Javier Hernández

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.

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.

Biotypes and DNA ribopatterns of thermophilic campylobacters from faeces and seawater in Eastern Spain

Twelve strains of thermophilic campylobacters isolated in Valencia from faeces of infants and young children with diarrhoea, and from seawater, were biotyped and examined by analysis of chromosomal DNA HaeIII ribopatterns. The strains were identified as Campylobacter jejuni and C. coli, and all had different ribopatterns except for three identical faecal isolates of C. jejuni biotype I. No correlation was found between Lior biotype and HaeIII ribopattern but several bands within ribopatterns were identified as possible species markers for use in diagnostic and epidemiological studies.

Comparison of six different methods for typing Pseudomonas aeruginosa strains isolated from bottled and well waters

Abstract Twenty-three strains of Pseudomonas aeruginosa isolated from bottled and well waters were biotyped, serotyped and examined by antimicrobial susceptibility, plasmid content, analysis of chromosomal DNA Eco RI ribopatterns (ribotyping) and by a PCR-based procedure. Phenotypic methods showed poor discrimination power between strains, and plasmids were detected in 29% of isolates. Southern blot hydridisation analysis showed genetic diversity between isolates, and six different ribotypes were defined. Arbitrarily primed polymerase chain reaction (AP-PCR) analysis also differentiated between the strains with higher discriminatory power than ribotyping, since 13 different profiles were obtained. Both DNA-based methods are valuable alternatives to traditional typing systems for P. aeruginosa , and AP-PCR could be particularly useful for epidemiological studies.

Determination of the reactivities and cross-reactivities of monoclonal antibodies against staphylococcal enterotoxin A by indirect ELISA and immunoblot including a semiautomated electrophoresis system

Eight murine monoclonal antibodies (Mabs) to staphylococcal enterotoxin A (SEA) were produced using standard hybridoma techniques. We studied reactivities and cross‐reactivities by indirect ELISA and immunoblotting. Two of these Mabs (A5 and A7) reacted with five serovars (A–E) of SE in both systems. Only Mab A1 reacted specifically with the homologous toxin, while four Mabs reacted with SEA and SEE. Mabs A5 and A7 could be used to detect all five serovars of SEs in a single assay.