Unveiling therapeutic potential of bacteriophage treatment in acinetobacter baumannii-infected zebrafish embryo model

Introduction: There is an urgent demand for the development of new therapeutic approachesto combat multidrug-resistant Acinetobacter baumannii, and bacteriophages appear to be a highly promising solution. Phages are suitable to precisely target the infection-causing bacteria without disrupting the beneficial microbiota. The zebrafish (Danio rerio) embryo model represents an insightful animal model for preclinical studying of various infectious diseases and for discovery of novel safe and effective antimicrobial drugs. Methods: Systemic bacterial infection was established by microinjection of 2000 cells of nosocomial carbapenem-resistant A. baumannii strain 6077/12 into the bloodstream of 48 hour old zebrafish embryos. Infected embryos were treated by parenteral administration of 4 different doses (10, 50, 100, 500 PFU) of bacteriophage vB_AbaM_ISTD at 6 hours after infection (hpi). Efficacy of treatment was evaluated according to embryo survival, morphological malformations and bacterial burden (CFU) over a 3- day period. Results: A. baumannii-infected embryos treated with bacteriophage resulted with 100% survival rate, while 70% of untreated embryos survived to 24 hpi and none to the end of the experiment. Viable bacterial cell count and embryo morphology observations indicated that the administered phage effectively reduced A. baumanii infection in vivo. The most effective dose was 500 PFU, decreasing the bacterial load by 3.09 log units during 24 hpi, while lower bacteriophage doses(10, 50 and 100 PFU) produced less prominent, but also significant bacterial reduction of 2.10, 2.19 and 2.67 log units, respectively. Conclusion: Parentheral administration of phage ISTD demonstrated potent therapeutic activity against A. baumannii infection in every investigated dose

Lactococcin B Is Inactivated by Intrinsic Proteinase PrtP Digestion in Lactococcus lactis subsp. lactis BGMN1-501

In our previous study we demonstrated that proteinase PrtP is able to impair bacteriocin LcnB activity, despite being produced by the same organism and encoded by the same plasmid. However, precise mechanism of this action, i.e., the exact cleavage site within LcnB bacteriocin, as well as its effect on antimicrobial activity of the resulting peptide remained vague. Here we further explored the interplay between these two proteins and defined, using mass spectrometry, that this unusual hydrolysis indeed occurs in vivo, between the sixth and seventh amino acid on the N terminus of LcnB. To address whether the cleaved form of LcnB retains any level of activity, both recombinant and chemically synthesized variant of truncated LcnB were engineered and produced, but demonstrated no antimicrobial activity. When LcnB was recombinantly overexpressed and subjected to PrtP digestion, the change in its antimicrobial activity was monitored and the degradation products analyzed with reverse-phase high-pressure liquid chromatography. The results confirmed the inactivity of the truncated LcnB and additionally corroborated the PrtP cleavage site in LcnB bacteriocin. In addition, it was demonstrated that, once truncated, LcnB is not able to bind its receptor and is susceptible to additional hydrolysis. This is the first report on proteolytic inactivation of bacteriocins inside the same bacterial host

Enrichment of Larval Fish Feed with Free Amino Acids and Proteins by Coating with Lactobacillus paracasei subsp. paracasei BGHN14 Homogenate

This research aimed to test whether coating with Lactobacillus paracasei subsp. paracasei BGHN14 homogenate may increase soluble protein and amino acid amount on larval fish feed surface. Total amino acid and protein, as well as taste stimulating and growth promoting amino acid amounts were analyzed in coated feed samples. Results indicated that coating with BGHN14 homogenate increased the amount of soluble proteins and free Glycine on feed surface, as well as the availability of protein-bound (hydroxy-)Proline. This lab-scale research provides the basis for use of lactobacilli as resource efficient source of soluble nutrients for animal feedstuffs

Proteinase PrtP impairs lactococcin LcnB activity in Lactococcus lactis BGMN1-501: new insights into bacteriocin regulation

Proteinases and bacteriocins are of great importance to the dairy industry, but their interactions have not been studied so far. Lactococcus lactis subsp. lactis BGMN1-5 is a natural isolate from homemade semi-hard cheese which produces two bacteriocins (Lactococcin B and LsbB), as well as proteinase PrtP. A medium-dependent increase in the bacteriocin LcnB activity of L. lactis BGMN1-501, a derivate of L. lactis subsp. lactis BGMN1-5, was shown to be accompanied by a decrease in its promoter activity. A similar effect of media components on gene expression was reported for proteinase PrtP whose gene is co-localized on the same plasmid as the IcnB gene. Thus, the PrtP-LcnB interplay was investigated. Single gene knockout mutants were constructed with disrupted prtP or IcnB genes. PrtP mutants showed higher bacteriocin activity that had lost its growth medium dependence, which was in contrast to the original strain. When LcnB from this mutant was combined with proteinase from the LonB(-) mutant in vitro, its activity was rendered to the original level, suggesting that proteinase reduces bacteriocin activity. We propose a new model of medium dependent expression of these genes with regard to the effects of their interaction in vivo

