Ubericin K, a New Pore-Forming Bacteriocin Targeting mannose-PTS.

Bovine mastitis infection in dairy cattle is a significant economic burden for the dairy industry globally. To reduce the use of antibiotics in treatment of clinical mastitis, new alternative treatment options are needed. Antimicrobial peptides from bacteria, also known as bacteriocins, are potential alternatives for combating mastitis pathogens. In search of novel bacteriocins against mastitis pathogens, we screened samples of Norwegian bovine raw milk and found a Streptococcus uberis strain with potent antimicrobial activity toward Enterococcus, Streptococcus, Listeria, and Lactococcus. Whole-genome sequencing of the strain revealed a multibacteriocin gene cluster encoding one class IIb bacteriocin, two class IId bacteriocins, in addition to a three-component regulatory system and a dedicated ABC transporter. Isolation and purification of the antimicrobial activity from culture supernatants resulted in the detection of a 6.3-kDa mass peak by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, a mass corresponding to the predicted size of one of the class IId bacteriocins. The identification of this bacteriocin, called ubericin K, was further confirmed by in vitro protein synthesis, which showed the same inhibitory spectrum as the purified antimicrobial compound. Ubericin K shows highest sequence similarity to the class IId bacteriocins bovicin 255, lactococcin A, and garvieacin Q. We found that ubericin K uses the sugar transporter mannose phosphotransferase (PTS) as a target receptor. Further, by using the pHlourin sensor system to detect intracellular pH changes due to leakage across the membrane, ubericin K was shown to be a pore former, killing target cells by membrane disruption. IMPORTANCE Bacterial infections in dairy cows are a major burden to farmers worldwide because infected cows require expensive treatments and produce less milk. Today, infected cows are treated with antibiotics, a practice that is becoming less effective due to antibiotic resistance. Compounds other than antibiotics also exist that kill bacteria causing infections in cows; these compounds, known as bacteriocins, are natural products produced by other bacteria in the environment. In this work, we discover a new bacteriocin that we call ubericin K, which kills several species of bacteria known to cause infections in dairy cows. We also use in vitro synthesis as a novel method for rapidly characterizing bacteriocins directly from genomic data, which could be useful for other researchers. We believe that ubericin K and the methods described in this work will aid in the transition away from antibiotics in the dairy industry.

Bacteria and bacterial anticancer agents as a promising alternative for cancer therapeutics.

Cancer is the leading cause of deaths worldwide, though significant advances have occurred in its diagnosis and treatment. The development of resistance against chemotherapeutic agents, their side effects, and non-specific toxicity urge to screen for the novel anticancer agent. Hence, the development of novel anticancer agents with a new mechanism of action has become a major scientific challenge. Bacteria and bacterially produced bioactive compounds have recently emerged as a promising alternative for cancer therapeutics. Bacterial anticancer agents such as antibiotics, bacteriocins, non-ribosomal peptides, polyketides, toxins, etc. These are adopted different mechanisms of actions such as apoptosis, necrosis, reduced angiogenesis, inhibition of translation and splicing, and obstructing essential signaling pathways to kill cancer cells. Also, live tumor-targeting bacteria provided a unique therapeutic alternative for cancer treatment. This review summarizes the anticancer properties and mechanism of actions of the anticancer agents of bacterial origin and antitumor bacteria along with their possible future applications in cancer therapeutics.

Alternatives to Conventional Antibiotics in the Era of Antimicrobial Resistance.

As more antibiotics are rendered ineffective by drug-resistant bacteria, focus must be shifted towards alternative therapies for treating infections. Although several alternatives already exist in nature, the challenge is to implement them in clinical use. Advancements within biotechnology, genetic engineering, and synthetic chemistry have opened up new avenues towards the search for therapies that can substitute for antibiotics. This review provides an introduction to the various promising approaches that have been adopted in this regard. Whilst the use of bacteriophages and antibodies has been partly implemented, other promising strategies, such as probiotics, lysins, and antimicrobial peptides, are in various stages of development. Propitious concepts such as genetically modified phages, antibacterial oligonucleotides, and CRISPR-Cas9 are also discussed.

