An update on the taxonomy and functional properties of the probiotic Enterococcus faecium SF68.

Enterococcus faecium SF68 (SF68) is a well-known probiotic with a long history of safe use. Recent changes in the taxonomy of enterococci have shown that a novel species, Enterococcus lactis, is closely related with E. faecium and occurs together with other enterococci in a phylogenetically well-defined E. faecium species group. The close phylogenetic relationship between the species E. faecium and E. lactis prompted a closer investigation into the taxonomic status of E. faecium SF68. Using phylogenomics and ANI, the taxonomic analysis in this study showed that probiotic E. faecium SF68, when compared to other E. faecium and E. lactis type and reference strains, could be re-classified as belonging to the species E. lactis. Further investigations into the functional properties of SF68 showed that it is potentially capable of bacteriocin production, as a bacteriocin gene cluster encoding the leaderless bacteriocin EntK1 together with putative Lactococcus lactis bacteriocins LsbA, and LsbB-like putative immunity peptide (LmrB) were found located in an operon on plasmid pF9. However, bacteriocin expression was not studied. Competitive exclusion experiments in co-culture over 7 days at 37 °C showed that the probiotic SF68 could inhibit the growth of specific E. faecium and Listeria monocytogenes strains, while showing little or no inhibitory activity towards an entero-invasive Escherichia coli and a Salmonella Typhimurium strain, respectively. In cell culture experiments with colon carcinoma HT29 cells, the probiotic SF68 was also able to strain-specifically inhibit adhesion and/or invasion of enterococcal and L. monocytogenes strains, while such adhesion and invasion inhibition effects were less pronounced for E. coli and Salmonella strains. This study therefore provides novel data on the taxonomy and functional properties of SF68, which can be reclassified as Enterococcus lactis SF68, thereby enhancing the understanding of its probiotic nature.

Characterization of coagulase-negative staphylococci and macrococci isolated from cheese in Germany.

Cheese, especially ripened varieties, harbor a very complex and heterogeneous microbiota. In addition to the desired microorganisms (starter cultures) added during cheese production, potentially harmful bacteria may also enter the production chain. Regarding the latter, the focus of this study was on coagulase-negative staphylococci (CNS) and Macrococcuscaseolyticus. Both are known to harbor a variety of genes coding for antibiotic resistance, including mecA, mecB, mecC, and mecD. Coagulase-negative staphylococci or macrococci carrying such genes or other virulence factors should not be present in cheese. Cheese samples (101 in total) were collected from retail sources. Coagulase-negative staphylococci and M. caseolyticus were isolated utilizing selective agars, and species were identified by phenotypical tests and partial sequencing of the sodA gene. The results allowed identification of 53 CNS strains and 19 M. caseolyticus strains. Among the CNS, 11 isolates of Staphylococcus saprophyticus and one Staphylococcus epidermidis isolate were obtained. Both species are potential human pathogens and may thus adversely affect the safety of these food products. Screening for antimicrobial resistance was performed by application of disc diffusion tests, a gradient strip-test, and 14 different PCR tests. Evidence for methicillin resistance (by either positive disc diffusion assay for cefoxitin or by mec PCR) was found in CNS isolates and M. caseolyticus (9 isolates each). Regarding other virulence factors, no genetic determinants for coagulase or the most common staphylococcal enterotoxins sea, seb, sec, sed, and see were detected in any of the CNS or M. caseolyticus isolates by PCR testing. In conclusion, the presence of facultatively pathogenic CNS and carriers of genes for antibiotic resistance in both groups of microorganisms, especially mec genes, and the respective food safety issues need further evaluation and surveillance.

Soluble Lactobacillus delbrueckii subsp. bulgaricus 92059 PrtB proteinase derivatives for production of bioactive peptide hydrolysates from casein.

