Detection of hemolysin variants of Shiga toxin-producing Escherichia coli by PCR and culture on vancomycin-cefixime-cefsulodin blood agar.

The presence of a hemolysin-encoding gene, elyA or hlyA, from Shiga toxin-producing Escherichia coli (STEC) was detected by PCR in each of 95 strains tested. PCR products of elyA from human STEC isolates of serovars frequently detected in Germany, such as O157:H-, O103:H2, O103:H-, O26:H11, and O26:H-, showed nucleotide sequences identical to previously reported ones for O157:H7 and O111:H- strains. Compared to them, four elyA amplicons derived from human isolates of rare STEC serovars showed identity of about 98% but lacked an AluI restriction site. However, the nucleotide sequence of an amplicon derived from a porcine O138:K81:H- STEC strain was identical to the corresponding region of hlyA, encoding alpha-hemolysin, from E. coli. This hlyA amplicon showed 68% identity with the nucleotide sequence of the corresponding elyA fragment. It differed from the elyA PCR product in restriction fragments generated by AluI, EcoRI, and MluI. Of the 95 representative STEC strains, 88 produced hemolysin on blood agar supplemented with vancomycin (30 mg/liter), cefixime (20 micrograms/liter), and cefsulodin (3 mg/liter) (BVCC). The lowest added numbers of two to six STEC CFU per g of stool or per ml of raw milk were detectable on BVCC plates after seeding of the preenrichment broth, modified tryptic soy broth (mTSB) supplemented with novobiocin (10 mg/liter), with 16 STEC strains. These strains represented the seven prevailing serovars diagnosed from German patients. However, with ground-beef samples, PCR was essential to identify the lowest added numbers of two to six STEC CFU among colonies of hemolyzing Enterobacteriaceae, such as Serratia spp. and alpha-hemolysin-producing E. coli. We conclude that preenrichment of stool and food samples in mTSB for 6 h followed by overnight culturing on BVCC is a simple method for the isolation and presumptive identification of STEC.

Blinded, externally controlled multicenter evaluation of light microscopy and PCR for detection of microsporidia in stool specimens. The Diagnostic Multicenter Study Group on Microsporidia.

The quality parameters for the detection of microsporidia in identical sets of 50 stool samples were determined for six laboratories where technicians used light microscopy and for six laboratories where technicians used PCR. The average overall sensitivities were 67% (89% for patient samples only) for the PCR laboratories and 54% (80% for patient samples only) for the light microscopy laboratories. Specificities were 98 and 95%, respectively. Differences in results were most apparent between the individual laboratories rather than between the two major methods used.

Nationwide outbreak of human salmonellosis in Germany due to contaminated paprika and paprika-powdered potato chips.

Between April and September 1993, a nationwide outbreak of salmonellosis occurred in Germany which was traced to contaminated paprika and paprika-powdered potato chips. Of the estimated 1000 cases, children below 14 years were principally affected. Levels of 0.04-0.45 organisms per gram were found in the snacks. The infective dose was estimated at 4-45 organisms with an attack rate of 1 in 10,000 exposed persons. The unique feature of the outbreak was the variety of serovars involved. S. saintpaul, S. rubislaw and S. javiana were isolated during the same time period from paprika powder, spice mixtures, snacks and patients. Their clonal identity was confirmed by molecular typing methods. Furthermore, monophasic and non-motile strains of rare salmonella O-groups were isolated from both paprika products and patients. This is the largest documented outbreak due to contaminated spices which proved that even extremely low numbers of salmonellae adapted to the dry state were able to cause illness.

An outbreak due to enterohaemorrhagic Escherichia coli O157:H7 in a children day care centre characterized by person-to-person transmission and environmental contamination.

An outbreak of gastrointestinal disease and haemolytic uraemic syndrome caused by Escherichia coli O157:H7 was investigated. The outbreak occurred in a day care centre located in northern Germany in August 1992 and involved 39 children and two adults. Furthermore, four asymptomatic infections were detected among the staff. Initial and secondary cases were reported over a 30-day interval, with cases occurring in three waves. Person-to-person contact and environmental contamination were assumed to be the main mode of transmission. The source of the outbreak has remained unknown but it is likely that primary or secondary contamination of the day care centre's kitchen, too, played a role in the spread of infections. The organisms were isolated from two open packs of deep-frozen stuffed cabbage rolls and turkey scallops in batter, and furthermore from swabs from two kitchen utensils. Of the 39 cases with diarrhoea, three developed a haemolytic uraemic syndrome; one of the latter patients died. In 8 of the cases as well as in four healthy adult employees, E. coli O157:H7 was isolated from stool samples, and in two stool culture-negative cases the presence of IgM antibody to O157 LPS indicated recent infection. The E. coli O157:H7 isolates from the cases and the kitchen were of identical phage type and yielded identical biochemical reactions. All E. coli O157:H7 isolates harboured stable slt-II genes. However, slt-I genes could only be demonstrated in the primary cultures and were lost during subcultivation. This is the largest outbreak caused by enterohaemorrhagic E. coli O157:H7 that has been documented in Germany so far. The high infectivity of the organism which was demonstrated by person-to-person transmission and propagation within certain groups of children stresses the need for strict hygienic measures and early case reporting when such infections occur in susceptible settings like day care centres, nursing homes, or hospitals.

Characterization and phylogeny of mcrII, a gene cluster encoding an isoenzyme of methyl coenzyme M reductase from hyperthermophilic Methanothermus fervidus.

