Evaluation of 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS) as a Vulnerable Target in Mycobacterium tuberculosis.

Tuberculosis (TB) remains one of the leading causes of death due to a single pathogen. The emergence and proliferation of multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) represent compelling reasons to invest in the pursuit of new anti-TB agents. The shikimate pathway, responsible for chorismate biosynthesis, which is a precursor of important aromatic compounds, is required for Mycobacterium tuberculosis growth. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (MtbDAHPS) catalyzes the first step in the shikimate pathway and it is an attractive target for anti-tubercular agents. Here, we used a CRISPRi system to evaluate the DAHPS as a vulnerable target in M. tuberculosis. The silencing of aroG significantly reduces the M. tuberculosis growth in both rich medium and, especially, in infected murine macrophages. The supplementation with amino acids was only able to partially rescue the growth of bacilli, whereas the Aro supplement (aromix) was enough to sustain the bacterial growth at lower rates. This study shows that MtbDAHPS protein is vulnerable and, therefore, an attractive target to develop new anti-TB agents. In addition, the study contributes to a better understanding of the biosynthesis of aromatic compounds and the bacillus physiology. IMPORTANCE Determining the vulnerability of a potential target allows us to assess whether its partial inhibition will impact bacterial growth. Here, we evaluated the vulnerability of the enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) from M. tuberculosis by silencing the DAHPS-coding aroG gene in different contexts. These results could lead to the development of novel and potent anti-tubercular agents in the near future.

EPSP Synthase-Depleted Cells Are Aromatic Amino Acid Auxotrophs in Mycobacterium smegmatis.

The epidemiological importance of mycobacterial species is indisputable, and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important bacterial metabolites, represents a set of targets for inhibitors of Mycobacterium tuberculosis growth. The aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene disruption, gene knockdown, point mutations (D61W, R134A, E321N), and kinetic analysis to evaluate aroA gene essentiality and vulnerability of its protein product, EPSPS, from Mycolicibacterium (Mycobacterium) smegmatis (MsEPSPS). We demonstrate that aroA-deficient cells are auxotrophic for aromatic amino acids (AroAAs) and that the growth impairment observed for aroA-knockdown cells grown on defined medium can be rescued by AroAA supplementation. We also evaluated the essentiality of selected MsEPSPS residues in bacterial cells grown without AroAA supplementation. We found that the catalytic residues R134 and E321 are essential, while D61, presumably important for protein dynamics and suggested to have an indirect role in catalysis, is not essential under the growth conditions evaluated. We have also determined the catalytic efficiencies (Kcat/Km) of recombinant wild-type (WT) and mutated versions of MsEPSPS (D61W, R134A, E321N). Our results suggest that drug development efforts toward EPSPS inhibition may be ineffective if bacilli have access to external sources of AroAAs in the context of infection, which should be evaluated further. In the absence of AroAA supplementation, aroA from M. smegmatis is essential, its essentiality is dependent on MsEPSPS activity, and MsEPSPS is vulnerable. IMPORTANCE We found that cells from Mycobacterium smegmatis, a model organism safer and easier to study than the disease-causing mycobacterial species, when depleted of an enzyme from the shikimate pathway, are auxotrophic for the three aromatic amino acids (AroAAs) that serve as building blocks of cellular proteins: l-tryptophan, l-phenylalanine, and l-tyrosine. That supplementation with only AroAAs is sufficient to rescue viable cells with the shikimate pathway inactivated was unexpected, since this pathway produces an end product, chorismate, that is the starting compound of essential pathways other than the ones that produce AroAAs. The depleted enzyme, the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), catalyzes the sixth step of shikimate pathway. Depletion of this enzyme inside cells was performed by disrupting or silencing the EPSPS-encoding aroA gene. Finally, we evaluated the essentiality of specific residues from EPSPS that are important for its catalytic activity, determined with experiments of enzyme kinetics using recombinant EPSPS mutants.