The proteinase-encoding prtB gene of Lactobacillus (Lb.) delbrueckii (d.) subsp. bulgaricus 92059 was cloned and sequenced. Two soluble, secreted, C-terminally His-tagged derivatives were constructed and expressed in Lactococcus lactis by means of the NICE® Expression System. In both obtained derivatives PrtBb and PrtB2, the C-terminal, cell wall-binding domain was deleted. In addition, in derivative PrtB2, the C-terminal part of the B domain was deleted and the signal sequence was replaced by a lactococcal export signal. The affinity-purified derivatives were both proteolytically active. Peptide hydrolysates produced from casein with each of the derivatives showed identical peptide composition, as determined by liquid chromatography-mass spectrometry. Comparison of the peptides generated to those generated with living Lb. d. subsp. bulgaricus 92059 cells (Kliche et al. Appl Microbiol Biotechnol 101:7621-7633, 2017) showed that ß-casein was the casein fraction most susceptible to hydrolysis and that some significant differences were observed between the products obtained by either the derivatives or living Lb. d. subsp. bulgaricus 92059 cells. When tested for biological activity, the hydrolysate obtained with PrtBb showed 50% inhibition of angiotensin-converting enzyme at a concentration of 0.5 mg/ml and immunomodulation/anti-inflammation in an in vitro assay of TNF-α induced NFκB activation at concentrations of 5 and 2.5 mg/ml, respectively. The enzymatically obtained hydrolysate did not show any pro-inflammatory or cytotoxic activity.

Epidermal-specific deletion of CD44 reveals a function in keratinocytes in response to mechanical stress.

CD44, a large family of transmembrane glycoproteins, plays decisive roles in physiological and pathological conditions. CD44 isoforms are involved in several signaling pathways essential for life such as growth factor-induced signaling by EGF, HGF or VEGF. CD44 is also the main hyaluronan (HA) receptor and as such is involved in HA-dependent processes. To allow a genetic dissection of CD44 functions in homeostasis and disease, we generated a Cd44 floxed allele allowing tissue- and time-specific inactivation of all CD44 isoforms in vivo. As a proof of principle, we inactivated Cd44 in the skin epidermis using the K14Cre allele. Although the skin of such Cd44Δker mutants appeared morphologically normal, epidermal stiffness was reduced, wound healing delayed and TPA induced epidermal thickening decreased. These phenotypes might be caused by cell autonomous defects in differentiation and HA production as well as impaired adhesion and migration on HA by Cd44Δker keratinocytes. These findings support the usefulness of the conditional Cd44 allele in unraveling essential physiological and pathological functions of CD44 isoforms.

Screening, identification and characterization of bacteriocins produced by wine-isolated LAB strains.

AIMS: To screen and identify wine-isolated LAB strains for bacteriocin production, and to identify and characterize bacteriocins. METHODS AND RESULTS: One hundred and fifty-five LAB strains isolated from South African red wines undergoing spontaneous malolactic fermentation were screened for bacteriocin production. Eight isolates were identified to be bacteriocin producers and were identified as Enterococcus faecium. All eight isolates had the same phenotypic and genotypic profiles. The peptides were preliminarily identified as enterocin P using mass spectrometry and further confirmed by PCR-amplifying enterocin P gene. The enterocin activity was inhibited by α-Chymotrypsin, papain and proteinase K treatments. It was heat stable at 37, 60, 80 and 100°C and showed activity over a broad pH range of 2-10. The production of the enterocin followed that of primary metabolite kinetics and, it showed bactericidal effect to some wine spoilage LAB strains. CONCLUSIONS: Our study identified the presence of the enterocin-producing Enterococcus in wine. The enterocin was heat stable; with broad pH range and bactericidal effects to sensitive strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of very few studies that isolated Enterococcus species from wine. It is, however, the first to report presence of bacteriocin-producing Enterococcus in wine fermentation.

Modulation of the murine microbiome with a concomitant anti-obesity effect by Lactobacillus rhamnosus GG and Lactobacillus sakei NR28.

