Characterization of a bacteriocin produced by Enterococcus faecium GM-1 isolated from an infant

AIM: To partially characterize the bacteriocin produced by the GM-1 strain of Enterococcus faecium, isolated from the faeces of a newborn human infant. METHODS AND RESULTS: The bacteriocin produced by E. faecium GM-1 showed a broad spectrum of activity against indicator strains of Escherichia coli, Staphylococcus aureus, Vibrio spp., Salmonella typhimurium, Listeria monocytogenes, Lactobacillus acidophilus, and Streptococcus thermophilus. Treatment of the GM-1 bacteriocin with proteolytic enzymes reduced its inhibitory activities. The bacteriocin was stable at 100 degrees C for 20 min and displayed inhibitory activity at neutral pH. The optimal production of bacteriocin from E. faecium GM-1 was obtained when the culture conditions were pH 6.0-6.5 and 35-40 degrees C. The inhibitory activity of the bacteriocin was not substantially changed by the use of different carbon sources in the media, except when galactose was substituted for glucose. The use of a sole nitrogen source caused a decrease in inhibitory activity. A bacteriocin gene similar to enterocin P was identified from the total DNA of E. faecium GM-1 by PCR and direct sequencing methods. CONCLUSION: E. faecium GM-1, which was isolated from the faeces of a newborn baby, produces an enterocin P-like bacteriocin with inhibitory activity against Gram-positive and Gram-negative bacteria, including food-borne pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: E. faecium GM-1, isolated from infant faeces, produces a new bacteriocin that is similar to enterocin P. This bacteriocin is heat stable and has a broad antibacterial spectrum that includes both Gram-positive and Gram-negative bacteria.     

Characteristics and identification of enterocins produced by Enterococcus faecium JCM 5804T

AIMS: To screen bacteriocin-producing lactic acid bacteria (LAB) in 52 type and reference strains, which have not previously been studied, with respect to bacteriocins, and to characterize the presence of bacteriocins. METHODS AND RESULTS: Only Enterococcus faecium JCM 5804T showed bacteriocin-like activity. It inhibited the growth of Lactobacillus spp., Enterococcus spp., Clostridium spp., Listeria monocytogenes, and vancomycin resistant Enterococcus (VRE). However, it was not effective against Gram-negative strains, Weisella spp., Leuconostoc spp., Lactococcus spp., or methicillin resistant Staphylococcus aureus (MRSA). The inhibitory activity of Ent. faecium JCM 5804T was inactivated by proteinase K, trypsin, alpha-chymotrypsin, and papain, but not by lysozyme, lipase, catalase, or beta-glucosidase. The inhibitory activity was stable at 100 degrees C for 30 min, and had a pH range from 2 to 10. The molecular weight of the partially purified bacteriocin(s) was approx. 4.5 kDa, according to tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Polymerase chain reaction and direct sequencing methods identified three different types of bacteriocins produced by Ent. faecium JCM 5804T, enterocin A, enterocin B, and enterocin P-like bacteriocin. CONCLUSION: Enterococcus faecium JCM 5804T produced three different types of bacteriocins, and they inhibited LAB and pathogens. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report of enterocin A, enterocin B, and enterocin P-like bacteriocin, detected in Ent. faecium JCM 5804T among LAB type and reference strains.     

Antilisterial activity of lactic acid bacteria isolated from rigouta, a traditional Tunisian cheese

AIMS: Screening for lactic acid bacteria (LAB) producing bacteriocins and other antimicrobial compounds is of a great significance for the dairy industry to improve food safety. METHODS AND RESULTS: Six-hundred strains of LAB isolated from 'rigouta', a Tunisian fermented cheese, were tested for antilisterial activity. Eight bacteriocinogenic strains were selected and analysed. Seven of these strains were identified as Lactococcus lactis and produced nisin Z as demonstrated by mass spectrometry analysis of the purified antibacterial compound. Polymerase chain reaction experiments using nisin gene-specific primers confirmed the presence of nisin operon. Plasmid profiles analysis suggests the presence of, at least, three different strains in this group. MMT05, the eighth strain of this antilisterial collection was identified, at molecular level, as Enterococcus faecalis. The purified bacteriocin produced by this strain showed a molecular mass of 10 201.33 +/- 0.85 Da. This new member of class III bacteriocins was termed enterocin MMT05. CONCLUSIONS: Seven lactococcal strains producing nisin Z were selected and could be useful as bio-preservative starter cultures. Additional experiments are needed to evaluate the promising strain MMT05 as bio-preservative as Enterococci could exert detrimental or beneficial role in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a few antibacterial strains isolated from traditional African dairy products were described. The new eight strains described herein contribute to the knowledge of this poorly studied environment and constitute promising strains for fermented food safety.     

