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the end product. However, quality control of gelatine producing factories pointed out that thermotrophic, aero-bic, proteolytic endosporeforming bacteria might be pre-0723-2020/02/25/04-611 $ 15.00/0Abbreviations: UHT, ultra high temperature; FAME, fatty acid methyl ester analysis; MIS, microbial identification system; BSE,bovine spongiform encephalopathyBacillus senso lato group, consisting of the genus Bacillus and some of the genera that were recently derived from it are considered to be the most common and often the only contaminants in UHT-treated gelatine batches. Some Bacillus species have been mentioned in food poisoning incidents. B. cereus is considered as the most important Bacillus contaminant in foods [23].Species belonging to Bacillus or related genera are known to produce different proteases, including gelati-nases [30]. Enzymatic degradation of gelatine would af-fect the viscosity and therefore the quality of the product itself and its derivatives. Moreover, because of this degradation, essential nutrients may become available for gelatinase negative contaminants, promoting their growth. Furthermore, some of the s e contaminants may be pathogenic for men, which is of great concern, espe-cially for the applications of gelatine in food and phar-maceutical products. In order to preserve the technical properties of gelatine, the UHT-treatment step cannot be extended, nor can sterilisation temperatures be increased to further diminish the survival of bacterial spores.The aim of this study is to unravel the diversity of the bacterial contamination in a gelatine production process.As contamination due to Bacillus or related genera is the major concern, characterisation was worked out to a greater extent for these contaminants. Additional charac-terisation via rep-PCR and 16S rDNA sequence analysis was performed.Materials and MethodsSampling and isolation procedureSamples were taken at five crucial points (a–e) along a gela-tine production chain (Fig. 1). One production line using pig skins and one line using bovine bones as raw material were sampled. Also water samples from water tanks used for extrac-tion and cooling were included in the study. Isolation and growth media were prepared based on gelatine extracts from the corresponding sampling points. Therefore, gelatine extracts from the sampling points were simply sterilised and used as liq-uid media for enrichment of contaminants (see below). Colony isolation was performed on plates by adding 2% agar (LAB M)to these media. More concentrated gelatine extracts (sampling points d and e) were diluted three times and to the extracts at sampling point c, d and e, 0.8 g/l protease (Acid Fungal Pro-tease, Genencor) was added to prevent solidification of the liq-uid media at room temperature and clogging of the solid media on addition of agar. One ml of sample was added to 9 ml corre-sponding liquid medium as well as to 9 ml of liquid media cor-responding with downstream sampling points, in order to ob-tain a first estimation of possible hot spots for contamination along the production chains. Water samples were inoculated in medium corresponding to the gelatine extract at sampling point e. After enrichment during 24 hours at 4 different tem-peratures (37 °C, 45 °C, 55 °C and 70 °C), samples were plat-ed on the corresponding solid media using a spiral plater. If necessary, dilution series were made. As many different colony types as visually distinguishable were picked up, purified and stored in Microbank™ tubes (PRO-LAB diagnostics) at –80 °C. Each combination sample-medium-temperature was performed in duplicate.Fatty acid methyl ester analysis (FAME)Analysis was performed as described by Yang et al. [29].Strains that were able to grow on TSA medium at 28 °C or 52 °C were identified using the standardized Microbial Identifi-cation System (MIS, Microbial ID, Newark, DE, USA). Howev-er, the majority of the isolates did not grow in conformity with the standard conditions. Therefore, all isolates were grown on Nutrient Agar (Oxoid) supplemented with 1.2% gelatine at 45 °C. The obtained FAME profiles were subjected to cluster