Isolation of Campylobacter spp. from Three Species of Antarctic Penguins in Different Geographic Locations.

The presence of Campylobacter species was studied in three Antarctic penguin species, Adélie (Pygoscelis adeliae), chinstrap (Pygoscelis antarctica) and gentoo (Pygoscelis papua). A total of 390 penguins were captured in 12 different rookeries along the Antarctic Peninsula with differences in the amount of human visitation: six colonies were highly visited [Stranger Point, King George Island (P. papua and P. adeliae); Hannah Point, Livingston Island (P. papua and P. antarctica); Deception Island (P. antarctica); and Paradise Bay, Antarctic Peninsula (P. papua)], and six colonies were rarely visited [Devil's Point, Byers Peninsula, Livingston Island (P. papua); Cierva Cove, Antarctic Peninsula (P. papua); Rongé Island (P. papua and P. antarctica); Yalour Island (P. adeliae); and Avian Island (P. adeliae)]. A total of 23 strains were isolated from penguins from nine different rookeries. Campylobacter lari subsp. lari was isolated from eight samples (seven from P. papua and one from P. adeliae); C. lari subsp. concheus from 13 (ten from P. adeliae and three from P. antarctica) and C. volucris from two samples (both from P. papua). We did not find any significant differences in the prevalence of Campylobacter spp. between the populations in highly and rarely visited areas. This is the first report of C. lari subsp. concheus and C. volucris isolation from penguins in the Antarctic region.

Cluster investigation of mixed O76:H19 Shiga toxin-producing Escherichia coli and atypical enteropathogenic E. coli infection in a Spanish household.

A Spanish household was identified through a Public Health follow up on a Shiga toxin-producing Escherichia coli (STEC)-positive 14-month-old girl reporting bloody diarrhoea, with the four household members experiencing either symptomatic or asymptomatic STEC and/or atypical enteropathogenic E. coli (aEPEC) shedding. In total, two different O76:H19 STEC strains and six aEPEC strains belonging to multiple serotypes were isolated and characterized in the household during a 5-month period. Prolonged asymptomatic shedding of O76:H19 STEC and O51:H49 aEPEC was detected in two family members. Although there was no conclusive evidence, consumption of vegetables fertilized with sheep manure was the suspected source of infection. This study highlights the risk of cross-infections posed by prolonged asymptomatic carriage and close household contact between family members, and illustrates the importance of molecular epidemiology in understanding disease clusters.

Cytotoxicity and genotoxicity of sewage treatment plant effluents in rainbow trout cells (RTG-2).

The cytotoxicity and genotoxicity of 11 organic fractions from sewage treatment plant (STP) effluents were tested using the RTG-2 rainbow trout permanent cell line. An automated in vitro micronucleus assay developed for RTG-2 cells was used to test the genotoxicity, whereas neutral red uptake, kenacid blue protein assay and ATP content were used to evaluate cytotoxicity. The induction of micronuclei (MN) and alterations in the cell cycle were analysed in these cells by flow cytometry after exposure to the organic fractions for 72 h. More than half of the organic extracts tested demonstrated a significant increase in the MN frequency, thus indicating that most of them can be considered to be genotoxic. The extracts were analysed chemically by GC/MS. Although the most frequently detected compounds in the effluents were bisphenol A (BPA), octylphenol (OP), di-2-ethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP), as well as other possible mutagens, the concentrations cannot explain the genotoxicity of the individual chemicals, thereby suggesting a mixture effect. The results obtained support the need to apply effect-based tests to monitor complex mixtures as the most accurate means of assessing the genotoxicity of environmental samples.

Cytotoxic and genotoxic effect in RTG-2 cell line exposed to selected biocides used in the disinfection of cooling towers.

