Antibacterial activity of Lactobacillus sake isolated from meat.

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.     

Antibacterial activity of Lactobacillus sake isolated from meat

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.     

Influence of complex nutrient source on growth of and curvacin a production by sausage isolate Lactobacillus curvatus LTH 1174

Lactobacillus curvatus LTH 1174, a fermented sausage isolate, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of a complex nutrient source were investigated in vitro during laboratory fermentations with modified MRS medium. A modification of the nutrient depletion model was used to fit the data describing growth and bacteriocin production. Both cell growth and bacteriocin activity were influenced by changes in the complex nutrient source concentration. Standard MRS medium clearly limited the growth of L. curvatus LTH 1174. Higher nutrient concentrations, up to a certain degree, led to improved growth, a higher attainable biomass concentration, and a higher bacteriocin activity in the supernatant. A lower concentration of complex nutrient source caused severe growth inhibition, leading to a lower biomass concentration but a much higher specific bacteriocin production. When examining the separate components of the complex nutrient source, a stimulating effect of bacteriological peptone on growth was found without an adverse effect on bacteriocin production, resulting in increased curvacin A activity. Furthermore, specific depletion of the amino acids tyrosine, serine, and asparagine/aspartic acid was observed for this strain.     

Genome sequence of the bacteriocin-producing Lactobacillus curvatus strain CRL705

Lactobacillus curvatus is one of the most prevalent lactic acid bacteria found in fermented meat products. Here, we present the draft genome sequence of Lactobacillus curvatus CRL705, a bacteriocin producer strain isolated from an Argentinean artisanal fermented sausage, which consists of 1,833,251 bp (GC content, 41.9%) and two circular plasmids of 12,342 bp (pRC12; GC, 43.9%) and 18,664 bp (pRC18; GC, 34.4%).     

Biochemical and genetic characterization of mundticin KS,an antilisterial peptide produced by Enterococcus mundtii NFRI 7393

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.     

Characterization of plantaricin KW30, a bacteriocin produced by Lactobacillus plantarum

A protease-sensitive antibacterial substance, produced by a strain of Lactobacillus plantarum isolated from fermented corn, was classified as a bacteriocin and designated plantaricin KW30. The bacteriocin was stable to heat, pH and treatment with surfactants, and unaffected by α-amylase, lipase or lysozyme. Plantaricin KW30 exhibited a bactericidal and non-bacteriolytic mode of action against indicator cells, and inhibitory activity was limited to other lactobacilli. Maximum production was in MRS broth, and coincided with the onset of stationary phase under conditions of low pH and high cell numbers. In a complex medium bacteriocin production was enhanced by the presence of sodium acetate and Tween 80. Curing experiments gave derivatives that no longer produced the bacteriocin but retained immunity to it. These Bac derivatives showed the same plasmid pattern as the parent strain suggesting a chromosomal location for the genes for bacteriocin production.     

The curing agent sodium nitrite,used in the production of fermented sausages,is less inhibiting to the bacteriocin-producing meat starter culture Lactobacillus curvatus LTH 1174 under anaerobic conditions

Curvacin A is a listericidal bacteriocin produced by Lactobacillus curvatus LTH 1174, a strain isolated from fermented sausage. The response of this strain to an added curing agent (sodium nitrite) in terms of cell growth and bacteriocin production was investigated in vitro by laboratory fermentations with modified MRS broth. The strain was highly sensitive to nitrite; even a concentration of 10 ppm of curing agent inhibited its growth and both volumetric and specific bacteriocin production. A meat simulation medium containing 5 ppm of sodium nitrite was tested to investigate the influence of the gas phase on the growth and bacteriocin production of L. curvatus LTH 1174. Aerating the culture during growth had no effect on biomass formation, but the oxidative stress caused a higher level of specific bacteriocin production and led to a metabolic shift toward acetic acid production. Anaerobic conditions, on the other hand, led to an increased biomass concentration and less growth inhibition. Also, higher maximum volumetric bacteriocin activities and a higher level of specific bacteriocin production were obtained in the presence of sodium nitrite than in fermentations under aerobic conditions or standard conditions of air supply. These results indicate that the inhibitory effect of the curing agent is at least partially masked under anaerobic conditions.     

