Gaëtan Muyldermans

Neonatal infections with Pseudomonas aeruginosa associated with a water-bath used to thaw fresh frozen plasma

In our 15-bed neonatal intensive care unit (NICU), four new-borns were found to be colonized or infected with Pseudomonas aeruginosa within a period of one week. To identify the outbreak source, three independent studies were performed: epidemiological investigation, environmental surveillance and genotypic typing of isolates. Although epidemiological investigation by a case-control study revealed no conclusive results, the transfusion of fresh frozen plasma (FFP) and human albumin (HA) appeared to be the factor with highest risk. Environmental surveillance and random amplification of polymorphic DNA (RAPD) of isolates identified a water-bath used to warm FFP and HA as the likely reservoir for the outbreak. Further spread of the organism did not occur after elimination of this water-bath from the NICU. RAPD identified in addition an isolate from an infant hospitalized in the NICU five months before the outbreak with a pattern matching the one of the outbreak cluster.

Molecular Typing of Bordetella pertussis Isolates Recovered from Belgian Children and Their Household Members

Recently, a moderate increase in the prevalence of pertussis, possibly contracted from adults, has been observed among unvaccinated children. During a 3-year period, we prospectively enrolled 93 index patients with a polymerase chain reaction (PCR) and/or culture result positive for Bordetella pertussis. Among 63 household contacts of 28 index patients, PCR and culture for B. pertussis identified 25 B. pertussis-positive persons. Nineteen of 25 B. pertussis-positive household contacts were asymptomatic. Isolates were available from 10 families of both index patients and household contacts for molecular typing by pulsed-field gel electrophoresis (PFGE) and for genotyping of pertactin and pertussis toxin by sequence-specific PCR and sequencing. PFGE demonstrated homogeneity among the isolates recovered from within each family but heterogeneity among the isolates recovered from different families. B. pertussis isolates recovered from index patients and their household contacts were indistinguishable by molecular typing, demonstrating that identical strains can cause full pertussis disease in children and asymptomatic infection in adults and adolescents.

Development of monoclonal antibodies against Ureaplasma urealyticum serotypes and their use for serotyping clinical isolates.

ABSTRACT Monoclonal antibodies (MAbs) against Ureaplasma urealyticum serotype 2, 5, 7, 8, 10, 11, 12, and 13 reference strains were developed. The reactivities of these MAbs with the 14 serotype reference strains was verified by colony immunofluorescence assay and Western blot assay. MAbs against serotypes 2, 7, 10, 11, and 12 were serotype specific, whereas MAbs against serotypes 5, 8, and 13 showed cross-reactivity. All MAbs against serotype 5 were cross-reactive with serotype 2, and one showed, in addition, cross-reactivity to serotypes 9 and 10. Mutual cross-reactivities were observed between MAbs against serotypes 8 and 13. The usefulness of the MAbs for the serotyping of U. urealyticum strains was evaluated by serotyping 21 selected clinical isolates. A complete set of MAbs (the newly developed MAbs and the previously described MAbs against serotypes 1, 3, 4, 6, 9, and 14) as well as a complete set of polyclonal antibodies (PAbs), PAbs 1 to 14, were used. MAbs were able to identify 18 of 21 isolates including 2 isolates with mixed serotypes. Polyreactivity, which occurred with 19 of the 21 isolates with PAbs, was not observed by the use of MAbs. MAbs seem to be a more valuable tool than PAbs for serotyping and could help in investigating a possible link between the expression or variability of the serotype-specific antigens and pathogenicity.

Simple Algorithm for Identification of Bordetella pertussis Pertactin Gene Variants

ABSTRACT Studies performed in several countries have demonstrated the recent emergence and subsequent dominance of circulating Bordetella pertussis strains harboring pertactin and pertussis toxin variants not included in pertussis vaccines. Determination of the pertactin gene variants is commonly performed using a time-consuming and expensive sequence analysis. We developed a simple and reliable pertactin typing algorithm suitable for large-scale screening. The assay correctly identified all pertactin alleles in representative strains. The typing of 231 clinical strains of B. pertussis routinely isolated in Belgium showed that this algorithm was adequate to identify less-frequent prn types like prn9 and prn11.

