Inmaculada Amorós

Cryptosporidium oocysts and giardia cysts on salad products irrigated with contaminated water.

A field study in Valencia, Spain, was done to determine the occurrence of Giardia and Cryptosporidium on salad products that are frequently eaten raw, such as lettuces and Chinese cabbage, and in irrigation waters. Four water samples were taken weekly 1 month before harvesting the vegetables. All water samples were analyzed using techniques included in the U.S. Environmental Protection Agency Method 1623. Standard methods for detecting protozoan parasites on salad vegetables are not available. Published techniques for the isolation of parasites from vegetables generally have low and variable recovery efficiencies. In this study, vegetables were analyzed using a recently reported method for detection of Cryptosporidium oocysts and Giardia cysts on salad products. The waters tested were positive for both Cryptosporidium and Giardia. Of 19 salad products studied, we observed Cryptosporidium in 12 samples and Giardia in 10 samples. Recoveries of the Texas Red-stained Cryptosporidium and Giardia, which were used as internal controls, were 24.5% +/- 3.5% for Cryptosporidium and 16.7% +/- 8.1% for Giardia (n = 8). This study provides data on the occurrence of Cryptosporidium and Giardia in salad products in Spain. The method was useful in the detection of Cryptosporidium oocysts and Giardia cysts on the vegetables tested, and it provides a useful analytical tool for occurrence monitoring.

Standard and new faecal indicators and pathogens in sewage treatment plants, microbiological parameters for improving the control of reclaimed water

This study involved collaboration between three centres with expertise in viruses, bacteria and protozoa. The focus of the research was the study of the dissemination and removal of pathogens and faecal indicators in two sewage treatment plants (STP1 and STP2) using tertiary treatments. Samples were collected over a period of five months through the sewage treatment processes. Analysis of the samples revealed that the plants were not efficient at removing the faecal indicators and pathogens tested during the study. From entry point (raw sewage) to effluent level (tertiary treatment effluent water), the experimental results showed that the reduction ratios of human adenoviruses were 1.2 log₁₀ in STP1 and 1.9 log₁₀ in STP2. Whereas for Giardia spp. and Cryptosporidium spp. the reduction ratios were 2.3 log₁₀ for both pathogens in STP1, and 3.0 and 1.7 log₁₀ in STP2, respectively. Furthermore, the presence of faecal indicators and pathogens at different sampling points was evaluated revealing that the tested pathogens were present in reclaimed water. Human adenovirus and Arcobacter spp. showed positive results in infectivity assays for most of the tertiary effluent water samples that comply with current legislation in Spain. The pathogens detected must be evaluated using a risk assessment model, which will be essential for the development of improved guidelines for the re-use of reclaimed water.

Quantitative determination of E. coli, and fecal coliforms in water using a chromogenic medium

Abstract A new medium, Chromocult Coliform® Agar (CC agar) developed by E. Merck AG (Darmstadt, Germany) was compared with the Standard Methods, membrane filtration fecal coliform (mFC) medium for fecal coliform detection and enumeration. In the CC agar, non‐E. coli, fecal coliforms (Klebsiella, Enterobacter, and Citrobacter,), (KEC) were identified by the production of a salmon to red colour from p‐galactosidase (LAC) cleavage of the substrate Salmon‐GAL, while E. coli, colonies were detected by the blue colour, produced by the cleavage of X‐glucuronide by β‐ glucuronidase (GUS). Statistically, there was no significant differences between fecal coliform counts obtained with the two media (CC agar and mFC agar) and two incubation procedures (2h‐37°C plus 22h‐44.5°, and 44.5°C) as determined by variance analysis. In our study K coli, represented, on average 70.5–92.5% of the fecal coliform population. A high incidence of false negative KEC (19.5%) and E. coli, (29.6%) colonies was detected at 44.5°C. Two K...

Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples

Cryptosporidium and Giardia are major causes of diarrheal disease in humans worldwide and are major causes of protozoan waterborne diseases. Two DNA TaqMan PCR-based Giardia and Cryptosporidium methods targeting a 74-bp sequence of the β-giardin Giardia gene and a 151-bp sequence of the COWP Cryptosporidium gene, respectively, were used as models to compare two different LNA/DNA TaqMan probes to improve the detection limit in a real-time PCR assay. The LNA probes were the most sensitive resulting in 0.96 to 1.57 lower Ct values than a DNA Giardia TaqMan probe and 0.56 to 2.21 lower than a DNA Cryptosporidium TaqMan probe. Evaluation of TaqMan Giardia and Cryptosporidium probes with LNA substitutions resulted in real-time PCR curves with an earlier Ct values than conventional DNA TaqMan probes. In conclusion, the LNA probes could be useful for more sensitive detection limits.

