Isolation and characterization of the Aspergillus niger trpC gene.

The Aspergillus niger trpC gene was isolated by complementation experiments with an Escherichia coli trpC mutant. Plasmid DNA containing the A. niger trpC gene transforms an Aspergillus nidulans mutant strain, defective in all three enzymatic activities of the trpC gene, to Trp+, indicating the presence of a complete and functional trpC gene. Southern blot analysis of DNA from these Trp+ transformants showed that plasmid DNA was present but that this DNA was not integrated at the site of the chromosomal trpC locus. The A. niger trpC gene was localized on the cloned fragment by heterologous hybridization experiments and sequence analysis. These experiments suggest that the organization of the A. niger trpC gene is identical to that of the analogous A. nidulans trpC and the Neurospora crassa trp-1 genes.

Typing of Listeria strains by random amplification of polymorphic DNA.

The polymerase chain reaction (PCR) was used to obtain randomly amplified polymorphic DNA (RAPD) profiles for typing of Listeria strains. In this procedure, whole cells were incubated in the reaction mixture. The discriminating ability of a randomly designed 10-mer primer, HLWL74, was assessed. A total of 60 collection strains of Listeria, encompassing all 7 Listeria species and all known serovars was submitted to PCR with the primer HLWL74. Upon agarose gel electrophoresis, 29 different banding profiles were reproducibly obtained. No common profiles were recorded for strains from different Listeria species. For various groups of strains sharing the same serotype (e.g. 4b, 1/2a, 1/2b), RAPD analysis could generate further subdivision. On the other hand, some strains from different serotypes produced identical RAPD profiles with the primer HLWL74. The RAPD typing method from whole cells is proposed as an attractive alternative for other Listeria typing systems, and the 10-mer HLWL74 as a primer to include in a forthcoming set of standard primers for RAPD typing of Listeria isolates.

A comparative study of randomly amplified polymorphic DNA analysis and conventional phage typing for epidemiological studies of Listeria monocytogenes isolates.

The analysis of RAPD profiles generated by PCR with a single 10-mer, HLWL74, was compared to bacteriophage susceptibility data for epidemiological typing of Listeria monocytogenes strains. A total of 104 L. monocytogenes strains was screened, all from serogroup 1 or serotype 4b. Of these, 53 had been isolated during 6 different listeriosis outbreaks. The remaining 51 strains were chosen randomly from our collection. A total of 38 RAPD types were observed, although each epidemic group of strains isolated during one of these outbreaks displayed a specific RAPD profile. For 98% of the strains isolated during outbreaks, the correlation between RAPD typing and phage typing was complete. Only one strain, typed as epidemic by phage typing, was clearly distinguishable from the others by RAPD analysis. Among the 51 strains not related to an outbreak, 12 were linked to epidemic groups by RAPD analysis. Two of these rearrangements were supported by phage typing. The remaining 10 strains could be excluded by phage typing from any of the epidemic groups studies. Considering all 104 isolates, the decision to relate a strain to a particular epidemic group or to exclude a strain from any epidemic group was the same for 92 isolates, using either phage typing or RAPD analysis. The RAPD analysis, which is quick, simple and suited for automation, is proposed as an attractive alternative for phage typing in epidemiological studies of listeriosis.

Evaluation and interpretation of data obtained with immunoassays and DNA-DNA hybridization techniques.

During the last decade several new analytical techniques have been developed for testing food products and clinical samples. One technique uses sensitive immunoassays such as enzyme-linked immunosorbent assay (ELISA) and latex agglutination. The most important step in developing sensitive immunoassays is the evaluation of the assay for specificity, cross-reactivity and sensitivity. False-negative results can easily be detected by adding known quantities of antigen to the sample. The most appropriate way to detect false-positive results is the specific inhibition of the immunological reaction by addition to the test-sample of either synthetic epitopes or anti-idiotype antibodies. The progress in recombinant DNA techniques now offers opportunities for application as analytical tools in food and clinical microbiology. Methods are being developed to detect microorganisms by their nucleic acid sequence using the so-called hybridization procedure. With this technique, labelled DNA fragments (probes) are hybridized with a complementary base sequence present in the microorganism. Foodborne pathogens can be detected by using a probe with a complementary base sequence which codes for toxin production. DNA-DNA hybridization techniques may replace the traditional cultural techniques for assaying pathogenic micro-organisms. However, more experience with these techniques is needed before further evaluation can be given.

