María A. Ferrús

Molecular detection of pathogens in water - the pros and cons of molecular techniques.

Pollution of water by sewage and run-off from farms produces a serious public health problem in many countries. Viruses, along with bacteria and protozoa in the intestine or in urine are shed and transported through the sewer system. Even in highly industrialized countries, pathogens, including viruses, are prevalent throughout the environment. Molecular methods are used to monitor viral, bacterial, and protozoan pathogens, and to track pathogen- and source-specific markers in the environment. Molecular techniques, specifically polymerase chain reaction-based methods, provide sensitive, rapid, and quantitative analytical tools with which to study such pathogens, including new or emerging strains. These techniques are used to evaluate the microbiological quality of food and water, and to assess the efficiency of virus removal in drinking and wastewater treatment plants. The range of methods available for the application of molecular techniques has increased, and the costs involved have fallen. These developments have allowed the potential standardization and automation of certain techniques. In some cases they facilitate the identification, genotyping, enumeration, viability assessment, and source-tracking of human and animal contamination. Additionally, recent improvements in detection technologies have allowed the simultaneous detection of multiple targets in a single assay. However, the molecular techniques available today and those under development require further refinement in order to be standardized and applicable to a diversity of matrices. Water disinfection treatments may have an effect on the viability of pathogens and the numbers obtained by molecular techniques may overestimate the quantification of infectious microorganisms. The pros and cons of molecular techniques for the detection and quantification of pathogens in water are discussed.

Specific Detection of Arcobacter and Campylobacter Strains in Water and Sewage by PCR and Fluorescent In Situ Hybridization

ABSTRACT The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting Arcobacter and Campylobacter strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 103 cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples.

Use of fluorescent in situ hybridization to evidence the presence of Helicobacter pylori in water

We have evaluated the use of a fluorescent in situ hybridization (FISH) technique for the detection of Helicobacter pylori in water (river and wastewater) samples. The assay was compared with PCR detection and isolation of cells on selective media. 16S rRNA and UreA+B sequence data were used as oligonucleotide probe and specific primers for FISH and PCR, respectively. Using FISH technique, H. pylori was detected in two river water and one wastewater samples, while PCR yielded only one positive result. H. pylori culture was not possible from any sample. According to these results, FISH technique has the potential to be used as a quick and sensitive method for detection of H. pylori in environmental samples.

Direct Detection and Identification of Arcobacter Species by Multiplex PCR in Chicken and Wastewater Samples from Spain

Twenty-two chicken livers, 10 chicken carcasses, and 15 wastewater samples were processed and analyzed for Arcobacter by PCR and traditional culture methods. Samples were enriched for 24 and 48 h, incubated at 30 degrees C under aerobic conditions, and streaked on blood selective media. To determine the best isolation conditions, 20 samples also were processed under microaerophilic conditions at 37 degrees C. Simple and multiplex PCR assays were used directly with enrichment broths and isolated strains. Seventeen Arcobacter strains were isolated from chicken samples, and A. butzleri was the only Arcobacter species identified. The direct PCR assay revealed that 29 of the 32 chicken samples were contaminated with Arcobacter. A. butzleri was the most frequently detected species, although Arcobacter cryaerophilus also was present in some of the samples and Arcobacter skirrowii occasionally was detected. All the wastewater samples were positive by PCR assay for Arcobacter after 24 h of enrichment. A. butzleri and A. cryaerophilus were detected with the multiplex PCR assay. Fourteen Arcobacter strains were isolated from 10 of the 15 water samples analyzed; 7 were identified as A. butzleri and the remaining 7 were A. cryaerophilus. Both for chicken and water samples, Arcobacter detection rate for PCR amplification was higher than for culture isolation. These results indicate the high prevalence of Arcobacter in chicken and wastewater and the inadequacy of available cultural methods for its detection. The species-specific multiplex PCR assay is a rapid method for assessing Arcobacter contamination in chicken and wastewater samples and is a viable alternative to biochemical identification of isolated strains.

Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

ABSTRACT We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells.

Detection of Vibrio vulnificus in seafood, seawater and wastewater samples from a Mediterranean coastal area.

Vibrio vulnificus is an opportunistic human pathogen that may cause gastroenteritis, severe necrotizing soft-tissue infections and primary septicaemia, with a high lethality rate. Illness is associated to ingestion of seafood or to the exposure of contaminated water. The aim of this work was to determine the occurrence of V. vulnificus in water and seafood samples from a coastal area near the Mediterranean (Valencia, Spain). A TaqMan probe-based real-time PCR assay was optimised and applied to 22 sea water, 42 raw sewage and 40 seafood samples. Results were compared with those obtained for culture isolation. The detection level of the PCR assay was 10 CFU g⁻¹ in inoculated samples. Seven seawater, four shellfish and six wastewater samples were positive by real time PCR. V. vulnificus was isolated from two oyster, three sea water and two wastewater samples. All the strains were obtained after 20 h enrichment, except for wastewater strains, which were isolated directly from the sample. To our knowledge, this is the first report on the isolation of V. vulnificus from sewage in Spain. Our results about the presence of V. vulnificus in food and environmental samples are strong enough to consider that the organism may represent a human health hazard in our geographical area.

