Association between virulence factors and antimicrobial resistance from enterotoxigenic escherichia coli isolated from pigs with diarrhea in Brazil

Background: Enterotoxigenic Escherichia coli (ETEC) is the etiologic agent of post weaning colibacillosis, one of the most important diseases in pig farming. The pathogenesis of the disease is associated with two virulence factors (VF), fimbriae and enterotoxins. In veterinary medicine, the use of antibiotics can lead to the selection of resistant bacteria. The association of VF and antibiotic resistance is an important mechanism for bacterial survival under adverse conditions. This study aimed to determine the VF and antimicrobial susceptibility of ETEC isolates from piglets with diarrhea and analyze the association between these factors. Materials, Methods & Results: A total of 185 rectal swabs were collected from weaned piglets in Brazilian farms of the states of Rio Grande do Sul, Santa Catarina, Paraná, Minas Gerais and Goiás. The isolation of ETEC was carried out on blood and MacConkey Agar and characterization by biochemical tests and detection by PCR of fimbrial genes F4, F45, F6, F18 and F41, and toxins genes LT, STa, STb and STx2e. Antimicrobial susceptibility profiles were determined by Agar diffusion test for amoxicillin, ampicillin, ceftiofur, ciprofloxacin, colistin, doxycycline, enrofloxacin, florfenicol, gentamicin, neomycin, norfloxacin, oxytetracycline, streptomycin, tetracycline, lincomycin + spectinomycin and sulfamethoxazole + trimethoprim. The association between VF and antimicrobials resistance results was determined by Chi-square and Fisher test (P ≤ 0.05). A total of 376 isolates were analyzed. The frequencies of fimbriae and toxins amplified were: F4 (31.6%), F18 (18.9%), F5 (4.2%) and toxins STa (43.1%), STb (24.7%), LT (21.8%) and STx2e (5.3%). Antibiotic resistance was higher to tetracycline (96.3%), florfenicol (95.2%), oxytetracycline (93.62%) and doxycycline (90.7%). Lowest levels of resistance were to ceftiofur (2.1%), colistin (9.8%), lincomycin + spectinomicin (15.4%) and neomycin (23.1%). The association of VF and resistance was significant for fimbriae F4 and streptomycin and sulfamethoxazole + trimethoprim; F5 and enrofloxacin, ciprofloxacin and norfloxacin; F18 and amoxicillin, ampicillin, doxycycline, enrofloxacin, norfloxacin, ciprofloxacin and sulfamethoxazole + trimethoprim. Among the toxins LT and tetracycline, streptomycin and florfenicol; STa and amoxicillin, ampicillin, oxytetracycline, enrofloxacin and sulfamethoxazole + trimethoprim; STb and colistin and streptomycin. Discussion: Resistance to all antimicrobials was observed, with higher levels for tetracyclines and lower to ceftiofur and lincomycin + spectinomycin. Several studies have reported ETEC virotype variation which can be influenced by management differences in pig farms. The presence of VF and acquisition of antimicrobial resistance mechanisms might be related to evolutionary factors, infection pressure and intensive use of antibiotics. E. coli is considered one of the most versatile bacterial species and its diversity in adaptability is due to its genomic plasticity, influencing the capacity to colonize numerous host species. This is possible by mechanisms such as gain or loss of genes through lateral transfer of plasmids, transposons and integrons, which vary regarding the environment to which they are exposed. More studies are needed to correlate genetically the interaction of antimicrobial resistance and virulence genes to elucidate if the virulence expression is affected by chromosomal mutations that lead to specific resistance or/ and both determinants are inserted into the same mobile genetic element, such as a conjugative plasmid

