Enterocin P selectively dissipates the membrane potential of Enterococcus faecium T136

Enterocin P is a pediocin-like, broad-spectrum bacteriocin which displays a strong inhibitory activity against Listeria monocytogenes. The bacteriocin was purified from the culture supernatant of Enterococcus faecium P13, and its molecular mechanism of action against the sensitive strain E. faecium T136 was evaluated. Although enterocin P caused significant reduction of the membrane potential (DeltaPsi) and the intracellular ATP pool of the indicator organism, the pH gradient (DeltapH) component of the proton motive force (Deltap) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from E. faecium T136-derived liposomes.     

Enterocin P Selectively Dissipates the Membrane Potential of Enterococcus faecium T136

Enterocin P is a pediocin-like, broad-spectrum bacteriocin which displays a strong inhibitory activity against Listeria monocytogenes. The bacteriocin was purified from the culture supernatant of Enterococcus faecium P13, and its molecular mechanism of action against the sensitive strain E. faecium T136 was evaluated. Although enterocin P caused significant reduction of the membrane potential (ΔΨ) and the intracellular ATP pool of the indicator organism, the pH gradient (ΔpH) component of the proton motive force (Δp) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from E. faecium T136-derived liposomes.     

一株猪源屎肠球菌HDRsEf1益生特性研究

本研究旨在筛选一株具有优良生物特性的屎肠球菌,作为微生态制剂候选菌株;重点研究其与肠道相关的抗逆性能和对病原菌的抑菌活性。以自健康通城猪直肠内容物分离得到的屎肠球菌HDRsEf1为研究对象,通过耐受试验、抑菌试验研究该菌的益生特性。发现屎肠球菌HDRsEf1能够耐受浓度为0.5%的胆盐,并能在pH为2.0的酸中存活;对粘蛋白有显著的粘附作用(P0.05);能耐受70℃的温度。该菌发酵上清液能够抑制食源致病菌和畜禽临床主要致病菌的生长,结果表明屎肠球菌HDRsEf1可作微生态制剂菌株。     

Enterococcus faecium AL 41: Its Enterocin M and Their Beneficial Use in Rabbits Husbandry

Enterococci are ubiquitous microbiota constituting a large proportion of autochthonous microflora in animals. Some produce bacteriocins mostly enterocins; some of bacteriocin-producing strains also possess probiotic properties. Enterococcus faecium AL 41, Ent M-producing strain was tested for beneficial effect in rabbits. Five-week-old animals (72, Hycole) were divided into experimental groups (E1, E2) and control (C); 24 animals in each. Rabbits in E1 were administered AL 41 (500???l per animal/day, 10⁹?cfu/ml) in water for 21?days; rabbits in E2 were administered Ent M (50???l/animal/day, activity 12,800?AU/ml) in water for 21?days. Rabbits in C fed a commercial diet. The experiment lasted 42?days. Sampling of faeces and blood was provided on day 0?C1 and 21, 42; 3 animals per group were slaughtered. Caecum and appendix were separated. AL 41 colonized rabbits intestines?<1.0 (log10) cfu/g, but stimulation of immunity was noted (P?<?0.01; P?<?0.001). Antimicrobial activity of both was noticed in faeces and/or caecum against pseudomonads. Significant decrease of coliform bacteria in faeces of E1 was noted on day 42 comparing with E2 (P?<?0.05). On day 21, S. aureus cells were not detected in E1, E2. On day 42, S. aureus was not found in E2; in E1 their counts were?<1.0?cfu/g, while in C it was in the count more than 1.0?cfu/g. In appendix, on day 21, significant decrease of not specified bacteria was found in E1, E2 comparing with C (P?<?0.01). Administration of additives has not evoked oxidative stress. Biochemical parameters were not influenced. Higher average daily weight gains were detected by both, AL 41 and Ent M.     

Atypical Genetic Locus Associated with Constitutive Production of Enterocin B by Enterococcus faecium BFE 900

A purified bacteriocin produced by Enterococcus faecium BFE 900 isolated from black olives was shown by Edman degradation and mass spectrometric analyses to be identical to enterocin B produced by E. faecium T136 from meat (P. Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernández, and H. Holo, Microbiology 143:2287–2294, 1997). The structural gene was located on a 2.2-kb HindIII fragment and a 12.0-kb EcoRI chromosomal fragment. The genetic characteristics and production of EntB by E. faecium BFE 900 differed from that described so far by the presence of a conserved sequence like a regulatory box upstream of the EntB gene, and its production was constitutive and not regulated. The 2.2-kb chromosomal fragment contained the hitherto undetected immunity gene for EntB in an atypical orientation that is the reverse of that of the structural gene. Typical transport and other genes associated with bacteriocin production were not detected on the 12.0-kb chromosomal fragment containing the EntB structural gene. This makes the EntB genetic system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of Carnobacterium piscicola LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal peptide, and expressed by the general secretory pathway in Enterococcus faecalis ATCC 19433.     

