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.

Associations of Knowledge, Attitude, and Practices toward Anemia with Anemia Prevalence and Height-for-Age Z-Score among Indonesian Adolescent Girls.

BACKGROUND: Crucial gaps persist in knowledge, attitude, and practice (KAP) of adolescent girls that affect anemia and linear growth failure. OBJECTIVE: To understand the role of KAP as a risk factor of anemia and linear growth problem in adolescent girls. METHODS: We conducted a cross-sectional survey of 335 adolescent girls selected by clustered random sampling. The KAP questionnaire had 18 variables consisting of 9 knowledge, 3 attitude, and 6 practice components. Twelve variables addressed nutrition, dietary diversity, and health environments related to both anemia and stunting. The questionnaire was adapted from the 2014 Food and Agriculture Organization nutrition-related KAP guidelines for anemia. Dietary practice was evaluated from 2-day 24-hour recalls and a semi-quantitative food-frequency questionnaire. Associations between KAP and anemia, and height-for-age z-score (HAZ), were analyzed using multivariate logistic and linear regression models, respectively. RESULTS: The mean hemoglobin (Hb) level was 119.7 g/L, with 44% of the adolescent girls being anemic (Hb < 120 g/L) and mean height was 151.0 cm with 25% being stunted (HAZ < -2 standard deviation [SD]). The median KAP score was 7 and ranged from 3 to 10. Low to moderate KAP scores were not significantly associated with being anemic (adjusted odds ratio [AOR] = 1.26; P = .43), however 1-point KAP score increment was associated with an increase of HAZ by 0.037 SD (P = .012). CONCLUSIONS: The KAP related to diet and healthy environments was not associated with anemia prevalence, but was positively associated with increased HAZ among adolescent girls. Strategy to reduce anemia risk in this population should combine KAP improvement with other known effective nutrition interventions.

Associations of meal patterning, dietary quality and diversity with anemia and overweight-obesity among Indonesian school-going adolescent girls in West Java.

BACKGROUND: Poor diet is a risk factor for anemia, overweight, and obesity among adolescent girls. However, comprehensive assessment on dietary quality and habits in this population is limited. We assessed the association of meal patterning, dietary quality, and dietary diversity with both anemia and overweight-obesity. METHODS: We conducted a cross-sectional survey in 335 school-going adolescent girls aged 12-19 years from three districts in West Java using multi-stage cluster sampling. Meal patterning, Dietary Quality Index for Adolescents (DQI-A), and Dietary Diversity Score (DDS) were determined using 2-day 24-h recall. RESULTS: Of the girls, 45% were anemic and 17% overweight or obese. Eating occasions of 3-4 times (AOR 2.68, 95% CI 1.21-5.98) and >4 times (AOR 2.43, 95% CI 1.01-5.83) were associated with greater odds of developing anemia compared to eating occasions of <3 times. Adolescent girls who skipped dinner had greater odds of being overweight or obese (AOR 2.13, 95% CI 1.10-4.10) and were less likely to be anemic (AOR 0.56, 95%CI 0.33-0.95) compared to those who did not skip dinner. Difference in energy intake was found between girls who had dinner and skipped dinner (p = 0.05). Mean total DQI-A score was 44.4% ± 7.71% and DDS was 4.0 out of 9.0. DQI-A score was significantly higher in non-anemic compared to anemic girls. Moreover, each unit increment of 1% of total DQI-A score was associated with a 3.967 g/dL increases of hemoglobin after adjustment for confounders. We found differences in total DQI-A score between normal-weight and overweight or obese girls. DDS score was not significantly different between groups, although lower meat, chicken, and fish consumption were correlated with anemia (p<0.01). CONCLUSIONS: Overall, the girls had poor dietary quality and diversity. The findings therefore indicated the importance of improving dietary quality and diversity in a regular meal pattern, especially meal frequency and meal skipping, to reduce the risk of anemia and overweight-obesity among adolescent girls.

Enterococcus faecium SF68 as a model for efficacy and safety evaluation of pharmaceutical probiotics.

