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Biomedical subjects

Lieve Herman

Publications and source records attributed to Lieve Herman.

18 recordsLinked to original sources
PubMed2006

Survival and stress induced expression of groEL and rpoD of Campylobacter jejuni from different growth phases.

Although Campylobacter jejuni is the leading cause of bacterial diarrhoeal disease in humans worldwide, its potential to adapt to the stressful conditions and survive in extra-intestinal environment is still poorly understood. We tested the effect of heat shock (55 degrees C, 3 min) and oxidative stress (3 mM H2O2 for 10 min or prolonged incubation at atmosphere oxygen concentration) on non-starved and starved cells of Campylobacter jejuni from different growth phases. Viability as assessed with the Bacterial Viability Kit LIVE/DEAD BacLighttrade mark dying before fluorescent microscopy and culturability of the cells (CFU ml(-1)) from both growth phases showed that starvation increased heat but not oxidative resistance. High temperature and oxidative stress invoked quick transformation from culturable spiral shaped to nonculturable spiral and coccoid cells. Despite physiological changes of the cells we were not able to document clear differences in the expression of heat shock and starvation genes (dnaK, htpG, groEL), oxidative (ahpC, sodB), virulence (flaA) and housekeeping genes (16S rRNA, rpoD) after heat treatment (55 degrees C, 3 min) or oxidative stresses applied. When starving, no induction of expression of any of these genes was noticed, chloramphenicol had no influence on their gene expression. Quantitative real-time PCR analyses showed that at least 10-20 min of heat shock was necessary to evidently increase the amount of groEL and rpoD transcripts.

Adaptation, Physiological↗
PubMed2006

Differential inlA and inlB expression and interaction with human intestinal and liver cells by Listeria monocytogenes strains of different origins.

In this study, a number of Listeria monocytogenes strains of different origins were evaluated for in vitro invasion capacity for various human cell types (monocytic THP-1, enterocytic Caco-2, and hepatocytic HepG2 cells) and for expression levels of specific virulence genes. For THP-1 cells, no differences between clinical and nonclinical L. monocytogenes strains in invasion capacity or in production of the proinflammatory cytokine interleukin-8 (IL-8) were observed, whereas for the Caco-2 and HepG2 cells, significant differences in invasion capacity were noticed. On average, the clinical strains showed a significantly lower invasion capacity than the nonclinical L. monocytogenes strains. Furthermore, it was shown that the clinical strains induce lower IL-8 levels in HepG2 cells than do the nonclinical strains. This observation led us to study the mRNA expression levels of inlA, inlB, and ami, important virulence genes mediating adhesion and invasion of eukaryotic cells, by real-time reverse transcription-PCR for 27 clinical and 37 nonclinical L. monocytogenes strains. Significant differences in inlA and inlB expression were observed, with clinical strains showing a lower expression level than nonclinical strains. These observations were in accordance with in vitro invasion of Caco-2 and HepG2 cells, respectively. The results of this study indicate that differential expression levels of inlA and inlB possibly play a role in the virulence capacities of L. monocytogenes strains. The lower capacity of clinical strains to invade HepG2 cells and to induce IL-8 is possibly a mechanism of immune evasion used by specific L. monocytogenes strains.

Agglutination Tests↗
PubMed2006

Influence of eggshell condensation on eggshell penetration and whole egg contamination with Salmonella enterica serovar enteritidis.

Shells of agar-filled and whole eggs were inoculated with 10(3) to 10(4) CFU of Salmonella enterica serovar Enteritidis per eggshell. The agar-filled eggs were used to study bacterial eggshell penetration, and the whole egg results were used to characterize contamination of the egg contents. In each group, half of the eggs were stored for 21 days at 20 degrees C and 60% relative humidity (RH), and the other half was stored for 24 h at 6 degrees C and then for 20 days at 20 degrees C. The latter conditions resulted in condensation on the eggshell for 30 min from the moment the eggs were placed in the 20 degrees C chamber. Taking into account the ages at which hens were studied (39, 53, and 67 weeks), an average of 62% of the eggshells with condensate were penetrated compared with 43% for the control group; this difference was significant (P < 0.01). No significant difference in whole egg contamination was found; 18% of the control eggs were contaminated compared with 22% of the condensate eggs. Whole egg contamination was significantly higher for eggs from the hens at an older age (67 weeks). This difference probably was not due to a higher penetration potential because differences were not observed for the corresponding agar-filled eggs. Condensation on the eggshell seemed to encourage bacterial penetration of the eggshell but had a smaller impact on whole egg contamination.

