Specific Detection of a Verotoxin 2 Variant,VT2vp1, by a Bead-Enzyme-Linked Immunosorbent Assay

A bead-enzyme-linked immunosorbent assay to specifically detect a Verotoxin 2 variant, VT2vpl, was developed. The sensitivity of the bead-ELISA was 200 pg/ml of the purified VT2vpl and it did not react with 20 ng/ml of the purified VT2. The specificity of the bead-ELISA was examined with 107 strains of Verocytotoxin-producing Escherichia coli that include VT1-, VT2-, VT2vha-, VT2vhb- and VT2vp1-producing E. coli, and only VT2vp1-producing E. coli that were confirmed by VT2vp1-specific polymerase chain reaction gave positive results. It was noted that all 58 VT2vp1-producing E. coli strains were from pigs, but not from cows and humans.     

Detection of Various Variant Verotoxin Genes in Escherichia coli by Polymerase Chain Reaction

We constructed common primers for the polymerase chain reaction to detect the genes for various Verotoxins reported, that is, VT1 (or SLT-I), VT2 (or SLT-II), VT2vha, VT2vhb, SLT-IIv (or VT2vp1, VTe) and SLT-IIva (or VT2vp2). A total of 80 Verocytotoxin-producing Escherichia coli strains isolated from humans, domestic animals and meats gave a positive result by PCR with the designed common primers. Digestion by restriction endonucleases BglII and EcoT14I of the amplicon of the VT2vp2 gene gave specific bands of the expected sizes, but not of the amplicons of other VT genes, suggesting a possible method for identification of the VT2vp2 gene. Application of the PCR with the designed primers in diagnostic and epidemiological studies on VTEC infection is also discussed.     

Construction of Mutant Genes for a Non-Toxic Verotoxin 2 Variant (VT2vp1) of Escherichia coli and Characterization of Purified Mutant Toxins

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.     

Functional and structural homology among regulatory cistrons of pili-adhesin determinants in Escherichia coli

Summary Expression of the digalactoside-binding Pap pili involves two trans-acting regulatory genes, papB and papI. Using pap-lac operon fusions and DNA hybridization probes derived from pap DNA we tested whether or not other pili-adhesin determinants from different Escherichia coli strains encode homologs to the pap regulatory genes. Digalactoside-specific clones of serotypes F7₂ and F11 complemented papB and papI mutants of the Pap (serotype F13) clone and DNA hybridization analysis showed that the clones are homologous in the DNA sequences encoding the two regulatory genes. Similar results were obtained with an S-pili determinant which mediates binding to sialic acid-containing receptors and the findings suggest that the regulatory regions may be more conserved than other genes in different pili-adhesin gene clusters. Determinants for type 1-pili (mannose-specific binding) and for pili associated with enterotoxigenic E. coli (K88, K99, CFAI, CFAII) did not appear to contain DNA sequences homologous to papB or papI. E. coli strain J96, which was the origin of the pap DNA, was found to carry two additional copies of papB-papI homologous sequences in the chromosome. In strains expressing more than one kind of pili the trans-active gene products thereby may allow for regulatory interaction between separate pili-adhesin gene systems.     

Genome sequence analyses of two isolates from the recent <Emphasis Type="Italic">Escherichia coli</Emphasis> outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EAHEC)

The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum β-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic E scherichia c oli (EAHEC).     

通辽地区犊牛腹泻大肠杆菌耐药性检测及一株多重耐药菌全基因组测序分析

【背景】大肠杆菌(Escherichia coli)是引起犊牛腹泻的最主要病原菌,其耐药性菌株的不断出现引起广泛关注。【目的】了解内蒙古自治区通辽市犊牛腹泻大肠杆菌耐药性及耐药基因流行情况。【方法】从通辽市多个旗县采集犊牛腹泻样品40份,经细菌分离纯化及16S rRNA基因测序,最终鉴定出20株大肠杆菌。采用药敏试验和PCR方法对分离菌进行耐药性及耐药基因检测分析,并对其中1株多重耐药菌株进行全基因组测序。【结果】20株分离菌均具有多重耐药性,对链霉素、环丙沙星、恩诺沙星和复方新诺明的耐药率达80%以上。所检耐药基因中,aphA1、strB、TEM-1和qnrS检出率达100%。通过对代表性菌株TL-13全基因组测序发现,其基因组大小为4897185bp,GC含量为50.68%,同时携带2个质粒,大小分别为108288bp(pTL13-1)和64018bp(pTL13-2)。质粒中共携带18个可移动耐药基因。【结论】通辽地区犊牛腹泻大肠杆菌多重耐药性普遍存在,4种常见耐药基因普遍流行。     