Description of durancin TW-49M, a novel enterocin B-homologous bacteriocin in carrot-isolated Enterococcus durans QU 49

Aims:  To characterize the novel bacteriocin produced by Enterococcus durans .
Methods and Results:  Enterococcus durans QU 49 was isolated from carrot and expressed bactericidal activity over 20–43°C. Bacteriocins were purified to homogeneity using the three-step purification method, one of which, termed durancin TW-49M, was an enterocin B-homologous peptide with most identical residues occurring in the N-terminus. Durancin TW-49M was more tolerant in acidic than in alkali. DNA sequencing analysis revealed durancin TW-49M was translated as a prepeptide of the double-glycine type. Durancin TW-49M and enterocin B expressed similar antimicrobial spectra, in which no significant variation due to the diversity in their C-termini was observed.
Conclusions:  Durancin TW-49M, a novel nonpediocin-like class II bacteriocin, was characterized to the amino acid and genetic levels. The diverse C-terminal parts of durancin TW-49M and enterocin B were hardly to be suggested as the place determining the target cell specificity.
Significance and Impact of the Study:  This is the first and comprehensive study of a novel bacteriocin produced by Ent. durans . The high homology at the N-terminal halves between durancin TW-49M and enterocin B makes them suitable to study the structure-function relationship of bacteriocins and their immunity proteins.     

Partial characterization of enterocin MR99 from a corn silage isolate of Enterococcus faecalis

AIMS: To assess the inhibitory activity on Gram-positive and Gram-negative bacteria of several species of enterococci recovered from a natural corn silage. METHODS AND RESULTS: The inhibitory activity of strains of Enterococcus faecalis (58), Enterococcus faecium (35), Enterococcus gallinarum (3) and Enterococcus casseliflavus (4) were studied employing indicator strains from various sources (clinical, food and ATCC). Enterococcus faecalis MR99, the only strain with inhibitory activity, inhibited other enterococci, Listeria spp., Staphylococcus aureus, Clostridium spp., Bacillus spp., Escherichia coli, Shigella sonnei and Shigella flexneri. The bacterium contained only one conjugative pheromone-responsive plasmid. The partially chromatography-purified MR99 enterocin (PPE) had a molecular weight of approx. 5000 Da and a pI of 6.2, was sensitive to proteolytic enzymes and could be extracted in benzene and butanol. It appeared stable to adjustment of pH 4.0, 5.0, 6.0, 7.0 and 8.0 and was resistant to heat. Inactivation was at 15 min at 121 degrees C. Enterocin MR99 was bactericidal on strains of Listeria monocytogenes, Staph. aureus, and bovine mastitis agents, it was bacteriostatic on E. coli. Although enterocins MR99 and AS48 have inhibitory activity on Gram-negative bacilli, PCR studies demonstrated a lack of relationship between them. CONCLUSIONS: The active component had a protein nature, was resistant to heat and presented a wide inhibitory spectrum. SIGNIFICANCE AND IMPACT OF THE STUDY: The biological properties of Ent. faecalis MR99 suggest that this strain merits further investigations so it can be applied in human and veterinary health programmes.     

