Virginie Storms

Application and Evaluation of the Interlaboratory Reproducibility of tRNA Intergenic Length Polymorphism Analysis (tDNA-PCR) for Identification of Streptococcus Species

ABSTRACT The discriminatory power, speed, and interlaboratory reproducibility of tRNA intergenic length polymorphism analysis (tDNA-PCR) combined with capillary electrophoresis was evaluated for the identification of streptococci. This method was carried out in three different laboratories under highly standardized conditions for 54 strains belonging to 18 different species. It was concluded that interlaboratory reproducibility of tDNA fingerprints produced by means of capillary electrophoresis was sufficiently high to permit the exchange between different laboratories and the construction of common libraries which can be consulted for comparison with fingerprints obtained independently in separate laboratories. In a second step, 17 other species were included in the study and examined in one of the participating laboratories. All Streptococcus species studied, except S. mitis, S. oralis, S. parasanguinis, S. pneumoniae, S. thermophilus, and S. vestibularis, showed distinguishable tDNA fingerprints. A database of well-characterized strains was constructed to enable computer-aided identification of unknown streptococcal isolates.

Polyphasic characterisation of Burkholderia cepacia-like isolates leading to the emended description of Burkholderia pyrrocinia.

Twenty-five Burkholderia cepacia-like isolates of human and environmental origin, comprising five different recA RFLP types, were examined by using a polyphasic taxonomic approach, including recA gene sequence analysis, 16S rRNA gene sequence analysis, DNA:DNA hybridisation studies, tDNA-PCR, fatty acid analysis and biochemical analysis. The results of the present study demonstrated that twenty-three of these strains belong to Burkholderia pyrrocinia, a B. cepacia complex species thus far comprising one single soil isolate only. An emended description of Burkholderia pyrrocinia is proposed. The taxonomic status of the remaining two isolates requires further analysis.

In Vitro Biodegradation of Polyester-Based Plastic Materials by Selected Bacterial Cultures

A simple and rapid in vitro test was designed for the assessment of the biodegradation of polyester-based plastics by selected biodegrading bacterial strains. Variovorax paradoxus LMG 16137 was used for the degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and Acidovorax avenae subsp. avenae LMG 17238 fo the synthetic-based polyesters poly(ε-caprolactone) (PCL), poly(butylene succinate-co-butylene adipate), and a starch-PCL blend. Degradation by the bacteria was studied in liquid medium with the plastics (films, granules, and injection-molded test bars) as sole sources of carbon. Degradation was followed through gravimetry, growth of the culture, and tensile testing. The effects of incubation time, inoculum density, aeration, incubation temperature, and pH of the medium on the mass loss were investigated and conditions optimized. The test allowed to obtain reproducible results on the mass loss of plastic samples in less than 3 weeks and yielded excellent partially degraded samples for further analysis.

Biodegradation of Poly (3 -hYdroxYalkanoates in Anaerobic Sludge and Characterization of a Poly (3 -hYdroxYalkanoates Degrading Anaerobic Bacterium

Summary The degradation of samples of poly (3-hydroxybutyrate) [P(3HB)], copolymers of 3-hydroxybutyrate and 10 and 20% 3-hydroxyvalerate [P(3HB-co-3HV)], and poly (3-hydroxyoctanoate-co-3-hydroxydecanoate) [P(3H0-co-3HD)] in anaerobic sludge was investigated by gravimetry, changes in molecular weight and mechanical properties. After 123 days, the mass of P(3HB) decreased by 15%. P(3HB-co-3HV) showed 7 and 11% mass loss, while P(3H0-co-3HD) showed no change in mass. The average molecular weight of all samples decreased with incubation time but this decrease was not correlated to the loss of mass, and is to be attributed to abiotic hydrolysis. The tensile strength of the P(3HB) and P(3HB-co-3HV) samples decreased with incubation time, but elongation remained practically unchanged. By enumeration, it was shown that anaerobic P(3HB) degrading bacteria were enriched during the incubation period. An anaerobic bacterium LMG 16094, able to degrade P(3HB) and P(3HB-co-19% 3HV) in pure culture, but not P(3HB-co-97%3HV), P(3H0-co-3HD) or poly (e-caprolactone), was isolated and characterized by fatty acid analysis. Its cells contained mainly straight chain, saturated and unsaturated fatty acids, with a high portion of fatty acids with 16 carbons, and did not contain hydroxylated or branched fatty acids. Its 16S rDNA gene sequence was found to be related to those of members of Clostridium group I ( Collins et al., 1994). In a simple in vitro test using strain LMG 16094 in pure culture, an extruded P(3HB-co-12% 3HV) film lost 10% of its initial mass within 21 days of anaerobic incubation.

