Multilocus sequence types of invasive Corynebacterium diphtheriae isolated in the Rio de Janeiro urban area, Brazil.

Invasive infections caused by Corynebacterium diphtheriae in vaccinated and non-vaccinated individuals have been reported increasingly. In this study we used multilocus sequence typing (MLST) to study genetic relationships between six invasive strains of this bacterium isolated solely in the urban area of Rio de Janeiro, Brazil, during a 10-year period. Of note, all the strains rendered negative results in PCR reactions for the tox gene, and four strains presented an atypical sucrose-fermenting ability. Five strains represented new sequence types. MLST results did not support the hypothesis that invasive (sucrose-positive) strains of C. diphtheriae are part of a single clonal complex. Instead, one of the main findings of the study was that such strains can be normally found in clonal complexes with strains related to non-invasive disease. Comparative analyses with C. diphtheriae isolated in different countries provided further information on the geographical circulation of some sequence types.

Similarity of rpoB gene sequences of sucrose-fermenting and non-fermenting Corynebacterium diphtheriae strains.

During the last decades, the majority of Brazilian Corynebacterium diphtheriae isolates were shown to be capable to metabolize sucrose, sometimes leading to erroneous identification as a non-diphtheric Corynebacterium species. The sequencing of the polymorphic region of the RNA polymerase beta subunit-encoding gene (rpoB) is an important taxonomic tool for identification of corynebacteria. The present study aimed to investigate the rpoB gene polymorphic features of sucrose-fermenting and non sucrose-fermenting strains. The results showed that sucrose-fermenting strains presented rpoB gene polymorphic regions with more than 98% similarity with the sequences deposited in the gene bank corresponding to non sucrose-fermenting strains. Data indicate that sucrose-fermenting isolates may act as a variant of C. diphtheriae biotype mitis. In addition we alert that sucrose-fermenting strains should not be discarded as contaminants mainly in countries where the possibility of isolation of this variant is higher.

Plasticity of Corynebacterium diphtheriae pathogenicity islands revealed by PCR.

Despite the existence of a vaccine against diphtheria, this disease remains endemic and is reemerging in several regions due to many factors, including variations in genes coding for virulence factors. One common feature of virulence factors is their high concentration in pathogenicity islands (PAIs), very unstable regions acquired via horizontal gene transfer, which has lead to the emergence of various bacterial pathogens. The 13 putative PAIs in Corynebacterium diphtheriae NCTC 13129 and the reemergence of this disease point to the great variability in the PAIs of this species, which may reflect on bacterial life style and physiological versatility. We investigated the relationships between the large number of PAIs in C. diphtheriae and the possible implications of their plasticity in virulence. The GenoFrag software was used to design primers to analyze the genome plasticity of two pathogenicity islands of the reference strain (PiCds 3 and 8) in 11 different strains. We found that PiCd 3 was absent in only two strains, showing genes playing putative important roles in virulence and that only one strain harbored PiCd 8, due to its location in a putative "hotspot" for horizontal gene transfer events.

Stable isotopes to study sulfur amino acid utilization in broilers.

Nutritionists have been discussing whether the dietary supplementation of cyst(e)ine is required as a part of the dietary methionine (Met) in the total sulfur amino acid (TSAA) requirement to achieve optimum performance in broilers. Part of Met is converted to cysteine (Cys) to meet the Cys requirement, especially for feather growth. The TSAA requirement has been determined by using graded levels of free Met in the diet, without supplementation of free cyst(e)ine. It has also been argued that the Met to Cys ratio (Met : Cys) changes with age and even with different Met sources. The objective of this study was to evaluate the two sources of Met, while determining the proportion of Met and Cys in total dietary TSAA that optimize the performance of broilers. A performance assay was carried out in a factorial arrangement (5 × 2) using 1080 broilers from 42 to 56 days of age fed diets having different dietary proportions of Met and Cys (44 : 56, 46 : 54, 48 : 52, 50 : 50 or 52 : 48) while maintaining the same dietary TSAA in the diets. Two synthetic Met sources (dl-Met or l-Met) were used for each of the diets with different dietary Met : Cys ratios. Twenty-one broilers of the same age were fed the diets 44 : 56, 48 : 52 and 52 : 48 by supplementing the diet with L-(15N) Met or L-(15N2) Cystine to study the metabolism of TSAA. No differences were observed between Met sources for feed intake, BW gain and feed conversion ratio (FCR; P > 0.05); however, FCR was numerically improved at 50 : 50 Met : Cys. Regarding TSAA utilization, the conversion of Met to Cys increased with increase in Met : Cys ratios, but the concentration of Met intermediates decreased. Broiler chickens responded to different dietary proportions of sulfur amino acids by altering their sulfur amino acid metabolism, and diets containing 50 : 50 Met : Cys is recommended for broilers of age 42 to 56 days.