Prevalence of Nosocomial Infection Microorganisms and the Presence of Antimicrobial Multi-Resistance / Prevalência de Microrganismos de Infecção Hospitalar e a Presença de Multirresistência Antimicrobiana / Prevalencia de Microorganismos de Infección Hospitalaria y Presencia de Multirresistencia Antimicrobiana

JUSTIFICATIVA E OBJETIVOS: hospitais representam o local onde bactérias resistentes se concentram e a partir de onde se espalham dentro da própria instituição e para a comunidade. Assim, esta pesquisa teve como objetivo traçar o perfil dos microrganismos relacionados à infecção hospitalar e analisar a existência de multirresistência no Hospital de Caridade de Santo Ângelo/RS, hospital de maior porte na cidade. MÉTODOS: foram utilizados os dados de infecção hospitalar de 100 prontuários, de agosto/2016 a março/2017, mantidos pela Comissão de Controle de Infecção Hospitalar. A identificação das bactérias foi realizada pelo Laboratório de Microbiologia do próprio hospital através de métodos morfo-tintoriais e bioquímicos, e a sensibilidade foi determinada pelo método de difusão em disco. A multirresistência bacteriana foi considerada a partir da resistência a três ou mais classes de antimicrobianas. RESULTADOS: os três microrganismos com maior prevalência foram Acinetobacter baumannii (17%), Escherichia coli (16%) e Staphylococcus aureus (8%). A ala hospitalar com maior número de isolados foi a Unidade de Terapia Intensiva adulta, com 41% dos casos. As amostras que forneceram o maior número de isolados foram: secreção traqueal, urina e secreção de ferida. Foram identificadas 16 diferentes espécies/grupos de bactérias multirresistentes: Morganella morganii (100%), Hafnia alvei (100%), Enterobacter sakazaki (100%), Serratia spp. (100%), Enterobacter aerogenes (100%), Proteus vulgaris (100%), Acinetobacter baumannii (100%), Klebsiella pneumoniae (83%), Enterobacter spp. (75%), Klebsiella ozaenae (66%), Staphylococcus coagulase negativa (66%), Escherichia coli (56%), Serratia rubidaea (50%), Serratia marcensces (50%), Staphylococcus aureus(37%) e Pseudomonas aeruginosa (28%). CONCLUSÃO: o grande número de isolados multirresistentes reforça a importância de estratégias de isolamento e restrição para evitar a contaminação cruzada dentro e até mesmo para fora do hospital.(AU)

JUSTIFICACIÓN Y OBJETIVOS: Los hospitales representan el lugar que más concentra bacterias resistentes que se propagan para la comunidad. Por lo tanto, esta investigación tuvo como objetivo esbozar el perfil de microorganismos relacionados con la infección nosocomial y analizar la existencia de resistencia a múltiples fármacos en el Hospital de Caridade de Santo Ângelo (Rio Grande do Sul, Brasil), el más grande de la ciudad. MÉTODOS: se utilizaron los datos de infección hospitalaria de 100 registros médicos, referidos al período de agosto/2016 a marzo/2017, mantenidos por la Comisión de Control de Infecciones del Hospital. La identificación de la bacteria fue realizada por el Laboratorio de Microbiología del propio hospital, utilizando métodos morfológicos, colorantes y bioquímicos, y la sensibilidad fue determinada por el método de disco difusión. La multirresistencia bacteriana se consideró basada en la resistencia a tres o más clases de antimicrobianos. RESULTADOS: los tres microorganismos más prevalentes fueron: Acinetobacter baumannii (17%), Escherichia coli (16%) y Staphylococcus aureus (8%). El ala del hospital con el mayor número de aislamientos fue la Unidad de Cuidados Intensivos (UCI) para adultos con el 41% de los casos. Las muestras que proporcionaron el mayor número de aislamientos fueron: secreción traqueal, orina y secreción de heridas. Se identificaron 16 especies/grupos diferentes de bacterias multirresistentes: Morganella morganii (100%), Hafnia alvei (100%), Enterobacter sakazakii (100%), Serratia spp. (100%), Enterobacter aerogenes (100%), Proteus vulgaris (100%), Acinetobacter baumannii (100%), Klebsiella pneumoniae (83%), Enterobacter spp. (75%), Klebsiella ozaenae (66%), Staphylococcus coagulase negativa (66%), Escherichia coli (56%), Serratia rubidaea (50%), Serratia marcescens (50%), Staphylococcus aureus (37%) y Pseudomonas aeruginosa (28%). CONCLUSIONES: la gran cantidad de aislados resistentes a múltiples fármacos refuerza la importancia de las estrategias de aislamiento y restricción para evitar la contaminación cruzada dentro del hospital e incluso fuera del mismo.(AU)