Considerações sobre o tratamento das mastites / Considerations on the treatment of mastitis

A mastite é considerada o maior problema dos animais destinados à produção de leite. Altera a sua composição e aumenta a contagem de células somáticas (CCS). Os micro-organismos envolvidos na doença podem ser origem infecciosa, como Staphylococcus aureus, ou ambiental, tal como Escherichia coli. A cultura bacteriana é uma ferramenta de diagnóstico e auxilia na detecção do patógeno causador da mastite. No entanto, fatores como fagocitose podem desencadear um resultado negativo. Quando estabelecido um programa de controle de mastite, o diagnóstico precoce e o início do tratamento adequado dos casos clínicos são fundamentais para se atingir os objetivos e seu sucesso, está relacionado com o patógeno envolvido. A indicação do tratamento de longa duração, ou terapia estendida, tem melhorado a resposta ao tratamento em casos de mastite por S. aureus, no entanto, com 30-50% de cura. Do ponto de vista do manejo dos animais, devido a alta contagiosidade deste patógeno, sua persistência no rebanho e custo em função ao tratamento, muitas vezes, o descarte do animal tem sido priorizado a fim de controlar os casos de mastite em propriedades. As medidas de controle são muito importantes para contribuir com a redução de casos de mastite por este patógeno. A indicação do tratamento intramamário associada com sistêmico tem poder efetivo em casos de mastite por E. coli, cujos casos agudos apresentam-se com sepse e toxemia. São abordados ainda aspectos de tratamentos alternativos das mastites, utilizados principalmente no processo orgânico de produção leiteira.(AU)

Mastites are considered a major problem on animals for milk production. Changes the milk composition and increases the somatic cell count (SCC). The microorganism involved in the disease may be infectious origin such as Staphylococcus aureus or environmental such as Escherichia coli. Bacterial culture is a diagnostic tool and aids in the detection of pathogenic causing masitis. However factor such as phagocytosis may result a negative result. When established a mastitis control program early diagnose and the initiation of appropriate treatment of clinical cases are fundamental for achieving the goals and success is related to the pathogen involved. The indication of treatment of long duration, or extended therapy has improved the response to the treatment in cases of matitis by S. aureus, however with 30-50% of cure. From the view point of handling of animals, given the high infectiousness of this pathogen, its persistence in the herd and cost-effective as a function of response to treatment often has prioritized the animal's discard in order to control the mastitis cases in properties. Control measures are very important to contribute the reduction of cases of mastitis symptoms by this pathogen. The indication of intramammary treatment associated with systemic has power effective in cases of mastitis by ­E. coli, whose acute cases present with sepsis and toxemia. Also address aspects of alternative treatments of mastitis, mainly used in the organic process of milk production.(AU)

The bacteriocin sublancin attenuates intestinal injury in young mice infected with Staphylococcus aureus.

Sublancin, a bacteriocin, has bactericidal activity against a broad spectrum of gram-positive bacteria. However, studies have not been conducted to determine its in vivo efficacy against potential pathogens. The objective of this study was to investigate the effects of sublancin in a Staphylococcus aureus (S. aureus) infected mouse model which induced intestinal injury. A total of 160, 4-week-old mice were randomly assigned to one of eight treatments. Mice in the control group were injected intraperitoneally with 0.5 mL of 0.9% saline. Mice in the other seven groups were given an intraperitoneal injection of 0.5 mL saline containing 1.0 × 10(10) colony-forming units (CFU)/mL S. aureus. Six hours after inoculation, mice in the control group were again injected with 0.5 mL of 0.9% saline. Mice in the other seven groups were injected intraperitoneally with 0.5 mL of 0.9% saline containing 0, 0.5, 1.0, 2.0, or 4.0 mg/kg body weight (BW) sublancin or 1.0 or 2.0 mg/kg BW ampicillin. The results showed that 4.0 mg/kg sublancin and 2.0 mg/kg ampicillin significantly reduced mice mortality from 55 to 10%. The height and the number of proliferated cells from the intestinal villi in the sublancin and ampicillin treated mice were higher than in the control. We conclude that sublancin has potent antibacterial activity against S. aureus. Therefore, sublancin could find use as an alternative antimicrobial agent for the treatment of gram-positive bacterial infections.

