Molecular characterization of a Shiga toxigenic Escherichia coli O113:H21 strain lacking eae responsible for a cluster of cases of hemolytic-uremic syndrome.

Shiga toxigenic Escherichia coli (STEC) strains are a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, certain strains appear to have greater virulence for humans. STEC strains carrying eae and belonging to serogroup O157 or O111 have been responsible for the vast majority of outbreaks of STEC disease reported to date. Here we describe a STEC O113:H21 strain lacking eae that was responsible for a cluster of three cases of hemolytic-uremic syndrome. This strain produces a single Stx2-related toxin and adheres efficiently to Henle 407 cells.

PCR for the detection of Campylobacter spp. in clinical specimens.

The suitability of PCR (based on the amplification of the 16S rRNA gene) for use as a diagnostic test for the detection of Campylobacter spp. in human faecal specimens was assessed. A total of 493 faecal specimens from patients with symptoms of enteritis were tested for the presence of campylobacters using PCR. Results were compared with those obtained from the analyses of the same specimens by culture techniques, using chi 2 square with Fisher's exact test. PCR was found to detect significantly more positive specimens than culture (chi 2 = 200.086; P < 0.0001). The sensitivity and specificity of PCR when compared with the culture technique were found to be 91 and 97%, respectively. It is proposed that the PCR is a reliable and sensitive method which may be used as a routine diagnostic technique for the detection of campylobacters in clinical specimens.

Sequence-based classification scheme for the genus Legionella targeting the mip gene.

The identification and speciation of strains of Legionella is often difficult, and even the more successful chromatographic classification techniques have struggled to discriminate newly described species. A sequence-based genotypic classification scheme is reported, targeting approximately 700 nucleotide bases of the mip gene and utilizing gene amplification and direct amplicon sequencing. With the exception of Legionella geestiana, for which an amplicon was not produced, the scheme clearly and unambiguously discriminated among the remaining 39 Legionella species and correctly grouped 26 additional serogroup and reference strains within those species. Additionally, the genotypic classification of approximately 150 wild strains from several continents was consistent with their phenotypic classification, with the exception of a few strains where serological cross-reactivity was complex, potentially confusing the latter classification. Strains thought to represent currently uncharacterized species were also found to be genotypically unique. The scheme is technically simple for a laboratory with even basic molecular capabilities and equipment, if access to a sequencing laboratory is available.

Interspecies sequence differences in the Mip protein from the genus Legionella: implications for function and evolutionary relatedness.

The nucleotide sequence of the mip genes and their inferred amino acid sequences were determined from 35 Legionella species and compared with the published sequences for L. pneumophila, L. micdadei and L. longbeachae. The sequences were 69-97% conserved at the nucleotide level and 82-99% at the amino acid level, with total conservation of amino acids determined to be associated with sites known to be involved in peptidyl prolyl cis-trans isomerase activity. No apparent difference could be determined in the arrangement of amino acids that would predict a functional difference in Mip from species associated with disease and Mip from species isolated only from the environment. Additionally, a phylogenetic comparison of the sequences with published 16S RNA sequences, using both genetic distance and maximum parsimony methods, was performed. Few relationships were apparent that were well supported by both data sets, the most robust being a clade comprising ([(cincinnatiensis, longbeachae, sainthelensi, santicrucis) gratiana] (moravica, quateirensis, shakespearei, worsleiensis) anisa, bozemanii, cherrii, dumoffii, gormanii, jordanis, parisiensis, pneumophila, steigerwaltii, tucsonensis, and wadsworthii).

Molecular microbiological investigation of an outbreak of hemolytic-uremic syndrome caused by dry fermented sausage contaminated with Shiga-like toxin-producing Escherichia coli.

Shiga-like toxin-producing Escherichia coli (SLTEC) strains are a diverse group of organisms which are known to cause diarrhea and hemorrhagic colitis in humans. This can lead to potentially fatal systemic sequelae, such as hemolytic-uremic syndrome (HUS). Strains belonging to more than 100 different O:H serotypes have been associated with severe SLTEC disease in humans, of which only O157 strains (which are uncommon in Australia) have a distinguishable cultural characteristic (sorbitol negative). During an outbreak of HUS in Adelaide, South Australia, a sensitive PCR assay specific for Shiga-like toxin genes (slt) was used to test cultures of feces and suspected foods. This enabled rapid confirmation of infection and identified a locally produced dry fermented sausage (mettwurst) as the source of infection. Cultures of feces from 19 of 21 HUS patients and 7 of 8 mettwurst samples collected from their homes were PCR positive for slt-I and slt-II genes. SLTEC isolates belonging to serotype O111:H- was subsequently isolated from 16 patients and 4 mettwurst samples. Subsequent restriction fragment length polymorphism analysis of chromosomal DNA from these isolates with slt-specific probes indicated that at least three different O111:H- genotypes were associated with the outbreak. Pulsed-field gel electrophoresis of genomic DNA restricted with XbaI showed that two of these restriction fragment length polymorphism types were closely related, but the third was quite distinct. However, SLTEC strains of other serotypes, including O157:H-, were also isolated from some of the HUS patients.