BACKGROUND AND OBJECTIVES: hospitals represent the place where multidrug-resistant bacteria are concentrated and from where they spread within the institution and to the community. Thus, this research aimed to verify the profile of microorganisms related to nosocomial infection and to analyze the existence of multidrug-resistant bacteria at the Hospital de Caridade de Santo Ângelo/RS, the largest hospital in the city. METHODS: hospital infection data from 100 medical records, from August/2016 to March/2017, maintained by the Hospital Infection Control Committee were used. Bacterial identification was carried out by the Microbiology Laboratory of the hospital using morpho-tinting and biochemical methods; and antimicrobial susceptibility was determined by the disk diffusion method. Bacterial multidrug-resistance was considered based on resistance to three or more classes of antimicrobials. RESULTS: the three most prevalent microorganisms were Acinetobacter baumannii (17%), Escherichia coli (16%), and Staphylococcus aureus (8%). The hospital unit with the highest number of isolates was the adult Intensive Care Unit, with 41% of the cases. Tracheal secretion, urine, and wound secretion samples provided the highest number of isolates. Sixteen different species/groups of multidrug-resistant bacteria were identified, as follows: Morganella morganii (100%), Hafnia alvei (100%), Enterobacter sakazaki (100%), Serratia spp. (100%), Enterobacter aerogenes (100%), Proteus vulgaris (100%), Acinetobacter baumannii (100%), Klebsiella pneumoniae (83%), Enterobacter spp. (75%), Klebsiella ozaenae (66%), coagulase-negative Staphylococcus (66%), Escherichia coli (56%), Serratia rubidaea (50%), Serratia marcensces (50%), Staphylococcus aureus (37%) e Pseudomonas aeruginosa (28%). CONCLUSION: the large number of multidrug-resistant isolates reinforces the importance of isolation and restriction strategies to avoid cross-contamination to inside and outside the hospital.(AU)

Characterization of intestinal Escherichia coli isolated from calves with diarrhea due to rotavirus and coronavirus.

PURPOSE: To address more information about changes in commensal Escherichia coli during virus intestinal infection, we characterized 30 faecal E. coli isolates from calves (21 to 60 days old) with diarrhea due to rotavirus and coronavirus, which received, before diagnosis, tetracycline, gentamicin and enrofloxacin drugs. METHODOLOGY: Clermont's phylogenetic classification; presence of genes for curli, cellulose, fimbriae (F4, F5, F6, F18, F41); and antimicrobial susceptibility were used to characterize the isolates. Disk diffusion technique and PCR were used as methodologies. RESULTS: E. coli isolates from calves with diarrhea were phylogenetically classified as B1 (70%, 21/30), B2 (3.33%, 1/30), C (3.33%, 1/30), D (3.33%, 1/30), E (13.33%, 4/30) and unknown (6.7 %; 2/30), whereas E. coli isolates from the control group were classified only as B1 (83.3%, 25/30), E (10 %; 3/30) and unknown (6,7 %; 2/30). E. coli isolates from calves with diarrhea showed a much higher resistance profile with 16 (53.3%) multiresistant isolates. Only isolates (30%-9/30) from diarrheic calves were also positive for fimbriae, specifically 16.7% (5/30) for F5 and 13.3% (4/30) for F18. CONCLUSION: To sum up, E. coli isolates from calves with diarrhea showed differences in relation to the control group, confirming changes in commensal E. coli during virus intestinal infection. It can be emphasized that some care should be taken to manage diarrheic calves: the pathological agent must be diagnosed prior to treatment; antibacterial treatment should be with antimicrobials with a different mechanism of action; and finally, treated animals should be maintained separately from others because they can carry micro-organisms with a resistant profile.

