Rational design, synthesis and testing of novel tricyclic topoisomerase inhibitors for the treatment of bacterial infections part 1.

The alarming reduction in drug effectiveness against bacterial infections has created an urgent need for the development of new antibacterial agents that circumvent bacterial resistance mechanisms. We report here a series of DNA gyrase and topoisomerase IV inhibitors that demonstrate potent activity against a range of Gram-positive and selected Gram-negative organisms, including clinically-relevant and drug-resistant strains. In part 1, we present a detailed structure activity relationship (SAR) analysis that led to the discovery of our previously disclosed compound, REDX05931, which has a minimum inhibitory concentration (MIC) of 0.06 µg mL-1 against fluoroquinolone-resistant Staphylococcus aureus. Although in vitro hERG and CYP inhibition precluded further development, it validates a rational design approach to address this urgent unmet medical need and provides a scaffold for further optimisation, which is presented in part 2.

Rational design, synthesis and testing of novel tricyclic topoisomerase inhibitors for the treatment of bacterial infections part 2.

Building on our previously-reported novel tricyclic topoisomerase inhibitors (NTTIs), we disclose the discovery of REDX07965, which has an MIC90 of 0.5 µg mL-1 against Staphylococcus aureus, favourable in vitro pharmacokinetic properties, selectivity versus human topoisomerase II and an acceptable toxicity profile. The results herein validate a rational design approach to address the urgent unmet medical need for novel antibiotics.

Increasing incidence of All-Terrain Vehicle trauma admissions in the pediatric and adult populations: An evaluation of injury types and severity.

OBJECTIVES: All Terrain Vehicles (ATVs) are increasing in popularity and becoming larger and faster at a production level. As a Level I Trauma Center, we perceived a disproportionately high volume of ATV-related admissions. Our goal was to study injury patterns and severity in adult and pediatric populations. METHODS: All ATV-related trauma admissions at a single Level I trauma center were retrospectively analyzed over a seven-year period. RESULTS: On-road incidents were more likely to result in a higher average Injury Severity Score (ISS) (p < 0.05). Higher ISS also occurred in children, un-helmeted, and impaired rider groups (p < 0.05). The pediatric population was more likely to have a major head injury (62.5% of children versus 31.8% of adults, p < 0.05) while thoracic injury was more common in adults (43.4% of adults versus 16.7% of children, p < 0.05). Death rates were similar in both adult and pediatric populations. CONCLUSION: ATV-related injuries vary depending on incident characteristics and patient populations. On-road use incurs a significant increase in injury severity. The pediatric population is significantly more likely to incur a severe injury and the presenting injury pattern differs from the adult population. Knowledge of population and presentation trends can help direct trauma care providers in the care and management of injured ATV riders.

The ability of low-magnitude mechanical signals to normalize bone turnover in adolescents hospitalized for anorexia nervosa.

We sought to determine whether low-magnitude mechanical stimulation (LMMS) normalizes bone turnover among adolescents hospitalized for anorexia nervosa (AN). Brief, daily LMMS prevents the decline in bone turnover typically seen during bed rest in AN. LMMS may have application for patients with AN in the inpatient setting to protect bone health. INTRODUCTION: Malnourished adolescents with AN requiring medical hospitalization are at high risk for rapid reduction in skeletal quality. Even short-term bed rest can suppress normal patterns of bone turnover. We sought to determine whether LMMS normalizes bone turnover among adolescents hospitalized for complications of AN. METHODS: In this randomized, double-blind trial, we prospectively enrolled adolescent females (n = 41) with AN, age 16.3 ± 1.9 years (mean ± SD) and BMI 15.6 ± 1.7 kg/m2. Participants were randomized to stand on a platform delivering LMMS (0.3 g at 32-37 Hz) or placebo platform for 10 min/day for 5 days. Serum markers of bone formation [bone-specific alkaline phosphatase (BSAP)], turnover [osteocalcin (OC)], and bone resorption [serum C-telopeptides (CTx)] were measured. From a random coefficients model, we constructed estimates and confidence intervals for all outcomes. RESULTS: BSAP decreased by 2.8% per day in the placebo arm (p = 0.03) but remained stable in the LMMS group (p = 0.51, pdiff = 0.04). CTx did not change with placebo (p = 0.56) but increased in the LMMS arm (+6.2% per day, p = 0.04; pdiff = 0.01). Serum OC did not change in either group (p > 0.70). CONCLUSIONS: Bed rest during hospitalization for patients with AN is associated with a suppression of bone turnover, which may contribute to diminished bone quality. Brief, daily LMMS prevents a decline in bone turnover during bed rest in AN. Protocols prescribing strict bed rest may not be appropriate for protecting bone health for these patients. LMMS may have application for these patients in the inpatient setting.

