Inhibition of Staphylococcus aureus by the commensal bacteria of human milk

AIMS: To study the bacterial diversity in expressed human milk with a focus on detecting bacteria with an antimicrobial activity against Staphylococcus aureus, known as a causative agent of maternal breast infections and neonatal infections. METHODS AND RESULTS: Random isolates (n = 509) were collected from breast milk samples (n = 40) of healthy lactating women, genotypically identified, and tested for antimicrobial activity against Staph. aureus. Commensal staphylococci (64%) and oral streptococci (30%), with Staph. epidermidis, Strep. salivarius, and Strep. mitis as the most frequent isolates, were the predominant bacterial species in breast milk. One-fifth of Staph. epidermidis and half of Strep. salivarius isolates suppressed growth of Staph. aureus. Enterococci (Ent. faecalis), isolated from 7.5% of samples, and lactic acid bacteria (LAB) (Lactobacillus rhamnosus, Lact. crispatus, Lactococcus lactis, Leuconoctoc mesenteroides), isolated from 12.5% of samples, were also effective against Staph. aureus. One L. lactis isolate was shown to produce nisin, a bacteriocin used in food industry to prevent bacterial pathogens and spoilage. CONCLUSIONS: Expressed breast milk contains commensal bacteria, which inhibit Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains inhibitory against the pathogen Staph. aureus have potential use as bacteriotherapeutic agents in preventing neonatal and maternal breast infections caused by this bacterium.     

A rapid procedure for isolating chromosomal DNA from Lactobacillus species and other Gram-positive bacteria

This study describes a rapid procedure for the isolation of genomic DNA from various Gram-positive bacteria. Species tested included Lactobacillus delbrueckii subsp. lactis ATCC 4797, Lact. acidophilus N2, Staphylococcus aureus, Staph. epidermidis, Propionibacterium jensenii P126, Bacillus pumilus and Enterococcus faecalis. Our technique for chromosomal DNA isolation circumvents the need for phenol-chloroform extractions and caesium chloride gradients. Isolated DNA is consistently greater than 25 kb in size and can be used directly for subcloning, polymerase chain reaction amplification, restriction digestions and library construction.     

Mode of antimicrobial action of vanillin against Escherichia coli, Lactobacillus plantarum and Listeria innocua

AIMS: To investigate the mode of action of vanillin, the principle flavour component of vanilla, with regard to its antimicrobial activity against Escherichia coli, Lactobacillus plantarum and Listeria innocua. METHODS AND RESULTS: In laboratory media, MICs of 15, 75 and 35 mmol l(-1) vanillin were established for E. coli, Lact. plantarum and L. innocua, respectively. The observed inhibition was found to be bacteriostatic. Exposure to 10-40 mmol l(-1) vanillin inhibited respiration of E. coli and L. innocua. Addition of 50-70 mmol l(-1) vanillin to bacterial cell suspensions of the three organisms led to an increase in the uptake of the nucleic acid stain propidium iodide; however a significant proportion of cells still remained unstained indicating their cytoplasmic membranes were largely intact. Exposure to 50 mmol l(-1) vanillin completely dissipated potassium ion gradients in cultures of Lact. plantarum within 40 min, while partial potassium gradients remained in cultures of E. coli and L. innocua. Furthermore, the addition of 100 mmol l(-1) vanillin to cultures of Lact. plantarum resulted in the loss of pH homeostasis. However, intracellular ATP pools were largely unaffected in E. coli and L. innocua cultures upon exposure to 50 mmol l(-1) vanillin, while ATP production was stimulated in Lact. plantarum cultures. In contrast to the more potent activity of carvacrol, a well studied phenolic flavour compound, the extent of membrane damage caused by vanillin is less severe. CONCLUSIONS: Vanillin is primarily a membrane-active compound, resulting in the dissipation of ion gradients and the inhibition of respiration, the extent to which is species-specific. These effects initially do not halt the production of ATP. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mode of action of natural antimicrobials may facilitate their application as natural food preservatives, particularly for their potential use in preservation systems employing multiple hurdles.     

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.     

