Molecular typing of Clostridium perfringens isolated from swine in slaughterhouses from Sao Paulo State, Brazil

Clostridium perfringens is an anaerobic Gram-positive bacterium known as common pathogen for humans, for domestic and wildlife animals. Although infections caused by C. perfringens type C and A in swine are well studied, just a few reports describe the genetic relationship among strains in the epidemiological chain of swine clostridioses, as well as the presence of the microorganism in the slaughterhouses. The aim of the present study was to isolate C. perfringens from feces and carcasses from swine slaughterhouses, characterize the strains in relation to the presence of enterotoxin, alpha, beta, epsilon, iota and beta-2 toxins genes. using polymerase chain reaction (PCR) and comparing strains by means of Pulsed field gel electrophoresis (PFGE). Clostridium perfringens isolation frequencies in carcasses and finishing pig intestines were of 58.8% in both types of samples. According to the polymerase chain reaction assay, only, alfa toxin was detected, being all isolates also negative to enterotoxin and beta2 toxin. Through PFGE technique, the strains were characterized in 35 pulsotypes. In only one pulsotype, the isolate from carcass sample was grouped with fecal isolate of the same animal, suggesting that the risk of cross-contamination was low. Despite the high prevalence of C. perfringens in swine carcasses from the slaughterhouses assessed, the risk of food poisoning to Brazilian pork consumers is low, since all strains were negative to cpe-gene, codifying enterotoxin.

Genetic features and molecular epidemiology of Enterococcus faecium isolated in two university hospitals in Brazil

The global emergence of vancomycin-resistant Enterococcus faecium (VREfm) has been characterized by a clonal spread of strains belonging to clonal complex 17 (CC17). Genetic features and clonal relationships of 53 VREfm isolated from patients in 2 hospitals in Ribeirao Preto, Sao Paulo, Brazil, during 2005-2010 were determined as a contribution to the Brazilian evolutionary history of these nosocomial pathogens. All isolates were daptomycin susceptible, vancomycin-resistant, and had the vanA gene. The predominant virulence genes were acm and esp. Only 5 VREfm isolated in 2005-2006 had intact Tn1546, while 81% showed Tn1546 with deleted left extremity and insertion of IS1251 between the vanS and vanH genes. Multilocus sequence typing analysis permitted the identification of 9 different sequence types (STs), with 5 being new ones (656, 657, 658, 659, and 660). Predominant STs were ST412 and ST478, all belonging to CC17, except ST658. This is the first report of the ST78 in Brazil. (c) 2012 Elsevier Inc. All rights reserved.

Molecular typing of Clostridium perfringens isolated from swine in slaughterhouses from São Paulo State, Brazil

Clostridium perfringens is an anaerobic Gram-positive bacterium known as common pathogen for humans, for domestic and wildlife animals. Although infections caused by C. perfringens type C and A in swine are well studied, just a few reports describe the genetic relationship among strains in the epidemiological chain of swine clostridioses, as well as the presence of the microorganism in the slaughterhouses. The aim of the present study was to isolate C. perfringens from feces and carcasses from swine slaughterhouses, characterize the strains in relation to the presence of enterotoxin, alpha, beta, epsilon, iota and beta-2 toxins genes, using polymerase chain reaction (PCR) and comparing strains by means of Pulsed field gel electrophoresis (PFGE). Clostridium perfringens isolation frequencies in carcasses and finishing pig intestines were of 58.8% in both types of samples. According to the polymerase chain reaction assay, only alfa toxin was detected, being all isolates also negative to enterotoxin and beta2 toxin. Through PFGE technique, the strains were characterized in 35 pulsotypes. In only one pulsotype, the isolate from carcass sample was grouped with fecal isolate of the same animal, suggesting that the risk of cross-contamination was low. Despite the high prevalence of C. perfringens in swine carcasses from the slaughterhouses assessed, the risk of food poisoning to Brazilian pork consumers is low, since all strains were negative to cpe-gene, codifying enterotoxin.

Semantic information extraction from images of complex documents

Even though the digital processing of documents is increasingly widespread in industry, printed documents are still largely in use. In order to process electronically the contents of printed documents, information must be extracted from digital images of documents. When dealing with complex documents, in which the contents of different regions and fields can be highly heterogeneous with respect to layout, printing quality and the utilization of fonts and typing standards, the reconstruction of the contents of documents from digital images can be a difficult problem. In the present article we present an efficient solution for this problem, in which the semantic contents of fields in a complex document are extracted from a digital image.

