Colony Blot Hybridization Research Articles

Rapid identification of Escherichia coli microcin J25 producing strains using polymerase chain reaction and colony blot hybridization

To screen, isolate, and characterize bacterial populations producing microcin J25, we report here two rapid, reliable, and sensitive methods, using polymerase chain reaction and colony blot hybridization with a digoxigenin-labelled probe. A sample of 26 Escherichia coli strains isolated from poultry intestinal contents was evaluated to detect the sequence of mcjA, the gene encoding the MccJ25 precursor. The two molecular techniques were compared with the commonly used cross-immunity tests. They generate accurate data with no obvious cross-reactions with other microcins. The results display that the producers of MccJ25 were widely distributed in the poultry intestinal habitat. The applications of these molecular methods will be useful in future studies of microcinogenic populations, and thus contribute to understand the relationships within the complex intestinal microbial ecosystem.

Association between intimin (eae) and EspB gene subtypes in attaching and effacing Escherichia coli strains isolated from diarrhoeic lambs and goat kids.

Attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic lambs and goat kids were characterized for intimin (eae) and EspB (espB) gene subtypes by PCR and sequencing, and for genetic relatedness by PFGE. Fifty (23 ovine and 27 caprine) AEEC strains of 398 (246 ovine and 152 caprine) analysed were detected by colony blot hybridization. These strains were epidemiologically unrelated since they were isolated from different outbreaks of neonatal diarrhoea over a long period. Ovine AEEC strains belonged to serogroups O2, O4, O26, O80, O91 or were untypable, and caprine strains belonged to serogroups O3, O153 and O163. Two intimin subtypes were detected among the ovine and caprine strains studied. Most of the strains (43/50) had the beta type intimin gene, but seven ovine strains possessed a variant gamma type intimin gene (gamma(V)). Analysis of deduced amino acid sequences of the eae gene revealed that the sequences of beta intimin of ovine and caprine strains were virtually identical to those of beta intimin of rabbit EPEC, human EPEC clone 2 and swine AEEC, whereas the gamma(V) intimin present in seven ovine strains had 75-76% identity with gamma intimin of human EHEC clone 1 strains, and 96% of identity with intimin of the human EHEC strain 95NR1 of serotype O111:H-. A PCR test was developed to identify the three different espB gene subtypes, espB of human EPEC clone 1 (espBalpha), espB of human EHEC clone 1 (espBgamma) and espB of rabbit EPEC and human EPEC clone 2 (espBbeta). There was close correlation between the intimin beta type and the espBbeta gene subtype in the ovine and caprine AEEC strains. The seven ovine strains possessing the gamma(V) intimin gene possessed the espBalpha gene subtype. None of the strains studied possessed the espBgamma gene found in human O157:H7 EHEC strains. PFGE analysis of genomic DNA of selected strains showed a great diversity among strains. Cluster analysis of PFGE patterns showed greater divergence between strains with the gamma(V) intimin gene than between strains with the beta intimin gene. This study showed that most of the AEEC strains isolated from diarrhoeic lambs and goat kids possessed beta intimin and espB genes identical to those of rabbit EPEC, and they may be associated with enteric disease in small ruminants.

Crenarchaeota and Euryarchaeota in temperate estuarine sediments.

Application of molecular techniques to ecological studies has unveiled a wide diversity of micro-organisms in natural communities, previously unknown to microbial ecologists. New lineages of Archaea were retrieved from several non-extreme environments, showing that these micro-organisms are present in a large variety of ecosystems. The aim was therefore to assess the presence and diversity of Archaea in the sediments of the river Douro estuary (Portugal), relating the results obtained to ecological data. Total DNA was extracted from sediment samples obtained from an estuary deprived of vegetation, amplified by PCR and the resulting DNA fragments cloned. The archaeal origin of the cloned inserts was checked by Southern blot, dot blot or colony blot hybridization. Recombinant plasmids were further analysed by restriction with AvaII and selected for sequencing. Phylogenetic analyses of 14 sequences revealed the presence of members of the domain Archaea. Most of the sequences could be assigned to the kingdom Crenarchaeota. Most of these sequences were closely related to those obtained from non-extreme Crenarchaeota members previously retrieved from diverse ecosystems, such as freshwater and marine environments. The presence of archaeal 16S rDNA sequences in temperate estuarine sediments emerges as a valuable contribution to the understanding of the complexity of the ecosystem.

