Demonstration of a Bactericidal Substance Against β-Hemolytic Streptococci in Supernatant Fluids of Staphylococcal Cultures

Staphylococcal skin isolates belonging to phage type 71 were found to produce a bactericidal substance against some streptococci, pneumococci, and corynebacteria. Fifteen strains of group A streptococci belonging to 13 different M types, group C streptococci, and group D streptococci were uniformly inhibited on solid media and in broth by membrane-filtered supernatant fluids of the staphylococcal broth cultures. Inhibition of group G streptococci and other staphyloccoci was variable, and no inhibition of group B streptococci or of a variety of gram-negative rods was demonstrable. A quantitative variation observed to exist among susceptible organisms was a function of the inoculum size of the inhibited strains. The bactericidal substance could be detected best from 24 to 48 hr after inoculation of the staphylococci in tryptic soy broth or in a dialysate of tryptic soy broth. Little or no bactericidal activity was noted when the organisms were grown in several other liquid media. The bactericidal substance was nondialyzable and could be precipitated with ammonium sulfate. It was heat-stable and its activity was not altered within a pH range of 4.0 to 8.5. Pronase and three times crystallized trypsin totally abolished its activity. The concentrated ammonium sulfate precipitate could be fractionated on a Sephadex G-100 column into several peaks, with the bactericidal activity localized to a single peak.     

Identification and characterization of helveticin V-1829, a bacteriocin produced by Lactobacillus helveticus 1829.

Lactobacillus helveticus 1829 produced an antimicrobial agent, designated helveticin V-1829, that demonstrated antagonistic activity against closely-related species. The agent was excreted into MRS agar, and was present in the supernatant fluids from both overnight broth and clotted milk cultures. It was heat labile (inactivated by 50 degrees C for 30 min) and was stable over the pH range 2.5 to 6.5. Production of the substance was pH-dependent and maximum yields were obtained in MRS broth cultures maintained at pH 5.5. Helveticin V-1829 was partially purified following growth of the producing strain in a semi-defined MRS medium and precipitating the cell-free filtrate with ammonium sulphate to 30% saturation. The cleared supernatant fluid was then brought to 60% saturation and the resulting precipitate pelleted and dialysed in 0.3 mol/l phosphate buffer. The partially purified inhibitor was sensitive to several proteolytic enzymes, and it was bactericidal in its mode of action against indicator cells of Lact. helveticus 1844 and Lact. delbrueckii subsp. bulgaricus 1489, indicating that it was a bacteriocin. A DNA probe specific for the helveticin J structural gene failed to hybridize to total genomic DNA of Lact. helveticus 1829, indicating that helveticin V-1829 is not significantly related to helveticin J.     

Identification and characterization of helveticin V-1829, a bacteriocin produced by Lactobacillus helveticus 1829

Lactobacillus helveticus 1829 produced an antimicrobial agent, designated helveticin V-1829, that demonstrated antagonistic activity against closely-related species. The agent was excreted into MRS agar, and was present in the supernatant fluids from both overnight broth and clotted milk cultures. It was heat labile (inactivated by 50°C for 30 min) and was stable over the pH range 2.5 to 6.5. Production of the substance was pH-dependent and maximum yields were obtained in MRS broth cultures maintained at pH 5.5. Helveticin V-1829 was partially purified following growth of the producing strain in a semi-defined MRS medium and precipitating the cell-free filtrate with ammonium sulphate to 30% saturation. The cleared supernatant fluid was then brought to 60% saturation and the resulting precipitate pelleted and dialysed in 0.3 mol/l phosphate buffer. The partially purified inhibitor was sensitive to several proteolytic enzymes, and it was bactericidal in its mode of action against indicator cells of Lact. helveticus 1844 and Lact. delbrueckii subsp. bulgaricus 1489, indicating that it was a bacteriocin. A DNA probe specific for the helveticin J structural gene failed to hybridize to total genomic DNA of Lact. helveticus 1829, indicating that helveticin V-1829 is not significantly related to helveticin J.     

