RESUMO
OBJECTIVES: To clarify the transmission mechanism of the blaCTX-M-64 gene between Escherichia coli and Salmonella isolates from food animals. METHODS: A total of 329 E. coli and 60 Salmonella isolates collected from food animals in 2016 were screened for the presence of blaCTX-M-64 genes. The blaCTX-M-64-positive isolates were typed and plasmid and chromosome DNA was sequenced to determine the genetic context of blaCTX-M-64 and the plasmid types present. RESULTS: The blaCTX-M-64 gene was identified in only three E. coli isolates but was the predominant gene in the Salmonella isolates (n = 9). These 12 CTX-M-64-positive isolates were all resistant to ampicillin, cefotaxime, ceftiofur, ceftriaxone, ceftazidime and florfenicol and 9 were resistant to ciprofloxacin. The blaCTX-M-64 gene was located on transferable IncI2 plasmids and an IncHI2 plasmid in three E. coli and one Salmonella isolate, respectively. The remaining eight Salmonella isolates contained blaCTX-M-64 integrated into the chromosome. Different genetic contexts of blaCTX-M-64 genes were found among the 12 isolates: ISEcp1-blaCTX-M-64-orf477-A/C on IncI2 plasmids of 3 E. coli isolates; ΔISEcp1-blaCTX-M-64-orf477-A/C in the chromosome of 1 Salmonella isolate; and ISEcp1-blaCTX-M-64-orf477 on the IncHI2 plasmid and chromosome of 8 Salmonella isolates. CONCLUSIONS: To the best of our knowledge, this is the first report of chromosomally encoded CTX-M-64 in Salmonella isolates. ISEcp1-mediated transposition is likely to be responsible for the spread of blaCTX-M-64 between different plasmids and chromosomes in Enterobacteriaceae especially E. coli and Salmonella.
Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Animais , Antibacterianos/farmacologia , Escherichia coli/genética , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Plasmídeos/genética , Salmonella/genética , beta-Lactamases/genéticaRESUMO
Maternal high-fat diet (HFD) during pregnancy is linked to cardiovascular diseases in postnatal life. The current study tested the hypothesis that maternal HFD causes myocardial changes through angiotensin II receptor (AGTR) expression modulation in fetal and neonatal rat hearts. The control group of pregnant rats was fed a normal diet and the treatment group of pregnant rats was on a HFD (60% kcal fat). Hearts were isolated from embryonic day 21 fetuses (E21) and postnatal day 7 pups (PD7). Maternal HFD decreased the body weight of the offspring in both E21 and PD7. The ratio of heart weight to body weight was increased in E21, but not PD7, when compared to the control group. Transmission electron microscopy revealed disorganized myofibrils and effacement of mitochondria cristae in the treatment group. Maternal HFD decreased S-phase and increased G1-phase of the cellular cycle for fetal and neonatal cardiac cells. Molecular markers of cardiac hypertrophy, such as Nppa and Myh7, were found to be increased in the treatment group. There was an associated increase in Agtr2 mRNA and protein, whereas Agtr1a mRNA and AGTR1 protein were decreased in HFD fetal and neonatal hearts. Furthermore, maternal HFD decreased glucocorticoid receptors (GRs) binding to glucocorticoid response elements at the Agtr1a and Agtr2 promoter, which correlated with downregulation of GR in fetal and neonatal hearts. These findings suggest that maternal HFD may promote premature termination of fetal and neonatal cardiomyocyte proliferation and compensatory hypertrophy through intrauterine modulation of AGTR1 and AGTR2 expression via GR dependent mechanism.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Fenômenos Fisiológicos da Nutrição Materna , Miocárdio/metabolismo , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Receptores de Angiotensina/genética , Animais , Animais Recém-Nascidos , Cardiomegalia/congênito , Cardiomegalia/embriologia , Cardiomegalia/genética , Cardiomegalia/metabolismo , Células Cultivadas , Gorduras na Dieta/farmacologia , Feminino , Feto/efeitos dos fármacos , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Coração/efeitos dos fármacos , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Ratos , Ratos Sprague-Dawley , Receptores de Angiotensina/metabolismoRESUMO
