Bacteriocin-producing strain of Enterococcus faecium EK 13 with probiotic character and its application in the digestive tract of rabbits

Enterococcus faecium EK 13 is a bacteriocin-enterocin A producing strain with probiotic properties. In this study its colonization, stability and effect on microflora in rabbits was studied as well as its influence on zootechnical parameters. Fifty rabbits of both sexes (HYPLUS, 30-day old; after weaning) were divided into control (CG) and experimental (EG) groups. They were fed a standard diet. Moreover, 25 rabbits in EG were fed daily (for 4 weeks) 15 g (separate doses ∼1.6 g) of lyophilized EK13 strain (rifampicin resistant variant — rif^R; 10⁹ cfu/g) dissolved in drinking water. After cessation of EK13 (rif^R) strain application, the rabbits in both groups were fed a standard diet for the next 2 weeks. Sampling was performed in double on day 0 (at the beginning of experiment), weekly during EK13 (rif^R) strain application as well as on week 1 and 2 after cessation of EK13 (rif^R) strain application. The counts of EK13 (rif^R) strain reached 7.1 ± 2.6 log₁₀ cfu/g after 4 weeks and even on week 2 after its cessation the counts 5.6 ± 2.3 log₁₀ cfu/g were determined. The total counts of enterococci in the rabbits were already increased in EG comparing with CG (p < 0.05); even 2 weeks after EK13 (rif^R) strain cessation, their counts in EG were 7.2 ± 2.6 log₁₀ cfu/g (p < 0.001). Enterococci in CG reached at the same time the value 3.7 ± 2.6 log₁₀ cfu/g. The counts of E. coli were significantly reduced in EG during 4 weeks (p < 0.05, p < 0.001). Even 2 weeks after EK13 (rif^R) strain cessation significant difference in E. coli counts between CG and EG was detected (p < 0.001). Enterobacteria in EG were significantly reduced (p < 0.001). Average daily gain in EG was 41.0 ± 3.83 in comparison to CG (40.6 ± 3.72); it means almost the same; although rabbits in EG showed higher feed intake per kg of gain than rabbits in CG. Preliminary results demonstrated that EK13 is a perspective probiotic candidate for rabbits. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Modulation of virulence and antibiotic susceptibility of enteropathogenic Escherichia coli strains by Enterococcus faecium probiotic strain culture fractions

The increasing rate of antimicrobial resistance drastically reduced the efficiency of conventional antibiotics and led to the reconsideration of the interspecies interactions in influencing bacterial virulence and response to therapy. The aim of the study was the investigation of the influence of the soluble and cellular fractions of Enterococcus (E.) faecium CMGB16 probiotic culture on the virulence and antibiotic resistance markers expression in clinical enteropathogenic Escherichia (E.) coli strains.The 7 clinical enteropathogenic E. coli strains, one standard E. coli ATCC 25,922 and one Bacillus (B.) cereus strains were cultivated in nutrient broth, aerobically at 37 °C, for 24 h. The E. faecium CMGB16 probiotic strain was cultivated in anaerobic conditions, at 37 °C in MRS (Man Rogosa Sharpe) broth, and co-cultivated with two pathogenic strains (B. cereus and E. coli O28) culture fractions (supernatant, washed sediment and heat-inactivated culture) for 6 h, at 37 °C. After co-cultivation, the soluble and cellular fractions of the probiotic strain cultivated in the presence of two pathogenic strains were separated by centrifugation (6000 rpm, 10 min), heat-inactivated (15 min, 100 °C) and co-cultivated with the clinical enteropathogenic E. coli strains in McConkey broth, for 24 h, at 37 °C, in order to investigate the influence of the probiotic fractions on the adherence capacity and antibiotic susceptibility. All tested probiotic combinations influenced the adherence pattern of E. coli tested strains. The enteropathogenic E. coli strains susceptibility to aminoglycosides, beta-lactams and quinolones was increased by all probiotic combinations and decreased for amoxicillin-clavulanic acid. This study demonstrates that the plurifactorial anti-infective action of probiotics is also due to the modulation of virulence factors and antibiotic susceptibility expression in E. coli pathogenic strains.     