Age Factors↗
PubMed2005

Real-time reverse transcription PCR for the quantification of the mntH expression of Salmonella enterica as a function of growth phase and phagosome-like conditions.

This article presents an experimental design for measuring the mRNA expression in Salmonella enterica of the mntH gene in phagosome-mimicking conditions. The expression of mntH was quantified by real-time reverse transcription PCR for different S. enterica strains of porcine origin under different biological growth conditions which mimicked the environment inside the phagosome. The expression of mntH and the different control genes (16S rRNA, rpoD and gmk) varied according to the growth phase. For mntH a maximum in the expression was detected in the early exponential phase. To obtain an accurate quantification and reliable comparison of the mntH expression in different S. enterica strains under various biological conditions, the ratio mntH mRNA level to the normalization factor was determined. The latter is the geometric mean of the RNA level of three housekeeping genes 16S rRNA, rpoD and gmk calculated by the geNorm program. MntH was basally expressed in all tested S. enterica strains and induced by hydrogen peroxide (H(2)O(2)) or ethylenediaminetetraacetic acid (EDTA) in Brain Heart Infusion. Under the nutrient limiting conditions of Sauton medium, the basal mntH expression was higher than in BHI, whereas H(2)O(2) induced the expression 40 times. A similar induction was obtained for Salmonella in porcine peripheral blood monocytes (PBM).

Animals↗
PubMed2005

Improved detection of Mycobacterium paratuberculosis in milk.

At present there is no rapid microbiological method for the detection of viable Mycobacterium paratuberculosis in milk. By combining an extensive milk sample pretreatment with solid phase cytometry as the detection technique we were able to demonstrate viable mycobacterial cells in 50 ml of artificially contaminated pasteurized milk in less than one working day.

Animals↗
PubMed2005

Incidence and diversity of potentially highly heat-resistant spores isolated at dairy farms.

The presence of highly heat-resistant spores of Bacillus sporothermodurans in ultrahigh-temperature or sterilized consumer milk has emerged as an important item in the dairy industry. Their presence is considered undesirable since they hamper the achievement of commercial sterility requirements. By using a selective 30-min heat treatment at 100 degrees C, 17 Belgian dairy farms were screened to evaluate the presence, sources, and nature of potentially highly heat-resistant spores in raw milk. High numbers of these spores were detected in the filter cloth of the milking equipment and in green crop and fodder samples. About 700 strains were isolated after the selective heating, of which 635 could be screened by fatty acid methyl ester analysis. Representative strains were subjected to amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, percent G+C content, and DNA-DNA reassociations for further identification. The strain collection showed a remarkable diversity, with representatives of seven aerobic spore-forming genera. Bacillus licheniformis and Bacillus pallidus were the most predominant species overall. Twenty-three percent of the 603 spore-forming isolates proved to belong to 18 separate novel species. These findings suggest that the selective heating revealed a pool of unknown organisms with a higher heat-resistant character. This study showed that high spore counts can occur at the dairy farm and that feed and milking equipment can act as reservoirs or entry points for potentially highly heat-resistant spores into raw milk. Lowering this spore load by good hygienic measures could probably further reduce the contamination level of raw milk, in this way minimizing the aerobic spore-forming bacteria that could lead to spoilage of milk and dairy products. Assessment and characterization of this particular flora are of great importance to allow the dairy or food industry to adequately deal with newly arising microbiological problems.

Animals↗
PubMed2005

Molecular and biochemical analysis of the galactose phenotype of dairy Streptococcus thermophilus strains reveals four different fermentation profiles.

Lactose-limited fermentations of 49 dairy Streptococcus thermophilus strains revealed four distinct fermentation profiles with respect to galactose consumption after lactose depletion. All the strains excreted galactose into the medium during growth on lactose, except for strain IMDOST40, which also displayed extremely high galactokinase (GalK) activity. Among this strain collection eight galactose-positive phenotypes sensu stricto were found and their fermentation characteristics and Leloir enzyme activities were measured. As the gal promoter seems to play an important role in the galactose phenotype, the galR-galK intergenic region was sequenced for all strains yielding eight different nucleotide sequences (NS1 to NS8). The gal promoter played an important role in the Gal-positive phenotype but did not determine it exclusively. Although GalT and GalE activities were detected for all Gal-positive strains, GalK activity could only be detected for two out of eight Gal-positive strains. This finding suggests that the other six S. thermophilus strains metabolize galactose via an alternative route. For each type of fermentation profile obtained, a representative strain was chosen and four complete Leloir gene clusters were sequenced. It turned out that Gal-positive strains contained more amino acid differences within their gal genes than Gal-negative strains. Finally, the biodiversity regarding lactose-galactose utilization among the different S. thermophilus strains used in this study was shown by RAPD-PCR. Five Gal-positive strains that contain nucleotide sequence NS2 in their galR-galK intergenic region were closely related.

