Biomedical subjects

N Klijn

Publications and source records attributed to N Klijn.

7 recordsLinked to original sources

PubMed2001

Heat inactivation data for Mycobacterium avium subsp. paratuberculosis: implications for interpretation.

AIMS: We discuss several factors that are critical for heat inactivation experiments and which should be taken into account for future research. METHODS AND RESULTS: On the basis of examples from the literature we discuss critical factors influencing the calculated heat inactivation of Mycobacterium avium subsp. paratuberculosis (MAP). Furthermore, using a modelling approach, we show that tailing of the inactivation curve of MAP is caused by the presence of cell clumps and not by a more heat-resistant cell fraction. CONCLUSIONS: The experimental conditions of the MAP heat inactivation studies of different research groups vary significantly and lead to considerable differences in results and conclusions. Therefore, a more consensual approach should be employed in future studies. In addition, our model on clumping of MAP can be used to predict the decimal reduction of MAP during heat treatment and to study the effect of clumping on other lethal effects. SIGNIFICANCE AND IMPACT OF THE STUDY: We discuss several factors that should be carefully considered in heat resistance experiments. This is essential for a thorough interpretation of results from experiments and should be given proper attention in future experiments and publications on this topic.

Animals↗

PubMed2000

Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods.

BACKGROUND: An obvious difference between breast-fed and formula-fed newborn infants is the development of the intestinal flora, considered to be of importance for protection against harmful micro-organisms and for the maturation of the intestinal immune system. In this study, novel molecular identification methods were used to verify the data obtained by traditional culture methods and to validate the culture independent fluorescent in situ hybridization (FISH) technique. METHODS: From each of six breast-fed and six formula-fed newborn infants, six fecal samples were obtained during the first 20 days of life. The microbial compositions of the samples were analyzed by culturing on specific media and by FISH, by using specific 16S rRNA-targeted oligonucleotide probes. The colonies growing on the media were identified by random amplified polymorphic DNA pattern analysis and by polymerase chain reaction amplification and subsequent analysis of the 16S rRNA gene. RESULTS: Molecular identification of the colonies showed that the selective media are insufficiently selective and unsuitable for quantitative analyses. Qualitative information from the culturing results combined with the data obtained by the FISH technique revealed initial colonization in all infants of a complex (adult-like) flora. After this initial colonization, a selection of bacterial strains began in all infants, in which Bifidobacterium strains played an important role. In all breast-fed infants, bifidobacteria become dominant, whereas in most formula-fed infants similar amounts of Bacteroides and bifidobacteria (approximately 40%) were found. The minor components of the fecal samples from breast-fed infants were mainly lactobacilli and streptococci; samples from formula-fed infants often contained staphylococci, Escherichia coli, and clostridia. CONCLUSIONS: This study confirms the differences in development of intestinal flora between breast-fed and formula-fed infants. The results obtained from the FISH technique were consistent. Although the repertoire of probes for this study was not yet complete, the FISH technique will probably become the method of reference for future studies designed to develop breast-fed-like intestinal flora in formula-fed infants.

Bacteroides↗

PubMed1997

Discrimination between Bacillus cereus and Bacillus thuringiensis using specific DNA probes based on variable regions of 16S rRNA.

Identification of Bacillus cereus and differentiation between B. cereus and closely related species are currently based on biochemical tests. The main problem is to discriminate between B. cereus and B. thuringiensis. Sequencing part of the 16S rRNA showed that several B. cereus isolates present in food and involved in food poisoning, confirmed according to the classical biochemical methods, were in fact B. thuringiensis. As this organism is the most commonly used microbial insecticide worldwide, the results of this study emphasize the need for accurate identification methods and for careful screening of strains for use as insecticides. Therefore, specific DNA probes based on the variable region VI of 16S rRNA of B. cereus and B. thuringiensis were designed. The probes were used in hybridization experiments with the variable region amplified using the polymerase chain reaction. In this way, a rapid and sensitive method was developed to distinguish B. cereus and B. thuringiensis.

