Production Of Antibacterial Substances Research Articles

Probiotics as Means of Diseases Control in Aquaculture, a Review of Current Knowledge and Future Perspectives.

Along with the intensification of culture systems to meet the increasing global demands, there was an elevated risk for diseases outbreak and substantial loss for farmers. In view of several drawbacks caused by prophylactic administration of antibiotics, strict regulations have been established to ban or minimize their application in aquaculture. As an alternative to antibiotics, dietary administration of feed additives has received increasing attention during the past three decades. Probiotics, prebiotics, synbiotics and medicinal plants were among the most promising feed supplements for control or treatments of bacterial, viral and parasitic diseases of fish and shellfish. The present review summarizes and discusses the topic of potential application of probiotics as a means of disease control with comprehensive look at the available literature. The possible mode of action of probiotics (Strengthening immune response, competition for binding sites, production of antibacterial substances, and competition for nutrients) in providing protection against diseases is described. Besides, we have classified different pathogens and separately described the effects of probiotics as protective strategy. Furthermore, we have addressed the gaps of existing knowledge as well as the topics that merit further investigations. Overall, the present review paper revealed potential of different probiont to be used as protective agent against various pathogens.

Pilot-scale production of antibacterial substances by the marine diatom Phaeodactylum tricornutum Bohlin

The production and extraction of antibacterial substances from Phaeodactylum tricornutum biomass were tested on a pilot scale in a photo-bioreactor greenhouse installation. Using column chromatography two antibacterial active fractions were obtained from the algal biomass: a pure fraction of eicosapentaenoic acid (EPA) and a potentially new antibacterial substance(s) (identified as fraction B) produced by P. tricornutum. Both fractions exhibited antibacterial activities against three human pathogens: Vibrio vulnificus, V. parahaemolyticus, and V. cholerae. Minimal inhibitory concentrations against Bacillus subtilis of active fractions and one antibacterial commercial product were determined revealing a higher antibacterial activity of EPA in comparison to the commercial product. Consequently, EPA as well as the raw algal biomass with both EPA and the fraction B compounds could be used as food additives and feeds in aquaculture, poultry, and livestock breeding. Two types of phytohormones, methyl jasmonate and salicylic acid, as well as one phytotoxin, coronatine, were tested for induction of the defense system of P. tricornutum. However, only a minor change in the fatty acids profile and antibacterial effects of the treated cultures indicated that P. tricornutum must employ another strategy to regulate its antibacterial defense system.

Mechanisms of resistance to linalool in Salmonella Senftenberg and their role in survival on basil.

Fresh produce contaminated with human pathogens raises vital and ecological questions about bacterial survival strategies. Such occurrence was basil harboring Salmonella enterica serovar Senftenberg that caused an outbreak in 2007. This host was unanticipated due to its production of antibacterial substances, including linalool. We show that linalool perforates bacterial membranes, resulting in increased permeability and leakage of vital molecules. It also inhibits cell motility and causes bacterial aggregation. Linalool-resistance was investigated by identification and characterization of S. Senftenberg mutants that perform altered resistance. Resistance mechanisms include selective permeability, regulated efflux/influx and chemotaxis-controlled motility. Moreover, survival of S. Senftenberg on basil leaves was substantially affected by McpL, a putative chemotaxis-related receptor, and RfaG, a component of the lipopolysaccharide production pathway, both have a role in resistance to linalool. Results reveal that adaptation to linalool occurs in nature by concurrent mechanisms. This adaption raises concerns about pathogens adaptation to new hosts including antimicrobial-compound-producing plants.