Bacterial Proteins↗
PubMed2005

Welfare, health, and hygiene of laying hens housed in furnished cages and in alternative housing systems.

The aim of this review was to compare welfare, health, and hygienic status of laying hens housed in furnished cages and in alternative systems. In alternative systems (floor housing and aviaries), birds have more freedom of movement and a more complex environment than in furnished cages. However, housing birds in much larger flocks in alternative systems leads to an increased risk of feather-pecking. Furthermore, air quality can be poorer in alternative systems than in furnished cages. This can affect health and hygienic status. There are only limited data on a direct comparison between furnished cages and alternative systems. Therefore, there is a need for an on-farm comparison of welfare, health, and hygienic status in these systems.

Aggression↗
PubMed2004

Comparison of selective and nonselective primary enrichments for the detection of Listeria monocytogenes in cheese.

A completely selective enrichment procedure was compared with two partially nonselective ones for the detection of Listeria monocytogenes in cheeses. After enrichment for approximately 48 h, the enrichment media were streaked on selective agars and presumptive Listeria colonies were confirmed using PCR. In some cases, PCR was also performed directly on the enrichment broth. The conventional, completely selective enrichment procedure was not always the best choice for the detection of stressed L. monocytogenes in cheeses. Especially in the case of semi-hard cheeses from pasteurized milk and soft cheeses of the blue veined and the red smear types, the methods that incorporated a nonselective enrichment step gave better results than the completely selective method. For mold ripened, soft cheeses, the results were highly dependent on the brand of cheese and time of sampling, but the best results were obtained using the completely selective enrichment procedure.

Agar↗
PubMed2004

Paenibacillus lactis sp. nov., isolated from raw and heat-treated milk.

Endospore-forming bacteria were recovered from individual packages from different processing lines in a dairy plant during a tenacious periodical contamination of their UHT-milk production. Two colony types were seen, one of which was identified as Bacillus sporothermodurans. Analysis of the 16S rRNA gene of the second colony type placed these isolates within the genus Paenibacillus, with Paenibacillus lautus as the closest known relative. Moreover, over 99 % similarity was observed to the 16S rDNA sequence of MB 2035, a strain isolated previously from raw milk during a survey at dairy farms for very heat-resistant spore-forming bacteria. Nine other potentially closely related strains among the dairy farm isolates were found using rep-PCR typing. The taxonomic positions of these 19 isolates were further investigated using 16S rRNA gene sequencing and DNA-DNA hybridizations of representative strains. All 19 isolates shared a high degree of phenotypic similarity and were easily distinguished from closely related members of the genus. Anteiso-C(15 : 0), C(16 : 0) and iso-C(15 : 0) were among the major fatty acids and the genomic DNA G+C content was 51.6-51.7 mol%. Therefore, based on their phenotypic, phylogenetic and genomic distinctiveness, these 19 strains, isolated from both raw and heat-treated milk, are placed in the genus Paenibacillus as Paenibacillus lactis sp. nov. The type strain is MB 1871(T) (=LMG 21940(T)=DSM 15596(T)).

Animals↗
PubMed2004

Bacillus farraginis sp. nov., Bacillus fortis sp. nov. and Bacillus fordii sp. nov., isolated at dairy farms.

