Intraspecies genomic groups in Enterococcus faecium and their correlation with origin and pathogenicity

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of SmaI restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.     

Biochemical and genetic characterization of the two-peptide bacteriocin enterocin 1071 produced by Enterococcus faecalis FAIR-E 309

The structural genes for the two-peptide bacteriocin enterocin 1071 (Ent1071) in Enterococcus faecalis FAIR-E 309 were cloned. DNA sequence analysis showed that the enterocin 1071A (Ent1071A) peptide of strain FAIR-E 309 differed by two amino acids from the Ent1071A reported for E. faecalis BFE 1071 (E. Balla, L. M. T. Dicks, M. Du Toit, M. J. van der Merwe, and W. H. Holzapfel, Appl. Environ. Microbiol. 66:1298-1304, 2000), while the Ent1071B gene encoded identical peptides in these strains. However, resequencing of ent1071A from E. faecalis BFE 1071 showed that the Ent1071A peptide sequence reported previously was incorrect in two amino acids. Also, ent1071B in E. faecalis FAIR-E 309 encoded a prepeptide that was three amino acids shorter than that previously reported for E. faecalis BFE 1071 Ent1071B. A presumptive immunity gene (eni1071) was located downstream of the bacteriocin structural genes. This gene was cloned into the heterologous host E. faecalis ATCC 19433 and was shown to confer immunity. A truncated ABC transporter gene was located upstream of the Ent1071 structural genes.     

Bacteriocin production ofLeuconostoc carnosum LA54A at different combinations of pH and temperature

Summary Leuconostoc carnosum LA54A produces a bacteriocin which is active againstListeria monocytogenes andListeria innocua. The ability ofLc. carnosum to produce the bacteriocin at various combinations of the growth parameters pH and temperature was analyzed. In the case of this strain bacteriocin production seems to be coupled to growth rate. This fact enables the prediction ifLc. carnosum can produce the bacteriocin at a given set of growth parameters, simply by predicting the growth rate of this organism. In addition we have analyzed the growth behavior of the target organismL. innocua WS2258 at the same set of growth parameters.     

Determination of layer-specific mechanical properties of human coronary arteries with nonatherosclerotic intimal thickening and related constitutive modeling

At autopsy, 13 nonstenotic human left anterior descending coronary arteries [71.5 +/- 7.3 (mean +/- SD) yr old] were harvested, and related anamnesis was documented. Preconditioned prepared strips (n = 78) of segments from the midregion of the left anterior descending coronary artery from the individual layers in axial and circumferential directions were subjected to cyclic quasi-static uniaxial tension tests, and ultimate tensile stresses and stretches were documented. The ratio of outer diameter to total wall thickness was 0.189 +/- 0.014; ratios of adventitia, media, and intima thickness to total wall thickness were 0.4 +/- 0.03, 0.36 +/- 0.03, and 0.27 +/- 0.02, respectively; axial in situ stretch of 1.044 +/- 0.06 decreased with age. Stress-stretch responses for the individual tissues showed pronounced mechanical heterogeneity. The intima is the stiffest layer over the whole deformation domain, whereas the media in the longitudinal direction is the softest. All specimens exhibited small hysteresis and anisotropic and strong nonlinear behavior in both loading directions. The media and intima showed similar ultimate tensile stresses, which are on average three times smaller than ultimate tensile stresses in the adventitia (1,430 +/- 604 kPa circumferential and 1,300 +/- 692 kPa longitudinal). The ultimate tensile stretches are similar for all tissue layers. A recently proposed constitutive model was extended and used to represent the deformation behavior for each tissue type over the entire loading range. The study showed the need to model nonstenotic human coronary arteries with nonatherosclerotic intimal thickening as a composite structure composed of three solid mechanically relevant layers with different mechanical properties. The intima showed significant thickness, load-bearing capacity, and mechanical strength compared with the media and adventitia.     

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.     