Antagonistic activity against pathogenic bacteria by human vaginal lactobacilli

This study attempted to isolate lactobacilli strains from healthy vaginal ecosystem to search for a new effective antibacterial probiotic strain. The strains were identified and characterized for their probiotic properties including bile salt and acid tolerance, growth at acidic pH, their ability to utilize protein, starch, and lipid, the production of hydrogen peroxide and bacteriocin as well as their antibiotic resistance patterns. The antibacterial activity of the culture supernatant of these strains were tested against a wide range of Gram-positive and Gram-negative pathogenic bacteria including Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae. Salmonella typhi, and Salmonella typhimurium. None of the strains inhibited the growth of Gram-negative bacteria. Contrastly, the culture supernatant of strain L 22, identified as Lactobacillus reuteri, significantly inhibited all of the clinical isolates of methicillin-resistant S. aureus (MRSA). The antibacterial effect of the selected strain L 22 was further investigated. In the presence of L 22, the bacterial growth was assessed in vitro by viable bacterial counting. The numbers of viable cells were significantly lower in L 22-containing broth than those in the control by 6h. This finding clearly demonstrates that strain L 22 can produce an anti-MRSA effect. The antibacterial ability of the strain L 22 was fundamentally attributed to their bacteriocin production which can cause both cell inhibition and cell death.     

Influence of nutrients on growth and bacteriocin production by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442

The aim of this study was to investigate the effect of complex nutrients on microbial growth and bacteriocin production, in order to improve bacteriocin synthesis during the growth cycle of Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442. The fermentations were conducted at the optimum pH and temperature for bacteriocin production (pH 5.5+/-0.1 and temperature 25+/-0.1 degrees C). Because of their association with the final biomass, conditions favouring the increase of the produced biomass resulted in the increase of bacteriocin activity in the growth medium. Since the produced final biomass and the final concentration of the bacteriocins were associated with the amount of the carbon (glucose) and nitrogen source, better growth of the lactic acid bacterial strains favoured the increase of the specific bacteriocin production. Additionally, the bacteriocin production was influenced by carbon/nitrogen ratio.     

C55 bacteriocin produced by ETB‐plasmid positive Staphylococcus aureus strains is a key factor for competition with S. aureus strains

Exfoliative toxin (ET) produced by Staphylococcus aureus is closely associated with the onset of bullous impetigo. To date, three ETs (ETA, ETB and ETD) have been identified. The gene encoding ETB is located in a plasmid designated pETB. Bacteriocin synthesis genes are also located in this plasmid and pETB‐positive strains reportedly produce the C55 bacteriocin. In this study, the antibacterial activity against S. aureus strains of the bacteriocin produced by the pETB‐positive strain TY4 was investigated. This bacteriocin demonstrated antibacterial activity against all pETB‐negative but not pETB‐positive strains, including TY4. Additionally, a TY4? strain from which the pETB plasmid had been deleted exhibited susceptibility to the bacteriocin. Further experiments revealed that two immunity factors (orf 46‐47 and orf 48) downstream of the bacteriocin synthesis genes in the pETB plasmid are associated with immunity against the bacteriocin produced by TY4. The TY4? with orf46‐47 strain exhibited complete resistance to bacteriocin, whereas the TY4? with orf48 strain exhibited partial resistance. Whether bacteriocin affects the proportion of each strain when co‐cultured with S. aureus strains was also investigated. When TY4 or TY4? was co‐cultured with 209P strain, which is susceptible to the bacteriocin, the proportion of 209P co‐cultured with TY4 was significantly less than when 209P was co‐cultured with TY4?, whereas the proportion of TY4? with orf46‐48 co‐cultured with TY4 was greater than with TY4?. These results suggest that the C55 bacteriocin produced by pETB‐positive strains affects the proportion of each strain when pETB‐positive and ‐negative strains co‐exist.

