RESUMO
The aim of this study was to compare the efficacy of conservation by freezing the strains of Haemophilus influenzae at -20 degrees C and -70 degrees C. Skim milk supplemented with glucose, yeast extract and glycerol allowed highest viability of H. influenzae both at -20 degrees C and -70 degrees C from the media analyzed. Trypticase soy broth and brain heart infusion broth supplemented with glycerol, allowed excellent recovery. Use of cotton swaps as supporting material, with or without addition of cryoprotective agents, did not modify H. influenzae viability after six months of storage. Concentration of the initial inoculum positively affected viability when stored at -20 degrees C. Initial concentration did not influence survival after storage at -70 degrees C. Thawing at room temperature should not exceed 3 h as to get highest survival percentage.
Assuntos
Criopreservação/métodos , Haemophilus influenzae , Crioprotetores/farmacologia , Meios de Cultura , Haemophilus influenzae/efeitos dos fármacos , Humanos , Fatores de TempoRESUMO
The effect of estrogen on the microbial colonization of the urogenital tract is widely discussed, mainly in regard to women with a high incidence of Urinary Tract Infections (UTI). The aim of this work was to study the effect of estradiol on the microbial colonization of lactobacilli and E. coli in mice. Female BALB/c mice were intramuscularly (i.m.) treated with beta-estradiol (one or three doses). The next day, L. fermentum was inoculated intraurethrally with three doses of 10(7) CFU (Colony Forming Units). Later, mice were challenged with uropathogenic E. coli (1 x 10(8) CFU). The hormone levels in sera increased to values 10 times higher than in control animals. Increased differentiation of desquamated vaginal cells and keratinization of the vaginal surface were also observed. The hormonal treatment produced an increased E. coli colonization in the whole tract and a higher level of L. fermentum in kidneys on the 6th day. In mice treated with hormones and lactobacilli, one dose of estradiol was enough to protect animals against the challenge with E. coli. Three doses of estradiol produced a more pronounced protection with a lower number of E. coli. No histological modifications were produced by L.fermentum, while lymphocytic proliferation at submucosal level was observed in E. coli-challenged animals.
Assuntos
Escherichia coli/efeitos dos fármacos , Estradiol/farmacologia , Lactobacillus/efeitos dos fármacos , Sistema Urogenital/microbiologia , Animais , Escherichia coli/crescimento & desenvolvimento , Estradiol/sangue , Feminino , Lactobacillus/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Sistema Urogenital/anatomia & histologia , Vagina/citologia , Vagina/efeitos dos fármacosRESUMO
beta-Lactamase was isolated from Neisseria gonorrhoeae, obtained from male patients with gonococcic urethritis. Biochemical properties of the enzyme were studied. The enzyme was purified 38-fold by ammonium sulphate precipitation and using Sephadex G75 and DEAE-cellulose columns. The purified extract exhibited a single band by polyacrylamide gel electrophoresis. Maximum enzyme activity was obtained at 37 degrees C and pH 7.0-7.2 in 50 mM phosphate buffer. Addition of Ni2+, Fe2+, Fe3+, Mn2+ and p-chloromercurybenzoate to the reaction buffer partially inhibited beta-lactamase activity, whereas Hg2+ and EDTA produced complete inhibition. The molecular weight was estimated to be 35,000 Da and the pI of the enzyme was 5.4.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Gonorreia/microbiologia , Neisseria gonorrhoeae/enzimologia , Uretrite/microbiologia , beta-Lactamases/isolamento & purificação , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Cátions/farmacologia , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Masculino , Peso Molecular , Desnaturação Proteica , Especificidade por Substrato , Temperatura , Inibidores de beta-Lactamases , beta-Lactamases/metabolismo , Ácido p-Cloromercurobenzoico/farmacologiaRESUMO
The structural gene determinants of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705, have been amplified from a plasmid of approximately 35 kb and sequenced. Lactocin 705 is a class IIb bacteriocin, whose activity depends upon the complementation of two peptides (705alpha and 705beta) of 33 amino acid residues each. These peptides are synthesized as precursors with signal sequences of the double-glycine type, which exhibited high identities with the leader peptides of plantaricin S and J from Lactobacillus plantarum, brochocin C from Brochotrix campestris, sakacin P from Lactobacillus sake, and the competence stimulating peptides from Streptococcus gordonii and Streptococcus mitis. However, the two mature bacteriocins 705alpha and 705beta do not show significant similarity to other sequences in the databases.
Assuntos
Bacteriocinas/biossíntese , Bacteriocinas/genética , Genes Bacterianos , Lacticaseibacillus casei/metabolismo , Sequência de Aminoácidos , Bacteriocinas/química , Bacteriocinas/classificação , Sequência de Bases , Lacticaseibacillus casei/genética , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Plasmídeos/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Administration of Lactobacillus reuteri CRL 1098 (10(4) cells/d) to mice for 7 d before inducing hypercholesterolemia (by feeding mice with a fat-enriched diet for the subsequent 7 d) was evaluated. At this low dose, L. reuteri was effective in preventing hypercholesterolemia in mice, producing a 17% increase in the ratio of high-density lipoprotein to low-density lipoprotein. Total cholesterol and triglycerides decreased by 22 and 33%, respectively, in the group that was not fed the lactobacilli. The hypocholesterolemic effect produced by L. reuteri CRL 1098 might be considered as indirect evidence of the permanency of the lactobacilli in the gut.