The Periplasmic Trehalase Affects Type 1 Fimbria Production and Virulence of Extraintestinal Pathogenic Escherichia coli Strain MT78.

Extraintestinal pathogenic Escherichia coli (ExPEC) is responsible for various infections outside the gastrointestinal tract in humans and other animals. ExPEC strain MT78 is invasive to various nonphagocytic cells and highly virulent in vivo To identify genes required for invasion of nonphagocytic cells by this strain, we applied signature-tagged mutagenesis to generate a library of mutants and tested them for invasion of avian fibroblasts. Mutants showing reduced cellular invasion included those with insertions in the fim operon, encoding type 1 fimbriae. Another attenuated mutant showed a disruption in the treA gene, which encodes a periplasmic trehalase. The substrate of TreA, trehalose, can be metabolized and used as a carbon source or can serve as an osmoprotectant under conditions of osmotic stress in E. coli K-12. We generated and characterized mutant MT78ΔtreA In contrast to the wild type, MT78ΔtreA was able to grow under osmotic stress caused by 0.6 M urea but not in minimal M9 medium with trehalose as the only carbon source. It presented decreased association and invasion of avian fibroblasts, decreased yeast agglutination titer, and impaired type 1 fimbria production. In a murine model of urinary tract infection, MT78ΔtreA was less able to colonize the bladder. All phenotypes were rescued in the complemented mutant. Our results show that the treA gene is needed for optimal production of type 1 fimbriae in ExPEC strain MT78 and that loss of treA significantly reduces its cell invasion capacity and colonization of the bladder in a murine model of urinary tract infection.

Virulence factors and antimicrobial susceptibility profile of extraintestinal Escherichia coli isolated from an avian colisepticemia outbreak.

In this study an avian colisepticemia outbreak was investigated. Two isolates from a chicken with colisepticemia were characterized for antimicrobial susceptibility and virulence factors profile. For this purpose 7 antimicrobial and 29 genes (fimH, hrlA/hek, iha, papC, sfa/focCD, tsh, mat, tia, gimB, ibeA, chuA, fyuA, ireA, iroN, irp2, iucD, sitD. chr., sitD. ep., iss, neuC, ompA, traT, astA, hlyA, sat, vat, pic, malX, cvi/cva) were tested. The outbreak happened in a hick chicken breeding located in the northwestern region of Rio Grande do Sul state in South of Brazil and caused 28.3% (102 deads of a total of 360 chickens) of mortality rate. Escherichia coli isolates obtained from the avian spleen and liver belong to the same phylogenetic group A and present resistance to all antimicrobials tested (ampicillin, tetracycline, gentamicin, neomycin, sulfa + trimethoprim, enrofloxacin, and norfloxacin). Both isolates harbor virulence factors related to adhesion (fimH, papC, mat), invasion (tia), iron acquisition system (iroN) and serum resistance (iss, ompA, traT), showing that these groups are important for Avian Pathogenic E. coli (APEC). However, they present different virulence profiles for some genes, whereas liver-isolate carries more hrlA/hek (adhesin), gimB (invasin), sitD ep. (iron acquisition system), sat (toxin) and hylA (toxin) genes, the spleen-isolate harbors fyuA (iron acquisition system) gene. Here, we highlight a coinfection by different strains of APEC in the same animal with colisepticemia, the great antimicrobial resistance of these bacterial isolates and the genetic traits that modulate the virulence for high mortality rate of chickens for human consumption.