Maternal high-fat diet (HFD) is associated with cardiovascular disease later in life. This study tested the hypothesis that maternal HFD causes programming of increased cardiac angiotensin II receptor type 2 (AGTR2) expression, resulting in heightened cardiac susceptibility to ischemic injury in male offspring in a sex-dependent manner. Pregnant rats were divided between control and HFD (HFD-fed during gestation) groups. Maternal HFD resulted in cardiac hypertrophy in only male offspring, but had no effect on cardiac systolic and diastolic function in both male and female offspring. In addition, maternal HFD increased heart susceptibility to ischemia-reperfusion injury in adult male offspring, but not female offspring. There was an increase in Agtr2 mRNA and protein abundance in male, but not female offspring. However, maternal HFD had no effect on angiotensin II receptor type 1 (AGTR1) expression in both male and female offspring. HFD resulted in decreased glucocorticoid receptors (GRs) binding to the glucocorticoid response elements at the Agtr2 promoter, which was due to decreased GRs in the hearts of adult male offspring. Inhibition of AGTR2 with PD123319 abrogated maternal HFD-induced increase in cardiac ischemic vulnerability in male adult rats. The results demonstrate that maternal HFD causes programming of increased Agtr2 gene expression in the heart by downregulation of GR, contributing to the heightened cardiac vulnerability to ischemic injury in adult male offspring in a sex-dependent manner.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Cardiopatias/induzido quimicamente , Receptor Tipo 2 de Angiotensina/biossíntese , Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/genética , Feminino , Desenvolvimento Fetal , Predisposição Genética para Doença/genética , Cardiopatias/genética , Imidazóis/farmacologia , Masculino , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Isquemia Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/genética , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor Tipo 2 de Angiotensina/genética , Caracteres SexuaisRESUMO
We investigated the molecular characteristics and antimicrobial susceptibility of Clostridium difficile isolated from animals in China. We obtained 538 rectal swabs from pigs, chickens and ducks in 5 provinces during 2015 and 2016. C. difficile isolates were characterized by detection of toxin genes, multilocus sequence typing and ribotyping. And antimicrobial susceptibility testing was performed using the agar dilution method. Out of 538 samples, 44 (8.2%) were C. difficile positive with high prevalence in pigs (n = 31). Among these, 39 (88.6%) were toxigenic including 14 (31.8%) that were A+B+CDT+ and 13 (29.5%) A+B+. The remaining 12 (27.3%) were A-B+. We identified 7 ST types and 6 PCR ribotypes. The most predominant type was ST11/RT078 with toxin profile A+B+CDT+ and all were isolated from piglets with diarrhea. ST109 isolates possessed two different toxigenic profiles (A-B-CDT- and A-B+CDT-) and although it was not the most prevalent sequence type, but it was widely distributed between chickens, ducks and pigs in the 5 provinces. All C. difficile isolates were fully susceptible to vancomycin, metronidazole, fidaxomicin, amoxicillin/clavulanate and meropenem but retained resistance to 4 or 5 of the remaining antibiotics, especially cefotaxime, tetracycline, ciprofloxacin, cefoxitin. The RT078/ST11 isolates were simultaneously resistant to cefotaxime, tetracycline, cefoxitin, ciprofloxacin and imipenem. This is the first report of the molecular epidemiology of C. difficile isolated from food animals in China. We identified the epidemic strain RT078/ST11 as the predominate isolate among the animals we screened in our study.
Assuntos
Clostridioides difficile/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Cefotaxima/análise , Cefoxitina/farmacologia , Galinhas , China , Ciprofloxacina/farmacologia , Clostridioides difficile/genética , Clostridioides difficile/isolamento & purificação , Patos , Imipenem/farmacologia , Metronidazol/farmacologia , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase , Ribotipagem , Suínos , Tetraciclina/farmacologia , Vancomicina/farmacologiaRESUMO
We studied mechanisms of drug resistance development in Escherichia coli strains lacking efflux pump components. E. coli K12 deletion mutants were subjected to increasing concentrations of ciprofloxacin (CIP) to determine the frequency of target gene mutations. We generated a series of mutants that were selected based on their minimum inhibitory concentrations (MICs) to CIP, as well as their corresponding point mutations in target genes. The mutants displayed a number of target modifications and, in particular, gyrA mutations altering codons Ser83Leu, Asp87Gly, and Asp87His as well as a change in parC at 78 (substitution of Gly for Asp). All these mutations were related to drug resistance. When exposed to CIP, mutants lacking efflux pump genes acrA and acrB demonstrated a low level of resistance that was because of point mutations in the target genes. High-level resistance was achieved with a 100- to 500-fold increase in expression of efflux pump genes acrE and acrF that compensated for the loss of AcrA and AcrB, and thus resulted in an obvious increase of CIP MIC. We demonstrate that an intact AcrAB-TolC efflux pump is crucial to the development of bacterial resistance. Its activity is complemented by expression of the alternative AcrEF efflux pump.