Cloning and Sequence Analysis of Another Shiga-Like Toxin IIe Variant Gene (slt-IIera) from an Escherichia coli R107 Strain Isolated from Rabbit

An Escherichia coli R107 strain (O26 serotype) producing a Shiga-like toxin IIe variant (SLT-IIera) was isolated from the mesenteric lymph node of a freshly dead rabbit carcass. The entire structural gene for this SLT-IIera was cloned from chromosomal DNA by PCR using primers based on previously published slt-IIe sequences. Nucleotide sequence analysis indicated that the slt-llera gene was very similar to slt-IIe (formerly called slt-IIy) from E. coli strains S1191 and 412; five and one nucleotide changes were detected in A and B subunits, respectively, which resulted in changes in amino acid sequences of the corresponding subunits by three and one residues. Recombinant SLT-IIera and SLT-IIe produced using an E. coli host-vector system showed similar cytotoxicity, suggesting that the variations in the structural gene of SLT-IIera have no significant effect on cytotoxic level.     

Production of enterotoxin,verotoxin, hemolysin and cytotoxic necrotizing factor byEscherichia coli of intestinal and extraintestinal origin

Seventy-sixEscherichia coli strains were examined for heat-labile enterotoxin (LT), verotoxin (VT), hemolysin (HLy) and cytotoxic necrotizing factor (CNF). Thirty-six strains were isolated from patients suffering from diarrhea and forty from different extraintestinal infections. The number of LT-producing strains was low (2.6%) (one of intestinal and one of extraintestinal origin). Verotoxin was produced only by one extraintestinal strain. Four intestinal strains were hemolytic (11.2%) and also positive for CNF. From 24 hemolytic strains of extraintestinal origin (60%), 17 produced also CNF. Most of the hemolytic (30%) as well as CNF-producing strains (22.5%) were isolated from urine. Our results are similar to those of other studies confirming the close association between hemolysin and CNF production as well as a possible role of these toxic factors in pathogenesis of extraintestinal, infections caused byE. coli.     

Adherence Patterns and DNA Probe Types of Escherichia coli Isolated from Diarrheal Patients in China

One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P. R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).     

Identification and Cloning of Peptide Synthetase Genes of Thermostable Bacilli Using the Polymerase Chain Reaction

Fourteen thermophilic and thermostable strains of the genus Bacillus were studied. Total DNA was isolated from these strains and used as a template to identify and clone peptide synthetase genes by means of polymerase chain reaction. Amplified DNA fragments were cloned into a phasmid vector, and nucleotide sequences of cloned fragments were determined. Stringent thermophilic strains were shown to lack genetic systems, which are responsible for the synthesis of secondary metabolites and homologous to the known peptide synthetase genes. On the contrary, thermostable strains had peptide synthetases and produced antimicrobial secondary metabolites. Analysis of nucleotide sequences and deduced amino acid sequences of cloned PCR fragments from B. licheniformis strains VK2, VK21, and VK2101 showed that they are absolutely identical. The cloned DNA fragment was found to be a portion of the open reading frame, which we termed ORF1. Data from analysis of a partial nucleotide sequence of the peptide synthetase gene of strain VK21 indicated that a 9.5-kb region of chromosomal DNA contains sequences of two genes homologous to the B. subtilis peptide synthetase genesdhbB and dhbF. Strains VK2, VK21, and VK2101 were shown to synthesize siderophores. A method for screening bacteria with peptide synthetase genes has been developed.     