Assuntos
Hipercolesterolemia/prevenção & controle , Lactobacillus/fisiologia , Probióticos , Animais , Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Gorduras na Dieta/administração & dosagem , Camundongos , Triglicerídeos/sangueRESUMO
Nutrition plays a key role in maintaining the balance of the intestinal microflora. Malnutrition disturbs the ecological barrier and induces histological damage. We evaluated modifications induced by renutrition with nonfat milk (NFM) and Lactobacillus casei administration (for 2 days) on the bacterial gut population and structural and ultrastructural gut modifications in malnourished mice. Balb/c mice suffering from a malnutrition process immediately after weaning (for 21 days) were divided into four groups and were given NFM for 0, 7, 14, and 21 days. Another group was treated in a similar way, but after different periods of NFM administration, mice in this group received L. casei for two consecutive days. All experimental animals were sacrificed by cervical dislocation, and both the microflora and the histological structure of the intestine were studied. In malnourished animals, a decrease in the numbers of Lactobacillus and anaerobic microorganisms was observed, whereas there was an increase in the number of Enterobacteriaceae. In animals treated with NFM and NFM plus L. casei, we could observe an important improvement in the microflora in the small and large intestines but no differences between both treatments. Structural and ultrastructural studies showed a slight improvement 7 days after treatment with NFM, and for 14 and 21 days after renutrition, the mice showed normal intestinal villi, whereas the additional feeding with L. casei for two consecutive days, after different periods of renutrition, yielded an earlier improvement (7 days).
Assuntos
Mucosa Intestinal/microbiologia , Lacticaseibacillus casei , Leite/microbiologia , Distúrbios Nutricionais/dietoterapia , Animais , Peso Corporal , Enterobacteriaceae , Mucosa Intestinal/ultraestrutura , Lacticaseibacillus casei/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Microscopia EletrônicaRESUMO
Lactobacillus casei CRL 705, isolated from a dry fermented sausage, produces an antibacterial peptide which is active against Listeria monocytogenes. Previous studies have shown that this compound is potentially useful to control food-borne pathogens in ground meat. In view of the potential application of this antimicrobial substance in food fermentation, a detailed biochemical analysis of this peptide is required. In this work, the purification and amino acid sequence of this bacteriocin is presented. The adsorption-desorption pH-dependent property of lactocin 705 was exploited for purification. The active extract was further subjected to RP-HPLC and SDS-PAGE. The active antimicrobial band was electroeluted from an SDS-PAGE gel and its amino acid sequence determined. Lactocin 705 had an estimated molecular weight of 3357.80 and an isoelectric point of 10.03. The peptide contains a high ratio of glycine residues and does not show any modified amino acids, like lanthionine or beta-methyllanthionine. The sequence was unique when compared to several databases.
Assuntos
Bacteriocinas/isolamento & purificação , Lacticaseibacillus casei/química , Sequência de Aminoácidos , Bacteriocinas/química , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Estrutura Secundária de ProteínaRESUMO
Enterocin CRL35 is an antibacterial polypeptide of 3.5 x 10(3) Da produced by Enterococcus faecium CRL35. A series of experiments are described that show the enterocin also had antiviral activity against thymidine-kinase positive (tk+) and deficient (tk-) strains of herpes simplex (HSV) type 1 and 2 in Vero and BHK-21 cells. This activity was observed at 100 microg/ml, 15-fold lower than the cytotoxic concentration. In both cell lines there was a 2 log inhibition of infectivity. The compound inhibited viral multiplication in a dose-dependent manner and had no virucidal effect. Enterocin CRL35 also inhibited the virion-associated host shutoff in infected Vero cells showing that intracellular viral multiplication was affected.
Assuntos
Antivirais/farmacologia , Bacteriocinas/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Aciclovir/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Cromatografia Líquida de Alta Pressão , Cricetinae , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 2/patogenicidade , Células VeroRESUMO
A stable microbial system in the respiratory tract acts as an important defense mechanism against pathogenic microorganisms. Perturbations in this system may allow pathogens to establish. In an ecological environment such as the respiratory tract, there are many diverse factors that play a role in the establishment of the indigenous flora. In the present work we studied the normal microbial flora of different areas of the respiratory tract of mice and their evolution from the time the mice were born. Our interest was to know which were the dominant groups of microorganisms in each area, which were the first capable of colonizing and which dominated over time to be used as probiotic microorganisms. Our results show that Gram negative facultatively anaerobic bacilli and strict anaerobic microorganisms were the last ones to appear in the bronchia, while aerobic and Gram positive cocci were present in all the areas of the respiratory tract. The number of facultative aerobes and strict anaerobes were similar in the nasal passage, pharynx instilled and trachea, but lower in bronchia. The dominant species were Streptococcus viridans and Staphylococcus saprophyticcus, followed by S. epidermidis, Lactobacilli and S. cohnii I which were present on every studied days but at different proportions. This paper is the first part of a research topic investigating the protective effect of the indigenous flora against pathogens using the mice as an experimental model.
Assuntos
Bactérias/isolamento & purificação , Sistema Respiratório/microbiologia , Animais , Contagem de Colônia Microbiana , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Swiss Albino mice were fed a diet enriched with fat to produce hypercholesterolemia. The further administration of Lactobacillus reuteri CRL 1098 (10(4) cells/d) to hypercholesterolemic mice for 7 d decreased total cholesterol by 38%, producing serum cholesterol concentrations similar to that of the control group (67.4 mg/ml). This low dose of L. reuteri caused a 40% reduction in triglycerides and a 20% increase in the ratio of high density lipoprotein to low density lipoprotein without bacterial translocation of the native microflora into the spleen and liver. These data suggest that L. reuteri CRL 1098 is an effective hypocholesterolemic adjuvant at a low cell concentration for mice.