Biomedical subjects

R J Willems

Publications and source records attributed to R J Willems.

At least 19 recordsLinked to original sources

PubMed2005

Streptococcal toxic shock syndrome by an iMLS resistant M type 77 Streptococcus pyogenes in the Netherlands.

An increasing number of group A streptococci (GAS) with constitutive or inducible resistance to macrolide-lincosamide-streptogramin B antibiotics (cMLS or iMLS phenotype) is observed in Europe, but MLS resistant GAS associated with streptococcal toxic shock syndrome (STSS) has not been reported. We describe a patient admitted with STSS caused by an iMLS resistant T28 M77 Streptococcus pyogenes carrying the ermA [subclass TR] gene. A 2-y retrospective analysis among 701 nationwide collected GAS strains revealed an incidence of 3.1% of this M type 77 GAS. Analysis of 17 available M77 strains (12 T28 and 5 T13) indicated that 2 (12%) were MLS resistant due to the ermA [TR] gene. Both MLS resistant strains were cultured from blood and belonged to T28 serotype. Multilocus sequence typing (MLST) showed that all M77 isolates belonged to sequence type 63. We conclude that 17 M77 GAS collected in the Netherlands in a 2-y period were associated with invasive disease and belonged to the same clonal complex. Since only 12% carried the ermA [TR] resistance gene, it is very likely that the gene has been acquired by horizontal transmission rather than from spread of a resistant circulating clone.

Adult↗

PubMed2004

[Vancomycin resistant enterococci in the Netherlands].

Enterococci (Enterococcus faecalis and Enterococcus faecium) are relatively avirulent enteric bacteria that usually only cause infections in immunocompromised patients. Antimicrobial treatment, however, is hampered as enterococci are intrinsically resistant to many antibiotics. For years, vancomycin was considered the last available antibiotic. Plasmid-mediated resistance against vancomycin among enterococci was first described in the nineteen-eighties and since then incidences of infection caused by vancomycin-resistant enterococci (VRE) have increased dramatically, especially in the United States. In 2000, three outbreaks of VRE occurred in hospitals in the Netherlands and a set of infection-control measures was proposed to limit further transmission. These measures were based on the simultaneous isolation of VRE from multiple patients. All three outbreaks were controlled by these measures and no new outbreaks in Dutch hospitals have been reported since then. Epidemiological studies have shown that hospital outbreaks on three continents were caused by a subpopulation of E. faecium, which is characterized by the presence of a potential virulence gene (variant esp) and resistance to amoxicillin. This 'hospital strain' of E. faecium has probably been prevalent within hospital settings for some time, but only became clinically relevant when it had acquired vancomycin-resistance. Current advice is to implement the set of infection control measures formulated in 2000, only in those patients colonized by amoxicillin-resistant VRE. The potential dangers of VRE were recently underlined by the proven transmission of the vancomycin-resistance gene from VRE to methicillin-resistant Staphylococcus aureus (MRSA) in two patients in the United States. It is in the interest of the patients that prevalence of VRE and MRSA in Dutch hospitals should be kept as low as possible.

Disease Outbreaks↗

PubMed2001

Variant esp gene as a marker of a distinct genetic lineage of vancomycin-resistant Enterococcus faecium spreading in hospitals.

In the USA, vancomycin-resistant Enterococcus faecium (VREF) is endemic in hospitals, despite lack of carriage among healthy individuals. In Europe, however, hospital outbreaks are rare, but VREF carriage among healthy individuals and livestock is common. We used amplified fragment-length polymorphism analysis to genotype 120 VREF isolates associated with hospital outbreaks and 45 non-epidemic isolates from the USA, Europe, and Australia. We also looked for the esp virulence gene in these isolates and in 98 VREF from animals. A specific E. faecium subpopulation genetically distinct from non-epidemic VREF isolates was found to be the cause of the hospital epidemics in all three continents. This subpopulation contained a variant of the esp gene that was absent in all non-epidemic and animal isolates. Identification of the variant esp gene will be important in guiding infection-control strategies, and the Esp protein could be a new target for antibacterial therapy.

