Multiplex real-time PCR for exhaustive detection of diarrhoeagenic Escherichia coli.

AIMS: The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real-time PCR assays for exhaustive detection of DEC. METHODS AND RESULTS: Primers and TaqMan probes were designed to amplify and quantify one gene (eae, stx1, stx2, elt, est, virB, aggR, astA, and afaB) from each of seven pathotypes of DEC, in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 860 strains including 88 DEC strains. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0.99. Subsequently, meat samples and enrichment broths were spiked with DEC and the assays used to detect the genes. The detection limits varied from 7.1 x 10(2) to 1.1 x 10(4) CFU ml(-1), depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU 10 g(-1)) were found to be positive by the method. CONCLUSIONS: The present system allows for the efficient and simultaneous determination of various DEC pathotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: This system makes epidemiological investigations for DEC sensitive and quick, and is a useful tool to clarify the source and routes of DEC.

Prevalence of enteric bacteria that inhibit growth of enterohaemorrhagic Escherichia coli O157 in humans.

Enterohaemorrhagic Escherichia coli O157 (O157) is infectious to humans, particularly children, at very low doses and causes not only haemorrhagic colitis but also other serious symptoms. To investigate an association between intestinal bacterial flora and resistance to such infections, we screened faecal samples for the presence of enteric bacteria that are able to suppress the growth of O157. Samples from 303 individuals, 35 children (aged < or =6 years) and 268 adults (aged 20-59 years), were examined. Colonies with different appearances on sorbitol MacConkey agar medium were screened for the production of bacteriocins inhibitory for O157 in an overlay agar plate assay. O157-inhibiting strains were isolated from 52 individuals. The prevalence of these bacteria tended to rise with age, and was significantly higher among 40- to 59-year-old adults (23/101, 22.8%) than among children (3/35, 8.6%; P<0.05). To test the hypothesis that these bacteriocin-producing strains contribute to resistance against O157 in human adults, we examined faecal samples of 25 healthy O157 carriers. Inhibitory bacteria were more prevalent among the latter (9/25, 36.0%) than among age-matched subjects who did not carry O157 (49/268, 18.3%). It appears, therefore, that inhibitory bacteria in the human gut may play a role in inhibiting propagation of O157 and/or suppressing expression of virulence factors by this pathogen.

Listeria monocytogenes isolated from cold-smoked fish products in Osaka City, Japan.

Listeria monocytogenes contamination of ready-to-eat seafood products commercially available in Osaka was examined between 1999 and 2000. L. monocytogenes was isolated from 12 (13%) of the 95 products tested. All positive samples were from cold-smoked fish with 9 being obtained during the summer. Thirteen isolates of L. monocytogenes were typed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction (PCR)-based typing methods. Isolates of the same serotype originating from the same manufacturer gave similar DNA profiles, irrespective of the type of sample or date of isolation. The finding suggest that persistent strains in each manufacturing facility proliferate during the summer and contaminate products during manufacturing processes.

An outbreak of gastroenteritis in Osaka, Japan due to Escherichia coli serogroup O166:H15 that had a coding gene for enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1).

In an outbreak of gastroenteritis on 23 July 1996, in Osaka, Japan, 54 of 91 persons who had attended a meeting the previous day became ill. Escherichia coli O166:H15 was isolated from stool specimens of patients (29/33, 88%). Laboratory tests for other bacterial pathogens and viruses were negative. The E. coli 0166 organisms did not adhere to HEp-2 cells in a localized, diffuse, or enteroaggregative manner. The organisms did not express known enterotoxigenic E. coli (ETEC) colonization factors. In polymerase chain reaction tests, the bacteria did not have coding genes for shigatoxin of enterohemorrhagic E. coli (EHEC), heat-labile, or heat-stable enterotoxin of ETEC, attachment and effacement (eaeA) of EPEC, or invasion (invE) of enteroinvasive E. coli (EIEC). Consequently, they could not be assigned to any of the recognized diarrhoeagenic groups of E. coli: EPEC, ETEC, EHEC, EIEC, enteroaggregative E. coli (EAggEC), or diffusely adhering E. coli. However, the organisms possessed the EAggEC heat-stable enterotoxin (EAST1) gene. To our knowledge, this is the first report of an outbreak caused by E. coli that did not have well-characterized virulence genes other than EAST1. The isolates showed the same DNA banding pattern in pulsed-field gel electrophoresis after digestion with the restriction enzymes XbaI or NotI. Three O166:H15 strains isolated from two sporadic cases and another outbreak during 1997-8 were distinct, indicating that multiple clones have spread already. We propose that diarrhoeal specimens should be examined for E. coli possessing the EAST1 gene.