Evaluation of NVB302 versus vancomycin activity in an in vitro human gut model of Clostridium difficile infection.

OBJECTIVES: First-line treatment options for Clostridium difficile infection (CDI) are limited. NVB302 is a novel type B lantibiotic under evaluation for the treatment of CDI. We compared the responses to NVB302 and vancomycin when used to treat simulated CDI in an in vitro gut model. METHODS: We used ceftriaxone to elicit simulated CDI in an in vitro gut model primed with human faeces. Vancomycin and NVB302 were instilled into separate gut models and the indigenous gut microbiota and C. difficile total viable counts, spores and toxin levels were monitored throughout. RESULTS: Ceftriaxone instillation promoted C. difficile germination and high-level toxin production. Commencement of NVB302 and vancomycin instillation reduced C. difficile total viable counts rapidly with only C. difficile spores remaining within 3 and 4 days, respectively. Cytotoxin was reduced to undetectable levels 5 and 7 days after vancomycin and NVB302 instillation commenced in vessel 2 and 3, respectively, and remained undetectable for the remainder of the experiments. C. difficile spores were unaffected by the presence of vancomycin or NVB302. NVB302 treatment was associated with faster resolution of Bacteroides fragilis group. CONCLUSIONS: Both NVB302 and vancomycin were effective in treating simulated CDI in an in vitro gut model. C. difficile spore recrudescence was not observed following successful treatment with either NVB302 or vancomycin. NVB302 displayed non-inferiority to vancomycin in the treatment of simulated CDI, and had less deleterious effects against B. fragilis group. NVB302 warrants further clinical investigation as a potentially novel antimicrobial agent for the treatment of CDI.

Novel therapeutic strategies to counter Pseudomonas aeruginosa infections.

Pseudomonas aeruginosa is a highly successful opportunistic pathogen that displays intrinsic multidrug resistance and has a tremendous capacity to acquire further resistance mechanisms. During chronic infection, the bacterium can form a protective biofilm therefore reducing the efficacy of existing antibiotics. P. aeruginosa also harbors an impressive range of virulence factors, many of which are controlled by the quorum-sensing system. Several novel therapeutics are under investigation such as those directed against biofilm formation and quorum-sensing systems along with bacteriophages and immunotherapies. Recent advances in next-generation sequencing and comparative genomics have opened the door to a new wave of smart drug design that could revolutionize P. aeruginosa treatment options.

Efficacy of the new lantibiotic NAI-107 in experimental infections induced by multidrug-resistant Gram-positive pathogens.

NAI-107 is a novel lantibiotic active against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), glycopeptide-intermediate S. aureus (GISA), and vancomycin-resistant enterococci (VRE). The aim of this study was to evaluate the in vivo efficacy of NAI-107 in animal models of severe infection. In acute lethal infections induced with a penicillin-intermediate Streptococcus pneumoniae strain in immunocompetent mice, or with MRSA, GISA, and VRE strains in neutropenic mice, the 50% effective dose (ED(50)) values of NAI-107 were comparable or lower than those of reference compounds, irrespective of the strain and immune status (0.51 to 14.2 mg/kg of body weight for intravenous [i.v.] NAI-107, 5.1 to 22.4 for oral linezolid, and 22.4 for subcutaneous [s.c.] vancomycin). In the granuloma pouch model induced in rats with a MRSA strain, intravenous NAI-107 showed a dose-proportional bactericidal activity that, at a single 40-mg/kg dose, compared with 2 20-mg/kg doses at a 12-h or 24-h interval, caused a 3-log(10)-CFU/ml reduction of viable MRSA in exudates that persisted for more than 72 h. Rat endocarditis was induced with a MRSA strain and treated for five consecutive days. In a first experiment, using 5, 10, or 20 mg/kg/day, and in a second experiment, when 10 mg/kg at 12-h intervals was compared to 20 mg/kg/day, intravenous NAI-107 was effective in reducing the bacterial load in heart vegetations in a dose-proportional manner. Trough plasma levels, as determined on days 2 and 5, were several times higher than the NAI-107 minimal bactericidal concentration (MBC). NAI-107 binding to rat and human serum ranges between 93% and 98.6%. The rapid bactericidal activity of NAI-107 observed in vitro was thus confirmed by the efficacy in several models of experimental infection induced by Gram-positive pathogens, supporting further investigation of the compound.