First Evidence of Bovine Viral Diarrhea Virus Infection in Wild Boars

Background: The farming of wild boars has growing due to the interest of the human consumption of this exotic meat. Such a development may pose an increased risk of disease transmission between boars and domestic animals. The wild boar population has increased in South America in the last years due the absence of predator causing economic losses due to direct damage to crops and risk of disease transmission. The genus Pestivirus within the family Flaviviridae are composed by four recognized species by the International Committee on the Taxonomy of Viruses (ICTV): classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhea virus type 1 (BVDV-1) and 2 (BVDV-2). Other putative species denoted as atypical pesitiviruses have been reported as ‘HoBi’-like virus, giraffe pestivirus, Bungowannah pestivirus, Pronghorn antelope virus, atypical porcine pestivirus (APPV), Norwegian rat pestivirus (NrPV) and Rhinolophus affinis bat pestivirus (RaPestV-1). CSFV is commonly detected in wild boars, but despite positive serology, bovine viral diarrhea virus (BVDV) was never detected in this animal species. Thereby, the present communication describes the first detection of BVDV in the lungs of captive boars using RT-PCR and DNA sequencing.Materials, Methods & Results: Forty lung samples from farmed wild boars were collected after slaughter in a commercial abattoir. The organs were crushed separately, centrifuged, and the supernatant was stored for further analysis. The total RNA was isolated using a phenol-based protocol and RT-PCR protocol that amplified 118 bp of 5’ untranslated region (5’UTR) was carried out. One out 40 samples resulted positive. The positive sample had partial fragments of 5’UTR and N terminal autoprotease (Npro) sequenced and analyzed. The strain LV Java/2012 presented 99% of identity in 5’UTR and 98% in Npro region with a BVDV-2 previously reported in bovines in Southern Brazil. In both 5’UTR and Npro phylogenetic analysis, the strain LV Java/2015 clustered with BVDV-2 strains and was most closely related to subtype 2b identified in bovines in Southern Brazil grouping in the same terminal node.Discussion: Wild boars are commonly associated to pathogen transmission to domestic animals. This animal species is considered a reservoir of the pestivirus CSFV and important keys in CSFV control and eradication programs in Europe. Despite indirect presence of BVDV was reported in wild boars by serology tests, the direct detection of the viral agent was never reported. The present study showed the presence of BVDV-2 genomic segments obtained by RT-PCR followed by DNA sequencing in captive wild boars. The reported data suggests a possible importance of this animal species in the epidemiology of ruminant pestiviruses which could interfere in control and eradication programs of these important pathogens for cattle worldwide. The strain LV Java/2012 was closely related to BVDV-2b and presented highest identity with a strain detected in cattle from Southern Brazil. This data suggests that wild boars and bovines could be sharing this pathogen due the similarity of the strains and that both were reported in the same region. It can lead to need of inclusion of wild swines in BVDV control programs since boars can circulate between different regions and carry this pathogen to different cattle herds. The present study reported the first molecular evidence of BVDV in wild boars in the literature. The data generated herein suggests a possible importance of boars in the epidemiology of ruminant pestiviruses

Pharmacogenomic–pharmacokinetic study of selective estrogen-receptor modulators with intra-patient dose escalation in breast cancer

BackgroundAn association between CYP2D6 polymorphisms and tamoxifen (TAM) efficacy has not been confirmed, partly due to unreliable prediction of active metabolite exposure solely by CYP2D6 activity. The efficacy of TAM dose escalation appears limited in poor TAM metabolizers. Since the chlorine atom on the side chain of toremifene (TOR) prevents 4-hydroxylation by CYP2D6, its contribution to active conversion of TOR is minor. We examined the role of TOR and its dose escalation among poor TAM metabolizers.MethodsThe pharmacokinetics (PK) and pharmacogenomics (PGx) of TAM and TOR were studied. Correlation between PK and CYP2D6 inhibitor use, smoking status, and PGx were examined by regression analysis. For patients showing low endoxifen levels, an intra-patient dose escalation of TOR was conducted, and TOR was increased from 40 to 120 mg for ≥ 24 weeks with PK sampling. Total activity was calculated as the sum of the concentration of each active metabolite adjusted by their respective in vitro activities.ResultsFifty and 11 of the 273 participating patients had endoxifen levels < 15 and < 7.5 ng/mL, respectively. The CYP2D6 genotype was the major determinant for TAM activity (p < 0.01). Smoking status (p = 0.07) and the CYP2C19 phenotype (p = 0.07), but not the CYP2D6 genotype (p = 0.61), showed marginally significant effects on TOR activity. TOR activity increased significantly with dose escalation, even among poor TAM metabolizers, and was maintained for ≥ 24 weeks.ConclusionTOR might be a valid alternative to TAM in patients predicted to be poor TAM metabolizers

Diversidade genética e virulência de amostras de Brachyspira hyodysenteriae isoladas de suínos assintomáticos e com doença clínica.