Haplosporidium littoralis sp. nov.: a crustacean pathogen within the Haplosporida (Cercozoa, Ascetosporea).

Previously, we described the pathology and ultrastructure of an apparently asporous haplosporidian-like parasite infecting the common shore crab Carcinus maenas from the European shoreline. In the current study, extraction of genomic DNA from the haemolymph, gill or hepatopancreas of infected C. maenas was carried out and the small subunit ribosomal DNA (SSU rDNA) of the pathogen was amplified by PCR before cloning and sequencing. All 4 crabs yielded an identical 1736 bp parasite sequence. BLAST analysis against the NCBI GenBank database identified the sequence as most similar to the protistan pathogen group comprising the order Haplosporida within the class Ascetosporea of the phylum Cercozoa Cavalier-Smith, 1998. Parsimony analysis placed the crab pathogen within the genus Haplosporidium, sister to the molluscan parasites H. montforti, H. pickfordi and H. lusitanicum. The parasite infecting C. maenas is hereby named as Haplosporidium littoralis sp. nov. The presence of a haplosporidian parasite infecting decapod crustaceans from the European shoreline with close phylogenetic affinity to previously described haplosporidians infecting molluscs is intriguing. The study provides important phylogenetic data for this relatively understudied, but commercially significant, pathogen group.

The occurrence of haplosporidian parasites, Haplosporidium nelsoni and Haplosporidium sp., in oysters in Ireland.

The phylum Haplosporidia is a group of obligate protozoan parasites that infect a number of freshwater and marine invertebrates. Haplosporidian parasites have caused significant mortalities in commercially important shellfish species worldwide. In this study, haplosporidia were detected in Pacific oysters Crassostrea gigas originating in Ireland and were subsequently identified independently in laboratories both in Ireland and in Spain as Haplosporidium nelsoni. In Ireland, H. nelsoni plasmodia were also observed in the heart tissue of a single Ostrea edulis. A range of techniques including heart smear screening, histology, standard polymerase chain reaction (PCR), direct sequencing and in situ hybridisation with an H. nelsoni specific DNA probe were carried out to confirm diagnosis. This is the first reporting of H. nelsoni in oysters in Ireland and this is the first reporting of the detection of this haplosporidian in O. edulis. In Ireland, another haplosporidian was also observed in a single O. edulis during heart smear screening. PCR and DNA sequencing were carried out and indicated the presence of a Haplosporidium sp., most likely Haplosporidium armoricanum. The low prevalence and intensity of infection of both haplosporidian species in Irish C. gigas and in particular O. edulis may indicate that their presence is inconsequential.

Haplosporidium raabei n. sp. (Haplosporidia): a parasite of zebra mussels, Dreissena polymorpha (Pallas, 1771).

Extensive connective tissue lysis is a common outcome of haplosporidian infection. Although such infections in marine invertebrates are well documented, they are relatively rarely observed in freshwater invertebrates. Herein, we report a field study using a comprehensive series of methodologies (histology, dissection, electron microscopy, gene sequence analysis, and molecular phylogenetics) to investigate the morphology, taxonomy, systematics, geographical distribution, pathogenicity, and seasonal and annual prevalence of a haplosporidian observed in zebra mussels, Dreissena polymorpha. Based on its genetic sequence, morphology, and host, we describe Haplosporidium raabei n. sp. from D. polymorpha - the first haplosporidian species from a freshwater bivalve. Haplosporidium raabei is rare as we observed it in histological sections in only 0·7% of the zebra mussels collected from 43 water bodies across 11 European countries and in none that were collected from 10 water bodies in the United States. In contrast to its low prevalences, disease intensities were quite high with 79·5% of infections advanced to sporogenesis.

Molecular diagnostics, field validation, and phylogenetic analysis of Quahog Parasite Unknown (QPX), a pathogen of the hard clam Mercenaria mercenaria.

