New PCR ribotypes of Clostridium difficile detected in children in Brazil

A total of 35 Brazilian isolates of Clostridium difficile from faecal stools and four isolates from hospital environments were analyzed by PCR ribotyping. A whole cell protein profile (as an alternative for serogrouping), in vitro toxin production and susceptibility to vancomycin, metronidazole and clindamycin were also investigated. All strains were typeable by both phenotypic and genotypic methods, and a total of 13 different PCR ribotypes were identified, of which seven (132, 133, 134, 135, 136, 142 and 143) were considered new types and accounted for 78.5% of all samples evaluated (including hospital environments). A non-toxigenic C. difficile PCR ribotype 133 was detected in all children groups examined (inpatients, outpatients and healthy children), whilst toxigenic PCR ribotypes 015, 131, 134 and 135 were associated mostly with symptomatic children. Serogroups G and D were disseminated both in patients from the community and from the pediatric hospital, with group G prevalent among outpatient children. All strains were susceptible to vancomycin and metronidazole but high levels of resistance to clindamycin were found, especially among serogroups G and D. Co-existence of different ribotypes and serogroups in the same individual was observed. The new seven ribotypes found in this investigation may represent strains characteristic of this region of Brazil.     

Proteome of the phytopathogen <Emphasis Type="Italic">Xanthomonas citri</Emphasis> subsp. <Emphasis Type="Italic">citri</Emphasis>: a global expression profile

Background  

Citrus canker is a disease caused by Xantomonas citri subsp.citri (Xac), and has emerged as one of the major threats to the worldwide citrus crop because it affects all commercial citrus varieties, decreases the production and quality of the fruits and can spread rapidly in citrus growing areas. In this work, the first proteome of Xac was analyzed using two methodologies, two-dimensional liquid chromatography (2D LC) and tandem mass spectrometry (MS/MS).     

Effects of deer,mice and dwarf bamboo on the emergence,survival and growth of <Emphasis Type="Italic">Abies homolepis</Emphasis> (Piceaceae) seedlings

In the subalpine mixed forest of Mt. Ôdaigahara, mid-western Japan, the understory is dominated by dwarf bamboo (Sasa nipponica), which is the major forage of overly populous sika deer (Cervus nippon). In the present study, we monitored the survival and growth of Abies homolepis seedlings over 5years to determine how they responded to the experimental removal of dwarf bamboo and to the exclusion of sika deer and mice (Apodemus argenteus and A.speciosus). Deer and dwarf bamboo reduced the survival of seedlings but had different effects on growth. The stems of seedlings were shorter in the presence of deer, indicating that taller seedlings were apt to be browsed by deer, whereas the diameters of seedlings were smaller in the presence of dwarf bamboo, mainly owing to its shading effect. The presence of mice decreased the number of seedlings germinating in a particular site, but had no effect on seedling survival after germination. There was no significant indirect effect whereby the survival of seedlings was predicted to be facilitated by the decreased biomass of bamboo because of grazing by deer. We supposed that this might be because the direct negative effect of deer was so large as to conceal the positive indirect effect.     

DNA methylation changes associated with risk factors in tumors of the upper aerodigestive tract

Cancers of the upper aerodigestive tract (UADT) are common forms of malignancy associated with tobacco and alcohol exposures, although human papillomavirus and nutritional deficiency are also important risk factors. While somatically acquired DNA methylation changes have been associated with UADT cancers, what triggers these events and precise epigenetic targets are poorly understood. In this study, we applied quantitative profiling of DNA methylation states in a panel of cancer-associated genes to a case-control study of UADT cancers. Our analyses revealed a high frequency of aberrant hypermethylation of several genes, including MYOD1, CHRNA3 and MTHFR in UADT tumors, whereas CDKN2A was moderately hypermethylated. Among differentially methylated genes, we identified a new gene (the nicotinic acetycholine receptor gene) as target of aberrant hypermethylation in UADT cancers, suggesting that epigenetic deregulation of nicotinic acetycholine receptors in non-neuronal tissues may promote the development of UADT cancers. Importantly, we found that sex and age is strongly associated with the methylation states, whereas tobacco smoking and alcohol intake may also influence the methylation levels in specific genes. This study identifies aberrant DNA methylation patterns in UADT cancers and suggests a potential mechanism by which environmental factors may deregulate key cellular genes involved in tumor suppression and contribute to UADT cancers.Key words: DNA methylation, upper aerodigestive tract, cancer, risk factors, biomarkers     

Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis

Background

Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species.

Methodology/Principal Findings

For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay.

Conclusions/Significance

The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.     

New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Background  

Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.     

Escape flights of yellowhammers and greenfinches: more than just physics

Wintering birds increase their fat reserves throughout the day, and impaired escape performance is often considered to be an important cost of fat reserves. Since lifting a larger mass requires more energy, if birds escape at maximum power output, an increase in mass will impair the escape flight. In this study we did not find support for mass-dependent escape performance for yellowhammers, Emberiza citrinella, and greenfinches, Carduelis chloris, with natural daily mass increases of 7-8%. This suggests either that the birds were not performing at maximum output at dawn, when light, or that maximum power output was higher at dusk, when heavy. Either way, the birds seemed to be able to put more effort into their escape flight when heavier. In both species, when alarmed, birds took off significantly faster and at a steeper angle than when not alarmed. Yellowhammers escaped at a higher speed and angle than greenfinches, and reacted faster to the predator model. This suggests that predator escape is more than just Newtonian physics, and may be influenced by behavioural, as well as morphological, adjustments. Different species may have evolved different responses to predation risk. Our results seem to be in disagreement with recent ideas about mass-dependent predation risk. However, to build up reserves, birds have to increase exposure time, which increases predation risk. This cost may be more important than impaired escape performance when relatively small, daily, changes in body mass are considered. Copyright 2000 The Association for the Study of Animal Behaviour.     

Detection of cross-infection associated to a Brazilian PCR-ribotype of <Emphasis Type="Italic">Clostridium difficile</Emphasis> in a university hospital in Rio de Janeiro,Brazil

Clostridium difficile is an important nosocomial enteric pathogen and is the etiological agent of pseudomembranous colites. Recently, the rates of C. difficile infection (CDI) have increased worldwide, but in Brazil few data about this situation and the incidence of clonal types of C. difficile exist. This study aimed to isolate and characterize C. difficile strains from samples obtained of a university hospital (HUCFF) in Rio de Janeiro city, Brazil. CDI was identified by ELISA in 27.1% of HUCFF-in-patients enrolled in the study, and the bacterium was recovered from eight of these fecal samples. All strains, except one, presented tcdA and tcdB genes and presented neither the cdtA and cdtB genes nor any significant deletions in the tcdC gene. All strains were sensitive to metronidazole, vancomycin and moxifloxacin, and resistant to clindamycin, ciprofloxacin and levofloxacin. PCR-ribotyping and PFGE revealed four different clonal types among the isolates. The Brazilian PCR-ribotype 133 accounted for 50% of strains isolated, and PCR-ribotype 233 strains were obtained from 25% of the in-patients. The prevalence and resurgence of the Brazilian PCR-ribotype 133 among the hospitalized patients of HUCFF was established, and cross-infection of different patients associated to the same PCR-ribotypes was detected. Our results emphasize the importance of the diagnosis and control of CDI in order to prevent the emergence of specific clones that can lead to C. difficile-associated outbreaks in Brazilian hospitals.