RESUMO
Between August and December 2013, the offshore cages of a commercial marine farm culturing red drum Sciaenops ocellatus in Campeche Bay Mexico were affected by an outbreak of an ulcerative granulomatous disease with up to 70% cumulative mortality. Thirty-one adults displaying open ulcers on the skin were submitted for diagnosis. At necropsy, multiple white-yellowish nodules (0.1-0.5 cm in diameter) were present in all internal organs, where the kidney and the spleen were the most severely affected. Histopathology evinced typical systemic granulomatous formations. Gram and Ziehl-Neelsen stains on tissue imprints, bacterial swabs and tissue sections revealed Gram-positive, acid-fast, branching beaded long rod filamentous bacteria. Tissue samples resulted positive for nocardiosis with a Nocardia genus-specific nested PCR. Definite identification at the species level and taxonomic positioning of the fastidious pathogen were achieved through a specific Nocardia seriolae PCR and by sequencing the gyrB gene of pure isolates. After administration of antibiotics during fry production, a posterior follow-up monitoring (from 2014 to 2017) detected mild but recurrent outbreaks of the bacteria with no seasonality pattern. To the extent of our knowledge, this is the first report of piscine nocardiosis in Mexico and the first time this disease is detected in red drum.
Assuntos
Doenças dos Peixes/diagnóstico , Peixes , Nocardiose/veterinária , Nocardia/isolamento & purificação , Animais , Doenças dos Peixes/microbiologia , México , Nocardia/classificação , Nocardia/genética , Nocardiose/diagnóstico , Nocardiose/microbiologiaRESUMO
The design, synthesis and structure-activity relationships of a series of oxazole-benzamide inhibitors of the essential bacterial cell division protein FtsZ are described. Compounds had potent anti-staphylococcal activity and inhibited the cytokinesis of the clinically-significant bacterial pathogen Staphylococcus aureus. Selected analogues possessing a 5-halo oxazole also inhibited a strain of S. aureus harbouring the glycine-to-alanine amino acid substitution at residue 196 of FtsZ which conferred resistance to previously reported inhibitors in the series. Substitutions to the pseudo-benzylic carbon of the scaffold improved the pharmacokinetic properties by increasing metabolic stability and provided a mechanism for creating pro-drugs. Combining multiple substitutions based on the findings reported in this study has provided small-molecule inhibitors of FtsZ with enhanced in vitro and in vivo antibacterial efficacy.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Benzamidas/farmacologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Desenho de Fármacos , Oxazóis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Benzamidas/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Oxazóis/química , Staphylococcus aureus/química , Relação Estrutura-AtividadeRESUMO
A series of dual-targeting, alcohol-containing benzothiazoles has been identified with superior antibacterial activity and drug-like properties. Early lead benzothiazoles containing carboxylic acid moieties showed efficacy in a well-established in vivo model, but inferior drug-like properties demanded modifications of functionality capable of demonstrating superior efficacy. Eliminating the acid group in favor of hydrophilic alcohol moieties at C(5), as well as incorporating solubilizing groups at the C(7) position of the core ring provided potent, broad-spectrum Gram-positive antibacterial activity, lower protein binding, and markedly improved efficacy in vivo.
Assuntos
Antibacterianos/farmacologia , Benzotiazóis/química , Benzotiazóis/farmacologia , DNA Bacteriano/química , DNA Bacteriano/efeitos dos fármacos , DNA Super-Helicoidal/efeitos dos fármacos , Haemophilus influenzae/efeitos dos fármacos , Álcoois/química , Antibacterianos/síntese química , Antibacterianos/química , Benzotiazóis/síntese química , Relação Dose-Resposta a Droga , Descoberta de Drogas , Testes de Sensibilidade Microbiana , Estrutura Molecular , Staphylococcus , Relação Estrutura-AtividadeRESUMO
Campylobacter is a leading cause of food-borne gastroenteritis worldwide, linked to the consumption of contaminated poultry meat. Targeting this pathogen at source, vaccines for poultry can provide short-term caecal reductions in Campylobacter numbers in the chicken intestine. However, this approach is unlikely to reduce Campylobacter in the food chain or human incidence. This is likely as vaccines typically target only a subset of the high genomic strain diversity circulating among chicken flocks, and rapid evolution diminishes vaccine efficacy over time. To address this, we used a genomic approach to develop a whole-cell autogenous vaccine targeting isolates harbouring genes linked to survival outside of the host. We hyper-immunised a whole major UK breeder farm to passively target offspring colonisation using maternally-derived antibody. Monitoring progeny, broiler flocks revealed a near-complete shift in the post-vaccination Campylobacter population with an ~50% reduction in isolates harbouring extra-intestinal survival genes and a significant reduction of Campylobacter cells surviving on the surface of meat. Based on these findings, we developed a logistic regression model that predicted that vaccine efficacy could be extended to target 65% of a population of clinically relevant strains. Immuno-manipulation of poultry microbiomes towards less harmful commensal isolates by competitive exclusion, has major potential for reducing pathogens in the food production chain.