As normal inhabitants of diverse ecosystems, including the human gastrointestinal tract, the enterococci, and especially the two species Enterococcus faecalis and Enterococcus faecium, can be considered ubiquitous with regard to our natural environment. E. faecium has gained special importance thanks to beneficial strains marketed as probiotics, and because of its beneficial role in traditional fermented foods such as artisanal cheeses in some Southern European countries. Yet, following reports on the increasing association of some enterococcal strains with nosocomial infections such as endocarditis and bacteraemia, it became evident that strains from clinical origin are frequently highly resistant to 'last-defence-line' antibiotics such as the glycopeptide derivatives. For this reason enterococci have been classified in risk group 2 in the European Directive 93/88. With this paper it is intended to clarify the uncertain situation around the safety of the species E. faecium, also with referring to intra-species heterogeneity. In fact, well established scientific and surveillance data support the safety of some probiotic E. faecium strains for both human and animal applications. As a model, summarising yet extensive information is provided on the efficacy and safety of E. faecium SF68®, a pharmaceutical probiotic with a long history of safe use. We propose the approach presented in this review as a model for the evaluation of safety of probiotic strains of this species.

Oral mucosa of coeliac disease patients produces antiendomysial and antitransglutaminase antibodies: the diagnostic usefulness of an in vitro culture system.

BACKGROUND: Antiendomysial (EmA) and antitransglutaminase (anti-tTG) antibodies are the most specific indirect marker of coeliac disease (CD). It is not known whether the oral mucosa of patients with CD is able to produce these antibodies or not. AIMS: To evaluate the ability of the oral mucosa of patients with CD to produce antibodies in an in vitro culture system. PATIENTS AND METHODS: Twenty-eight patients with new diagnosis of CD (15 adults and 13 children) and 14 adult subjects with other diseases (controls) were studied. All underwent oral mucosa biopsy and subsequent EmA and anti-tTG assays on the mucosa culture medium. RESULTS: Sensitivity and specificity of EmA and anti-tTG assayed in the oral mucosa culture medium for CD diagnosis were 54% and 100% and 57% and 100%, respectively. The CD clinical presentation, such as the presence of oral mucosa lesions, did not influence the results of the EmA and anti-tTG assays in the oral mucosa culture medium. There was an association between positivity of antibodies and greater severity of the oral mucosa lymphocyte infiltration. CONCLUSION: This study demonstrates that the oral mucosa contributes to EmA and anti-tTG production in untreated patients with CD.

Metal detoxification and gene expression regulation after a Cd and Zn contamination: an experimental study on Danio rerio.

This study aimed to demonstrate the recovery potential of Danio rerio after Cd and Zn contaminations. Fish demonstrated high accumulation capacities of Cd with concentrations reaching 3716.4±578.6 µg Cd/kg FW in gills after 15 d of contamination. The 75-day decontamination failed to completely eliminate Cd (93.4% and 82.2% eliminated respectively in the gills of fish exposed to Cd and Cd/Zn) whereas Zn, poorly accumulated, was quickly depurated. The fast Cd depuration in the gills likely resulted from a metal transfer to the liver. MT response was clearly correlated to the Zn contamination, while genetic responses were more pronounced in case of Cd contamination. Cd induced over-expressions of genes involved against oxidative stress (sod, sodmt), and involved in detoxification mechanisms (mt1, mt2), mitochondrial mechanisms (cox1) and DNA repair (rad51 and gadd45). Zn binary contamination with Cd was demonstrated to provide protective effects on Cd-induced toxicity in D.rerio. Results highlighted that the genetic response was metal- and tissue-dependent. The brain and the muscles showed very few genetic responses, probably due to the low bioaccumulations measured in these tissues. Conversely, genes expressed in gills and liver of fish exposed to Cd were strongly affected (sod×3 and×12, mt1×11 and ×30 at T3 respectively in gills and liver). However, after 14-30 d of depuration, genes were no longer over expressed in response to Cd contamination in gills and liver of fish exposed to Cd and Cd/Zn conditions, suggesting an gene expression regulation of fish to the residual Cd contamination.

Lack of hotspot targets: a constraint for IS30 transposition in Salmonella.