Isolation, taxonomic identification and hydrogen peroxide production by Lactobacillus delbrueckii subsp. lactis T31, isolated from Mongolian yoghurt: inhibitory activity on food-borne pathogens

AIMS: The aim of this work was to isolate lactic acid bacteria (LAB) strains from Mongolian tarag (a traditionally homemade yoghurt) displaying antimicrobial activities against food-borne pathogens, identify inhibitory substances and study the kinetics of their production. METHODS AND RESULTS: Inhibitory substance-producing bacterial strains were isolated from tarag. From 300 bacterial clones, 31 were able to inhibit the growth of the indicator strain Lactobacillus bulgaricus 340. One of the most active strains was identified as Lactobacillus delbrueckii subsp. lactis strain T31 by using cluster analysis of amplified fragment length polymorphism (AFLP) DNA fingerprints. The antimicrobial substance was inactivated by catalase, demonstrating the production of hydrogen peroxide (H(2)O(2)). Production of H(2)O(2) was studied under aerated and nonaerated culture conditions. The amount of H(2)O(2) in the culture supernatant increased during bacterial growth and reached a maximum (5.12 mmol l(-1)) at the early stationary phase under aerated conditions (agitated cultures). H(2)O(2) was not detected in the culture performed without agitation. In mixed cultures performed in milk with either Lact. delbrueckii subsp. lactis T31 in the presence of Escherichia coli, or Lact. delbrueckii subsp. lactis T31 in the presence of Listeria innocua under aerated and nonaerated conditions, a significant decrease in pathogen count was observed in aerated cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The significant decrease in Listeria viability observed in aerated mixed cultures of Lact. delbrueckii subsp. lactis T31 is mainly because of H(2)O(2) production. Lactobacillus delbrueckii subsp. lactis T31 could be used as a protective culture in food industries or as a probiotic to prevent intestinal and urogenital infections.     

Isolation from food sources, of lactic acid bacteria that produced antimicrobials

The potential of lactic acid bacteria, isolated from a variety of foods, to inhibit indicators representative of spoilage and pathogenic bacteria associated with food products was examined. Fruit and vegetables were a poor source of lactic acid bacteria but large numbers were readily isolated on MRS agar from cheese, milk and meat samples. Approximately 1000 isolates from each of the food samples were examined by the deferred antagonism procedure to determine their ability to inhibit Staphylococcus aureus, Listeria innocua and Pseudomonas fragi. Listeria innocua was the bacterium predominantly inhibited by isolates from the cheese, milk and meats, but antagonism was also observed to a lesser extent against the other indicators. The only inhibition observed for isolates from vegetable material was directed against Staph. aureus. The majority of inhibitor producers were effective against only one of the indicators but a small number were isolated which inhibited two or three.     

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.     

Lactobacillus ruminis is a predominant lactic acid producing bacterium in the caecum and rectum of the pig

AIMS: To identify the predominant lactic acid producing bacteria in the small intestine, caecum and the rectum of the healthy pig. METHODS AND RESULTS: Samples obtained from the large intestine of healthy pigs post-mortem were cultured using a modified agar-MRS medium in roll tubes. Thirteen isolates were selected on the basis of their morphological characteristics and Gram stain reaction for gene sequencing. These isolates were characterized by DNA sequence analysis of 16S rDNA. Eight isolates were identified as Lactobacillus ruminis, two as Enterococcus faecium, one as Mitsuokella multiacidus and two as Escherichia coli. CONCLUSION: This is the first report of Lact. ruminis as the dominant lactic acid bacteria in the large intestine of the pig. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that Lact. ruminis is a dominant bacterium in the large intestine of the healthy pig. Future work should focus on the role of this bacterium in relation to the physiological function of the intestine and the health of the animal.     

Antimicrobial activity of lactic acid bacteria isolated from Tenerife cheese: initial characterization of plantaricin TF711, a bacteriocin-like substance produced by Lactobacillus plantarum TF711

AIMS: The screening and initial characterization of bacteriocins produced by lactic acid bacteria (LAB) from raw Tenerife goats' cheese with possible application as biopreservatives or ripening accelerators for Tenerife cheese. METHODS AND RESULTS: One hundred and eighty LAB of the genera Lactobacillus (95), Leuconostoc (64) and Lactococcus (21) isolated from raw Tenerife goats' cheese were screened for the production of antimicrobial substances. Lactobacillus plantarum TF711, which had the broadest spectrum of antimicrobial activity, was selected for further characterization. The antimicrobial compound was determined as a proteinaceous substance, as it was sensitive to proteases. The bacteriocin-like substance, which we called plantaricin TF711, was active against the Gram-positive bacteria Bacillus cereus, Clostridium sporogenes and Staphylococcus aureus; and against the Enterobacteriaceae Shigella sonnei and Klebsiella pneumoniae. It was stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 1 and 9. Plantaricin TF711 exhibited primary metabolite kinetics, a bacteriostatic mode of action and a molecular mass of c. 2.5 kDa as determined by tricine SDS-PAGE. CONCLUSIONS: Lact. plantarum TF711 produces a low molecular mass bacteriocin-like compound with a wide spectrum of activity and interesting technological properties (thermostability, good pH stability and stability against surfactants and organic solvents). SIGNIFICANCE AND IMPACT OF THE STUDY: Plantaricin TF711 was found to have potential for use as a biopreservative in the food industry.     