Characterization of Haemophilus parasuis isolated from Brazilian swine through serotyping, AFLP and PFGE

Haemophilus parasuis infection in pigs is characterized by fibrinous polyserositis, arthritis and meningitis. Despite the fact that traditional diagnosis is based on herd history, clinical signs, bacterial isolation and serotyping, molecular-based methods are alternatives for species-specific tests and epidemiological studies. The aim of this study was to characterize H. parasuis field strains from different states of Brazil, employing serotyping and genotyping methods. Serotyping revealed that serovar 4 was the most prevalent (26.1%), followed by serovars 5(17.4%), 14(8.7%), 13 (4.4%) and 2 (4.4%), whereas 39% of the strains were considered as untypeable. AFLP with a single enzyme and PFGE were able to type all isolates tested, generating 34 and 20 different profiles, respectively, including untypeable strains. Besides the slightly higher discrimination index presented by AFLP, PFGE with Not I restriction enzyme showed a better correlation with epidemiological data, grouping strains of the same serovar, animal or farm origin. The results indicated AFLP and PFGE as valuable tools for typing H. parasuis isolates collected in Brazil. (C) 2011 Elsevier Ltd. All rights reserved.

Lack of beta(2)-AR improves exercise capacity and skeletal muscle oxidative phenotype in mice

beta(2)-adrenergic receptor (beta(2)-AR) agonists have been used as ergogenics by athletes involved in training for strength and power in order to increase the muscle mass. Even though anabolic effects of beta(2)-AR activation are highly recognized, less is known about the impact of beta(2)-AR in endurance capacity. We presently used mice lacking beta(2)-AR [beta(2)-knockout (beta(2) KO)] to investigate the role of beta(2)-AR on exercise capacity and skeletal muscle metabolism and phenotype. beta(2) KO mice and their wild-type controls (WT) were studied. Exercise tolerance, skeletal muscle fiber typing, capillary-to-fiber ratio, citrate synthase activity and glycogen content were evaluated. When compared with WT, beta 2KO mice displayed increased exercise capacity (61%) associated with higher percentage of oxidative fibers (21% and 129% of increase in soleus and plantaris muscles, respectively) and capillarity (31% and 20% of increase in soleus and plantaris muscles, respectively). In addition, beta 2KO mice presented increased skeletal muscle citrate synthase activity (10%) and succinate dehydrogenase staining. Likewise, glycogen content (53%) and periodic acid-Schiff staining (glycogen staining) were also increased in beta 2KO skeletal muscle. Altogether, these data provide evidence that disruption of beta(2)AR improves oxidative metabolism in skeletal muscle of beta 2KO mice and this is associated with increased exercise capacity.

Determination of flagellar types by PCR-RFLP analysis of enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) strains isolated from animals in Sao Paulo, Brazil

This study evaluated the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) analysis of fliC for typing flagella antigen (H) of Shiga toxin-producing Escherichia coil (STEC) and enteropathogenic E. coli (EPEC) strains isolated from different animals. The molecular typing of the H type was efficient in the determination of 93 (85%) strains. Two nonmotile (H-) E. coil strains showed a PCR-RFLP electrophoretic profile that did not match known H type patterns. The fliC nucleotide sequence of strains B2N and 4a revealed a nucleotide substitution at the restriction site and a nucleotide insertion that generated a stop codon, respectively. The results of this study showed that PCR-RFLP analysis of fliC is faster, less laborious and as efficient for the determination of H type E. coli isolated from animals, compared to serotyping and that it is useful in determining H type in nonmotile strains and strains expressing non-reactive H antigens. Moreover, the fliC sequence of strain B2N suggests that we could have found a new flagellin antigen type. (C) 2010 Elsevier Ltd. All rights reserved.

Genetic diversity, virulence genes and antimicrobial resistance of Salmonella Enteritidis isolated from food and humans over a 24-year period in Brazil

Salmonellosis is a major health problem worldwide. Serovar Enteritidis has been a primary cause of Salmonella outbreaks in many countries. In Brazil, few molecular typing studies have been performed. The aims of this study were to molecularly type Salmonella Enteritidis strains isolated in Brazil in order to determine the genetic relationship between strains of food and human origin, as well as, to assess their pathogenic potential and antimicrobial resistance. A total of 128 S. Enteritidis strains isolated from human feces (67) and food (61) between 1986 and 2010 were studied. The genotypic diversity was assessed by ERIC-PCR and PFGE using Xbal, the antimicrobial resistance by the disc-diffusion assay and the presence of the SPI-1, SPI-2 and pSTV virulence genes assessed by PCR. The ERIC-PCR results revealed that 112 strains exhibited a similarity of >85.4% and the PFGE that 96 strains exhibited a similarity of >80.0%. Almost all strains (97.6%) harbored all 13 virulence genes investigated. Thirty-six strains (28.12%) were resistant to nalidixic acid. In conclusion, the nalidixic acid resistance observed after 1996 is indicative of an increase in the use of this drug. It may be suggested that these 128 strains might have descended from a common ancestor that differed little over 24 years and has been both contaminating food and humans and causing disease for more than two decades in Brazil. (c) 2012 Elsevier Ltd. All rights reserved.