Prevalence of enteropathogens in suckling and weaned piglets with diarrhoea in southern Germany.

Faecal samples from suckling (n = 205) and weaned piglets (n = 82) with diarrhoea from 24 farms in Southern Germany were examined for shedding of important metazoic parasitic, viral and bacterial pathogens using culture, microscopic and electronmicroscopic methods. Escherichia coli isolates were tested further for the enterotoxin genes est-Ia and elt-I by colony blot hybridization. Isospora suis was diagnosed in 26.9% and Cryptosporidium parvum in 1.4% of the piglets investigated. The proportion of coronavirus-positive animals was 13.4% and 4% were positive for rotavirus. It was found that 17.6% of the animals were infected with enterotoxigenic E. coli (ETEC; 10.1% ETEC-ST-Ia and 8.6% ETEC-LT-I, respectively). The occurrence of the pathogens was significantly associated with the age of the animals examined (P < 0.001). Isospora suis was predominantly isolated from suckling piglets (in the second and third week of life), while in weaned piglets (fourth week of life) rotavirus and ETEC were most prevalent. On 22 of the 24 piglet production farms examined at least one of the investigated pathogens was detected. Coronavirus was diagnosed in 66.7%, I. suis in 62.5%, rotavirus in 20.8% and C. parvum in 8.3% of the farms. These results underline the fact that despite the hygienic, technical and immune preventive efforts during the last years, enteropathogens are still common in German piglet production units.

Prevalence of Enteropathogens in Suckling and Weaned Piglets with Diarrhoea in Southern Germany

Faecal samples from suckling (n=205) and weaned piglets (n=82) with diarrhoea from 24 farms in Southern Germany were examined for shedding of important metazoic parasitic, viral and bacterial pathogens using culture, microscopic and electronmicroscopic methods. Escherichia coli isolates were tested further for the enterotoxin genes est‐Ia and elt‐I by colony blot hybridization. Isospora suis was diagnosed in 26.9 % and Cryptosporidium parvum in 1.4 % of the piglets investigated. The proportion of coronavirus‐positive animals was 13.4 % and 4 % were positive for rotavirus. It was found that 17.6 % of the animals were infected with enterotoxigenic E. coli (ETEC; 10.1 % ETEC‐ST‐Ia and 8.6 % ETEC‐LT‐I, respectively). The occurrence of the pathogens was significantly associated with the age of the animals examined (P < 0.001). Isospora suis was predominantly isolated from suckling piglets (in the second and third week of life), while in weaned piglets (fourth week of life) rotavirus and ETEC were most prevalent. On 22 of the 24 piglet production farms examined at least one of the investigated pathogens was detected. Coronavirus was diagnosed in 66.7 %, I. suis in 62.5 %, rotavirus in 20.8 % and C. parvum in 8.3 % of the farms. These results underline the fact that despite the hygienic, technical and immune preventive efforts during the last years, enteropathogens are still common in German piglet production units.

Characterization of Shiga toxin producing E. coli and O157 serotype E. coli isolated in France from healthy domestic cattle