Antagonistic effect of enterocin CCM 4231 from Enterococcus faecium on “bryndza”, a traditional Slovak dairy product from sheep milk

“Bryndza” is a traditional Slovak dairy product (type of soft cheese) made from sheep cheese which was ripened for 14 days. Because its manufacture, transporting and/or storing represent conditions which facilitate contamination, the effect of enterocin CCM4231 in “bryndza” was investigated with the aim to reduce the contaminant agents. “Bryndza” was divided into equal portions (50 g). The experimental sample (ES) as well as the control sample one (C1) were inoculated with Listeria innocua Li1 strain. The other control samples C2 and C3 were without Li1 strain. C3 control was selected as a reference control. ES and C2 portions were treated with purified enterocin CCM4231 in a concentration of 6400 AU/ml. Before the experimental inoculation, “bryndza” was checked for the presence of contaminant agents. The experiment lasted 1 week and the samples were stored in the refrigerator at 4 °C. Sampling was performed on day 1, on day 4 and on day 7. The control samples C2 and C3 were checked only on day 1 and then after 1 week. The following contaminant agents were detected in “bryndza” before its experimental inoculation with L. innocua Li1 strain: Escherichia coli in the amount 10³ cfu/ml/g, Staphylococcus aureus (10² cfu/ml/g) and enterococci (10⁴ cfu/ml/g). In the control sample C2, the number of E. coli was reduced to 10² cfu/ml/g. Enterococci and staphylococci were totally eliminated there. Concerning C3 control, natural decrease of bacteria was found and/or their unchanged counts. The value of pH (5) was stable during the whole experiment. In the experimental sample inoculated with Li1 strain, its counts were decreased immediately after enterocin CCM4231 addition approximately by one order of magnitude. This inhibitory effect was also detectable on day 4 by the difference of one order of magnitude between ES and C1. On day 7, 10³ cfu/ml/g of Li1 strain were detected in both samples (ES, C1). The difference by one order of magnitude indicated, an inhibitory effect of enterocin CCM4231 in “bryndza”. However, bacteriocin activity was not determined by laboratory analyses.     

Phloroglucinol Derivatives from Myrtus communis ‘Variegata’ and Their Antibacterial Activities

Myrtucomvalones D–F, three new triketone‐phloroglucinol‐triketone adducts, and three known ones (myrtucommulone E, myrtucommulone D and callistenone D) were obtained from Myrtus communis ‘Variegata’. Myrtucomvalone D is a pair of enantiomers which was further resolved into (+)‐myrtucomvalone D and (?)‐myrtucomvalone D by chiral HPLC. Their structures and complete stereochemistry were established from interpretation of NMR and crystallographic data and chemical calculations. Myrtucomvalone F, myrtucommulone D and callistenone D showed significant antibacterial activities.     

Clonal groups of high-level gentamicin-resistant Enterococcus faecium isolated from municipal wastewater and clinical samples in Tehran, Iran

Aims:  Clonality among high-level gentamicin-resistant Enterococcus faecium (HLGR-EF) isolates obtained from clinical and sewage treatment plants (STP) were investigated using PhePlate system (PhP), ribotyping and pulsed-field gel electrophoresis (PFGE).
Methods and Results:  During 1 year study (September 2005–2006), a total of 106 HLGR-EF isolates were collected from clinical ( n  = 48) and STP ( n  = 58) samples in Tehran, Iran. Biochemical fingerprinting of these isolates using the PhP showed the presence of 21 PhP types (diversity index, Di  = 0·97) among the clinical and 21 PhP types ( Di  = 0·91) among the STP isolates. Representative isolates of each PhP type ( n  = 42) were further characterized by the ribotyping method. Sixteen ribotypes were identified among the isolates with five types shared between the clinical and STP isolates. PFGE recognized 24 clonal types among these isolates with three pulsotypes shared between the clinical and STP isolates. Combination of the two techniques (PFGE and ribotyping) resulted in 24 ( Di  = 0·96) and 16 ( Di  = 0·93) types among the strains isolated from clinical and STP samples, respectively.
Conclusions:  We concluded that the combination of PhP typing, ribotyping and PFGE could be extremely discriminatory when examining HLGR-EF isolates.
Significance and Impact of the Study:  The emergence of highly diverse HLGR-EF population in Iran is of serious concern especially because of their multi-resistances.     