Antibacterial potential of streptomycete strains from Antarctic soils

The exploration of habitats with unusual environment and poorly explored areas such as Antarctica is one of the strategies for discovery of new biologically active substances and/or new producers. The aim of this study was to identify the actinomycetes isolated from the soils of the island Livingston – Antarctica and to investigate their potential to synthesize antibacterial agents against phytopathogens. Twenty-three actinomycete strains were the object of this study. Using PCR (polymerase chain reaction) amplification all strains were affiliated to genus Streptomyces.The sequencing of the 16S rRNA for three of the strains showed greatest similarity to Streptomyces tendae for one of them, and revealed that the other strains had closest relations to streptomycetes isolated from anthropogenically unaltered regions including Antarctica.The isolates were studied for production of antibacterial substances both by molecular and culture methods. PCR targeting specific biosynthetic genes involved in the production of some groups of antibiotics was performed. The screening showed that all strains possessed the gene for Type-II polyketide synthase, 11 strains – for non-ribosomal peptide synthetase; 6 strains – for polyene antibiotics; and 4 strains – for glycopeptide antibiotics. The production of antibacterial substances by the strains was tested in vitro against phytopathogenic bacteria. The strains differed in the number of inhibited test – bacteria and in their spectrum of action. Four strains showed a wide range of activity against Gram-positive and Gram-negative phytopathogens.The results obtained revealed that the Antarctic soils are potential source for isolation of streptomycetes producing antibiotics from different groups.

Pathogen removal during wastewater treatment by vermifiltration

The study was designed to determine the effect of earthworms and the mechanism behind removal of pathogens during wastewater treatment by vermifiltration. The experimental phase continued for 10 weeks, starting after the initial stabilization phase of one week. Significant organic matter degradation and coliform removal were observed during vermifiltration of domestic wastewater. It was observed that vermifilter (VF) reduced biochemical oxygen demand and chemical oxygen demand by 84.8% and 73.9%, respectively. Significant log removal was observed for total coliforms (TC), faecal coliforms (FC), faecal streptococci (FS) and Escherichia coli with a mean log removal value (K) of 2.92, 2.20, 1.85 and 1.68, respectively. The decay rate constant (k) for indicator organisms (TC, FC and FS) was observed to be high as 8.04, 6.59 and 5.55 m day−1, respectively. The population of total heterotrophic bacteria, total fungi and actinomycetes reduced remarkably by 3.14, 1.29 and 2.13 log units, respectively. Antibacterial activity of the isolated microorganisms from VF against gram-positive Staphylococcus aureus (ATCC 29213) and gram-negative E. coli (ATCC 25922) was observed which indicated the existence of a mechanism that may be responsible for pathogen removal during wastewater treatment. This demonstrated the production of antibacterial substance, from the microorganisms associated with earthworms, that causes inhibition of other microbes, specifying the effect of earthworms for pathogen removal. Overall, the present study contributes to the understanding of mechanism for pathogen removal during vermifiltration through antibacterial action of microflora.

Screening antibacterial activity of entomopathogenic bacteria isolated from pests of hazelnut

Thirty-seven entomopathogenic bacteria isolated from six common pests of hazelnut in the Black Sea Region of Turkey have been screened for their potential of antibacterial substance production against indicator bacteria by the agar spot assay and well diffusion assay. Results indicated that 13.5% of entomopathogenic bacteria, Pseudomonas fluorescens (Pf-Xd1), Bacillus polymyxa (Bp-Ar2), Bacillus thuringiensis (Bt-Bn1), Serratia marcescens (Sm-Mm3) and Pseudomonas flourescens (Pf-Aa4) isolated from pests of Xyleborus dispar, Anoplus roboris, Balaninus nucum, Melolontha melolontha and Agelastica alni, respectively, showed significant levels of inhibitor activities against indicator bacteria. Well diffusion assay showed that supernatants of Bp-Ar2, Bt-Bn1 and Sm-Mm3 have antibacterial activity, whereas Pf-Xd1 and Pf-Aa4 did not show any activity. Furthermore, the antibacterial activity of the substance produced by the Bp-Ar2 has a narrow spectrum, whereas those of Bt-Bn1 and Sm-Mm3 exhibit broad spectrum. The production of these antibacterial substances were similarly determined at early logarithmic phase in the growth cycle of three bacteria and continued until the beginning of the stationary phase as primer metabolite. In addition, optimal pH (at 7-9 forBt-Bn1 and 5-9 forSm-Mm3), medium (Muller Hinton broth forBt-Bn1 and Luria Bertani broth forSm-Mm3), temperature (25 ◦ Cf or Bt-Bn1 and Sm-Mm3) and production time (24h forBt-Bn1 and 72h forSm-Mm3) of these substances were determined. Our results demonstrate that entomopathogenic bacteria are a potential source of antibacterial substances.