Exportado OPUSMade available in DSpace on 2019-08-14T13:59:40Z (GMT). No. of bitstreams: 1 jos__paulo_hiroji_sato.pdf: 1713577 bytes, checksum: ba39f6cd58fecab564a783cb0597d55a (MD5) Previous issue date: 29Esta tese foi dividida em dois estudos. Primeiro objetivou-se a tipificação e avaliação molecular de cepas de Brachyspira hyodysenteriae isoladas de suínos no Brasil. Foi aplicada a técnica de tipagem de sequências multilocus (MLST) de sete genes housekeeping em 46 isolados obtidos dos surtos de disenteria suína que ocorreram entre 2011 a 2015 nos estados de Minas Gerais, São Paulo, Mato Grosso, Rio Grande do Sul e Santa Catarina e isolados históricos da década de 90 de granjas do Rio Grande do Sul. Foi detectado um perfil nacional independente das sequencias globais depositadas no banco de dados de Brachyspira spp. Na análise entre as amostras brasileiras, foi constatada relação epidemiológica molecular intra-granja, entre granjas e entre os estados. O segundo projeto teve como objetivo, em experimento in vivo em suínos, avaliar a patogenicidade, características clínicas e moleculares de uma cepa de B. hyodysenteriae isolada de uma granja sem sinais clínicos de disenteria suína comparada com outra cepa isolada de animal com sinal clínico da doença. Nesta avaliação foram observadas diferenças significativas entre os grupos, considerando o início dos sinais clínicos, número de animais afetados e severidade das lesões macroscópicas e microscópicas.This thesis comprised the results of two studies. First, molecular typing and epidemiological evaluation of strains of Brachyspira hyodysenteriae isolated from swine in Brazil were performed. Multilocus Sequence Typing (MLST) analysis of seven housekeeping genes was performed in 46 Brazilian B. hyodysenteriae isolates obtained from pigs in farms during outbreaks of swine dysentery. Bacterial isolation was obtained between 2011 to 2015 in cases originated from the states of Minas Gerais, São Paulo, Mato Grosso, Rio Grande do Sul and Santa Catarina, and historical 1990s isolates from farms in the state Rio Grande do Sul. An independent MLST Brazilian MLST profile was observed compared with sequences deposited in the global Brachyspira spp database. Among the Brazilian strains, molecular relationship was observed in intra-farm profiles, among farms and among states. The second project used an in vivo challenge trial in pigs to evaluate the pathogenicity, clinical and molecular characteristics of a B. hyodysenteriae strain isolated from a pig with no clinical signs of swine dysentery, and a strain obtained from an animal with typical clinical signs of the disease. Significant differences were observed between groups, regarding onset of clinical signs, proportion ofclinically affected animals and severity of gross and microscopic lesions

Frequency and association of virulence factors in escherichia coli strains isolated in postweaning pigs