The tyrosine decarboxylation test does not differentiate Enterococcus faecalis from Enterococcus faecium

According to the current edition of the Bergey's Manual of Systematic Bacteriology [11] the tyrosine decarboxylation test allows the differentiation of enterococci. Tyrosine is decarboxylated to the biogenic amine tyramine by E. faecalis and not by E. faecium strains. In the present study we sequenced the16S rDNA of two tyramine-producing strains, BIFI-56 and BIFI-58, presumptively classified as E. faecalis. Their 16S rDNA were identical to the same fragment from the E. faecium type strain. Several E. faecium strains were then checked for their ability to decarboxylate tyrosine and also a putative tyrosine decarboxylase-coding gene was PCR amplified from these strains. All the strains confirmed as E. faecium produced tyramine and possessed a DNA fragment coding for a putative tyrosine decarboxylase. The concordance of the two methods allows us to conclude that the tyrosine decarboxylase test cannot be used in the differentiation of E. faecalis from E. faecium since at least some E. faecium strains are tyramine producers.     

Chemical structure of wall teichoic acid isolated from Enterococcus faecium strain U0317

Wall teichoic acid (WTA) was isolated from Enterococcus faecium strain U0317 and structurally characterized using ¹H, ¹³C, and ³¹P NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, HMQC, and HMBC experiments. Further compositional determination was undertaken using classical chemical methods and HF treatment followed by GLC and GLC–MS analyses. The repeating unit of WTA consisted of two residues of 2-acetamido-2-deoxy-d-galactose, glycerol (Gro), and phosphate, and has the structure shown below:→6)-α-d-GalpNAc-(1→3)-β-d-GalpNAc-(1→2)-Gro-(3→P→     

Selenium enhances the antimutagenic activity of probiotic bacterium Enterococcus faecium M-74

The antibacterial activity of the probiotic bacterium Enterococcus faecium M-74 was assessed on De Man–Rogosa–Sharpe (MRS), Todd–Hewitt (T–H), M17 (M-17) and brain heart infusion (BHI) media with sodium selenite pentahydrate (+Se) and without sodium selenite pentahydrate (–Se) under aerobic or anaerobic conditions against nine bacterial pathogens. The highest antibacterial activity was found to be in the MRS medium under anaerobic conditions. There were no differences in the antibacterial activity between MRS(+Se) and MRS(–Se) media. The antimutagenic activity of MRS(+Se) and MRS(–Se) extracts after culture with E. faecium M-74 as well as of live and killed cells of E. faecium M-74 grown in the presence or absence of Se against the genotoxicity of ofloxacin (OFL) and acridine orange (AO) was determined in the Euglena gracilis assay. The MRS(+Se) extracts showed a significantly higher activity in reducing the genotoxicity of OFL and AO than MRS(–Se) extracts. The live cells of the probiotic strain M-74 exhibited higher antimutagenic activity than the killed bacterial cells, but differed depending on the mutagen used. However, the live bacterial cells grown in the presence of Se showed significantly higher antimutagenic activity. These results suggest a potential benefit for the future development of new Se-enriched probiotics exhibiting higher antimutagenic properties.     

Impact of Enterococcus faecium on specific activity of disaccharidases in small intestine of gnotobiotic pigs

The aim of the present work was to study the changes in the activity of disaccharidase enzymes (lactase. maltase, saccharase) in the small intestine of gnotobiotic pigs aged 0–35 days and inoculated with Enterococcus faecium. The continual decrease of lactase activity was observed from the 14^th day of age up to the end of the experiment. The most significant decrease of specific lactase activity in the duodenum (2.1 μmol/mg protein/hour) was noted from the 21^st to the 28^th day of age. On the other hand, the specific saccharase activity increased moderately during the post weaning period and maltase activity maintained a constant level. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Isolation and characterization of a new bacteriocin,termed enterocin M,produced by environmental isolate <Emphasis Type="Italic">Enterococcus faecium</Emphasis> AL41

The new bacteriocin, termed enterocin M, produced by Enterococcus faecium AL 41 showed a wide spectrum of inhibitory activity against the indicator organisms from different sources. It was purified by (NH₄)₂SO₄ precipitation, cation-exchange chromatography and reverse phase chromatography (FPLC). The purified peptide was sequenced by N-terminal amino acid Edman degradation and a mass spectrometry analysis was performed. By combining the data obtained from amino acid sequence (39 N-terminal amino acid residues was determined) and the molecular weight (determined to be 4 628 Da) it was concluded that the purified enterocin M is a new bacteriocin, which is very similar to enterocin P. However, its molecular weight is different from enterocin P (4 701.25). Of the first 39 N-terminal residues of enterocin M, valine was found in position 20 and a lysine in position 35, while enterocin P has tryptophane residues in these positions.     

