Molecular characterisation of bacterial contamination in semi-final gelatine extracts, using denaturing gradient gel electrophoresis.

Contamination of gelatine may affect the safety and/or quality of its applications. Characterisation of bacterial isolates from semi-final gelatine batches revealed thermotolerant, aerobic, endosporeforming contaminants. In this paper, bacterial contamination in gelatine batches is analysed without previous isolation, by means of denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA sequences. V9 and V6-V8 regions of the 16S rDNA gene were found more suitable for this purpose than V1 or V3 regions. Bacillus fumarioli, Bacillus licheniformis, members of the 'Bacillus cereus group', Bacillus subtilis, Bacillus shackletonii, Brevibacillus borstelensis and Brevibacillus agri were detected.

Paenibacillus cineris sp. nov. and Paenibacillus cookii sp. nov., from Antarctic volcanic soils and a gelatin-processing plant.

Seven strains of aerobic, endospore-forming bacteria were found in soil taken from an active fumarole on Lucifer Hill, Candlemas Island, South Sandwich archipelago, Antarctica, and four strains were from soil of an inactive fumarole at the foot of the hill. Amplified rDNA restriction analysis, 16S rDNA sequence comparisons, SDS-PAGE and routine phenotypic tests support the proposal of two novel species of Paenibacillus, Paenibacillus cineris sp. nov. and Paenibacillus cookii sp. nov., the type strains of which are LMG 18439T (=CIP 108109T) and LMG 18419T (=CIP 108110T), respectively. A further strain, isolated from a gelatin-production process, showed more than 99% 16S rDNA sequence similarity to the proposed P. cookii type strain and, although the gelatin isolate was atypical when compared with the fumarole isolates by repeated element primed-PCR, SDS-PAGE and phenotypic analyses, it was shown by DNA-DNA reassociation studies to belong to the same species. Strains of P. cookii produce spreading growth with motile microcolonies. Both species produce swollen sporangia that are typical for the genus, they both show 97.6% 16S rDNA sequence similarity to Paenibacillus azoreducens, they have 51.5-51.6 mol% G+C in their DNA and their major fatty acid is anteiso-C(15 : 0); however, fatty acids C(16 : 0) and anteiso-C(17 : 0) represent, respectively, 18 and 10 % of the total in P. cineris, but 11 and 20% in P. cookii.

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.

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.

Anoxybacillus contaminans sp. nov. and Bacillus gelatini sp. nov., isolated from contaminated gelatin batches.

Aerobic, endospore-forming bacteria that are attributed to the genus Bacillus or related genera constitute a hazard to the quality of gelatin. During repetitive extragenic palindromic DNA (rep)-PCR screening of gelatin isolates, a group of five isolates (group 1) and a group of 66 isolates (group 2) that did not match any pattern in our database were found. On the basis of 16S rDNA sequence analysis, representative strains of the different rep-PCR fingerprint types of group 1 were shown to be related most closely to Anoxybacillus species, but with sequence similarity of <97 %. Likewise, representative strains of group 2 were shown to be related most closely to Bacillus species, with 16S rDNA sequence similarity of <97 %. DNA-DNA reassociation values of isolates that displayed the most divergent rep-PCR profiles revealed that strains within each group belonged to a single species, according to recommendations for species delineation. A mean fatty acid profile could be calculated for each group. Isolates within a single group had similar patterns of results in API and other phenotypic tests; no correlation of patterns of results with rep-PCR groups was seen. Physiological characterization of group 1 isolates allows their distinction from other Anoxybacillus species. Despite the weak reaction of group 2 isolates in API tests, physiological characterization allows distinction between Bacillus species that react weakly in API tests. Two novel species are therefore proposed, with the names Anoxybacillus contaminans sp. nov. (type strain, LMG 21881(T)=DSM 15866(T)) and Bacillus gelatini sp. nov. (type strain, LMG 21880(T)=DSM 15865(T)).

Polyphasic characterization of Bacillus coagulans strains, illustrating heterogeneity within this species, and emended description of the species.