A study was carried out in France in collaboration with the meat industry to investigate the occurrence and characteristics of Shiga toxin-producing E. coli (STEC) and O157 E. coli in a population of healthy bovines representative of French livestock. A total of 851 animals belonging to three bovine classes (106 young bulls, 374 dairy cows and 371 meat cows) were included in the study. Samples of feces and of the corresponding carcasses were collected from March 97 to August 97 in seven abattoirs spread throughout the national territory. STEC cultures from the 1702 samples were screened using PCR for the presence of stx genes. Positive samples were further subjected to colony blot hybridization and to O157-specific immunomagnetic separation. Probe-positive colonies and O157 colonies were then analyzed for the presence of virulence genes and phenotypic characters (serotype, Stx production). In 154 (18.1%) feces and 91 (10.7%) carcass samples stx genes were detected. Two hundred and twenty-two STEC colonies were isolated from 67 (7.9%) feces and 16 (1.9%) carcass samples, with 183 STEC isolated from feces and 39 from carcasses. Only eight O157 isolates were collected from feces samples. None of these O157 E. coli isolates presented stx genes and thus could not be considered as pathogenic regarding hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). In 3.2% of STEC isolated from feces and in 10.2% of STEC from carcasses eae genes were detected. In 17% of STEC from feces and in 30.7% from carcasses ehx genes were detected. Using these data, the 222 STEC colonies could be classified in 11 different ‘virulence patterns’ (presence/absence of stx1, stx2, eae and ehx genes), showing that more than 77% of isolates presented only one virulence factor. Only three STEC on 222 colonies (1.3%) presented the three virulence factors stx, eae and ehx in association, none of them reacting with antisera specific for enterohemorrhagic E. coli. (EHEC). These data, together with the fact that only five isolates on the 222 (2.2%) reacted with such antisera (three O111 and two O26 isolates) demonstrated that the natural bacterial populations isolated during this study were clearly distinct from EHEC.

Novel tellurite-amended media and specific chromosomal and Ti plasmid probes for direct analysis of soil populations of Agrobacterium biovars 1 and 2.

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K(2)TeO(3) was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K(2)TeO(3) concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K(2)TeO(3)-amended medium. The bona fide agrobacterium colonies growing on media amended with K(2)TeO(3) were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10(3) to 10(4) agrobacteria. g of dry soil(-1) in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.

Rapid identification of &lt;i&gt;Escherichia coli&lt;/i&gt; microcin J25 producing strains using polymerase chain reaction and colony blot hybridization

To screen, isolate, and characterize bacterial populations producing microcin J25, we report here two rapid, reliable, and sensitive methods, using polymerase chain reaction and colony blot hybridization with a digoxigenin-labelled probe. A sample of 26 Escherichia coli strains isolated from poultry intestinal contents was evaluated to detect the sequence of mcjA, the gene encoding the MccJ25 precursor. The two molecular techniques were compared with the commonly used cross-immunity tests. They generate accurate data with no obvious cross-reactions with other microcins. The results display that the producers of MccJ25 were widely distributed in the poultry intestinal habitat. The applications of these molecular methods will be useful in future studies of microcinogenic populations, and thus contribute to understand the relationships within the complex intestinal microbial ecosystem.

Use of Genetic and Immunological Probes for Pediocin PA-1 Gene Detection and Quantification of Bacteriocin Production in Pediococcus acidilactici Strains of Meat Origin

PCR amplification analysis with pediocin PA-1 (pedA1) specific primers, a gene probe covering the pedA1 structural gene, and an immunological probe based on anti-peptide antibodies with predetermined specificity for pedA1, have been evaluated for pedA1 structural gene detection and quantification of bacteriocin production in Pediococcus acidilactici strains of meat origin. PCR products were obtained from colonies and purified plasmid DNA. The gene probe was used in dot-blot and colony-blot hybridization analysis. The production of pedA1 was evaluated by a non-competitive indirect ELISA (NCI-ELISA), a competive indirect ELISA (CI-ELISA), a competitive direct ELISA (CD-ELISA), and by immunoblotting. The approaches taken in the generation and performance of the reported probes could be useful for the rapid detection and identification of other bacteriocin-producing lactic acid bacteria (LAB) from many substrates and reservoirs and for quantification of bacteriocin production in food and environmental isolates and heterologous hosts.

Characterization of Escherichia coli strains from cases of childhood diarrhea in provincial southwestern Nigeria.

