Prevalence and risk factor assessment of Tropheryma whipplei in a rural community in Gabon: a community-based cross-sectional study.

Tropheryma whipplei is the causative agent of Whipple's disease and has been detected in stools of asymptomatic carriers. Colonization has been associated with precarious hygienic conditions. There is a lack of knowledge about the epidemiology and transmission characteristics on a population level, so the aim of this study was to determine the overall and age-specific prevalence of T. whipplei and to identify risk factors for colonization. This molecular epidemiological survey was designed as a cross-sectional study in a rural community in Central African Gabon and inhabitants of the entire community were invited to participate. Overall prevalence assessed by real-time PCR and sequencing was 19.6% (95% CI 16-23.2%, n=91) in 465 stool samples provided by the study participants. Younger age groups showed a significantly higher prevalence of T. whipplei colonization ranging from 40.0% (95% CI 27.8-52.2) among the 0-4 year olds to 36.4% (95% CI 26.1-46.6) among children aged 5-10 years. Prevalence decreased in older age groups (p<0.001) from 12.6% (95% CI 5.8-19.4%; 11-20 years) to 9.7% (95% CI 5.7-13.6) among those older than 20. Risk factor analysis revealed young age, male sex, and number of people sharing a bed as factors associated with an increased risk for T. whipplei carriage. These results demonstrate that T. whipplei carriage is highly prevalent in this part of Africa. The high prevalence in early life and the analysis of risk factors suggest that transmission may peak during childhood facilitated through close person-to-person contacts.

Biofilms augment the number of free-living amoebae in dental unit waterlines.

Freshwater amoebae are ubiquitous. Some species can cause infections in humans while others can ingest and protect opportunistic bacteria. Although the presence of free-living amoebae in various water sources has been reported, few studies have looked at their concentration, which may be clinically relevant, especially if they are present in healthcare devices. A simple technique was used to detect, observe, and evaluate the concentration of free-living amoebae in dental unit and tap water samples. Fifty-three water samples were collected from 35 dental units (air/water syringes) and 18 water taps. The technique was based on the ability of waterborne bacteria to create a biofilm and serve as substratum for the development of amoebae naturally present in the water samples. Laboratory-grown freshwater biofilms support the proliferation of a wide variety of free-living amoebae. All the dental unit water samples tested contained amoebae at concentrations up to 330/mL, or more than 300 times the concentration in tap water from the same source. Hartmanella, Vanella, and Vahlkampfia spp. were the most frequently encountered. Naegleria and Acanthamoeba spp. were also present in 40% of the samples. Four of the samples collected from dental units, but none from water taps, contained amoebae able to proliferate at 44 degrees C. Biofilms that form inside some dental instruments can considerably increase the concentration of free-living amoebae, some of which are potential human pathogens.

Increasing resistance of planktonic and biofilm cultures of Burkholderia cepacia to ciprofloxacin and ceftazidime during exponential growth.

The change in resistance of Burkholderia cepacia to ceftazidime and to ciprofloxacin during the exponential phase and up to the onset of stationary phase was assessed along the growth curve in batch culture. B. cepacia was grown in planktonic culture and in a biofilm on a membrane support. Resistance increased progressively during the exponential phase, being increased by ten-fold about every four generations. Bacteria grown in a biofilm were about 15 times more resistant than equivalent planktonic-grown bacteria. The growth rate was not the key factor for the development of resistance. The growth phase and the mode of growth have a fundamental impact on the susceptibility of B. cepacia towards antimicrobial agents. Bacteria growing at the same rate may differ greatly in their resistance to antimicrobial agents.

Generation of a reproducible nutrient-depleted biofilm of Escherichia coli and Burkholderia cepacia.

An in vitro method of growing bacteria as a defined nutrient-depleted biofilm is proposed. The medium was defined nutritionally in terms of the quantitative composition and by the total amount of nutrient required to achieve a defined population size. Escherichia coli and Burkholderia cepacia were incubated on a filter support placed on a defined volume of solid medium. The change of biomass of the biofilm population was compared with the change in a planktonic culture. The size of the population in stationary phase was proportional to the concentration of limiting substrate up to 40 mumol cm-1 glucose for E. coli and up to 2.7 x 10(-9) mol cm-2 iron for B. cepacia. Escherichia coli growing exponentially had a growth rate of mu = 0.30 h-1 in a biofilm and mu = 0.96 h-1 in planktonic culture. The growth rate, mu, for exponentially growing B. cepacia in a biofilm was 1.12 h-1 and in planktonic culture 0.78 h-1. This method allows the limitation of the size of a biofilm population to a chosen value.