The microbiota of the gastrointestinal tract (GIT) constitutes the major part of the total human microbiome and is considered to be an important regulator of human health and host metabolism. Numerous investigations in recent years have focused on the connection between the human microbiota and metabolic diseases such as obesity, type II diabetes and atherosclerosis. Yet, little is known about the impact of probiotic consumption on the GIT microbial population and the potential effect on chronic diseases. In this study, the modulation of the microbial community in the murine small intestine resulting from probiotic feeding was investigated and was found to be associated with an anti-obesity effect. Changes in the microbiota of the mouse faeces and small intestine were monitored using quantitative real-time PCR and by following the mRNA expression levels of various obesity-related biomarkers following probiotic feeding in a mouse model. Lactobacillus rhamnosus GG and Lactobacillus sakei NR28 (a putative probiotic strain isolated from kimchi) were administered at a daily level of approximately 1×10(8) viable bacteria per mouse (C57BL/6J mice) for up to three weeks. Feeding these strains resulted in a significant reduction of epididymal fat mass, as well as obesity-related biomarkers like acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase-1 in the liver. The total number and ratio of the microbial groups, i.e. Firmicutes, Bacteroidetes, Clostridium cluster I and XIVab, and Lactobacillus spp. were modulated in the small intestine, and the Firmicutes:Bacteroidetes ratio was decreased. In contrast, no noticeable effect of probiotic feeding could be detected on the faecal microbiota, neither quantitatively, nor with regard to the bacterial groups (Firmicutes, Bacteroidetes, Clostridium cluster I and XIVab, and Lactobacillus spp.) studied.

Modulation of innate and adaptive immunity by the probiotic Bifidobacterium longum PCB133 in turkeys.

The use of protective, probiotic cultures in poultry farming may serve as a useful strategy to improve food product safety from the beginning of the food chain and thus to protect consumer health. The objective of this study was to investigate the effect of the probiotic strain Bifidobacterium longum PCB133 on innate and adaptive immune responses in turkeys beginning at 2 wk of age, under farming conditions. The vaccination efficiency against Newcastle disease virus served as the primary endpoint. At 2 wk of age, male turkeys (British United Turkey Big 6 strain) were randomly assigned to the control (n = 25) or probiotic group (n = 25). Turkeys in the probiotic group received the probiotic B. longum PCB133 (at least 3 × 10(7) cfu/d) incorporated into the daily feed ration for 5 wk, until slaughter at 7 wk of age. At the beginning of the probiotic intervention, birds in both groups were vaccinated against Newcastle disease. Birds were weighed weekly throughout the intervention period, and finally blood sera and heparinized blood were collected for immune function tests (lymphocyte proliferation, phagocytosis, respiratory burst), and for the determination of Newcastle disease virus antibody titers. No effects on BW gain and on the proliferation of blood lymphocytes were elicited by the 5-wk intervention with the probiotic. Concerning the primary endpoint of the study (i.e., specific antibody production as a response to vaccination against Newcastle disease), no adjuvant effect of the probiotic could be determined. In addition, innate immune functions tested were not significantly affected. In conclusion, first scientific evidence on the application of the probiotic strain B. longum PCB133 in turkeys beginning at 2 wk of age does not support an improvement in live performance, humoral immunity, or innate immunity.

Anti-listerial activity of bacteriocin-producing Lactobacillus curvatus CWBI-B28 and Lactobacillus sakei CWBI-B1365 on raw beef and poultry meat.

AIM: The study aimed to evaluate the effect of the bacteriocins produced by Lactobacillus sakei CWBI-B1365 and Lactobacillus curvatus CWBI-B28 on the growth and survival of Listeria monocytogenes in raw beef and poultry meat. METHODS AND RESULTS: The sakacin P and sakacin G structural genes were identified in Lact. curvatus CWBI-B28 and Lact. sakei CWBI-B1365 using PCR amplification, respectively. The effect of the two bacteriocinogenic strains either alone or together, and that of the nonbacteriocin-producing strain Lact. sakei LMG17302, on the growth of L. monocytogenes was evaluated in beef and poultry meat. In raw beef, the pathogenic bacteria were inhibited by the bacteriocinogenic strains. The bacteriocinogenic strains had no activity in raw chicken meat when inoculated separately, while they showed a clear anti-Listeria effect when applied together. CONCLUSION: Sakacin G producing Lact. sakei and sakacin P producing Lact. curvatus may be applied in raw beef to inhibit L. monocytogenes. In poultry meat, the inhibition of L. monocytogenes could only be achieved by a combined application of these bacteriocin-producing strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In some meat products, the combined application of different class IIa bacteriocin producing lactic acid bacterium can enhance the anti-listerial activity.

