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
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
At present, most Neisseria gonorrhoeae testing is done with beta-lactamase and agar dilution tests with common therapeutic agents. Generally, in bacteriological diagnosis laboratories in Argentina, study of antibiotic susceptibility of N. gonorrhoeae is based on beta-lactamase determination and agar dilution method with common therapeutic agents. The National Committee for Clinical Laboratory Standards (NCCLS) has recently described a disk diffusion test that produces results comparable to the reference agar dilution method for antibiotic susceptibility of N. gonorrhoeae, using a dispersion diagram for analyzing the correlation between both techniques. We obtained 57 gonococcal isolates from patients attending a clinic for sexually transmitted diseases in Tucumán, Argentina. Antibiotic susceptibility tests using agar dilution and disk diffusion techniques were compared. The established NCCLS interpretive criteria for both susceptibility methods appeared to be applicable to domestic gonococcal strains. The correlation between the MIC's and the zones of inhibition was studied for penicillin, ampicillin, cefoxitin, spectinomycin, cefotaxime, cephaloridine, cephalexin, tetracycline, norfloxacin and kanamycin. Dispersion diagrams showed a high correlation between both methods.
Assuntos
Contagem de Colônia Microbiana , Imunodifusão , Testes de Sensibilidade Microbiana/métodos , Neisseria gonorrhoeae/efeitos dos fármacos , Sensibilidade e EspecificidadeRESUMO
The objective of the present study was to find an explanation for the biological effect of the bacteria present in a biotherapeutic milk (Lactobacillus casei CRL 431 and Lactobacillus acidophilus CRL 730). The ability of bacterial cell walls to induce an immune response when introduced into an organism is well known. In this paper we specifically analyzed the morphology of these cell walls. Besides the two bacterial strains used in the fermented milk, two other lactic acid bacteria, belonging to another genus and unable to induce an immune system response, as well as a strain of Propionibacterium, of which the immune modulating capacity is known, were used in this work. We found a structural particularly in strains with immunostimulating capacity (L. casei CRL 431 and P. acidopropionici CRL 1198): molecules which protrude from the cell surface. In L. casei CRL 431 these molecules were identified as lectins because they are able to agglutinate yeast cells treated with glutaraldehyde and glycine. The structures protruding from P. acidipropionici CRL 1198 cells were teichoic acids. Teichoic acid and lectin-like structures can participate in adhesion to intestinal cells. The immunostimulation observed can be induced by the adhesion phenomenon.
Assuntos
Proteínas de Bactérias/imunologia , Parede Celular/imunologia , Lacticaseibacillus casei/imunologia , Lactobacillus acidophilus/imunologia , Propionibacterium/imunologia , Adjuvantes Imunológicos/farmacologia , Testes de Aglutinação , Animais , Bile/microbiologia , Adesão Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Intestinos/efeitos dos fármacos , Lactobacillus acidophilus/ultraestrutura , Lacticaseibacillus casei/ultraestrutura , Lectinas , Proteínas de Membrana/imunologia , Leite/microbiologia , Propionibacterium/ultraestrutura , Ácidos Teicoicos/farmacologiaRESUMO
Beta-lactamase activity was studied in Neisseria gonorrhoeae strains. Optimum temperature was found to be 37 degrees C. The enzyme was inactivated at temperatures higher than 60 degrees C, but remained active during storage at low temperatures (4 degrees C, -30 degrees C and -70 degrees C) for two months. Enzyme activity was observed within a pH range of 5.8-8.0, while the optimum pH was 7.0-7.2. Addition of Ni2+, Fe2+, Mn2+ and p-chloromercurybenzoate to the reaction buffer exerted a negative effect upon the activity, whereas Hg2+ and ethylene diamine tetra-acetic acid produced complete inhibition. These results would indicate the presence of -SH groups at the catalytic site of the enzyme.