IS30 is an insertion element common in E. coli strains but rare or absent in Salmonella. Transfer of the IS30-flanked transposon Tn2700 to Salmonella typhimurium was assayed using standard delivery procedures of bacterial genetics (conjugation and transduction). Tn2700 'hops' were rare and required transposase overproduction, suggesting the existence of host constraints for IS30 activity. Sequencing of three Tn2700 insertions in the genome of S. typhimurium revealed that the transposon had been inserted into sites with a low homology to the IS30 consensus target, suggesting that inefficient Tn2700 transposition to the Salmonella genome might be caused by a lack of hotspot targets. This view was confirmed by the introduction of an IS30 'hot target sequence', whose sole presence permitted Tn2700 transposition without transposase overproduction. Detection of IS30-induced DNA rearrangements in S. typhimurium provided further evidence that the element undergoes similar activities in E. coli and S. typhimurium. Thus, hotspot absence may be the main (if not the only) limitation for IS30 activity in the latter species. If these observations faithfully reproduce the scenario of natural populations, establishment of IS30 in the Salmonella genome may have been prevented by a lack of DNA sequences closely related to the unusually long (24 bp) IS30 consensus target.

Radiozinc spermatogenesis test: follow-up semen analysis.

PIP: Semen analysis was performed on 8 men, every 2 months, for a period of 8 months, after the application of the radiozinc spermatogensis test. Semen was examined for volume, cell count, motility, and abnormal forms. Results showed no evidence of any changes in semen parameters. Student's test was used to compare the data before the test with the data after the test, and no significant differences were seen. The radiozinc spermatogensis test seems safe, from a radiologic point of view, does not have an adverse effect on semen, and unlike testicular biopsy, saves the patient from surgical exposure. The radiozinc spermatogensis test can be applied as an important adjuvant measure in the diagnosis and prognosis of male infertility.^ieng

What are the outcomes of an industrial remediation on a metal-impacted hydrosystem? A 2-year field biomonitoring of the filter-feeding bivalve Corbicula fluminea.

The Riou-Mort watershed (southwest France) exhibits high metal contaminations (Cd and Zn) related to an important mining past. In this context, a remediation process has been implemented in 2007 to reduce the watershed contamination. The aim of this study was to assess the early effectiveness of the remediation process on the hydrosystem contamination state. A biomonitoring was realized over two years (2008-2010) with the filter-feeding bivalve Corbicula fluminea, exposed along a contamination gradient. Several biological parameters were monitored: (1) Cd and Zn bioaccumulation, (2) Metallothionein (MT) production as detoxification mechanism, (3) differential gene expression (cat, sod, gst, 12S, cox1, mt). The physicochemical data highlighted strong metal contamination persistence in the river water and failed to demonstrate a significant decrease of metal contamination during the 2-year monitoring. The bioaccumulation results confirmed the persistence of a water contamination despite remediation works, with maximum values measured downstream from the industrial site (Joany). The bioaccumulation increased in 2010, reaching 69.3±5.3 µg Cd g(-1) DW at Joany in July 2010, whereas it did not exceed 1.4±0.2 µg Cd g(-1) DW at the reference site throughout the biomonitoring. MT concentrations were closely related to the contamination gradient, especially at Joany, demonstrating their strong involvement in the detoxification processes. The mt gene induction was strongly correlated to the MT and metal concentrations. The gene inductions of cat, sod, gst and 12S were correlated to both the metal concentrations and the seasonal variations, especially temperatures. This suggests that environmental factors require serious consideration for the interpretation of bioaccumulation kinetics and thus for the assessment of the remediation effectiveness. Consequently, the whole results did not yet highlight strong beneficial effects of remediation work on the hydrosystem contamination state. First benefits of that process should be progressively felt, once the remediation achieved, and should grandly accelerate the decontamination process of the contaminated area.

Detoxification and recovery capacities of Corbicula fluminea after an industrial metal contamination (Cd and Zn): a one-year depuration experiment.

This study aimed to assess the recovery capacity of the freshwater bivalve Corbicula fluminea subjected to industrial metal discharges (Cd, Zn). After a 24-day exposure in a metal-contaminated river, bivalves were transferred and maintained in the laboratory for one year under metal-free conditions. Metal accumulation, metallothionein production and genetic expressions of genes involved in metal stress were studied. Results demonstrated the high persistence of Cd in tissues (only 73% eliminated after 365 days) whereas Zn was rapidly depurated. The Cd half-life was estimated around 240 days. Metallothioneins were strongly induced within the 28 first days of decontamination, then decreased by 45% after 365 days. The metal exposure of bivalves led to a significant gene induction. After 28 days, most of the genes were no longer overexpressed, suggesting that the bivalves may withstand small amounts of non-essential metals in their tissues without showing signs of detrimental effects on the tested genes.