Study of the intra- and interlaboratory reproducibility of partial Single Base C-Sequencing of the 16S rRNA gene and its applicability for the identification of members of the genus Streptococcus

The use of Single Base C-Sequencing of the first 500 bases of the 16S rRNA-gene (SBCS) combined with capillary electrophoresis was evaluated for the identification of reference strains of 30 different species within the genus Streptococcus. For SBCS, only dd-CTPs are used in the sequencing reactions instead of the four dideoxy bases and the primer is fluorescently labeled. The reproducibility, interlaboratory exchangeability and discriminative power of this method were studied by comparing the patterns obtained in three laboratories under highly standardized conditions. The interlaboratory reproducibility proved to be high, enabling the construction of a common database for the identification of strains belonging to the streptococcal species studied. Most of the examined species generated distinguishable profiles. SBCS did not differentiate between the closely related species S. constellatus and S. intermedius. Also S. thermophilus and S. vestibularis as well as S. mitis and S. pneumoniae showed highly resembling profiles. The previously reported heterogeneity within the species S. equinus was reflected by SBCS. For all other species, strains belonging to the same species generated indistinguishable patterns. In conclusion, Single Base C-sequencing of the first 500 bases of the 16S rRNA-gene could be a useful and widely applicable method for the identification of bacteria at the species level, with the added advantage of being more rapid and easier to automatize than full sequence determination.

Evaluation of tRNA intergenic length polymorphism (tDNA-pCR) for the differentiation of the members of the Burkholderia cepacia complex.

Nowadays, tentative identification of B. cepacia complex bacteria in routine diagnostic laboratories is based on a combination of selective media, conventional biochemical reactions, commercial test systems and PCR-based assays. Some of these assays have the capacity to discriminate reliably among several members of the B. cepacia complex, however one single method differentiating all B. cepacia-like organisms is not available. In this study, the applicability of tDNA-PCR for the differentiation and rapid identification of the different members of the B. cepacia complex was evaluated. For B. gladioli and most of the B. cepacia genomovars, differentiable patterns were obtained. For some of the members of the B. cepacia complex however, the tDNA-PCR patterns were very similar and sometimes multiple patterns existed within in a single genomovar. No distinction could be made between the tDNA-PCR patterns of B. vietnamiensis and B. pyrrocinia and of B. cepacia genomovars I and VIII respectively. We could conclude that, although tDNA-PCR is not sufficient as a single method to identify all the members of the B. cepacia complex unambiguously or to replace the currently used methods, it is a very fast and easily applicable method that could be a very useful tool for the differentiation and identification of B. cepacia-like organisms.

The three cornerstones for biological resource centres

Biological resource centres (BRCs) are an essential part of the infrastructure underpinning life sciences and biotechnology. The Organization for Economic Co-Operation and Development (OECD) taskforce on BRCs (1999-2004), has put in a great effort of thought to define the new BRCs and forms the basis for the future development of the actual culture and reference collections. The effort, which has taken so many years and was, from the beginning, inspired by many WFCC members, has resulted in an important visionary document.