The large plasmids found in enterohemorrhagic and enteropathogenic Escherichia coli constitute a related series of transfer-defective Inc F-IIA replicons

Forty-six of 52 (88.5%) enterohemorrhagic Escherichia coli (EHEC) strains screened carried a “common” plasmid of about 90 kb which encoded sequences homologous to the Inc F-IIA replicon. A similarly high incidence of Inc F-IIA plasmid-containing strains was observed in other groups of diarrheagenic E. coli, but not in random environmental coliform isolates. Enteropathogenic E. coli (EPEC) contain plasmids of similar properties and share a 23-kb DNA fragment with plasmids from EHEC. The common region encodes the F-IIA replication region and sequences homologous to the transfer operon of the Inc F-II plasmid R1. Sequence homology varied between plasmids isolated from different EHEC/EPEC strains with >80% showing homology to the regions encoding the rep and par genes. Only 5% of plasmids from EHEC strains had intact sequences homologous to the DNA between these two regions, including the oriT site. Some plasmids with an apparently intact tra operon still failed to plaque F-pilus-specific phages. This is consistent with observations that the large plasmids of EHEC and EPEC are phenotypically nonconjugative. These results suggest that the large plasmids of EHEC/EPEC constitute a family of transfer-deficient Inc F-IIA plasmids with varying degrees of deletion in tra function. The evolutionary ramifications of this finding are considered.     

Typing of Verotoxins by DNA Colony Hybridization with Poly- and Oligonucleotide Probes,a Bead-Enzyme-Linked Immunosorbent Assay,and Polymerase Chain Reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.     

Isolation of rfb Gene Clusters Directing the Synthesis of O Polysaccharides Consisting of Mannose Homopolymers and Serological Analysis of Lipopolysaccharides

Four serotypes of two genera, Escherichia coli O8 and O9 and Klebsiella O3 and O5, produce the O polysaccharides consisting of mannose homopolymers. Previously we reported the isolation and expression of E. coli O9 rfb in E. coli K-12 strains (Kido et al, J. Bacteriol., 171: 3629–3633, 1989). In this study, R' plasmids carrying his-rfb region of the other three strains were isolated and expressed in E. coli K-12 strain. Serological study of lipopolysaccharides (LPS) synthesized in E. coli K-12 strain was carried out. His-linked rfb genes from E. coli O9 and Klebsiella O3 directed the synthesis of O polysaccharides with the same antigenicity as those of the parental strains in E. coli K-12 strain. On the other hand, rfb genes from E. coli O8 and Klebsiella O5 directed the synthesis of O polysaccharides which were antigenically not identical but partially common to those of the parental strains. A rough strain derived from E. coli O8 synthesized LPS which showed the identical antigenicity as the wild strain when the his-rfb region of E. coli O8 was introduced. The results suggest that some genes located distantly from his are additionally required to complete the synthesis of O polysaccharides of E. coli O8 and Klebsiella O5.     

Isolation and characterization ofEscherichia coli O157:H7 strains in China

Five strains ofEscherichia coli O157:H7 were isolated from 486 stool specimens collected in 1986, 1987, and 1988 from patients with diarrhea in Xuzhou City, Jiangsu Province, China; 21 of the specimens were from patients with bloody diarrhea. The biochemical reactions of all five strains were almost identical with those of the well-knownE. coli O157:H7 strain 933. All of the strains were found to carry a 60 Md plasmid and two small plasmids. The plasmid DNA Hind 111 restriction patterns were identical. The strains were lysed byE. coli typing phage E1, E2, and E3, but not by E4 or E5. Data suggested that it might belong to a single phage or plasmid group. All strains produced vero toxin and caused diarrhea and death in infant rabbits and mice.     

Analysis of the flanking regions from different haemolysin determinants of Escherichia coli