Immunological methods for detection of foodborne pathogens and their toxins.

Improved methods to detect microorganisms and their toxins introduced during the last decade involve among others recombinant DNA techniques and various immuno-assays such as the enzyme-linked immunosorbent assay and the latex agglutination. Immuno-assays are based on a quantitative reaction of an antigen (bacterial metabolite, e.g., toxin) with its antibody. Therefore, they are suited for detection of microorganisms based on their production of specific antigens and for quantitative detection of bacterial toxins. Sensitivity and specificity of immuno-assays are mainly determined by the antiserum used. In this respect the use of well selected monoclonal antibodies can be of advantage. With the enzyme-linked immunosorbent assay and latex agglutination test quantities of 0.1-1 ng of antigen/ml can be detected. Of both techniques the latex agglutination method has several advantages; the method is simple, inexpensive and rapid. Since each immuno-assay is sensitive to non-specific reactions, recognition of false positive results is necessary. The most appropriate method for this is to add an inhibitor to the test sample which blocks specifically the paratope of the immunoglobulin. Another general disadvantage of immuno-assays is that only the antigenicity is determined and this may differ from the actual toxicity. Therefore, antibodies should be used that react with the toxic centre(s) of the molecule, which can be accomplished by using well selected monoclonal antibodies.

The presence of enterotoxigenic Clostridium perfringens strains in faeces of various animals.

The presence of Clostridium perfringens in faeces of horses, cattle, poultry and pigs was determined. C. perfringens was detected in 24%, 36%, 80% and 2% of the faecal samples, respectively. Faecal samples containing enterotoxigenic strains as assessed by colony hybridization amounted to 14%, 22%, 10% and 0% respectively.

DNA hybridization and latex agglutination for detection of heat-labile- and shiga-like toxin-producing Escherichia coli in meat.

DNA-hybridization and latex-agglutination tests were used for screening of a group of Escherichia coli isolates for heat-labile enterotoxin (LT)- and shiga-like toxin (SLT1 or VT1) -producing strains, respectively. Strains tested originated from 162 meat samples (poultry, pigs and beef) chosen at random. Additionally LT- and SLT1-producing reference strains were tested. The DNA-hybridization technique allowed screening of large numbers of strains, whereas large scale testing of strains by latex agglutination was laborious. Of 800 E. coli strains tested by DNA hybridization none contained the gene encoding LT. Production of LT as tested by latex agglutination was not found. The gene encoding SLT1 was detected in 10 of the 800 isolates tested. None of these strains, however, showed cytotoxicity on Vero cells. Serotyping was done with sorbitol-negative E. coli strains, first by using the latex-agglutination test for O157 followed by complete serotyping. No E. coli of serogroup O157 were found. Therefore the results obtained also indicate that routine screening of E. coli isolated randomly from food for toxin production is not useful and should be limited to food-borne disease outbreaks with an etiology resembling an E. coli infection.

Detection of genes encoding for enterotoxins and determination of the production of enterotoxins by HBL blood plates and immunoassays of psychrotrophic strains of Bacillus cereus isolated from pasteurised milk.

