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.

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.

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.

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).

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.

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.

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.

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.

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.

[Prevalence in Tucumán of Chlamydia trachomatis and Ureaplasma urealyticum in sexually active women]. / Prevalencia en Tucumán de Chlamydia trachomatis y Ureaplasma urealyticum en mujeres sexualmente activas.

A group of 298 sexually active women who consulted for cervicitis, sterility or infertility were studied to detect the prevalence of Chlamydia trachomatis and Ureaplasma urealyticum and also to correlate the presence of these pathogens with tube damage. Chlamydial antigens were detected with a fluorescent commercial reagent with specific monoclonal antibodies. The isolation of U. urealyticum was made following the Shepard and Lunceford (19) techniques. In all samples of endocervical canal, we observed a higher incidence of chlamydial antigens (42.8%) with respect to U. urealyticum (5.7%). In peritoneal liquid the values were 38.8% and 5.5%, respectively. In the group of infertile women, we observed a higher incidence of C. trachomatis (49.5%), with 31.7% in the sterile group and 53.2% in those women with cervicitis without failures in reproduction. The frequency of U. urealyticum was 11.1%, 6.1% and 4%, respectively. According to the results obtained we can postulate that there is no correlation between infections produced by C. trachomatis and U. urealyticum and failures in reproduction.

Prevalencia en Tucumán de Chlamydia trachomatis y Ureaplasma urealyticum en mujeres sexualmente activas / [Prevalence in Tucumán of Chlamydia trachomatis and Ureaplasma urealyticum in sexually active women]

A group of 298 sexually active women who consulted for cervicitis, sterility or infertility were studied to detect the prevalence of Chlamydia trachomatis and Ureaplasma urealyticum and also to correlate the presence of these pathogens with tube damage. Chlamydial antigens were detected with a fluorescent commercial reagent with specific monoclonal antibodies. The isolation of U. urealyticum was made following the Shepard and Lunceford (19) techniques. In all samples of endocervical canal, we observed a higher incidence of chlamydial antigens (42.8

) with respect to U. urealyticum (5.7

). In peritoneal liquid the values were 38.8

, respectively. In the group of infertile women, we observed a higher incidence of C. trachomatis (49.5

), with 31.7

in the sterile group and 53.2

in those women with cervicitis without failures in reproduction. The frequency of U. urealyticum was 11.1

, respectively. According to the results obtained we can postulate that there is no correlation between infections produced by C. trachomatis and U. urealyticum and failures in reproduction.

Prevalencia en Tucumán de Chlamydia trachomatis y Ureaplasma urealyticum en mujeres sexualmente activas / [Prevalence in Tucumán of Chlamydia trachomatis and Ureaplasma urealyticum in sexually active women]

A group of 298 sexually active women who consulted for cervicitis, sterility or infertility were studied to detect the prevalence of Chlamydia trachomatis and Ureaplasma urealyticum and also to correlate the presence of these pathogens with tube damage. Chlamydial antigens were detected with a fluorescent commercial reagent with specific monoclonal antibodies. The isolation of U. urealyticum was made following the Shepard and Lunceford (19) techniques. In all samples of endocervical canal, we observed a higher incidence of chlamydial antigens (42.8

) with respect to U. urealyticum (5.7

). In peritoneal liquid the values were 38.8

, respectively. In the group of infertile women, we observed a higher incidence of C. trachomatis (49.5

), with 31.7

in the sterile group and 53.2

in those women with cervicitis without failures in reproduction. The frequency of U. urealyticum was 11.1

, respectively. According to the results obtained we can postulate that there is no correlation between infections produced by C. trachomatis and U. urealyticum and failures in reproduction.

Prevalencia en Tucumán de Chlamydia trachomatis y Ureaplasma urealyticum en mujeres sexualmente activas. / [Prevalence in Tucumán of Chlamydia trachomatis and Ureaplasma urealyticum in sexually active women]

A group of 298 sexually active women who consulted for cervicitis, sterility or infertility were studied to detect the prevalence of Chlamydia trachomatis and Ureaplasma urealyticum and also to correlate the presence of these pathogens with tube damage. Chlamydial antigens were detected with a fluorescent commercial reagent with specific monoclonal antibodies. The isolation of U. urealyticum was made following the Shepard and Lunceford (19) techniques. In all samples of endocervical canal, we observed a higher incidence of chlamydial antigens (42.8

) with respect to U. urealyticum (5.7

). In peritoneal liquid the values were 38.8

and 5.5

, respectively. In the group of infertile women, we observed a higher incidence of C. trachomatis (49.5

), with 31.7

in the sterile group and 53.2

in those women with cervicitis without failures in reproduction. The frequency of U. urealyticum was 11.1

, 6.1

and 4

, respectively. According to the results obtained we can postulate that there is no correlation between infections produced by C. trachomatis and U. urealyticum and failures in reproduction.

Prevalencia en Tucumán de Chlamydia trachomatis y Ureaplasma urealyticum en mujeres sexualmente activas. / [Prevalence in Tucumán of Chlamydia trachomatis and Ureaplasma urealyticum in sexually active women]

A group of 298 sexually active women who consulted for cervicitis, sterility or infertility were studied to detect the prevalence of Chlamydia trachomatis and Ureaplasma urealyticum and also to correlate the presence of these pathogens with tube damage. Chlamydial antigens were detected with a fluorescent commercial reagent with specific monoclonal antibodies. The isolation of U. urealyticum was made following the Shepard and Lunceford (19) techniques. In all samples of endocervical canal, we observed a higher incidence of chlamydial antigens (42.8

) with respect to U. urealyticum (5.7

). In peritoneal liquid the values were 38.8

and 5.5

, respectively. In the group of infertile women, we observed a higher incidence of C. trachomatis (49.5

), with 31.7

in the sterile group and 53.2

in those women with cervicitis without failures in reproduction. The frequency of U. urealyticum was 11.1

, 6.1

and 4

, respectively. According to the results obtained we can postulate that there is no correlation between infections produced by C. trachomatis and U. urealyticum and failures in reproduction.