Nocardia nova causing empyema necessitatis afterlung re-transplantation: a case report.

We report herein a case of thoracic infection due to Nocardia nova following lung re-transplantation performed for emphysema related to alpha-1-antitrypsin deficiency. The infection extended from the lung into the pleural space, thoracic wall, and mediastinum, presenting as pericarditis and empyema necessitatis. Nocardia nova was identified by 16S ribosomal deoxyribonucleic acid (rDNA) sequencing and phylogenetic analysis. According to a literature search of PubMed, LILACS and MEDLINE databases, we describe herein the first case of empyema necessitatis caused by N. nova species in a transplanted patient.

Nocardia nova causing empyema necessitatis afterlung re-transplantation: a case report

Abstract: We report herein a case of thoracic infection due to Nocardia nova following lung re-transplantation performed for emphysema related to alpha-1-antitrypsin deficiency. The infection extended from the lung into the pleural space, thoracic wall, and mediastinum, presenting as pericarditis and empyema necessitatis. Nocardia nova was identified by 16S ribosomal deoxyribonucleic acid (rDNA) sequencing and phylogenetic analysis. According to a literature search of PubMed, LILACS and MEDLINE databases, we describe herein the first case of empyema necessitatis caused by N. nova species in a transplanted patient.

Pyogranulomatous dermatitis and panniculitis due to Nocardia nova in a cat / Dermatite e paniculite piogranulomatosas causadas por Nocardia nova em um gato

This report describes the clinical, pathological and microbiological findings of an uncommon infection in a cat by Nocardia nova. A 3-year-old male domestic short hair cat with an ulcerated and exudative cutaneous wound was presented for clinical examination. Samples were collected for histopathology and bacteriology diagnosis. Microscopically, the lesion was diagnosed as pyogranulomatous dermatitis and panniculitis with large and irregular colonies of branching filamentous bacterium. Skin bacteriological culture showed gram-positive rods and partially acid-fast branching filaments by gram and kinyoun staining, respectively. The identity of Nocardia novawas confirmed by 16S rDNA sequencing and phylogenetic analysis. This is the first case of pyogranulomatous dermatitis and panniculitis in a cat caused byNocardia novareported in Brazil.

Este relato descreve os achados clínicos, patológicos e microbiológicos de uma infecção incomum por Nocardia novaem um gato. Um gato macho, sem raça definida, de 3 anos de idade, apresentou ferida cutânea exsudativa e ulcerada. Amostras da lesão foram coletadas para histopatologia e bacteriologia. Histologicamente, a lesão consistiu de dermatite e paniculite piogranulomatosas associadas a colônias grandes e irregulares de bactérias filamentosas e ramificadas. O cultivo bacteriológico revelou bacilos filamentosos, gram-positivos, parcialmente ácido resistentes, visualizados pelas colorações de Gram e Kinyoun, respectivamente. A identificação da Nocardia novafoi confirmada pelo sequenciamento 16S rDNA e análise filogenética. Este é o primeiro caso de paniculite e dermatite piogranulomatosas em um gato causado por Nocardia novaregistrado no Brasil.

Phylogenetic analysis and genetic diversity of 3' region of rtxA gene from geographically diverse strains of Moraxella bovis, Moraxella bovoculi and Moraxella ovis.

