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.

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.

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.

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.

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.

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.

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.

Evaluation of the standardized disk diffusion and agar dilution antibiotic susceptibility test methods by using strains of Neisseria gonorrhoeae from Tucumán, Argentina.

At present, most Neisseria gonorrhoeae testing is done with beta-lactamase and agar dilution tests using 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 using common therapeutic agents. The National Committee for Clinical Laboratory Standards (NCCLS) recently described a disk diffusion test that produces results similar to the reference agar dilution method for antibiotic susceptibility of N. gonorrhoeae. 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 minimum inhibitory concentration (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, with a sensitivity of 89% and specificity of 91%.

Bactericidal activity against Vibrio cholerae of chemical products used in lemon production in Tucumán, Argentina.

The present research was set up to verify whether the chemical products used in lemon production (from cultivation until packaging) have a bactericidal or a bacteriostatic ability against Vibrio cholerae O1. The studied products were: copper oxychloride, benomil (a carbamate), active chlorine, sodium-o-phenylphenoate, guazatine (a polyamine mixture), imazalil (an imidazole) and lemon peel. The latter was studied with and without treatment using the above mentioned chemicals. Different dilutions of these products were tried out with varying exposure times against the bacterium V. cholerae Serogroup O1, Biotype E1 Tor, Serotype Inaba. The concentrations of the microorganism ranged from 10(2) to 10(8) CFU ml-1, the latter one being considered an infectious dose. The following results were obtained: 1) active chlorine (chlorinated water) showed bactericidal activity at concentrations of 50, 100 and 200 ppm 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 during its cultivation the fruit is successively sprayed with several chemical products, it could be that the result of the successive treatments is superior to the result of a repeated treatment with each of the individual products. This consideration should be taken into account when evaluating the eventual protection of the lemon.

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.

Chemical composition of the cell wall of lactic acid bacteria and related species.

In order to examine the relationship between biological activities and the cell wall content, the murein type and the teichoic acid of the cell wall from five strains of bacteria were studied. Two of these Lactobacillus casei CRL 431 and L. acidophilus CRL 730, are used in a commercial fermented milk (BIO MILK), which is believed to be beneficial for health. The other strains, Lactococcus lactis CRL 526, Pediococcus pentosaceus CRL 923 and Propionibacterium acidipropionici CRL 1198 were included in order to compare the cell wall structures of active and inactive strains. A method was designed to confirm the amino acids of the peptidoglycan in impure substrates. Four of the studied strains, L. casei, L. acidophilus, L. lactis and P. acidipropionici, contained glycerol teichoic acids. L. casei, L. acidophilus, P. pentosaceus and L. lactis contained A4 alpha type murein, while P. acidipropionici contained A3 gamma type. The capacity of orally administered peptidoglycans of the studied strains to stimulate phagocytosis by mouse peritoneal macrophages was analyzed. Only the PG of L. casei showed this activity. No differences were observed between active and inactive strains with respect to the chemical composition of the peptidoglycan. Therefore the biological activity is unlikely to be due to the peptidoglycan structure.

Agar dilution method for susceptibility testing of Neisseria gonorrhoeae.

The antibiotic susceptibilities of Neisseria gonorrhoeae isolates obtained from patients attending a clinic for sexually transmitted diseases in Tucumán, Argentina, were determined by the agar dilution method (MIC). 3.5% of the isolates produced beta-lactamase. A total of 96.5% of beta-lactamase negative isolates tested were susceptible to penicillin (MIC < or = 2 micrograms/ml-1); 14.03% of the tested isolates were resistant to tetracycline (MIC < or = 2 micrograms/ml-1), and 98% of the tested isolates were susceptible to spectinomycin (MIC < or = 64 micrograms/ml-1). The MICs for 95% of the isolates, tested for other drugs were: < or = 2 micrograms/ml-1 for cefoxitin, < or = 0.06 microgram/ml-1 for cefotaxime, < or = 0.25 microgram/ml-1 for norfloxacin, < or = 10 micrograms/ml-1 for cephaloridine, < or = 10 micrograms/ml-1 for cephalexin, and < or = 50 micrograms/ml-1 for kanamycin. Antibiotic resistance among N. gonorrhoeae isolates from Tucumán, Argentina, appeared to be primarily limited to penicillin and tetracycline, which has been a general use against gonorrhoeae in Tucumán since 1960. Periodic monitoring of the underlying susceptibility profiles of the N. gonorrhoeae strains prevalent in areas of frequent transmission may provide clues regarding treatment options and emerging of drug resistance.

Inducible transport of citrate in Lactobacillus rhamnosus ATCC 7469.

Lactobacillus rhamnosus ATCC 7469 exhibited diauxie when grown in a medium containing both glucose and citrate as energy source. Glucose was used as the primary energy source during the glucose-citrate diauxie. Uptake of citrate was carried out by an inducible citrate transport system. The induction of citrate uptake system was repressed in the presence of glucose. This repression was reversible and mediated by cAMP.

Purification and N-terminal amino acid sequence of Enterocin CRL 35, a 'pediocin-like' bacteriocin produced by Enterococcus faecium CRL 35.

Enterocin CRL 35, a bacteriocin produced by Enterococcus faecium CRL 35 that inhibits food-borne pathogens, was purified by precipitation with (NH4)2SO4, gel filtration, ion exchange and reverse phase chromatography. The partial N-terminal amino acid sequence indicated a strong homology with other 'pediocin-like bacteriocins' previously described.