The cytotoxic and genotoxic effects induced by trichloroisocyanuric acid, Oxone, and sodium bromide, active principles included in formulations for cleaning and disinfection of cooling towers, were studied on RTG-2 cell line. Neutral red assay was used to determine the cellular viability. Toxicity ranking based on IC(50) values found that trichloroisocyanuric acid was the most cytotoxic biocide tested followed by Oxone, whereas sodium bromide resulted in a very low cytotoxicity. DNA damage has been evaluated on RTG-2 cultures by means of an in vitro assay based on the ability of PicoGreen fluorochrome to interact preferentially with dsDNA, and the results indicated that trichloroisocyanuric acid induced DNA strand breaks at concentrations above 1.2 mg/l, equivalent to 1/50-EC(50(48)), whereas exposures to Oxone and sodium bromide did not induce DNA damage at the maximal concentrations tested (1/10-EC(50(48))). These results confirm the suitability of this method for the screening of genotoxic effects of this type of aquatic pollutants, and we suggest their use in environmental risk assessment procedures.

Correlation between the presence of symptoms and the Giardia duodenalis genotype.

Clinical manifestations of Giardia duodenalis infection vary from asymptomatic infection to chronic diarrhoea. We study the correlation between the presence of symptoms and the G. duodenalis genotype in 108 patients with giardiasis. Patient age ranged from 2 to 72 years old. We found a correlation between assemblage AII and symptomatic infections, and between assemblage B and asymptomatic infections in the overall patient group and in patients less than five years of age. Nevertheless, if only patients of more than five years of age were considered, no statistically significant relationship between assemblage and symptomatic or asymptomatic Giardia infections was found. In these patients, host factors may affect the presence of clinical manifestations more than Giardia assemblage.

Genotoxic effects of selected biocides on RTG-2 fish cells by means of a modified Fast Micromethod Assay.

A sensitive in vitro assay for detecting DNA damage in RTG-2 cells culture is described. This assay employs a dye, PicoGreen double stranded DNA (dsDNA) quantitation reagent, which becomes intensely fluorescent upon binding nucleic acids. The assay includes a simple and rapid 50-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 11.6 after NaOH addition. The time course and the extent of DNA denaturation are followed in a microplate fluorescence reader at room temperature for less than 1h. Comparative studies between suspension and fixed RTG-2 cells indicated that it is possible to apply this methodology in both cases with good results. Neutral red assay was used for to determine the cellular viability when RTG-2 cultures were exposed to tetrakis(hydroxymethyl) phosphonium chloride (THPC) and benzalkonium chloride (BC), as biocides used in the disinfection of cooling towers. The results obtained by neutral red assay indicate IC(50(48)) values of 0.017 (0.011-0.028) and 2.71 (1.91-3.86) mg/L for tetrakis(hydroxymethyl) phosphonium chloride and benzalkonium chloride, respectively. DNA damage has been evaluated for both disinfectants in RTG-2 culture, by exposure to 1/10-, 1/25-, 1/50-, and 1/100-IC(50(48)) value, and the results obtained indicate a strain scission factor (SSF) of 0.126+/-0.014, 0.181+/-0.014, 0.217+/-0.013, and 0.245+/-0.013 in cell suspensions, and 0.077+/-0.019, 0.107+/-0.014, 0.151+/-0.014, and 0.202+/-0.015 in attached cells for tetrakis(hydroxymethyl) phosphonium chloride; while the SSF values for benzalkonium chloride are 0.023+/-0.009, 0.033+/-0.017, 0.068+/-0.012, and 0.088+/-0.015 in cell suspensions, and 0.033+/-0.010, 0.044+/-0.011, 0.080+/-0.009, and 0.093+/-0.010 in attached cells. Thus, the assay proposed in this study has made it possible to show DNA damage in RTG-2 cells when exposed to 0.2(1/100 IC(50(48))) and 300(1/10 IC(50(48))) Hg/L of tetrakis(hydroxymethyl) phosphonium chloride and benzalkonium chloride, respectively. The results obtained indicate that the Fast Micromethod Assay, applied on RTG-2 cell line cultures, is a fast and sensitive method for the early DNA damage detection in the aquatic environment.