In a study carried out in small-town and rural primary health care centers in southwestern Nigeria, 330 Escherichia coli strains isolated from 187 children with diarrhea and 144 apparently healthy controls were examined for virulence traits. Based on the results of colony blot hybridization, strains were categorized as enteropathogenic E. coli (1.8%), enterotoxigenic E. coli (2.4%), enteroinvasive E. coli (1.2%), enterohemorrhagic E. coli (0.6%), enteroaggregative E. coli (10.3%), diffusely adherent E. coli (7.9%), cell-detaching E. coli (6.9%), and cytolethal distending toxin-producing E. coli (0.9%). E. coli strains that hybridized with a Shiga toxin gene probe but lacked other characteristics usually present in enterohemorrhagic E. coli constituted 8.4% of the isolates. Ninety-seven E. coli isolates adhered to HEp-2 cells in an aggregative fashion but did not hybridize with any of the probes employed in the study. Overall the pathotypes, apart from cytolethal distending toxin-producing E. coli, were recovered both from children with diarrhea and from children without diarrhea, though to a lower extent from the healthy children. All diarrheagenic E. coli strains were associated with diarrhea (P < 0.02). Heat-stable-enterotoxin-producing enterotoxigenic E. coli showed significant association with diarrhea (P < 0.02), as did strains that demonstrated aggregative adherence to HEp-2 cells (P < 0.04), but not those that hybridized with the CVD432 enteroaggregative probe.

PER-1 extended-spectrum beta-lactamase production in an Alcaligenes faecalis clinical isolate resistant to expanded-spectrum cephalosporins and monobactams from a hospital in Northern Italy.

An Alicaligenes faecalis (FL-424/98) resistant to expanded-spectrum cephalosporins and aztreonam was isolated from the urine of an inpatient at the Intensive Care Unit of the Varese Hospital (Northern Italy) after antimicrobial chemotherapy with cefazolin, vancomycin, and amikacin. Clavulanic acid restored the activity of expanded-spectrum cephalosporins, suggesting the production of an extended-spectrum beta-lactamase (ESbetaL). A crude extract of FL-424/98 showed the presence of two beta-lactamase activities focusing at pH 5.3 and 7.6, respectively. The ESbetaL activity, purified by means of three chromatographic steps, was found to correspond to the pI 5.3 enzyme. Determination of kinetic parameters confirmed that the enzyme efficiently hydrolyzed expanded-spectrum cephalosporins and aztreonam. A colony-blot hybridization revealed the presence of blaPER-related sequences in FL-424/98, and sequencing confirmed the identity of this determinant with blaPER-1, previously detected in Pseudomonas aeruginosa, Acinetobacter, and Salmonella clinical isolates from Turkey. Finding of blaPER-1 in a species that can be part of the resident human microbiota raises the possibility that it could be an efficient shuttle for spreading of this resistance gene among other opportunistic pathogens that are normally members of the resident microbiota. Kinetic parameters determined for the PER-1 enzyme with some cephalosporin substrates were somewhat different from those previously reported.

Identification of a third metalloprotease toxin gene in extraintestinal isolates of Bacteroides fragilis.

To further understand the epidemiology of enterotoxigenic Bacteroides fragilis (ETBF), 89 extraintestinal B. fragilis strains from Seoul, Korea, were examined for secretion of B. fragilis toxin (BFT) by the HT29/C1 biologic assay and for the B. fragilis toxin gene (bft) by colony blot hybridization and PCR. Complete agreement between the three techniques was found. Overall, 34 B. fragilis strains (38%) were identified as ETBF. Eleven of the 34 ETBF strains (32%) expressed a new isoform of BFT (Korea-BFT). This new isoform is more related to BFT-2 than to BFT-1. Like BFT-1 and BFT-2, Korea-BFT cleaves E-cadherin, the zonula adherens protein.

Generation of Vector-Insensitive Dig-Labeled Probes from Large Cosmid Library Inserts Using PCR

We have devised a general method for producing vector-insensitive probes from clones in which insert DNA (ca. 40 kb) could not be amplified in one piece nor be excised from the vector sequence. The method involves PCR and vector-specific primers in combination with restriction digestion and ligation. It yields specific PCR products that could subsequently be labeled using DIG-11-dUTP in a single-cycle PCR. In colony blot hybridization, the probes were specific for the insert DNA from which the probe was derived and, importantly, did not detect vector sequences.

Cloning and nucleotide sequence of retroposons specific to hominoid primates derived from an endogenous retrovirus (HERV-K).