Reproductive value in a complex life cycle: heat tolerance of the pitcher-plant mosquito, Wyeomyia smithii

Because mortality accumulates with age, Fisher proposed that the strength of selection acting on survival should increase from birth up to the age of first reproduction. Hamilton later theorized that the strength of selection acting on survival should not change from birth to age at first reproduction. As organisms in nature do not live in uniform environments but, rather, experience periodic stress, we hypothesized that resistance to environmental stress should increase (Fisher) or remain constant (Hamilton) from birth to age at first reproduction. Using the pitcher-plant mosquito, Wyeomyia smithii, we imposed heat stress by simulating the passage of a warm-weather front at different pre-adult and adult stages. Contrary to either Fisher or Hamilton, stress tolerance declined from embryos to larvae to pupae to adults. Consequently, reproductive value appears to have been of little consequence in the evolution of stage-specific tolerance of heat stress in W. smithii.     

Initial licking responses of mice to sweeteners: effects of tas1r3 polymorphisms

Recent studies have established that the T1R3 receptor plays a central role in the taste-mediated ingestive response to sweeteners by mice. First, transgenic mice lacking the gene for T1R3, Tas1r3, show dramatically reduced lick responsiveness to most sweeteners. Second, strains with the taster allele of Tas1r3 (T strains) are more sensitive to low sweetener concentrations than strains with the nontaster allele (NT strains) and consume greater quantities of low- to midrange concentrations of sweeteners during 24-h tests. We asked how Tas1r3 polymorphisms influence the initial licking responses of four T strains (FVB/NJ, SWR/J, SM/J, and C57BL/6J) and four NT strains (BALB/cJ, 129P3/J, DBA/2J, and C3H/HeJ) to two sweeteners (sucrose and SC-45647, an artificial sweetener). We used the initial licking response as a measure of the taste-mediated ingestive response because its brief duration minimizes the potential contribution of nontaste factors (e.g., negative and positive postingestive feedback). Further, we used two complimentary short-term intake tests (the brief-access taste test and a novel 1-min preference test) to reduce the possibility that our findings were an epiphenomenon of a specific testing procedure. In both tests, the T strains were more responsive than the NT strains to low concentrations of each sweetener. At higher concentrations, however, there was considerable overlap between the T and NT strains. In fact, the initial licking response of several NT strains was more vigorous than (or equivalent to) that of several T strains. There was also considerable variation among strains with the same Tas1r3 allele. We conclude that Tas1r3 polymorphisms contribute to strain differences in initial lick responsiveness to low but not high concentrations of sweeteners.     

Diversity and technological properties of predominant lactic acid bacteria from fermented cassava used for the preparation of Gari, a traditional African food

Traditional fermentation of cassava is dominated by a lactic acid bacteria (LAB) population. Fermentation is important for improving product flavour and aroma as well as safety, especially by reduction of its toxic cyanogenic glucosides. The production of Gari from cassava in Benin typically occurs on a household or small industrial scale, and consequently suffers from inconsistent product quality and may not always be safe for consumption. Therefore, the diversity of LAB from a typical cassava fermentation for the preparation of Gari, and their technologically relevant characteristics were investigated with a view towards selection of appropriate starter cultures. A total of 139 predominant strains isolated from fermenting cassava were identified using phenotypic tests and genotypic methods such as rep-PCR and RAPD-PCR. DNA-DNA hybridisation and sequencing of the 16S rRNA genes were done for selected strains. Lactobacillus plantarum was the most abundantly isolated species (54.6% of isolates), followed by Leuconostoc fallax (22.3%) and Lactobacillus fermentum (18.0%). Lactobacillus brevis, Leuconostoc pseudomesenteroides and Weissella paramesenteroides were sporadically isolated. The L. plantarum strains were shown to be better acid producers and capable of faster acid production than the L. fallax or L. fermentum strains. The incidence of beta-glucosidase (linamarase) activity was also highest among strains of this species. Production of antagonistic substances such as H2O2 and bacteriocins, however, was more common among L. fallax and L. fermentum strains. Strains of all three species were capable of utilising the indigestible sugars raffinose and stachyose. Therefore, a starter culture containing a mixture of strains from all three species was recommended.     

Characterization and Cloning of the Genes Encoding Enterocin 1071A and Enterocin 1071B, Two Antimicrobial Peptides Produced by Enterococcus faecalis BFE 1071

The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Göttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100°C, 60 min; 50% of activity remained after 15 min at 121°C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A and ent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the α and β peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.