RESUMO
The bacterial cell division protein FtsZ is an attractive target for small-molecule antibacterial drug discovery. Derivatives of 3-methoxybenzamide, including compound PC190723, have been reported to be potent and selective antistaphylococcal agents which exert their effects through the disruption of intracellular FtsZ function. Here, we report the further optimization of 3-methoxybenzamide derivatives towards a drug candidate. The in vitro and in vivo characterization of a more advanced lead compound, designated compound 1, is described. Compound 1 was potently antibacterial, with an average MIC of 0.12 µg/ml against all staphylococcal species, including methicillin- and multidrug-resistant Staphylococcus aureus and Staphylococcus epidermidis. Compound 1 inhibited an S. aureus strain carrying the G196A mutation in FtsZ, which confers resistance to PC190723. Like PC190723, compound 1 acted on whole bacterial cells by blocking cytokinesis. No interactions between compound 1 and a diverse panel of antibiotics were measured in checkerboard experiments. Compound 1 displayed suitable in vitro pharmaceutical properties and a favorable in vivo pharmacokinetic profile following intravenous and oral administration, with a calculated bioavailability of 82.0% in mice. Compound 1 demonstrated efficacy in a murine model of systemic S. aureus infection and caused a significant decrease in the bacterial load in the thigh infection model. A greater reduction in the number of S. aureus cells recovered from infected thighs, equivalent to 3.68 log units, than in those recovered from controls was achieved using a succinate prodrug of compound 1, which was designated compound 2. In summary, optimized derivatives of 3-methoxybenzamide may yield a first-in-class FtsZ inhibitor for the treatment of antibiotic-resistant staphylococcal infections.
Assuntos
Antibacterianos/farmacocinética , Proteínas de Bactérias/antagonistas & inibidores , Benzamidas/farmacocinética , Proteínas do Citoesqueleto/antagonistas & inibidores , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxazóis/farmacocinética , Pró-Fármacos/farmacocinética , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus epidermidis/efeitos dos fármacos , Succinatos/farmacocinética , Administração Oral , Animais , Antibacterianos/síntese química , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Benzamidas/síntese química , Benzamidas/química , Benzamidas/farmacologia , Disponibilidade Biológica , Contagem de Colônia Microbiana , Citocinese/efeitos dos fármacos , Proteínas do Citoesqueleto/genética , Farmacorresistência Bacteriana Múltipla , Feminino , Injeções Intravenosas , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Camundongos , Testes de Sensibilidade Microbiana , Mutação , Oxazóis/síntese química , Oxazóis/farmacologia , Pró-Fármacos/síntese química , Pró-Fármacos/farmacologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/crescimento & desenvolvimento , Succinatos/síntese química , Succinatos/farmacologia , Ácido Succínico/química , Coxa da Perna/microbiologia , Resultado do TratamentoRESUMO
The type II topoisomerases DNA gyrase (GyrA/GyrB) and topoisomerase IV (ParC/ParE) are well-validated targets for antibacterial drug discovery. Because of their structural and functional homology, these enzymes are amenable to dual targeting by a single ligand. In this study, two novel benzothiazole ethyl urea-based small molecules, designated compound A and compound B, were evaluated for their biochemical, antibacterial, and pharmacokinetic properties. The two compounds inhibited the ATPase activity of GyrB and ParE with 50% inhibitory concentrations of <0.1 µg/ml. Prevention of DNA supercoiling by DNA gyrase was also observed. Both compounds potently inhibited the growth of a range of bacterial organisms, including staphylococci, streptococci, enterococci, Clostridium difficile, and selected Gram-negative respiratory pathogens. MIC90s against clinical isolates ranged from 0.015 µg/ml for Streptococcus pneumoniae to 0.25 µg/ml for Staphylococcus aureus. No cross-resistance with common drug resistance phenotypes was observed. In addition, no synergistic or antagonistic interactions between compound A or compound B and other antibiotics, including the topoisomerase inhibitors novobiocin and levofloxacin, were detected in checkerboard experiments. The frequencies of spontaneous resistance for S. aureus were <2.3 × 10(-10) with compound A and <5.8 × 10(-11) with compound B at concentrations equivalent to 8× the MICs. These values indicate a multitargeting mechanism of action. The pharmacokinetic properties of both compounds were profiled in rats. Following intravenous administration, compound B showed approximately 3-fold improvement over compound A in terms of both clearance and the area under the concentration-time curve. The measured oral bioavailability of compound B was 47.7%.