Use of gene amplification to detect Clostridium difficile in clinical specimens.

The combined use of an enrichment broth and gene amplification following simple DNA extraction to detect toxigenic strains of Clostridium difficile in feces was investigated by examining feces from 329 cases of suspected C. difficile infection. DNA was extracted by heating the washed centrifuged deposit from the broth in a microwave oven. For comparison, specimens were tested concurrently using standard methods for culture and cytotoxin testing. Amplified fragments were identified by molecular weight estimation, restriction enzyme digestion patterns and Southern blot hybridization. The combination of an enrichment broth followed by gene amplification was shown to be a sensitive, specific and rapid method for detecting toxigenic strains of C. difficile in feces. Use of the method in diagnostic laboratories may require the development of improved detection and verification systems for the amplified gene fragment.

Rapid identification of mycobacteria by the Gen-Probe Accuprobe system.

Chemiluminescent acridinium ester-labelled (AE)-DNA probes (Gen-Probe, Inc., San Diego, Calif.) for the identification of Mycobacterium tuberculosis complex (MTBC) and the M. avium-M. intracellulare complex (MAC) were evaluated using 184 mycobacterial isolates cultured in BACTEC 12B vials (Becton Dickinson and Co., Towson, Md.). A 1.5 mL aliquot from BACTEC 12B vials containing acid-fast bacilli and a Growth Index of > 200 was concentrated 15-fold using a centrifugation step prior to performing the test procedure. When 184 mycobacterial isolates were tested (42MTBC/142 nontuberculous mycobacteria) using the AE-MTBC probe, there was 100% sensitivity and specificity when compared with conventional identification procedures. Criteria for using the AE-MAC probe were defined to optimize results whilst minimizing laboratory costs. Ninety-one (64%) of AE-MTBC probe negative isolates failed to meet selection criteria and were not tested. When 51 (36%) of the AE-MTBC-probe negative mycobacterial isolates were tested, the AE-MAC probe was found to be 88% sensitive and 100% specific. The non-isotopic Gen-Probe test is a rapid and practical alternative to current procedures for differentiation of mycobacteria.

Legionella adelaidensis, a new species isolated from cooling tower water.

A Legionella-like organism (strain 1762-AUS-E) was isolated from a cooling tower of an air-conditioning system in Adelaide, South Australia, Australia. The isolate was presumptively identified as a Legionella strain by its growth requirement for L-cysteine and its cellular branched-chain fatty acids. Strain 1762-AUS-E was serologically distinct in the slide agglutination test with absorbed antisera. DNA hybridization confirmed that it is a new Legionella species for which the name Legionella adelaidensis is proposed.

Epidemiology of Salmonella sofia in Australia.

In recent years, the incidence of isolation of Salmonella sofia in Australia has risen from 33% of all poultry isolates in 1982 to a peak of 49% of isolates in 1988. A parallel rise has not been seen in S. sofia isolated from humans. In Israel, however, S. sofia has been commonly isolated from both humans and poultry. We investigated the possibility that the Israeli strains may belong to a different clonal group and express virulence determinants not seen in the Australian isolates, accounting for the apparent differences in the virulence seen within this species. A number of S. sofia isolates from Australian chickens and humans, as well as from Israeli humans and chickens, were compared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of outer membrane proteins, plasmid profiles, and restriction fragment length polymorphism analysis. No reproducible differences could be detected by analysis of outer membrane proteins. A small 6.4-kb plasmid, pIMVS2, was detected in all Australian isolates from chickens but not in the Israeli isolates. Restriction fragment length polymorphism studies with cosmid clones as probes provided the most discrimination among isolates, allowing us to divide them into seven groups. This technique revealed that significant differences exist between Australian and Israeli isolates and provided additional insights into the epidemiology of these Salmonella isolates.

Genetic relatedness of Legionella longbeachae isolates from human and environmental sources in Australia.

