Protozoan parasites and free-living amoebae contamination in organic leafy green vegetables and strawberries from Spain.

In this study, the presence of Acanthamoeba spp., Blastocystis sp., Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia sp., Toxoplasma gondii and Vermamoeba vermiformis was assessed in organic leafy green vegetables (lettuce, spinach, cabbage) and fruits (strawberry), which are usually consumed raw. A total of 110 organic samples were collected in Valencia (Spain). Protozoa were concentrated before detection by immunofluorescence (Cryptosporidium spp. and Giardia sp.) or real-time qPCR (Acanthamoeba spp., Blastocystis sp., C. cayetanensis, E. histolytica, T. gondii and V. vermiformis). The most abundant protozoa in organic vegetables and berry fruits were Acanthamoeba (65.5%), followed by T. gondii (37.2%), V. vermiformis (17.3%), C. cayetanensis (12.7%), Cryptosporidium spp. (6.8%), Blastocystis sp. (1.8%) and Giardia sp. (1.7%). E. histolytica was not found in any of the organic samples. Thus, results showed that consumers can be exposed to protozoan parasites by consuming organic vegetables and berry fruits. This is the first report in Spain describing the presence of the protozoan pathogens Acanthamoeba spp., Blastocystis sp., C. cayetanensis, T. gondii and V. vermiformis, Cryptosporidium spp. and Giardia sp. in organic fresh produce. The results of this research will help determine the risk of foodborne protozoan parasites on organic leafy greens and strawberries that are available at local markets.

Genotyping and molecular characterization of antimicrobial resistance in thermophilic Campylobacter isolated from poultry breeders and their progeny in Eastern Spain.

Thermophilic Campylobacter spp. are recognized as a major cause of acute bacterial diarrhea in humans, with broiler meat being the most common source of human infection. Antibiotic therapy is usually necessary for severe or prolonged infections, especially in immunocompromised populations such as young or elderly individuals. However, different studies have demonstrated a close association between antibiotic use in animal production and antimicrobial resistance (AMR) in humans. In this sense, there is social pressure to reduce antibiotic administration and find adequate alternatives to control the presence of bacterial infections in farms. However, there is a lack of information related to Campylobacter AMR dynamics through the entire production system from breeders to their progeny. It is unknown if resistance genes are a result of adaptation through chromosomal mutation or through horizontal gene transfer, instead of vertical transmission of DNA from the parent to their progeny. Thus, the main objectives of this study were to assess the main AMR rates present in a poultry production system, to study the relationship between Campylobacter AMR profiles from breeders and their progeny, and to study the presence and distribution of antibiotic resistance genes in poultry production. Regarding AMR rates, ciprofloxacin was classified as extremely high, followed by nalidixic acid and tetracyclines that were classified as very high. Moreover, this study demonstrated a relationship between the AMR patterns and genes found from Campylobacter strains isolated in breeders and those present in their progeny.

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.

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.

Development of a simple and rapid method based on polymerase chain reaction-based restriction fragment length polymorphism analysis to differentiate Helicobacter, Campylobacter, and Arcobacter species.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of amplified DNA fragment of the 16S and 23S rRNA genes was performed on 35 Helicobacter, 24 Campylobacter, and 15 Arcobacter strains. PCR amplification generated a 1004-bp fragment of 16S rDNA and a 2.6-Kbp fragment of 23S rDNA from each strain. The amplicons were digested with DdeI and HpaII, respectively. For both assays, distinctive profiles were obtained for each genus. 23S rDNA PCR-RFLP analysis with HpaII enzyme identified Campylobacter and Helicobacter strains at the species level. Analysis of 16S rRNA gene with DdeI enzyme was not useful for the specific identification of Campylobacter and Arcobacter, although it discriminated among Helicobacter species. The PCR-RFLP technique allowed for the discrimination among these three related genus with only one restriction enzyme; therefore it can be a simple, rapid, and useful method for routine identification.

Antimicrobial peptides are among the antagonistic metabolites produced by Bifidobacterium against Helicobacter pylori.