The presence of genes for the production of the three components of the HBL enterotoxin complex and enterotoxin-T in Bacillus cereus was evaluated by PCR tests for strains isolated from milk. In addition enterotoxin production of B. cereus was evaluated by means of the HBL blood agar plate and two commercially available toxin tests. All three genes for the HBL enterotoxin complex were detected in 55% of the 86 strains tested, the enterotoxin-T gene was detected in 62% of the strains. A few strains showed a weak reaction in the PCR tests for the L1 or L2 components of the HBL enterotoxin complex. Many strains that were found to contain the genes for the HBL complex gave negative or doubtful results in the HBL blood agar plate test. All strains that contain the L2 part of the HBL complex showed a titer of at least 8 in the Oxoid RPLA test. Two strains that did not contain the L2 part of the HBL enterotoxin complex gave high titers (= 64) in the RPLA test.

Probes and polymerase chain reaction for detection of food-borne bacterial pathogens.

DNA-hybridization and the polymerase chain reaction (PCR) are techniques commonly used to detect pathogenic bacteria. In this paper, the use of these techniques for detection of Salmonella, E. coli, V. cholerae, non-O1 Vibrio, Yersinia enterocolitica, Campylobacter, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, and C. botulinum is reviewed with emphasis on application in food microbiology. In food control, DNA-techniques have most often been used in a 'culture confirmation' fashion, i.e. bacteria are enriched and sometimes even purified by traditional culture procedures and thereafter identified by the use of DNA-based methods. The most desirable approach is, however, to detect organisms directly in the food, but major problems remain to be solved before this can be routinely performed.

The WHO multicenter study on Listeria monocytogenes subtyping: random amplification of polymorphic DNA (RAPD).

As part of a WHO multicenter study on Listeria monocytogenes subtyping methods the random amplification of polymorphic DNA (RAPD)-technique was evaluated. Six participants were asked to use a standard protocol to analyse a set of 80 L. monocytogenes strains. This set contained 22 groups of epidemiologically linked isolates and 11 pairs of duplicate strains. Using three different 10-mer primers the median reproducibility of the RAPD-results obtained by the six participants was 86.5% (range 0-100%). Failure in reproducibility was mainly due to results obtained with one particular primer. The number of epidemiological groups found to be homogeneous varied from 1-22 (median 16). However, for some groups an inhomogeneity was found by the majority of participants. The overall correlation between the results from the different participants ranged from 32 to 85%.

Four cases of hemolytic uremic syndrome--source contaminated swimming water?

In June '93, 4 children, aged 1.5-3.5 years, all living in one town, were admitted to our hospital with the diagnosis hemolytic uremic syndrome (HUS) within one week. In cooperation with the local health authorities a common source was searched for. Questionnaires indicated that the single condition shared by all patients was swimming water. The patients were not acquainted, visited different daycares, and had no food resources in common. All 4 patients bathed in the same, shallow, recreational lake within a period of 5 days. During this time the air temperature was high according to Dutch standards (around 27 degrees C), and many people visited the lake, estimated several hundreds a day. The water level was lower than normal. Diarrhea followed 3-11 days after swimming and the first clinical symptoms of HUS developed 6-7 days after the onset of diarrhea. The lake was closed for swimming when the fourth HUS patient was diagnosed and the possibility of transmission by way of the lake was mentioned. E. coli O157: H7 was demonstrated in the fecal samples of 2 index patients. The samples were taken 9-20 days after the start of diarrhea. Antibodies to O157 and verotoxin 2 were strongly positive in all patients. A local outbreak of diarrheal illness was not registered. Of 16 family members who also swam in the same lake, 7 developed symptoms of enteritis, 3 had positive cultures of their fecal samples and 5 had positive serology. Pulsed-field gel electrophoresis of the E. coli isolates of the patients and family members showed an identical pattern. No O157: H7-DNA could be detected in filter concentrated lake water samples using polymerase chain reaction (PCR) enhancement. These samples were, however, taken 16 days after the latest possible date of contamination of our patients, 15 days after decrease of the air temperature to 15-17 degrees C, and 14 days after the inlet from water from the environment. It could thus very well be that the microorganism was no longer present. This third report of swimming water associated HUS should direct environmental surveys in similar cases of local HUS outbreaks.