Isolation and characterization of bacteriocin‐producing bacteria from the intestinal microbiota of elderly Irish subjects

Aims

To isolate and characterize bacteriocins produced by predominant species of lactic acid bacteria from faeces of elderly subjects.

Methods and Results

Screening over 70 000 colonies, from faecal samples collected from 266 subjects, using the indicator organisms Lactobacillus bulgaricus LMG 6901 and Listeria innocua DPC 3572, identified 55 antimicrobial‐producing bacteria. Genomic fingerprinting following ApaI digestion revealed 15 distinct strains. The antimicrobial activities associated with 13 of the 15 strains were sensitive to protease treatment. The predominant antimicrobial‐producing species were identified as Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus and Enterococcus spp. A number of previously characterized bacteriocins, including ABP‐118 and salivaricin B (from Lact. salivarius), enterocin B (Enterococcus faecium), lactacin B (Lact. acidophilus), gassericin T and a variant of gassericin A (Lact. gasseri), were identified. Interestingly, two antimicrobial‐producing species, not generally associated with intestinally derived microorganisms were also isolated: Lactococcus lactis producing nisin Z and Streptococcus mutans producing mutacin II.

Conclusion

These data suggest that bacteriocin production by intestinal isolates against our chosen targets under the screening conditions used was not frequent (0·08%).

Significance and Impact of the Study

The results presented are important due to growing evidence indicating bacteriocin production as a potential probiotic trait by virtue of strain dominance and/or pathogen inhibition in the mammalian intestine.     

Bacteriocin production by spray-dried lactic acid bacteria

AIMS: Cell survival and antagonistic activity against Listeria innocua, Listeria monocytogenes and Staphylococcus aureus were investigated after spray-drying three bacteriocin-producing strains of lactic acid bacteria: Carnobacterium divergens, Lactobacillus salivarius and Lactobacillus sakei. METHODS AND RESULTS: Bacterial cell concentrates were spray-dried and stored at 4 degrees C and 18 degrees C and 0.3% ERH (equilibrium relative humidity). Enumeration and antagonistic activity were evaluated before and after spray-drying and at regular intervals during storage. CONCLUSIONS: A higher survival rate was obtained when survival was performed at 4 degrees C. With the exception of Carnobacterium divergens which lost the inhibitory activity against Staph. aureus after drying, antagonistic production was not affected by the process nor by the storage. Of the three species studied, Lact. salivarius showed the highest resistance to the spray-drying and storage processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Spray-drying is a potentially useful process for large scale production of dried powders containing viable organisms with antagonistic activity against pathogens.     

Inhibitory activity of 2-nitropropanol against select food-borne pathogens in vitro

AIMS: To test the inhibitory activity of 2-nitro-1-propanol (2NPOH) against Salmonella Typhimurium, Escherichia coli O157:H7 and Enterococcus faecalis. METHODS AND RESULTS: Specific growth rates (h(-1)) of S. Typhimurium, E. coli O157:H7 and Ent. faecalis were determined during culture in tryptic soya broth (TSB) supplemented with 0-10 mm 2NPOH. Growth rates were inhibited by 2NPOH, with nearly complete inhibition observed with 10 mm. Studies with S. Typhimurium revealed that its survivability during culture in TSB containing 5 or 10 mm 2NPOH was lower (P < 0.05) under aerobic than anaerobic conditions. The survivability of Salmonella during anaerobic culture in TSB containing 2.5 mm 2NPOH was less at pH 5.6 than at pH 7.0 and 8.0. No Salmonella survived anaerobic incubation in TSB supplemented with 10 mm 2NPOH regardless of pH. When incubated in suspensions of freshly collected populations of ruminal and faecal bacteria, Salmonella concentrations were lower (P < 0.05) in suspensions containing 10 mm 2NPOH than in suspensions containing no 2NPOH. CONCLUSIONS: 2NPOH inhibited S. Typhimurium, E. coli O157:H7 and Ent. faecalis. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that 2NPOH may be a useful antimicrobial supplement to reduce carriage of certain food-borne pathogens in food animals.     