Evaluation of GABA Production and Probiotic Activities of Enterococcus faecium BS5

Probiotics and Antimicrobial Proteins - Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of...     

Detection and antimicrobial spectrum of a bacteriocin-like substance produced by Enterococcus faecium CCM4231

Enterococcus faecium CCM4231, isolated from the rumen content of a calf, produced an antimicrobial agent active against Gram-positive and Gram-negative indicator organisms. After 100-fold concentration by ultrafiltration, the diameters of zones of inhibition ranged from 2 to 10 mm. The agent was sensitive to pronase and trypsin and resistant to heating at 60°C for 30 min.     

Immunochemical Characterization of Temperature-Regulated Production of Enterocin L50 (EntL50A and EntL50B), Enterocin P, and Enterocin Q by Enterococcus faecium L50

Polyclonal antibodies with specificity for enterocin L50A (EntL50A), enterocin L50B (EntL50B), and enterocin Q (EntQ) produced by Enterococcus faecium L50 have been generated by immunization of rabbits with chemically synthesized peptides derived from the C terminus of EntL50A (LR1) and EntL50B (LR2) and from the complete enterocin Q (EntQ) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of these antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect ELISA (CI-ELISA). The NCI-ELISA was valuable for detecting anti-EntL50A-, anti-EntL50B-, and anti-EntQ-specific antibodies in the sera of the LR1-KLH-, LR2-KLH-, and EntQ-KLH-immunized animals, respectively. Moreover, these antibodies and those specific for enterocin P (EntP) obtained in a previous work (J. Gutiérrez, R. Criado, R. Citti, M. Martín, C. Herranz, M. F. Fernández, L. M. Cintas, and P. E. Hernández, J. Agric. Food Chem. 52:2247-2255, 2004) were used in an NCI-ELISA to detect and quantify the production of EntL50A, EntL50B, EntP, and EntQ by the multiple-bacteriocin producer E. faecium L50 grown at different temperatures (16 to 47°C). Our results show that temperature has a strong influence on bacteriocin production by this strain. EntL50A and EntL50B are synthesized at 16 to 32°C, but production becomes negligible when the growth temperature is above 37°C, whereas EntP and EntQ are synthesized at temperatures ranging from 16 to 47°C. Maximum EntL50A and EntL50B production was detected at 25°C, while EntP and EntQ are maximally produced at 37 and 47°C, respectively. The loss of plasmid pCIZ1 (50 kb) and/or pCIZ2 (7.4 kb), encoding EntL50A and EntL50B as well as EntQ, respectively, resulted in a significant increase in production and stability of the chromosomally encoded EntP.     

Microbiota,Phagocytic Activity,Biochemical Parameters and Parasite Control in Horses with Application of Autochthonous,Bacteriocin-Producing,Probiotic Strain Enterococcus faecium EF 412

Probiotics and Antimicrobial Proteins - The beneficial influence of bacteriocin-producing, probiotic, mostly non-autochthonous bacteria has already been reported in various animals. However, their...     

First Complete Genome Sequence of a Probiotic Enterococcus faecium Strain T-110 and Its Comparative Genome Analysis with Pathogenic and Non-pathogenic Enterococcus faecium Genomes

<正>Enterococci bacteria are important in environmental,food and clinical microbiology.Enterococcus faecium is a nosocomial pathogen that causes bacteremia,endocarditis and other infections.It is among the most prevalent organisms encountered in hospital-associated infections accounting for approximately 12%of nosocomial infections in the USA(Linden and Miller,1999).However,certain strains of E.faecium are not only non-pathogenic but also have beneficial     

Comparison of Two Methods for Purification of Enterocin B,a Bacteriocin Produced by Enterococcus faecium W3

This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography, and sequential reverse-phase high-performance liquid chromatography. The latter approach exploited a pH-mediated cell adsorption–desorption method to extract cell-bound bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide were confirmed again by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) analysis. Although both approaches were satisfactory to obtain a sufficient amount of enterocin B for use in MS and amino acid sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B.     

