A comparative study of preservation and storage of Haemophilus influenzae.

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.

Experimental administration of estradiol on the colonization of lactobacillus fermentum and escherichia coli in the urogenital tract of mice.

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.

Purification and characterization of beta-lactamase from Neisseria gonorrhoeae from clinical samples.

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.

Enteric bifidobacteria: isolation from human infants and challenge studies in mice.

Bifidobacteria from breast-fed infants, formula-fed infants, or premature babies fed by parenteral methods were isolated and identified. The persistence of these microorganisms in the gastrointestinal tract of mice, after oral administration, was studied to determine the optimal dose and frequency of translocation to the liver and spleen. The rate of isolation among infants varied between 19 and 82% depending on the origin of the samples, with the highest values seen in breast-fed babies. The predominant species found in all cases was Bifidobacterium adolescentis. The optimal dose for oral administration of bifidobacteria to mice was 10(7) cells per day per animal for up to 2, 5, or 7 days. These bacteria remained up to 5 days postfeeding, even if feeding was interrupted. The results of bacterial translocation assays showed differences for the different strains and doses tested.

Bactericidal activity of lemon juice and lemon derivatives against Vibrio cholerae.

Food products can be possible vectors of the agent responsible for cholera epidemics, because some of these products allow Vibrio cholerae O1 to develop to concentrations above the dangerous level. This study deals with the behaviour of essential oils, natural and concentrated lemon juice and fresh and dehydrated lemon peel against V. cholerae O1 biotype Eltor serotype Inaba tox+. Our aim was to evaluate whether these products, used at different dilutions, exhibit bactericidal or bacteriostatic activity against the microorganism, when present at concentrations of 10(2), 10(4), 10(6) and 10(8) colony forming units (CFU) ml(-1), and after different exposure times. 10(8) CFU ml(-1) was considered an infectious dose. Concentrated lemon juice and essential oils inhibited V. cholerae completely at all studied dilutions and exposure times. Fresh lemon peel and dehydrated lemon peel partially inhibited growth of V. cholerae. Freshly squeezed lemon juice, diluted to 10(-2), showed complete inhibition of V. cholerae at a concentration of 10(8) CFU ml(-1) after 5 min of exposure time; a dilution of 2 x 10(-3) produced inhibition after 15 min and a dilution of 10(-3) after 30 min. It can be concluded that lemon, a natural product which is easily obtained, acts as a biocide against V. cholerae, and is, therefore, an efficient decontaminant, harmless to humans.

Identification and nucleotide sequence of genes involved in the synthesis of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705.

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.

Effect of Lactobacillus reuteri on the prevention of hypercholesterolemia in mice.

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.

Gut mucosa morphology and microflora changes in malnourished mice after renutrition with milk and administration of Lactobacillus casei.

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).

Purification and amino acid sequence of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705.

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.

Antiviral activity of enterocin CRL35 against herpesviruses.

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.

Microbial flora variations in the respiratory tract of mice.

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.

A clinical and bacteriological study of children suffering from haemolytic uraemic syndrome in Tucuman, Argentina.

Haemolytic uraemic syndrome (HUS) is a disease with serious consequences for children, such as terminal chronic renal failure. During the last few years there have been numerous studies undertaken to determine whether there is a relationship between this disease and the presence of Shiga toxin-producing bacteria. Escherichia coli (E. coli) O157:H7 is one of the most frequent etiologic agents of HUS. It acts through cytotoxins called Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2) and carries a 90-Kb plasmid codified for an adhesion fimbria which is part of its pathogenicity. The objectives of this study were to: 1). confirm whether there exists a relationship between severity and clinical presentation of HUS; 2). prove the existence of Stx1 and/or Stx2 in the faeces of HUS patients; and 3). detect the presence of Stx1- and/or Stx2-producing E. coli. Our results did not show any difference in the average age, sex or clinical behavior between children with diarrhea positive (D+) HUS and diarrhea negative (D-) HUS. Male patients were predominant, as was incidence during summer, considering all cases. Nor could we find any relationship between severity and HUS type. E. coli O157:H7 was isolated in 40% of the patients with (D+) HUS and in 50% of patients with (D-) HUS. Another serotype, O55:K59, was also isolated (7%). Stx1 and/or Stx2 were found in all HUS cases. The following virulence factors of E. coli strains isolated from 12 patients were found: Adhesion fimbria (100%), Stx1 (16%), Stx2 (32%), and Stx1 + Stx2 (50%). None of these factors was found in control patients. Sixty-three percent of the HUS cases showed seroconversion for lipopolysaccharides of E. coli O157. We drew the following conclusions: 1). there is no significant relationship between seriousness of HUS and type of disease; 2). an association exists between HUS and the production of Stx1 and Stx2; 3). the incidence of E. coli O157:H7 was high in Tucuman, Argentina; and 4). Stx2 alone or in association with Stx1 was the predominant toxin.

Evidence for hypocholesterolemic effect of Lactobacillus reuteri in hypercholesterolemic mice.

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.