Detection of secreted antimicrobial peptides isolated from cell-free culture supernatant of Paenibacillus alvei AN5

An antimicrobial substance produced by the Paenibacillus alvei strain AN5 was detected in fermentation broth. Subsequently, cell-free culture supernatant (CFCS) was obtained by medium centrifugation and filtration, and its antimicrobial activity was tested. This showed a broad inhibitory spectrum against both Gram-positive and -negative bacterial strains. The CFCS was then purified and subjected to SDS-PAGE and infrared spectroscopy, which indicated the proteinaceous nature of the antimicrobial compound. Some de novo sequencing using an automatic Q-TOF premier system determined the amino acid sequence of the purified antimicrobial peptide as Y-S-K-S-L-P-L-S-V-L-N-P (1,316 Da). The novel peptide was designated as peptide AN5-1. Its mode of action was bactericidal, inducing cell lysis in E. coli ATCC 29522 and S. aureus, and non-cell lysis in both S. marcescens and B. cereus ATCC 14579. Peptide AN5-1 displayed stability at a wide range of pH values (2–12) and remained active after exposure to high temperatures (100 °C). It also maintained its antimicrobial activity after incubation with chemicals such as SDS, urea and EDTA.     

Characterization and partial purification of a broad spectrum antibiotic AS-48 produced by Streptococcus faecalis

Streptococcus faecalis S-48 produces a broad spectrum antibiotic, active against Gram-positive and Gram-negative bacteria. This substance is produced in solid and liquid media and also in a defined basal medium. It is sensitive to protease, pronase, or trypsin, heating at 70 degrees C, and alkaline pH, but resistant to treatment with lipase, lysozyme, alkaline phosphatase, DNAase, RNAase, acidic or neutral pHs, and also lower temperatures (60 degrees C). Several organic solvents cause precipitation, but not inactivation. This antibiotic has been partially purified by gel filtration and further ion-exchange chromatography. Its molecular weight has been estimated close to 2000. The biological activity of this antagonistic substance against the selected indicator strains, Streptococcus faecalis S-47 and Escherichia coli U-9, is bactericidal. The characterization of this substance, initially classified as a bacteriocin, indicates that it is an antibiotic of peptidic nature. The significance of antibiotic occurrence in group D of the genus Streptococcus is also discussed.     

Inhibition of adhesion of food-borne pathogens to Caco-2 cells by Lactobacillus strains

AIMS: To select adhesive strains among strains of Lactobacillus and to apply them to inhibit adhesion of food-borne pathogens. METHODS AND RESULTS: Twelve Lactobacillus strains (10 from intestine) were examined for adhesion using Caco-2 cell cultures. The two most adhesive strains, Lactobacillus crispatus JCM 8779 and Lact. reuteri JCM 1081, were used to test antiadhesion activity against enterotoxigenic Escherichia coli, Salmonella typhimurium and Enterococcus faecalis strains. Adhesion of the pathogens was inhibited by both Lactobacillus strains. Adhesion of Ent. faecalis was especially strongly inhibited by JCM 8779. Although antimicrobial activity was not detected in the culture supernatant fluid by agar well diffusion assay, the supernatant fluid obtained from the harvested JCM 8779 cell suspension showed bactericidal activity against Ent. faecalis. CONCLUSION: The strong antiadhesion activity of JCM 8779 against Ent. faecalis appears to be due to the combined effect of both bactericidal activity and competition for attachment site. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that Lact. crispatus produces a bactericidal substance.     

Production of an extracellular ribonuclease by Pseudomonas maltophilia.

As part of a screening program for pseudomonad enzymes having an industrial interest, we selected ribonuclease (RNase) producing strains. Of the 150 pseudomonads screened, 6 were found to produce an extracellular RNase activity when grown on solid medium. In broth culture, the RNase activity from these six species remained bound to the cells unless gelatin was added to the medium. Gelatin was essential for the release of RNase in the broth culture, but the pH of the medium, addition of potential inducers such as nucleic acids, or addition of cations did not affect this release. However, gelatin did not appear to induce the synthesis of the enzyme. Strain B-88, identified as Pseudomonas maltophilia, was selected for further study of the enzyme. The extracellular RNase isolated from B-88 broth cultures could be separated in two fractions on the basis of the molecular weight by the ultrafiltration technique. The low molecular weight fraction reacts optimally at temperatures between 55 and 60 degrees C and optimal pH values varying from 7.4 to 9.5. At neutral or alkaline pH, the enzyme was stable at temperatures below 37 degrees C but was inactivated at 55 degrees C. The RNase was inhibited by mercury and cobalt and stimulated by magnesium.     