Lactobacilli hydrolysis of cows' milk proteins abrogates their humoral immunoreactivity in patients with immune-mediated diseases

The level of humoral immunoreactivity to total cows' milk proteins (TCMP) in sera of patients suffering from recurrent oral ulcerations, gastrointestinal diseases or haematological malignancies was investigated. TCMP were also hydrolysed with two different species of lactobacilli and dramatic changes in the levels of specific IgG and IgE were found with statistically significant decreases in the levels of specific antibodies in sera from all patient groups. The levels below cut-off values of IgG specific for TCMP hydrolysates were detected in sera from all patients, while values of IgE for hydrolysates obtained with Lactobacillus helveticus BGRA43 and Lactobacillus zeae LMG17315were below cut-off in 85% and 97% of patients, respectively. Competitive ELISA confirmed the specificity of antibodies for immunogenic TCMP epitopes, demonstrating that lactobacilli hydrolyse TCMP by degrading immunogenic epitopes, and could therefore be used in processing of milk proteins to obtain products suitable for patients with altered immune response on TCMP.This is the peer reviewed version of the paper: Vukotic, G., Matic, I., Begovic, J., Besu, I., Kojic, M., Djokic, J., Juranic, Z., & Strahinic, I. (2016). Lactobacilli hydrolysis of cows’ milk proteins abrogates their humoral immunoreactivity in patients with immune-mediated diseases. International Dairy Journal, 63, 1–7. [https://doi.org/10.1016/j.idairyj.2016.07.009

LraI from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of EcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity

Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. Lactococcus raffinolactis BGTRK10-1 expresses LraI Type II restriction-modification enzyme, whose activity is similar to that shown for EcoRI; LraI methyltransferase protects DNA from EcoRI cleavage. The gene encoding LraI endonuclease was cloned and overexpressed in E. coli. Purified enzyme showed the highest specific activity at lower temperatures (between 13 degrees C and 37 degrees C) and was stable after storage at -20 degrees C in 50% glycerol. The concentration of monovalent ions in the reaction buffer required for optimal activity of LraI restriction enzyme was 100 mM or higher. The recognition and cleavage sequence for LraI restriction enzyme was determined as 5'-G/AATTC-3', indicating that LraI restriction enzyme is an isoschizomer of EcoRI. In the reaction buffer with a lower salt concentration, LraI exhibits star activity and specifically recognizes and cuts another alternative sequence 5'-A/AATTC-3', leaving the same sticky ends on fragments as EcoRI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin of LraI restriction-modification system with previously described EcoRI-like restriction-modification systems

EMPOWERING ANTIFUNGAL DRUGS DISCOVERY THROUGH THE ZEBRAFISH-INFECTIOUS DISEASES MODELLING

Fungal infections, once considered a rare disease, have become an everyday problem in modern societies, posing major challenges to global health. It is estimated that more than one billion people are affected by fungal infections and 1.6 million people succumb to these diseases every year. Of the 600 species of fungi capable of causing infections in humans, species of the genus Candida cause more than 85% of infections, especially C. albicans, which has become a serious threat to human health in immunocompromised and immunosuppressed individuals. Unfortunately, the current arsenal of clinical drugs relies on only four classes of approved drugs (polyenes, azoles, echinocandins and allylamines), which are only partially effective, resulting in incomplete eradication of the fungal infection. In addition, the serious side effects, ranging from systemic or organ-specific toxicity to poor bioavailability and low activity, significantly hamper the clinical use of antifungals. These problems call for new effective and safe antifungal agents,but also for appropriate preclinical models to accurately study potential adverse effects on the human population and test their efficacy against fungal infections. In this sense, zebrafish (Danio rerio) embryos have become one of the most powerful preclinical animal models in infection biology and drug discovery, offering the unique opportunity to simultaneously monitor the safety and efficacy of the applied molecule in real time. With the aim of providing a preclinical platform for the identification of new safe antifungal drugs to effectively control C. albicans infection, we comprehensively tested the toxicity of 13 clinical antifungal drugs in the zebrafish embryo model. The 21 toxicity endpoints, including survival, teratogenicity, cardiotoxicity and hepatotoxicity, were evaluated and compared with adverse effects described in rats and humans. Of the clinical drugs, the efficacy of fluconazole and voriconazole was evaluated in the zebrafish - C. albicans model of systemic and wound biofilm infection.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi

Antioxidant activity of pea protein hydrolysates produced by batch fermentation with lactic acid bacteria

Nine Lactobacillus strains known for surface proteinase activity were chosen from our collection and tested for their ability to grow in pea seed protein-based medium, and to hydrolyze purified pea proteins in order to produce peptides with antioxidant (AO) activity. Two strains, Lactobacillus rhamnosus BGT10 and Lactobacillus zeae LMG17315, exhibited strong proteolytic activity against pea proteins. The AO activity of the pea hydrolysate fraction, MW lt 10 kDa, obtained by the fermentation of purified pea proteins with Lactobacillus rhamnosus BGT10, was tested by standard spectrophotometric assays (DPPH, ABTS, Fe3+-reducing capacity) and the recently developed direct current (DC) polarographic assay. The low molecular weight fraction of the obtained hydrolysate was separated using ion exchange chromatography, while the AO activity of eluted fractions was determined by means of a sensitive DC polarographic assay without previous concentration of samples. Results revealed that the fraction present in low abundance that contained basic peptides possessed the highest antioxidant activity. Based on the obtained results, it can be concluded that Lactobacillus rhamnosus BGT10 should be further investigated as a candidate strain for large-scale production of bioactive peptides from legume proteins

Solid state treatment with Lactobacillus paracasei subsp. paracasei BGHN14 and Lactobacillus rhamnosus BGT10 improves nutrient bioavailability in granular fish feed

The aim of this research was to improve nutritive value of fishmeal-based feed by lactobacilli in order to achieve satisfactory nutrient availability needed to support fish development. Feed was solid-state treated at a laboratory scale with the combination of Lactobacillus paracaseisubsp. paracasei BGHN14 and Lactobacillus rhamnosus BGT10 in different experimental settings, which included the variation of strain ratio, total lactobacilli concentration, percentage of moisture and duration of incubation. Short peptides, soluble proteins, phospho-, neutral and unsaturated lipids were quantified. Differences among treated and control feeds were evaluated by Student t-test, while Gaussian process regression (GPR) modeling was employed to simulate the incubation process and define the optimal treatment combination in the context of overall feed nutritional profile. Treatment duration was shown to be the critical determinant of final outcome, either as single factor or via interaction with strain ratio. Optimal nutrient balance was achieved with 12 h incubation period, 260% moisture, 75:25 and 50:50 BGHN14:BGT10 ratios and 200 mg of lactobacilli per g of dry feed. This study should serve as the basis for large-scale tests which would simulate on-farm production of both fishmeal-based and unconventional, lower cost aquafeed with added value

Characterization, Antibiofilm, and Depolymerizing Activity of Two Phages Active on Carbapenem-ResistantAcinetobacter baumannii

Acinetobacter baumanniiis a leading cause of healthcare-associated infections worldwide. Its various intrinsic and acquired mechanisms of antibiotic resistance make the therapeutic challenge even more serious. One of the promising alternative treatments that is increasingly highlighted is phage therapy, the therapeutic use of bacteriophages to treat bacterial infections. Two phages active against nosocomial carbapenem-resistantA. baumanniistrain 6077/12, vB_AbaM_ISTD, and vB_AbaM_NOVI, were isolated from Belgrade wastewaters, purified, and concentrated using CsCl gradient ultracentrifugation. The phages were screened against 103 clinical isolates ofA. baumanniifrom a laboratory collection and characterized based on plaque and virion morphology, host range, adsorption rate, and one-step growth curve. Given that phage ISTD showed a broader host range, better adsorption rate, shorter latent period, and larger burst size, its ability to lyse planktonic and biofilm-embedded cells was tested in detail. Phage ISTD yielded a 3.5- and 2-log reduction in planktonic and biofilm-associated viable bacterial cell count, respectively, but the effect was time-dependent. Both phages produced growing turbid halos around plaques indicating the synthesis of depolymerases, enzymes capable of degrading bacterial exopolysaccharides. Halos tested positive for presence of phages in the proximity of the plaque, but not further from the plaque, which indicates that the observed halo enlargement is a consequence of enzyme diffusion through the agar, independently of the phages. This notion was also supported by the growing halos induced by phage preparations applied on pregrown bacterial lawns, indicating that depolymerizing effect was achieved also on non-dividing sensitive cells. Overall, good rates of growth, fast adsorption rate, broad host range, and high depolymerizing activity, as well as antibacterial effectiveness against planktonic and biofilm-associated bacteria, make these phages good candidates for potential application in combatingA. baumanniiinfections