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Benzotiazóis/farmacologia , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerases Tipo II/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Inibidores da Topoisomerase/farmacologia , Ureia/análogos & derivados , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Antibacterianos/química , Antibacterianos/farmacocinética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzotiazóis/química , Benzotiazóis/farmacocinética , Sobrevivência Celular/efeitos dos fármacos , DNA Topoisomerase IV/genética , DNA Topoisomerase IV/metabolismo , DNA Topoisomerases Tipo II/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/crescimento & desenvolvimento , Células Hep G2 , Humanos , Interleucina-33 , Interleucinas , Levofloxacino/farmacologia , Masculino , Testes de Sensibilidade Microbiana , Novobiocina/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Inibidores da Topoisomerase/química , Inibidores da Topoisomerase/farmacocinética , Ureia/química , Ureia/farmacocinética , Ureia/farmacologiaRESUMO
The discovery and optimisation of a new class of benzothiazole small molecules that inhibit bacterial DNA gyrase and topoisomerase IV are described. Antibacterial properties have been demonstrated by activity against DNA gyrase ATPase and potent activity against Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes and Haemophilus influenzae. Further refinements to the scaffold designed to enhance drug-likeness included analogues bearing an α-substituent to the carboxylic acid group, resulting in excellent solubility and favourable pharmacokinetic properties.
Assuntos
Benzotiazóis/química , Benzotiazóis/farmacologia , DNA Topoisomerase IV/antagonistas & inibidores , Desenho de Fármacos , Ácidos Isonipecóticos/química , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Benzotiazóis/síntese química , DNA Girase/química , DNA Girase/metabolismo , DNA Topoisomerase IV/metabolismo , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/enzimologia , Ativação Enzimática/efeitos dos fármacos , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/enzimologia , Meia-Vida , Camundongos , Testes de Sensibilidade Microbiana , Ratos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Streptococcus pyogenes/efeitos dos fármacos , Streptococcus pyogenes/enzimologia , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacocinéticaRESUMO
In late 2018, unusual patterns of very high mortality (>50% production) were reported in intensive tilapia cage culture systems across Lake Volta in Ghana. Samples of fish and fry were collected and analysed from two affected farms between October 2018 and February 2019. Affected fish showed darkening, erratic swimming and abdominal distension with associated ascites. Histopathological observations of tissues taken from moribund fish at different farms revealed lesions indicative of viral infection. These included haematopoietic cell nuclear and cytoplasmic pleomorphism with marginalization of chromatin and fine granulation. Transmission electron microscopy showed cells containing conspicuous virions with typical iridovirus morphology, that is enveloped, with icosahedral and/or polyhedral geometries and with a diameter c.160 nm. PCR confirmation and DNA sequencing identified the virions as infectious spleen and kidney necrosis virus (ISKNV). Samples of fry and older animals were all strongly positive for the presence of the virus by qPCR. All samples tested negative for TiLV and nodavirus by qPCR. All samples collected from farms prior to the mortality event were negative for ISKNV. Follow-up testing of fish and fry sampled from 5 additional sites in July 2019 showed all farms had fish that were PCR-positive for ISKNV, whether there was active disease on the farm or not, demonstrating the disease was endemic to farms all over Lake Volta by that point. The results suggest that ISKNV was the cause of disease on the investigated farms and likely had a primary role in the mortality events. A common observation of coinfections with Streptococcus agalactiae and other tilapia bacterial pathogens further suggests that these may interact to cause severe pathology, particularly in larger fish. Results demonstrate that there are a range of potential threats to the sustainability of tilapia aquaculture that need to be guarded against.