Quahog Parasite Unknown (QPX) is a protistan parasite that causes disease and mortality in the hard clam Mercenaria mercenaria. PCR primers and DNA oligonucleotide probes were designed and evaluated for sensitivity and specificity for the QPX organism specifically and for the phylum Labyrinthulomycota in general. The best performing QPX-specific primer pair amplified a 665 bp region of the QPX small-subunit ribosomal DNA (SSU rDNA) and detected as little as 1 fg cloned QPX SSU rDNA and 20 fg QPX genomic DNA. The primers did not amplify DNA of uninfected hard clams M. mercenaria or of the thraustochytrids Schizochytrium aggregatum, Thraustochytrium aureum, and T. striatum. The general labyrinthulomycete primers, which were designed to offer broader specificity than the QPX primers, amplified a 435 bp region of SSU rDNA from QPX, and a 436 to 437 bp region of SSU rDNA from S. aggregatum, T. aureum, and T. striatum, but did not amplify that of the clam M. mercenaria. Field validation of the QPX-specific primer pair, through comparative sampling of 224 clams collected over a 16 mo period from a QPX endemic site in Virginia, USA, indicated that the PCR assay is equivalent to histological diagnosis if initially negative PCR products are reamplified. Oligonucleotide DNA probes specific for QPX and the phylum Labyrinthulomycota were evaluated for in situ hybridization assays of cell smears or paraffin-embedded tissues. Two DNA probes for QPX offered limited sensitivity when used independently; however, when used together as a probe cocktail, sensitivity was greatly enhanced. The probe cocktail hybridized to putative QPX organisms in tissues of hard clams collected from Virginia, New Jersey, Massachusetts and Canada, suggesting that the QPX organisms in these areas are either very closely related or the same species. The QPX probe cocktail did not hybridize with clam tissue or with the thraustochytrids S. aggregatum, T. aureum, and T. striatum. The labyrinthulomycete DNA probe hybridized with QPX and the 3 thraustochytrids, with no background hybridization to clam tissue. SSU rDNA sequences were obtained for the putative QPX organisms from geographically distinct sites. Phylogenetic analyses based on the QPX and Labyrinthulomycota sequences confirmed earlier reports that QPX is a member of this phylum, but could not definitively demonstrate that all of the QPX organisms were the same species.

Aneurysmal SAH: cognitive outcome and structural damage after clipping or coiling.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (SAH) and surgical clipping of intracranial aneurysms are associated with substantial morbidity and mortality. OBJECTIVE: To compare cognitive outcome and structural damage in patients with aneurysmal SAH treated with surgical clipping or endovascular coiling. METHODS: Forty case-matched pairs of patients with aneurysmal SAH treated by surgical clipping or endovascular coiling were prospectively assessed by use of a battery of cognitive tests. Twenty-three case-matched pairs underwent MRI 1 year after the procedure. Matching was based on grade of SAH on admission, location of aneurysm, age, and premorbid IQ. RESULTS: Both groups were impaired in all cognitive domains when compared with age-matched healthy control subjects. Comparison of cognitive outcome between the two groups indicated an overall trend toward a poorer cognitive outcome in the surgical group, which achieved significance in four tests. MRI showed focal encephalomalacia exclusively in the surgical group. This group also had a significantly higher incidence of single or multiple small infarcts within the vascular territory of the aneurysm, but both groups had similar incidence of large infarcts and global ischemic damage. CONCLUSION: Endovascular treatment may cause less structural brain damage than surgery and have a more favorable cognitive outcome. However, cognitive outcome appears to be dictated primarily by the complications of SAH.

Haplosporidiosis in the Pacific oyster Crassostrea gigas from the French Atlantic coast.

Two cases of haplosporidian infection occurred during 1993 in Pacific oysters Crassostrea gigas from the French Atlantic coast. The localization and ultrastructure of the plasmodia are described. In situ hybridization of infected tissue sections was conducted with DNA probes for oyster-infecting haplosporidians. The Haplosporidium nelsoni-specific DNA probe MSX1347 hybridized with the C. gigas parasite, and the H. costale-specific probe SSO1318 did not hybridize. Total genomic DNA was extracted from the infected tissue sections for polymerase chain reaction (PCR) amplification of the haplosporidian. PCR amplifications with H. nelsoni-specific primers and with 'universal' actin primers did not yield the expected products of 573 and 700 bp, respectively. A series of primers was designed to amplify short regions of small subunit ribosomal DNA (SSU rDNA) from most haplosporidians. The primers encompass a highly variable region of the SSU rDNA and did not amplify oyster DNA. PCR amplification of the infected C. gigas genomic DNA with these primers yielded the expected-sized product from the primer pair targeting the shortest region (94 bp). This PCR product was sequenced and it was identical to the corresponding SSU rDNA region of H. nelsoni.

