Occurrence of Shiga-like toxin-producing Escherichia coli in retail fresh seafood, beef, lamb, pork, and poultry from grocery stores in Seattle, Washington.

Fresh meat, poultry, and seafood purchased from Seattle area grocery stores were investigated for the presence of Shiga-like toxin-producing Escherichia coli by using DNA probes for Shiga-like toxin (SLT) genes I and II. Of the 294 food samples tested, 17% had colonies with sequence homology to SLT I and/or SLT II genes.     

Shiga-like-toxin-producing Escherichia coli in retail meats and cattle in Thailand.

Specific DNA probes were used to identify Shiga-like toxin I (SLT I)- and SLT II-producing Escherichia coli in vegetables, meats, cattle, and farm animals in Thailand. SLT-producing E. coli was isolated from 9% of market beef specimens, from 8 to 28% of fresh beef specimens at slaughterhouses, and from 11 to 84% of fecal specimens from cattle. Animals were frequently infected with several different SLT-producing E. coli types that hybridized with either the SLT I, SLT II, or both SLT probes. Of 119 SLT-producing E. coli isolates, 24% hybridized with the SLT I probe, 31% hybridized with the SLT II probe, and 44% hybridized with both SLT probes. The enterohemorrhagic E. coli plasmid probe hybridized with 64% (68 of 106) of SLT-producing E. coli isolates from food and cattle and with 8% (17 of 201) of E. coli isolates from pigs. No SLT-producing E. coli was detected in pigs. Seventy-six percent (26 of 34) of E. coli isolates that hybridized with the SLT II probe were cytotoxic to Vero but not to HeLa cells, suggesting that they produced the variant of SLT II. The high prevalence of SLT-producing E. coli in beef-producing animals suggests that exposure to animals and eating beef may pose a health risk for acquiring enterohemorrhagic E. coli infections in Thailand.     

Shiga-like-toxin-producing Escherichia coli in retail meats and cattle in Thailand

Specific DNA probes were used to identify Shiga-like toxin I (SLT I)- and SLT II-producing Escherichia coli in vegetables, meats, cattle, and farm animals in Thailand. SLT-producing E. coli was isolated from 9% of market beef specimens, from 8 to 28% of fresh beef specimens at slaughterhouses, and from 11 to 84% of fecal specimens from cattle. Animals were frequently infected with several different SLT-producing E. coli types that hybridized with either the SLT I, SLT II, or both SLT probes. Of 119 SLT-producing E. coli isolates, 24% hybridized with the SLT I probe, 31% hybridized with the SLT II probe, and 44% hybridized with both SLT probes. The enterohemorrhagic E. coli plasmid probe hybridized with 64% (68 of 106) of SLT-producing E. coli isolates from food and cattle and with 8% (17 of 201) of E. coli isolates from pigs. No SLT-producing E. coli was detected in pigs. Seventy-six percent (26 of 34) of E. coli isolates that hybridized with the SLT II probe were cytotoxic to Vero but not to HeLa cells, suggesting that they produced the variant of SLT II. The high prevalence of SLT-producing E. coli in beef-producing animals suggests that exposure to animals and eating beef may pose a health risk for acquiring enterohemorrhagic E. coli infections in Thailand.     

Extraction of lithium from spodumene by bioleaching

A multiplex PCR assay specifically detecting Escherichia coli O157 : H7 was developed by employing primers amplifying a DNA sequence upstream of E. coli O157 : H7 eaeA gene and genes encoding Shiga-like toxins (SLT) I and II. Analysis of 151 bacterial strains revealed that all E. coli O157 : H7 strains were identified simultaneously with the SLT types and could be distinguished from E. coli O55 : H7 and E. coli 055 : NM, and other non-O157 SLT-producing E. coli strains. Primer design, reaction composition (in particular, primer quantity and ratios), and amplification profile were most important in development of this multiplex PCR. This assay can serve not only as a confirmation test but also potentially can be applied to detect the pathogen in food.     