     

Sodium chloride reduces production of curvacin A, a bacteriocin produced by Lactobacillus curvatus strain LTH 1174, originating from fermented sausage

Lactobacillus curvatus LTH 1174, a strain originating in fermented sausage, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of salt (sodium chloride) were investigated in vitro during laboratory fermentations using modified MRS medium. A model was set up to describe the effects of different NaCl concentrations on microbial behavior. Both cell growth and bacteriocin activity were affected by changes in the salt concentration. Sodium chloride clearly slowed down the growth of L. curvatus LTH 1174, but more importantly, it had a detrimental effect on specific curvacin A production (k(B)) and hence on overall bacteriocin activity. Even a low salt concentration (2%, wt/vol) decreased bacteriocin production, while growth was unaffected at this concentration. The inhibitory effect of NaCl was mainly due to its role as an a(w)-lowering agent. Further, it was clear that salt interfered with bacteriocin induction. Additionally, when 6% (wt/vol) sodium chloride was added, the minimum biomass concentration necessary to start the production of curvacin A (X(B)) was 0.90 g (cell dry mass) per liter. Addition of the cell-free culture supernatant or a protein solution as a source of induction factor resulted in a decrease in X(B), an increase in k(B), and hence an increase in the maximum attainable bacteriocin activity.     

Antimicrobial activity of lactic acid bacteria isolated from sour doughs: purification and characterization of bavaricin A, a bacteriocin produced by Lactobacillus bavaricus MI401

Three hundred and thirty-five lactic acid bacteria were isolated from sour doughs and screened for antagonistic activity. Of these 145 showed activity against one or several of the indicator strains used in the screening. The antimicrobial activity of 18 isolates were due to a proteinaceous compound. These 18 isolates belonged to three different Lactobacillus species: Lactobacillus bavaricus, Lactobacillus curvatus and Lactobacillus plantarum. The spectrum of antimicrobial activity for the three species suggested that the inhibitory components were different. The inhibitory compound from Lact. bavaricus MI401 was chosen for further study. The proteinaceous nature, antimicrobial activity against closely-related species, heat resistance and sensitivity to alkaline treatment strongly indicated that this substance was a bacteriocin, which we designated bavaricin A. The bacteriocin was purified to homogeneity by ammonium sulphate precipitation, ion exchange, hydrophobic interaction and reverse-phase chromatography. The purification resulted in 193000-fold increase in specific activity. SDS-PAGE of bavaricin A showed a molecular weight of 3500—4000 Da. By amino acid sequencing 41 amino acids were determined. Bavaricin A had a bactericidal mode of action and inhibited nine out of 10 Listeria monocytogenes. Lactobacillus bavaricus MI401 produced bavaricin A at temperatures from 4°C to 30°C. The production of active bavaracin A was inhibited at increasing sodium chloride concentration. In the presence of 3% sodium chloride at 4°C no active bavaricin A could be detected. Nitrite (100 ppm) did not affect the production of active bavaricin A.     

Detection of bacteriocins of lactic acid bacteria isolated from foods and comparison with pediocin and nisin

A total of 663 533 colonies from 72 dairy and meat sources showed a detection rate of 0·2% for bacteriocin producers using direct plating techniques. A further 83 000 colonies from 40 fish and vegetable sources showed a detection rate of 3·4% for bacteriocin producers using selective enrichment procedures. A collection of seven purified isolates showing a different host spectrum of bacteriocin activity and with the ability to produce bacteriocins in broth culture were compared with nisin and pediocin (with respect to their inhibitory activity, determined by the critical dilution method), against various indicator bacteria in agar and broth. The sensitivity of Listeria species to various bacteriocins was influenced by the agar and broth test systems used. A Lactobacillus curvatus strain was found to be the most suitable indicator for quantitating antimicrobial effects of all the bacteriocins investigated in both agar and broth test systems. The bacteriocin-producing isolates were characterized by biochemical reactions and DNA restriction enzyme profiles and taxonomic identification revealed species of Lactobacillus , Carnobacterium and Lactococcus assigned on the basis of 16S rDNA sequences.     

