Wild birds and urban pigeons as reservoirs for diarrheagenic <Emphasis Type="Italic">Escherichia coli</Emphasis> with zoonotic potential

In order to describe the role of wild birds and pigeons in the transmission of shiga toxigenic Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) to humans and other animals, samples were collected from cloacae and oropharynx of free-living wild birds and free-living pigeons. Two STEC (0.8%) and five EPEC strains (2.0%) were isolated from wild birds and four EPEC strains (2.0%) were recovered from pigeons. Serogroups, sequence types (STs) and virulence genes, such as saa, iha, lpfA _O113, ehxA, espA, nleB and nleE, detected in this study had already been implicated in human and animal diseases. Multidrug resistance (MDR) was found in 25.0% of the pigeon strains and in 57.0% of the wild bird strains; the wild birds also yielded one isolate carrying extended-spectrum β-lactamases (ESBLs) gene bla _CTX-M-8. The high variability shown by PFGE demonstrates that there are no prevalent E. coli clones from these avian hosts. Wild birds and pigeons could act as carriers of multidrug-resistant STEC and EPEC and therefore may constitute a considerable hazard to human and animal health by transmission of these strains to the environment.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected.     

Synthesis of cholera toxin B subunit gene: cloning and expression of a functional 6XHis-tagged protein in Escherichia coli

Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending on the antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, we synthesized a versatile gene coding a 6XHis-tagged CTB (359bp). The sequence was designed according to codon usage of Escherichia coli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in which the polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, and expressed in E. coli in an insoluble form, reaching levels about 13 mg of purified active pentameric rCTB per liter of induced culture. Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxin activity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functional CTB through Ni(2+)-charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained from Vibrio cholerae cultures.     

13C-urea breath test to diagnose Helicobacter pylori infection in children aged up to 6 years

BACKGROUND: 13C-urea breath test (13C-UBT) is an accurate noninvasive tool for diagnosis of Helicobacter pylori infection. It is considered the best method for epidemiological studies, but there are few studies to evaluate the 13C-UBT in infants and toddlers. AIM: To evaluate the 13C-UBT performed with infrared spectroscopy in children aged up to 6 years. PATIENTS: Sixty-eight patients (6 months. to 5 years 11 months.) were evaluated prospectively and consecutively. METHODS: Helicobacter pylori infection was detected by positive culture, or rapid urease test and histological examination, both positive. 13C-UBT was performed with 50 mg of 13C-urea diluted in 100 ml of commercial orange juice. Two expired air samples were collected: before and 30 minutes after tracer ingestion. Cutoff of delta over baseline (DOB) was 4.0 per thousand and urea hydrolysis rate 10 microg/minute. RESULTS: Fifteen of 68 (22.1%) patients were H. pylori infected. Sensitivity was 93.3% (95% CI; 86.8%-99.7%) and specificity was 96.2% (95% CI; 93.6%-98.8%), and these values were equal for DOB and urea hydrolysis rate. Negative DOB values in noninfected patients ranged from -1.5 per thousand to 2.6 per thousand and positive DOB values ranged from 10.8 per thousand to 105.5 per thousand. There was no relationship between DOB values and age. Conclusion. 13C-UBT performed with infrared spectroscopy proved to be a reliable and accurate noninvasive diagnostic tool for H. pylori infection detection in children aged up to 6 years. Results far from cutoff value can clearly distinguish positive from negative 13C-UBT results in children up to 6 years old.     

Genetic variability in G2 and F2 region between biological clones of human respiratory syncytial virus with or without host immune selection pressure

Human respiratory syncytial virus (HRSV) is an important respiratory pathogens among children between zero-five years old. Host immunity and viral genetic variability are important factors that can make vaccine production difficult. In this work, differences between biological clones of HRSV were detected in clinical samples in the absence and presence of serum collected from children in the convalescent phase of the illness and from their biological mothers. Viral clones were selected by plaque assay in the absence and presence of serum and nucleotide sequences of the G2 and F2 genes of HRSV biological clones were compared. One non-synonymous mutation was found in the F gene (Ile5Asn) in one clone of an HRSV-B sample and one non-synonymous mutation was found in the G gene (Ser291Pro) in four clones of the same HRSV-B sample. Only one of these clones was obtained after treatment with the child''s serum. In addition, some synonymous mutations were determined in two clones of the HRSV-A samples. In conclusion, it is possible that minor sequences could be selected by host antibodies contributing to the HRSV evolutionary process, hampering the development of an effective vaccine, since we verify the same codon alteration in absence and presence of human sera in individual clones of BR-85 sample.     

Direct Detection by the Xpert MTB/RIF Assay and Characterization of Multi and Poly Drug-Resistant Tuberculosis in Guinea-Bissau,West Africa

BackgroundThis study aimed to evaluate the usefulness of the Xpert MTB/RIF assay for the rapid direct detection of M. tuberculosis complex (MTBC) strains and rifampicin resistance associated mutations in a resource-limited setting such as Guinea-Bissau and its implications in the management of tuberculosis (TB) and drug resistant tuberculosis, complementing the scarce information on resistance and genotypic diversity of MTBC strains in this West African country.ConclusionsThe Xpert MTB/RIF assay was reliable for the detection of rifampicin resistant MTBC strains directly from sputum samples of patients undergoing first-line treatment for two months, being more trustworthy than the simple presence of acid-fast bacilli in the smear. Its implementation is technically simple, does not require specialized laboratory infrastructures and is suitable for resource-limited settings when a regular source of electricity and maintenance is available as well as financial and operation sustainability is guaranteed by the health authorities. A high prevalence of MDR-TB among patients at risk of MDR-TB after two months of first-line treatment was found, in support of the WHO recommendations for its use in the management of this risk group.     

Turiasauria-like teeth from the Upper Jurassic of the Lusitanian Basin,Portugal

Turiasauria is a clade of eusauropods with a wide stratigraphic range that could extend from the Bathonian to the lower Aptian including Turiasaurus, Losillasaurus, Zby and putatively, Galveosaurus, Atlasaurus and isolated remains from Middle Jurassic-to-Lower Cretaceous. Some are characterised by the presence of heart-shaped teeth. Several tooth occurrences from the Portuguese Upper Jurassic with this type of morphology (SI: 1.1–1.8) are reported and discussed. If this morphology is regarded as synapomorphic of Turiasauria, the teeth will be tentatively related to this clade. From a sample of 43 teeth, three main morphotypes are described. Three hypotheses might explain the morphological variation: (1) the range of tooth morphologies indicates variation in the jaw, (2) the range of tooth morphologies indicates taxonomic variation or (3) a combination of both. The general wear pattern in morphotypes I and II starts with a distal facet, then the appearance of mesial/apical facet and finally a ‘V’-shaped facet. In morphotype III, the wear begins with a mesial facet. The variability observed for Portuguese Upper Jurassic specimens is congruent with the morphological variability along the tooth row shown by other sauropods with spatulate/spoon-shaped teeth and it is considered the most parsimonious hypothesis to explain it.