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.

Study of Arcobacter spp. contamination in fresh lettuces detected by different cultural and molecular methods

Arcobacters are considered potential emerging food and waterborne pathogens. However, there is no data on the presence of Arcobacter spp. in fresh vegetables. Therefore the objective of this research was to study the presence of Arcobacter spp. in fresh lettuces. Fifty fresh lettuces purchased from different local shops in Valencia (Spain) were analyzed. The assay was performed simultaneously by cultural and molecular methods. Isolates were identified by real-time, multiplex PCR and restriction fragment length polymorphism analysis of PCR-amplified DNA fragment (PCR-RFLP). Finally, all the isolates were genotyped using the randomly amplified polymorphic DNA (RAPD-PCR) method. Arcobacter sp. was detected in 10 of the 50 samples (20%) by real-time PCR, being A. butzleri the unique detected species by mPCR. The detection levels obtained by conventional PCR (7 samples/50, 14%) were slightly lower. These seven samples were found to be positive also by culture isolation. All 19 obtained isolates were identified as A. butzleri by multiplex PCR and PCR-RFLP. Great genetic heterogeneity among the isolates was observed by RAPD-PCR profiling. To our knowledge, this is the first study in which Arcobacter spp. is detected in fresh vegetables such as lettuces. Although these foods are generally considered safe, given the large quantities consumed and the fact that further cooking is absent, lettuce could be a source of Arcobacters of public health concern.

Amplified fragment length polymorphism (AFLP) and biochemical typing of Photobacterium damselae subsp. damselae

Aims: The aim of the present study was to characterize subspecifically Photobacterium damselae subsp. damselae strains isolated from cultured Sparus aurata and Dicentrarchus labrax by means of phenotypic and molecular typing techniques (amplified fragment length polymorphism, AFLP). 
Methods and Results: Seventy‐one strains of P. damselae subsp. damselae were isolated from 38 cultured fishes at different fish farms located on the Mediterranean coast near Valencia, Spain. Most fish studied were asymptomatic and some were recovered during infectious outbreaks. Phenotypic characterization revealed a considerable degree of variability within the subspecies, including some characters, such as production of urease, which are used to differentiate P. damselae subsp. damselae from P. damselae subsp. piscicida. Genetic characterization was conducted on a selection of 33 strains, including two reference strains. Dice coefficient (Sd) and the unweighted pair group method with average linkage (UPGMA) were used for numerical analysis of banding patterns. AFLP type was defined on the basis of 100% similarity in the dendrogram obtained, yielding 24 distinct AFLP profiles. At 70% similarity, 13 clusters were defined, thus confirming the great variability observed for the phenotypic traits. 
Conclusions: The AFLP variability shown by the isolates was high enough to discriminate between different strains which colonize the same fish. However, closely related AFLP types were usually derived from strains isolated at the same fish farm, indicating an epidemiological relationship. 
Significance and Impact of the Study: This study has confirmed that the AFLP technique allows discrimination of individual strains within P. damselae subsp. damselae for epidemiological studies, and that this subspecies exhibits greater variability than that described for subspecies piscicida.

Characterization of Lactobacillus sake isolates from dry-cured sausages by restriction fragment length polymorphism analysis of the 16S rRNA gene

Lactobacillus sake strains originally isolated from dry‐fermented sausages were characterized by phenotypic and genotypic methods, including DNA‐DNA hybridization, restriction fragment length polymorphism (RFLP), and 16S rDNA sequencing analysis, in order to establish their taxonomic position and relation to well defined reference species. Initially, isolates of Lact. sake showing a characteristic phenotype (melibiose‐positive, maltose‐ and arabinose‐negative) were identified by DNA‐DNA hybridization. Subsequently, RFLP studies using EcoRI and HindIII as restriction enzymes, and cDNA from Escherichia coli or 16S rDNA from Lact. sake strains as probes, showed distinct polymorphism levels. Thus, EcoRI‐digested DNA probed with cDNA from E. coli disclosed the presence of a unique cluster for the meat isolates tested, allowing their differentiation from the reference type strain. When HindIII‐digested DNA was hybridized with the cDNA probe, strain‐specific patterns were obtained, showing a higher discrimination power. Considerable strain differentiation was also observed when EcoRI and HindIII digests were hybridized with 16S rDNA probes. Finally, sequence analysis of the 16S rDNA from one isolate also revealed a certain degree of genetic variability with respect to the reference strain of Lact. sake.