Production of Antimicrobial Substances by Strains of Myxobacteria Corallococcus and Myxococcus

We prepared culture extracts of 174 Corallococcus and 207 Myxococcus strains isolated in Korea, and compared their antimicrobial activity against Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus. The percentage of strains showing antifungal activity was lower in Corallococcus (7.5% [13 of the 174 strains]) than in Myxococcus (51.7% [107 of the 207 strains]). However, the percentage of strains exhibiting antibacterial activity was higher in Corallococcus (12.1% [21 strains]) than in Myxococcus (1% [2 strains]). The culture extracts of 6 Corallococcus strains inhibited both P. aeruginosa and S. aureus and displayed similar high-performance liquid chromatography chromatograms, although the shapes of their fruiting bodies were dissimilar. The rate of production of antibacterial substances was the highest when the strains were cultured in CYS medium for more than 6 days.

Evaluation of the probiotics Bacillus subtilis and Lactobacillus plantarum bioencapsulated in Artemia nauplii against vibriosis in European sea bass larvae (Dicentrarchus labrax, L.)

Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.

Probióticos, seus modos de ação e a produção animal

The use of antimicrobials in animal production has proven ability to improve animal performance, but the use of these additives has been restricted due to the possibility of leaving residues in meat, and induction of cross-resistance to pathogenic bacteria in humans. Preparing to meet the consumer market that increasingly demand safe and quality foods, have sought alternatives to antibiotic use among these probiotics, live microorganisms administered in adequate amounts which confer health benefits to the host, and many mechanisms are suggested on the mode of action of probiotics, among them competition for binding sites or competitive exclusion, stimulating the immune system, nutritional effect, production of antibacterial substances and enzymes. It has also been studied another very important aspect, the viability of the probiotic micro-organisms used in animal production, ie the importance of assessing the viability of micro-organisms is an essential condition for the action that probiotics can have on body or if the inactivation process reduces or inhibits this action

Characterization of the probiotic strain Vagococcus fluvialis in the protection of European sea bass (Dicentrarchus labrax) against vibriosis by Vibrio anguillarum

Aquaculture is one of the main sources of income in many countries worldwide. Intensive farms are often affected by different infectious diseases that can decrease their final production. To control this situation, several antibiotics are frequently used with known environmental consequences. The aim of this study was to analyze different bacterial strains isolated from of gilthead sea bream, sea bass, sole and meagre guts, for use as probiotics in aquaculture. The strains were evaluated in vitro through various mechanisms of selection, such as the production of antagonistic effects against pathogens, production of antibacterial substance, adhesion to the intestinal mucus, competition for nutrients or binding site, and growth in intestinal mucus. A total of 50 bacterial strains were analyzed and only one showed excellent in vitro results for consideration as a candidate to be analyzed in vivo. The strain, identified as Vagococcus fluvialis, showed good protection against Vibrio anguillarum 975-1 in vivo in the experimental challenge, showing a relative percent survival of 42.3% higher than positive control group. Therefore, in conclusion we consider this strain to be a good candidate for use as a future probiotic in aquaculture.

Production of Anti-Helicobacter pylori metabolite by the lichen-Forming fungus Nephromopsis pallescens

The present study was conducted to evaluate the antibacterial activity of lichen-forming fungi (LFF) against Helicobacter pylori, and to optimize the culture conditions of LFF for maximum production of natural antibiotics against H. pylori. To accomplish this, a screening assay was first conducted among 19 species of LFF. The extract of Nephromopsis pallescens (KOLRI-040516) exhibited the strongest anti-ff. pylori activity. Bioautograghic TLC and HPLC analysis identified usnic acid as the main antibacterial substance produced by JV. pallescens. The growth of JV. pallescens and production of antibacterial substances produced by the fungus were then investigated under several culture conditions including the culture media, initial medium pHs, incubation temperatures, and the degree of aeration. The results indicated that culture in MY medium with an initial pH of 6.0, a temperature of 15°C and a low degree of aeration supported the largest usnic acid production of the fungus (16.4 ug usnic acid/g dry biomass). Especially, aeration was found to be an important factor that affect both growth and usnic acid production of N. pallescens.