Characteristics of E.coli O157 strains isolated in Poland from clinical material and food samples

E. coli belonging to the O157 serological group are among the organisms isolated most frequently out of all the so called entero-hemorrhagic E. coli strains (EHEC). Since several years they have been isolated also in Poland. The purpose of the present study was determination on selected phenotypic and genotypic properties of E. coli O157 strains isolated in our country from clinical material samples and from food. The serotype of the strains was determined, together with the following properties regarded as pathogenicity markers of verotoxic E. coli strains such as absence of beta-glucuronidase activity and sorbitol fermentation ability, as well as production of verotoxins SLT I and/or SLT II and entero-hemolysin. Besides that, by the PCR method the fragments of the genes coding for verotoxins, intimin and enterohaemolysin were amplified. The products of PCR were analysed by the restriction enzyme analysis (RFLP). All verotoxic E. coli O157 strains isolated in Poland were analysed by the pulsed field gel electrophoresis of genomic DNA (PFGE). The studied group comprised E. coli O157 strains, among them 40 strains were isolated from human faeces and 5 from food. The remaining strains were the reference E. coli O157:H7 EDL 933 and G 5244 strains and strains from NIH collection. The obtained results showed that the tested strains were a very varying population. 21 of them (all isolated from food, 11 from faeces and 5 reference strains) belonged to serotype O157:H7, five were not peritrichous O157:NM and the remaining ones had other ciliary antigen than H7. All strains isolated from food, reference strains and only 3 O157:NM strains isolated from humans were verotoxic. The strains from food and two reference strains produced only SLT II, 2 of 3 strains isolated from humans and one reference strain also produced only SLT II and the other produced both verotoxins. Apart from these 13 verotoxic strains all remaining strains caused sorbitol fermentation.     

Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction.

A rapid and sensitive method for detection of Shiga-like toxin (SLT)-producing Escherichia coli (SLT-EC) with the polymerase chain reaction (PCR) is described. Two pairs of oligonucleotide primers homologous to SLTI and SLTII genes, respectively, were used in multiplex PCR assays. The first pair generated a ca. 600-bp PCR product with DNA from all SLTI-producing E. coli tested but not from E. coli strains that produce SLTII or variants of SLTII. The second pair generated a ca. 800-bp PCR product with DNA from E. coli strains that produce SLTII or variants of SLTII but not from SLTI-producing E. coli. When used in combination, the SLTI and SLTII oligonucleotide primers amplified DNA from all of the SLT-EC tested. No PCR products were obtained with SLT primers with DNA from 28 E. coli strains that do not produce SLT or 44 strains of 28 other bacterial species. When ground beef samples were inoculated with SLT-EC strains 319 (O157:H7; SLTI and SLTII), H30 (O26:H11; SLTI), and B2F1/3 (O91:H21; SLTII variants VT2ha and VT2hb) and cultured in modified Trypticase soy broth for 6 h at 42 degrees C, an initial sample inoculum of as few as 1 CFU of these SLT-EC strains per g could be detected in PCR assays with DNA extracted from the broth cultures.     

Multiplex PCR for detection of the heat-labile toxin gene and shiga-like toxin I and II genes in Escherichia coli isolated from natural waters.

A triplex PCR method was developed to simultaneously amplify a heat-labile toxin sequence (LT) of 258 bp, a shiga-like toxin I sequence (SLT I) of 130 bp, and a shiga-like toxin II sequence (SLT II) of 346 bp from toxigenic strains of Escherichia coli. This method was used to screen 377 environmental E. coli isolates from marine waters or estuaries located in Southern California and North Carolina for enterotoxigenic or enterohemorrhagic E. coli strains. Of the 377 E. coli screened, one isolate was found to belong to the enterotoxigenic group, since it contained a LT homologous sequence, and one isolate was found to belong to the enterohemorrhagic group, since it contained a SLT I homologous sequence. None was found to contain SLT II homologous sequences. The pathogenicity of the positive environmental E. coli isolates was confirmed by standard bioassays with Y-1 adrenal cells and Vero cells to confirm toxin production. Our results suggest that toxigenic E. coli occurs infrequently in environmental waters and that there is a low public health risk from toxigenic E. coli in coastal waters.     

Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction.

A rapid and sensitive method for detection of Shiga-like toxin (SLT)-producing Escherichia coli (SLT-EC) with the polymerase chain reaction (PCR) is described. Two pairs of oligonucleotide primers homologous to SLTI and SLTII genes, respectively, were used in multiplex PCR assays. The first pair generated a ca. 600-bp PCR product with DNA from all SLTI-producing E. coli tested but not from E. coli strains that produce SLTII or variants of SLTII. The second pair generated a ca. 800-bp PCR product with DNA from E. coli strains that produce SLTII or variants of SLTII but not from SLTI-producing E. coli. When used in combination, the SLTI and SLTII oligonucleotide primers amplified DNA from all of the SLT-EC tested. No PCR products were obtained with SLT primers with DNA from 28 E. coli strains that do not produce SLT or 44 strains of 28 other bacterial species. When ground beef samples were inoculated with SLT-EC strains 319 (O157:H7; SLTI and SLTII), H30 (O26:H11; SLTI), and B2F1/3 (O91:H21; SLTII variants VT2ha and VT2hb) and cultured in modified Trypticase soy broth for 6 h at 42 degrees C, an initial sample inoculum of as few as 1 CFU of these SLT-EC strains per g could be detected in PCR assays with DNA extracted from the broth cultures.     

Prevalence of nptII and Tn5 in kanamycin-resistant bacteria from different environments

Abstract Kanamycin (Km)-resistant bacterial populations in different soil, river water, sewage and pig manure slurry samples were enumerated and their prevalence in the total populations determined. About 350 Km-resistant Gram-negative colonies grown in the presence of kanamycin were identified using a rapid presumptive identification scheme. They were then screened for the presence of Tn5 and npt II sequences using hybridization of cells in dot blots, of Southern-blotted genomic DNA extracts and of PCR amplification products. Colonies reacting positively with a 2.7 kb probe of the central region of Tn5, or with a 925 bp npt II specific probe were primarily obtained from sewage samples, whereas fewer were obtained from pig manure slurry, river water and soil. However, in soil samples bacteria containing Tn5 or npt II were not found. Transposon Tn5 carrying the npt II gene could be unequivocally demonstrated in 3 isolates from sewage, identified as Aeromonas spp. (2x) and Escherichia coli . Hin dIII digests of chromosomal DNA obtained from these strains were cloned and shown to confer Km resistance to a sensitive E. coli strain. Further, various strains revealed the presence of npt II homologous sequences in a non-Tn5 background. The occurence of Tn5 and npt II in the samples was also assessed via PCR analysis of total community DNA extracts obtained from the aforementioned environmental samples. Evidence for the occurence of npt II was obtained for sewage, pig manure slurry, for 2 (out of 3) river water (Avon, Rhine) and 3 (out of 6) soil (Flevo silt loam, Westmaas silt loam, Ahlum rhizosphere) samples. Tn5 was not detectable via PCR in any of these environmental DNA extracts but it was found in Ede loamy sand and Flevo silt loam samples taken from a field microplot 2 and 4 weeks after release of a Tn5-containing genetically modified organism.     

PCR differentiation of Escherichia coli from other Gram-negative bacteria using primers derived from the nucleotide sequences flanking the gene encoding the universal stress protein

A PCR-based test was developed to differentiate Escherichia coli from other Gram-negative bacteria. The assay employed primers derived from the nucleotide sequences flanking the gene encoding the universal stress protein ( uspA ) and was found to be highly specific for E. coli . An 884 base pair (bp) specific product was amplified from all of the E. coli tested ( n  ₌ 45), whereas no amplification was associated with non- E. coli Gram-negative bacteria ( n  ₌ 11).     