Bacillus cereus↗

PubMed1995

Detection and characterization of lactose-utilizing Lactococcus spp. in natural ecosystems.

The presence of lactose-utilizing Lactococcus species in nondairy environments was studied by using identification methods based on PCR amplification and (sub)species-specific probes derived from 16S rRNA sequences. Environmental isolates from samples taken on cattle farms and in the waste flow of a cheese production plant were first identified to the genus level, using a Lactococcus genus-specific probe. Isolates which showed a positive signal with this probe were further identified to the (sub)species level. Lactococcus lactis isolates were also characterized at the phenotypic level for the ability to hydrolyze arginine, to ferment citrate, and to produce proteases and bacteriocins. With specific PCR amplifications, the presence of sequences related to citP, coding for citrate permease; prtP, coding for protease; and nisA or nisZ, the structural genes for production of nisin A or nisin Z, respectively, was verified. By these methods, it was possible to isolate lactococci from various environmental sources, such as soil, effluent water, and the skin of cattle. The strains of L. lactis isolated differed in a number of properties, such as the ability to hydrolyze arginine or the absence of citP-related sequences, from those found in industrial starter cultures. The results indicate that the majority of the industrially produced lactococci do not survive outside the dairy environment, although natural niches are available. However, from those niches strains with the potential to be developed into novel starter cultures may be isolated.

Animals↗

PubMed1995

Genetic marking of Lactococcus lactis shows its survival in the human gastrointestinal tract.

A human feeding study was performed with Lactococcus lactis TC165.5, which is genetically marked by insertion of the sucrose-nisin conjugative transposon Tn5276 and chromosomal resistance to rifampin and streptomycin. The fate of strain TC165.5 and its nucleic acids was monitored by conventional plating methods and by molecular detection techniques based on specific PCR amplification of the nisin (nisA) gene from DNA extracted from human feces. A method was developed for the efficient extraction of microbial DNA from human feces. The results show that a fraction of viable cells of L. lactis TC165.5 survived passage through the human gastrointestinal tract. Only cells that passed within 3 days of ingestion could be recovered from the feces of the volunteers, and they accounted for approximately 1% of the total number of cells consumed. The presence of nisA in DNA extracted from feces could be detected up to 4 days, when viable cells were no longer present.

Base Sequence↗

PubMed1995

Identification of Clostridium tyrobutyricum as the causative agent of late blowing in cheese by species-specific PCR amplification.

Butyric acid fermentation, the late-blowing defect in cheese, caused by the outgrowth of clostridial spores present in raw milk, can create considerable loss of product, especially in the production of semihard cheeses like Gouda cheese, but also in grana and Gruyère cheeses. To demonstrate the causative relationship between Clostridium tyrobutyricum and late blowing in cheese, many cheesemaking experiments were performed to provoke this defect by using spores from several strains of the major dairy-related clostridia. A method of PCR amplification of a part of the 16S rRNA gene in combination with hybridization with species-specific DNA probes was developed to allow the specific detection of clostridial sequences in DNAs extracted from cheeses. The sensitivity was increased by using nested PCR. Late blowing was provoked in experimental cheeses with 28 of the 32 C. tyrobutyricum strains tested, whereas experimental cheeses made with spores from C. beijerinckii, C. butyricum, and C. sporogenes showed no signs of butyric acid fermentation. In all experimental and commercial cheeses with obvious signs of late blowing, DNA from C. tyrobutyricum was detected; in some cheeses, signals for C. beijerinckii were also found. It was concluded that only C. tyrobutyricum strains are able to cause butyric acid fermentation in cheese.

Base Sequence↗

PubMed1991

Identification of mesophilic lactic acid bacteria by using polymerase chain reaction-amplified variable regions of 16S rRNA and specific DNA probes.

Specific DNA probes based on variable regions V1 and V3 of 16S rRNA of lactic acid bacteria were designed. These probes were used in hybridization experiments with variable regions amplified by using the polymerase chain reaction. In this way, a rapid and sensitive method was developed for the identification and classification of Lactococcus and Leuconostoc species.

Base Sequence↗