analysis allowing to determine the similarities in fatty acid com-position between isolates that could and could not grow under standardised growth conditions of MIS.Gelatinase testsGelatinase activity of the isolates was investigated by two dif-ferent tests.For the first test, an analogous procedure was followed as in the quality control of the production plant. In this test, per-formed in tubes, a small amount of cells of a pure culture is in-oculated in 5 ml medium, consisting of 0.25% yeast extract,0.5% bactopeptone, 0.5% glucose, 0.1% MgSO 4·7H 2O and 12% gelatine, suspended in 0.02 M phosphate buffer pH 7(0.3% KH 2PO 4and 1% Na 2HPO 4·12H 2O). After incubation during one week at 37 °C, gelatinase activity is revealed after an612 E. De Clerck and P. De VosFig. 1.Schematical overview of a gelatine production process,with indication of the sampling points (a–e).extra 24 hours incubation at room temperature, by liquefaction of the medium. The second test, performed on plates, is based on the method described by Smibert and Krieg [24]. Bacterial cells are streaked as a single line across the centre of a plate with Nutrient Agar supplemented with 1.2% gelatine. After incuba-tion during one week at the optimal growth temperature, the medium is overlaid with a HCl/HgCl2solution (10% HCl, 15% HgCl2). A clear zone around the growth of the bacteria reveals gelatinase activity.Repetitive element genomic fingerprinting (Rep-PCR) Template DNA was prepared using a slight modification of the method of Pitcher et al. [21], as described by Heyndrickx et al. [9]. The BOX A1R primer (5′CTACGGCAAGGC-GACGCTGACG-3′) and (G TG)5primer (5′-GTGGTGGTG-GTGGTG-3′) were used, both described by Versalovic et al. [26]. PCR amplifications were performed with a DNA thermal cycler (Perkin Elmer 9600) as described by Versalovic et al. [26], using G oldstar DNA polymerase (Eurogentec, Belgium). The PCR products were electrophoresed in a 1.5% agarose gel (15 ×20 cm) for 16 h at a constant voltage of 1.9 V cm–1in 1 ×TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) at 4 °C. The rep-PCR pro-files were visualized after staining with ethidium bromide under ultraviolet light, followed by digital image capturing using a CCD camera. The resulting fingerprints were analyzed by the BioNumerics V2.0 software package (Applied Maths, Belgium). The similarity between digitized profiles was calculated using Pearson correlation coefficient and shown as an average linkage (UPGMA) dendrogram.16S rDNA sequencingA fragment of the 16S rRNA gene was sequenced using an Applied Biosystems 377 Sequencer, as described by Heyrman and Swings [10]. The sequencing primers were 5′-CTCC-TACGGGAGGCAGCACT-3′(forward primer, corresponding to positions 339–358 in E. coli numbering), 5′-AACTCAAAG-GAATTGACGG-3′(forward, 908–926), 5′-AGTCCCGCAAC-GAGCGCAAC-3′(forward, 1093–1112), 5′-ACTGCTGCCTC-CCGTAGGAG-3′(reverse, 358–339), 5′-GTATTACCGCGGCT GCTG-3′(reverse, 536–519) and 5′-GTTGCGCTCGTTGCG GGACT-3′(reverse, 1112–1093). The FASTA program [17] was applied to find the closest related sequences from the EMBL database.Results and DiscussionIn the present study, 132 isolates contaminating a gela-tine production process were obtained.Identification of the isolates to the genus level, selection of representatives of the genus Bacillusand related endosporeforming generaFatty acid analysis was performed. This is a relatively rapid technique provided with a commercial database for identification under standardized conditions of growth of a broad range of genera. Only 50 isolates of 132 could be grown conform to the standard conditions necessary for database comparison. The majority of them showed sig-nificant similarities with data entries in the MIS database for genus assignment. However, FAME analysis could be performed for all isolates by adjusting the growth condi-tions. As a consequence, identification with the database was no longer possible. Nevertheless, FAME data ob-tained under these non-standardised conditions were compared and allowed indirect assignment of another 14 isolates at the genus level in combination with the FAME data of the 50 isolates that could be analysed under stan-dardised