Detección in vivo mediante RAPD de alteraciones en el ADN producidas por benzo(a)pireno / In vivo detection of DNA alterations produced by Benzo(a)pyrene using the RAPD technique

L a técnica de RAPD (Amplificación al Azar de ADN Polimórfico) permite detectar alteraciones inespecíficas en el ADN procedente de células que poseen una dotación genética idéntica, como son las líneas celulares establecidas, mediante la comparación del patrón de bandas de las células expuestas y no expuestas a la acción de genotóxicos.En los últimos años hemos desarrollado una metodología sensible y reproducible utilizando la línea celular RTG-2, derivada de trucha arco iris (Oncorhynchus mykiss). Sin embargo, es preciso comprobar la capacidad predictiva de este ensayo mediante estudios in vivo. La línea celular RTG-2, como se ha evidenciado en trabajos anteriores, presenta una gran similitud genética con la especie de la que procede. Por ello, en este trabajo, se ha llevado a cabo -una -exposición -subletal a benzo(a)pireno mediante inyección intraperitoneal de 69 mg/g de p.c. en alevines de trucha arco iris, valorando la aparición de mutaciones mediante la comparación del patrón de bandas obtenido a partir del ADN de células de sangre periférica, a diferentes tiempos (1 - 3 meses). Debido a que la presencia de bandas polimórficas dificulta el análisis entre los grupos de individuos tratados y no tratados, las comparaciones se realizaron en un mismo individuo antes y después del tratamiento. Los análisis cualitativos y cuantitativos mostraron tanto la aparición de nuevas bandas, como alteraciones en Su intensidad confirmando, de esta manera, los resultados que previamente habíamos obtenido in vitro tras exposiciones a este mismo genotóxico (AU)

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Postantifungal effect and effects of sub-MIC concentrations on previously treated Candida sp. influence of growth phase.

OBJECTIVES: This study evaluates the influence of growth phase on the postantifungal effect (PAFE) and on the effect of sub-MIC concentrations (1/4x MIC) on Candida sp. in PAFE stage (PAFSE). METHODS: This stage was induced by pre-treatments of 1.5 h of C. albicans or C. glabrata in their exponential or stationary phase, with 1x, 4x or 8x MIC of four antifungal agents that are fundamental for modern candidiasis therapy. RESULTS: Ketoconazole and fluconazole induced longer PAFSEs on microorganisms in logarithmic growth phase. However, this influence did not exist in the case of PAFSEs induced by AmB and 5-Fc or with the postantifungal effect induced by the four antifungal agents. In any way, significant PAFEs were always observed for Amphotericin B and 5-fluorocytosine (0.8-4.8 and 0.5-3 h, respectively, depending on the treatment dose). These values were increased (2.3-3.6 and 1.4-3.2 h respectively, depending on the pre-treatment dose) by posterior exposition to 1/4x MIC of the respective antifungal agent. In the case of ketoconazole and fluconazole, both antimycotics were not able to induce significant PAFEs, but posterior treatments to 1/4x MIC of each of the two azoles led in both yeast species to significant PAFSE of up to 2.6 h duration with ketoconazole, and 0.8 h with fluconazole, depending on the pre-treatment concentration. CONCLUSION: The growth phase of microorganisms should be considered in the planning of dosage protocols with azoles, because if the concentration applied is not high enough, the sub-MIC effects could be no significant for fungi in stationary phase of large wounds. Amphotericin B and 5-fluorocytosine induced significant postantifungal effect onCandida sp. This effect was increased by posterior exposition to sub-MIC concentration of the antifungal agents. Ketoconazole and fuconazole were not able to induce significant PAFEs at the concentrations tested, but posterior treatments to sub-MIC concentrations led to significant PAFSE. The growth phase of the culture at the time of its pre-treatment did not influence the length of the PAFE induced in it. However, the effect of the sub-MIC concentrations of Kz or Flu in yeast in PAFE phase was greater on yeast in exponential phase than for cultures in stationary phase.

Evaluation of an immunochromatographic dip-strip test for the detection of Cryptosporidium oocysts in stool specimens.

The study presented here examined the efficacy of a commercially available qualitative immunochromatographic assay for detecting Cryptosporidium oocysts in stool samples. A total of 75 samples were tested, including 50 positive for Cryptosporidium spp. by acid-fast stain, 20 positive for other parasites ( Blastocystis hominis, Endolimax nana, Entamoeba coli, Giardia lamblia, Ascaris lumbricoides, Strongyloides stercoralis and Trichuris trichiura), and five negative samples. The observed sensitivity was 98%, while specificity was 100%; the detection threshold was near 1000 oocysts/ml. Correctly diagnosed positive samples included Cryptosporidium parvum genotypes 1 and 2, whereas the single false-negative sample corresponded to a Cryptosporidium meleagridis infection.