Separation of bacteria using agglutinins isolated from invertebrates

The agglutination of a selection of Gram-positive and Gram-negative bacteria by the haemolymph and coelomic fluid from several invertebrates was studied. The haemolymph from Lumbricus terrestris and Limulus polyphemus caused the strongest agglutination of most of the bacteria studied. When the agglutinating fraction of Lim. Polyphemus was liganded to magnetic microspheres 53% of the cells in pure cultures of Listeria monocytogenes C200, 15% of Salmonella enteritidis 37782, 92% of Staphylococcus aureus NCDO 949, 19% of Escherichia coli E4936/76 and 65% of E. coli W2–2 were adsorbed to the beads. The immobilized haemolymph from Lumb. terrestris adsorbed 42% of Salm. enteritidis 37782, 64% of E. coli 4936/76 and 27% of Staph. aureus NCDO 1499 cells and the coelomic fluid from Haemopsis sanguisuga adsorbed 42, 48 and 50% of these cultures respectively. With immobilized Haem. sanguisuga agglutinins, 21–27% of Staph. aureus NCDO 2044 cells were recovered from full-fat pasteurized milk and 20–51% from braising steak. Immobilized Lim. polyphemus agglutinins recovered 17–34% of Staph. aureus cells from raw egg. The potential of agglutinins isolated from invertebrates for enhancing rapid microbiological assays of foods is discussed.     

The antimicrobial activity of the appetite peptide hormone ghrelin

The present study examined the antimicrobial activity of the peptide ghrelin. Both major forms of ghrelin, acylated ghrelin (AG) and desacylated ghrelin (DAG), demonstrated the same degree of bactericidal activity against Gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), while bactericidal effects against Gram-positive Staphylococcus aureus (S. aureus) and Enterococcus faecalis (E. faecalis) were minimal or absent, respectively. To elucidate the bactericidal mechanism of AG and DAG against bacteria, we monitored the effect of the cationic peptides on the zeta potential of E. coli. Our results show that AG and DAG similarly quenched the negative surface charge of E. coli, suggesting that ghrelin-mediated bactericidal effects are influenced by charge-dependent binding and not by acyl modification. Like most cationic antimicrobial peptides (CAMPs), we also found that the antibacterial activity of AG was attenuated in physiological NaCl concentration (150mM). Nonetheless, these findings indicate that both AG and DAG can act as CAMPs against Gram-negative bacteria.     

Production of Antimicrobial Substances by Bacteria Isolated from Fermented Table Olives

Summary The production of antimicrobial substances was studied among 195 bacterial isolates from retail table olives. A total 86 isolates tested positive, and they clustered in 10 groups according to their inhibitory spectra. Many isolates (38.37%) produced strong inhibition against all bacteria tested (Listeria innocua, Lactococcus lactis, Bacillus cereus, B. megaterium, Staphylococcus aureus, Micrococcus luteus, Enterococcus faecalis, and Escherichia coli). The selected bacterial isolates were Gram-positive bacteria with rod morphology (62.67%), short rods (26.65%) or cocci (10.67%). Isolates producing antimicrobial substances may be useful as starters to enhance control of table olive fermentation and improve the safety of retail table olives.     

Monitoring of antibiotic resistance in bacteria isolated from laboratory animals

The drug resistance of microorganisms isolated from laboratory animals never treated with antibiotics is being reported consistently, while the number of laboratory animals used in medicine, pharmacy, veterinary medicine, agriculture, nutrition, and environmental and health science has increased rapidly in Korea. Therefore, this study examined the development of antimicrobial resistance in bacteria isolated from laboratory animals bred in Korea. A total of 443 isolates (7 species) containing 5 Sphingomonas paucimobilis, 206 Escherichia coli, 60 Staphylococcus aureus, 15 Staphylococcus epidermidis, 77 Enterococcus faecalis, 27 Citrobacter freundii, 35 Acinetobacter baumannii were collected from the nose, intestine, bronchus and reproductive organs of ICR mice and SD rats. Of these species, Acinetobacter baumannii and Enterococcus faecalis showed significant antimicrobial resistance according to the minimum inhibition concentration (MIC) in E-test. In case of Acinetobacter baumannii, several isolates showed MIC values 16-128 μg/mL for cefazolin and cefoxitin, and higher resistance (128-512 μg/mL) to nitrofurantoin than that of standard type. Resistance to cefazolin, cefoxitin and nitrofurantoin was detected in 17.14, 20.00, and 8.57% of the Acinetobacter baumannii isolates, respectively. In addition, 44.1% of the Enterococcus faecalis isolates collected from the laboratory animals were resistant to oxacillin concentration of 16-32 μg/mL range, while MIC value of standard type was below oxacillin concentration of 6 μg/mL. These results suggest that in rodent species of laboratory animals, Acinetobacter baumannii are resistance to cefazolin, cefoxitin and nitrofurantoin, whereas those of Enterococcus faecalis were resistance to oxacillin.     