Forty-eight bacterial strains were isolated at dairy farms from raw milk, the milking apparatus, green fodder or feed concentrate after a heat treatment of 30 min at 100 degrees C. In this way, spore-forming bacteria with a very high intrinsic heat resistance were selected for. The aerobic spore-forming isolates were subjected to a polyphasic taxonomical study, including repetitive element sequence-based PCR typing, whole-cell protein profiling, 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base composition, fatty acid analysis, and morphological and biochemical characteristics. A comparison of the REP- and (GTG)5-PCR and whole-cell protein SDS-PAGE profiles resulted in three clusters of similar strains. Analysis of the 16S rDNA sequences and DNA-DNA relatedness data showed that these clusters represented three novel species. The highest 16S rDNA similarity to a recognized species found for the three groups was around 94% with Bacillus lentus and Bacillus sporothermodurans. Further phenotypic characterization supported the proposal of three novel species in the genus Bacillus, Bacillus farraginis, Bacillus fortis and Bacillus fordii. The respective type strains are R-6540T (=LMG 22081T=DSM 16013T), R-6514T (=LMG 22079T=DSM 16012T) and R-7190T (=LMG 22080T=DSM 16014T); their G+C DNA base contents are 43.7, 44.3 and 41.9 mol%, respectively. Although in variable amounts, a predominance of the branched fatty acids iso-C(15 : 0) and anteiso-C(15 : 0) was observed in all three novel species.

Aerobiosis↗
PubMed2004

Phenotypic and molecular typing of Salmonella strains reveals different contamination sources in two commercial pig slaughterhouses.

This study aimed to define the origin of Salmonella contamination on swine carcasses and the distribution of Salmonella serotypes in two commercial slaughterhouses during normal activity. Salmonellae were isolated from carcasses, from colons and mesenteric lymph nodes of individual pigs, and from the slaughterhouse environment. All strains were serotyped; Salmonella enterica serotype Typhimurium and Salmonella enterica serotype Derby isolates were additionally typed beyond the serotype level by pulsed-field gel electrophoresis (PFGE) and antibiotic resistance profiling (ARP); and a subset of 31 serotype Typhimurium strains were additionally phage typed. PFGE and ARP had the same discriminative possibility. Phage typing in combination with PFGE could give extra information for some strains. In one slaughterhouse, 21% of the carcasses were contaminated, reflecting a correlation with the delivery of infected pigs. Carcass contamination did not result only from infection of the corresponding pig; only 25% of the positive carcasses were contaminated with the same serotype or genotype found in the corresponding feces or mesenteric lymph nodes. In the other slaughterhouse, 70% of the carcasses were contaminated, and only in 4% was the same genotype or serotype detected as in the feces of the corresponding pigs. The other positive carcasses in both slaughterhouses were contaminated by genotypes present in the feces or lymph nodes of pigs slaughtered earlier that day or from dispersed sources in the environment. In slaughterhouses, complex contamination cycles may be present, resulting in the isolation of many different genotypes circulating in the environment due to the supply of positive animals and in the contamination of carcasses, probably through aerosols.

Abattoirs↗
PubMed2004

Prevalence and typing of Listeria monocytogenes in ready-to-eat food products on the Belgian market.

Listeria monocytogenes is a major concern to producers of ready-to-eat foods because of the high mortality rate associated with listeriosis and the widespread nature of the organism. To investigate the prevalence of this pathogen in different ready-to-eat food products on the Belgian market, a variety of 252 ready-to-eat food products, mainly fish and meat products, were analyzed. Overall, L. monocytogenes was detected in 23.4% of the samples. The highest prevalence of L. monocytogenes was found in prepared minced meat (42.1%) and smoked halibut (33.3%). Contamination levels were in most cases low (<10 CFU/g); however, levels higher than 100 CFU/g were detected in some samples of smoked salmon, smoked halibut, and prepared minced meat. A high prevalence of Listeria innocua (15.8%) and Listeria welshimeri (36.8%) was detected in prepared minced meat. L. monocytogenes strains isolated from different contaminated products were subjected to repetitive element sequence-based PCR (REP-PCR) typing to determine possible associations with product type, producer, or market. REP-PCR patterns were analyzed using BioNumerics software, and seven different groups with at least 90% similarity were identified. The cluster analysis indicates that cross-contamination occurred at the producer and retail level. Serotype identification of the strains by PCR revealed that most belonged to the 1/2a(3a) serotype group.

Animals↗
PubMed2003

Inactivation of Salmonella enteritidis during boiling of eggs.