Summary The haemolysin (hly) determinant of the plasmid pHly152 contains an IS2 element at 469 bp upstream of the hlyC gene. The sequence at the other (right-hand) end (RS) also shows multiple hybridization with the plasmid pHly152 and the chromosome of some Escherichia coli strains but the nucleotide sequence of this region does not reveal the typical properties of an IS element. Similar arrangements in the regions flanking the hly determinant are also found on various Hly plasmids from uropathogenic E. coli strains. Chromosomal hly determinants lack both flanking sequences (IS2 and RS) in the immediate vicinity pf the hly genes. The sequences immediately upstream of the hlyC gene have been determined from several chromosomal hly determinants and compared with the corresponding sequence of the hly determinant of the plasmid pHly152. We show that these sequences, which contain one promoter (left promoter, phly _L) in all hly determinants tested, vary considerably although common sequence elements can still be identified. In contrast, only relatively few nucleotide exchanges have been detected in the adjacent structural hlyC genes. The A+T content of the 200 bp sequence upstream of hlyC is very high (72 mol% A+T) but even the structural hly genes show a considerably higher A+T content (about 60 mol%) than the E. coli chromosome on average (50 mol% A+T) suggesting that the hly determinant may not have originated in E. coli.Dedicated to Prof. F. Lingens on the occasion of his 60th anniversary     

Evaluation of Enzyme-Labeled Oligonucleotide Probes to Identify Enterohaemorrhagic Escherichia coli

Alkaline phosphatase-conjugated oligonucleotide probes were developed to detect the gene coding for Vero toxin 1 (VT1) and Vero toxin 2 (VT2). Using these probes, 3 hr was enough to detect VT genes when suspicious colonies of enterohaemorrhagic Escherichia coli (EHEC) were obtained on an agar plate. The results of a hybridization test with 144 isolates of EHEC O157 and one isolate of Shigella dysenteriae Type 1 agreed exactly with the immunological detection, reversed passive latex agglutination (RPLA) test, of VTs in their culture supernatants. The sensitivity levels of these probes for the detection of VT genes were 100%. The specificity of these probes were also tested with a total of 1,002 strains of Escherichia coli other than EHEC and 8 strains of Shigella sp. other than Shigella dysenteriae Type 1; the results showed 100% specificity.     

Coat Protein Gene of Cymbidium mosaic virus from Vanilla fragrans in Reunion Island

Nucleotide and amino acid sequences of the coat protein (CP) of 12 isolates of Cymbidium mosaic virus from Vanilla fragrans in Reunion Island (CyMV‐R) were compared with each other and with those of previously described Asian strains. Alignment revealed that CyMV‐R isolates were highly homologous, suggesting that one strain is prevalent in Reunion. This strain also showed high homology with the Korean CyMV‐K2 and Singapore CyMV‐S2 strains, but nucleotide additions resulted in the carboxy‐terminal ends of the CP sequences differing from those of the Korean CyMV‐K1 and Singapore CyMV‐K1 strains.     

Identification and Characterization of Heat-Stable Enterotoxin II-Producing Escherichia coli from Patients with Diarrhea

Stock strains of Eschericia coli isolated from patients with traveller's diarrhea were examined for production of heat-stable enterotoxin II (STII). Of 400 strains examined, 3 were found to produce STII. The nucleotide sequence of the STII gene of these human strains was shown to be identical to that of porcine strains. Cultured cells of these strains induced fluid accumulation in ligated mouse intestinal loops and the activity was neutralized by anti-STII antiserum. These results suggest that STII-produciing enterotoxigenic E. coli can cause human diarrhea.     

Evolutionary implications of potential frameshift hot-spots, methylation sites, and codon usage differences between genes and between organisms in Escherichia coli and Salmonella typhimurium

The published nucleotide sequences of the E. coli and S. typhimurium trp A and trp B genes show a high degree of similarity between homologous genes of the two organisms, and an even greater degree of similarity between the amino acid sequences of the gene products. In spite of this, analysis of the nucleotide sequences reveals that there are marked differences between E. coli and S. typhimurium genes with respect to potential frameshift mutation hot-spots and dam and mec, mutationally important, methylation sites. Such existing differences may well lead to divergent evolution of these two, presently closely related, bacteria. Codon usage patterns in the trp A and trp B genes of E. coli and S. typhimurium, and the lac I gene of E. coli, have been re-analysed in terms of AT-rich, GC-rich, neutral, or unique codons and marked preferences found. In some cases particular amino acids are most often specified by AT-rich, in others by the GC-rich, alternative codons. In still other cases the codon preference depends on the gene studied. These patterns can be interpreted in terms of enteric bacterial evolution, via hybridizations, from ancestral bacteria with AT- or GC-rich DNA.     

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.