RESUMO
We developed a simple new selective LB-based medium, named CYP broth, suitable for recovering long-term stored Y. pestis subcultures and for isolation of Y. pestis strains from field-caught samples for the Plague surveillance. It aimed to inhibit the growth contaminating microorganisms and enrich Y. pestis growth through iron supplementation. The performance of CYP broth on microbial growth from different gram-negative and gram-positive strains from American Type Culture Collection (ATCC®) and other clinical isolates, field-caught rodent samples, and more importantly, on several vials of ancient Y. pestis subcultures was evaluated. Additionally, other pathogenic Yersinia species such as Y. pseudotuberculosis and Y. enterocolitica were also successfully isolated with CYP broth. Selectivity tests and bacterial growth performance on CYP broth (LB broth supplemented with Cefsulodine, Irgasan, Novobiocin, nystatin and ferrioxamine E) were evaluated in comparison with LB broth without additive; LB broth/CIN, LB broth/nystatin and with traditional agar media including LB agar without additive, and LB agar and Cefsulodin-Irgasan-Novobiocin Agar (CIN agar) supplemented with 50 µg/mL of nystatin. Of note, the CYP broth had a recovery twofold higher than those of the CIN supplemented media or other regular media. Additionally, selectivity tests and bacterial growth performance were also evaluated on CYP broth in the absence of ferrioxamine E. The cultures were incubated at 28 °C and visually inspected for microbiological growth analysis and O.D.625 nm measurement between 0 and 120 h. The presence and purity of Y. pestis growth were confirmed by bacteriophage and multiplex PCR tests. Altogether, CYP broth provides an enhanced growth of Y. pestis at 28 °C, while inhibiting contaminant microorganisms. The media is a simple, but powerful tool to improve the reactivation and decontamination of ancient Y. pestis culture collections and for the isolation of Y. pestis strains for the Plague surveillance from various backgrounds. KEY POINTS: ⢠The newly described CYP broth improves the recuperation of ancient/contaminated Yersinia pestis culture collections ⢠CYP broth was also efficient in reducing environmental contamination in field-capture samples, improving Y. pestis isolation ⢠CYP broth can also be used for the isolation of Y. enterocolitica and Y. pseudotuberculosis.
Assuntos
Peste , Yersinia pestis , Humanos , Ágar , Peste/microbiologia , Novobiocina/farmacologia , Nistatina , Meios de Cultura/farmacologia , Cefsulodina/farmacologiaRESUMO
Although homologs of the ATP-dependent Lon protease exist in all domains of life, the relevance of this protease in archaeal physiology remains a mystery. In this study, we have constructed and phenotypically characterized deletion and conditional lon mutants in the model haloarchaeon Haloferax volcanii to elucidate the role of the unusual membrane-bound LonB protease in archaea. Hvlon could be deleted from the chromosome only when a copy of the wild type gene was provided in trans suggesting that Lon is essential for survival in this archaeon. Successful complementation of the lethal phenotype of ΔHvlon was attained by expression of the heterologous protease gene Nmlon from the haloalkaliphilic archaeon Natrialba magadii, meaning that the biological function of Lon is conserved in these organisms. Suboptimal cellular levels of Lon protein affected growth rate, cell shape, cell pigmentation, lipid composition and sensitivity to various antibiotics. The contents of bacterioruberins and some polar lipids were increased in the lon mutants suggesting that Lon is linked to maintenance of membrane lipid balance which likely affects cell viability in this archaeon. The phenotypes associated to a membrane-bound LonB protease mutant were examined for the first time providing insight on the relevance of this protease in archaeal physiology.