Synthetic oligodeoxyribonucleotide probes to detect verocytotoxin-producing Escherichia coli in diseased pigs

Oligonucleotide probes constructed from the sequences published for Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) genes and antibody against the purified toxins were used to study the SLT (SLT-IIp) produced by porcine E. coli O138 and O139 strains. By DNA hybridization assays no homology was observed between SLT-I and SLT-IIp. By contrast the oligonucleotide probe derived from the slt-II A gene detected porcine strains of E. coli producing SLT-IIp and E. coli strains associated with human disease producing SLT-II. Homology of nucleotide sequences between SLT-IIp and SLT-II is reflected by serological cross-reactivity as demonstrated by a dot blot ELISA and neutralization of SLT-IIp with anti-SLT-II. The toxins were distinguishable in their ability to kill HeLa S-3 cells. The oligonucleotide probe and anti-SLT-II can facilitate identification of SLT-IIp producing E. coli to further clarify their role in diseased pigs.     

Studies on the interaction of homologues of spermine with deoxyribonucleic acid and with bacterial protoplasts

Four homologues of the naturally occurring polyamine spermine, of the type H(2)N.[CH(2)](3).NH.[CH(2)] (n).NH.[CH(2)](3).NH(2) where n=2, 3, 5 and 6, have been synthesized. Their ability to stabilize Escherichia coli protoplasts against osmotic lysis was compared with that of spermine. All homologues were approximately as effective as spermine. The effect of low concentrations of the homologues on the T(m) of calf thymus DNA and of Aerobacter aerogenes DNA in 0.03m-sodium chloride-1mm-potassium dimethylglutarate buffer, pH6.2, was tested. The increase in T(m) for a given concentration of amine was found to be n=5>n=4 and n=6> n=3>n=2. When calf thymus DNA in 0.15m-sodium chloride-15mm-sodium citrate was used spermine gave the highest increase in T(m). It is concluded that the stabilization of E. coli protoplasts by tetra-amines is a non-specific effect independent of chain length, whereas the elevation of T(m) of DNA is a more specific effect which depends on chain length.     

Molecular cloning and nucleotide sequence of another variant of the Escherichia coli Shiga-like toxin II family

Escherichia coli strain H.I.8 (O128:B12) produces low levels of a Shiga-like toxin (SLT) which we have called SLTIIva because of its close relationship with SLTIIv. The Vero cell cytotoxicity of SLTIIva is neutralized by antisera against SLTII and SLTIIv but not by antisera against SLTI. These data indicate that the SLT of strain H.I.8 is a member of the SLTII family. Since SLTIIva shares with SLTIIv the property of having low cytotoxicity to HeLa cells compared with Vero cells, it is appropriate to consider both toxins as variants of SLTII. SLTIIva differs from SLTIIv in that it is more heat-stable. Further, SLTIIv-producing strains of E. coli have only been isolated from pigs while the SLTIIva-producing E. coli strain examined in this study was isolated from a human infant with diarrhoea. The genes for this SLT were cloned from a cosmid library of total cellular DNA by screening recombinants for Vero cell toxicity and with a DNA probe derived from SLTIIv structural genes. Nucleotide sequence analysis was performed on a 2.0 kb AvaII-HincII fragment which encodes the toxin gene. The nucleotide sequence data confirm the close relationship between SLTIIva and SLTIIv: they have 98% nucleotide sequence homology in the B subunit gene and 70.6% homology in the A subunit gene. Comparison of DNA sequences indicated that SLTIIva was most closely related to SLTIIv, closely related to SLTII and less closely related to SLTI.     