Appropriate coating methods and other conditions for enzyme-linked immunosorbent assay of smooth, rough, and neutral lipopolysaccharides of Pseudomonas aeruginosa.

Smooth, rough, and neutral forms of lipopolysaccharide (LPS) from Pseudomonas aeruginosa were used to assess the appropriate conditions for effective enzyme-linked immunosorbent assay (ELISA) of LPS. Each of these forms of well-defined LPS was tested for the efficiency of antigen coating by various methods as well as to identify an appropriate type of microtiter plate to use. For smooth LPS, the standard carbonate-bicarbonate buffer method was as efficient as the other sensitivity-enhancing plate-coating methods compared. The rough LPS, which has an overall hydrophobic characteristic, was shown to adhere effectively, regardless of the coating method used, to only one type of microtiter plate, CovaLink. This type of plate has secondary amine groups attached on its polystyrene surface by carbon chain spacers, which likely favors hydrophobic interactions between the rough LPS and the well surfaces. Dehydration methods were effective for coating microtiter plates with the neutral LPS examined, which is composed predominantly of a D-rhamnan. For the two dehydration procedures, LPS suspended in water or the organic solvent chloroform-ethanol was added directly to the wells, and the solvent was allowed to dehydrate or evaporate overnight. Precoating of plates with either polymyxin or poly-L-lysine did not give any major improvement in coating with the various forms of LPS. The possibility of using proteinase K- and sodium dodecyl sulfate-treated LPS preparations for ELISAs was also investigated. Smooth LPS prepared by this method was as effective in ELISA as LPS prepared by the hot water-phenol method, while the rough and neutral LPSs prepared this way were not satisfactory for ELISA.

Genetic analysis of the gene cluster encoding nonfimbrial adhesin I from an Escherichia coli uropathogen.

The chromosomally encoded nonfimbrial adhesion I (NFA-I) from Escherichia coli urinary tract isolate 827 (O83:K1:H4) mediates agglutination of human erythrocytes. Subclones were constructed from an NFA-I-expressing recombinant E. coli K-12 clone, derived from a genomic library of E. coli 827. Minicell analysis and nucleotide sequencing revealed that proteins of 30.5, 9, 80, 15, and 19 kDa encoded on a stretch of approximately 6 kb are involved in the expression of NFA-I. NFA-I exhibits a polymeric structure, which disintegrates with elevated temperature into a 19-kDa monomer but with some relatively stable dimers. By using gold-conjugated monoclonal antibodies directed against NFA-I in electron microscopy, the adhesin could be localized on the outer surface of the recombinant E. coli K-12 bacteria. The nucleotide sequence of the nfaA gene encoding the monomeric structural subunit of the adhesin was determined. An open reading frame of 184 amino acids encoding the NfaA precursor, which is processed to the mature protein, was found; it consisted of 156 amino acids with a calculated molecular weight of 16,000. Peptide sequencing of the NFA-I subunit protein confirmed that this open reading frame corresponds to the NfaA coding locus. Furthermore, the nucleotide sequence of the open reading frame termed NfaE, located at the proximal part of the DNA stretch responsible for NFA-I expression, was elaborated. NfaE consists of 247 amino acids, including a presumptive 29-amino-acid signal peptide, leading to a molecular weight of 24,000 for the mature protein. The nfaE sequence shares homology with the 27-kDa CS3 protein, which is involved in the assembly of CS3 fibrillae, and might encode the 30.5-kDa protein, detected in minicells.

Localization and quantification of Wnt-2 gene expression in mouse mammary development.