Complete Sequence of the Enterocin Q-Encoding Plasmid pCIZ2 from the Multiple Bacteriocin Producer Enterococcus faecium L50 and Genetic Characterization of Enterocin Q Production and Immunity

The locations of the genetic determinants for enterocin L50 (EntL50A and EntL50B), enterocin Q (EntQ), and enterocin P (EntP) in the multiple bacteriocin producer Enterococcus faecium strain L50 were determined. These bacteriocin genes occur at different locations; entL50AB (encoding EntL50A and EntL50B) are on the 50-kb plasmid pCIZ1, entqA (encoding EntQ) is on the 7.4-kb plasmid pCIZ2, and entP (encoding EntP) is on the chromosome. The complete nucleotide sequence of pCIZ2 was determined to be 7,383 bp long and contains 10 putative open reading frames (ORFs) organized in three distinct regions. The first region contains three ORFs: entqA preceded by two divergently oriented genes, entqB and entqC. EntqB shows high levels of similarity to bacterial ATP-binding cassette (ABC) transporters, while EntqC displays no significant similarity to any known protein. The second region encompasses four ORFs (orf4 to orf7), and ORF4 and ORF5 display high levels of similarity to mobilization proteins from E. faecium and Enterococcus faecalis. In addition, features resembling a transfer origin region (oriT) were found in the promoter area of orf4. The third region contains three ORFs (orf8 to orf10), and ORF8 and ORF9 exhibit similarity to the replication initiator protein RepE from E. faecalis and to RepB proteins, respectively. To clarify the minimum requirement for EntQ synthesis, we subcloned and heterologously expressed a 2,371-bp fragment from pCIZ2 that encompasses only the entqA, entqB, and entqC genes in Lactobacillus sakei, and we demonstrated that this fragment is sufficient for EntQ production. Moreover, we also obtained experimental results indicating that EntqB is involved in ABC transporter-mediated EntQ secretion, while EntqC confers immunity to this bacteriocin.     

Purification and N-terminal amino acid sequence of Enterocin CRL 35, a 'pediocin-like' bacteriocin produced by Enterococcus faecium CRL 35

M.E. FARÍAS, R.N. FARÍAS, A.P. DE RUIZ HOLGADO AND F. SESMA. 1996. Enterocin CRL 35, a bacteriocin produced by Enterococcus faecium CRL 35 that inhibits food-borne pathogens, was purified by precipitation with (NH₄)₂SO₄, gel filtration, ion exchange and reverse phase chromatography. The partial N-terminal amino acid sequence indicated a strong homology with other 'pediocin-like bacteriocins' previously described.     

Combined Effect of Enterocin and Lipase From Enterococcus faecium NCIM5363 Against Food Borne Pathogens: Mode of Action Studies

Food borne diseases have a major impact on public health whose epidemiology is rapidly changing. The whole cells of pathogens involved or their toxins/metabolites affect the human health apart from spoiling sensory properties of the food products finally affecting the food industry as well as consumer health. With pathogens developing mechanisms of antibiotic resistance, there has been an increased need to replace antibiotics as well as chemical additives with naturally occurring bacteriocins. Bacteriocins are known to act mainly against Gram-positive pathogens and with little or no effect towards Gram-negative enteric bacteria. In the present study, combination effect of lipase and bacteriocin produced by Enterococcus faecium NCIM5363, a highly lipolytic lactic acid bacterium against various food pathogens was assessed. The lipase in combination with enterocin exhibited a lethal effect against Gram-negative pathogens. Scanning electron microscopy studies carried out to ascertain the constitutive mode of action of lipase and enterocin revealed that the lipase degrades the cell wall of Gram-negative bacteria and creates a pore through which enterocin enters thereby resulting in cell death. The novelty of this work is the fact that this is the first report revealing the synergistic effect of lipase with enterocin against Gram-negative bacteria.     

粪肠球菌和屎肠球菌耐药性分析

目的 监测我院肠球菌中粪肠球菌株和屎肠球菌株的耐药性,为临床合理应用抗菌药物提供依据。方法 采用法国生物梅里埃公司的GPI板进行细菌鉴定及药敏试验,应用whonet5软件统计粪肠球菌和屎肠球菌的耐药率。结果 粪肠球菌和屎肠球菌对氯霉素、呋喃妥因、万古霉素有较好体外抗菌活性,耐药率都在50%以下,对万古霉素的耐药率在1%以下。粪肠球菌对青霉素、高水平庆大霉素、环丙沙星、利福平、红霉素等大部分抗菌素的耐药率有逐年下降趋势,而屎肠球菌对环丙沙星、利福平、呋喃妥因等抗菌素的耐药率则有上升趋势,屎肠球菌对大多数抗菌素耐药率都高于粪肠球菌。结论 粪肠球菌和屎肠球菌呈多重耐药,临床用药应结合药敏试验结果合理选择抗菌药物。     

Characterization and Heterologous Expression of the Genes Encoding Enterocin A Production, Immunity, and Regulation in Enterococcus faecium DPC1146