Characterisation and biochemical properties of predominant lactic acid bacteria from fermenting cassava for selection as starter cultures.

A total of 375 lactic acid bacteria were isolated from fermenting cassava in South Africa, Benin, Kenya and Germany, and were characterised by phenotypic and genotypic tests. These could be divided into five main groups comprising strains of facultatively heterofermentative rods, obligately heterofermentative rods, heterofermentative cocci, homofermentative cocci and obligately homofermentative rods, in decreasing order of predominance. Most of the facultatively heterofermentative rods were identified by phenotypic tests as presumptive Lactobacillus plantarum-group strains, which also comprised the most predominant bacteria (54.4% of strains) isolated in the study. The next predominant group of lactic acid bacteria (14.1% of total isolates) consisted of obligately heterofermentative rods belonging either to the genus Lactobacillus or Weissella, followed by the heterofermentative cocci (13.9% of isolates) belonging to the genera Weissella or Leuconostoc. Homofermentative cocci were also isolated (13.3% of isolates). Biochemical properties such as production of alpha-amylase, beta-glucosidase, tannase, antimicrobials (presumptive bacteriocin and H(2)O(2)-production), acidification and fermentation of the indigestible sugars raffinose and stachyose, were evaluated in vitro for selection of potential starter strains. A total of 32 strains with one or more desirable biochemical properties were pre-selected and identified using rep-PCR fingerprinting in combination with 16S rRNA sequencing of representative rep-PCR cluster isolates. Of these strains, 18 were identified as L. plantarum, four as Lactobacillus pentosus, two each as Leuconostoc fallax, Weissella paramesenteroides and Lactobacillus fermentum, one each as Leuconostoc mesenteroides subsp. mesenteroides and Weissella cibaria, while two remained unidentified but could be assigned to the L. plantarum-group. These strains were further investigated for clonal relationships, using RAPD-PCR with three primers, and of the 32 a total of 16 strains were finally selected for the development as starter cultures for Gari production.

Molecular characterization, technological properties and safety aspects of enterococci from 'Hussuwa', an African fermented sorghum product.

AIMS: To identify enterococci from Hussuwa, a Sudanese fermented sorghum product, and determine their technological properties and safety for possible inclusion in a starter culture preparation. METHODS AND RESULTS: Twenty-two Enterococcus isolates from Hussuwa were identified as Enterococcus faecium by using phenotypic and genotypic tests such as 16S rDNA gene sequencing, RAPD-PCR and restriction fragment length polymorphism of the 16S/23S intergenic spacer region fingerprinting. Genotyping revealed that strains were not clonally related and exhibited a considerable degree of genomic diversity. Some strains possessed useful technological properties such as production of bacteriocins and H2O2 or utilization of raffinose and stachyose. None produced alpha-amylase or tannase. A safety investigation revealed that all strains were susceptible to the antibiotics ampicillin, gentamicin, chloramphenicol, tetracycline and streptomycin, but some were resistant to ciprofloxacin, erythromycin, penicillin and vancomycin. Production of biogenic amines or presence of genes encoding virulence determinants occurred in some strains. CONCLUSIONS: Enterococcus faecium strains are associated with fermentation of Sudanese Hussuwa. Some strains exhibited useful technological properties such as production of antimicrobial agents and fermentation of indigestible sugars, which may aid in stabilizing and improving the digestibility of the product respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococci were shown to play a role in the fermentation of African foods. While beneficial properties of these bacteria are indicated, their presence in this food may also imply a hygienic risk as a result of antimicrobial resistances or presence of virulence determinants.

Antibiotic resistance and virulence traits of enterococci isolated from Baylough, an Irish artisanal cheese.