Phage typing and DNA-based comparison of strains of enterohemorrhagic Escherichia coli O157 from apparently sporadic infections in Osaka City, Japan, 1996.

A marked increase in sporadic cases of enteritis due to enterohemorrhagic Escherichia coli serogroup O157 occurred in Osaka City, Japan, during 1996. To elucidate why the number of cases had increased, the isolates were classified using phage typing, random amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis (PFGE). Fifty-seven percent of the isolates (105/184) belonged to the same phage type (PT-32) and gave the same PFGE pattern; the clone had been isolated during a 3-week period, with a peak on July 15. It was concluded that the majority of the cases identified in July 1996 formed an outbreak, although epidemiological links to a possible common source were not established. The possibility that this outbreak was part of a huge regional outbreak including children at primary schools in Sakai City was discussed.

Processing of beta-dystroglycan by matrix metalloproteinase disrupts the link between the extracellular matrix and cell membrane via the dystroglycan complex.

The dystroglycan complex is a membrane-spanning complex composed of two subunits, alpha- and beta-dystroglycan. alpha-dystroglycan is a cell surface peripheral membrane protein which binds to the extracellular matrix (ECM), whereas beta-dystroglycan is an integral membrane protein which anchors alpha-dystroglycan to the cell membrane. The dystroglycan complex provides a tight link between the ECM and cell membrane. Dysfunction of the dystroglycan complex has commonly been implicated in the molecular pathogenesis of severe forms of hereditary neuromuscular diseases, including Duchenne muscular dystrophy, Fukuyama-type congenital muscular dystrophy and sarcoglycanopathy (LGMD2C, -D, -E and -F). To begin to clarify the pathway by which the dysfunction of the dystroglycan complex could lead to muscle cell degeneration, we investigated the proteolytic processing of the dystroglycan complex in this study. We demonstrate that (i) a 30 kDa fragment of beta-dystroglycan is expressed in peripheral nerve, kidney, lung and smooth muscle, but not skeletal muscle, cardiac muscle or brain, and (ii) this fragment is the product of proteolytic processing of the extracellular domain of beta-dystroglycan by the membrane-associated matrix metalloproteinase (MMP) activity. Importantly, furthermore, we demonstrate that this processing disintegrates the dystroglycan complex. Our results indicate that the processing of beta-dystroglycan by MMP causes the disruption of the link between the ECM and cell membrane via the dystroglycan complex, which could have profound effects on cell viability. Based on these and previously reported findings, we propose a hypothesis that this processing may play a crucial role in the molecular pathogenesis of sarcoglycanopathy.

Expression of dystroglycan complex in satellite cells of dorsal root ganglia.

In Schwann cells, the transmembrane glycoprotein beta-dystroglycan composes the dystroglycan complex together with the extracellular glycoprotein alpha-dystroglycan, which binds laminin-2 (alpha2/beta1/gamma1), a major component of the Schwann cell basal lamina. In the Schwann cell cytoplasm, beta-dystroglycan is anchored to a dystrophin isoform, Dp116. In this study, we investigated the expression of beta-dystroglycan, Dp116 and the laminin-alpha2 chain in satellite cells of rat dorsal root ganglia (DRGs). Immunohistochemical study showed that immunoreactivities for beta-dystroglycan and Dp116 were both localized to the outer rim of neuron-satellite cell and axon-Schwann cell units, indicating that both satellite and Schwann cells expressed these proteins in DRGs. Immunoreactivity for the laminin-alpha2 chain was detected in a similar location, indicating that the basal lamina surrounding satellite and Schwann cells in DRGs contained laminin-2. Ultrastructurally, immunoreactivity for the cytoplasmic domain of beta-dystroglycan as well as that for Dp116 was most intense in the cytoplasm just underlying the outer membrane of satellite cells. The immunoreactivity for laminin was associated with the outer surface of those cells, suggesting that it was localized in the surrounding basal lamina. These results indicate that the dystroglycan complex is expressed in the satellite cell outer membrane and involved in the adhesion with the basal lamina through the interaction with laminin-2.

Transgenic mice expressing mutant caveolin-3 show severe myopathy associated with increased nNOS activity.