Bacterial Proteins↗

PubMed2001

A reappraisal of pacemaker timing cycles pertaining to automatic mode switching.

Automatic mode switching algorithms of dual chamber pacemakers require fundamental changes in the operation of pacemaker timing cycles to optimize detection of supraventricular tachyarrhythmias. The timing cycles related to mode switching are basically independent of the algorithm design. Blanking periods (when the sensing amplifier is temporarily disabled) should be optimized to a relatively small fraction of the pacing cycle to enhance atrial sensing and prevent far-field sensing. This review explains the function of the timing cycles pertaining to mode switching and proposes simpler terminology to facilitate the understanding of pacemaker function and electrographic interpretation of complex recordings.

Algorithms↗

PubMed2001

Molecular analysis of Tn1546-like elements in vancomycin-resistant enterococci isolated from patients in Europe shows geographic transposon type clustering.

Resistance mechanism relatedness was studied in 18 clinical, European vanA vancomycin-resistant enterococci. Molecular analysis revealed 10 Tn1546-like elements, suggesting two evolutionary lineages. Lineage I dominated the European mainland, and lineage II dominated the United Kingdom and Israel. Geographic clustering reflected different types of meat consumption between countries, since each lineage is associated with colonization of different animals.

Adolescent↗

PubMed2001

Multilocus sequence typing system for Campylobacter jejuni.

The gram-negative bacterium Campylobacter jejuni has extensive reservoirs in livestock and the environment and is a frequent cause of gastroenteritis in humans. To date, the lack of (i) methods suitable for population genetic analysis and (ii) a universally accepted nomenclature has hindered studies of the epidemiology and population biology of this organism. Here, a multilocus sequence typing (MLST) system for this organism is described, which exploits the genetic variation present in seven housekeeping loci to determine the genetic relationships among isolates. The MLST system was established using 194 C. jejuni isolates of diverse origins, from humans, animals, and the environment. The allelic profiles, or sequence types (STs), of these isolates were deposited on the Internet (http://mlst.zoo.ox.ac.uk), forming a virtual isolate collection which could be continually expanded. These data indicated that C. jejuni is genetically diverse, with a weakly clonal population structure, and that intra- and interspecies horizontal genetic exchange was common. Of the 155 STs observed, 51 (26% of the isolate collection) were unique, with the remainder of the collection being categorized into 11 lineages or clonal complexes of related STs with between 2 and 56 members. In some cases membership in a given lineage or ST correlated with the possession of a particular Penner HS serotype. Application of this approach to further isolate collections will enable an integrated global picture of C. jejuni epidemiology to be established and will permit more detailed studies of the population genetics of this organism.

Alleles↗

PubMed2000

Host specificity of vancomycin-resistant Enterococcus faecium.

Amplified-fragment length polymorphism (AFLP) analysis was used to investigate the genetic relationships among 255 vancomycin-resistant Enterococcus faecium (VREF) strains isolated from hospitalized patients, nonhospitalized persons, and various animal sources. Four major AFLP genogroups (A-D) were discriminated. The strains of each taxon shared >/=65% of the restriction fragments. Most isolates recovered from nonhospitalized persons (75%) were grouped together with all pig isolates in genogroup A. Most isolates from hospitalized patients (84%), a subset of veal calf isolates (25%), and all isolates from cats and dogs clustered in genogroup C. Most isolates from chickens (97%) and turkeys (86%) were grouped in genogroup B, whereas most veal calf isolates (70%) clustered in genogroup D. Therefore, VREF strains are predominantly host-specific, and strains isolated from hospitalized patients are genetically different from the prevailing VREF strains present in the fecal flora of nonhospitalized persons.

Animals↗

PubMed2000

Antimicrobial resistance of Neisseria gonorrhoeae and emerging ciprofloxacin resistance in the Netherlands, 1991 to 1998.