The characteristics of VIM-1-producing Klebsiella pneumoniae from South Africa

A study was designed to characterize a carbapenem-resistant Klebsiella pneumoniae (KPSA01) isolated from a patient in Gauteng, South Africa without recent travel outside South Africa. Molecular characterization was done using isoelectric focusing, polymerase chain reaction and sequencing for bla(VIM), bla(IMP), bla(NDM), bla(CTX-Ms), bla(OXAs), bla(TEMs), and bla(SHV), plasmid-mediated quinolone resistance determinants, multilocus sequencing typing, plasmid replicon typing, and addiction factors. KPSA01 produced VIM-1 and belonged to the newly described sequence type ST569. The plasmid that harboured bla(VIM) typed within the narrow host range IncF replicon group, contained the aadA1 gene cassette, and tested positive for the vagCD and ccdAB addiction systems. This is the first report of VIM-1-producing K. pneumoniae outside Europe. It is important that surveillance studies be undertaken in Africa to determine if VIM-1-producing K. pneumoniae are present in significant numbers.

Capsule Locus Polymorphism among Distinct Lineages of Enterococcus faecalis Isolated from Canals of Root-filled Teeth with Periapical Lesions

Introduction: Although Enterococcus faecalis is a member of the normal microbiota, it is also a major cause of nosocomial infections. Some strains of E. faecalis produce capsule, which contributes to pathogenesis through evasion of host defenses, and its production is dependent on the capsule (cps) operon polymorphism. This study investigated cps locus polymorphism in distinct lineages of E. faecalis isolated from canals of root-filled teeth with periapical lesions. Methods: Twenty-two E. faecalis isolates were evaluated regarding the cps operon polymorphism and genetic diversity. The 3 known CPS types were determined by polymerase chain reaction. This information was correlated with multilocus sequence typing data, which were used to define genetic lineages. Results: cpsA and cpsB were the only detected genes within the cps operon in 62.5% of E. faecalis strains (14/22), indicative of genotype CPS 1, which lacks capsule expression. The essential genes in the cps operon for capsule production were detected in the remaining strains, whereas 3 belonged to genotype CPS 5 and 5 strains to genotype CPS 2. A total of 14 sequence types (STs) were resolved in 22 E. faecalis isolates. Comparison with the E. faecalis international multilocus sequence typing database revealed that 9 STs were previously found, and that the 5 STs were novel. Conclusions: Certain E. faecalis genotypes from canals of root-filled teeth with periapical lesions belong to lineages associated with capsule expression and production of multiple virulence factors, which might account for their increased pathogenic potential. (J Endod 2012;38:58-61)

Expression of nonclassical molecule human leukocyte antigen-G in oral lesions

Introduction: Human leukocyte antigen (HLA)-G is a nonclassic class I molecule that acts as a modulator of immune responses, and the expression of these molecules in virus-infected cells has been associated with subversion of the immune response. Objective: In this study, we performed a cross-sectional study, systematically comparing the expression of the HLA-G in benign, premalignant, and malignant oral lesions and correlating it with the presence of high-risk and low-risk human papillomavirus (HPV) types. Specimens and Methods: Oral biopsies were collected from 51 patients and analyzed by immunohistochemistry using anti HLA-G antibody. Human papillomavirus detection and typing from oral biopsies were obtained by polymerase chain reaction using GP5+/GP6+ and specific primers. Results: The 51 biopsies were stratified into 3 groups according to lesion grade: oral benign lesions (oral hyperplasia and papilloma, n = 16), oral premalignant lesions (oral leukoplakia with dysplasia and lichen planus, n = 17), and malignant lesions (oral squamous cell carcinoma, n = 18). Human leukocyte antigen G overexpression was mainly observed in benign and premalignant oral lesions but was not related to HPV infection (P>.05). On the other hand, HPV DNA was detected in 24 (47%) oral lesions, mainly in benign and premalignant lesions, with the most frequent type detected being high-risk HPV type. Conclusion: The HLA-G molecule was expressed in a significant number of benign oral lesions and was not correlated with HPV infection or oral cancer. Crown Copyright (C) 2012 Published by Elsevier Inc. All rights reserved.