Characterization of bacteriocin produced by Pediococcus pentosaceus from wine

A.M. STRASSER DE SAAD AND M.C. MANCA DE NADRA. 1993. Twenty strains of Pediococcus pentosaceus isolated from wine were examined for production of bacteriocins. Only two of them showed inhibitory activity, Ped. pentosaceus N4p against the indicator strains of the same species and N5p against 19 strains of the three genera of lactic acid bacteria from wine. The antimicrobial substance from N5p strains was removed by membrane (0.2 μm) filtration, destroyed by organic solvents and proteolytic enzymes. It was stable for 60 min at 100C. The bacteriocin was produced early in the growth cycle and its production was maximum after 48 h of culture in tomato juice medium at an initial pH of 6.5. The bactericidal effect was observed.     

Purification of a kappa-carrageenase from marine Cytophaga species

A mixture of extracellular carrageenases was isolated from the cell-free medium of a culture of marine Cytophaga sp. 1k-C783 grown on ZoBell 2216 E broth with 0.1% commercial carrageenan. A single active peak of kappa-carrageenase was separated and purified from the mixture by ammonium sulfate precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration chromatography. Molecular weight of the purified kappa-carrageenase was estimated as 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified kappa-carrageenase had pH optimum 7.6 and temperature optimum 25 C.     

Amylose-like polysaccharide accumulation and hyphal cell-surface structure in relation to citric acid production by Aspergillus niger in shake culture

When 120 mg glucose/ml was used as a carbon source, in shake culture Aspergillus niger Yang no. 2 maximally produced only 15.4 mg citric acid/ml but accumulated 3.0 mg extracellular polysaccharide/ml. The polysaccharide secreted by mycelia of Yang no. 2 in shake culture was confirmed to be an amylose-like alpha-1,4-glucan by hydrolysis analysis with acid, amylase and glucoamylase. However, in static cultures, such as semisolid and surface cultures free from physical stresses caused by shaking damage, Yang no. 2 produced more citric acid but did not accumulate the polysaccharide. With cultivation time in shake culture, the amount of extracellular polysaccharide and the viscosity of the culture broth increased. The increase of shaking speed caused a remarkable increase in the accumulation of extracellular polysaccharide, e.g. 11.2 mg extracellular polysaccharide/ml was accumulated in the medium at a shaking speed of 200 rpm. The addition of 2.0 mg carboxymethylcellulose (CMC)/ml as a viscous additive to the medium reduced drastically the amount of extracellular polysaccharide accumulated to 1.5 mg/ml, but increased the citric acid produced to 52.0 mg/ml. However, intracellular polysaccharide accumulation kept up a steady rate of 0.26 microgram/mg dried mycelium through the entire period of cultivation. The addition of 3.0 mg polysaccharide/ml purified from the culture broth to the medium at the start of a culture resulted in a decrease of extracellular polysaccharide accumulation but an increase of citric acid accumulation. From electronmicroscopic observation, cell surfaces of hyphae cultivated with CMC were smooth, while hyphae cultivated without CMC had fibrous and granular polysaccharide on the cell surface. These results suggested that Yang no. 2 secreted the polysaccharide on the cell surface as a viscous substance and/or a shock absorber to protect itself from physical stresses caused by shaking damage in shake culture.     

Binding of complement inhibitor C4b-binding protein contributes to serum resistance of Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the alpha-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.     

Production of antifungal recombinant peptides in Escherichia coli

Antifungal peptides derived from the human bactericidal/permeability-increasing protein (BPI) were produced in Escherichia coli as fusion proteins with human BoneD. Bacterial cultures transformed with the gene encoding the fusion protein were grown to a high cell density (OD(600)>100), and induced with L-arabinose to initiate product expression. Fusion protein accumulated into cytoplasmic inclusion bodies and recombinant peptide was released from BoneD by acid hydrolysis at an engineered aspartyl-prolyl dipeptide linker. Acid hydrolysis of purified inclusion bodies at pH <2.6 followed Arrhenius kinetics and did not require prior inclusion body solubilization in detergents or denaturants. Surprisingly, at pH <2.6 and 85 degrees C, cell lysis and aspartyl-prolyl hydrolysis with concomitant peptide release occurred simultaneously. Bacterial cultures were, therefore, adjusted to approximately pH 2.6 with HCl directly in the bioreactor and incubated at elevated temperature. Peptide, which is soluble in the aqueous acidic environment, was separated from the insoluble material and purified using column separation techniques. Recombinant peptide was separated from the hydrolyzed bioreactor culture with >76% recovery and a final peptide purity of >97%. Antifungal peptide prepared by recombinant and solid phase synthesis methods demonstrated similar activity against Candida sp. in a broth microdilution assay.     