Surveillance of antibiotic resistance in Neisseria gonorrhoeae showed a decrease in the percentage of beta-lactamase-producing isolates but an increase in intermediately penicillin-resistant strains and strains resistant to a high level of tetracycline. MICs for the ciprofloxacin-resistant isolates that emerged increased, and these isolates had mutations in gyrA and parC similar to those observed in the Far East.

Anti-Infective Agents↗

PubMed1999

A comparison of ELISA assays as routine diagnostic test for detection of autoantibodies against extractable nuclear antigens.

OBJECTIVE: In an analytical evaluation, commercially available ELISA test kits for detection of antibodies directed against extractable nuclear antigens (ENA) were compared with the currently used combination of counterimmunoelectrophoresis and immunoblotting. DESIGN: Three screening ELISAs and two typing ELISAs were tested. These methods were fairly simple, easy to perform and "user friendly," because most of the reagents were ready to use. RESULTS: The agreement with the current methods was good, but the screening as well as typing ELISAs proved to be more sensitive, especially with regard to detection of SS-A auto-antibodies. The cut-off range of one screening assay was not well established and one typing assay suffered from problems with inaccuracy of package insert, purity of antigen and standardisation of reactivity (possibly caused by differences in amount of coated antigen). The other three ELISAs were reliable and sensitive for detection of ENA auto-antibodies. CONCLUSIONS: The ELISA ENA screen assays ENA-LISA polyvalent and Milenia ENA screen and typing assays ENA-LISA are reliable and sensitive for detection of autoantibodies in clinical specimens without substantial false negatives.

Autoantibodies↗

PubMed1999

Molecular diversity and evolutionary relationships of Tn1546-like elements in enterococci from humans and animals.

We report on a detailed study on the molecular diversity and evolutionary relationships of Tn1546-like elements in vancomycin-resistant enterococci (VRE) from humans and animals. Restriction fragment length polymorphism (RFLP) analysis of the VanA transposon of 97 VRE revealed seven different Tn1546 types. Subsequent sequencing of the complete VanA transposons of 13 VRE isolates representing the seven RFLP types followed by sequencing of the identified polymorphic regions in 84 other VanA transposons resulted in the identification of 22 different Tn1546 derivatives. Differences between the Tn1546 types included point mutations in orf1, vanS, vanA, vanX, and vanY. Moreover, insertions of an IS1216V-IS3-like element in orf1, of IS1251 in the vanS-vanH intergenic region, and of IS1216V in the vanX-vanY intergenic region were found. The presence of insertion sequence elements was often associated with deletions in Tn1546. Identical Tn1546 types were found among isolates from humans and farm animals in The Netherlands, suggesting the sharing of a common vancomycin resistance gene pool. Application of the genetic analysis of Tn1546 to VRE isolates causing infections in Hospitals in Oxford, United Kingdom, and Chicago, Ill., suggested the possibility of the horizontal transmission of the vancomycin resistance transposon. The genetic diversity in Tn1546 combined with epidemiological data suggest that the DNA polymorphism among Tn1546 variants can successfully be exploited for the tracing of the routes of transmission of vancomycin resistance genes.

Animals↗

PubMed1999

Role of Bordetella pertussis virulence factors in adherence to epithelial cell lines derived from the human respiratory tract.

During colonization of the respiratory tract by Bordetella pertussis, virulence factors contribute to adherence of the bacterium to the respiratory tract epithelium. In the present study, we examined the roles of the virulence factors filamentous hemagglutinin (FHA), fimbriae, pertactin (Prn), and pertussis toxin (PT) in the adherence of B. pertussis to cells of the human bronchial epithelial cell line NCI-H292 and of the laryngeal epithelial cell line HEp-2. Using B. pertussis mutant strains and purified FHA, fimbriae, Prn, and PT, we demonstrated that both fimbriae and FHA are involved in the adhesion of B. pertussis to laryngeal epithelial cells, whereas only FHA is involved in the adherence to bronchial epithelial cells. For PT and Prn, no role as adhesion factor was found. However, purified PT bound to both bronchial and laryngeal cells and as such reduced the adherence of B. pertussis to these cells. These data may imply that fimbriae play a role in infection of only the laryngeal mucosa, while FHA is the major factor in colonization of the entire respiratory tract.