A series of inactivation curves for Salmonella enteritidis were determined for boiling eggs using different conditions of time and temperature. No significant influence of egg weight could be found on the temperature evolution in the yolk. The inactivation curves consistently showed an initial slow decline in bacterial number at lower temperatures, after which a very rapid inactivation took place. It was not possible to reproduce this behavior using a traditional inactivation model. A pragmatic model existing in two parts was therefore constructed. When the temperature is below a certain threshold, the inactivation follows a second order temperature dependence. Above the temperature threshold, standard Bigelow inactivation kinetics are assumed. This model could describe the data reasonably well, provided that the decimal reduction time in the Bigelow model was assumed to be different for a fast or slow heating process, respectively. The results suggest that the bacteria are more resistant towards a slower heating process, which is confirmed by analyzing the raw data. A fail-safe model can be obtained by using the parameters associated with the slow heating process. The statistical properties of the calibrated model are satisfactory, and a cross-validation shows that it can be used for egg boiling conditions outside its calibration range.

Animals↗
PubMed2003

Detection and characterization of verotoxigenic Escherichia coli by a VTEC/EHEC multiplex PCR in porcine faeces and pig carcass swabs.

To determine whether pigs and pig carcasses are a reservoir of verotoxigenic-producing Escherichia coli, a sensitive and rapid isolation method was developed. A VTEC/EHEC multiplex PCR method was developed to test all samples taken at the farm (n=289) and at the slaughterhouse (n=233). From the 137 positive VTEC/EHEC multiplex PCR farm samples, 54 strains were isolated that carried one virulence gene. Of these, thirty-one strains carried the vt2e variant of the vt2 gene, which causes oedemic disease in young pigs, four strains contained the hlyA gene and 19 the eaeA gene. All strains isolated were further identified with an enterotoxigenic E. coli (ETEC) multiplex PCR developed to detect the LTI, STIa, STII and vt2 genes in a single reaction. One-third of the vt2e-positive strains also contained the STIa gene. A small fraction of the 132 carcasses tested were found positive for VTEC or the E. coli O157 serotype. We conclude that none of the isolated strains carried a combination of virulence genes indicative of potential human pathogenic characteristics.

Abattoirs↗
PubMed2002

Genetic heterogeneity in Bacillus sporothermodurans as demonstrated by ribotyping and repetitive extragenic palindromic-PCR fingerprinting.

Thirty-eight strains of Bacillus sporothermodurans isolated from ultra-high-temperature (UHT)-treated milk or sterilized milk (UHT isolates) and from animal feed or raw milk (farm isolates) were characterized by automated ribotyping and by repetitive extragenic palindromic (REP)-PCR fingerprinting. By investigating the genetic relationships among isolates from these various sources, the relative importance of different contamination sources could be evaluated. The results of the separate clustering analyses of the PvuII and EcoRI ribopatterns and the REP-PCR patterns were largely consistent with each other and revealed the existence of two main clusters; there was one homogeneous group containing all (REP-PCR) or most (ribotyping) of the UHT isolates, and there was a second more diverse group comprising the farm isolates. A combined three-dimensional analysis of all data showed that three German UHT isolates did not belong to the compact group containing the majority of the UHT isolates. These results demonstrate that B. sporothermodurans is more heterogeneous than previously assumed and that most of the UHT isolates form a genetically distinct subgroup and are capable of producing highly heat-resistant spores. The close genetic relationship of these UHT isolates suggests a clonal origin of a few predominant strains of B. sporothermodurans that can be found in UHT-treated or sterilized milk products.

Bacillus↗
PubMedDate not supplied

Genomic and phenotypic comparison of Bacillus fumarioli isolates from geothermal Antarctic soil and gelatine.

Bacillus fumarioli was originally isolated from geothermal soils in continental and maritime Antarctica, and recently, it has been shown to be a frequent contaminant of gelatine extracts obtained from European and American production plants. These habitats are geographically widely separated, share similar temperature and pH conditions, but have substantially different organic loads. Because of the prevalence in gelatine extracts and the dissimilarity of this habitat to geothermal soil, a comparative study was performed to assess the diversity among B. fumarioli strains and reveal possible intraspecies differences that might correspond to their niches of origin. Genomic (rep-PCR, 16S rDNA sequencing, DNA-DNA hybridisations) and phenotypic techniques (analysis of fatty acid content, total cellular proteins, metabolic and morphological traits) illustrate the very close relationship between isolates from the two niches. An abundant protein band was demonstrated for gelatine isolates only. This band was shown to result from a protein with high similarity to a stress response protein. Furthermore, subtractive hybridisation revealed genomic differences between Antarctic and gelatine isolates that may indicate adaptive evolution to a specific environment.

Antarctic Regions↗