Inhibitory and lethal effects of chlorhexidine and a polymeric biguanide on some strains of Providencia stuartii

The effects of chlorhexidine diacetate and vantocil IB on the viability of Providencia stuartii strains are described. Exposure of Prov. stuartii strains to different concentrations of chlorhexidine in broth culture resulted in a decrease in viability over the first 6 h, followed by regrowth. During incubation, bacteria adhered to the surface of the culture vessel and multiplied despite the presence of a bactericidal concentration of the drug in the medium. It is concluded that the phenomenon of 'regrowth' results from adhesion to glass containers and the subsequent dispersal of some of these cells into the culture medium.     

Isolation and characterization of a new bacteriocin from Lactobacillus gasseri KT7

A bacteriocin-producing Lactobacillus gasseri strain, KT7, was isolated from infant faeces. The supernatant fluid showed inhibitory activity not only against some lactic acid bacteria but also, against some pathogenic and food-spoilage species, including Clostridium, Listeria and Enterococcus. An antimicrobial peptide designated gassericin KT7 was isolated from Lactobacillus gasseri KT7. It was purified to homogeneity by a single four-step procedure: a crude supernatant fluid obtained from early stationary-phase culture in MRS medium was subjected to ammonium sulphate fractionation, CM-Sephadex cation-exchange chromatography, Phenyl-Sepharose hydrophobic chromatography and reverse-phase HPLC chromatography. Gassericin KT7 was sensitive to proteolytic enzymes, resistant to heat, active over a wide range of pH, and migrated as a 4.5-5.0 kDa peptide on SDS-PAGE. The bacteriocin was produced constitutively during exponential growth. It was bactericidal to sensitive cells and the bactericidal effect was not produced by cell lysis. The amino acid composition of the bacteriocin was determined and no modified amino acid was found among the residues identified.     

Growth Stimulating Substance for Microorganisms Produced by Escherichia coli Causing the Reduction of the Lag Phase in Microbial Growth and Identity of the Substance with Pyrroloquinoline Quinone

The finding that most strains of microbes produce a growth stimulating substance for microorganisms was demonstrated and confirmed with the culture broth of Escherichia coli grown on a glucose-mineral medium. Addition of culture broth of E. coli to the culture media of the others markedly reduced the lag phase in microbial growth but not growth rate in the subsequent exponential phase nor the total cell yield in the stationary phase. The growth stimulation causing reduction of the lag phase was dependent on the amount of culture broth added. Occurrence of cell growth was essential for the excretion of the growth stimulating substance by E. coli. Under identical inoculum size, even with a heavy inoculum, a further reduction of the lag phase was observed by the addition of culture broth of E. coli. The substance was only effective at the initial growth phase but inert when the substance was added to a growing culture at the exponential phase. Finally, the substance was identified as pyrroloquinoline quinone, a newly established coenzyme, through chromatographic, spectroscopic and enzymatic criteria.     

Analysis of bacteriocinogenic properties of Yersinia enterocolitica strains

The aim of the study was the investigation of bacteriocinogenic properties of 102 Yersinia enterocolitica strains. The influence of selected factors on the production of bacteriocins by Y. enterocolitica and properties of jersiniacin 44JPSBKOH were also investigated. Bacteriocinogenic properties of Y. enterocolitica strains were tested by using the delayed cross-streaking method. It was found that the production of bacteriocins by Y. enterocolitica depended on the type of media on which the producer and indicator strains were grown. It turned out that some strains of Y. enterocolitica showed bacteriocinogenic properties at 25 degrees C, 30 degrees C and 37 degrees C irrespective of the presence of manganese ions in medium. In the presence of iron ions these strains showed bacteriocinogenic properties only at 25 degrees C. Y. enterocolitica strains which required Mn2+ or Mn7+ ions for bacteriocins production showed this activity only at 25 degrees C but in presence of Fe3+ ions they had no bacteriocinogenic properties. The partially purified jersiniacin 44JPSBKOH is a protein, its molecular weight was estimated to be 40 kDa. Yersiniacin 44JPSBKOH was active in the pH range of 3 to 9. Its bactericidal activity was rapidly lost when heated to 100 degrees C and treated with proteolytic enzymes. Yersiniacin 44JPSBKOH showed bactericidal activity against other Y. enterocolitica strains and some strains of Pseudomonas aeruginosa isolated from humans.     