Resistencia antibiótica en cepas clínicas de Salmonella enterica aisladas en Zaragoza / Antimicrobial resistance of clinical strains of Salmonella enterica isolated in Zaragoza

Para determinar la evolución de las frecuencias de los serotipos de Salmonella enterica y su resistencia a distintos antimicrobianos, hemos realizado un estudio restrospectivo de todas las serovariedades aisladas de muestras fecales en el Hospital Clínico Universitario Lozano Blesa de Zaragoza durante el periodo 1997-2000. Se observó un aumento en el número de aislamientos de Salmonella, así como de Campylobacter, en detrimento del resto de los enteropatógenos. El serotipo más frecuentemente aislado (55,2 por ciento) fue enteritidis, con una tendencia creciente (desde el 44,1 por ciento en 1997 hasta el 60,6 por ciento en 2000). Los serotipos que mostraron una mayor tasa de resistencia al ácido nalidíxico fueron hadar, glostrup y virchow, aunque enteritidis muestra un importante incremento (desde un 17,6 por ciento en 1997 hasta un 41,4 por ciento en 2000). El serotipo que mostró una mayor tasa de resistencia a ampicilina, cloranfenicol y cotrimoxazol fue typhimurium. No se ha detectado resistencia a las fluoroquinolonas ni a la cefotaxima, salvo en un 0,5 por ciento de las cepas de S. enteritidis, que fueron resistentes a las fluoroquinolonas. (AU)

Post-antifungal effect and effects of sub-MIC concentrations on previously treated Candida spp.: influence of exposure time and concentration.

This study evaluates the influence of exposure time and concentration on the post-antifungal effect (PAFE) and the effect of sub-MIC concentrations (1/4 x MIC) on Candida albicans and C. glabrata in the PAFE stage (PAFSE). This stage was induced by pretreatment for 1.5, 3 or 12 h with 1 x, 4 x or 8 x MIC of 4 antifungal agents fundamental to modern candidiasis therapy. The length of the 2 effects studied was dependent on the concentration of the antifungal agent applied during pretreatment, as well as on the exposure time. An increase in the dose and/or longer pretreatment prolonged the duration of the PAFE and PAFSE in both species and with all the antifungal agents. Significant PAFEs were always observed for amphotericin B and 5-fluorocytosine (0.8-13 h and 0.6-10.8 h, respectively). These values were increased (by 2.3-8.7 h and 1.5-7.8 h, respectively) by posterior exposure to 1/4 x MIC of the respective antifungal agent. Neither ketoconazole nor fluconazole were able to induce significant PAFEs, even with exposures of up to 12 h duration and a dose of 8 x MIC. However, treatment with 1/4 x MIC of each of the 2 azoles led to significant PAFSEs in both yeast species, of up to 6.5 h duration with ketoconazole and 1.7 h with fluconazole, if the concentrations and/or exposure times were sufficiently high.

Detection of cytogenetic alterations and blood cell changes in natural populations of carp.

A field study was conducted to investigate the appearance of alterations in the peripheral blood cells of wild populations of fish. Two aspects were evaluated: the appearance of cytogenetic effects, measured as increases on micronuclei frequencies, and the appearance of haematological effects by checking changes in the relative proportion of the different blood cell types. For this purpose common carp (Cyprinus carpio) were caught from four areas along a Spanish river. Three areas were located under the influence of chemical industries and/or a nuclear power plant. The fourth was a clean reference area. Flow cytometry was used to quantify the appearance of micronuclei on the same day of sampling and also after two and twelve months. The alterations in the relative proportion of cell types were counted in blood smears stained with Giemsa. Increases in micronuclei frequencies were observed in fish living in supposedly polluted areas. Alterations of the relative proportions of blood cells were manifested as an increase in white blood cells and as a decrease in red blood cells vs. control area. Since accidental spills have not been reported over this period of time, the alterations observed could suggest that fish are suffering chronic effects due to low level contamination associated with the sampled areas.