Adhesins, Bacterial↗

PubMed1998

The efficacy of a whole cell pertussis vaccine and fimbriae against Bordetella pertussis and Bordetella parapertussis infections in a respiratory mouse model.

Due to local and systemic side-effects, the currently used, highly effective, whole-cell pertussis vaccines (WCVs) will be replaced by acellular vaccines (ACVs) in some countries. These ACVs contain detoxified pertussis toxin, either alone or in combination with the filamentous haemagglutinin, pertactin and fimbriae. Ongoing clinical trials show that ACVs are clearly less reactogenic than WCVs and that ACVs comprised of three to five proteins are highly efficacious in inducing protection against Bordetella pertussis infections. An important unresolved question is, what the effect will be of the switch from WCVs to ACVs on the incidence of Bordetella parapertussis infections, the second causative agent of pertussis. A comparison of the efficacy of WCVs and ACVs against B. parapertussis infection is required to answer this question. We show that the Dutch WCV, although prepared from B. pertussis strains, protects against B. parapertussis infection in a murine respiratory model, although less efficiently than against B. pertussis infection. It was shown previously that the ACV components pertussis toxin, FHA and pertactin did not protect against B. parapertussis infection in a murine respiratory model. We have investigated the efficacy of two other ACV components, B. pertussis serotype-2 and -3 fimbriae against B. parapertussis infection in the murine model. The B. pertussis fimbriae protected mice against B. parapertussis infection although less efficiently than against B. pertussis infection. This result indicates that B. pertussis and B. parapertussis fimbriae are antigenically distinct. B. pertussis fimbriae were found to be as efficacious as the WCV against B. pertussis infection. Our results are discussed in the light of the switch from WCVs to ACVs.

Animals↗

PubMed1998

Polymorphism in the Bordetella pertussis virulence factors P.69/pertactin and pertussis toxin in The Netherlands: temporal trends and evidence for vaccine-driven evolution.

The Bordetella pertussis proteins P.69 (also designated pertactin) and pertussis toxin are important virulence factors and have been shown to confer protective immunity in animals and humans. Both proteins are used in the new generation of acellular pertussis vaccines (ACVs), and it is therefore important to study the degree of antigenic variation in these proteins. Sequence analysis of the genes for P.69 and the pertussis toxin S1 subunit, using strains collected from Dutch patients in the period 1949 to 1996, revealed three P.69 and three S1 variants which show differences in amino acid sequence. Polymorphism in P.69 was confined to a region comprised of repeats and located proximal to the RGD motif involved in adherence to host tissues. Variation in S1 was observed in two regions previously identified as T-cell epitopes. P.69 and S1 variants, identical to those included in the Dutch whole-cell pertussis vaccine (WCV), were found in 100% of the strains from the 1950s, the period when the WCV was introduced in The Netherlands. However, nonvaccine types of P.69 and S1 gradually replaced the vaccine types in later years and were found in approximately 90% strains from 1990 to 1996. These results suggest that vaccination has selected for strains which are antigenically distinct from vaccine strains. Analysis of strains from vaccinated and nonvaccinated individuals indicated that the WCV protects better against strains with the vaccine type P.69 than against strains with non-vaccine types (P = 0.024). ACVs contain P.69 and S1 types which are found in only 10% of recent Dutch B. pertussis isolates, implying that they do not have an optimal composition. Our findings cast a new light on the reemergence of pertussis in highly vaccinated populations and may have major implications for the long-term efficacy of both WCVs and ACVs.

Amino Acid Sequence↗

PubMed1998

Identification and characterization of heparin binding regions of the Fim2 subunit of Bordetella pertussis.