[Enterohemorrhagic Escherichia coli O157:H7, an underestimated food pathogen? A literature review]. / Enterohemorragische Escherichia coli O157:H7, een onderschatte voedselpathogeen? Een literatuuroverzicht.

Escherichia coli O157:H7 has recently been recognised as a human pathogen involved in outbreaks and sporadic cases of food-borne diseases in North America and Great Britain. At present, less is known about the significance of E. coli O157:H7 as a cause of food-borne diseases in the Netherlands. This article will review current literature about this serotype, which is a predominant representative of the enterohaemorrhagic E. coli (EHEC) group. Biochemical features, toxins, pathogenesis, clinical characteristics, epidemiology and methods of isolation will also be discussed.

The use of the Listeria monocytogenes DTH gene for the detection of pathogenic biovars in food.

A gene probe encoding a Listeria monocytogenes delayed type hypersensitivity factor (DTH) was used for the detection of pathogenic biovars/serovars of the genus Listeria isolated from soft cheese. A total of 187 strains, isolated by plating techniques, were screened. The probe reacted with all isolated serovars of L. monocytogenes (except one 4b strain) and did not react with any of the Listeria innocua strains. Using the mouse bio-assay, as described by Kaufmann, a significant correlation was found between probe reaction and pathogenicity.

Synthetic enterotoxin B DNA probes for detection of enterotoxigenic Staphylococcus aureus strains.

DNA-DNA colony hybridization experiments with three different synthetic probes were carried out with 210 strains of Staphylococcus aureus. The synthetic probes encoded the amino acids 15 to 29 (probe 1), 179 to 192 (probe 2), and 207 to 219 (probe 3) of staphylococcal enterotoxin B (SEB). The amino acid sequences of these parts of SEB are identical to those of SEC1. All 21 SEB-producing strains tested reacted with each of the three probes. Of the 69 SEC-producing strains, 21 reacted with probe 1, none reacted with probe 2, and all 69 reacted with probe 3. With other strains no hybridization signals were obtained. The results presented here show that with a single synthetic DNA probe (probe 3) both SEB- and SEC-producing strains are detectable.

Cloning and sequencing of the Clostridium perfringens enterotoxin gene.

Several gene banks of Clostridium perfringens in E. coli were constructed. Using a mixture of synthetic 29-mer DNA probes clones were selected containing inserts from the C. perfringens gene coding for the enterotoxin. This has allowed sequencing of the complete gene and its flanking regions. The decuded amino acid sequence (320 a.a.) was found to differ at several sites from the sequence published previously by others. Two 40-mer DNA-probes were used to detect the toxin gene in C. perfringens strains isolated from the faeces of different non-symptomatic animals. Only 6% of the strains were found to possess the gene.

Successful approach for detection of low numbers of enterotoxigenic Escherichia coli in minced meat by using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used as a tool for the detection of enterotoxigenic Escherichia coli in minced meat. With two synthetic 29-mer oligonucleotides, a 195-bp fragment from the E. coli heat-labile enterotoxin (LT) gene could be amplified specifically. When 6 CFU was added to the reaction mixture as a template, the PCR yielded sufficient amplified product for visualization on an agarose gel. Prior to PCR amplification, the minced meat samples were subjected to enrichment culturing for E. coli. From these cultures, 10 microliters was used in the PCR assay. All 20 25-g samples that were examined in this assay were negative for E. coli LT. However, when 3 CFU of E. coli LT was added to the 25-g samples of minced meat prior to enrichment culturing, the PCR assay yielded positive results.

Terminal uridylyl transferase of Vigna unguiculata: purification and characterization of an enzyme catalyzing the addition of a single UMP residue to the 3'-end of an RNA primer.