Replication origin mutations affecting binding of pSC101 plasmid-encoded Rep initiator protein.

To investigate the role of binding sites for Rep initiation protein in the replication of pSC101, a series of plasmids was constructed which carried different combinations of mutations in three binding sites within the minimal origin of replication. Mutation of all three sites reduced the affinity of purified Rep protein for the origin by 100-fold, as measured by a competition binding assay. Mutations in individual binding sites prevented binding of Rep protein to the mutant site but not to adjacent wild-type sites. Transformation efficiency, copy number, and stability over 150 generations were measured for each of the mutant plasmids. Unlike other similar plasmids related to pSC101, the Rep binding sites were found not to be equivalent. A mutation in the site RS1, proximal to repeated sequences which serve as DnaB helicase entry sites in oriC, had a severe effect on replication activity. A similar mutation in the distal site RS3 caused a reduction in copy number, but the mutant plasmid was stably maintained despite a broadened distribution of copy number within the population. A mutation in the middle RS2 site had no significant effect on pSC101 replication.

An antisense RNA in IS30 regulates the translational expression of the transposase.

Two functional promoters had previously been identified in the mobile genetic element IS30 of Escherichia coli. One, P30A, controls the transcription of ORF-A, whose product is the transposase; the other, located within the ORF-A sequence but on the opposite strand, is called P30C, but the nature and function of its product had remained unknown. We identified this product as an RNA about 150 nucleotides long (called RNA-C) that functions as an untranslated antisense transcript. Indeed, biochemical evidence indicates that ORF-C, which is completely contained on RNA-C, is not translated at detectable levels. Mutational analysis of P30C revealed that overproduction of RNA-C resulted in a decrease of IS30 transposition, while a reduction in the promoter strength resulted in an increase of transposition, as measured by the rate of cointegrate formation. We showed that the translation of ORF-A, but not transcription, is negatively affected by the presence of antisense RNA-C. In contrast to other antisense RNAs acting inhibitorily on translation, RNA-C does not seem to affect translation initiation. Most likely its hybridization to the transposase mRNA in the complementary region located in the central part of ORF-A inhibits the ribosomes in their progression, thus reducing the number of completely translated transposase molecules.

[Epidemiology of Neisseria gonorrhoeae isolated in Switzerland: sensitivity to antibiotics and auxotyping]. / Epidémiologie de Neisseria gonorrhoeae isolées en Suisse: sensibilité aux antibiotiques et auxotypie.

In a study to determine the epidemiological situation of N. gonorrhoeae (NG) in Switzerland, 149 strains isolated from different regions over the period September 1981 to February 1982 were analyzed for sensitivity to various antibiotics (minimum inhibitory concentration [MIC] for penicillin G, ampicillin, cefuroxime, tetracycline, thiamphenicol, and spectinomycin) and for their auxotype. From the results obtained it can be concluded that (1) the PPNG (penicillinase producing NG) strains are not a problem at present in Switzerland; (2) 15% of the non-penicillinase producing NG strains are "less sensitive" to penicillin G (MIC equal to or greater than 0.5 micrograms/ml); (3) sensitivity level of the NG strains to the various antibiotics assayed and the auxotypes do not show any significant peculiarity when compared to the data reported in other countries; (4) penicillin G should still be used as the first choice antibiotic at present in Switzerland, with spectinomycin, thiamphenicol and cefuroxime as second choice antibiotics; (5) when compared according to the geographical origin of their isolation, the NG strains show specific peculiarities, a fact which could lead to an antibiotic therapy policy more suited to specific regions.

In vitro activity of norfloxacin against Neisseria gonorrhoeae.

