PubMed HealthSearch

Biomedical subjects

H Hofstra

Publications and source records attributed to H Hofstra.

At least 19 recordsLinked to original sources

A comparison of immunomagnetic and surface adhesion immunofluorescent techniques for the rapid detection of Listeria monocytogenes and Listeria innocua in meat.

An immunomagnetic immunofluorescent method was investigated for the rapid detection of Listeria monocytogenes and Listeria innouca. This technique involved enrichment of the suspect sample at 30 degrees C overnight. Listeria monocytogenes cells were isolated from the enriched sample using immunomagnetic separation and Listeria were subsequently visualized using an immunofluorescent microscopy technique. This technique was used in the detection of Listeria cells from pure culture, inoculated beef mince samples and naturally contaminated retail beef mince samples. A detection level of approximately 1 x 10(3) cfu ml-1 was achieved. When compared with traditional detection methods no false negatives or positives were recorded for L. monocytogenes or L. innocua. The immunomagnetic immunofluorescent technique had a detection level similar to a previously described surface adhesion immunofluorescent technique. Isolation of the Listeria cells by surface adhesion involved dipping a membrane attached to a microscope slide into the enriched sample for 10 min. This was quicker and simpler to perform than the immunomagnetic separation technique which took 2 h to carry out.

Animals

DNA based typing, identification and detection systems for food spoilage microorganisms: development and implementation.

The rapid identification of spoilage microorganisms is of eminent importance to the food industry. It provides the food industry with the opportunity to reduce economical losses by designing adequate intervention measures. The use of identification systems based on biochemical and physiological characteristics resulted often in disappointing identification results and misidentifications. This will inevitably lead to inappropriate strategies to prevent spoilage. This review discusses the potential of the DNA based identification technology including the polymerase chain reaction (PCR) for the identification and specific detection of microorganisms. Fingerprinting methods based on the DNA-probe technology enable a clear insight in the identity of microorganisms on different levels, varying from genus to strain level depending on the systems used. Discrimination between subspecies and strain level is shown to be helpful for investigating routes and sources of contamination. Differentiation at the species level is demonstrated to be essential in order to design a highly specific detection system enabling to signalize a microorganism that belongs to a particular species. Also indicated in this review is the necessity and the technical approach to detect microorganisms that display a particular undesirable trait.

DNA

Evaluation of molecular typing techniques to assign genetic diversity among Saccharomyces cerevisiae strains.

Discrimination of strains within the species Saccharomyces cerevisiae was demonstrated by the use of four different techniques to type 15 strains isolated from spoiled wine and beer. Random amplified polymorphic DNA with specific oligonucleotides and PCR fingerprinting with the microsatellite oligonucleotide primers (GAC)5 and (GTG)5 enabled discrimination between the strains tested. Additionally, restriction enzyme analysis, with TaqI and MseI, of PCR-amplified fragments from the complete internal transcribed spacer and nontranscribed spacer, both present in the rRNA-encoding gene cluster, proved to be suitable for generating intraspecies-specific patterns. Random amplified polymorphic DNA with primers 24 and OPA-11 and PCR fingerprinting with primer (GTG)5 appeared to generate the highest degree of diversity. However, the results indicated that there was no single PCR-mediated typing technique enabling discrimination on the strain level. Discrimination of each individual strain was nevertheless possible by combining the results obtained with all typing techniques.

Base Sequence

Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: two identification techniques for food-borne yeasts.

The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be adequate tools for yeast identification. Since the RAPD does provide less stable patterns than restriction enzyme analysis of PCR amplified rDNA, and a large amount of data had to be compared without data reduction, Principal Component Analysis (PCA) was applied successfully for clustering the RAPD patterns. The success of PCA is highly influenced by the primer used in RAPD and the amount of reference samples. A large amount of reference samples improves the performance of clustering in PCA. The primer of choice was shown to be important with respect to the discriminatory power of the RAPD method. Some primers used enabled discrimination on the subspecies level. The results collected with both typing methods justify the conclusion that the present typing system can be applied for taxonomical purposes.

Base Sequence

Development of a detection system for histidine decarboxylating lactic acid bacteria based on DNA probes, PCR and activity test.