Sixty acid-resistant Bifidobacterium isolates were recovered from human faeces and identified by genus-specific PCR and RAPD-PCR. Helicobacter pylori strains were isolated from gastric biopsies and identified by species-specific PCR. Twenty-four of the 60 Bifidobacterium isolates were considered to be different strains by RAPD-PCR. Six of the twenty-four different strains were shown to inhibit H. pylori. These antagonistic effects were related to heat-stable proteinaceous compounds, resistant to heating at 100 degrees C for 10 min, but sensitive to proteases. H. pylori stains showed variable resistance to therapeutic antibiotics (metronidazole and clarithromycin), while all the selected bifidobacteria showed intrinsic resistance to metronidazole. These potentially probiotic bifidobacteria were able to inhibit the growth of both antibiotic sensitive and resistant H. pylori strains. Thus, the synthesis of antimicrobial peptides could be one of the mechanisms of bifidobacteria to combat H. pylori infections.

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.

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.

Direct detection of thermotolerant campylobacters in chicken products by PCR and in situ hybridization.

We have evaluated the use of PCR and fluorescent in situ hybridization (FISH) techniques for the detection of thermotolerant campylobacters in naturally contaminated chicken products. 16S rRNA sequence data was used to design two specific primers and an oligonucleotide probe for PCR and FISH analyses, respectively. The PCR protocol amplified a 439-bp fragment corresponding to a portion of specific 16S RNA gene from thermotolerant campylobacters. The detection range of the PCR assay varied between 10 cells (after enrichment) to 10(2) cells per mL (without enrichment). FISH probes were able to identify thermotolerant Campylobacter species in 'spiked' and 'unspiked' naturally contaminated samples. PCR and FISH were performed on naturally contaminated samples and compared with the isolation of cells on selective media. The in situ hybridization technique was less sensitive than PCR, although its sensitivity of detection was increased considerably after 22 h of enrichment. These results confirm the usefulness of 16S rRNA-based techniques for the direct detection of campylobacters in food samples.

Genetic diversity in Helicobacter pullorum from human and poultry sources identified by an amplified fragment length polymorphism technique and pulsed-field gel electrophoresis.

Helicobacter pullorum was first isolated from the faeces and carcasses of poultry and has been associated with human gastroenteritis. The aim of this study was to examine interstrain genetic diversity within H. pullorum. Two fingerprinting techniques were used: amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoretic (PFGE) analysis. The 20 strains examined were from four countries and comprised 13 human isolates and seven poultry isolates. Their identity was confirmed by a species-specific PCR assay. The human and poultry isolates had distinct genotypes and most strains showed a high degree of genetic diversity. Genotyping also indicated a clonal origin for two strains from the same poultry flock, and established a close relatedness between three chicken carcass isolates from a processing plant. It is concluded that these two genotyping techniques will provide a useful basis for future epidemiological investigations of H. pullorum in poultry, and may provide a link with its possible causal role in human gastrointestinal infections.

A rapid procedure for the isolation of plasmid DNA from environmental bacteria.

The INSTA-MINI-PREP method, a rapid protocol for plasmid DNA extraction, was originally developed to prepare plasmid DNA from 1 to 3 ml miniprep Escherichia coli cultures. Direct extraction of plasmid DNA is achieved by a two-phase solution which is separated by centrifugation in the presence of the INSTA-PREP gel barrier material. This method has been successfully tested on various environmental Salmonella strains, although it was not suitable for Pseudomonas aeruginosa and enterococci strains. The INSTA-MINI-PREP method is a new alternative procedure to screen plasmid contents of Salmonella and E. coli strains rapidly and easily.

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.

Ribotyping of Pseudomonas aeruginosa from infected patients: evidence of common strain types.