O presente estudo teve como objetivo avaliar a frequência dos fatores de virulência em amostras de Escherichia (E.) coli isoladas de leitões desmamados. Para a classificação de cepas de E. coli do patotipo ETEC, foram coletados suabes retais de leitões com idade entre 25-40 dias (fase de creche) com sinal clínico de diarreia, em granjas dos estados do Rio Grande do Sul, Santa Catarina e Paraná, totalizando 456 amostras. Para isolamento e caracterização bacteriana, foram utilizados métodos fenotípicos e moleculares. Foi realizada a análise estatística da frequência dos fatores de virulência e dos virotipos, para as fímbrias F4, F5, F6, F18, F41 e toxinas LT, STa, STb e STx2e. Das 456 amostras analisadas, 287 apresentaram crescimento significativo de E. coli. As maiores frequências observadas foram para as fímbrias F4 e F18 e para as enterotoxinas LT, STa e STb. Os virotipos mais frequentes foram F18-STa, F4-LT-STa-STb, F4-STa, F4-LT-STb e F18-STa-STx2e. Noventa e três amostras (32,4%) não apresentaram nenhum gene dos fatores analisados. Foi observada a associação significativa entre amostras positivas para a fímbria F4 e as toxinas LT, STa e STb; entre a fímbria F18 e as toxinas STa e STx2e; entre a fímbria F5 e todas as toxinas. Os genes de toxinas mais detectados (LT, STb) apresentaram associação significativa (P<0,02). A beta-hemólise foi observada em 47,4% das amostras e houve associação (P<0,0001) entre as amostras hemolíticas e os fatores de virulência F4, F18, STa e STx2e. Em relação à consistência das fezes, observou-se associação entre as fezes com consistência líquida e F4 e STa. Com base nos resultados obtidos, conclui-se que a ETEC é um importante agente etiológico de diarreia em leitões desmamados. Em diversas amostras (32,4%) não foram detectados fatores de virulência, sugerindo a ação de outros fatores e/ou agentes desencadeantes de diarreia. E além destes, existem várias hipóteses para explicar esta discrepância, as quais foram revisadas neste trabalho.The frequency of virulence factors in strains of Escherichia (E.) coli isolated from weaned piglets was assessed. For classification of the strains of enterotoxigenic pathotypes of E. coli, rectal swabs were collected from 25-40 days old nursery piglets with clinical signs of diarrhea in the states of Rio Grande do Sul, Santa Catarina and Paraná, totaling 456 samples. For isolation and bacterial characterization of the samples, phenotypic and molecular tests were used. The results of frequency of virulence factors and virotypes to fimbriae F4, F5, F6, F18, F41 and toxins LT, STa, STb and STx2e were submitted to statistical analysis. Out of 456 samples, 287 showed significant growth of E. coli. The highest frequency was observed for F4 and F18 fimbriae and LT, STa and STb enterotoxins. The most prevalent virotypes were F18-STa, F4-LT-STa-STb, STa-F4, F4-LT-STa-STb and F18-STx2e. Ninety-three samples (32.4%) were negative for virulence genes. There was a significant association between positive samples for F4 fimbriae and enterotoxins LT, STa and STb; between the F18 fimbriae and STa and STx2e; between F5 fimbriae and all enterotoxins. The most frequent toxins (LT, STb) presented significant association (P<0.02). Beta-hemolysis was observed in 47.4% of the samples and there was a association (P<0.0001) between hemolysis and fimbriae F4, F18, STa and STx2e. Regarding stool consistency, it was observed a association between liquid consistency and F4 and STa. Based on the results, it can be concluded that ETEC is an important agent of diarrhea in weaned pigs. In several samples (32.4%) virulence factors were not detected, suggesting the action of other factors and/or agents inducing diarrhea. And besides these, there are other hypotheses to explain this discrepancy, which were reviewed in this work

Pumpkin Seed Oil Extracted From Cucurbita maxima Improves Urinary Disorder in Human Overactive Bladder

The pumpkin seed oil obtained from Cucurbita pepo has been shown to be useful for the treatment of nocturia in patients with urinal disorders in several western countries. In this study, we evaluated the effect of the pumpkin seed oil from Cucurbita maxima on urinary dysfunction in human overactive bladder (OAB). Forty-five subjects were enrolled in this study. An extract of pumpkin seed oil from C. maxima (10 g of oil/day) was orally administrated for 12 weeks. After 6 and 12 weeks, urinary function was evaluated using Overactive Bladder Symptom Score (OABSS). Pumpkin seed oil from C. maxima significantly reduced the degree of OABSS in the subjects. The results from our study suggest that pumpkin seed oil extracts from C. maxima as well as from C. pepo are effective for urinary disorders such as OAB in humans