A quantitative competitive polymerase chain reaction assay for the oyster pathogen Perkinsus marinus.

A quantitative competitive polymerase chain reaction (QCPCR) assay was developed for the oyster parasite Perkinsus marinus. PCR primers for the rRNA gene region of P. marinus amplified DNA isolated from P. marinus but not from Perkinsus atlanticus, Crassostrea virginica, or the dinoflagellates Peridinium sp., Gymnodinium sp., or Amphidinium sp. A mutagenic primer was used to create a competitor plasmid molecule identical to the P. marinus target DNA sequence except for a 13-bp deletion. Both P. marinus and competitor DNA amplified with equivalent efficiencies. Each of 25 oysters was processed by 5 P. marinus diagnostic methods--Ray's fluid thioglycollate medium (FTM) tissue assay, FTM hemolymph assay, whole oyster body burden assay, QCPCR of combined gill and mantle (gill/mantle) tissue, and QCPCR of hemolymph. The QCPCR assay enabled detection of 0.01 fg of P. marinus DNA in 1.0 microg of oyster tissue. QCPCR of gill/mantle tissue or hemolymph as well as the body burden assay detected infections in 24 of 25 oysters. Ray's FTM tissue assay detected only 19 infections. The FTM hemolymph assay detected only 22 infections. Regression analysis of QCPCR results and FTM results indicated that the QCPCR assays were effective in quantitating P. marinus infections in oyster tissues.

Increased Virulence in an Introduced Pathogen: Haplosporidium nelsoni (MSX) in the Eastern Oyster Crassostrea virginica.

The protistan parasite Haplosporidium nelsoni has caused extensive mortality in the eastern oyster Crassostrea virginica along the mid-Atlantic coast of the United States since 1957. The origin of H. nelsoni has remained unresolved. Molecular diagnostic tools were used to examine the hypothesis that a haplosporidian parasite in the Pacific oyster C. gigas is H. nelsoni. A DNA probe specific for H. nelsoni reacted positively in in situ hybridizations with haplosporidian plasmodia from C. gigas collected in Korea, Japan, and California. Primers that specifically amplify H. nelsoni DNA in the polymerase chain reaction amplified product from Californian C. gigas infected with the haplosporidian parasite. The DNA sequence of the 565-base pair amplified product was identical to the H. nelsoni sequence except for a single nucleotide transition, a similarity of 99.8%. These results are conclusive evidence that the parasite in C. gigas is H. nelsoni and strongly support previous speculation that the parasite was introduced into Californian populations of C. gigas from Japan. Results also support previous speculation that H. nelsoni was introduced from the Pacific Ocean to C. virginica on the East Coast of the United States, likely with known importations of C. gigas. These results document greatly increased virulence in a naive host-parasite association and reinforce potential dangers of intentional, but improper, introductions of exotic marine organisms for aquaculture or resource restoration.

Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity.

Mechanosensitive channels are ubiquitous amongst bacterial cells and have been proposed to have major roles in the adaptation to osmotic stress, in particular in the management of transitions from high to low osmolarity environments. Electrophysiological measurements have identified multiple channels in Escherichia coli cells. One gene, mscL, encoding a large conductance channel has previously been described, but null mutants were without well-defined phenotypes. Here, we report the characterization of a new gene family required for MscS function, YggB and KefA, which has enabled a rigorous test of the role of the channels. The channel determined by KefA does not appear to have a major role in managing the transition from high to low osmolarity. In contrast, analysis of mutants of E.coli lacking YggB and MscL shows that mechanosensitive channels are designed to open at a pressure change just below that which would cause cell disruption leading to death.

A student research review of the mouthbreathing habit: discussing measurement methods, manifestations and treatment of the mouthbreathing habit.

Mouthbreathing as an oral habit is seldom discussed in detail and as a consequence has tended to be overlooked by dental professionals. This review paper is an in-depth look at the current research on the mouthbreathing habit. This report aims to inform the dental professional of the most current definition of mouthbreathing and the methods of measuring the habit, including both observational and quantitative techniques. The various factors that can cause a mouthbreathing habit, such as asthma, allergies and enlarged glandular tissue, are discussed in detail. A review of current data on the skeleto-facial, dental and gingival changes that occur in mouthbreathing individuals is given, with the intention of raising the awareness of dental professionals to the special needs of these patients.