A 5.7 kb region of chromosomal DNA from Methanothermus fervidus, harbouring a second mcr gene cluster, was cloned and sequenced. This gene cluster, termed mcrII, encodes an isoenzyme of methyl coenzyme M reductase (MCR). In contrast to the known mcr gene clusters from other methanogens, mcrII lacks mcrC, a gene of unknown function. But the remaining mcrII genes B, D, G and A are arranged in the same order as in previously sequenced mcr gene clusters. The mcrII genes from M. fervidus are located 3' to the open reading frame (ORF) B of the methylviologen-reducing hydrogenase (mvh) gene cluster. The genes of mcrII are cotranscribed, resulting in an mRNA of 4500 nucleotides. The transcriptional initiation and termination sites were identified. Phylogenetic reconstructions reveal that the mcr gene clusters fall into two different types, I and II, and that the ancestral mcr gene cluster was duplicated before the segregation of methanogens into three major orders occurred.

Cloning, sequencing and expression of the gene encoding 2-phosphoglycerate kinase from Methanothermus fervidus.

The gene encoding 2-phosphoglycerate kinase (2PGK), which catalyses the first step in the biosynthesis of cyclic 2,3-diphosphoglycerate in methanogens, was cloned and sequenced from the hyperthermophilic Methanothermus fervidus. The 2pgk gene codes for 304 amino acids, corresponding to a relative molecular mass of 35040. The 2pgk mRNA was estimated to be 1600 nucleotides in size. Putative transcription signals and the ribosome-binding site of 2pgk are discussed. Production of 2PGK from M. fervidus in Es-cherichia coli reveals the same apparent molecular weights for the native enzyme and its denatured subunit as those shown by the 2PGK purified from M. fervidus. Also the kinetic parameters of 2PKG produced in E. coli correspond well with those from the enzyme isolated from the natural host M. fervidus.

Cloning, sequencing and transcript analysis of the gene encoding formylmethanofuran: tetrahydromethanopterin formyltransferase from the hyperthermophilic Methanothermus fervidus.

The formylmethanofuran:tetrahydromethanopterin formyltransferase (FTR) from Methanothermus fervidus was partially purified and its N-terminal amino acid sequence determined. Using as probe a mixture of oligonucleotides derived from the FTR N-terminus, the corresponding gene (ftr) was cloned and sequenced. The ftr gene codes for 297 amino acids, corresponding to a molecular mass of 31,836 daltons, in contrast to the 41,000 daltons estimated for the protein by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the hyperthermophilic FTR from M. fervidus is 76% identical to the thermophilic FTR from Methanobacterium thermoautotrophicum and has a larger number of lysine residues. A putative ATP-binding site of the FTR is reported. The size of the ftr mRNA was estimated as 1000 nucleotides indicating monocistronic transcription of the 891 bp gene. The ftr mRNA starts 27 bp downstream of the centre of a putative archaeal box A motif and terminates at an oligo-dT stretch. In vitro transcription of the ftr gene, utilizing a transcription system developed for the distantly related Sulfolobus shibatae, is discussed with respect to the functional conservation of the basal transcription apparatus of Archaea.

Biosynthesis of cyclic 2,3-diphosphoglycerate. Isolation and characterization of 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase from Methanothermus fervidus.

Starting from 2-phosphoglycerate the biosynthesis of cDPG comprises two steps: (i) the phosphorylation of 2-phosphoglycerate to 2,3-diphosphoglycerate and (ii) the intramolecular cyclization to cyclic 2,3-diphosphoglycerate. The involved enzymes, 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase, were purified form Methanothermus fervidus. Their molecular and catalytic properties were characterized.

Comparative kinetics of D-xylose and D-glucose isomerase activities of the D-xylose isomerase from Thermus aquaticus HB8.

The D-xylose isomerase from T. aquaticus accepts, besides D-xylose, also D-glucose, and, with lower efficiency, D-ribose, and D-arabinose as alternative substrates. The activity of the enzyme is strictly dependent on divalent cations. Mn2+ is most effective in the D-xylose isomerase reaction and Co2+ in the D-glucose isomerization. Mg2+ is active in both reactions, Zn2+ only in the further one. The enzyme is strongly inhibited by Cu2+, and weakly by Ni2+, Fe2+, and Ca2+. A hyperbolic dependence of the reaction velocity of the D-xylose isomerase on the concentration of D-xylose xylose and of D-glucose was found, while biphasic saturation curves were obtained by variation of the metal ion concentrations. The D-glucose isomerization reaction shows normal behaviour with respect to the metal ions. A kinetic model was derived on the basis of the assumption of two binding sites for divalent cations, one cofactor site with higher affinity and a second, low affinity site, which modulates the activity of the enzyme.

Expression of the glyceraldehyde-3-phosphate dehydrogenase gene from the extremely thermophilic archaebacterium Methanothermus fervidus in E. coli. Enzyme purification, crystallization, and preliminary crystal data.

The gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extremely thermophilic archaebacterium Methanothermus fervidus (growth optimum 82 degrees C) was cloned in vector pJF118EH and expressed in E. coli cells. As shown by molecular mass determination, protein sequencing, heat stability, and substrate saturation kinetics, the enzyme synthesized in E. coli is identical to the original enzyme from M. fervidus. The high thermostability of the E. coli-produced M. fervidus GAPDH allows rapid purification to homogeneity. From this enzyme protein crystals were grown which proved to be suitable for X-ray analysis. The crystals are of tetragonal space group P4(1)22 and contain a dimer per asymmetric unit.