Cloning of a novel G protein-coupled receptor, SLT, a subtype of the melanin-concentrating hormone receptor

A DNA fragment encoding an amino acid sequence possessing common features to the G protein-coupled receptor (GPCR) superfamily was found in the human genomic sequence, and from this information, the full-length cDNA of a novel GPCR, designated SLT, was cloned from the human hippocampus cDNA library. SLT showed the highest homology to the melanin-concentrating hormone (MCH) receptor, SLC-1 (31.5% identity), and to a lesser extent, to the somatostatin (SST) receptor subtypes. MCH exhibited agonistic behavior when applied to the SLT-expressing CHO cells at subnanomolar doses whereas more than 200 known peptides, including SST and cortistatin, did not. These results indicated that MCH is the cognate ligand of the SLT receptor and that this newly cloned GPCR is the second subtype of the MCH receptor. Quantitative polymerase chain reaction analysis of the SLT gene expression in human tissues showed that the SLT receptor is expressed mainly in brain areas including the cerebral cortex, amygdala, hippocampus, and corpus callosum, as well as in a limited number of peripheral tissues. The distribution of the SLT nearly overlapped that of SLC-1, suggesting that some of the neural functions of MCH may be mediated by both of these receptor subtypes.     

Indeno[1,2-c]isoquinolin-5,11-diones conjugated to amino acids: Synthesis, cytotoxicity, DNA interaction, and topoisomerase II inhibition properties

Three series of indeno[1,2-c]isoquinolin-5,11-dione-amino acid conjugates were designed and synthesized. Amino acids were connected to the tetracycle through linkers with lengths of n=2 and 3 atoms using ester (series I), amide (series II), and secondary amine (series III) functions. DNA binding was evaluated by thermal denaturation and fluorescence measurements. Lysine and arginine substituted derivatives with n=3 provided the highest DNA binding. Arginine derivative 32 (n=2, series II) and glycine derivative 34 (n=2, series III) displayed high topoisomerase II inhibition. Incrementing the length of the N-6 side chain from two to three methylene units provided a significant increase in DNA affinity but a substantial loss in topoisomerase II inhibition. The most cytotoxic compounds toward HL60 leukemia cells were 19, 33, and 34 displaying micromolar IC(50) values. When tested with the topoisomerase II-mutated HL60/MX2 cell line, little variation of IC(50) values was found, suggesting that topoisomerase II might not be the main target of these compounds and that additional targets could be involved.     

Enzyme-linked immunosorbent assays for detecting antibodies to Shiga-like toxin I,Shiga-like toxin II,andEscherichia coli O157:H7 lipopolysaccharide in human serum

Shiga-like toxin-producingEscherichia coli O157:H7 are important causes of bloody diarrhea and hemolytic uremic syndrome. To facilitate the epidemiologic study of these organisms, we developed enzyme-linked immunosorbent assays (ELISAs) for antibodies to Shiga-like toxin I (SLT I), Shiga-like toxin II (SLT II), andE. coli O157 lipopolysaccharide (LPS). We tested serum samples from 83 patients in two outbreaks ofE. coli O157:H7 diarrhea and from 66 well persons. Forty-three patients (52%) had at least one serum sample positive for anti-O157 LPS antibodies; among 26 culture-confirmed patients, 24 (92%) had at least one positive serum sample. Two (3%) of 66 control sera had positive anti-O157 LPS titers. ELISA results for SLT I and II were compared with those of HeLa cell cytotoxicity neutralization assays on both patient and control sera. Neutralization assays detected anti-SLT I antibodies in at least one serum sample from each of 17 (20%) patients and 7 (10.6%) controls, while 16 (19%) patients and 7 controls had positive titers by anti-SLT I ELISA. Although all serum samples, including control sera, showed nonspecific neutralization of SLT II, no antibody titers to SLT II were detected by either neutralization or ELISA. These results indicate that ELISAs for SLT I and SLT II antibodies are comparable to HeLa cell cytotoxicity neutralization assays. Both the ELISAs and neutralization assays are insensitive in detecting infected patients. However, the ELISA for antibodies toE. coli O157 LPS is both sensitive and specific, and may be more useful than assays for antitoxic antibodies in detecting persons withE. coli O157:H7 infection.     