An enzyme which catalyzes the addition of a single UMP residue from UTP to the 3'-end of an RNA primer and which is referred to as terminal uridylyl transferase (TUT) has been extensively purified from the membrane fraction of vigna unguiculata leaves. The purification procedure involved (i) solubilization by cation depletion (ii) DEAE-Sepharose CL-6B column chromatography (iii) affinity chromatography of poly(U)-Sepharose 4B and (iv) glycerol gradient centrifugation. The molecular weight of the native enzyme was approximately 50,000 as determined by velocity sedimentation. Under conditions that were optimal for UMP-incorporation (5 mM Mg2+, low salt, 30 degrees C) TUT displayed a marked specificity for UTP as substrate, was unable to incorporate deoxyribonucleoside triphosphates and required a single-stranded oligo- or polyribonucleotide as primer. When oligoA20, tRNAasp of E. coli or alfalfa mosaic virus RNA 4 were used as primers at various substrate to primer ratio's, the vast majority of the product appeared to consist of primer molecules elongated with a single UMP residue as shown by polyacrylamide gelelectrophoresis and nearest neighbour analysis. We believe TUT to be a novel enzyme which has not been reported before and which may be a feasible tool in RNA sequencing as it enables the specific 3'-terminal labeling of RNA molecules.

The complete sequence of botulinum neurotoxin type A and comparison with other clostridial neurotoxins.

The seven serologically different botulinum neurotoxins are highly potent protein toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. The activated toxins consist of the toxifying A-subunits (Mr approximately 50,000) linked by a disulfide bond to the receptor-binding BC-subunits (Mr approximately 100,000). We have established the complete sequence of botulinum neurotoxin type A (BoNT/A; 1,296 amino acid residues, Mr = 149,425) and a partial sequence of botulinum neurotoxin type E (273 amino acid residues) as deduced from the corresponding nucleotide sequences of the chromosomally located structural genes. The promoter of the BoNT/A gene is inactive in Escherichia coli. Primer extension experiments indicated that initiation of transcription of the BoNT/A gene occurred 118 nucleotides upstream from the ATG codon. A comparison of the protein sequence revealed an overall identity of 33.8% to that of tetanus toxin. No significant similarity to other known proteins including ADP-ribosylating toxins could be detected. Three of the six histidine residues of the A-subunit of BoNT/A were found in the peptide sequence H223ELIHXXH230 within a domain of predicted alpha-helical secondary structure. This motif is also found in similar positions of the A-subunits of tetanus toxin and BoNT/E.

Genetic analysis of Aspergillus nidulans AmdS+ transformants.

To correlate the genetic background of the Aspergillus nidulans amdS deletion strain MH1277 with the integrational behaviour of transforming vectors, classical genetic methods were used to construct AmdS- strains in which whole chromosomes had been exchanged with those of a master strain. Progeny strains were transformed to the AmdS+ phenotype with vector p3SR2. From Southern analysis it was concluded that transformants from all constructions contained tandemly repeated, multiple copy inserts of vector DNA as found for MH1277-derived AmdS+ transformants. AmdS+ transformants of MH1277 were analysed genetically to prove that the transformant phenotype is genome linked and that transformation by integration can take place on various chromosomes. In one case the AmdS+ property showed linkage to both chromosomes II and IV, due to a chromosomal translocation. Sexual analysis of two transformants with AmdS+ insertions on the same chromosome revealed a considerable instability of the AmdS+ phenotype in one of the strains upon selfing. Due to this instability no decisive answer could be given for the degree of linkage between the AmdS+ insertions in these transformants.

Suitability of the prfA gene, which encodes a regulator of virulence genes in Listeria monocytogenes, in the identification of pathogenic Listeria spp.

The pathogenesis of listerial infections is complex and involves a number of virulence factors expressed by virulent Listeria species. We have recently described a regulator gene, prfA, that positively regulates the expression of a number of virulence factors in Listeria monocytogenes. When the prfA gene was used as a DNA probe, we found it to be extremely specific for the pathogenic species L. monocytogenes. No reaction was obtained with strains of all other species of this genus. By using this information, an oligonucleotide primer pair was developed that specifically amplifies the prfA gene in L. monocytogenes strains of all known serotypes.