The genetic relatedness of Legionella longbeachae isolated in Australia since 1987 was investigated by restriction fragment length polymorphism (RFLP) analysis and allozyme electrophoresis. Three radiolabeled probes were used in Southern hybridizations for the RFLP studies. They were Escherichia coli 16S and 23S rRNA and cloned fragments of L. longbeachae selected empirically from genomal banks in lambda and a cosmid. The legionellae included in the study comprised 11 Legionella longbeachae serogroup 1 organisms isolated from humans, 28 L. longbeachae serogroup 1 isolates from environmental sources, and 3 L. longbeachae serogroup 2 environmental isolates. These were compared with the American Type Culture Collection reference strains of both serogroups and some other related Legionella species. Results of allozyme and RFLP analysis showed that all the isolates from humans and all but three of the environmental L. longbeachae serogroup 1 isolates were closely related. They were also closely related to L. longbeachae serogroup 1 ATCC 33462. There was wider variation among the three L. longbeachae serogroup 2 environmental isolates. One of these was closely related to L. longbeachae serogroup 2 ATCC 33484. RFLP studies with the rRNA probe provided the most discrimination among isolates but did not distinguish between the two serogroups.

Problems associated with identification of Legionella species from the environment and isolation of six possible new species.

Following investigation of an outbreak of legionellosis in South Australia, numerous Legionella-like organisms were isolated from water samples. Because of the limited number of commercially available direct fluorescent-antibody reagents and the cross-reactions found with some reagents, non-pneumophila legionellae proved to be difficult to identify and these isolates were stored at -70 degrees C for later study. Latex agglutination reagents for Legionella pneumophila and Legionella anisa developed by the Institute of Medical and Veterinary Science, Adelaide, Australia, were found to be useful as rapid screening aids. Autofluorescence was useful for placing isolates into broad groups. Cellular fatty acid analysis, ubiquinone analysis, and DNA hybridization techniques were necessary to provide definitive identification. The species which were isolated most frequently were L. pneumophila, followed by L. anisa, Legionella jamestowniensis, Legionella quinlivanii, Legionella rubrilucens, Legionella spiritensis, and a single isolate each of Legionella erythra, Legionella jordanis, Legionella birminghamensis, and Legionella cincinnatiensis. In addition, 10 isolates were found by DNA hybridization studies to be unrelated to any of the 26 currently known species, representing what we believe to be 6 possible new species.

Localization of a 23,000 MW antigen of Cryptosporidium by immunoelectron microscopy.

Rabbit antiserum was raised against a 23,000 molecular weight (MW) antigen prepared from Cryptosporidium oocysts by electro-elution from polyacrylamide gels. The antiserum was tested for specificity by immunoblotting against solubilized oocyst preparations. Several antigens including the 23,000 MW antigen were recognized suggesting that it shared common epitopes with higher MW proteins. The antiserum was then used in conjunction with a protein A-colloidal gold conjugate to locate antigenic sites within exogenous and endogenous developmental stages of Cryptosporidium. The pellicles of both sporozoites and merozoites exhibited specific labelling, particularly around their anterior ends. No specific labelling was observed for any other membrane determinants or organelles in these or other life cycle stages.

Legionella longbeachae pneumonia: report of two cases.

Legionella longbeachae serogroup 1 was isolated from the respiratory secretions of two patients with community-acquired pneumonia. One patient had a mild infection without evidence of the involvement of other organs and recovered, in spite of inappropriate antibiotic therapy. The other patient was severely-ill on presentation with multisystem failure and died soon after admission to hospital. The organisms were identified by the immunofluorescence technique and by quantitative DNA-hybridization studies. The sources of the infection in these patients are unknown as the organism has never been isolated from the SA environment.

Electrophoretic and immunoblot analysis of Cryptosporidium oocysts.

Cryptosporidium oocysts were recovered by density gradient centrifugation from diarrhoeal faeces of four human patients and one goat kid. Goat-derived oocysts were further treated with excystation medium and the excysted oocyst walls purified by isopycnic ultracentrifugation. Soluble extracts from intact oocysts and the oocyst wall preparation were analysed by SDS-PAGE. Fifty-one polypeptide bands were detected in intact oocyst preparations: 48 were in the range 14,000-200,000 molecular weight (MW), two bands were less than 14,000 MW and one band was above 200,000 MW. Twenty-one bands were detected in the oocyst wall preparation, all within the range 14,000-200,000 MW. Immunoblot analysis of Cryptosporidium polypeptides using acute or convalescent human and goat sera revealed a large number of reactive bands. Varying degrees of heterogeneity were observed within and between the two serum groups. Nine of the 10 human sera and all of the goat kid sera reacted with a 23,000 MW and 32,000 MW antigen. A 15,500 MW antigen was also detected by all the goat and four of the 10 human sera. Both serum groups reacted with various antigens above 40,000 MW. Surface labelling of three human isolates of Cryptosporidium oocysts with 125I was performed using the Bolton and Hunter reagent. The solubilized preparations were separated by SDS-PAGE on 12% and 18% slab gels and autoradiographed. Common bands were seen at 15,500, 32,000, 47,500, 79,000 and 96,000 MW. Some variation in the molecular weight of polypeptides labelled with 125I was observed among the three isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of enterotoxigenic Escherichia coli isolates with enzyme-labeled synthetic oligonucleotide probes.