On the basis of the comparison of the nucleotide sequences of the histidine decarboxylase genes (hdcA) of Lactobacillus 30A and Clostridium perfringens and the amino acid sequences of these histidine decarboxylases and those of Lactobacillus buchneri and Micrococcus, oligonucleotides unique to the hdcA genes were synthesized and used in PCR. All histidine-decarboxylating lactic acid bacteria gave a signal with primer set JV16HC/JV17HC in PCR. In addition to this primer set, CL1/CL2 and CL1/JV17HC were also useful for the detection of histamine-forming Leuconostoc aenos strains in PCR. The 150 base pair amplification product of the decarboxylating Leuc. aenos strain generated with primer set CL1/CL2 was sequenced. Alignment studies showed a high degree of relatedness among the hdcA gene products of Gram-positive bacteria. The amplification products of the hdcA genes from Lac. buchneri and Leuct. aenos were used to serve as a DNA probe in hybridization studies. All histidine-decarboxylating lactic acid bacteria gave a hybridization signal with the DNA probes. In hybridization only one false-positive signal with a Lactobacillus lindneri strain was observed, which was anticipated to contain a truncated hdcA gene. In addition to these DNA probe tests, a simple and reliable activity test is presented, which can be used during starter selection to test strains for histidine decarboxylase activity.

Amino Acid Sequence

RAPD analysis: a rapid technique for differentiation of spoilage yeasts.

Techniques for the identification of the spoilage yeasts Saccharomyces cerevisiae and members of the Zygosaccharomyces genus from food and beverages sources were evaluated. The use of identification systems based on physiological characteristics resulted often in incomplete identification or misidentification. Also the cellular fatty acid analysis failed on differentiating species within the Zygosaccharomyces genus. However, the Random Amplified Polymorphic DNA (RAPD) assay, using selected 10-mer oligonucleotides, allowed discrimination between all species tested. For this RAPD assay, a simple and reproducible method of DNA isolation from spoilage yeast cells is described.

Base Sequence

Microbes in food processing technology.

There is an increasing understanding that the microbial quality of a certain food is the result of a chain of events. It is clear that the microbial safety of food can only be guaranteed when the overall processing, including the production of raw materials, distribution and handling by the consumer are taken into consideration. Therefore, the microbiological quality assurance of foods is not only a matter of control, but also of a careful design of the total process chain. Food industry has now generally adapted quality assurance systems and is implementing the Hazard Analysis Critical Control Point (HACCP) concept. Rapid microbiological monitoring systems should be used in these cases. There is a need for rapid and simple microbiological tests which can be adapted to the technology and logistics of specific production processes. Traditional microbiological methods generally do not meet these high requirements. This paper discusses the tests, based on molecular biological principles, to detect and identify microbes in food-processing chains. Tests based on DNA technology are discussed, including in vitro DNA amplification like the polymerase chain reaction (PCR) method and identifications based on RFLP, RAPD and DNA fingerprinting analysis. PCR-based methodology can be used for the rapid detection of microbes in food manufacturing environments. In addition, DNA fingerprinting methods are suitable for investigating sources and routes of microbial contamination in the food cycle.

Bacterial Typing Techniques

Polymerase chain reaction for the specific detection of Escherichia coli/Shigella.

The outer membrane protein PhoE of members of the family Enterobacteriaceae consists of conserved membrane-spanning segments and hypervariable surface-exposed regions. Two oligonucleotides based on DNA sequences encoding two different cell-surface-exposed regions of the Escherichia coli K12 PhoE protein were tested for their specificity in polymerase chain reactions. They reacted with all strains of the species E. coli/Shigella tested, except for strain S. boydii serovar 13, which is known to represent a different DNA-relatedness group. The probes did not react with any other Enterobacteriaceae tested, including strains of Escherichia blattae, Escherichia hermanii, Escherichia vulneris and Escherichia adecarboxylata, except for an Escherichia fergusonnii strain, which is most closely related to E. coli. Therefore, the primer couple showed a high degree of species-specificity. In addition, a second primer couple based on two conserved regions of the phoE genes was tested. This primer couple recognized a broad group of closely related enteric bacteria including Salmonella and Shigella.

Bacterial Outer Membrane Proteins

The production and partial characterization of a monoclonal IgG antibody specific for moulds belonging to the order Mucorales.