Inhibition of Listeria innocua and Brochothrix thermosphacta in vacuum-packaged meat by addition of bacteriocinogenic Lactobacillus curvatus CRL705 and its bacteriocins

AIMS: To evaluate the inhibition effectiveness of Lactobacillus curvatus CRL705 used as a bioprotective culture and of its bacteriocins, lactocin 705 and lactocin AL705, against Listeria innocua, Brochothrix thermosphacta and indigenous lactic acid bacteria (LAB) in vacuum-packaged meat stored at 2 degrees C. METHODS AND RESULTS: The live culture of Lact. curvatus CRL705 as well as synthetic lactocin 705 and purified lactocin AL705 were shown to be similarly effective in preventing the growth of B. thermosphacta and L. innocua in meat discs in contrast to control samples in which these micro-organisms grew rapidly, their numbers increasing by 3.0- and 2.1-log cycles respectively. In addition, indigenous LAB population showed a lower growth rate in the presence of lactocin 705. Bacteriocin activity was detected in the meat discs during 36 days at 2 degrees C irrespective of the biopreservation strategy applied. Changes in pH were not significantly different in meat discs treated with the protective culture when compared with control samples. CONCLUSIONS: Lactobacillus curvatus CRL705 and the produced bacteriocins, lactocin 705 and lactocin AL 705, were effective in inhibiting L. innocua and B. thermosphacta. The use of the bioprotective culture in refrigerated vacuum-packaged fresh meat would be more feasible from an economic and legal point of view. SIGNIFICANCE AND IMPACT OF THE STUDY: Establishment of biopreservation as a method to ensure the microbiological safety of vacuum-packaged fresh meat at 2 degrees C.     

Composition and antimicrobial activity of the essential oil of Dicyclophora persica Boiss. from Iran

The chemical composition of the essential oil of Dicyclophora persica Boiss. was identified by GC and GC-MS analysis. The analysis of the oil resulted in the identification of forty-five components constituting 98.6% of the total oil. The main constituents were a-pinene (31.5%), (Z)-beta-ocimene (23.3%), p-cymene (6.7%) and (E)-beta-ocimene (5.4%). The antimicrobial activity of the oil was tested by the disk diffusion method against four Gram-positive (Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis) and three Gram-negative (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) bacteria together with a fungus (Aspergillus niger). The oil showed strong inhibition activity toward all the tested microorganisms except for Pseudomonas aeruginosa.     

Antimicrobial activity of essential oils and other plant extracts

The antimicrobial activity of plant oils and extracts has been recognized for many years. However, few investigations have compared large numbers of oils and extracts using methods that are directly comparable. In the present study, 52 plant oils and extracts were investigated for activity against Acinetobacter baumanii, Aeromonas veronii biogroup sobria, Candida albicans, Enterococcus faecalis, Escherichia col, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype typhimurium, Serratia marcescens and Staphylococcus aureus, using an agar dilution method. Lemongrass, oregano and bay inhibited all organisms at concentrations of < or = 2.0% (v/v). Six oils did not inhibit any organisms at the highest concentration, which was 2.0% (v/v) oil for apricot kernel, evening primrose, macadamia, pumpkin, sage and sweet almond. Variable activity was recorded for the remaining oils. Twenty of the plant oils and extracts were investigated, using a broth microdilution method, for activity against C. albicans, Staph. aureus and E. coli. The lowest minimum inhibitory concentrations were 0.03% (v/v) thyme oil against C. albicans and E. coli and 0.008% (v/v) vetiver oil against Staph. aureus. These results support the notion that plant essential oils and extracts may have a role as pharmaceuticals and preservatives.     

Characterization of reutericyclin produced by Lactobacillus reuteri LTH2584

Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains of Escherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C(8) chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. H?ltzel, M. G. G?nzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766-2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of L. reuteri LTH2584 corresponded to that of purified as well as synthetic reutericyclin.