Eight representative Enterococcus strains from a collection of over 600 previously isolated from an Irish artisanal cheese were subjected to phenotypic and genotypic analysis of antibiotic resistance and virulence properties. Genes encoding resistance to tetracycline (tet(M) and tet(L)) and/or erythromycin (erm(B)) were detected in five strains. In addition, all strains contained two or more of the virulence genes tested (agg, gel, cyl, esp, ace, efaAfs, and efaAfm). Further investigation into the transferability and environmental dissemination of these resistance and virulence traits will allow risk assessment and safety evaluation of artisanal cheeses.

Lactobacillus versmoldensis sp. nov., isolated from raw fermented sausage.

Lactobacillus versmoldensis sp. nov. (KU-3T) was isolated from raw fermented sausages. The new species was present in high numbers, and frequently dominated the lactic acid bacteria (LAB) populations of the products. 16S rDNA sequence data revealed that the isolates are closely related to the species Lactobacillus kimchii DSM 13961T, Lactobacillus paralimentarius DSM 13238T, Lactobacillus alimentarius DSM 20249T and Lactobacillus farciminis DSM 20184T. DNA-DNA reassociation data, however, clearly distinguished the new isolates from these species; they showed a low degree of DNA relatedness with the type strains of this group of phylogenetically closely related lactobacilli. These results warrant separate species status for strain KU-3T, for which the name Lactobacillus versmoldensis sp. nov. is proposed. The type strain is KU-3T (=DSM 14857T =NCCB 100034T =ATCC BAA-478T).

Incidence of virulence factors and antibiotic resistance among Enterococci isolated from food.

The incidence of virulence factors among 48 Enterococcus faecium and 47 Enterococcus faecalis strains from foods and their antibiotic susceptibility were investigated. No strain was resistant to all antibiotics, and for some strains, multiple resistances were observed. Of E. faecium strains, 10.4% were positive for one or more virulence determinants, compared to 78.7% of E. faecalis strains. Strains exhibiting virulence traits were not necessarily positive for all traits; thus, the incidence of virulence factors may be considered to be strain specific.

Population dynamics and antagonistic potential of enterococci colonizing the phyllosphere of grasses.

AIMS: To investigate the spatial and temporal dynamics of enterococci colonizing forage grass and their ability to produce bacteriocins. METHODS AND RESULTS: Enterococci could be detected on above-ground plant parts throughout the growing season, with high continuity but low cell numbers (2.60 x 101-6.16 x 104 cfu g-1 fresh matter). A total of 750 strains were isolated and identified by their whole-cell protein patterns as Enterococcus faecalis (7.9%), Ent. mundtii (7.9%), Ent. casseliflavus (5.5%), Ent. faecium (5.2%) and Ent. sulfureus (0.1%). The vast majority of the strains (69.7%) formed a homogeneous 16S rDNA genotype that differed from those of known enterococci. A screening for antagonistic activity using an agar spot test revealed that 18.4% of all isolates were potential antagonists. Partially-purified proteins extracted from cell-free culture supernatant fluids of various species were characterized as pH- and heat-stable bacteriocins active against a wide range of lactic acid bacteria, clostridia and Listeria. The producing strains were antagonistically active even on 'phylloplane agar' at temperatures between 4 and 37 degrees C. CONCLUSION: Enterococci are a common part of the epiphytic microflora of grasses, displaying probably some antagonistic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide new information on the distribution, species diversity and antagonistic potential of enterococci in the phyllosphere.

Bile salt hydrolase activity of Enterococci isolated from food: screening and quantitative determination.

One hundred seventeen enterococcal strains isolated from food (47 Enterococcus faecium, 48 Enterococcus faecalis, 16 Enterococcus durans, 2 Enterococcus gallinarum, 3 Enterococcus casseliflavus, and 1 Enterococcus malodoratus) were screened for bile salt hydrolase (BSH) activity on de Man, Rogosa, and Sharpe agar medium containing taurocholic acid and calcium chloride. The highest incidence of BSH-active strains was observed for E. faecalis (81%) followed by E. faecium (50%) and E. durans (44%). Isolates were grouped into four putative activity groups (no, low, medium, and high activity) based on the size of precipitation zones observed in the screening experiment. Our results showed that assumptions on BSH activity based on the size of bile precipitation zones in screening experiments did not correlate with actual activity as quantified by high-pressure liquid chromatography, but the screening assay is useful for assessing the presence or absence of BSH activity.