Caveolin-3 is the muscle-specific isoform of the caveolin protein family, which is a major component of caveolae, small membrane invaginations found in most cell types. Caveolins play important roles in the formation of caveola membranes, acting as scaffolding proteins to organize and concentrate lipid-modified signaling molecules, and modulate a signaling pathway. For instance, caveolin-3 interacts with neuronal nitric oxide synthase (nNOS) and inhibits its catalytic activity. Recently, specific mutations in the caveolin-3 gene, including the Pro104Leu missense mutation, have been shown to cause an autosomal dominant limb-girdle muscular dystrophy (LGMD1C), which is characterized by the deficiency of caveolin-3 in the sarcolemma. However, the molecular mechanism by which these mutations cause the deficiency of caveolin-3 and muscle cell degeneration remains elusive. Here we generated transgenic mice expressing the Pro104Leu mutant caveolin-3. They showed severe myopathy accompanied by the deficiency of caveolin-3 in the sarcolemma, indicating a dominant negative effect of mutant caveolin-3. Interestingly, we also found a great increase of nNOS activity in their skeletal muscle, which, we propose, may play a role in muscle fiber degeneration in caveolin-3 deficiency.

Relationship of genetic type of Shiga toxin to manifestation of bloody diarrhea due to enterohemorrhagic Escherichia coli serogroup O157 isolates in Osaka City, Japan.

One hundred sixty-nine strains of enterohemorrhagic Escherichia coli serogroup O157 were examined for the correlation between the genotype of their Shiga toxin genes (stx) and manifestation of bloody diarrhea (BD). It was shown that the strains carrying only stx2vha were probably less virulent and caused BD less frequently.

Sarcoglycan complex: a muscular supporter of dystroglycan-dystrophin interplay?

In striated muscle, the cytoskeletal protein dystrophin, the protein product of the Duchenne muscular dystrophy gene, is associated with a number of sarcolemmal glycoproteins to form a large oligomeric complex, the dystrophin-glycoprotein complex (DGC). Over the last 10 years, four of these sarcolemmal glycoproteins, alpha-, beta-, gamma- and delta-sarcoglycans, have been shown to form a distinct subcomplex, the sarcoglycan complex, in the DGC. Furthermore, the genetic defects of alpha-, beta-, gamma- and delta-sarcoglycans have been identified as the causes of four distinct forms of muscular dystrophies, which are now collectively called sarcoglycanopathy. Current studies are beginning to focus on the biological functions of the sarcoglycan complex and the molecular mechanism by which its dysfunction leads to muscle cell degeneration.

Distinct regions specify the nuclear membrane targeting of emerin, the responsible protein for Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy is a neuromuscular disorder that has three characteristics: (a) early contracture of the elbows, Achilles tendons and postcervical muscles; (b) slowly progressive wasting and weakness of skeletal muscle; and (c) cardiomyopathy with severe conduction block. The responsible gene for the X-linked recessive form of this disease encodes an inner nuclear membrane protein named emerin. Although emerin is absent in tissues from patients with this disorder, it remains obscure why the loss of this widely expressed protein affects selectively skeletal muscle, heart and joints. As the first step to address this question, we examined the molecular regions of emerin that are essential for nuclear membrane targeting and stability of the protein. We found that the C-terminal hydrophobic region was necessary, but not sufficient, for nuclear membrane anchoring and stability of the protein. In the absence of this transmembrane domain, the upstream nucleoplasmic domain showed no firm association with the nuclear rim, but showed the tendency to accumulate at the nucleolus-like structures. Furthermore, proper targeting of emerin to the nuclear membrane required the latter half of the nucleoplasmic domain. These characteristics are distinct from those of lamina-associated polypeptide 2. Our findings indicate that emerin has distinct interactions with the inner nuclear membrane components that may be required for the stability and function of rigorously moving nuclei in tissues such as skeletal muscle, heart and joints.

Expression of human GFR alpha-1 (GDNF receptor) at the neuromuscular junction and myelinated nerves.

Motor neurons have been known to require a wide variety of neurotrophic factors for their survival. As one of the target-derived trophic factors, glial cell line-derived neurotrophic factor (GDNF) has been shown to exert its effects on motor neurons via a receptor complex including GDNF receptor alpha 1 (GFR alpha-1). Immunoreactivity of GFR alpha-1 was observed at myelinated peripheral nerves and neuromuscular junction (NMJ) of human skeletal muscles. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that mRNA of GFR alpha-1 existed in the ventral horn of human spinal cord, but not in the skeletal muscles. The results suggested that GFR alpha-1 might play a key role for uptake and internalization of GDNF at the human NMJ.