Commercially available kits containing alkaline phosphatase-labeled oligonucleotide probes for Escherichia coli heat-stable enterotoxins (STI-H, STI-P, and STII) and the heat-labile enterotoxin were compared with bioassays and radiolabeled recombinant DNA probes to identify enterotoxigenic E. coli from 100 clinical isolates. There was very good agreement between the three methods.

Plasmid profile analysis of a salmonellosis outbreak and identification of a restriction and modification system.

After an outbreak of salmonellosis in humans caused by Salmonella typhimurium bacteriophage type 135, 62 isolates from human, animal, and water sources were retained for further analysis. Most of the isolates (92%) could be placed in one of five plasmid pattern groups, with a majority containing a common 60-kilobase plasmid and a smaller 3.8-kilobase-pair plasmid. This small plasmid, pIMVS1, was labeled with [32P]phosphate and used as a probe in subsequent colony and Southern hybridization studies. We concluded that pIMVS1 from isolates obtained from humans was genetically different from plasmids of a similar size found in isolates from chickens. Studies to characterize pIMVS1 were undertaken to determine if it codes for known virulence factors. It did not appear to be associated with the formation of attachment pili or major outer membrane proteins. By using transposon mutagenesis techniques, Tn3(Apr) was inserted into pIMVS1, and the existence of a restriction and modification system was deduced.

Enterotoxigenic Escherichia coli in Central Australia: diagnosis using cloned and synthetic nucleic acid probes.

Feces from 169 children admitted with diarrhea to Alice Springs Hospital, were screened for enterotoxigenic E. coli (ETEC) using specific DNA probes. E. coli which hybridized with probes for ST-P, ST-H and LT were confirmed by bioassay for toxin production. Fifty children were shown to excrete ETEC. The probes for LT correlated well with bioassay; however, probes for ST-H and ST-P hybridized with more E. coli than were shown to produce toxin by bioassay. When probing for ST was repeated using a synthetic oligonucleotide probe, only those specimens which were bioassay-positive hybridized with the probe.

Ultrastructure of the attachment of Cryptosporidium sporozoites to tissue culture cells.

The attachment of Cryptosporidium sporozoites to Madin-Darby canine kidney (MDCK) cells was examined using transmission electron microscopy. As the anterior end of the sporozoite came into close proximity to the MDCK cell, the host cell membrane evaginated around the sporozoite, forming a parasitophorous vacuole. A dense band formed below the host cell membrane at the site nearest to the conoid. Variably electron-dense material was apparently released from the conoid and a large membrane-bound vacuole was formed in the anterior end of the sporozoite, displacing the typical anterior electron-dense organelles (rhoptries and micronemes). The outer membrane of the sporozoite pellicle then fused with the host cell membrane immediately adjacent to the conoid. The membrane surrounding the anterior vacuole was also fused with the common host-parasite membrane, forming Y-shaped membrane junctions where each limb was a unit membrane. A direct link was thereby established between the anterior vacuole of the sporozoite and the host cell cytoplasm. The anterior vacuole membrane separating the sporozoite and the host cell cytoplasm was the precursor of the feeder organelle.

Enzyme-linked synthetic oligonucleotide probes: non-radioactive detection of enterotoxigenic Escherichia coli in faecal specimens.

Synthetic oligonucleotides, complementary to unique sequences in the heat stable enterotoxin gene of Escherichia coli specific for humans, were prepared with a 30-atom spacer arm and a 3' terminal sulfhydryl group which was coupled to bromoacetyl-derivatized alkaline phosphatase. The resulting direct enzyme-linked oligonucleotide probes, containing one enzyme molecule per oligonucleotide, successfully diagnosed enterotoxigenic Escherichia coli in clinical specimens by using a modified colony hybridization method and a colorimetric assay. The procedure is rapid, simple and reliable with a sensitivity equivalent to that using 5'-terminally labelled [32p]-oligonucleotide probes. The results indicate that the enzyme-labelled oligonucleotide probes should be applicable to the routine diagnosis of enterotoxigenic Escherichia coli and possess the potential for the detection of other microbial pathogens.