Use of gene probes and adhesion tests to characterise Escherichia coli belonging to enteropathogenic serogroups isolated in the United Kingdom.

Nine hundred and twenty-five Escherichia coli isolates from cases of diarrhoea in the United Kingdom and belonging to enteropathogenic E. coli (EPEC) O serogroups were examined for virulence properties. The tests included adhesion to HEp-2 cells, the fluorescence actin staining (FAS) test (which correlates with the ability to cause attaching and effacing lesions) and DNA hybridisations with probes to detect sequences for eaeA (E. coli attaching and effacing factor), EAF (EPEC adherence factor), verocytotoxins VT1 and VT2, enteroaggregative E. coli and diffusely adherent E. coli. The O serogroups examined were 18, 26, 44, 55, 86, 111, 114, 119, 125, 126, 127, 128 and 142. Six hundred and sixty strains (71.4%) hybridised with at least one of the DNA probes. Over 80% of strains in O serogroups 26, 55, 119, 125, 127 and 142 and 41% of strains of serogroups 86, 111, 114, 126 and 128 hybridised with the eae probe and most showed localised attachment and were FAS-positive. However, <10% of these eae probe-positive strains hybridised with the EAF probe. Eighty-four of 232 strains in O serogroups 44, 86, 111, and 126 were enteroaggregative. VT genes were detected in 57 of 402 strains in O serogroups 26, 55, 111 and 128. Identification of EPEC by serogrouping was shown to be an effective method of identifying strains with pathogenic potential, although the organisms were diverse in their properties.

A sensitive and specific DNA probe for the oyster pathogen Haplosporidium nelsoni.

Haplosporidium nelsoni is a pathogen of the eastern oyster, Crassostrea virginica, along the middle Atlantic coast of the U.S. Genomic DNA was extracted from H. nelsoni plasmodia and small subunit (SSU) rDNA was amplified by PCR, cloned and sequenced. The sequence of H. nelsoni SSU rDNA was aligned with that of another haplosporidian, Minchinia teredinis, and with SSU rDNA data of C. virginica and various protists in GenBank. A 21-base oligonucleotide unique to H. nelsoni, designated MSX1347, was commercially synthesized and tested for sensitivity and specificity. In dot blot hybridizations the probe detected 100 pg of cloned H. nelsoni rDNA and the presence of H. nelsoni in 1 microgram of genomic DNA from an infected oyster. It did not hybridize with 1 microgram of genomic DNA from uninfected C. virginica or with cloned SSU rDNA of M. teredinis. The probe was further tested for specificity with in situ hybridizations on AFA-fixed, paraffin-embedded tissue sections. The probe hybridized well with H. nelsoni plasmodia and immature spores, but poorly with mature spores. The probe did not hybridize with oyster tissue, with other common oyster parasites such as P. marinus or Nematopsis sp., or with the haplosporidians Haplosporidium louisiana from mud crabs (Panopeus spp.), Haplosporidium costale from C. virginica or M. teredinis from shipworms (Teredo spp.).

Molecular phylogenetic evidence that the phylum Haplosporidia has an alveolate ancestry.

The phylogenetic position of the phylum Haplosporidia among other protists was investigated with the complete 16S-like rRNA gene sequences from two species in the phylum: Haplosporidium nelsoni, a parasite of oysters, and Minchinia teredinis, a parasite of shipworms. Because the lack of obvious morphological homologies with other protists hampered decisions regarding taxonomic composition for sequence alignment and phylogenetic analysis, the complete sequences for these two haplosporidians were directed as search queries to the blast/ncbi.nlm.nih.gov electronic mail server. The results of this heuristic similarity search provided a basis for constructing a preliminary higher-taxonomic-level analysis comparing the haplosporidians with species from the slime molds, fungi, algae, amoebae, ciliates, dinoflagellates, and apicomplexans. Maximum parsimony yielded equivocal results, whereas transversionally weighted parsimony suggested an affinity with the alveolates (i.e., the ciliates, dinoflagellates, and apicomplexans). Multiple alignment of the two haplosporidian sequences against 17 taxa in a secondary analysis focusing on the alveolates and subsequent parsimony analysis placed the phylum Haplosporidia as a monophyletic group within the Alveolata and as a taxon of equal rank with the other three alveolate phyla. The precise placement within the Alveolata was sensitive to weighting.