[Use of enterocin S760 for prevention and treatment of experimental Salmonella infection in mice].

AIM: To demonstrate treatment efficacy of bacteriocin S760 synthesized by Enterococcus faecium 760 for septic Salmonella infection in mice. MATERIALS AND METHODS: One hundred mice, which were intraperitoneally inoculated with 100 LD50 of Salmonella enteritidis strain 92 Rif(r), received bacteriocin 1 hour (prevention) or 48 hours (treatment) after inoculation in doses 25, 50, or 100 mg/kg every 6 hours during 5 or 10 days. RESULTS: Use of peptide S760 for prophylaxis in dose 50 mg/kg during 10 days prevented lethal infection in 100% of animals, whereas its use for treatment cured 70% of animals with generalized salmonellosis. Shortening of treatment course from 10 to 5 days and reducing dose of bacteriocin led to less pronounced treatment effect but in all animals it was expressed by increase of mean length of life compared to control (not treated). CONCLUSION: Obtained results demonstrated high treatment efficacy of bacteriocin S760 during septic salmonellosis and perspectives of its use in medicine and animal health.

In vitro antimicrobial effect and in vivo preventive and therapeutic effects of partially purified lantibiotic lacticin NK34 against infection by Staphylococcus species isolated from bovine mastitis.

Lantibiotics are small (<5 kDa), polycyclic peptides produced by gram-positive bacteria; they are also known as gram-positive bacteriocins. The high antimicrobial activity of lacticins and the continuing appearance of antibiotic-resistant bacteria in recent years have resulted in a renewed interest in lantibiotics. A partially purified form of lacticin NK34 (a Lactococcus lactis product isolated from the Korean fermented fish jeotgal) was tested to determine its antimicrobial effects against Staphylococcus aureus (n=20) and coagulase-negative Staphylococcus (CNS, n=20) strains isolated from the raw milk of cows with subclinical bovine mastitis in the present study. The spot-on-lawn assay was used to identify the 2 strains from each group with the greatest lacticin NK34 susceptibility, and the minimal lethal dose (MLD) was measured in ICR (imprinting control region) mice. The preventive and therapeutic effects of lacticin NK34 on the mouse infection model were determined for the first time. Lacticin NK34 demonstrated antimicrobial effects in 14 of 20 (70%) S. aureus indicator strains and in 18 of 20 (90%) CNS strains. Staphylococcus aureus 69 and S. simulans 55 demonstrated the greatest susceptibility to lacticin NK34 in the spot-on-lawn assay. The S. aureus 69 MLD was measured at 1.53 x 10(9) cfu/mouse, whereas the S. simulans 55 MLD was 3.59 x 10(9) cfu/mouse. Mice infected experimentally with S. aureus 69 MLD or S. simulans 55 MLD were treated with lacticin NK34. Treated mice demonstrated an 80% survival rate (48 of 60 mice) compared with a survival rate of 7.5% (3 of 40 mice) in control mice treated with distilled water. These data suggest that lacticin NK34 might be useful in the control of bovine mastitis and systemic bacterial infection.

Further characterization of a bacteriocin produced by Lactobacillus paracasei HL32.