[Antimicrobial resistance of clinical strains of Salmonella enterica isolated in Zaragoza]. / Resistencia antibiótica en cepas clínicas de Salmonella enterica aisladas en Zaragoza.

In order to identify any changes in the incidence of Salmonella enterica serotypes and their resistance to a variety of antimicrobial agents, we conducted a retrospective study of all the strains isolated from stool samples at Hospital Clínico Universitario Lozano Blesa in Zaragoza from 1997 to 2000. We observed an increase in the number of isolates of Salmonella and Campylobacter and a decrease in other enteropathogens. Enteritidis was the most frequently isolated serotype (55.2%), showing an increasing tendency (from 44.1% in 1997 to 60.6% in 2000). Hadar, glostrup and virchow showed the highest rate of resistance to nalidixic acid. Enteritidis also showed an important increase in resistance to nalidixic acid (from 17.6% in 1997 to 41.4% in 2000). Typhimurium showed the highest resistance levels to ampicillin, chloramphenicol and cotrimoxazole. No resistance to fluoroquinolones or to cefotaxime was detected, with the exception of 0.5% of the S. enteritidis strains, which showed resistance to fluoroquinolones.

The eaeA gene is not found in Hafnia alvei from patients with diarrhea in Aragón, Spain.

A total of 102 Hafnia alvei clinical strains isolated from different patients with diarrhea has been tested, using polymerase chain reaction and dot-blot hybridization, for the enteropathogenic Escherichia coli attaching and effacing A (eaeA) gene to establish their role as a causative agent of diarrhea in our environment. None of them was positive for the eaeA gene. We cannot consider the eaeA gene as the virulence-associated factor implicated in the H. alvei strains isolated from diarrheal feces in our region.

Influence of temperature and concentration on the postantifungal effect and the effects of sub-MIC concentrations of four antifungal agents on previously treated Candida species.

BACKGROUND: The objective of this study was to investigate the impact of different temperatures (22, 35 and 37 degrees C) on the postantifungal effect (PAFE) and the effect of sub-MIC concentrations (1/4 x MIC) on Candida albicans and Candida glabrata in PAFE stage (PAFSE). METHODS: This stage was induced by a 1.5-hour pretreatment with different doses (1 x, 4 x and 8 x MIC) of four antifungal agents that are fundamental to modern candidiasis therapy. RESULTS: The temperature, as well as the dose of the antifungal agent that was applied during the pretreatment, determined the duration of the two studied effects. An increase in the temperature and/or the dose prolonged the duration of the PAFE and PAFSE in both species, independent of the applied antifungal agent. Amphotericin B and 5-fluorocytosine always induced significant PAFEs (0.5-4.8 h and 0.5-3.0 h, respectively), which were increased (0.7-3.4 h and 0.5-3. 2 h, respectively) by posterior exposure to 1/4 x MIC of the respective antifungal agent. In the case of ketoconazole and fluconazole, temperature and concentration were especially important. Although neither antimycotics was able to induce a significant PAFE, posterior exposure to 1/4 x MIC of each of the two azoles led in both yeast species to a significant PAFSE of up to 0.8 h (if the concentrations and/or the temperatures were high enough). CONCLUSION: Factors such as temperature and concentration could be important when choosing an antifungal agent.

The use of alternative systems for the ecotoxicological screening of complex mixtures on fish populations.

This paper presents the results of the use of alternative systems in a screening study of four complex mixtures. The following tests were performed: in vitro induction of micronuclei in a rainbow trout-derived cell line by flow cytometry, and hatching percentage, time of hatching and teratogenic alterations on the embryolarval development on medaka fish eggs. The results obtained with the proposed tests in this study allows an increase in the information level in a short period of time (2 weeks), using very low sample volumes (< 100 ml). Inclusion of chronic and specific effects (genotoxicity and teratogenicity) allows the selection of the most sensitive endpoint to increase security factors in the ecotoxicological assessment of complex mixtures, so that detailed studies can be focused only on those samples which require further research.

Flow cytometric detection of micronuclei and cell cycle alterations in fish-derived cells after exposure to three model genotoxic agents: mitomycin C, vincristine sulfate and benzo(a)pyrene.