Small subunit ribosomal RNA gene sequence of Minchinia teredinis (Haplosporidia: Haplosporidiidae) and a specific DNA probe and PCR primers for its detection.

Minchinia teredinis is a pathogen of wood-boring molluscs (shipworms), Teredo spp., along the middle Atlantic coast of the U.S. Genomic DNA was extracted from M. teredinis spores and small subunit (SSU) rDNA was amplified by PCR, cloned, and sequenced. The sequence of M. teredinis SSU rDNA was aligned with that of Haplosporidium nelsoni and various protists in GenBank. A 22-base oligonucleotide probe unique to M. teredinis, designated MIN702, was commercially synthesized and tested for sensitivity and specificity. In dot-blot hybridizations the probe detected 500 pg of cloned M. teredinis rDNA. The probe did not hybridize with cloned SSU rDNA of Teredo spp. or H. nelsoni. The probe was further tested for specificity with in situ hybridizations on AFA-fixed, paraffin-embedded tissue sections. The probe hybridized well with M. teredinis plasmodia and immature spores, but poorly with mature spores. The probe did not hybridize with shipworm tissue or with the haplosporidians Haplosporidium louisiana from mud crabs (Panopeus spp.) or H. nelsoni and H. costale from Crassostrea virginica. The probe and a second 18-base oligonucleotide, when used as PCR primers, amplified a 536-bp fragment of the M. teredinis SSU rRNA gene. The PCR assay was able to detect 10 fg of the cloned gene and also detected the presence of M. teredinis DNA in shipworms in which infections were observed microscopically. The 536-bp amplification product was not obtained in one Teredo sp. or in one Bankia gouldi, both categorized as uninfected after microscopic inspection. The DNA probe and PCR primers appear to be specific for M. teredinis and should be useful as diagnostic tools and for life cycle investigations.

Effects of reactive hydrocarbons and hydrogen peroxide on antioxidant activity in cherry leaves.

One-year-old cherry trees were fumigated with propene and gas-phase hydrogen peroxide, singly and in combination, in controlled-environment chambers for an 8-week period during the summer season. A UV light source was included with the combined propene and hydrogen peroxide regime to provide a source of hydroxyl radicals and ozone, and thus all the constituents of a photochemical smog. Measurements were made of soluble protein concentration and of glutathione reductase activity in leaf extracts from two or three leaf classes in plants from each treatment regime at the end of each fumigation period. Significant increases in soluble protein concentration with respect to the controls were found in plants fumigated with propene and hydrogen peroxide. The occurrence and extent of these differences depended on the leaf class and on the timing of the fumigation period over the summer with respect to bud break. The activity of glutathione reductase was found to be significantly increased in mature lower leaves of plants which had been fumigated with hydrogen peroxide. This effect was independent of the timing of fumigation with respect to bud break. Enzyme activity was also increased in propene and in propene plus hydrogen peroxide treatments, but only when plants were fumigated early in the growth season.

Virulence properties of Escherichia coli strains belonging to serogroups O26, O55, O111 and O128 isolated in the United Kingdom in 1991 from patients with diarrhoea.

Some strains of Escherichia coli belonging to serogroups O26, O55, O111 or O128 produce Vero cytotoxin (VT). These serogroups are included in the range of enteropathogenic E. coli (EPEC) serogroups for which commercial antisera are available. In an attempt to obtain information on VT-producing strains other than those of serogroup O157, 122 strains belonging to these four serogroups and isolated in 1991 from patients with diarrhoea in the United Kingdom were tested for hybridization with VT probes. Only 18 of the 122 strains were VT-positive and these were O26 or O128. However 90 strains hybridized with the E. coli attaching and effacing (eae) probe (including 14 VT-positive strains) and 17 with the enteroaggregative E. coli (EAggEC) probe. For 78 eae-positive and 9 EAggEC-positive strains, tissue culture tests correlated with the probe results as the strains gave, respectively, either localized adhesion and a positive fluorescent-actin staining test or a characteristic aggregative attachment. A total of 111 of the 122 strains belonging to serogroups O26, O55, O111 or O128 possessed properties that may be associated with the ability to cause human diarrhoeal disease, and similar studies are needed on strains from the other classical EPEC serogroups.