conditions. Results of the identification are shown in Table 1.In gelatine samples originating from bovine bones, members of Bacillus s.l.,Burkholderia, Enterococcus, Yersinia, Brevundimonas, Enterobacter and Kluyvera were found.In gelatine samples originating from pig skin, mem-bers of Bacillus s.l., Enterococcus, Burkholderia, Kluyvera, Staphylococcus, Streptococcus, Pseudomonas and Salmonella were found.Finally, in the water samples representatives of the gen-era Bacillus and Pseudomonas were found.Based on the results of the fatty acid analysis, supple-mented with morphologic data (not shown), representa-tives of Bacillus or related endosporeforming genera were selected for further analysis.Bacillus Contamination in a Gelatine Production Process613Table 1.Identification of isolates based on fatty acid analysis Source1Genus identification Number of isolatesB/a Bacillus s.l.1Burkholderia10B/b Bacillus s.l.4Burkholderia1B/c Burkholderia10Enterococcus1Yersinia1B/d Brevundimonas2Enterococcus1B/e Bacillus s.l.1Burkholderia1Enterobacter1Kluyvera2P/a Bacillus s.l.1Enterococcus4P/b Bacillus s.l.3Burkholderia1Kluyvera2Staphylococcus2Streptococcus1P/c Bacillus s.l.3Burkholderia3Pseudomonas1Salmonella1P/d Enterococcus1W Bacillus s.l.2Pseudomonas31Isolation source:B: gelatine extracts originating from bovine bonesP: gelatine extracts originating from pig skinW: water samplesa–e: sampling points as indicated in Fig. 1.Characterisation, typing and identification of Bacillus s.l. isolatesAll selected isolates from the present study were subject-ed to both gelatinase tests and scored positive in at least one of them. Both tests are considered complementary. In-deed, the test in tubes cannot always be performed at opti-mal growth temperature, because at temperatures of 45 °C or higher, gelatine liquefies spontaneously. The test in plates can be performed at higher temperatures, but visual-isation of the gelatinase activity is not always evident. Ob-servation of a gelatinase activity by means of at least one of these tests is an indication for the gelatine deteriorating effects of the contaminating Bacillus and related en-dosporeformers. Moreover, gelatinase positive organisms could be able to liberate nutrients for gelatinase negative organisms and in this way stimulating their growth.Bacillus s.l. isolates were further characterised by rep-PCR. Gelatinase positive strains, assigned to Bacillus s.l.,isolated in a previous analogous study performed in 1995were included. In the 1995 study, all gelatinase positive isolates identified as Bacillus s.l. were obtained from the bovine bone production line. Clear banding patterns were obtained with the BOX A1R primer as well as with the (GTG)5 primer (not shown). However, with the (GTG)5primer a pattern could be obtained for all isolates. The results of numerical analysis of the generated (G TG )5banding patterns are shown in a dendrogram (Fig. 2). For isolates with identical rep-PCR banding patterns, one or more representatives were selected to unravel further species affiliation (Table 2), as rep-PCR is known to dis-criminate at the subspecies level [26].16S rDNA sequencing was performed and according to Stackebrandt and G oebel [25], two organisms that614 E. De Clerck and P . De VosFig. 2.Cluster analysis of digitized banding patterns, generated by rep-PCR using the (GTG)5primer, of gelatine isolates expected to belong to the genus Bacillus or related genera.The dendrogram was constructed using the unweighted pair-group method using arithmetic averages with correlation levels ex-pressed as percentage values of the Pearson correlation coefficient.1Identification based on FASTA analysis of 16S rDNA sequences at the EMBL database.2Isolation sourceB: gelatine extracts originating from bovine bones P: gelatine extracts originating from pig skin W: water samplesa-e: sampling points as indicated in Fig.1show 16S rDNA sequence homologies of 97% or lower,will have less than 70% DNA-DNA relatedness andshould be considered belonging to different species [27].A hypervariable region (HV region, nucleotide positions70–344) of the 16S rDNA cistron is most informative for rapid identification of Bacillus