Use of a 'Miniprep' for rapid extraction of plasmids from vancomycin- and gentamicin-resistant Enterococcus faecium

Conventional methods of plasmid extraction are largely unsuited to diagnostic laboratories. The 'Miniprep' is a rapid method that utilises a centrifugable column to separate plasmid DNA from chromosomal DNA. We have modified this technique to extract plasmid DNA from seven strains of vancomycin- and gentamicin-resistant Enterococcus faecium (VGREF): 1% mannitol was added to the growth medium and cell lysis was achieved by incubation in 10 mg of lysozyme/ml in 10 mM Tris, 1 mM EDTA and 25% sucrose at pH 8.0. RNase A was added to plasmid eluate rather than at the lytic step. In comparison to a standard phenol/chloroform method, Miniprep completely eliminated chromosomal interference in gel electrophoresis but otherwise produced identical plasmid profiles. Plasmids obtained from the VGREF ranged from 42 to 1.3 Md. Band densities on a single elution from the Miniprep varied from 8.3 to 106.3 relative units. Double elution increased band densities from the same preparation from 30.4 to 196 relative units; mean percentage increases per track between 7.0 and 34.6%. This method is suitable to achieve plasmid DNA extraction from VGREF within 1 h, making the process more suitable for diagnostic laboratories.     

Characterization and enhanced production of enterocin HJ35 byEnterococcus faecium HJ35 isolated from human skin

A strain named as HJ35 was isolated from the skin of sixty-five men and fourteen women for acne therapy, in order to find an effective antimicrobial agent againstPropionibacterium acnes. Isolate HJ35 was identified asEnterococcus faecium based on 16 rDNA sequence and produced enterocin HJ35 having antimicrobial activities against most lactic acid bacteria,Enterococcus spp.,Staphylococcus aureus, S. epidermidis, Clostridium perfringens, some bacilli,Micrococcus flavus, Listeria monocytogenes, L. ivanovii, Escherichia coli, Pseudomonas fluorescens andPropionibacterium acnes, in the modified well diffusion method. Especially, enterocin HJ35 showed a bactericidal activity againstPropionibacterium acnes P1. The antimicrobial activity of enterocin HJ35 was disappeared completely with the use of protease XIV. But enterocin HJ35 activity is very stable at high temperature (up to 100°C for 30 min), in wide range of pH 3.0∼9.0), and by treatment with organic solvents. The apparent molecular mass of enterocin HJ35 was estimated to be approximately 4∼4.5 kDa on detection of its bactericidal activity after SDS-PAGE. In batch fermentation ofE. faecium HJ35, enterocin HJ35 was produced at the midlog growth phase, and its maximum production was obtained up to 2,300 AU/mL at the late stationary phase. By employing fed-batch fermentation, the enhanced production of enterocin HJ35 was achieved up to 12,800 AU/mL by feeding with 10 g/L glucose or 6 g/L lactate.     

Activities and regulation of the enzymes involved in the first and the third steps of the aspartate biosynthetic pathway in Enterococcus faecium

The enzymes aspartokinase and homoserine dehydrogenase catalyze the reaction at key branching points in the aspartate pathway of amino acid biosynthesis. Enterococcus faecium has been found to contain two distinct aspartokinases and a single homoserine dehydrogenase. Aspartokinase isozymes eluted on gel filtration chromatography at molecular weights greater than 250,000 and about 125,000. The molecular weight of homoserine dehydrogenase was determined to be 220,000. One aspartokinase isozyme was slightly inhibited by meso-diaminopimelic acid. Another aspartokinase was repressed and inhibited by lysine. Although the level of diaminopimelate-sensitive (DAP^s) enzyme was not much affected by growth conditions, the activity of lysine-sensitive (Lys^s) aspartokinase disappeared rapidly during the stationary phase and was depressed in rich media. The synthesis of homoserine dehydrogenase was controlled by threonine and methionine. Threonine also inhibited the specific activity of this enzyme. The regulatory properties of aspartokinase isozymes and homoserine dehydrogenase from E. faecium are discussed and compared with those from Bacillus subtilis.     