Production of Antibacterial Like Substances from Some Clinical Isolates of Trichophytic Genera of Dermatophytes

Nine clinical and six soil isolates of Trichophytic genera of dermatophytes were demonstrated for their in vitro production of antibacterial substances. Clinical isolates were isolated from superficial skin of patients suffering from dermatomycoses and soil isolates were procured from soil using hair-baiting technique. Among nine clinical isolates of dermatophytes tested for their ability to produce antibacterial substances, five were found to be producers and among six soil isolates, three produces the antibacterial substances. The qualitative identification of the produced antibiotics reveals five types; a penicillin-like substance produced by Trichophyton concentricum, and two different types of unknown substances obtained from Trichophyton mentagrophytes and T megninii, the kojic acid-like antibacterial substance produced by Trichophyton verrucosum. The fusidic acid like substances was obtained from T tonsurans and T simii.

Changes in phenolic acids content, phenylalanine ammonia-lyase and peroxidase activities in sugarcane leaves induced by elicitors isolated fromXanthomonas albilineans

Xanthomonas albilineans is the agent responsible for a serious disease of sugarcane called leaf scald disease. Symptoms of the disease vary from a white line on the leaves to plant death. This paper correlates leaf scald resistance to changes in the concentration of phenolic compounds and phenylalanine ammonia-lyase and peroxidase activities in sugarcane leaves which were induced by a putative elicitor isolated from X. albilineans within different SE-chromatography fractions. The interaction was studied in leaf discs obtained from cultivars Louisiana 55-5 (susceptible to leaf scald) and Mayari 55-14 (resistant to leaf scald). SE-chromatography fractions containing only proteins enhanced the production of hydroxycinnamic acids (HCA) in cv. Mayari whereas fractions containing both proteins and carbohydrates enhanced the production of hydroxybenzoic acids (HBA) in cv. Louisiana. The eliciting protein(s) induced a level of PAL activity that increased the concentration of p-coumaric and caffeic acids, whereas ferulic acid, later synthesised in the host plant defence cycle, is used for the synthesis of syringic acid and for reinforcing the cell walls through peroxidase activation in the resistant cultivar. These facts justify the increase of p-coumaric, caffeic and syringic acids and the decrease of ferulic acid concentrations in this cultivar. Bacterial carbohydrates not only elicit the accumulation of p-coumaric acid, but also induce synthesis and accumulation of some hydroxybenzoic acids (HBA), such as gallic, protocatechuic and syringic acids and impede the production of antibacterial substances in the susceptible cultivar.

Diverse Antibacterial Activity of Pectobacterium carotovorum subsp.carotovorum Isolated in Korea

Fifty-four Pectobacterium carotovorum subsp. carotovorum strains isolated in Korea were characterized by a spectrum of antibacterial activities against 7 indicator strains chosen to represent various regions and host plants. All P. carotovorum subsp. carotovorum isolates tested could be grouped into 4 classes depending on the pattern of antibacterial substance production. All tested strains had DNA fragment(s) homologous to the genes encoding carotovoricin and 21 of them had genes homologous to DNA invertase. Sixteen strains had genes homologous to the genes encoding carocin S1. Several isolates produced antibacterial substances active against strains in Brenneria, Pantoea, and Pectobacterium genera that belonged formerly to the genus Erwinia. Strains in Pseudomonas or Xanthomonas sp. were not sensitive to the antibacterial substances produced by P. carotovorum subsp. carotovorum, except for X. albilineans that was sensitive to antibacterial substances produced by most strains in P. carotovorum subsp. carotovorum and P. betavasculorum KACC10056. These results demonstrated the diverse patterns of antibacterial substance production and the possibility of the existence of new antibacterial substance(s) produced by P. carotovorum subsp. carotovorum isolated in Korea.