Bordetella pertussis fimbriae bind to sulfated sugars such as heparin through the major subunit Fim2. The Fim2 subunit contains two regions, designated H1 and H2, which show sequence similarity with heparin binding regions of fibronectin, and the role of these regions in heparin binding was investigated with maltose binding protein (MBP)-Fim2 fusion proteins. Deletion derivatives of MBP-Fim2 showed that both regions are important for binding to heparin. The role of H2 in heparin binding was confirmed by site-directed mutagenesis in which basic amino acids were replaced by alanine. These studies revealed that Lys-186 and Lys-187 are important for heparin binding of MBP-Fim2, whereas Arg-179 is not required. Peptides derived from H1 and H2 (pepH1 and pepH2) also showed heparin binding activity. Using a series of peptides, in each of which a different basic amino acid was substituted for alanine, we demonstrated that the structural requirements for heparin binding differ significantly among pepH1 and pepH2 peptides. A Pepscan analysis of Fim2 revealed regions outside H1 and H2 which bind heparin and showed that not only basic amino acids but also tyrosines may be important for binding to sulfated sugars. A comparison of the heparin binding regions of Fim2 with homologous regions of Fim3 and FimX, two closely related but antigenically distinct fimbrial subunits, showed that basic amino acids and tyrosines are generally conserved. The major heparin binding regions identified in Fim2 are part of epitopes recognized by human antibodies, suggesting that the heparin binding regions are exposed at the fimbrial surface and are immunodominant. Since B. pertussis fimbriae show weak serological cross-reactivity, the differences in primary structure in the heparin binding regions of Fim2, Fim3, and FimX may affect antibody binding but not heparin binding, allowing the bacteria to evade antibody-mediated immunity by switching the fimbrial gene expressed.

Amino Acid Sequence↗

PubMed1997

Role of the Bordetella pertussis minor fimbrial subunit, FimD, in colonization of the mouse respiratory tract.

Bordetella pertussis fimbriae are composed of a major subunit, Fim2 or Fim3, and the minor subunit FimD. Using immunoelectron microscopy, we provide evidence that FimD is located at the fimbrial tip. The role of FimD in colonization of the mouse respiratory tract was studied by using two fimbrial mutants: a mutant completely devoid of fimbriae (designated FimD-) and a mutant devoid of the major fimbrial subunits but still producing the minor subunit (designated FimD+). The ability of the two fimbrial mutants to colonize the nasopharynx, trachea, and lungs was compared with those of the wild type parental strain and a filamentous hemagglutinin (FHA) mutant. Of the three mutants studied, the FimD- mutant showed the greatest defect, colonizing less well in the nasopharynx, trachea, and lungs. The most pronounced defect in colonizing ability of the three mutants was observed in the trachea. However, the colonizing defect of the FHA and FimD+ mutants in the trachea was observed only during the first 3 days of infection. After 10 days, the colonization level was nearly restored to wild-type levels. The FHA and FimD+ mutants showed a slight colonization defect in the nasopharynx but no defect in the lungs. A maltose binding protein-FimD fusion protein and a peptide derived from FimD were able to bind to heparin, a member of a class of sulfated sugars which are ubiquitous in the respiratory tract. Recently it was shown (W. L. W. Hazenbos, C. A. W. Geuijen, B. M. van den Berg, F. R. Mooi, and R. van Furth, J. Infect. Dis. 171:924-929, 1995) that FimD also binds to the integrin VLA-5, and our results suggest that the binding of B. pertussis to these two molecules plays an important role in colonization of the respiratory tract of the mouse.

Adhesins, Bacterial↗

PubMed1996

Genetic organization and functional analysis of the otn DNA essential for cell-wall polysaccharide synthesis in Vibrio cholerae O139.

In 1992 a new Vibrio cholerae strain, designated V. cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh. Previously, we have shown that this strain arose from a V. cholerae O1 strain by the acquisition of novel DNA. Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains. The mosaic structure of rfaDVCO139 indicated that it was one of the regions involved in recombination between donor and acceptor DNA. However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second recombination site between donor and O1-acceptor DNA is probably located downstream of rfbDVCO139. The DNA region between rfaDVCO139 and rfbQRSVCO139, designated otn, contained seven open reading frames (ORFs). Two ORFs, otnA and otnB, showed homology with genes involved in cell-wall polysaccharide synthesis. Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis. The otn DNA is also found in V. cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain. However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains. No antigenic relationship was found between the different V. cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA. The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide-synthetic genes in several bacterial species. Furthermore, a repeat motif was observed in extragenic regions. A number of observations suggest that these sequences may facilitate gene flow between V. cholerae strains and the assembly of clusters of functionally related genes.