First detection of Corynebacterium ulcerans producing a diphtheria-like toxin in a case of human with pulmonary infection in the Rio de Janeiro metropolitan area, Brazil.

The frequency and severity of human infections associated with Corynebacterium ulcerans appear to be increasing in different countries. Here, we describe the first C. ulcerans strain producing a diphtheria-like toxin isolated from an elderly woman with a fatal pulmonary infection and a history of leg skin ulcers in the Rio de Janeiro metropolitan area.

A description of genes of Corynebacterium pseudotuberculosis useful in diagnostics and vaccine applications.

Corynebacterium pseudotuberculosis, a Gram-positive intracellular pathogen, is the etiological agent of caseous lymphadenitis or CLA. This bacterium infects goats and sheep and causes great economic losses worldwide annually, mainly for goat producers. Despite its importance, CLA is still poorly characterized. However, with advances in the genomic field, many C. pseudotuberculosis genes have already been characterized, mainly those related to virulence such as phospholipase D. Here, we examined the use of the several available genes of C. pseudotuberculosis and reviewed their applications in vaccine construction, more efficient diagnostics for CLA, and control of this disease, among other applications.

High seroprevalence of caseous lymphadenitis in Brazilian goat herds revealed by Corynebacterium pseudotuberculosis secreted proteins-based ELISA.

We conducted a seroepidemiological survey to determine the prevalence of caseous lymphadenitis (CLA) in goat herds in Minas Gerais state, Brazil. Serum samples were collected from goats (n=676) from 108 rural properties in 2001, covering most of the sub-regions of this ca. 586,500 square kilometer state. Antibodies against Corynebacterium pseudotuberculosis secreted proteins were detected by an indirect enzyme-linked immunosorbent assay (ELISA). Most of the animals (78.9%) tested positive for CLA; 98% of flocks presented at least one seropositive animal. Goats managed under an extensive production system had a significantly higher seroprevalence of CLA than those in intensive and semi-intensive operations. The age distribution of the animals in the flocks affected the prevalence of this disease; however, goat breed did not. We found seropositivity against C. pseudotuberculosis to be highly prevalent in these Brazilian goat herds; consequently, appropriate management practices for the control of CLA should be implemented.

A description of genes of Corynebacterium pseudotuberculosis useful in diagnostics and vaccine applications

Corynebacterium pseudotuberculosis, a Gram-positive intracellular pathogen, is the etiological agent of caseous lymphadenitis or CLA. This bacterium infects goats and sheep and causes great economic losses worldwide annually, mainly for goat producers. Despite its importance, CLA is still poorly characterized. However, with advances in the genomic field, many C. pseudotuberculosis genes have already been characterized, mainly those related to virulence such as phospholipase D. Here, we examined the use of the several available genes of C. pseudotuberculosis and reviewed their applications in vaccine construction, more efficient diagnostics for CLA, and control of this disease, among other applications.

First detection of Corynebacterium ulcerans producing a diphtheria-like toxin in a case of human with pulmonary infection in the Rio de Janeiro metropolitan area, Brazil

The frequency and severity of human infections associated with Corynebacterium ulcerans appear to be increasing in different countries. Here, we describe the first C. ulcerans strain producing a diphtheria-like toxin isolated from an elderly woman with a fatal pulmonary infection and a history of leg skin ulcers in the Rio de Janeiro metropolitan area.

Update of microbial genome programs for bacteria and archaea

Since the Haemophilus influenzae genome sequence was completed in 1995, 172 other prokaryotic genomes have been completely sequenced, while 508 projects are underway. Besides pathogens, organisms important in several other fields, such as biotechnology and bioremediation, have also been sequenced. Institutions choose the organisms they wish to sequence according to the importance that these species represent to them, the availability of the microbes, and based on the similarity of a species of interest with others that have been sequenced previously. Improvements in sequencing techniques and in associated methodologies have been achieved; however, scientists need to continue working on the development of this field. In Brazil, a multicentered, centrally coordinated and research-focused network was adopted and successfully used for the sequencing of several important organisms. We analyzed the current status of microbial genomes, the trends for criteria used to choose new sequencing projects, the future of microbial sequencing, and the Brazilian genome network.