loading in the liver with mild gross and histological lesions of organs, erosion, and necrosis of microvilli and low mortality. The bacteria entered the body system from the intestinal tract through the interepithelial clefts of the M-like M cells of the cecal tonsil.
METHODS: Immune antibody libraries are designed to isolate specific and high affinity antibodies against disease antigens. The pre-exposure of the host to an infection results in the production of a skewed population of antibodies against the particular infection.
RESULTS: This characteristic takes advantage of the in vivo editing machinery to generate bias and specific immune repertoire. The skewed but diverse repertoire of immune libraries has been adapted successfully in the generation of antibodies against a wide range of diseases.
CONCLUSION: We envisage immune antibody libraries to play a greater role in the discovery of antibodies for diseases in the near future.
MATERIALS AND METHODS: Twelve varieties of commercial wound coverings based on biopolymers of natural and synthetic origin, a biological preparation Staphylophag produced by scientific-industrial association Microgen (Russia), registration certificate P N001973/01, and the S. aureus 3196 test strain (GenBank JARQZO000000000) isolated from a patient with a burn wound have been used in our work. The ability of commercial biological wound coatings to absorb solutions was examined by immersing them in a physiological solution (pH 7.0-7.2) followed by weighing. The lytic activity of three bacteriophage series against the test strain was studied using the Appelman method and a spot test. The lytic activity of the bacteriophage in the wound samples was studied within 7 days after its absorption by the wound coatings.
RESULTS: The greatest volume of fluid was absorbed by the LycoSorb, NEOFIX FibroSorb Ag, Biatravm, and Chitocol-S wound coatings. All bacteriophage series have been found to have a high lytic activity against the test strain. It has also been shown that Chitocol-S, Collachit-FA, Algipran, and Aquacel Ag Extra possessed their own inherent antibacterial activity under in vitro conditions stable for 7 days; moreover, the lysis zones of the test strain increased after their saturation with bacteriophage. On day 0, a high level of bacteriophage lytic activity with the maximum size of the test strain lysis zones from 49 to 59 mm have been found to remain in all samples of the wound coverings. The bacteriophage activity persisted for 1 day in the samples of Hydrofilm, Polypran, and NEOFIX FibroCold Ag coatings, up to 4 days in Algipran, Nano-Aseptica, and Biatravm coatings; and for 7 days in the Chitocol-S, Collachit-FA, Opsite Post-Op Visible, NEOFIX FibroSorb Ag, Aquacel Ag Extra, and LycoSorb samples.
CONCLUSION: Modern commercial wound dressings based on chitosan-collagen complex (Chitocol-S, Collachit-FA), polyurethane (Opsite Post-Op Visible, LycoSorb, NEOFIX FibroSorb Ag), and Hydrofiber (Aquacel Ag Extra) have a sufficient level of bacteriophage solution absorption, provide a stable preservation of the bacteriophage lytic activity under in vitro conditions up to 7 days. Thus, the in vitro studies prove the possibility of their use as a carrier matrix for bacteriophages.
RESULTS: The constructed integration system comprises of a lactococcal promoter (PnisA or P170), phage attachment site (attP) from bacteriophage TP901-1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP344 anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P170 promoter with USP45 signal peptide which gave very low signals in whole cell ELISA.
CONCLUSION: The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.
METHODS: A synthetic human scFv phage antibody library was used to select phage-displayed antibody fragments that recognized CD1b-Ac2SGL using CD1b-transfected THP-1 cells loaded with Ac2SGL.
RESULTS: One clone, D11-a single, light-variable domain (kappa) antibody (dAbκ11)-showed high relative binding to the Ac2SGL-CD1b complex.
CONCLUSION: A ligand recognizing the Ac2SGL-CD1b complex was obtained, which is a potential candidate to be further tested for diagnostic and therapeutic applications.