A monoclonal antibody (mAb) was raised against extracellular polysaccharides from Mucor racemosus after intrasplenic immunization of mice. An indirect ELISA and a dot-blot assay were developed with this mAb. The IgG antibody was found to be very specific for all mould species tested belonging to the order of Mucorales, except species belonging to the genus Mortierella sensu stricto. No cross-reactions were observed with other moulds or yeasts. The immunoreactivity of the polysaccharides of these moulds with this mAb is based on carbohydrate epitopes, in which fucose residues probably play an important role. The mAb may be suited for specific detection of species of the genera Mucor, Rhizopus, Rhizomucor, Thamnidium, Absidia, Syncephalastrum and species belonging to the Mortierella isabellina group in food, and possibly for diagnosis of mucormycosis in humans.

Animals

Characterization of the Citrobacter freundii phoE gene and development of C. freundii-specific oligonucleotides.

The phoE gene of Citrobacter freundii, encoding a pore-forming outer membrane protein, was cloned and its nucleotide sequence was determined. The homologies in terms of identical amino acids between the C. freundii PhoE protein and those of Escherichia coli, E. cloacae and Klebsiella pneumoniae were 90%, 86% and 84%, respectively. Two synthetic oligonucleotides, corresponding to hypervariable, cell surface-exposed regions of the protein, were tested for their specificity in polymerase chain reactions. They were specific for the species C. freundii, i.e., no reaction was detected with 35 non-C. freundii strains tested, including 17 Salmonella, two C. amalonaticus and three C. diversus strains, whereas all five C. freundii strains tested were correctly recognized.

Amino Acid Sequence

Characterization of the Salmonella typhimurium phoE gene and development of Salmonella-specific DNA probes.

In Escherichia coli K-12, the phoE gene, encoding a phosphate-limitation-inducible outer membrane pore protein (PhoE), is closely linked to the genes proA and proB. When the corresponding fragment of the Salmonella typhimurium chromosome was transferred to E. coli K-12 using an RP4::miniMu plasmid, pULB113, no production of S. typhimurium PhoE could be detected. Nevertheless, DNA hybridization studies revealed that the corresponding plasmid did contain S. typhimurium phoE. Production of S. typhimurium PhoE in E. coli was detected only after subcloning the gene in a multicopy vector. Nucleotide (nt) sequence analysis showed extensive homology of S. typhimurium phoE to the E. coli gene and suggested possible explanations for the low expression of S. typhimurium phoE in E. coli. In addition, the sequence information was used to develop Salmonella-specific DNA probes. Two oligodeoxyribonucleotides were synthesized based on nt sequences encoding the fifth and eighth cell-surface-exposed regions of PhoE. When used in polymerase chain reactions, these probes turned out to be specific, i.e., no crossreactions occurred with the non-Salmonella strains, whereas 132 out of 133 tested Salmonella strains were recognized.

Amino Acid Sequence

Identification of Klebsiella pneumoniae by DNA hybridization and fatty acid analysis.

On the basis of the idea that DNA sequences encoding cell surface-exposed regions of outer membrane proteins are genus or species specific, two oligonucleotide probes which were based on the PhoE protein of Klebsiella pneumoniae were evaluated. In slot blot hybridizations and in polymerase chain reactions, no cross-hybridizations were observed with non-Klebsiella strains. When the probes were tested on 75 different K-antigen reference Klebsiella strains, 16 strains were not recognized although they did produce PhoE protein under phosphate starvation. To determine whether these 16 strains belong to (a) different species, the reference strains were also tested for the ability to produce indole and to grow at 10 degrees C and their whole-cell fatty acid patterns were analyzed by gas chromatography. A strong correlation was observed among (i) reaction with the probes, (ii) the inability to produce indole, (iii) the inability to grow at 10 degrees C, and (iv) the presence of the hydroxylated fatty acid C14:0-2OH. From these results we conclude that the two oligonucleotides are specific for the species K. pneumoniae. Furthermore, analysis of fatty acid patterns appears to be a useful tool to distinguish K. pneumoniae from other Klebsiella species.

Base Sequence

Development of enterobacterium-specific oligonucleotide probes based on the surface-exposed regions of outer membrane proteins.

Outer membrane proteins of members of the family Enterobacteriaceae consist of conserved membrane-spanning segments and hypervariable, surface-exposed regions. We demonstrate that the hypervariable DNA segments corresponding to the surface-exposed regions of these proteins can be used to develop specific DNA probes for the identification of members of the family Enterobacteriaceae.

Bacterial Outer Membrane Proteins

Heat-labile enterotoxin in Escherichia coli. Kinetics of association of subunits into periplasmic holotoxin.