Triatominae– Trypanosoma cruzi/ T. rangeli: Vector–parasite interactions

Of the currently known 140 species in the family Reduviidae, subfamily Triatominae, those which are most important as vectors of the aetiologic agent of Chagas disease, Trypanosoma cruzi, belong to the tribes Triatomini and Rhodniini. The latter not only transmit T. cruzi but also Trypanosoma rangeli, which is considered apathogenic for the mammalian host but can be pathogenic for the vectors. Using different molecular methods, two main lineages of T. cruzi have been classified, T. cruzi I and T. cruzi II. Within T. cruzi II, five subdivisions are recognized, T. cruzi IIa–IIe, according to the variability of the ribosomal subunits 24Sα rRNA and 18S rRNA. In T. rangeli, differences in the organization of the kinetoplast DNA separate two forms denoted T. rangeli KP1+ and KP1−, although differences in the intergenic mini-exon gene and of the small subunit rRNA (SSU rRNA) suggest four subpopulations denoted T. rangeli A, B, C and D. The interactions of these subpopulations of the trypanosomes with different species and populations of Triatominae determine the epidemiology of the human-infecting trypanosomes in Latin America. Often, specific subpopulations of the trypanosomes are transmitted by specific vectors in a particular geographic area. Studies centered on trypanosome–triatomine interaction may allow identification of co-evolutionary processes, which, in turn, could consolidate hypotheses of the evolution and the distribution of T. cruzi/ T. rangeli-vectors in America, and they may help to identify the mechanisms that either facilitate or impede the transmission of the parasites in different vector species. Such mechanisms seem to involve intestinal bacteria, especially the symbionts which are needed by the triatomines to complete nymphal development and to produce eggs. Development of the symbionts is regulated by the vector. T. cruzi and T. rangeli interfere with this system and induce the production of antibacterial substances. Whereas T. cruzi is only subpathogenic for the insect host, T. rangeli strongly affects species of the genus Rhodnius and this pathogenicity seems based on a reduction of the number of symbionts.

An investigation of the bacteriocinogenic potential of lactic acid bacteria associated with wheat ( Triticum durum) kernels and non-conventional flours

One hundred and thirty-seven lactic acid bacteria (LAB), previously isolated from wheat ( Triticum durum) grains and non-conventional flour samples, were tested for the production of antibacterial substances. A total of 16 strains (5 Enterococcus faecium, 5 Enterococcus mundtii, 4 Pediococcus pentosaceus, 1 Lactobacillus coryniformis and 1 Lactococcus garvieae) were found to inhibit the growth of Listeria innocua. The antibacterial activities were preliminarily investigated for their general behaviour with proteolytic (proteinase K, protease B and trypsin), amylolytic (α-amylase) and lipolytic (lipase) enzymes, after heat treatment, and exposure to different pHs and ethanol concentrations. Bacteriocin-like inhibitory substances (BLIS) were also characterized for their inhibition spectra against non-pathogenic and pathogenic food-associated and human pathogenic bacteria. LAB showing the best characteristics in terms of inhibition spectrum, inhibition activity and mode of action (bactericidal) belonged to the species Ent. mundtii. The high percentage (11.68%) of BLIS-producing strains detected confirmed previous observations that raw materials may harbour higher numbers of bacteriocinogenic LAB than fermented foods.