595 ENDOGENOUS RETROVIRUSES comprise approximately 1% of the human genome.1 Although most human endogenous retroviruses (HERVs) are defective, one class, HERV-K (homologous to mouse mammary tumor virus [MMTV]), encodes viral proteins and particles3 and is highly expressed in germ cell tumors. Retroposons (a retroelementso and a retrovirus-like elements,o which multiply in the genome by retrotransposition) are distinguished from retroviruses by the absence of one or both long terminal repeats (LTRs). These repetitive elements are classified into two groupsÐshort (up to 600 bp) interspersed nuclear elements (SINEs) and long (about 6 kg) interspersed nuclear elements (LINEs)Ð that include a number of families that have diverged in the course of mammalian evolution.4 One class of SINEs, the Alu sequences, is specific to primates. Another class, termed SINE-R (for retrovirus), which is derived from the HERV-K sequence, has apparently undergone modification in the hominoid lineage.5 Such elements are a potential agency of genome change.1,6,7 The human-specific retroposon SINE-R.C2, a member of the SINE-R retroposon family (present at 4000 to 5000 copies per haploid human genome), was discovered in an investigation of the variable number of tandem repeats (VNTR) locus within intron 3 of the gene for C2, the second component of complement, located in class III of the major histocompatibility complex on the short arm of human chromosome 6.8,9 Other members of the SINE-R family in the human genome, SINER11, -14, and -19, were isolated by colony blot hybridization with an HERV-K10 probe. By BLAST search, schizophrenic brain S11 cDNA (GenBank, accession No. AA772777) was also found to have high homology with the 39 LTR of HERV-K10. Therefore, specific polymerase chain reaction (PCR) primers, JS902 and JS903, were designed from S11 cDNA. Using these primers, the sequences HS307 (GenBank, accession No. AB016781) and HS408 (GenBank, accession No. AB016782), which have a high degree of homology to the SINE-R.C2, -R11, -R14, and -R19 retroposons, were identified in an investigation of the Xq21.3 region that has been tentatively linked to schizophrenia and schizo-affective disorder by Laval et al.10 and Kim et al. Each of these retroposons derives from the 3 9 long terminal repeat and the small upstream flanking regions of the human endogenous retrovirus HERV-K10,5 the entire sequence of which has been reported as a candidate autoimmune gene in type I diabetes.11 The HERV-K10 (present at 50 copies per haploid human genome) has been found to be present in all primates except for New World monkeys, whereas SINE-R.C2 is present only in Homo sapiens. Other members of the SINER family have been found to be present in the human, chimpanzee, and gorilla genomes by Southern blot analysis.12 To obtain sequence information relating to SINE-R-type retroposons in hominoid primates, genomic DNAs from the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and agile gibbon (Hylobates agilis) were amplified by PCR using primers JS902 (5 9 -GAGA ATGGGCCATGATGAC-3 9 , bases 4±22) and JS903 (59 GATCATTCTTGGATGTTTCT-3 9 , bases 466±485) derived from schizophrenic brain S11 cDNA (GenBank, accession No. AA772777). The PCR conditions followed were those of Kim et al. with an annealing temperature of 56°C. PCR products were separated on a 1.8% agarose gel, purified with the QIAEX II gel extraction kit (Qiagen, Chatsworth, CA) and cloned into the T-khs307 vector.14 Plasmid DNA was isolated by the alkali lysis method, using the High Pure plasmid isolation kit (Boehringer Mannheim, Indianapolis, IN). Individual plasmid DNAs were screened for inserts by PCR and positive samples were subjected to sequence analyses on both strands with T7 and M13 reverse primers using an automated DNA sequencer (model 373A; Applied Biosystems, Foster City, CA) and the DyeDeoxy terminator kit. Sequence analyses were done with the aid of GAP and PILEUP from the GCG program. The neighbor-joining phylogenetic analysis was performed with the CLUSTAL W program.15 We searched for sequence homology using the BLAST network server.16 Using genomic DNA from hominoid primates, SINE-R-type retroposons were amplified and nucleotide sequences deter-

Clonal diversity of Chilean isolates of enterohemorrhagic Escherichia coli from patients with hemolytic-uremic syndrome, asymptomatic subjects, animal reservoirs, and food products.