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.     

Comparison of the effects of Enterococcus faecium and aromatic oils from sage and oregano on growth performance and diarrhoeal diseases of weaned pigs

The influence of a probiotic additive and aromatic oils from medicinal and spicy plants on the basic production parameters and on the incidence of intestinal disorders of weaned pigs (weaning at the age of 30–32 days) was compared in two trials in a commercial pig fattening farm (Michalovce, Slovakia). In the first trial, an experimental (n _ex = 38) and a control group (n _co = 32) were used. A commercial probiotic additive containing Enterococcus faecium M-74 (6 × 10⁸ CFU/g) and IgY antibodies (920 mg/g dried activated egg matter) against pig Rotavirus and different strains of Escherichia coli (K88, K99, 987P, F18) were added to feed mixture of the experimental group from 30^th to 40^th day (12 × 10⁸ CFU/g) and subsequently from 40^th to 50^th day (6 × 10⁸ CFU/g) of age. The second trial was performed with three groups of pigs (n _ex1 = 28, n _ex2 = 24, n _co = 28). Aromatic oils isolated from sage (Salvia officinalis L., Labiateae) or oregano (Origanum vulgare L., Lamiaceae) were added to feed mixtures of the 1^st (90 mL/100 kg) and 2^nd experimental group (35 mL/100 kg), respectively, from 32^th to 53^th day of age. A statisticaly non-significant decrease of the incidence of diarrhoeal diseases was observed in the experimental groups of both trials as follows: in the 1^st trial by 43.41%, whereas in the 2^nd trial by 20.03% (1^st exp. group) and 6.72% (2^nd exp. group) in comparison with the control groups. The daily weight gains increased in the 1^st trial by 42.95 g/day, while in the 2^nd trial by 69.52 g/day (1^st exp. group) and 105.23 g/day (2^nd exp. group; p < 0.05) compared with controls. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Proteome changes underpin improved meat quality and yield of chickens (Gallus gallus) fed the probiotic Enterococcus faecium

Background

Supplementation of broiler chicken diets with probiotics may improve carcass characteristics and meat quality. However, the underlying molecular mechanism remains unclear. In the present study, 2D-DIGE-based proteomics was employed to investigate the proteome changes associated with improved carcass traits and meat quality of Arbor Acres broilers (Gallus gallus) fed the probiotic Enterococcus faecium.

Results

The probiotic significantly increased meat colour, water holding capacity and pH of pectoral muscle but decreased abdominal fat content. These meat quality changes were related to the altered abundance of 22 proteins in the pectoral muscle following E. faecium feeding. Of these, 17 proteins have central roles in regulating meat quality due to their biological interaction network. Altered cytoskeletal and chaperon protein expression also contribute to improved water holding capacity and colour of meat, which suggests that upregulation of chaperon proteins maintains cell integrity and prevents moisture loss by enhancing folding and recovery of the membrane and cytoskeletal proteins. The down-regulation of β-enolase and pyruvate kinase muscle isozymes suggests roles in increasing the pH of meat by decreasing the production of lactic acid. The validity of the proteomics results was further confirmed by qPCR.

Conclusions

This study reveals that improved meat quality of broilers fed probiotics is triggered by proteome alterations (especially the glycolytic proteins), and provides a new insight into the mechanism by which probiotics improve poultry production.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1167) contains supplementary material, which is available to authorized users.     

牦牛源产细菌素屎肠球菌的分离鉴定和益生特性

【背景】抗生素的滥用导致牦牛肠道常见病原菌耐药性增加,益生菌作为对抗耐药性细菌的新型武器,应用前景广阔。【目的】获取益生特性优良的牦牛源益生菌。【方法】将20份牦牛粪便样本在含0.5%CaCO3的MRS培养基上分离纯化,以大肠杆菌和金黄色葡萄球菌为指示菌,用牛津杯法筛选有抑菌活性的菌株;排除酸和过氧化氢后,经耐酸耐热试验和蛋白酶敏感试验筛选产细菌素菌株,用形态学和16S rRNA基因序列分析鉴定;通过对大肠杆菌、沙门氏菌等腹泻病原菌体外抑菌试验、耐模拟胃肠液、测定自聚集能力和疏水性及抗生素敏感试验分析益生特性。【结果】从20份牦牛粪便样本中共分离出11株产生溶钙圈的菌株,其中6株对大肠杆菌和金黄色葡萄球菌抑菌效果显著,经复筛得到2株产细菌素的乳酸菌SC6和SC9,经鉴定均为屎肠球菌(Enterococcus faecium)。其中SC9对腹泻病原菌抑菌效果明显,有良好的耐受性和肠道黏附能力,对5种常用抗生素均敏感。【结论】屎肠球菌SC9有一定的抗逆性和潜在的益生能力,具备作为益生菌的潜力。     