Norfloxacin, an analogue of nalidixic acid, is a novel antibacterial compound active against both gram-negative and gram-positive pathogens. 142 Neisseria gonorrhoeae isolates proved to be remarkably sensitive to it; the MIC50 and MIC90 values for norfloxacin were 0.03 microgram/ml and 0.06 microgram/ml, respectively, for the strains resistant to the penicillins; for the strains susceptible to the penicillins, the same value of 0.03 microgram/ml was found both for the MIC50 and MIC90. 100% of the strains were inhibited by 0.125 microgram/ml of the drug: this concentration is far below the serum levels (1.5 micrograms/ml) achievable following normal dosage. Clinical trials should be performed to establish whether the activity in vivo corresponds to that found in vitro.

pUB307 mobilizes resistance plasmids from Escherichia coli into Neisseria gonorrhoeae.

The plasmid pUB307, a derivative of RP1, is a conjugative, broad-host-range plasmid. We have shown that this element mobilizes gonococcal resistance plasmids from Escherichia coli to Neisseria gonorrhoeae, thus providing evidence that extrachromosomal elements can efficiently enter gonococci by conjugation. Furthermore, pUB307 can also be used as a helper element to mobilize the cloning vector pLES2 into N. gonorrhoeae. This finding significantly increases the usefulness of pLES2 as a shuttle vector between E. coli and gonococcus.

Specific enumeration of the probiotic strain Enterococcus faecium NCIMB 10415 in the intestinal tract and in faeces of piglets and sows.

The intestinal bacterium Enterococcus faecium NCIMB 10415 (E. faecium SF68) has been used for more than a decade as a probiotic strain in animal nutrition as well as in the prevention and treatment of diarrhoea in humans. Beneficial effects have been shown in feeding and clinical trials. However, the strain has no selective growth markers and monitoring in the intestinal tract is impossible by cultivation. Using specific nucleotide sequences, in this study a probe for colony hybridization was constructed in order to quantify this probiotic strain in feed and intestinal and faecal samples from piglets and sows. The probiotic strain showed almost constant amounts in sow faeces (1.8 x 10(5) cfu/g wet weight), while contents in digesta and piglet faeces varied on a lower level depending on gut section and piglet age. The ratio of specific probiotic counts and total enterococci was much lower than in sow faeces however the strain could be detected reliably in faeces already on the 14th day of life. The application of the colony hybridization method enables for the first time the selective detection of the widely used probiotic E. faecium NCIMB 10415 strain among total Enterococcus spp. counts of digesta, faeces and feed. It is now possible to monitor the presence of the probiotic in the intestinal tract and faeces. Results of this study have implications for the proposed modes of action of probiotics in animal nutrition.

A novel type of resistance plasmid in Haemophilus influenzae.

Resistance plasmids of a novel type were found in two Haemophilus influenzae clinical isolates. pPJ301 and pPJ302 are 10.0 kilobases in size, carry a Tn2-like transposable element, and are related only by their common beta-lactamase genes to the other two types of resistance plasmids known to occur in H. influenzae.

Terminal inverted repeats of insertion sequence IS30 serve as targets for transposition.

In the present study, we demonstrate that the terminal inverted repeats of the Escherichia coli insertion sequence IS30 are functional target sites for the transposition of the (IS30)2 dimer, which represents an intermediate structure in the transposition of IS30. Comparative analysis of various target regions revealed that the left and right ends differ in their "attractivity." In our experiments, the joined left and right ends, i.e., the (IS30)2 intermediate structure, was found to be the most preferred target. It was also shown that flanking sequences can influence the target activity of the terminal repeats. The functional part of the target region was localized in the inverted repeats by means of mutational analysis, and it corresponds to the binding site of IS30 transposase. Insertion of 1 bp into the right inverted repeat resulted in unusual target duplication accompanied by gene conversion. The choice of the terminal inverted repeats as targets in transposition leads to the reconstruction of the (IS30)2 structure, which may induce a cascade of further rearrangements. Therefore, this process can play a role in the evolution of the genome.

Radiation induced sarcomas following treatment for breast cancer: presentation of a series of 14 cases treated with an aggressive surgical approach.

According to the criteria established by Cahan et al., we report 14 radiation induced sarcomas occurring in the treated volume after radiation therapy for adenocarcinoma of the breast. All these sarcomas were treated by wide surgical resection. In spite of a macroscopic free margin, only patients who underwent major amputation could hope to be cured. All the centrally located sarcomas recurred.