To determine clonal relationship among Chilean enterohemorrhagic Escherichia coli (EHEC) strains from different sources (clinical infections, animal reservoirs, and food), 54 EHEC isolates (44 of E. coli O157, 5 of E. coli O111, and 5 of E. coli O26) were characterized for virulence genes by colony blot hybridization and by pulsed-field gel electrophoresis (PFGE). By colony blotting, 12 different genotypes were identified among the 44 E. coli O157 isolates analyzed, of which the genetic profile stx1+ stx2+ hly+ eae+ was the most prevalent. All human O157 strains that were associated with sporadic cases of hemolytic-uremic syndrome (HUS) carried both the stx1 and stx2 toxin-encoding genes and were eaeA positive. Only 9 of 13 isolates from human controls were stx1+ stx2+, and 8 carried the eaeA gene. Comparison of profiles obtained by PFGE of XbaI-digested genomic DNA showed a great diversity among the E. coli O157 isolates, with 37 different profiles among 39 isolates analyzed. Cluster analysis of PFGE profiles showed a wide distribution of clinical isolates obtained from HUS cases and asymptomatic individuals and a clonal relationship among O157 isolates obtained from HUS cases and pigs. Analysis of virulence genes showed that a correlation exists among strains with the genotype stx1+ stx2+ eae+ and pathogenic potential. A larger difference in the PFGE restriction patterns was observed among the EHEC strains of serogroups O26 and O111. These results indicate that several different EHEC clones circulate in Chile and suggest that pigs are an important animal reservoir for human infections by EHEC. Guidelines have been proposed for better practices in the slaughter of animals in Chile.

Isolation and characterization of enteroaggregative Escherichia coli (EAggEC) by genotypic and phenotypic markers, isolated from diarrheal children in Congo

OBJECTIVE: To determine the prevalence of enteroaggregative Escherichia coli (EAggEC) in African diarrheal children in Lwiro, Congo, to characterize EAggEC isolates by possible genotypic and phenotypic markers, and to evaluate the EAggEC probe pCVD432 in identifying EAggEC. METHODS: The Hep-2 cell adhesion assay and colony-blot hybridization assays were carried out for the identification of EAggEC. O:H serotyping, biotyping, antibiogram and plasmid-profile analysis were done. To detect the E. coli LT and ST, ELISA tests were used and, for VT, a vero cell assay was used. RESULTS: EAggEC strains were isolated from 56 out of 115 diarrheal children (48.7%): the organism was present alone and presumed to cause diarrhea in 22 (19.1%) cases. The rest of the cases were associated with two or more diarrheagenic E. coli strains. EAggEC strains were isolated from 25% of total diarrheal children (first day of isolation) and 8.86% of age-matched healthy individuals (p<0.03). This isolation rate was significantly higher than the one found for other diarrheagenic E. coli strains. In parallel, we evaluated the sensitivity and specificity of EAggEC probe pCVD432, and found that it had 56% sensitivity with 100% specificity compared with the Hep-2 cell test. EAggEC isolates were characterized by serotyping, biotyping, antibiotic resistance pattern, plasmid profiling and toxin production analysis. They did not produce any one of these classical toxins and nor did they relate to any particular serotypes. Plasmid analysis of the 79 EAggEC isolates (n=315) showed seven different profiles. Ten resistance patterns were identified and 34 strains were sensitive to all drugs. There was no association between plasmid profiles and antibiotic resistance patterns. All 16 classical E. coli biotypes were found in this small EAggEC population. CONCLUSIONS: EAggEC has been emerging as a cause of childhood diarrhea in African children in Congo. From the accumulated data it was found that there is a great heterogeneity in EAggEC populations.

Localization of nod, nif, and acidic exopolysaccharide determinants on a large plasmid in slow-growing cowpea Rhizobium sp. S2.

Slow-growing cowpea Rhizobium sp. S2, a pigeon pea isolate, showed excessive synthesis of exopolysaccharide (EPS) and nod factor(s) only when grown in the presence of corresponding host root exudate (RE) or naringenin (a flavonoid present in RE of pigeon pea). muc- strain, a plasmid-cured derivative of the parent strain, showed negligible EPS production and failed to synthesize the nod factor(s) under similar growth conditions. The nod factor(s) was extracted and partially purified from the total EPS of the culture supernatant of S2 grown in the presence of either RE or naringenin by the butanol-soluble, ethyl acetate-insoluble method. The parent strain also showed ex-planta nitrogenase activity, whereas in muc- it was not detectable. In contrast to observations of the location of nod, nif- and EPS-synthesizing genes on the chromosome in commonly studied cowpea Rhizobia and Bradyrhizobium sp. colony blot and dot blot hybridization studies revealed the presence of nod (common nod genes), nif (nif KDH); and EPS (pss) determinants to be on the large plasmid in the parent strain and therefore absent in muc-, which is a plasmid-cured derivative.