Sixty-nine strains of Pseudomonas aeruginosa isolated from infected patients at three hospitals in Valencia were serotyped and analyzed by comparison of restriction fragment length polymorphisms of ribosomal RNA genes (ribotyping). Genomic Southern blots of EcoRI restriction digests were hybridized with a universal rRNA gene probe from Escherichia coli 16S and 23S rRNA. Strains were genetically diverse and 12 different ribotypes of 2 to 7 bands between 5 and 21.5 kb were defined. All strains shared a common band of 6.0 kb. The predominant ribotypes were R1 and R2, representing 25% and 41% of all isolates, respectively. Ribotypes were not consistently associated with serotypes, but they clearly subtyped strains of the same serotype. This study demonstrated the prevalence of certain strain types associated with infected patients at Valencia hospitals, confirming the high typability and reproducibility of a single enzyme ribotyping for epidemiological studies of Pseudomonas aeruginosa. Ribotyping could be particularly useful if used in conjunction with serotyping.

[Ribotypes of strains of Pseudomonas aeruginosa in a patient with cystic fibrosis]. / Ribotipos de cepas de Pseudomonas aeruginosa en un paciente afectado de fibrosis quística.

BACKGROUND: Pseudomonas aeruginosa is the major causative agent of respiratory infection in cystic fibrosis (CF) patients, and its presence in sputum is related with important deterioration of lung function in affected patients, thus a methodical monitorization and control of such a microorganism is decisive in the clinical status of the patient. Due to the limited effectiveness of phenotypic subtyping techniques, it is necessary to use molecular characterization methods. MATERIAL AND METHODS: We have studied the respiratory secretions periodically obtained over a period of time of 19 months, screening for total bacterial counts, P. aeruginosa load and genomic analysis of isolates using a ribotyping protocol. RESULTS: This study has showed the chronical and transitory carriage of different strains and the relation of increased bacterial counts with clinical deterioration periods. CONCLUSIONS: We consider that the use of molecular markers can be of great interest in the epidemiological surveillance of P. aeruginosa in CF patients.

Arbitrary primed PCR fingerprinting and serotyping of clinical Pseudomonas aeruginosa strains.

Arbitrary primed PCR (AP-PCR) analysis was compared with serotyping as a means of high-resolution typing of Pseudomonas aeruginosa. Seventy-four isolates from 3 different hospitals and 18 reference strains were studied. Serotyping provided good index of discrimination, although eleven isolates could not be serotyped. Genomic DNA was amplified with a single 10 nucleotide primer (sequence 5'-AGG GGT CTT G-3'). The strains were genetically diverse and 61 different AP-PCR profiles of 2-7 bands between 0.3 and 2.4 kb were obtained. AP-PCR profiles were not consistently associated with serotypes, but they clearly subtyped strains of the same serotype. Numerical analysis of AP-PCR patterns defined 7 groups at the 55% similarity level, and identified predominant strains in each hospital. The results show that AP-PCR analysis provides a simple and practical approach to typing P. aeruginosa that is more discriminatory than traditional serotyping scheme. We suggest that maximum discrimination can be achieved by a combination of both methods.

Random amplified polymorphic DNA fingerprinting of Campylobacter jejuni and C. coli isolated from human faeces, seawater and poultry products.

The polymerase chain reaction (PCR) technique was used to obtain randomly amplified polymorphic DNA (RAPD) profiles from 64 type and serotype reference strains and 114 isolates of Campylobacter jejuni and C. coli from food, seawater and human faeces. Genetic diversity was detected among the strains as a total of 118 different RAPD profiles were obtained, each one containing from 4 to 11 bands between 0.30 and 1.50 kb. The discriminatory power of a random 10-mer primer (sequence 5'-CAATCGCCGT-3') was assessed. In general, no profiles were common to strains of the same Penner serogroup, but occasional strains from different Penner serotypes shared identical band profiles. RAPD analysis also differentiated between the species, and after numerical analysis, five main clusters were defined at the 40% similarity level, corresponding to C. jejuni, C. coli and C. lari with some exceptions. RAPD profiling of Campylobacter is highly discriminatory and is a valuable new alternative to traditional typing in epidemiological studies.