RESUMO
Cryopreservation is a recognized method for the maintenance of Leptospira collections. Although cryoprotectants are commonly used in order to prevent or reduce the adverse effects of freezing, there is no consensus regarding the protocols of cryopreservation. This study aimed to compare cryopreservation protocols for Leptospira using different glycerol and dimethyl sulfoxide (DMSO) concentrations. Leptospira interrogans serovar Icterohaemorrhagiae, L. interrogans serovar Bratislava, and L. borgpetersenii serovar Hardjo were used as the experimental strains. For each strain, three protocols were tested using 5% and 10% glycerol and 2.5% DMSO. For each protocol, 12 tubes containing 1.5 mL of serovar were frozen at -70°C on the same day. An aliquot of each serovar/protocol was thawed once a month throughout 1 year. The viability of leptospires was evaluated by the recovery of those at days 7, 14, and 21 after thawing. Although no significant difference was found among the leptospiral recovery rates for the 9 serovar/protocols tested, DMSO (2.5%) was shown to be slightly better than glycerol, and its use should be encouraged as a cryoprotectant for leptospires.
Assuntos
Criopreservação , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Leptospira/efeitos dos fármacos , Leptospira interrogans/efeitos dos fármacosRESUMO
INTRODUCTION: Leptospirosis has reemerged as significant threat particularly in developing countries, including those in Latin America. Data from Colombia is still limited and there are no published studies in the Western area of the country. METHODS: Data on suspected cases were collected over the study period (2008- 2012). Cases were diagnosed clinically and confirmed by ELISA IgG and microscopic agglutination test (MAT) (titers ≥1:400). RESULTS: During the study period 264 suspected cases of leptospirosis were found. From those, 8.33% (22 cases) were microbiologically confirmed. Number of suspected cases increased in the period from 20 (2008) (40 cases/100,000 consultations) to 58 (2012) (120 cases/100,000 consultations). Regard sex distribution, 62.5% were males, 14% in the age group 21-30 y-old, from confirmed cases 95% live in urban areas of Pereira, 25.7% own dogs and 13.2% cats, 32.3% reporting rats at home as well 22.7% at work places. From confirmed cases 72.7% were hospitalized. Clinical findings found were: fever (60.2%), myalgias (47%), and headache (41.9%), among others. All the cases corresponded to Leptospira interrogans. Regard the serovars, in these patients 6 were identified: Australis (54.5%), Icterohaemorrhagiae (45.5%), Canicola (45.5%), Panama (45.5%), Pomona (36.3) and Grippotyphosa (1%). Thirty nine percent of the patients received antimicrobial therapy, 50% ceftriaxone. No deaths occurred. CONCLUSION: Leptospirosis is an emerging infectious disease that has changed from an occupational disease of veterinarians, farmers, butchers, and other animal handlers to a cause of epidemics in poor and decayed urban communities in developing countries, including those in Latin America such as Colombia.
Assuntos
Doenças Transmissíveis Emergentes/epidemiologia , Instalações de Saúde , Leptospira interrogans/patogenicidade , Leptospirose/epidemiologia , Setor Privado , Saúde da População Urbana , Zoonoses/epidemiologia , Adulto , Animais , Antibacterianos/uso terapêutico , Técnicas Bacteriológicas , Gatos , Colômbia/epidemiologia , Doenças Transmissíveis Emergentes/diagnóstico , Doenças Transmissíveis Emergentes/tratamento farmacológico , Doenças Transmissíveis Emergentes/microbiologia , Doenças Transmissíveis Emergentes/transmissão , Estudos Transversais , Vetores de Doenças , Cães , Feminino , Humanos , Leptospira interrogans/efeitos dos fármacos , Leptospira interrogans/isolamento & purificação , Leptospirose/diagnóstico , Leptospirose/tratamento farmacológico , Leptospirose/microbiologia , Leptospirose/transmissão , Masculino , Animais de Estimação/microbiologia , Valor Preditivo dos Testes , Ratos , Estudos Retrospectivos , Fatores de Risco , Resultado do Tratamento , Adulto Jovem , Zoonoses/diagnóstico , Zoonoses/tratamento farmacológico , Zoonoses/microbiologia , Zoonoses/transmissãoRESUMO