Fermentation Science & Engineering

One hundred and thirty-seven lactic acid bacteria (LAB), previously isolated from wheat (Triticum durum) grains and non-conventional flour samples, were tested for the production of antibacterial substances. A total of 16 strains (5 Enterococcus faecium, 5 Enterococcus mundtii, 4 Pediococcus pentosaceus, 1 Lactobacillus coryniformis and 1 Lactococcus garvieae) were found to inhibit the growth of Listeria innocua. The antibacterial activities were preliminarily investigated for their general behaviour with proteolytic (proteinase K, protease B and trypsin), amylolytic (α-amylase) and lipolytic (lipase) enzymes, after heat treatment, and exposure to different pHs and ethanol concentrations. Bacteriocin-like inhibitory substances (BLIS) were also characterized for their inhibition spectra against non-pathogenic and pathogenic food-associated and human pathogenic bacteria. LAB showing the best characteristics in terms of inhibition spectrum, inhibition activity and mode of action (bactericidal) belonged to the species Ent. mundtii. The high percentage (11.68%) of BLIS-producing strains detected confirmed previous observations that raw materials may harbour higher numbers of bacteriocinogenic LAB than fermented foods.

Isolation and characterization of antibacterial substances produced by marine actinomycetes in the presence of seawater

Actinomycetes, which produced antibacterial substances against Gram-positive bacteria in the presence of seawater only, were isolated from marine environment and characterized. Of the 100 isolates from Otsuchi Bay in Iwate, 41 failed to produce any antibacterial substances in the absence of seawater. However, of the 41, two were found to produce antibacterial substances in the presence of seawater. Strain No. 18 exhibited higher activity than strain No. 28 and was, therefore, selected for further studies. Phylogenetic analysis and physiological characterization showed a high similarity between strain No. 18 and Micromonospora globosa. However, M. globosa JCM 3126 did not produce any antibiotics in the presence of seawater. Therefore, the effect of seawater on the growth of strain No. 18 and its production of antibacterial substances was investigated. Strain No. 18 grew in a medium prepared with artificial seawater at a concentration range of 0–140% (v/v). The optimal concentration of growth ranged from 10 to 30% (v/v). However, production of antibacterial substances was observed in the concentration range 60–110% (v/v) despite poor growth. Thus, the production of antibacterial substances is seawater-dependent. The purification and characterization of the substances is now in progress.

The in vitro inhibition of Gram-negative pathogenic bacteria by bifidobacteria is caused by the production of organic acids

Several Bifidobacterium strains, including commercial probiotics, were screened for the production of antibacterial substances active towards Gram-negative pathogenic bacteria. Agar spot assays, microtitre plate assays, and time-kill studies were performed. Bifidobacteria were found to exert strong inhibitory activity towards Gram-negative indicator bacteria, namely Salmonella enterica serovar Typhimurium SL1344 and Escherichia coli C1845. The inhibitory mechanism was investigated and was shown to be dependent on the lowering of the pH of the medium and the production of organic acids, in particular acetic acid and lactic acid. The production of other antibacterial substances was examined by concentrating the cell-free culture supernatants by ammonium sulphate precipitation and chloroform/methanol extraction. The extracts of cultures of seven Bifidobacterium strains inhibited the growth of Gram-positive indicator bacteria, indicating that they produced a bacteriocin. The extract of a culture of Bifidobacterium longum BB536 was active against E. coli as well. However, the contribution of such inhibitory substances to the antibacterial activity of bifidobacteria towards Gram-negative pathogenic bacteria was found to be negligible.

Growth and antibacterial activity of Lentinula edodes in liquid media supplemented with agricultural wastes

Antibacterial activity of Lentinula edodes against Bacillus subtilis was evaluated in cell-free filtrates obtained after growth in 14 different culture media. The highest B. subtilis growth inhibition was promoted by filtrates of growth media supplemented with rice bran, vermiculite or molasses. L. edodes dry mycelial biomass in liquid culture with 0.5% added rice bran was 3.2 mg/ml, after growth for 30 days at 25oC without shaking, and 4.3 mg/ml under orbital shaking (150 rpm). However, antibacterial activity, detected between 20 and 24 days of incubation of stationary cultures, was absent in filtrates of aerated cultures. Temperatures of 20-25oC enhanced both growth and antibacterial activity. Optimum pH for L. edodes mycelial growth was 3.0-3.5, while for production of antibacterial substance(s) it was 4.5. Our results indicated that incubation conditions that enhance mycelial growth are quite different from those necessary for production of antibacterial substance(s) by L. edodes.