The measurement of cytogenetic alterations in vitro is considered an initial step in the risk assessment procedures for genotoxic agents. The concern about genotoxic pollutants in natural fish population makes the use of fish-derived cells an useful tool for these purposes. The technological improvements in well-established cytogenetic endpoints, such as micronuclei (MN) estimations by means of flow cytometry, have been proposed in the later years using mammalian cells. In this work, we test the capability of flow cytometry to evaluate MN induction and cell cycle alterations in an established fish cell line (RTG-2) using three agent-inductor models at different concentrations and exposure periods. For mitomycin C, an inverse relationship between length of exposure period and concentrations was observed. A dose-response relationship was observed after exposing RTG-2 cells to vincristine sulfate and benzo(a)pyrene. As this study shows, RTG-2 cells respond to clastogenic and aneugenic effects of the tested chemicals through the induction of MN at similar doses to mammalian cells and without the addition of exogenous metabolic activity. The possibility to check cell cycle alterations, in the same sample, gives the opportunity to evaluate early signals of cytotoxicity. The use of flow cytometry improves the assay by means of its speed and objectivity, which makes the assay very useful for genotoxicity assessment of aquatic chemicals.

Influence of pH and concentration on the postantifungal effect and on the effects of sub-MIC concentrations of 4 antifungal agents on previously treated Candida spp.

This study investigates the impact of different pH values (5.5 and 7.4) on the postantifungal effect (PAFE) and the effect of sub-MIC concentrations (1/4 x MIC) on C. albicans and C. glabrata in the PAFE stage (PAFSE). The PAFE stage was induced by a 1.5 h pretreatment with different doses (1, 4 and 8 x MIC) of 4 antifungal agents. An increase in the pH and/or an increase in the dose of the antimycotic prolonged the duration of the PAFE induced by amphotericin B or 5-fluorocytosine and the PAFSE induced by all 4 antifungal agents in both species. 5-Fluorocytosine and amphotericin B (except for treatment with 1 x MIC at pH 5.5) induced significant PAFEs (0.5-3.0 h and 1.4-4.8 h, respectively), which were increased (to 0.9-3.2 h and 0.8-3.4 h, respectively) by posterior (PLEASE EXPLAIN WHAT YOU MEAN BY THE WORD "POSTERIOR" HERE) exposure to 1/4 X MIC of the respective antifungal agent. Although ketoconazole and fluconazole were not able to induce significant PAFEs, posterior exposure to 1/4 x MIC of each of these 2 azoles led to significant PAFSEs of up to 2.6 h in both yeast species when the concentrations and pH were high enough.

Activity of voriconazole: post-antifungal effect, effects of low concentrations and of pretreatment on the susceptibility of Candida albicans to leucocytes.

This study examined: (i) the post-antifungal effect (PAFE) of Voriconazole (UK 109,496) on Candida albicans, at 2 concentrations (MIC and 4 x MIC) in the presence or absence of 10% human serum; (ii) the activity of low concentrations of the drug (1/4 x MIC) on yeasts that had previously been exposed to Voriconazole (PAFSE) with or without 10% human serum; and (iii) the effect of Voriconazole pretreatment on the fungicidal activity of leucocytes and serum against C. albicans (PALE). Two concentrations (0.25 and 1 mg/l) of Voriconazole induced no PAFE against C. albicans between -4.3 and -1.4 h, but when the assays were performed in the presence of serum, positive and concentration-dependent PAFEs were obtained (0.2-4.1 h). Pretreated yeasts were more susceptible than untreated yeasts to low concentrations (0.0625 mg/l) of Voriconazole, so the drug showed positive PAFSE that was dependent on the concentration used in pretreatment without serum (0.3-1.9 h) or with 10% human serum (0.5-2.5 h). Pretreatment of the growing C. albicans cells with Voriconazole (0.25 mg/l) increased their vulnerability to killing by leucocytes during the last 2 h (p < 0.05), leading to PALE of 2 h. The results suggest that these effects might be used to evaluate the in vivo activity of an antifungal agent. The sum of the durations of these effects (PAFE, PAFSES and PALE) cause a considerable delay in yeast growth in treated cultures compared with control cultures.