The Wnt gene family encodes a group of proteins probably involved in cell-cell communication during several stages of vertebrate development. More than 10 members of this family have been identified and shown to be expressed mainly in developing neural tissue. Using a reverse transcription-polymerase chain reaction (RT-PCR)-based approach with degenerate oligonucleotides directed against conserved sequences in the Wnt genes, Wnt-2 transcripts were detected in RNA isolated from mammary glands of 4- to 6-week-old virgin C3H mice, a period characterized by extensive end bud and ductal proliferation. The spatial and temporal expression of Wnt-2 in the developing mouse mammary gland was studied by in situ hybridization, quantitative RT-PCR, and Northern analysis. Wnt-2 is expressed during the ductal phase of mammary gland development, primarily in the basal layer of mammary ducts and in the body cells of end buds. Wnt-2 RNA transcripts were readily detected in poly(A) RNA isolated from 5-week-old C3H and Balb/c mice. RNA transcript levels measured as molecules per nanogram of total RNA by RT-PCR decreased 10- to 40-fold within 2 days after the onset of pregnancy and remained low during pregnancy and lactation. This is in contrast to the patterns of expression of other Wnt family members, Wnt-5a and -5b, whose expression was either barely or not detectable in the 4- to 6-week-old mammary gland, but increased markedly during pregnancy. These results confirm the differential expression of Wnt gene family members during mammary gland development. Furthermore, they suggest that Wnt-2, as well as several other family members, may play a role in pattern formation during early mammary gland development.

Analysis of colonization factor antigen I, an adhesin of enterotoxigenic Escherichia coli O78:H11: fimbrial morphology and location of the receptor-binding site.

Colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli was dissociated into one type of subunit (15 kDa). The dissociation was achieved either by heating CFA/I in sodium dodecyl sulfate at 100 degrees C or by heating it for 20 min in water. Heating in water to 100 degrees C yielded only in the 15-kDa subunit, but heating to 85 degree C yielded small amounts of oligomers in addition. The monomeric subunits obtained after heating in water are stable, as demonstrated by gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis without heating prior to the electrophoretic run. These subunits inhibited CFA/I-induced hemagglutination, indicating that they had maintained their receptor-binding properties. When the hybridoma technique was used, two types of monoclonal anti-CFA/I antibodies were obtained. Antibodies obtained by immunization with the purified subunits were more reactive with subunits than with fimbriae, as shown by enzyme-linked immunosorbent assay. These antibodies strongly inhibited CFA/I-induced hemagglutination. When examined by immunoelectron microscopy, these antibodies seemed to label the fimbrial tips. A similar labeling pattern was obtained with gold particles modified with the receptor ganglioside GM2. Antibodies obtained by immunization with fimbriae reacted in enzyme-linked immunosorbent assays equally well with fimbriae and subunits. They inhibited CFA/I-induced hemagglutination only slightly. Immunoelectron microscopy revealed that these antibodies labeled the fimbriae densely and regularly over their entire lengths. In a coagglutination experiment with Staphylococcus aureus and monoclonal antibodies, the subunits retained their receptor-binding properties. From these results, we conclude that CFA/I fimbriae consist entirely of one type of adhesive subunit, of which only the one at the tip is accessible to the receptor.

Rabbit beta-casein promoter directs secretion of human interleukin-2 into the milk of transgenic rabbits.

To test the potential usefulness of transgenic rabbits as production systems for human proteins of pharmaceutical value, we cloned the rabbit beta-casein promoter and fused it to the genomic sequence of the human interleukin-2 (hIL2) gene. Four transgenic female rabbits were tested for expression and biological activity of the foreign protein in their milk. The milk of all four females proved to contain biologically active hIL2. The results show that transgenic rabbits may represent a convenient and economic system for the rapid production of biologically active protein in milk.

Absence of prognostic significance of skeletal involvement in acute lymphocytic leukemia and non-Hodgkin lymphoma in children.

Over a period of 6 years 88 children with acute lymphocytic leukemia and malignant non-Hodgkin lymphoma were treated according to the West-Berlin protocol. In 72 children skeletal surveys were performed initially and these showed leukemic bone changes in 31 patients. Follow-up was obtained in 70 patients for up to 8 years after diagnosis: 20 of these patients died and of these 8 showed initial skeletal involvement. In 17 children relapses occurred and 10 of them had bone lesions at first presentation. There was no significant correlation between the extent of the skeletal involvement and the survival time as calculated by life table analysis.