Prominent expression of glial cell line-derived neurotrophic factor in human skeletal muscle.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to exert neurotrophic effects on motor neurons as well as mesencephalic dopaminergic neurons. Because GDNF promotes survival of motor neurons in vivo and in vitro and rescues motor neurons from naturally occurring cell death, the potential use of GDNF for treatment of motor neuron diseases has been a major focus of recent research. The expression of GDNF in humans, however, has not been fully examined. In the present study, we examined the expression of GDNF in adult human muscle by Northern blot, reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemical analyses to address physiological roles of GDNF in humans. Northern blot analysis demonstrated high expression of GDNF mRNA in human skeletal muscle when compared to that of mouse. Intense GDNF immunoreactivity was observed in the vicinity of plasma membranes of skeletal muscle, particularly at neuromuscular junctions. GDNF immunoreactivity was also observed within the axons and surrounding Schwann cells of peripheral nerves. However, RT-PCR detected expression of GDNF mRNA only in skeletal muscle, and not within the anterior horn cells of human spinal cord. These results suggest that GDNF is produced by skeletal muscle and taken up at the nerve terminals for retrograde transport by axons. Thus, GDNF in human skeletal muscle may be involved in promoting motor neuron survival as a target-derived neurotrophic factor.

Up-regulation of glial cell line-derived neurotrophic factor (GDNF) expression in regenerating muscle fibers in neuromuscular diseases.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to exert a target-derived trophic factor for motor neurons. Immunohistochemical analyses revealed that expression of GDNF in regeneration muscle fibers was up-regulated in polymyositis (PM) and Duchenne type muscular dystrophy (DMD). Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that the full length GDNF was up-regulated in PM and DMD muscle; normal muscle exhibited mostly truncated GDNF. The results indicate that the GDNF expression is regulated in regeneration of human skeletal muscle.

Epidemiology and properties of heat-stable enterotoxin-producing Escherichia coli serotype O169:H41.

Enterotoxigenic Escherichia coli (ETEC) serotype O169:H41 organisms have become the most prevalent ETEC in Japan since the first outbreak in 1991. It was assumed that the outbreaks were due to clonal spread of this new ETEC serotype. The relationship of 32 strains isolated from 6 outbreaks were examined for biotype, antibiotic susceptibility, enterotoxigenicity, protein banding pattern, lipopolysaccharide banding pattern, plasmid analysis, and ribotyping. Further, the strains were examined by haemagglutination, surface hydrophobicity, and the ability to adhere to HEp-2 cells. The present study suggests that the outbreaks were caused by multiple clones of STp-producing O169:H41 since they showed differences in ribotype and outer membrane protein banding patterns. The strains did not agglutinate human or bovine red blood cells in a mannose-resistant manner. They adhered to HEp-2 cells in a manner resembling enteroaggregative E. coli. Five strains were examined by dot-blot tests for the colonization factor antigens CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS7, PCFO159, PCFO166 and CFA/III. Although four strains expressed CS6, no structure for CS6 was identified. A strain that the anti-CS6 MAbs did not react with could adhere to HEp-2 cells in mannose resistant manner; thus, it is unlikely that CS6 play an important role in the adhesion to the cells. Electron microscopy studies of the O169:H41 strains suggested that curly fimbriae, a possible new colonization factor, may be playing an important role in the adhesion of the bacteria to HEp-2 cells. In conclusion, outbreaks due to ETEC O169:H41 were caused by multiple clones, and the strains should be examined in detail for a possible new colonization factor.

Apoptosis by a cytosolic extract from Fas-activated cells.

Fas is a type I membrane protein and its activation by binding of the Fas ligand or an agonistic anti-Fas antibody induces apoptosis in Fas-bearing cells. In this report we prepared lysates from cells treated with anti-Fas antibody. The lysates induced apoptotic morphological changes in nuclei from normal mouse liver, accompanied by DNA degradation. The apoptosis-inducing activity was quickly generated in cells by anti-Fas antibody and was found in the soluble cytosolic fraction. Induction of the activity in cells was inhibited by a tetrapeptide, acetyl-Tyr-Val-Ala-Asp-chloromethylketone, a specific inhibitor of interleukin-1 beta converting enzyme. Addition of COS cell lysates containing Bcl-2 to the assay significantly inhibited the apoptotic process, indicating that the in vitro process reflected apoptosis that occurs in intact cells.