Simple method to identify bacteriocin induction peptides and to auto-induce bacteriocin production at low cell density.

The production of some bacteriocins by lactic acid bacteria is regulated by induction peptides (IPs) that are secreted by a dedicated secretion system. The IP gene cbaX, for carnobacteriocin A production by Carnobacterium piscicola LV17A, and a presumptive IP gene (orf6), associated with the genetic locus for enterocin B production in Enterococcus faecium BFE 900, were fused to the signal peptide of the bacteriocin divergicin A from Carnobacterium divergens LV13 to access the general secretory pathway. The culture supernatants of C. piscicola UAL26 and Lactococcus lactis MG1363 containing either of these constructs were used to induce bacteriocin production by Bac(-) cultures of C. piscicola LV17A or E. faecium CTC492. The cbaX fusion product induced bacteriocin production by Bac(-) C. piscicola LV17A, but the orf6 fusion product did not induce bacteriocin production by E. faecium CTC492. This represents a relatively simple method of confirming the role of presumptive IPs. The transformation of C. piscicola LV17A with the CbaX gene under expression of the P32 promoter from L. lactis resulted in constitutive production of bacteriocin by either the dedicated transport apparatus or the general secretory pathway.

Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces.

A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by alpha-chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS-PAGE, was approximately 3.4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B.

Enterococci at the crossroads of food safety?

Enterococci are gram-positive bacteria and fit within the general definition of lactic acid bacteria. Modern classification techniques resulted in the transfer of some members of the genus Streptococcus, notably some of the Lancefield's group D streptococci, to the new genus Enterococcus. Enterococci can be used as indicators of faecal contamination. They have been implicated in outbreaks of foodborne illness, and they have been ascribed a beneficial or detrimental role in foods. In processed meats, enterococci may survive heat processing and cause spoilage, though in certain cheeses the growth of enterococci contributes to ripening and development of product flavour. Some enterococci of food origin produce bacteriocins that exert anti-Listeria activity. Enterococci are used as probiotics to improve the microbial balance of the intestine, or as a treatment for gastroenteritis in humans and animals. On the other hand, enterococci have become recognised as serious nosocomial pathogens causing bacteraemia, endocarditis, urinary tract and other infections. This is in part explained by the resistance of some of these bacteria to most antibiotics that are currently in use. Resistance is acquired by gene transfer systems, such as conjugative or nonconjugative plasmids or transposons. Virulence of enterococci is not well understood but adhesins, haemolysin, hyaluronidase, aggregation substance and gelatinase are putative virulence factors. It appears that foods could be a source of vancomycin-resistant enterococci. This review addresses the issue of the health risk of foods containing enterococci.

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.

Characterisation and selection of probiotic lactobacilli for a preliminary minipig feeding trial and their effect on serum cholesterol levels, faeces pH and faeces moisture content.

Three out of 297 Lactobacillus strains isolated from pig faeces were selected for a feeding trial on account of their high bile-salt hydrolase (BSH) activity, bile-salt resistance, low pH tolerance and the production of antimicrobial substances. Two strains were identified as Lactobacillus johnsonii and one as Lactobacillus reuteri by DNA-DNA hybridisation. L. johnsoniii BFE 1061 produced a bacteriocin active against a range of lactic acid bacteria (LAB) and nonrelated bacteria including Clostridium perfringens. Six minipigs were maintained on a high-fat, high-cholesterol ('Western Style') diet for 17 weeks after which the diet was supplemented with the 'probiotic mixture' containing the above mentioned three Lactobacillus strains at 2 x 10(12) CFU per pig per day for five weeks. The mixture was given as a resuspended lyophilisate. During a two week follow-up period the minipigs received only the 'Western-style' diet without probiotic supplementation. A lowering effect on serum cholesterol levels was indicated after three weeks probiotic feeding, concomitant with an increase in the moisture content of the faeces and Lactobacillus cell numbers. Triglycerides, pH and number of lactic acid bacteria in faeces were not significantly influenced by probiotic supplementation.