Enterocin A is a small, heat-stable, antilisterial bacteriocin produced by Enterococcus faecium DPC1146. The sequence of a 10,879-bp chromosomal region containing at least 12 open reading frames (ORFs), 7 of which are predicted to play a role in enterocin biosynthesis, is presented. The genes entA, entI, and entF encode the enterocin A prepeptide, the putative immunity protein, and the induction factor prepeptide, respectively. The deduced proteins EntK and EntR resemble the histidine kinase and response regulator proteins of two-component signal transducing systems of the AgrC-AgrA type. The predicted proteins EntT and EntD are homologous to ABC (ATP-binding cassette) transporters and accessory factors, respectively, of several other bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia coli hemolysin A. Immediately downstream of the entT and entD genes are two ORFs, the product of one of which, ORF4, is very similar to the product of the yteI gene of Bacillus subtilis and to E. coli protease IV, a signal peptide peptidase known to be involved in outer membrane lipoprotein export. Another potential bacteriocin is encoded in the opposite direction to the other genes in the enterocin cluster. This putative bacteriocin-like peptide is similar to LafX, one of the components of the lactacin F complex. A deletion which included one of two direct repeats upstream of the entA gene abolished enterocin A activity, immunity, and ability to induce bacteriocin production. Transposon insertion upstream of the entF gene also had the same effect, but this mutant could be complemented by exogenously supplied induction factor. The putative EntI peptide was shown to be involved in the immunity to enterocin A. Cloning of a 10.5-kb amplicon comprising all predicted ORFs and regulatory regions resulted in heterologous production of enterocin A and induction factor in Enterococcus faecalis, while a four-gene construct (entAITD) under the control of a constitutive promoter resulted in heterologous enterocin A production in both E. faecalis and Lactococcus lactis.     

Enterocin AS-48RJ: a variant of enterocin AS-48 chromosomally encoded by Enterococcus faecium RJ16 isolated from food

The bacteriocinogenic strain RJ16 isolated from goat cheese has been identified as Enterococcusfaecium by species-specific PCR, DNA-rRNA hybridization and rDNA sequencing. Purified bacteriocin from strain RJ16 is a carboxypeptidase A-resistant peptide with a molecular mass (7125 Da) very close to the cyclic peptide enterocin AS-48. Bacteriocin from strain RJ16 and AS-48 show identical antibacterial spectra, although the former is slightly less active on strains of Listeria monocytogenes and Bacillus cereus. Producer strains show cross-immunity. PCR amplification of total DNA from strain RJ16 with primers for the AS-48 structural gene and sequencing of the amplified fragment revealed an almost identical sequence (99.5%), except for a single mutation that predicts the change of Glu residue at position 20 of AS-48 to Val. Therefore, bacteriocin produced by E. faecium RJ16 should be considered a variant of AS-48, which we call AS-48RJ. PCR amplification revealed that strain RJ16 contains the complete as-48. gene cluster. Hybridization with probes for as-48 gene cluster revealed a chromosomal location of as-48 genes in strain RJ16, being the first example of a chromosomal location of this bacteriocin trait. Strain RJ16 produced enzymes of interest in food processing (esterase, esterase lipase and phytase activities), and did not decarboxylate amino acids precursors for biogenic amines. Strain RJ16 did not exhibit haemolytic or gelatinase activities, and PCR amplification revealed the lack of genes encoding for known virulence determinants (aggregation substance, collagen adhesin, enterococcal surface protein, endocarditis antigens, as well as haemolysin and gelatinase production). Strain RJ16 was resistant to ciprofloxacin (MIC > 2 mgl(-1)) and levofloxacin (MIC > 4 mgl(-1)) and showed intermediate resistance to nitrofurantoin and erythromycin, but was sensitive to ampicillin, penicillin, streptomycin, gentamicin, rifampicin, chloramphenicol, tetracycline, quinupristin/dalfopristin, vancomycin and teicoplanin. Altogether, results from this study suggest that this broad-spectrum bacteriocin-producing strain may have a potential use in food preservation.     

Comparative study of vanA gene transfer from Enterococcus faecium to Enterococcus faecalis and to Enterococcus faecium in the intestine of mice

Vancomycin-resistant enterococci represent a large reservoir in animals because of the use of avoparcin as a growth promoter in Europe. These strains of animal origin enter the food chain and can either colonize the human gut or transfer their resistance genes to the human microbiota. In this study, we compared the transfer of vancomycin resistance from resistant animal Enterococcus faecium to sensitive human Enterococcus faecalis and E. faecium. We analysed these transfers in dibiotic mice and human faecal flora-associated mice. VanA transfer from animal E. faecium to human E. faecalis occurred in dibiotic mice. The transconjugants appeared rapidly and persisted at levels between 3.0 and 4.0 log10 colony-forming units g(-1) of faeces. In human faecal flora-associated mice, vanA gene transfer was not detected towards E. faecalis but was possible between E. faecium strains. Our experiments revealed the possibility of vanA transfer from animal E. faecium to human E. faecalis in vitro and in vivo in the intestine of dibiotic mice. However, intraspecies transfer of vanA gene seems more common than interspecies transfer among enterococci.