Study of antimicrobial activity amongLactobacillus helveticus strains using three different assays

The antimicrobial activity of 18Lactobacillus helveticus strains as well as one control strain, the bacteriocin producingLactobacillus helveticus 481, was tested with three different inhibition assays. FourLactobacillus helveticus strains had antimicrobial activity against seven otherLactobacillus helveticus strains and twoLactobacillus delbrueckil strains while the remaining sevenLactobacillus helveticus strains were indifferent. Inhibition was also observed againstLactococcus andLeuconostoc species, due to production of organic acids or hydrogen peroxide. The strains with the highest antimicrobial activity produced a heat sensitive proteinacious bacteriocin with a narrow species activity spectrum against only thermophilicLactobacillus strains.     

Characterization of Lactobacillus sakei by the type of stereoisomers of lactic acid produced

Lactobacillus sakei strains were characterized by the shift of the type of stereoisomers of lactic acid produced in the presence of 50 mM sodium acetate in a medium. Of 27 Lactobacillus sakei strains studied, 20 strains showed high levels of DNA-DNA similarity with L. sakei NRIC 1071(T), and were confirmed as L. sakei. The three remaining strains were identified as Lactobacillus curvatus by DNA-DNA similarity, and three other strains were included in the cluster of Lactobacillus plantarum/Lactobacillus pentosus/Lactobacillus paraplantarum and one strain in the cluster of Lactobacillus paracasei on the basis of 16S rRNA gene sequences. Of the 20 L. sakei strains, 19 strains shifted the type of stereoisomers of lactic acid produced from the DL-type to the L-type in the presence of 50 mM sodium acetate. L. curvatus strains and strains included in the cluster of L. plantarum/L. pentosus/L. paraplantarum and in the cluster of L. paracasei did not shift the type of stereoisomers of lactic acid produced. The change of the type of stereoisomers of lactic acid from the DL-type to the L-type in the presence of sodium acetate was concluded to be species-specific for L. sakei and useful for identification of strains in this species.     

Characterization of the amylovorin locus of Lactobacillus amylovorus DCE 471, producer of a bacteriocin active against Pseudomonas aeruginosa, in combination with colistin and pyocins

Lactobacillus amylovorus DCE 471 produces amylovorin L, a bacteriocin with an antibacterial activity against some strains of the Lactobacillus lineage. Based on the sequence of one active peptide, a gene encoding active amylovorin L was cloned and sequenced. Genome walking allowed us to sequence a larger fragment of 7577 bp of genomic DNA, with 12 predicted ORFs. The previously characterized amylovorin L peptide-encoding gene is preceded by another gene encoding a small polypeptide with a typical bacteriocin-processing double-glycine site, suggesting that amylovorin L is a two-component class IIb bacteriocin (amylovorin Lalpha/beta). Lalpha and Lbeta show the highest similarity to gassericin T from Lactobacillus gasseri SBT2055 and BlpN from Streptococcus pneumoniae R6, respectively, and to LafA and LafX, which form the lactacin F bacteriocin of Lactobacillus johnsonii NCC 533. As for other lactic acid bacteria bacteriocins, amylovorin L showed no activity against the Gram-negative opportunistic pathogen Pseudomonas aeruginosa on its own, but showed synergistic inhibitory activity when used in combination with the peptide antibiotic colistin, and, remarkably, with the P. aeruginosa soluble bacteriocins, pyocins S1 and S2.     