Filamentation of Escherichia coli K12 elicited by some monomeric, dimeric and trimeric complexes of ruthenium in various oxidation states

A number of ruthenium complexes were tested for their ability to induce filamentation in Escherichia coli. These included monomeric and dimeric complexes with ruthenium in the II or III oxidation states, as well as mixed-valence complexes with ruthenium in the (II,III) oxidation states. In general, dimeric mixed-valence Ru(II,III) complexes were the most active class of compound, although some complexes of this type were relatively inactive. These were pyrazine- or bipyridyl-bridged complexes which are known to involve strong metal-ligand interaction, which stabilizes the Ru(II) oxidation state. Some Ru(III) complexes were also significantly active in induction of filamentous growth in E. coli. One of these was [Ru(NH3)5Cl]Cl2, which did not inhibit electron transport, Mg2+-ATPase activity or DNA synthesis in E. coli, but like [Ru2(NH3)6Br3]Br2 X H2O was a potent inhibitor of respiration-driven calcium transport in the organism. Filament-inducing activity of the complex was reduced in the presence of NaCl, but not in the presence of added Ca2+, ethanol, calcium pantothenate, or E. coli 'division promoting extract'. This behaviour is also similar to that of [Ru2(NH3)6Br3]Br2 X H2O. It is suggested that both complexes may induce filamentation in E. coli by a common mechanism, which may involve interference with calcium metabolism, or a wall or membrane target, rather than interaction with DNA.     

Yeast DNA topoisomerase II is encoded by a single-copy, essential gene

The gene TOP2 encoding yeast topoisomerase II has been cloned by immunological screening of a yeast genomic library constructed in the phage lambda expression vector, lambda gt11. The ends of the message encoded by the cloned DNA fragment were delimited by the Berk and Sharp procedure (S1 nuclease mapping) for the 5' end and mapping of the polyA tail portion of a cDNA fragment for the 3' end. The predicted size of the message agrees with the length of the message as determined by Northern blot hybridization analysis. The identity of the gene was confirmed by expressing the gene in E. coli from the E. coli promoter lac UV5 to give catalytically active yeast DNA topoisomerase II. Disruption of one copy of the gene in a diploid yeast creates a recessive lethal mutation, indicating that the single DNA topoisomerase II gene of yeast has an essential function.     

Hin4II, a new prototype restriction endonuclease from Haemophilus influenzae RFL4: discovery, cloning and expression in Escherichia coli

The genes encoding restriction-modification system of unknown specificity Hin4II from Haemophilus influenzae RFL4 were cloned in Escherichia coli and sequenced. The Hin4II system comprises three tandemly arranged genes coding for m6A DNA methyltransferase, m5C DNA methyltransferase and restriction endonuclease, respectively. Restriction endonuclease was expressed in E. coli and purified to apparent homogeneity. The DNA recognition sequence and cleavage positions were determined. R.Hin4II recognizes the novel non-palindromic sequence 5'-CCTTC-3' and cleaves the DNA 6 and 5 nt downstream in the top and bottom strand, respectively. The new prototype restriction endonuclease Hin4II was classified as a potential candidate of HNH nuclease family after comparison against SMART database. An amino acid sequence motif 297H-X14-N-X8-H of Hin4II was proposed as forming a putative catalytic center.     

Directed evolution and identification of control regions of ColE1 plasmid replication origins using only nucleotide deletions

Genes can be mutated by altering DNA content (base changes) or DNA length (insertions or deletions). Most in vitro directed evolution processes utilize nucleotide content changes to produce DNA libraries. We tested whether gain of function mutations could be identified using a mutagenic process that produced only nucleotide deletions. Short nucleotide stretches were deleted in a plasmid encoding lacZ, and screened for increased beta-galactosidase activity. Several mutations were found in the origin of replication that quantitatively and qualitatively altered plasmid behavior in vivo. Some mutations allowed co-residence of ColE1 plasmids in Escherichia coli, and implicate hairpin structures II and III of the ColE1 RNA primer as determinants of plasmid compatibility. Thus, useful and unexpected mutations can be found from libraries containing only deletions.