Because of its food spoiling capacity on the one hand and its significant role in the production of industrially valuable products on the other, Bacillus coagulans is of economic concern. Several studies have revealed a great deal of diversity within the species and this has led to a number of taxonomic adjustments. The present study aims to clarify the diversity within Bacillus coagulans sensu stricto and determine the taxonomic status of the species. Therefore, a polyphasic study was performed on a set of B. coagulans strains from diverse habitats. Techniques as ARDRA, SDS-PAGE of whole cell proteins, FAME analysis, routine phenotypic tests and rep-PCR illustrate considerable intra-species heterogeneity, while 16S rDNA sequence comparison and DNA-DNA relatedness support the accommodation of these strains in one species. Although most techniques demonstrate appreciable heterogeneity among the Bacillus coagulans strains, the intraspecies groupings are not consistent throughout all the methods applied and are not supported by any economic, historic or practical traits. Therefore, a division in subspecies seems inappropriate. In attempt to achieve a better species delineation, an emended description of Bacillus coagulans is included.

Genotypic diversity among Bacillus licheniformis strains from various sources.

Bacillus licheniformis is exploited industrially for the production of enzymes and has been shown to exhibit pathogenic properties. Because of these divergent characteristics, questions arise concerning intraspecies diversity. A comparative study by means of combined repetitive polymerase chain reaction, rpoB and gyrA sequencing, 16S rDNA targeted probe analysis, DNA-DNA hybridizations, gelatinase tests and antibiotic susceptibility tests was performed on a set of strains from diverse sources, including strains with pathogenic potential. B. licheniformis was found to consist of two lineages that are distinguished genotypically.

Diversity of transconjugants that acquired plasmid pJP4 or pEMT1 after inoculation of a donor strain in the A- and B-horizon of an agricultural soil and description of Burkholderia hospita sp. nov. and Burkholderia terricola sp. nov.

We examined the diversity of transconjugants that acquired the catabolic plasmids pJP4 or pEMT1, which encode degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), in microcosms with agricultural soil inoculated with a donor strain (Dejonghe, W., Goris, J., El Fantroussi, S., Höfte, M., De Vos, P., Verstraete, W., and Top, E. M. Appl. Environ. Microbiol. 2000, p. 3297-3304). Using repetitive element PCR fingerprinting, eight different rep-clusters and six separate isolates could be discriminated among 95 transconjugants tested. Representative isolates were identified using 16S rDNA sequencing, cellular fatty acid analysis, whole-cell protein analysis and/or DNA-DNA hybridisations. Plasmids pJP4 and pEMT1 appeared to have a similar transfer and expression range, and were preferably acquired and expressed in soil by indigenous representatives of Ralstonia and Burkholderia. Two rep-clusters were shown to represent novel Burkholderia species, for which the names Burkholderia hospita sp. nov. and Burkholderia terricola sp. nov. are proposed. When easily degradable carbon sources were added together with the plasmid-bearing donor strain, also a significant proportion of Stenotrophomonas maltophilia isolates were found. The transconjugant collections isolated from A- (0-30 cm depth) and B-horizon (30-60 cm depth) soil were similar, except for B. terricola transconjugants, which were only isolated from the B-horizon.

Study of the bacterial load in a gelatine production process focussed on Bacillus and related endosporeforming genera.

Gelatine is an animal protein with many industrial applications. Previous studies pointed out that endosporeforming bacteria, belonging to the genus Bacillus or related genera, might contaminate and survive the production process of gelatine, leading to products of low quality and safety. The aim of this study is to determine the bacterial diversity of contaminants isolated from a gelatine production chain with emphasis on aerobic endosporeforming bacteria. Contaminants were isolated from samples taken at five crucial points along two different production lines of a gelatine production process and from water supplies used for extraction and cooling. Gaschromatographic methyl ester analysis of fatty acids was performed to differentiate isolates at the genus level. Apart from members of the genus Bacillus or related endosporeforming genera, also members of Salmonella, Kluyvera, Staphylococcus, Burkholderia, Enterococcus, Pseudomonas, Yersinia, Streptococcus and Brevundimonas could be detected. Isolates identified as belonging to Bacillus and related endosporeforming genera were further characterised by gelatinase tests, rep-PCR and 16S rDNA sequencing. All these isolates showed the ability to liquefy gelatine. Endosporeforming isolates were assigned to Bacillus licheniformis, B. fumarioli, members of the B. cereus group, B. badius, B. coagulans, B. subtilis, Brevibacillus agri, Alicyclobacillus acidocaldarius and a yet undescribed Paenibacillus species.