Assuntos
Proteínas Arqueais/genética , Haloferax volcanii/enzimologia , Lipídeos de Membrana/metabolismo , Peptídeo Hidrolases/genética , Antibacterianos/farmacologia , Proteínas Arqueais/metabolismo , Bacitracina/farmacologia , Sequência de Bases , Expressão Gênica , Regulação da Expressão Gênica em Archaea , Haloferax volcanii/efeitos dos fármacos , Lovastatina/farmacologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Novobiocina/farmacologia , Peptídeo Hidrolases/metabolismo , Pigmentação , Ligação Proteica , Puromicina/farmacologiaRESUMO
BACKGROUND: Microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide produced by Escherichia coli (E. coli). MccJ25 enters into the sensitive E. coli strains by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD and SbmA. The resistance of Salmonella enterica serovar Typhimurium (S. Typhimurium) to MccJ25 is attributed to the inability of its FhuA protein to incorporate the antibiotic into the cell. RESULTS: In this work we demonstrate that S. Typhimurium becomes notably susceptible to MccJ25 when replicating within macrophages. In order to determine the possible cause of this phenomenon, we studied the sensitivity of S. Typhimurium to MccJ25 at conditions resembling those of the internal macrophage environment, such as low pH, low magnesium and iron deprivation. We observed that the strain was only sensitive to the antibiotic at low pH, leading us to attribute the bacterial sensitization to this condition. A MccJ25-resistant E. coli strain in which fhuA is deleted was also inhibited by the antibiotic at low pH. Then, we could assume that the MccJ25 sensitivity change observed in both E. coli fhuA and S. Typhimurium is mediated by a MccJ25 uptake independent of the FhuA receptor. Moreover, low pH incubation also sensitized S. Typhimurium to the hydrophobic antibiotic novobiocin, which does not affect enteric bacteria viability because it is unable to penetrate the bacterial outer membrane. This observation supports our hypothesis about low pH producing a modification in the bacterial membrane permeability that allows an unspecific MccJ25 uptake. On the other hand, MccJ25 inhibited S. Typhimurium when cells were preincubated in acidic pH medium and then treated at neutral pH with the antibiotic. CONCLUSIONS: Our results suggest that acidic condition does not alter MccJ25 hydrophobicity but irreversibly modifies bacterial membrane permeability. This would allow an unspecific antibiotic uptake into the cell.From our data it is possible to infer that intracellular pathogenic strains, which are in vitro resistant to MccJ25, could become susceptible ones in vivo. Therefore, the MccJ25 action spectrum would be broader than what in vitro experiments indicate.
Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Macrófagos/microbiologia , Salmonella typhimurium/efeitos dos fármacos , Animais , Linhagem Celular , Permeabilidade da Membrana Celular , Meios de Cultura/química , Citosol/microbiologia , Escherichia coli/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Camundongos , Testes de Sensibilidade Microbiana , Modelos Teóricos , Novobiocina/farmacologiaRESUMO
The META cluster of Leishmania amazonensis contains both META1 and META2 genes, which are upregulated in metacyclic promastigotes and encode proteins containing the META domain. Previous studies defined META2 as a 48.0-kDa protein, which is conserved in other Leishmania species and in Trypanosoma brucei. In this work, we demonstrate that META2 protein expression is regulated during the Leishmania life cycle but constitutive in T. brucei. META2 protein is present in the cytoplasm and flagellum of L. amazonensis promastigotes. Leishmania META2-null replacement mutants are more sensitive to oxidative stress and, upon heat shock, assume rounded morphology with shortened flagella. The increased susceptibility of null parasites to heat shock is reversed by extra-chromosomal expression of the META2 gene. Defective Leishmania promastigotes exhibit decreased ability to survive in macrophages. By contrast, META2 expression is decreased by 80% in RNAi-induced T. brucei bloodstream forms with no measurable effect on survival or resistance to heat shock.