AIMS: Purification, identification and partial characterization of bacteriocin produced by Lactobacillus paracasei HL32. It has been shown to have activity against Porphyromonas sp. METHODS AND RESULTS: The purification of bacteriocin consisting of gel exclusion followed by anion exchange chromatography produced a single band upon an electrophoresis gel with a molecular weight corresponding to 56 kDa. The isolated protein contained 171 amino acids and the first 151 were sequenced. The bacteriocin contained a high percentage of cationic amino acids near the N-terminus, hydrophobic amino acids in the central region (Leu, Ile, Val, Phe, Trp and Gly) and hydrophilic residues (Ser, Asn and Gln) at the C-terminus. This structure did not match with that of previously reported bacteriocins. The antimicrobial activity of the bacteriocin was determined against some pathogens and normal microbiota (P. gingivalis, P. intermedia, T. forsythensis, S. salivarius and S. sanguinis) found in saliva and crevicular fluid. The bacteriocin was found to inhibit P. gingivalis at the minimum bactericidal concentration (MBC) of 0.14 mmol l(-1), but was found not to inhibit the other oral micro-organisms. The bacteriocin was found from transmission electron microscopy studies to cause pore formation in the cytoplasmic membranes of P. gingivalis at the pole and induce potassium efflux. Bacteriocin concentrations of two to four times of MBC were shown to induce haemolysis. The bacteriocin was heat-stable, surviving at 110 degrees C under pressure and possessed activity over a pH range of 6.8-8.5. Only a small reduction of activity was found to occur after incubation in biological fluids (saliva and crevicular fluid). CONCLUSIONS: A novel bacteriocin has been identified that has selective activity against Porphyromonas sp. associated with periodontal disease. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work gained the knowledge of specific antibacterial activity of bacteriocin against Porphyromonas gingivalis.

Real-time assessment of inflammation and treatment response in a mouse model of allergic airway inflammation.

Eosinophils are multifunctional leukocytes that degrade and remodel tissue extracellular matrix through production of proteolytic enzymes, release of proinflammatory factors to initiate and propagate inflammatory responses, and direct activation of mucus secretion and smooth muscle cell constriction. Thus, eosinophils are central effector cells during allergic airway inflammation and an important clinical therapeutic target. Here we describe the use of an injectable MMP-targeted optical sensor that specifically and quantitatively resolves eosinophil activity in the lungs of mice with experimental allergic airway inflammation. Through the use of real-time molecular imaging methods, we report the visualization of eosinophil responses in vivo and at different scales. Eosinophil responses were seen at single-cell resolution in conducting airways using near-infrared fluorescence fiberoptic bronchoscopy, in lung parenchyma using intravital microscopy, and in the whole body using fluorescence-mediated molecular tomography. Using these real-time imaging methods, we confirmed the immunosuppressive effects of the glucocorticoid drug dexamethasone in the mouse model of allergic airway inflammation and identified a viridin-derived prodrug that potently inhibited the accumulation and enzyme activity of eosinophils in the lungs. The combination of sensitive enzyme-targeted sensors with noninvasive molecular imaging approaches permitted evaluation of airway inflammation severity and was used as a model to rapidly screen for new drug effects. Both fluorescence-mediated tomography and fiberoptic bronchoscopy techniques have the potential to be translated into the clinic.

Treatment of plague: promising alternatives to antibiotics.

Plague still poses a significant threat to human health, and interest has been renewed recently in the possible use of Yersinia pestis as a biological weapon by terrorists. The septicaemic and pneumonic forms are always lethal if untreated. Attempts to treat this deadly disease date back to the era of global pandemics, when various methods were explored. The successful isolation of the plague pathogen led to the beginning of more scientific approaches to the treatment and cure of plague. This subsequently led to specific antibiotic prophylaxis and therapy for Y. pestis. The use of antibiotics such as tetracycline and streptomycin for the treatment of plague has been embraced by the World Health Organization Expert Committee on Plague as the 'gold standard' treatment. However, concerns regarding the development of antibiotic-resistant Y. pestis strains have led to the exploration of alternatives to antibiotics. Several investigators have looked into the use of alternatives, such as immunotherapy, non-pathogen-specific immunomodulatory therapy, phage therapy, bacteriocin therapy, and treatment with inhibitors of virulence factors. The alternative therapies reported in this review should be further investigated by comprehensive studies of their clinical application for the treatment of plague.

Antibacterial activity of a bacteriocin from Lactobacillus paracasei HL32 against Porphyromonas gingivalis.