Properties of beta-lactamase from Neisseria gonorrhoeae.

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.

Study of the morphology of the cell walls of some strains of lactic acid bacteria and related species.

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.

In vitro comparison of disk diffusion and agar dilution antibiotic susceptibility test methods for Neisseria gonorrhoeae.

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.

Association between hemolytic uremic syndrome and verotoxin-producing strains of E. coli.

One of the most frequent etiologic agents of Hemolytic Uremic Syndrome (HUS) is Escherichia coli O157H7, a microorganism that possesses virulence factors (Shiga-like Toxins I and II and adhesion fimbriae). The present study was set up to determine the relationship between HUS and the presence of Verotoxin in patients of "Niño Jesús" Children's Hospital. Tucumán, Argentina. 19 Children between 0 and 4 years old suffering from HUS (typical and atypical symptoms) and 15 control children of similar sex and age were selected. Presence of enterohemorrhagic E. coli was studied in both groups using molecular hybridization techniques. Free Verotoxin and Verotoxin-producing E. coli were analyzed in Vero cells. The following results were obtained: 1) The cytotoxic effect on Vero cells from fecal filtrates was observed in all children suffering from HUS 2) Verotoxin-producing E. coli was detected in only 12 of them 3) None of the filtrates of feces from control children presented a cytotoxic effect on Vero cells 4) In 8 of the patients suffering from HUS serotype O157H7 was isolated, in one O55K59 and in 3 typification of E. coli was not possible with the serums assayed 5) 77.5% of the strains isolated from HUS patients gave a positive molecular hybridization reaction, showing the following: Adhesion Fimbriae (AF) (25%); AF + Shiga-like Toxin I (13.75%); AF + Shiga-like Toxin II (20%); AF + Shiga-like Toxins I and II (41.25%). In patients suffering from atypical HUS a combination of AF + Shiga-like Toxins I and II was found. The 15 control children did not hybridize to the probes assayed. From the results obtained we may conclude that there exists a relationship between HUS and the presence of Verotoxin in the children suffering from HUS studied. The predominant serotype in our cases was O157H7 and Shiga-like Toxin II was found with highest frequency.

Action against Vibrio cholerae O1 Tox+ of chemical products used in the lemon production.

Tucuman is the first lemon exporting province in Argentina and the fourth lemon exporter in the world. The present work was set up to study the survival of Vibrio cholerae O1 Tox+ after application of different chemical products used in the lemon production (from its cultivation until its packing). The following products were studied: copper oxychloride, benomil (a carbamate), active chlorine, sodium-o-phenylphenoate, guazatine (a polyamine mixture), imazalil (an imidazole) and fresh and dehydrated lemon peel. Using different dilutions of the products above mentioned antimicrobial tests were carried out with different exposure times against V. cholerae Serogroup O1, Biotype El Tor, Serotype Inaba. The microorganism was used at concentrations of 10(2), 10(4), 10(6) and 10(8) CFU ml-1, the latter one being considered as an infectious dose. The following results were obtained: 1) Active chlorine (chlorinated water) showed bactericidal activity at concentrations of 0.5 x 10(-1), 10(-1), y 2 x 10(-1) g l-1 after 10 min of exposure time. 2) Copper oxychloride, sodium-o-phenylphenoate, guazatine and imazalil showed bactericidal activity against V. cholerae at concentrations of 10(2) and 10(4) CFU ml-1. 3) Due to the fact that the fruit is successively sprayed with several chemical products during its cultivation, it could be proposed that the result of the successive treatments is superior to the result of a treatment with each of the individual products. This consideration should be taken into account when evaluating the eventual protection of the lemon.

Proteolytic activity of Lactobacillus strains isolated from dryfermented sausages on muscle sarcoplasmic proteins.

The proteolytic activity of seven strains of Lactobacillus from two species isolated from dry cured sausages was assayed using a soluble muscle extract as a source of proteins, at a temperature of 30 °C. The results indicated that the strains of Lactobacillus plantarum tested had the more active proteolytic system, showing the highest amino acid release in the medium after 72 hr of incubation (L. plantarum CRL 681) when the microorganism was in the stationary phase of growth. The strains of L. casei showed a continued hydrolytic activity with a lower amino acids concentration along the studied period. The SDS-PAGE profiles showed that the major changes in sarcoplasmic proteins were produced in the 13 kDa and 36-40 kDa molecular weights region.

Influence of bile on beta-galactosidase activity and cell viability of Lactobacillus reuteri when subjected to freeze-drying.

The effect of bile on beta-galactosidase activity and cell viability was investigated using two strains of Lactobacillus reuteri that were subjected to freeze-drying. In the presence of 0.15% oxgall, beta-galactosidase activity of the whole cells was significantly increased. After lyophilization, the cultures that had been treated with oxgall showed a low survival rate without changes in beta-galactosidase activity. The poor resistance of the cells to damage from freeze-drying might be related to the presence of membranous structures containing simple folds and buds of the cell membrane, as was observed by transmission electron microscopy.