The cytotoxin A (MbxA) is one of the main virulence factors of Moraxella bovis involved in the pathogenesis of infectious bovine keratoconjunctivitis (IBK). Moraxella ovis and Moraxella bovoculi, suspected to be associated with infectious keratitis in sheep and cattle respectively, also have a gene that encodes the cytotoxin A (movA and mbvA, respectively). The aim of this study was to determine the molecular sequence of the 3' region of the cytotoxin gene of Moraxella spp. strains isolated from clinical cases to establish phylogenetic and evolutionary comparisons. PCR amplification, nucleotide sequencing (nt) and amino acid (aa) sequence prediction were performed, followed by the sequences comparison, identity level calculation and selective pressure analysis. The phylogenetic reconstruction based on nt and aa sequences clearly differentiate M. bovis (n=15), M. bovoculi (n=11) and M. ovis (n=7) and their respective reference strains. An alignment of 843nt revealed high similarity within bacterial species (MbxA=99.9% nt and aa; MbvA=99.3% nt and 98.8% aa; MovA=99.5% nt and 99.3% aa). The similarity of partial sequences (nt 1807-2649) of MbxA in relation to MbvA and MovA ranged from 76.3 to 78.5%; similarity between MbvA and MovA ranged from 95.7 to 97.5%. A negative selection on mbvA and movA sequences was revealed by the molecular evolution analysis. The phylogenetic analysis of movA and mbvA allowed different strains of Moraxella spp. to be grouped according to the period of isolation. Sequence analysis of cytotoxin may provide insights into genetic and evolutionary relationships and into the genetic/molecular basis of Moraxella spp.

Molecular characterization and clonal relationships among Escherichia coli strains isolated from broiler chickens with colisepticemia.

This study characterized 52 Escherichia coli isolates from distinct diseased organs of 29 broiler chickens with clinical symptoms of colibacillosis in the Southern Brazilian state of Rio Grande do Sul. Thirty-eight isolates were highly virulent and 14 were virtually avirulent in 1-day-old chicks, yet all isolates harbored virulence factors characteristic of avian pathogenic E. coli (APEC), including those related to adhesion, iron acquisition, and serum resistance. E. coli reference collection phylogenetic typing showed that isolates belonged mostly to group D (39%), followed by group A (29%), group B1 (17%), and group B2 (15%). Phylogenetic analyses using the Amplified Ribosomal DNA Restriction Analysis and pulse-field gel electrophoresis methods were used to discriminate among isolates displaying the same serotype, revealing that five birds were infected with two distinct APEC strains. Among the 52 avian isolates, 2 were members of the pandemic E. coli O25:H4-B2-ST131 clone.

Genotypes and pathogenicity of cellulitis isolates reveal traits that modulate APEC virulence.

We characterized 144 Escherichia coli isolates from severe cellulitis lesions in broiler chickens from South Brazil. Analysis of susceptibility to 15 antimicrobials revealed frequencies of resistance of less than 30% for most antimicrobials except tetracycline (70%) and sulphonamides (60%). The genotyping of 34 virulence-associated genes revealed that all the isolates harbored virulence factors related to adhesion, iron acquisition and serum resistance, which are characteristic of the avian pathogenic E. coli (APEC) pathotype. ColV plasmid-associated genes (cvi/cva, iroN, iss, iucD, sitD, traT, tsh) were especially frequent among the isolates (from 66.6% to 89.6%). According to the Clermont method of ECOR phylogenetic typing, isolates belonged to group D (47.2%), to group A (27.8%), to group B2 (17.4%) and to group B1 (7.6%); the group B2 isolates contained the highest number of virulence-associated genes. Clonal relationship analysis using the ARDRA method revealed a similarity level of 57% or higher among isolates, but no endemic clone. The virulence of the isolates was confirmed in vivo in one-day-old chicks. Most isolates (72.9%) killed all infected chicks within 7 days, and 65 isolates (38.1%) killed most of them within 24 hours. In order to analyze differences in virulence among the APEC isolates, we created a pathogenicity score by combining the times of death with the clinical symptoms noted. By looking for significant associations between the presence of virulence-associated genes and the pathogenicity score, we found that the presence of genes for invasins ibeA and gimB and for group II capsule KpsMTII increased virulence, while the presence of pic decreased virulence. The fact that ibeA, gimB and KpsMTII are characteristic of neonatal meningitis E. coli (NMEC) suggests that genes of NMEC in APEC increase virulence of strains.

Diversity of seM in Streptococcus equi subsp. equi isolated from strangles outbreaks.