Porphyromonas gingivalis infections cause problems in periodontal diseases and in certain systemic diseases. There is evidence that Lactobacillus spp. can control populations of P. gingivalis, but there are few data on the effects of purified bacteriocins from Lactobacillus paracasei HL32 on P. gingivalis. The objective of this study was to examine the antibacterial activity of a bacteriocin from L. paracasei HL32 and to relate this activity to its composition. A bacteriocin was purified from culture supernatants of Lactobacillus spp. using a dialysis technique followed by gel-permeation chromatography. Composition of the bacteriocin was characterised by ninhydrin tests, ultraviolet spectrophotometry, thin-layer chromatography, sodium-dodecyl sulphate-polyacrylamide gel electrophoresis, electrospray ionisation mass spectrometry and amino acid analysis. The amino acid sequence from the N-terminal of the bacteriocin was determined. Antibacterial activity was examined by the cylinder plate method, microtitre assay and scanning electron microscopy as compared with standard antibiotics. The bacteriocin had a molecular weight of approximately 56kDa, was comprised of 68% carbohydrate and 32% protein, and showed maximum peak absorbance at 214 and 254nm. The bacteriocin was found to be effective against P. gingivalis; it caused swelling and pore formation on the cell envelope at a minimum bactericidal concentration of 0.14mM, and caused death within 2h. Metronidazole killed P. gingivalis but did not affect the envelope, whereas tetracycline affected P. gingivalis with cell deformation. In conclusion, the bacteriocin from L. paracasei HL32 had the ability to kill P. gingivalis, suggesting that it could be a promising alternative chemotherapeutic agent for P. gingivalis infections.

In vivo activity of mutacin B-Ny266.

OBJECTIVES: The objective of this study was to assess the in vivo activity of mutacin B-Ny266 (a bacteriocin produced by Streptococcus mutans) in order to eventually use it as an antibiotic. METHODS: Intraperitoneal infection was induced with a methicillin-susceptible Staphylococcus aureus strain in mice. Some of the mice were simultaneously injected intraperitoneally with mutacin B-Ny266, some with the vehicle only and some with vancomycin. RESULTS: While there was 70 and 100% mortality in the control groups of mice, no mortality was observed in the mice injected with vancomycin or mutacin B-Ny266. CONCLUSIONS: The results presented here show, for the first time, the in vivo efficacy of a mutacin (B-Ny266) against an experimental intraperitoneal infection by S. aureus in a mouse model.

[The efficacy of the use of lactic acid lactobacterin enriched with zinc in the treatment of patients with celiac disease]. / Effektivnost' primeneniia kislomolochnogo laktobakterina, obogashchennogo tsinkom, v terapii bol'nykh tseliakiei.

Zinc sulfate-enriched lactic acid lactobacterin was used in the combined treatment of 23 children with celiac disease, aged from 1 to 10 years. A group of 23 children with celiac disease who received lactic acid lactobacterin without zinc were used as control. The patients treated with lactobacterin containing zinc showed a higher increase in body mass, total protein and zinc levels in the blood serum and elevated activity of metalloenzymes-ceruloplasmin and cytochrome oxidase.

[Therapeutic effectiveness of ristomycin in the visceral form of actinomycosis (experimental research)]. / Terapevticheskaia éffektivnost' ristomitsina pri vistseral'noi forme aktinomikoza (éksperimental'nye issledovaniia).

Therapeutic efficacy of ristomycin in visceral actinomycosis, a severe disease difficult for diagnosis requiring long-term complex therapy was studied on 24 rabbits. The animals were divided into 4 groups. A model of thoracal actinomycosis was developed. 7 rabbits were treated with ristomycin. The other 7 rabbits were treated with ristomycin in combination with actinolysate, a specific immune preparation. 5 rabbits were treated with actinolysate alone and the other 5 rabbits received no treatment. alone and especially in combination with actinolysate was an efficient therapeutic agent for treatment of severe visceral actinomycosis. This allowed to recommend ristomycin for clinical trials in treatment of the disease.