We have investigated the assembly of the heat-labile enterotoxin (LT) subunits after their processing and segregation into the periplasmic space as mature LT A and LT B polypeptides. LT B starts associating into oligomers during or immediately after translocation through the cytoplasmic membrane. Binding to LT A occurs immediately after oligomerization. Over 80% of the LT B subunits have oligomerized, and over 50% have associated with LT A into holotoxin within 1 min after synthesis. The fate of newly synthesized LT A is totally different. There is an extensive overproduction of LT A relative to LT B and after membrane translocation it becomes part of a periplasmic pool of free LT A. It is then bound by LT B oligomers or degraded at such a rate that the free periplasmic LT A disappears from the pool with a half-time of 20-25 min. About half of the LT A is incorporated into holotoxin, while the other half is degraded. We conclude that LT subunits are translocated and processed in a ratio of about 2 A to 5 B. Since free LT A is either degraded slowly or bound to newly synthesized LT B oligomers, the net result is a steady state of 1.4 to 1.7 A subunits to 5 B subunits in the periplasm. About 60% of this LT A is bound by LT B to form periplasmic holotoxin with a subunit ratio of about 1 A to 5 B. The remaining 40% of periplasmic LT A occurs free.

Bacterial Toxins

Heat-labile enterotoxin of Escherichia coli. Characterization of different crystal forms.

Heat-labile enterotoxin (LT) was obtained in large quantities (several-gram amounts) and great purity from Escherichia coli C600 carrying the LT-coding multicopy plasmid EWD299. By growing this strain on a medium that allows high cell densities in the early stationary phase, we increased the net LT production per milliliter by a factor of 200, compared to natural porcine enterotoxigenic E. coli. Adsorption and redesorption on Controlled-Pore Glass usually resulted in a 50-100-fold purification of LT in one simple step, which was followed by established purification procedures. LT shows a natural tendency to form large crystals, which, however, are disordered. After numerous trials, conditions were found which virtually eliminated the disorder. Much better crystals were obtained by employing CdCl2 and KF as coprecipitating agents. CdCl2 yielded crystals which did not contain A subunits as judged by electrophoresis of dissolved crystals. Adding KF results in beautifully shaped crystals which diffracted beyond 2 A and are suitable for a high resolution structure determination.

Bacterial Toxins

Kinetics of synthesis, processing, and membrane transport of heat-labile enterotoxin, a periplasmic protein in Escherichia coli.

We report the detection in vivo of precursors to the A and the B subunits of the heat-labile enterotoxin (LT) in Escherichia coli. Both pre-LT A (Mr = 29,500) and pre-LT B (Mr = 13,500) are present in the spheroplast fraction of the bacteria after separation of the cells in spheroplasts and periplasm. Two smaller LT A related polypeptides (17 and 23 kDa) were also detected in the spheroplast fraction. Both were degraded with a half-time of about 40 s. Mature subunits (Mr = 27,500 for LT A, and 11,500 for LT B) are released from the spheroplasts soon after processing and occur freely in the periplasm not associated with the cytoplasmic or the outer membranes. Processing occurs mainly post-translationally for both the A and the B subunits. However, they show different kinetics of processing and subsequent segregation into the periplasm. Whereas pre-LT B is processed and released within seconds after chain termination, pre-LT A is processed and released more slowly, and a subfraction of mature LT A may reside in the cytoplasmic membrane for several minutes.

Bacterial Toxins

Effect of beta-lactam antibiotics of the resistance of the digestive tract of mice to colonization.

Four broad-spectrum antibiotics-azlocillin, mezlocillin, cefuroxime, and moxalactam - were injected subcutaneously into mice twice a day. The animals divided into treatment groups for each antibiotic and then into subgroups, each subgroup receiving a different dose of antibiotic. The effect of treatment on the resistance of the digestive tract to colonization and the effect of treatment on endogenous gram-negative and intestinal streptococcal flora was studied: resistance to colonization decreased during treatment with approximately 0.9 mg of antibiotic per mouse per day. During treatment with this dose or a higher dose, Escherichia coli or a strain of Enterobacter resistant to the antibiotic being used grew to significantly higher numbers per gram of feces than in the control groups. The resistance of the digestive tract to colonization in mice decreases during systemic treatment with all four antibiotics at or above certain dose levels, a result also likely to occur in humans.

Animals