Polymerase chain reaction-mediated cloning and in vitro translation of the genes coding for the structural proteins of hog cholera virus

SummaryAfter amplification by PCR, the 5′ region of the genome of hog cholera virus (HCV) strain Alfort 187 was cloned and sequenced. The nucleotide and deduced amino acid sequences were compared with the ones of other pestiviruses. By in vitro translation experiments we were able to demonstrate the protease activity of the p 20 protein of HCV.

Prevalence of Fragilysin Gene in Bacteroides fragilis Isolates from Blood and Other Extraintestinal Samples

ABSTRACT Of 166 Bacteroides fragilis isolates, 26.2% of 103 isolates from blood and 20.6% of 63 extraintestinal isolates harbored the fragilysin gene (difference not statistically significant). Clinical characteristics and evolution were comparable in patients with B. fragilis bacteremia with or without this enterotoxin. Fragilysin seems not to be an important virulence factor in B. fragilis disease.

Recovery of Mycobacterium elephantis from sputum of a patient in Belgium

Mycobacterium elephantis was isolated from a human respiratory specimen in April 1999, demonstrating its presence in Europe. The biochemical reaction results, antimicrobial susceptibility pattern, and sequence data for this strain are all in agreement with those of M . elephantis strains isolated

Development of an Enzyme-Linked Immunosorbent Assay for Serotyping Ureaplasma urealyticum Strains Using Monoclonal Antibodies

ABSTRACT Ureaplasma urealyticum comprises 14 serotypes. The existing serotyping methods all use polyclonal antibodies. These methods are time-consuming and labor-intensive, and they cannot always be performed on primary isolates; in addition, the results are difficult to interpret. We developed a new enzyme-linked immunosorbent assay (ELISA) method using serotype-specific monoclonal antibodies (MAbs) to enable the serotyping of U. urealyticum isolates from primary broth cultures. Each of the 14 serotype reference strains was tested against 14 selected MAbs. Homologous reactions were very strong, while heterologous reactions were negligible. Three cross-reactions were observed: MAb 5 cross-reacted with serotype 2, MAb 14 cross-reacted with serotype 3, and MAb 8 cross-reacted with serotype 13. Despite the cross-reactions observed, all the serotype reference strains ofU. urealyticum could be identified and differentiated using a combination of MAbs. Reproducibility was analyzed with a fractionated antigenic preparation and with several freshly prepared antigens of the same strain. No significant interrun variation was found with the fractionated antigen, but significant variations in optical density (OD) values were found when freshly prepared antigens were tested. However, the variation in OD values did not influence the overall interpretation of the ELISA: reactions with homologous MAbs were always prominent compared to those of the negative controls. This newly developed ELISA using MAbs seems promising for serotyping of U. urealyticum clinical isolates.

Expression inE. coli and purification of the active autoprotease P20 of classical swine fever virus

The genes for Flaviviridae structural proteins are located at the 5′ terminus of the genome, while the 3′ terminus contains the genes for the non-structural proteins. The first protein product of the large ORF of pestiviruses, the p20 protein, is however a non-structural protein which possess an autoproteolytic activity. Here we report the cloning of the p20/p14 genes behind the strong Trc promotor and expression of the p20 at high levels inE. coli. The autoprotease p20 was responsible for its own release from the nascent polyprotein inE. coli and was further purified by chromatography techniques.

Characterization of structural and non-structural proteins of hog cholera virus by means of monoclonal antibodies

SummaryA panel of 15 monoclonal antibodies, produced against the hog cholera virus, were characterized by radioimmunoprecipitation assays. Using this panel, we were able to identify 4 sets of monoclonal antibodies precipitating each a different viral protein with relative molecular weight of 40, 46, 120 kDa, respectively, and a protein complex containing 15, 16, 27, and 55 kDa polypeptides which were further characterized. One monoclonal antibody recognized an antigenic determinant at the C-terminal cleavage product of the non-structural p 125 of BVDV. The 40 kDa protein was precipitated from the pelleted virions, indicating its structural importance. On the contrary the 46 kDa protein could only be precipitated from the cell lysate and not from the pelleted virions. The glycosylated 15/16 kDa-55 kDa proteins form a disulfide linked heterodimer on the virus particle with a relative molecular weight of 65 kDa.