Assuntos
Temperatura Alta/efeitos adversos , Leishmania mexicana/metabolismo , Estresse Oxidativo , Proteínas de Protozoários/fisiologia , Animais , Antiprotozoários/farmacologia , Western Blotting , Imunofluorescência , Regulação da Expressão Gênica , Leishmania mexicana/química , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/genética , Leishmaniose Cutânea/parasitologia , Macrófagos Peritoneais/parasitologia , Meglumina/farmacologia , Antimoniato de Meglumina , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Mutação , Novobiocina/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Compostos Organometálicos/farmacologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Interferência de RNA , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
Topoisomerases from trypanosomatids play key functions in the replication and organization of kinetoplast DNA (kDNA). Hence, they are considered as potential targets for anti-parasite drugs. In this paper, the effect of topoisomerase II inhibitors, such as nalidixic acid, novobiocin and etoposide, on the ultrastructure of trypanosomatids that present distinct kDNA arrangements was evaluated. Prokaryotic topoisomerase II inhibitors were more effective on growth arrest and ultrastructure changes than etoposide, a eukaryotic topoisomerase II inhibitor. With the exception of novobiocin, drug concentrations which inhibited cell proliferation also promoted kinetoplast ultrastructure alterations, including the redistribution of topoisomerase II. The data reinforce the concept that prokaryotic topoisomerase II inhibitors may offer greater selectivity in drug therapy of trypanosomatid infections.
Assuntos
DNA de Cinetoplasto/ultraestrutura , Inibidores da Topoisomerase II , Tripanossomicidas/farmacologia , Trypanosomatina/efeitos dos fármacos , Animais , Replicação do DNA , DNA Topoisomerases Tipo II/metabolismo , DNA de Cinetoplasto/metabolismo , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Ácido Nalidíxico/farmacologia , Novobiocina/farmacologia , Testes de Sensibilidade Parasitária , Trypanosomatina/enzimologia , Trypanosomatina/genética , Trypanosomatina/crescimento & desenvolvimentoRESUMO
The activity of antimicrobial agents frequently used for treating bovine mastitis was determined against 101 coagulase-positive staphylococci isolated from bovine mammary secretion. The isolates were obtained from 39 dairy farms located in the central dairy area of Argentina. The disk diffusion method was used and the following antimicrobial agents were tested: penicillin, ampicillin, oxacillin, cephacetrile, penicillin + novobiocin, erythromycin, pirlimycin, novobiocin and neomycin. The highest levels of resistance were observed against penicillin and ampicillin (47.6%), while the lowest against erythromycin (2%), pirlimycin (4%) and neomycin (2.9%). No resistant strains against oxacillin, cephacetrile and penicillin + novobiocin were detected.
Assuntos
Clindamicina/análogos & derivados , Indústria de Laticínios , Farmacorresistência Bacteriana Múltipla , Resistência a Medicamentos , Mastite Bovina/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Argentina , Bovinos , Cefacetrila/farmacologia , Clindamicina/farmacologia , Coagulase , Eritromicina/farmacologia , Feminino , Testes de Sensibilidade Microbiana , Neomicina/farmacologia , Novobiocina/farmacologia , Staphylococcus aureus/isolamento & purificaçãoRESUMO
The activity of antimicrobial agents frequently used for treating bovine mastitis was determined against 101 coagulase-positive staphylococci isolated from bovine mammary secretion. The isolates were obtained from 39 dairy farms located in the central dairy area of Argentina. The disk diffusion method was used and the following antimicrobial agents were tested: penicillin, ampicillin, oxacillin, cephacetrile, penicillin + novobiocin, erythromycin, pirlimycin, novobiocin and neomycin. The highest levels of resistance were observed against penicillin and ampicillin (47.6), while the lowest against erythromycin (2), pirlimycin (4) and neomycin (2.9). No resistant strains against oxacillin, cephacetrile and penicillin + novobiocin were detected.(AU)
Assuntos
Estudo Comparativo , Animais , Feminino , Bovinos , RESEARCH SUPPORT, NON-U.S. GOVT , Clindamicina/análogos & derivados , Indústria de Laticínios , Resistência a Medicamentos , Farmacorresistência Bacteriana Múltipla , Mastite Bovina/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/farmacologia , Argentina , Cefacetrila/farmacologia , Clindamicina/farmacologia , Coagulase , Eritromicina/farmacologia , Testes de Sensibilidade Microbiana , Neomicina/farmacologia , Novobiocina/farmacologia , Staphylococcus aureus/isolamento & purificaçãoRESUMO
A survey of the changes in populations of heterotrophic bacteria, coliform microorganisms and S. meliloti was conducted in samples taken from the water irrigation channels of the Neuquén River (Argentina). Fifty-six water samples were collected during the spring-summer seasons of 1997-1999 years. Both the heterotrophic plate count bacterial and the number of coliforms oscillated between 110-5050 CFU/ml and 8-1400 CFU/100 ml, respectively, during the period this study was carried out. Fecal coliforms were detected in 91.1% of the water samples investigated. Moreover, the results showed that S. meliloti capable of nodulating alfalfa (Medicago sativa L.) Cuf 101 were present in 68% of the water samples and in effectiveness studies, no isolate out of 25 evaluated could be classified as superior N fixers. That is, they did not produce plants equal in weight to nitrate-grown plants (KNO3 0.05%). All the S. meliloti strains were resistant to novobiocin and bacitracin, while 72% of the microsymbionts demonstrated resistance to between seven and ten antibiotics. Results presented in this study showed that irrigation waters of the Neuquén river could act as dispersal agents of both ineffective S. meliloti strains and thermotolerant coliform bacteria.