Antigens, Bacterial↗

PubMed1996

Characterization of the fim2 and fim3 fimbrial subunit genes of Bordetella bronchiseptica: roles of Fim2 and Fim3 fimbriae and flagella in adhesion.

With DNA probes derived from the fimbrial subunit genes fim2 and fim3 of Bordetella pertussis, two homologous subunit genes of Bordetella bronchiseptica were identified and cloned. The nucleotide sequences of these genes were determined. Comparison of these nucleotide sequences with the B. pertussis fimbrial fim2 and fim3 subunit genes showed a pronounced homology. Therefore, the B. bronchiseptica genes were also designated fim2 and fim3. Expression of the two B. bronchiseptica genes was demonstrated by Western blotting (immunoblotting) with polyclonal antiserum directed against the Fim2 and Fim3 fimbrial subunit proteins of B. pertussis and by enzyme-linked immunosorbent assay with monoclonal antibodies. After growth of B. bronchiseptica in the presence of MgSO4, no expression of both fimbrial subunit genes was observed. While no fimbriae were expressed, expression of flagella was observed under these circumstances. A longer C-stretch (up to 19 cytosine residues) than the one in front of the fim2 and fim3 genes of B. pertussis is present in front of the B. bronchiseptica fimbrial genes. In adherence experiments, fimbriated (Bvg+) as well as flagellated (Bvg-) B. bronchiseptica bacteria were able to adhere to HeLa cells, whereas nonflagellated B. pertussis did not. This suggests that fimbriae as well as other factors (possibly flagella) contribute to adherence of B. bronchiseptica to eukaryotic cells.

Amino Acid Sequence↗

PubMed1996

The major fimbrial subunit of Bordetella pertussis binds to sulfated sugars.

Bordetella pertussis fimbriae are composed of major and minor subunits, and recently it was shown that the minor fimbrial subunit binds to Vla-5, a receptor located on monocytes (W. Hazenbos, C. Geuijen, B. van den Berg, F. Mooi, and R. van Furth, J. Infect. Dis. 171:924-929, 1995). Here we present evidence that the major subunits bind to sulfated sugars, which are ubiquitous in the respiratory tract. Binding was observed to chondroitin sulfate, heparan sulfate, and dextran sulfate but not to dextran. Removal of the minor subunit from fimbriae did not significantly affect binding to sulfated sugars, indicating that the major subunit alone is sufficient for this binding. Fimbriae were also able to bind HEp-2 cells, which are known to display glycoconjugates on their surface. This binding was not dependent on the presence of the minor subunit. However, binding was dependent on the sulfation state of the glycoconjugates, since inhibition of the sulfation resulted in a significant reduction of fimbria binding. The specificity of fimbria binding was further characterized by using heparan sulfate-derived disaccharides in inhibition assays. Two disaccharides were highly effective inhibitors, and it was observed that both the degree of sulfation and the arrangement of the sulfate groups on the disaccharides were important for binding to fimbriae. B. pertussis bacteria also bound to sulfated sugars and HEp-2 cells, and analysis of B. pertussis mutants indicated that both filamentous hemagglutinin and fimbriae were required for this binding. A host protein present in the extracellular matrix, fibronectin, has binding activities similar to those of B. pertussis fimbriae, binding to both Vla-5 and sulfated sugars. Two regions in the major fimbrial subunit were identified which showed similarity with fibronectin peptides which bind to sulfated sugars. Thus, B. pertussis fimbriae exemplify molecular mimicry and may co-opt host processes by mimicking natural ligand-receptor interactions.

Amino Acid Sequence↗