Strangles is the main upper respiratory tract disease of horses. There are currently no studies on the changes in alleles of the M protein gene (seM) in Brazilian isolates of Streptococcus equi ssp. equi (S. equi). This study aimed to analyze and differentiate molecularly S. equi isolates from equine clinical specimens from southern Brazil, between 1994 and 2010. seM alleles were analyzed in 47 isolates of S. equi obtained from clinical cases of strangles (15 Thoroughbred horses, 29 Crioulo breed horses and three Brasileiro de Hipismo--BH). seM alleles characterization was performed by comparing variable region sequences of the seM gene. The alleles were also phylogenetically grouped by Neighbor-joining analysis, which demonstrated the geographic distribution of those in properties from southern Brazil. Fifteen alleles of the gene seM were found among the 47 S. equi isolates analyzed. Among these, only one allele (seM-61), which was identified in seven isolates (14.9%), was found in the database PubMLST-seM. Within the new alleles, allele seM-115 was the most prevalent, having been found in 13 isolates (27.7%), followed by allele seM-117 in 10 isolates (21.3%). In the Brazilian horse population studied, there is greater diversity of M protein alleles in S. equi isolates compared to worldwide data deposited in PubMLST-seM. Among the 15 seM alleles identified, only one allele sequence was previously published. The alleles identification is important to control the disease by guiding selection of strains for the manufacture of commercial and autogenous vaccines.

Avian pathogenic Escherichia coli MT78 invades chicken fibroblasts.

Avian pathogenic Escherichia coli (APEC) are responsible for extraintestinal diseases, called colibacillosis, in avian species. The most severe manifestation of the disease is colisepticemia that usually starts at the respiratory tract and may result in bird death. However, it is not yet clear how APEC cross the respiratory epithelium and get into the bloodstream. In this work, we studied the interaction between 8 APEC strains (UEL31, UEL17, UEL13, UEL29, MT78, IMT5155, IMT2470, A2363) and a chicken non-phagocytic cell, the fibroblast CEC-32 cell line. We investigated the association profile, the invasion capability, the cytotoxicity effect and the induction of caspase-3/7 activation in an attempt to understand the way the pathogen gains access to the host bloodstream. Association to cells was determined after 1 h of infection, while cell invasion was determined after 4 and 24 h of infection. The cytotoxic effect of bacterial infection was measured by lactate dehydrogenase (LDH) release and the activation of the apoptotic program was verified by caspase-3/7 activation. Also, the presence of genes for adhesins, invasins and other related virulence-associated factors was verified by PCR. All bacterial strains showed similarity in relation to adhesion, LDH release and caspase-3/7 activation. However, one APEC strain, MT78, showed high invasion capability, comparable to the invasive Salmonella typhimurium strain SL1344. Since an APEC strain was capable of invading non-phagocytic cells in vitro, the same may be happening with the epithelial cells of the avian respiratory tract in vivo. CEC-32 monolayers can also provide a useful experimental model to study the molecular mechanisms used by APEC to invade non-phagocytic cells.

Incidência de hipotireoidismo auto imune em pacientes atendidos na cidade de Três de Maio, RS / Incidence of hypothyroidism autoimmune in patients checked in Três de Maio City, RS

A TH (TH) é uma doença auto-imune considerada como causa habitual do hipotireoidismo primário. No presente estudoavaliou-se, retrospectivamente, o perfil clínico dos pacientes da Clínica São Vicente de Paula, Três de Maio, RS, atendidos no período de março a dezembro de 2003. Foram analisados 60 casos com hipotireoidismo diagnosticado, entre 2 a 96 anos de idade, sendo 54 do sexo feminino e 6 do sexo masculino. Destes, 22 casos (36,7) apresentaram anticorpos antitireiodianos, sendo 18 do sexo feminino e 4 do sexo masculino, numa proporção, aproximada, de 5:1, localizados na área rural (36,36...