Purification, characterization and gene cloning of the killer toxin produced by the marine-derived yeast Williopsis saturnus WC91-2

As the killer toxin produced by Williopsis saturnus WC91-2 could kill many sensitive yeast strains, including the pathogenic ones, the extracellular killer toxin in the supernatant of cell culture of the marine yeast strain was purified and characterized. The molecular mass of the purified killer toxin was estimated to be 11.0kDa according to the data from SDS-PAGE. The purified killer toxin had killing activity, but could not hydrolyze laminarin. The optimal conditions for action of the purified killer toxin against the pathogenic yeast Metschnikowia bicuspidate WCY were the assay medium with 10% NaCl, pH 3-3.5 and temperature 16°C. The gene encoding the killer toxin from the marine killer yeast WC91-2 was cloned and the ORF of the gene was 378bp. The deduced protein from the cloned gene encoding the killer toxin had 125 amino acids with calculated molecular weight of 11.6kDa. It was also found that the N-terminal amino acid sequence of the purified killer toxin had the same corresponding sequence deduced from the cloned killer toxin gene in this marine yeast, confirming that the purified killer toxin was indeed encoded by the cloned gene.     

Production of cutinase by Thermomonospora fusca ATCC 27730

Ten strains belonging to various Thermomonospora species were tested for their ability to hydrolyse the insoluble plant polyester cutin. One strain, the thermophile T . fusca ATCC 27730, was found to produce a highly inducible cutinase when grown in broth medium containing purified apple cv. Golden Delicious cutin. Apple pomace, tomato peel, potato suberin and commercial cork were also shown to induce cutinase production. Addition of glucose to the culture medium either at the beginning of fermentation or after 2 days of incubation in the presence of apple cutin led to repression of cutinase production. The cutinase was active against a wide range of cutins, including those isolated from other apple cultivars as well as tomato, cucumber, grapefruit, and green pepper. Cutinase activity in the induced culture supernatant fluids exhibited a half-life of over 60 min at 70 °C and a pH optimum of 11·0. Some potential applications for cutinases are discussed.     

Characterization of a bacteriocin produced by Streptococcus thermophilus ST134

A pH-dependent adsorption/desorption technique was used to screen Streptococcus thermophilus strains for the production of bacteriocins. Agar-diffusion tests with S. thermophilus strains as targets identified 13 out of 41 strains as producers of antibacterial activity. Thermophilin A, the bacteriocin-like substance present in the culture supernatant of S.thermophilus ST134 was purified to homogeneity by ammonium sulfate precipitation and ion-exchange chromatography, followed by ultrafiltration. Thermophilin A is a relatively heat-stable and apparently glycosylated bacteriocin with a bactericidal mode of action against sensitive cells.     

Antibacterial activity of lactic acid bacteria isolated from vacuum-packaged meats

Lactic acid bacteria isolated from vacuum-packaged fresh meat stored at 4°C were shown to produce antagonistic substances active against closely related bacteria. Growth medium, pH and growth temperature all affected the production of the inhibitory substances. Ten strains including aciduric Lactobacillus -type organisms, Carnobacterium spp. and Leuconostoc spp. were selected that produced protein-aceous substances that caused inhibition of indicator strains. These were considered to be bacteriocins or bacteriocin-like compounds based on their inactivation with protease, generally narrow spectra of antibacterial activity and bactericidal or bacte-riostatic modes of action. Activity was not lost from supernatant fluids as a result of heat treatment at 62°C for 30 min, except for the Leuconostoc strains. Inhibitory spectra of some strains included Enterococcus spp. and Listeria monocytogenes . Some strains were of interest because their inhibitory substances were detected in the supernatant fluid early in the growth cycle. The inhibitory substances differed in characteristics between strains and there is evidence that more than one bacteriocin-like substance may be produced by some strains.