Fenitrothion and 3‐methyl‐4‐nitrophenol degradation by two bacteria in natural waters under laboratory conditions

Abstract The ability of two bacterial strains, Flavobacterium sp. strain ATCC 27551 and Arthrobacter aurescens strain TW17, to degrade fenitrothion and the product of its hydrolysis 3‐methyl‐4‐nitrophenol (MNP) in natural water samples under laboratory conditions was studied. Fenitrothion was degraded by Flavobacterium sp whereas MNP was not degraded by this strain. In comparison, MNP was metabolized by A. aurescens and fenitrothion was not. The natural microbial populations did not decompose the fenitrothion and MNP, however Flavobacterium sp and R. aurescens degraded these compounds in the presence of the natural microbial population.

Molinate decontamination processes in effluent water from rice fields

The performance of aeration, photodecomposition and biological degradation processes as methods to reduce molinate contamination levels in effluent water from rice fields was studied. Aeration produced a molinate dissipation of 84%, as against 22% without aeration. Application of UV-light to clean water solutions achieved a molinate photodecomposition of 96% in 24 h. Maximal degradation obtained in algal cultures was 55% in 20 days and 78% in 40 days. In micro-organism cultures, kept in darkness and with a continuous flow of aqueous solution of molinate and inorganic salts, a degradation of 97% was achieved.

Quantitative determination of Escherichia coli in water using CHROMagar®E. coli

A new medium, CHROMagar® E. coli (CAEC), containing a combination of X-glucuronide and methyl-glucuronide for the detection of β-glucuronidase activity of Escherichia coli has been evaluated by the membrane filtration (MF) technique n fresh water samples. The CAEC agar was compared with conventional media, mFC agar and mLSB, for the enumeration of faecal coliforms. The variance analysis showed that CAEC was as sensitive as mFC agar and mLSB. A good correlation was found between E. coli versus those from faecal coliforms in the water sampling areas tested. Of 321 presumptively positive E. coli colonies (blue) and 154 presumptively negative E. coli colonies (white), only 8 (2.5%) false positive and 19 (12.4%) false negative colonies were found. Specificity of the CEAC agar in spanish samples was temperature dependent, false negative E. coli colonies occurred less frequently at 37°C (2.3%) than at 44.5°C (18.8%). The results of this study indicate that CAEC agar is efficient for the enumeration of E. coli from a wide range of environmental freshwater samples.

Salmonella detection in marine waters using a short standard method

Abstract A study was undertaken to compare two enrichment broths and to test a new plate medium, Rambach agar, for their suitability to detect Salmonella from marine recreational waters. The study shows that under conditions of low levels of fecal contamination, Salmonella spp was isolated more efficiently using NR10(10)/43 broth. While at higher levels of fecal contamination NR10/43 broth effectively limits the growth of accompanying flora. For the highest level of fecal contamination Rambach agar was less selective than Hektoen enteric agar but Rambach agar was shown to be better than Hektoen agar for the recovery of Salmonella strains from low contaminated waters. On Rambach agar the distinct color of salmonellas, seen in red, enhanced correct identification. Biochemical and serological confirmation of Salmonella isolates was performed using the API Z System and Oxoid Salmonella Rapid Latext test. The combination of enrichment broths (24 h at 42°C), isolation (24 h at 37°C) and identification systems described above (2 h or I min) allowed the detection of Salmonella from marine waters, with low or high levels of fecal contamination, in 48 h.

The occurrence of mesophilic aeromonads species in marine recreational waters of Valencia (Spain)

Abstract In this study aeromonads were isolated in the eight zones analyzed. The numbers of aeromonads varied from one zone to the other and reflected the proximity of the zone to the terrestrial effluents. The concentration of aeromonads exceeded those of fecal coliforms in several samples. No significant seasonal variations were observed in the aeromonads populations in the majority of zones analyzed and water temperature seemed to have little effect on their growth. A. caviae was found to be predominating, followed by A. hydrophila and A. sobria. In general, the correlation coefficients between mesophilic aeromonads and their standard biologic indicator bacteria were higher in fecal polluted zones than in zones with a low fecal pollution.

Prevalence of Cryptosporidium oocysts and Giardia cysts in raw and treated sewage sludges.

ABSTRACT Treated sludge from wastewater treatment plants (WWTPs) is commonly used in agriculture as fertilizers and to amend soils. The most significant health hazard for sewage sludge relates to the wide range of pathogenic microorganisms such as protozoa parasites.The objective of this study was to collect quantitative data on Cryptosporidium oocysts and Giardia cysts in the treated sludge in wastewater treatment facilities in Spain. Sludge from five WWTPs with different stabilization processes has been analysed for the presence of Cryptosporidium and Giardia in the raw sludge and after the sludge treatment. A composting plant (CP) has also been assessed. After a sedimentation step, sludge samples were processed and (oo)cysts were isolated by immunomagnetic separation (IMS) and detected by immunofluorescence assay (IFA). Results obtained in this study showed that Cryptosporidium oocysts and Giardia cysts were present in 26 of the 30 samples (86.6%) of raw sludge samples. In treated sludge samples, (oo)cysts have been observed in all WWTP’s analysed (25 samples) with different stabilization treatment (83.3%). Only in samples from the CP no (oo)cysts were detected. This study provides evidence that (oo)cysts are present in sewage sludge-end products from wastewater treatment processes with the negative consequences for public health.