副干酪乳杆菌HD1.7群体感应行为

【目的】Paracin1.7是从副干酪乳杆菌(Lactobacillus paracasei)HD1.7发酵液中提取的一种细菌素,本文主要研究菌株HD1.7在发酵过程中调控Paracin1.7代谢的群体感应机制。【方法】利用杯碟法检测不同生长条件下菌株HD1.7培养液的抑菌活性,通过调整培养基营养成分的多寡,控制培养液中细胞密度。【结果】菌株HD1.7的抑菌活性与其细胞密度密切相关,只有当细胞密度达到一定的阈值(OD600为0.8,菌体干重为0.331 1 g/L)时,菌株才能表现抑菌活性;以发酵上清液作为信号分子,当添加不同浓度信号分子至低于阈值浓度培养液后,菌株抑菌活性受到不同程度的影响,并且在去除信号分子后,菌株的抑菌活性明显降低。【结论】细菌素Paracin1.7是存在于HD1.7发酵液中的特殊的群体感应信号分子,可进行自我诱导。细菌素Paracin1.7的抑菌活性受到HD1.7群体感应系统的调控。     

Characterization and purification of a bacteriocin from <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">paracasei</Emphasis> BMK2005, an intestinal isolate active against multidrug-resistant pathogens

A strain of Lactobacillus paracasei subsp. paracasei BMK2005 isolated from healthy infant faeces has shown a remarkable antibacterial activity against 32 bacterial pathogenic strains of human clinical isolates. Among them, 13 strains belonging to species of Escherichia coli, Citrobacter freundii, Citrobacter diversus, Klebsiella oxytoca, Enterobacter cloacae and Pseudomonas aeruginosa were resistant to Cefotaxime (CTX) and Ceftazidime (CAZ), and 4 strains of Staphylococcus aureus were resistant to Methicillin (MRSA). This antibacterial activity was attributed to a bacteriocin designated as Paracaseicin A. It was heat-stable up to 120°C for 5 min and active within the pH range of 2–5. Its activity was lost when treated with proteases, which reveals its proteinaceous nature. This bacteriocin was successfully purified only by two steps of reversed phase chromatography. Its molecular mass, determined by mass spectrometry analysis, was 2,462.5 Da. To our knowledge, the present study is the first report on characterization and purification of a bacteriocin, produced by a L. paracasei subsp. paracasei strain exhibiting an antibacterial activity against various multidrug-resistant species of Gram-positive and Gram-negative bacteria, which reveals its potential for use in prevention or treatment of infections caused by multidrug-resistant species especially in cases of antibiotics-associated diarrhea (AAD).     

Bacteriocin production by Enterococcus faecium NA01 from 'wara'—a fermented skimmed cow milk product from West Africa

An Enterococcus faecium strain from Nigerian fermented skimmed cow milk ('wara') produced bacteriocin inhibitory towards Lactobacillus, Enterococcus and Listeria strains. The bacteriocin (designated enterocin 01) was inactivated by proteases, heat-stable at 100°C and active at pH 2.0–6.0. The Ent. faecium isolate harboured plasmids of ca 36.3 and 23.1 kb. Curing experiments with ethidium bromide resulted in a bacteriocin-negative mutant which had not lost immunity to the bacteriocin. Slight differences in plasmid profiles between wild-type and mutant indicated a possible plasmid-coded bacteriocin production.     

Purification and Partial Characterization of Novel Bacteriocin L23 Produced by <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">fermentum L</Emphasis>23

Lactobacillus fermentum strain L23 produced a small bacteriocin, designated bacteriocin L23, with an estimated molecular mass of < 7000 Da. Isolation, purification, and partial characterization of bacteriocin L23 are described. It displayed a wide inhibitory spectrum including both Gram-negative and Gram-positive pathogenic strains and two species of Candida. The antibacterial activity of cell-free culture supernatant fluid was not affected by catalase or urease but was abolished by the proteolytic enzymes trypsin and protease VI. Bacteriocin L23 was heat stable (60 min at 100°C) and showed inhibitory activity over a wide pH range (4.0 to 7.0). The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin sample was prepared by a process of ammonium sulfate precipitation, gel filtration, thin-layer chromatography, bioautography, and reversed-phase HPLC.