Novel approach for optimization of a ‘difficult’ peptide synthesis by utilizing quantitative reaction monitoring assays

Three different synthetic strategies were tested to synthesize EGFRvIII peptide (1), which is a functional variant of the epidermal growth factor receptor, a protein that has been well validated as a target for cancer therapy. The initial synthesis was performed with Applied Biosystem (Carlsbad, CA, USA) 433A peptide synthesizer and it indicated that the last three amino acids coupling and Fmoc removal rates were lower than the rest of the sequence. Purity of the crude peptide was 54.5%. The second synthesis was performed manually utilizing C S Bio (Menlo Park, CA, USA) synthesizer and the Kaiser test for reaction monitoring. Because of non‐optimized reaction conditions and an unexpected by‐product, lower purity crude peptide (40.5%) was obtained. Quantitative assays to monitor reactions were developed and demonstrated in gram scale synthesis with C S Bio synthesizer. The optimized synthetic conditions improved the peptide purity to 68.1%. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Aqueous Extract of the Pericarp of Sapindus trifoliatus Fruits: A Novel ‘Green’ Catalyst for the Aldimine Synthesis

The catalytic efficiency in organic synthesis of the aqueous extract of the pericarp of Sapindus trifoliatus fruits was evaluated. The synthesis of a series of aldimines from aromatic aldehydes and amines was successfully catalyzed by the extract, whereas aromatic ketones and amines did not yield ketimines under comparable reaction conditions, indicating the chemoselective catalysis of the extract. The catalytic activity of the extract is due to saponins, which have a common structural skeleton containing a pentacyclic triterpenoid part substituted with different carbohydrate side chains. The mild conditions, high yields, and short reaction times not only make this protocol a valuable alternative to the conventional methods, but it also becomes significant under the roof of environmentally greener and safer processes.     

Molecular Characterization of a Phytoplasma Associated with Potato Witches’‐broom Disease in Iran

Potato plants showing symptoms suggestive of potato witches’‐broom disease including witches’‐broom, little leaf, stunting, yellowing and swollen shoots formation in tubers were observed in the central Iran. For phytoplasma detection, Polymerase Chain Reaction (PCR) and nested PCR assays were performed using phytoplasma universal primer pair P1/P7, followed by primer pair R16F2n/R16R2. Random fragment length polymorphism analysis of potato phytoplasma isolates collected from different production areas using the CfoI restriction enzyme indicated that potato witches’‐broom phytoplasma isolate (PoWB) is genetically different from phytoplasmas associated with potato purple top disease in Iran. Sequence analysis of the partial 16S rRNA gene amplified by nested PCR indicated that ‘Candidatus Phytoplasma trifolii’ is associated with potato witches’‐broom disease in Iran. This is the first report of potato witches’‐broom disease in Iran.     

Facile synthesis of AgNPs@SNCDs nanocomposites as a fluorescent ‘turn on’ sensor for detection of glutathione

The present study illustrates the facile synthesis of silver nanoparticles capped with sulfur and nitrogen co‐doped carbon dots (AgNPs@SNCDs) nanocomposites and their application towards the sensitive and selective detection of glutathione (GSH) using a spectrofluorimetry method. SNCDs were synthesized using solvothermal treatment of cysteamine hydrochloride and p‐phenylenediamine. The as‐fabricated SNCDs were then utilized as capping and stabilizing agents for the preparation of AgNPs@SNCDs nanocomposites using wet chemistry. The size of AgNPs@SNCDs nanocomposites was characterized to be ~37.58 nm or even larger aggregates. Particularly, the quenched fluorescence of AgNPs@SNCDs nanocomposites could be significantly restored upon addition of GSH, and the colour of its solution changed to some extent. The fluorescence intensity ratio of AgNPs@SNCDs nanocomposites at ~450 nm and 550 nm was directly proportional to the GSH concentration within the ranges 8.35–66.83 μM and 66.83–200.5 μM, and the detection limit was 0.52 μM. Furthermore various common organic molecules had no obvious interference in the detection mode. The proposed nanosensor was successfully applied for GSH assay in actual water samples.