Assuntos
Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Lipoproteínas/genética , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Substituição de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Códon , DNA Girase/genética , DNA Girase/metabolismo , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipoproteínas/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos/deficiência , MutaçãoRESUMO
The aim of this study was to determine the expression of eight other functional transporter genes upon acrAB inactivation and also the expression of acrAB when the function of eight other transporters are impaired in Salmonella enterica. We used single- or multigene deletion mutants (i.e., ΔacrA, ΔacrB, ΔtolC, ΔacrAB, ΔacrEF, ΔacrD, ΔmdsABC, ΔmdtABC, ΔemrAB, ΔmacAB, ΔmdfA, ΔmdtK, ΔacrABramA, ΔacrABmarA, and ΔacrABsoxS) and real time (RT)-PCR to quantify the expression of different pump and regulator genes; infection ability was characterized by adhesion and invasion assays. The expression of acrAB operon was increased upon acrB inactivation. Single deletion of acrA or tolC also increased expression of acrB. The deletion of acrAB increased expression of eight other functional efflux pumps genes and vice versa, in which increased expression of ramA and marA was also detected. Mutants containing single deletions of functional pump genes were attenuated in cells. In conclusion, there is a feedback mechanism that coordinates regulation of AcrAB-TolC and eight other functional efflux pumps through the global transcriptional regulators ramA and marA in S. enterica serovar Typhimurium.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Salmonella typhimurium/genética , Transativadores/genética , Transcrição Gênica , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Retroalimentação Fisiológica , Deleção de Genes , Proteínas de Membrana Transportadoras/deficiência , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Óperon , Salmonella typhimurium/metabolismo , Sorogrupo , Transativadores/deficiência , Transativadores/metabolismoRESUMO
The aim of this study was to investigate the difference in resistance mechanisms and fitness of Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE) mutants selected during the evolution of resistance under exposure to increasing ciprofloxacin concentrations in vitro. Mutations in quinolone target genes were screened by PCR. Phenotypic characterization included susceptibility testing by the broth dilution method, investigation of efflux activity and growth rate, and determination of the invasion of human intestinal epithelium cells in vitro. The two Salmonella serotypes exhibited differences in target gene mutations and efflux pump gene expression during the development of resistance. In the parental strains, ST had a competitive advantage over SE. During the development of resistance, initially, the SE strain was more competitive. However, once ciprofloxacin resistance was acquired, ST once again became the more competitive strain. In the absence of bile salts or at 0.1% bile, the growth rate of SE was initially greater than that of ST, but once ciprofloxacin resistance was acquired, ST had higher growth rates. ST strains showed decreased invasion of epithelial cells in 0.1% bile. These data indicate that ciprofloxacin-resistant ST strains are more competitive than ciprofloxacin-resistant SE strains.
Assuntos
Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Farmacorresistência Bacteriana , Aptidão Genética , Mutação , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/genética , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Evolução Molecular , Testes de Sensibilidade Microbiana , Salmonella enteritidis/classificação , Salmonella typhimurium/classificação , Seleção Genética , SorogrupoRESUMO
One known cyclic peptide, beauvericin, was isolated from the secondary metabolites of mangrove endophytic fungi Fusarium sp. (No. DZ27) in South China Sea. Its structure was determined by spectral analyses and comparisons with reference data from literatures. Beauvericin inhibited growth of KB and KBv200 cells potently with IC50 values of 5.76 ± 0.55 and 5.34 ± 0.09 µM, respectively. Furthermore, beauvericin induced apoptosis through mitochondrial pathway, including decrease of relative oxygen species generation, loss of mitochondrial membrane potential, release of cytochrome c, activation of Caspase-9 and -3, and cleavage of PARP. Additionally, regulation of Bcl-2 or Bax was not involved in the apoptosis induced by beauvericin in KB and KBv200 cells.