High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil

Carbapenem resistance amongst Acinetobacter spp. has been increasing in the last decade. This study evaluated the outer membrane protein (OMP) profile and production of carbapenemases in 50 carbapenem-resistant Acinetobacter spp. isolates from bloodstream infections. Isolates were identified by API20NE. Minimum inhibitory concentrations (MICs) for carbapenems were determined by broth microdilution. Carbapenemases were studied by phenotypic tests, detection of their encoding gene by polymerase chain reaction (PCR) amplification, and imipenem hydrolysis. Nucleotide sequencing confirming the enzyme gene type was performed using MegaBACE 1000. The presence of OMPs was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and PCR. Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). All isolates were resistant to carbapenems. Moreover, 98% of the isolates were positive for the gene encoding the enzyme OXA-51-like, 18% were positive for OXA-23-like (only one isolate did not show the presence of the insertion sequence ISAba1 adjacent to this gene) and 76% were positive for OXA-143 enzyme. Five isolates (10%) showed the presence of the IMP-1 gene. Imipenem hydrolysing activity was detected in only three strains containing carbapenemase genes, comprising two isolates containing the bla(IMP) gene and one containing the bla(OXA-51/OXA-23-like) gene. The OMP of 43 kDa was altered in 17 of 25 strains studied, and this alteration was associated with a high meropenem MIC (256 mu g/mL) in 5 of 7 strains without 43 kDa OMP. On the other hand, decreased OMP 33-36 kDa was found in five strains. The high prevalence of OXA-143 and alteration of OMPs might have been associated with a high level of carbapenem resistance. (C) 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Repertoire, Genealogy and Genomic Organization of Cruzipain and Homologous Genes in Trypanosoma cruzi, T. cruzi-Like and Other Trypanosome Species

Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches.

Gametic phase disequilibrium between the syntenic multiallelic HTG4 and HMS3 markers widely used for parentage testing in Thoroughbred horses

Validation of parentage and horse breed registries through DNA typing relies on estimates of random match probabilities with DNA profiles generated from multiple polymorphic loci. Of the twenty-seven microsatellite loci recommended by the International Society for Animal Genetics for parentage testing in Thoroughbred horses, eleven are located on five chromosomes. An important aspect in determining combined exclusion probabilities is the ascertainment of the genetic linkage status of syntenic markers, which may affect reliable use of the product rule in estimating random match probabilities. In principle, linked markers can be in gametic phase disequilibrium (GD). We aimed at determining the extent, by frequency and strength, of GD between the HTG4 and HMS3 multiallelic loci, syntenic on chromosome 9. We typed the qualified offspring (n (1) = 27; n (2) = 14) of two Quarter Bred stallions (registered by the Brazilian Association of Quarter Horse Breeders) and 121 unrelated horses from the same breed. In the 41 informative meioses analyzed, the frequency of recombination between the HTG4 and HMS3 loci was 0.27. Consistent with genetic map distances, this recombination rate does not fit to the theoretical distribution for independently segregated markers. We estimated sign-based D' coefficients as a measure of GD, and showed that the HTG4 and HMS3 loci are in significant, yet partial and weak, disequilibrium, with two allele pairs involved (HTG4*M/HMS3*P, D'(+) = 0.6274; and HTG4*K/HMS3*P, D'(-) = -0.6096). These results warn against the inadequate inclusion of genetically linked markers in the calculation of combined power of discrimination for Thoroughbred parentage validation.

A dysflagellar mutant of Leishmania (Viannia) braziliensis isolated from a cutaneous leishmaniasis patient

Background: Parasites of the Leishmania genus alternate between the flagellated extracellular promastigote stage and intracellular amastigotes. Here we report the characterization of a Leishmania isolate, obtained from a cutaneous leishmaniasis patient, which presents peculiar morphological features. Methods: The parasite was cultured in vitro and characterized morphologically using optical and electron microscopy. Identification was performed based on monoclonal antibodies and internal ribosomal spacer typing. In vitro macrophage cultures, murine experimental models and sand fly infections were used to evaluate infectivity in vitro and in vivo. Results: The isolate was identified as Leishmania (Viannia) braziliensis. In the atypical promastigotes grown in culture, a short flagellum surrounded or interrupted by a protuberance of disorganized material was observed. A normal axoneme was present close to the basal body but without elongation much further outside the flagellar pocket. A disorganized swelling at the precocious end of the axoneme coincided with the lack of a paraflagellar rod structure. The isolate was able to infect macrophages in vitro, induce lesions in BALB/c mice and infect Lutzomyia longipalpis. Conclusions: Notwithstanding the lack of an extracellular flagellum, this isolate infects macrophages in vitro and produces lesions when inoculated into mice. Moreover, it is able to colonize phlebotomine sand flies. Considering the importance attributed to the flagellum in the successful infection and survival of Leishmania in the insect midgut and in the invasion of macrophages, these findings may bring new light into the infectious mechanisms of L. (V.) braziliensis.