Leptospira interrogans serogroup Icterohaemorrhagiae is the major serogroup infecting humans worldwide, and rodents and dogs are the most significant transmission sources in urban environments. Knowledge of the prevalent serovars and their maintenance hosts is essential to understand the epidemiology of leptospirosis. In this study, 20 Leptospira isolates were evaluated by pulsed-field gel electrophoresis (PFGE), variable number tandem-repeat analysis (VNTR), serotyping, and determination of antimicrobial resistance profile. Isolates, originated from bovine, canine, human, and rodent sources, were characterized by microscopic agglutination test with polyclonal and monoclonal antibodies and were identified as L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni. MICs of antimicrobials often used in veterinary medicine were determined by broth microdilution test. Most of tested antibiotics were effective against isolates, including penicillin, ampicillin, and ceftiofur. Higher MIC variability was observed for fluoroquinolones and neomycin; all isolates were resistant to trimethoprim/sulfamethoxazole and sulphadimethoxine. Isolates were genotyped by PFGE and VNTR; both techniques were unable to discriminate between serovars Copenhageni and Icterohaemorrhagiae, as expected. PFGE clustered all isolates in 1 pulsotype, indicating that these serovars can be transmitted between species and that bovine, rodent, and dogs can maintain them in the environment endangering the human population.
Assuntos
Antibacterianos/farmacologia , Leptospira interrogans/classificação , Leptospira interrogans/efeitos dos fármacos , Leptospirose/microbiologia , Leptospirose/veterinária , Tipagem Molecular , Testes de Aglutinação , Animais , Brasil , Bovinos , Análise por Conglomerados , Cães , Eletroforese em Gel de Campo Pulsado , Genótipo , Humanos , Leptospira interrogans/genética , Leptospira interrogans/isolamento & purificação , Testes de Sensibilidade Microbiana , Repetições Minissatélites , Fenótipo , Roedores , SorotipagemRESUMO
A lipopolysaccharide mutant of Leptospira interrogans (LaiMut) was obtained by growth in the presence of an agglutinating monoclonal antibody (mAb) against lipopolysaccharide. Agglutination reactions with anti-lipopolysaccharide mAbs and polyclonal antibodies showed that LaiMut had lost some serogroup Icterohaemorrhagiae agglutinating epitopes. However, LaiMut displayed an increased reactivity to antisera against related serogroups, suggesting that the disruption of some lipopolysaccharide epitopes resulted in greater exposure to cross-reactive epitopes, not accessible to antibodies in the wild type (LaiWT). Comparison of the nucleotide sequences of the lipopolysaccharide loci of LaiMut and Lai wild type (LaiWT) strains showed an inframe stop mutation in the gene encoding undecaprenyl-galactosyltransferase, a protein that provides a fundamental and nonredundant function essential for lipopolysaccharide biosynthesis. Despite this, the biosynthesis of lipopolysaccharide agglutinating antigens was not abolished by the mutation. Based on the phenotype of LaiMut and analysis of the domain structure of the undecaprenyl-galactosyltransferase in relation to the mutation, we propose that the mutation results in the expression of two functional proteins in place of the undecaprenyl-galactosyltransferase. We hypothesize that the loss of coordination of the coupled function afforded by the intact dual function protein present in the parent strain results in an inefficient production of lipopolysaccharide in LaiMut.
Assuntos
Anticorpos Monoclonais/farmacologia , Leptospira interrogans/genética , Lipopolissacarídeos/biossíntese , Mutação , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Leptospira interrogans/efeitos dos fármacos , Leptospira interrogans/crescimento & desenvolvimento , Leptospira interrogans/metabolismo , Lipopolissacarídeos/imunologia , Dados de Sequência MolecularRESUMO
A method to determine the minimum inhibitory concentration for leptospiras was developed, since there is not a standard method to measure it at the international level. Reference strains from the pathogenic complex L. interrogans and L. biflexa were used against penicillin, cyprofloxacine, chloramphenicol, rifampicine and tetracycline. The minimum inhibitory concentration was defined as the lowest concentration of antibiotic where it was observed the inhibition of the bacterial mobility by direct examination in dark field. Ranges for penicillin were from 0.095 to 152 microg/mL, for tetracycline from 0.156 to 3.13 microg/mL, for chloramphenicol, from 0.08 to 12.52 microg/mL, for rifampicine from 0.08 to 1.56 microg/mL, and for cyprofloxacine from 0.15 to 2.4 microg/mL. The antibiotics that showed the lowest values were cyprofloxacine, rifampicine and tetracycline, whereas the most elevated value was obtained against chloramphenicol and penicillin. The strains from the serogroups circulating more frequently in Cuba were used in this research. This study will allow in a near future to determine the antimicrobial susceptibility in autochthonous strains isolated from patients with Leptospirosis at the national level.