Assuntos
Ciclídeos , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/diagnóstico , Iridoviridae/isolamento & purificação , Animais , Aquicultura , Infecções por Vírus de DNA/diagnóstico , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/virologia , GanaRESUMO
3-Methoxybenzamide is a weak inhibitor of the essential bacterial cell division protein FtsZ. Exploration of the structure-activity relationships of 3-methoxybenzamide analogues led to the identification of potent anti-staphylococcal compounds.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Benzamidas/farmacologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Staphylococcus/efeitos dos fármacos , Antibacterianos/química , Benzamidas/química , Testes de Sensibilidade Microbiana , Relação Estrutura-AtividadeRESUMO
The synthesis and antibacterial activities of three chemotypes of DNA supercoiling inhibitors based on imidazolo[1,2-a]pyridine and [1,2,4]triazolo[1,5-a]pyridine scaffolds that target the ATPase subunits of DNA gyrase and topoisomerase IV (GyrB/ParE) is reported. The most potent scaffold was selected for optimization leading to a series with potent Gram-positive antibacterial activity and a low resistance frequency.
Assuntos
Anti-Infecciosos/farmacologia , Química Farmacêutica/métodos , DNA Topoisomerase IV/antagonistas & inibidores , Inibidores da Topoisomerase II , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Desenho de Fármacos , Enterococcus faecalis/metabolismo , Escherichia coli/metabolismo , Bactérias Gram-Positivas/metabolismo , Humanos , Imidazóis/química , Concentração Inibidora 50 , Piridinas/química , Staphylococcus aureus/metabolismo , Relação Estrutura-Atividade , Triazóis/químicaRESUMO
3-Methoxybenzamide (1) is a weak inhibitor of the essential bacterial cell division protein FtsZ. Alkyl derivatives of 1 are potent antistaphylococcal compounds with suboptimal drug-like properties. Exploration of the structure-activity relationships of analogues of these inhibitors led to the identification of potent antistaphylococcal compounds with improved pharmaceutical properties.
Assuntos
Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas do Citoesqueleto/antagonistas & inibidores , Piridinas/síntese química , Staphylococcus aureus/efeitos dos fármacos , Tiazóis/síntese química , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Disponibilidade Biológica , Proteínas Sanguíneas/metabolismo , Células CACO-2 , Divisão Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular , Hepatócitos/metabolismo , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Ligação Proteica , Piridinas/química , Piridinas/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/citologia , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacologiaRESUMO
FtsZ is an essential bacterial guanosine triphosphatase and homolog of mammalian beta-tubulin that polymerizes and assembles into a ring to initiate cell division. We have created a class of small synthetic antibacterials, exemplified by PC190723, which inhibits FtsZ and prevents cell division. PC190723 has potent and selective in vitro bactericidal activity against staphylococci, including methicillin- and multi-drug-resistant Staphylococcus aureus. The putative inhibitor-binding site of PC190723 was mapped to a region of FtsZ that is analogous to the Taxol-binding site of tubulin. PC190723 was efficacious in an in vivo model of infection, curing mice infected with a lethal dose of S. aureus. The data validate FtsZ as a target for antibacterial intervention and identify PC190723 as suitable for optimization into a new anti-staphylococcal therapy.
Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas do Citoesqueleto/antagonistas & inibidores , Piridinas/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Tiazóis/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/uso terapêutico , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , Cristalografia por Raios X , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana Múltipla , Ligantes , Resistência a Meticilina , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Piridinas/química , Piridinas/metabolismo , Piridinas/uso terapêutico , Staphylococcus aureus/química , Tiazóis/química , Tiazóis/metabolismo , Tiazóis/uso terapêutico , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMO
The continuous emergence of antibiotic resistance demands that novel classes of antibiotics continue to be developed. The division machinery of bacteria is an attractive target because it comprises seven or more essential proteins that are conserved almost throughout the bacteria but are absent from humans. We describe the development of a cell-based assay for inhibitors of cell division and its use to isolate a new inhibitor of FtsZ protein, a key player in the division machinery. Biochemical, cytological, and genetic data are presented that demonstrate that FtsZ is the specific target for the compound. We also describe the effects of more potent analogues of the original hit compound that act on important pathogens, again at the level of cell division. The assay and the compounds have the potential to provide novel antibiotics with no pool of pre-existing resistance. They have provided new insight into cytokinesis in bacteria and offer important reagents for further studies of the cell division machinery.