Comparison of genotypic and phenotypic cluster analyses of virulence determinants and possible role of CRISPR elements towards their incidence in Enterococcus faecalis and Enterococcus faecium

Enterococcus faecalis and Enterococcus faecium are human commensals frequently found in fermented foods or used as probiotics, but also recognized as opportunistic pathogens. We investigated 62 Enterococcus strains isolated from clinical, food and environmental origins towards a rationale for safety evaluation of strains in food or probiotic applications. All isolates were characterised with respect to the presence of the virulence determinants fsrB, sprE, gelE, ace, efaAfs/fm, as, esp, cob and the cytolysin operon. In addition RAPD-PCR was used to obtain genomic fingerprints that were clustered and compared to phenotypic profiles generated by MALDI-TOF-MS. The gelatinase phenotype (GelE) and the haemolytic activity (β-haemolysis) were analysed. E. faecium strains contained esp and efaAfm only, and none of them contained any CRISPR elements. The amenability of E. faecalis strains to acquisition of virulence factors was investigated along the occurrence of CRISPR associated (cas) genes. While distribution of most virulence factors, and RAPD versus MALDI-TOF-MS typing patterns were unrelated, 2 out of 5 RAPD clusters almost exclusively contained clinical E. faecalis isolates, and an occurrence of CRISPR elements versus reduced number of virulence factors was observed. The presence of the cytolysin operon, cob and as encoding pheromone and aggregation substance, respectively, significantly corresponded to absence of cas. As their production promote genetic exchange, their absence limits further gene acquisition and distribution. Thus, absence of the cytolysin operon, cob and as in a cas positive environment suggests itself as promising candidate for E. faecalis evaluation towards their occurrence in food fermentation or use as probiotics.     

屎肠球菌噬菌体vB_EfaS_29生物学特性及基因组分析

【背景】随着屎肠球菌耐药性越来越严重,亟需筛选获得新的、裂解效率高且遗传背景清晰的裂解性噬菌体,丰富噬菌体资源,为噬菌体疗法提供可用的菌株。【目的】从江苏省南京市西岗奶牛场粪便样品中分离出一株裂解性屎肠球菌噬菌体,研究其生物学特性及分析基因组学特征。【方法】通过双层平板法对其进行纯化,观察噬菌斑特征,透射电镜观察噬菌体形态,测定一步生长曲线、pH耐受性、温度耐受性以及最佳感染复数,对噬菌体进行全基因组测序及遗传进化分析。【结果】分离并纯化的一株裂解性屎肠球菌噬菌体命名为vB_EfaS_29,其噬菌斑圆形透亮,外周无晕环。该噬菌体头部呈二十面体,头部直径约52.4 nm,尾长约157.1 nm,为长尾噬菌体科。该噬菌体的最佳感染复数为0.1,最高耐受温度为70℃左右,当pH值在6.0-9.0时其效价稳定。一步生长曲线表明,其潜伏期为10 min,暴发期约为50 min,暴发量为80。基因组全长41 014 bp,GC含量为34.99%,共有63个开放阅读框(Open Reading Frame,ORF),其中36个被注释出已知基因,基因组中不含毒力基因、耐药基因及整合基因。进化分析显示以裂解酶编码氨基酸为靶标对比,该噬菌体与GenBank上的粪肠球菌噬菌体vB_EfaS_DELF1相似性大于99%,但是以噬菌体衣壳蛋白编码氨基酸为靶标比对发现,该噬菌体肠球菌与其他噬菌体遗传距离均较远。【结论】分离出一株裂解性屎肠球菌噬菌体,对其进行生物学特性及基因组分析,为噬菌体研究和应用提供理论依据及研究基础。     

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM

Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6′)-Ii that encodes a 6′-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m⁵C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m⁵C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 Å resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m⁵C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes'' respective specificities for nucleotides C1404 and C1407.     