Assuntos
Antibacterianos/farmacologia , Leptospira/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Cloranfenicol/farmacologia , Ciprofloxacina/farmacologia , Cuba , Previsões , Humanos , Leptospira interrogans/efeitos dos fármacos , Leptospira interrogans serovar canicola/efeitos dos fármacos , Leptospira interrogans serovar pomona/efeitos dos fármacos , Leptospirose/microbiologia , Penicilinas/farmacologia , Rifampina/farmacologia , Tetraciclina/farmacologiaRESUMO
Plasmid pGL4W15-96 was constructed from the pGL4W74 vector without promoter for kanamycin and a sequence of 480pb containing the supposed J15 promoter with the objective of confirming the role of J15 sequence as a promoter for Leptospira, which restored the transcription of the gene of resistance to kanamycin in Escherichia coli and Leptospira biflexa, corroborating this way the function of the insertion as a promoter for both organisms.
Assuntos
Proteínas de Bactérias , Farmacorresistência Bacteriana/genética , Canamicina/farmacologia , Leptospira interrogans/efeitos dos fármacos , Leptospira interrogans/genética , Lipopolissacarídeos/biossíntese , Plasmídeos/genética , Transcrição Gênica , Escherichia coli/efeitos dos fármacos , Leptospira interrogans/metabolismo , Antígenos O , Regiões Promotoras Genéticas/genéticaRESUMO
La leptospirosis es probablemente la zoonosis más extendida en el mundo. Se presenta más comúnmente en climas húmedos y cálidos. Es una fécción producida por una variedad de espiroqueta llamada Leptospira ínterrogans, de la cual se conocen más de 200 serovariedades. El contagio es infrecuente en el hombre, y se contrae a través del contacto directo o indirecto de la piel o mucosas con la orina de animales salvajes o domésticos infectados. Puede ser asintomática o presentarse como una enfermedad parecida a la influenza, a veces asociada a compromiso meníngeo. En una minoría de los casos se agregan ictericia, manifestaciones hemorrágicas e insuficiencia renal sintomática, constituyendo la enfermedad de Weil, que es la forma más grave y potencialmente fatal de la enfermedad. No son raros los cuadros clínicos incompletos o parciales. La transmisión tiene comúnmente lugar en ámbitos ocupacionales, recreacionales o domésticos. Los grupos ocupacionales más expuestos son aquellos que trabajan en contacto con suelos húmedos o con aguas estancadas, y en la esfera recreacional aquellos que se bañan, pescan o hacen deportes en aguas de poca movilidad. En el hogar tiene importancia la presencia de roedores, o el contacto estrecho con perros dentro de la casa. La mayoría de los casos se presenta en hombres, durante el verano u otoño. En su forma más común la leptospirosis adopta el aspecto clínico de un síndrome febril anictérico con múltiples síntomas inespecíficos, lo que hace difícil sospechar el diagnóstico. Sin embargo, la relevancia de la cefalea, fiebre o intensas mialgias en una persona con riesgo de exposición debe sugerir esta posibilidad. La inyección conjuntival, la presencia de meningitis aséptica, las alteraciones de sedimento urinario y la elevación frecuente de la CPK sérica pueden constituir claves clínicas valiosas. La presencia de ictericia y de insuficiencia renal clínicamente significativa definen el cuadro como enfermedad de Weil. El diagnóstico definitivo se hace por cultivo del agente causal, o más comúnmente por serología. El tratamiento antibiótico precoz y aún después de varios días de comienzo con penicilina, amoxicilina, ampicilina, tetraciclina o doxiciclina se ha demostrado como muy útil. Dado que el germen tiene una amplia sensibilidad, es posible que otros antimicrobianos también sean eficaces