Shiga Toxin-Producing Escherichia coli Infections in Germany

A prospective study was carried out in collaboration with two children's hospitals in Würzburg, Germany to assess the incidence and clinical manifestations of infections due to Shiga toxin-producing Escherichia coli (STEC) in children. Between 1991 and 1995, stool samples from 2788 children with enteritis were investigated for the occurrence of STEC. STEC cultures from stools were screened using PCR with primers complementary to Shiga toxin 1(Stx1) and Shiga toxin 2 (Stx2) genes. PCR-positive samples were further subjected to colony blot hybridization and probe positive colonies were serotyped and analyzed for the presence of virulence genes. There was an increase in the incidence of STEC infections from 0.4% in 1991 to 2.8% in 1994. In 1995 the number of infections remained nearly unchanged (2.5%). Infection with STEC was associated with painful nonbloody diarrhea in most patients. Among the 35 patients in this study with stools containing STEC, only 9 (25.7%) had O157 colonies of which 3 (8.6%) were O157:H7 and 6 (17.1%) were sorbitol-fermenting O157:H−. In an additional study in 1994/l995, STEC etiology in 88 patients with HUS from Germany was confirmed in our laboratories by culture of STEC from stools, and in 20 additional HUS cases by serological analysis. Of the strains from stools of HUS patients, 78% belonged to serogroup O157. The most frequently isolated non-O157 serogroups were O26 and O111. These results demonstrate that when analyzing stools of patients with bloody diarrhea, HUS, or painful nonbloody diarrhea, the occurrence of non-O157:H7 strains should be considered when classical microbiological analysis fails to yield a standard enteric pathogen, such as Campylobacter. E. coli O157:H7, Salmonella. Shigella, or Yersinia.

An ileX tRNA gene is located close to the Shiga toxin II operon in enterohemorrhagic Escherichia coli O157 and non-O157 strains

A cosmid library of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL 933 was constructed and clones carrying the stx 2 gene were identified by colony blot hybridization with a stx 2 B specific probe. Nucleotide sequencing upstream of the stx 2 A gene revealed high sequence identities of 89.5% to the ileX tRNA gene found in E. coli. The ileX gene was located 260 bp from the translational start codon of stx 2 A. PCR analysis with primers specific for this analyzed region showed that in 11 Stx2-producing EHEC strains from patients with hemolytic uremic syndrome, all PCR-positive strains carried the ileX tRNA gene. However, PCR analysis of the respective region in 11 Stx1-producing EHEC strains detected no ileX genes. Although the role of ileX in Stx2-producing EHEC strains is not clear, its function in regard to the use of rare codons and as an integration site is discussed.

Detection of the fimbrial gene F18 (F107) from swine enteritis Escherichia coli.

Virulent Escherichia coli (E. coli) strains can be significant etiologic agents in neonatal and post- weaning diarrhea of pigs. The virulence of certain isolates is associated with their ability to adhere to and/or colonize the intestine and also through the production of enterotoxins. One method of virulence determination is via hybridization to adhesin and toxin DNA probes. The University of Missouri, Veterinary Medical Diagnostic Laboratory has been using the colony blot hybridization method along with conventional serotyping by agglutination for the past six years as an aide in the determination of colibacillosis. Our focus here was porcine diarrhea and the role of hemolytic E. coli. Hemolytic E. coli from neonatal pigs may be K88:LT:STb positive. Post- weaning age pigs yielding hemolytic, K88 negative E. coli have stimulated the search for additional adhesins. Therefore the finding of the F18 (F107) family of adhesins in E. coli recovered from pigs with edema disease or post-weaning diarrhea has been of interest and we have incorporated the F18 probe into our panel of ten E. coli virulence factors.