Assuntos
Enterobacteriaceae/isolamento & purificação , Sinorhizobium meliloti/isolamento & purificação , Microbiologia da Água , Poluição da Água/análise , Argentina , Bacitracina/farmacologia , Resistência Microbiana a Medicamentos , Resistência a Múltiplos Medicamentos , Temperatura Alta , Medicago sativa/microbiologia , Fixação de Nitrogênio , Novobiocina/farmacologia , Estações do Ano , Sinorhizobium meliloti/efeitos dos fármacos , Sinorhizobium meliloti/fisiologiaRESUMO
A survey of the changes in populations of heterotrophic bacteria, coliform microorganisms and S. meliloti was conducted in samples taken from the water irrigation channels of the NeuquUn River (Argentina). Fifty-six water samples were collected during the spring-summer seasons of 1997-1999 years. Both the heterotrophic plate count bacterial and the number of coliforms oscillated between 110-5050 CFU/ml and 8-1400 CFU/100 ml, respectively, during the period this study was carried out. Fecal coliforms were detected in 91.1 of the water samples investigated. Moreover, the results showed that S. meliloti capable of nodulating alfalfa (Medicago sativa L.) Cuf 101 were present in 68 of the water samples and in effectiveness studies, no isolate out of 25 evaluated could be classified as superior N fixers. That is, they did not produce plants equal in weight to nitrate-grown plants (KNO3 0.05). All the S. meliloti strains were resistant to novobiocin and bacitracin, while 72 of the microsymbionts demonstrated resistance to between seven and ten antibiotics. Results presented in this study showed that irrigation waters of the NeuquUn river could act as dispersal agents of both ineffective S. meliloti strains and thermotolerant coliform bacteria.(AU)
Assuntos
RESEARCH SUPPORT, NON-U.S. GOVT , Enterobacteriaceae/isolamento & purificação , Sinorhizobium meliloti/isolamento & purificação , Microbiologia da Água , Poluição da Água/análise , Argentina , Bacitracina/farmacologia , Resistência Microbiana a Medicamentos , Resistência a Múltiplos Medicamentos , Temperatura Alta , Medicago sativa/microbiologia , Fixação de Nitrogênio , Novobiocina/farmacologia , Estações do Ano , Sinorhizobium meliloti/efeitos dos fármacos , Sinorhizobium meliloti/fisiologiaRESUMO
Experiments with novobiocin (NB) post-treatment were performed to verify its effect on the frequencies of micronuclei (MN) and chromosomal aberrations (CA) induced by g-irradiation (0.75, 1.5 and 3.0 Gy) in human lymphocytes at G0-phase. The frequencies of MN significantly decreased by 44 and 50 per cent, for the treatment with NB 50 µg/ml (30-min pulse) after radiation doses of 1.5 and 3.0 Gy, respectively. However, CA frequencies were not significantly affected. No significant effect on CA was observed when lymphocyte cultures were exposed to a single dose of 2.0 Gy at the G0-phase and posttreated with 25 µg/ml NB for three hours either immediately after irradiation (G0-phase) or after 24 h (S-phase). The significant suppressive effect of NB on MN frequencies supports the hypothesis that NB interaction with chromatin increases access to DNA repair enzymes.