Inhibitory activity of probiotics <Emphasis Type="Italic">Streptococcus phocae</Emphasis> PI80 and <Emphasis Type="Italic">Enterococcus faecium</Emphasis> MC13 against Vibriosis in shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Occurrence of widespread epizootics among larval and cultured shrimp has put on viable preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study, four probiotics bacteria were isolated from marine fish and shrimp intestine based on the antagonistic activity and nonpathogenic to the host. The isolates of probiotics strains Streptococcus phocae PI80, Enterococcus faecium MC13, Lactococcus garvieae LC149, B49 and one commercial probiotics (ECOFORCE) were fed to post larvae of Penaeus monodon obtained from two different hatcheries to analyze the growth and protection against Vibrio harveyi and V. parahaemolyticus. Growth of P. monodon post larvae fed with probiotic strain S. phocae PI80 was significantly (P < 0.001) higher when compared with control and other three strains in both experiments. The treatment of post larvae with B49 reduced the growth as well as Specific growth rate. Among the three probiotic strains S. phocae PI80 and E. faecium MC13 have effectively inhibited the pathogens. In experiment I high survival (92%) were observed in S. phocae PI80 treated post larvae when challenged with Vibrio harveyi followed by E. faecium MC13 (84%), B49 (76%) and ECOFORCE (68%) but PI80 did not protect the post larvae in the same experiment when they were exposed to V. parahaemolyticus. The probiotic isolate of MC13 has protected the post larvae against V. parahaemolyticus when compared to other probiotics and control. Similarly in the second experiment feeding of S. phocae enhanced the survival of larvae when challenged with V. harveyi. The laboratory studies proved that bacterial probionts S. phocae and E. faecium isolated from shrimp and brackishwater fish has potential applications for controlling pathogenic vibriosis in shrimp culture.     

Novel probiotic Enterococcus faecium IS-27526 supplementation increased total salivary sIgA level and bodyweight of pre-school children: A pilot study

Enterococcus faecium IS-27526 is a novel probiotic isolated from dadih, an Indonesian traditional fermented buffalo milk. A 90 days randomized double-blind placebo-controlled study ofpre-post trial was conducted in pre-school children with two groups, placebo and probiotic group. Ultra High Temperature low fat milk was used as a carrier in each group. The aims of this study were to investigate the effect of E. faecium IS-27526 in milk on humoral immune response and on bodyweight of pre-school children. Total serum IgA and total salivary sIgA were measured by sandwich ELISA. The bodyweight of young children was measured. The results showed that total serum IgA did not significantly increase in the probiotic group compared with the placebo group. Total salivary sIgA level and the bodyweight significantly increased (p < 0.05) in probiotic groups compared to the placebo. Changes of total salivary sIgA level were significantly higher in underweight children supplemented with probiotic. Weight gain was observed significantly in children with normal bodyweight supplemented with probiotic. Neither mortality nor weight loss was recorded throughout the study. Taken together, novel probiotic E. faecium IS-27526 has significant positive effects on humoral immune response, salivary sIgA, in underweight pre-school children, and on weight gain of pre-school children.     

Sensitivity to detergents and plasmid curing in Enterococcus faecalis

This research reports the sensitivity of a clinical isolate of Enterococcus faecalis to sodium N-lauroylsarcosinate (sarkosyl) and sodium dodecyl sulfate (SDS), as well as the efficiency of these detergents in curing the strain. Compared to Escherichia coli, Enterococcus faecalis was very sensitive to both detergents, with minimum inhibitory concentrations (MIC) for the latter being 100 times lower than for Escherichia coli. The clinical isolate of Enterococcus faecalis used in this study exhibited plasmid-borne resistance to kanamycin (MIC 2 mg/ml) and tetracycline (MIC 50 μg/ml); 3% curing was observed after growth in the presence of sarkosyl but no curing was observed after growth in the presence of either SDS or acridine orange. In contrast, 35% curing of plasmid-bearing Escherichia coli was observed after growth in the presence of either SDS or acridine orange, but none was observed after growth in the presence of sarkosyl.