species [7]. Therefore,partial sequences were obtained first, allowing a firsttentative species identification. For two Bacillus strains (R-10919 and R-11590) and strains identified as Alicy-clobacillus , Paenibacillus and Brevibacillus species (R-10926, R-11600 and R-12868 respectively), a morecomplete sequence of 16S rDNA was generated. The re-sults of the identification are included in Table 2. All partially sequenced strains, except R-11600, showed afirst match with similarity above 97% (the majorityeven above 99%). The strains where a more complete16S rDNA sequence was generated also showed a firstmatch with similarity above 97%. Isolates R-1168,R-1223, R-10758, R-10919, R-10925, R-10926, R-10940, R-10944, R-11590, R-11604, R-12868 and R-13753 showed a second match with significant lower similarity than the first match, which is an important in-dication for the species allocation. Isolates R-10947 and R-12866 showed 100% similarity with B. cereus , B. thuringiensis and B. anthracis . This is not surprising because Ash et al. [1] pointed out that 16S rRNA se-quence data alone do not permit to differentiate these species. They show a high degree of DNA-DNA reasso-ciation [11] and are called members of the ‘Bacillus cereus group’. Many authors have questioned their sta-tus as separate species [1, 8] but differences in their habitats, pathogenicity for mammals or insects and theirmorphologic and physiologic characteristics keep thisgroup divided in different species. The second best fit isB. mycoides (99.3% similarity), also a member of theB. cereus group. Isolate R-11600 could only be identi-fied to the genus level and may represent a yet unde-scribed Paenibacillus species. In Fig. 2 the identificationof all gelatinase positive Bacillus s.l. isolates is shown based on the combination of their rep-PCR profiles and16S rDNA sequencing.Isolates identified as Bacillus licheniformis were foundin gelatine samples, produced from bovine bones, both in1995 and in 2000. These isolates show highly similar rep-PCR banding patterns (Fig. 2). An important remark needs to be given here in respect to the eventual presence of B. licheniformis in gelatine. Indeed, toxin-producing Bacillus licheniformis strains have been associated with food poisoning incidents [22]. Furthermore, B. licheni-formis should also be considered as a potential pathogen in immunocompromised patients [3]. Since gelatine isused in pharmaceutical products, this kind of contamina-tion is of great concern.Members of the B. cereus group were found in the pre-sent study in gelatine samples originating from pig skin and in water samples. The presence of a member of the Bacillus cereus group in gelatine could also be harmful.Bacillus cereus is known as a causative agent in both gas-trointestinal and non-gastrointestinal diseases [12], while Bacillus anthracis causes the often lethal disease anthrax Bacillus Contamination in a Gelatine Production Process615Table 2.16S rDNA sequences determined in this studyStrain Sequence Accession Best match 3Similar-no.length 1no.2(accession no.)ity (%)R-1168454AJ438292Bacillus badius 99.6(X77790)R-1223476AJ438293Brevibacillus agri 99.6(AB039334)R-10758453AJ438294Bacillus subtilis 99.8(Z99104)R-10919438Bacillus fumarioli 100(AJ250058)1222AJ438295Bacillus fumarioli 99.9R-10925432AJ438296Bacillus fumarioli 99.8(AJ250058)R-10926462Alicyclobacillus 99.1acidocaldarius(AB059673)1417AJ438297Alicyclobacillus 99.5acidocaldarius(AB059674)R-10940454AJ438298Bacillus fumarioli 99.8(AJ250058)R-10944690AJ438299Bacillus fumarioli 99.9(AJ250058)R-10947456AJ438300Bacillus cereus 100(AF176322)Bacillus 100thuringiensis(AF155955)Bacillus anthracis 100(AF176321)R-11590452Bacillus licheniformis 98.7(AB039328)1507AJ438301Bacillus licheniformis 99.5(AB039328)R-11600408Paenibacillus sp.96.3(AJ272249)1446AJ438302Paenibacillus sp.97.6(AJ272249)R-11604452AJ438303Bacillus licheniformis 99.6(X68416)R-12866455AJ438304Bacillus cereus 100(AF176322)Bacillus thuringiensis 100(AF155955)Bacillus anthracis 100(AF176321)R-12868585Brevibacillus sp.98.8(AF228763)1455AJ438305Brevibacillus agri 99.3(AB039334)R-13753384AJ438306Bacillus coagulans 98.2(D16267)1For most strains only partial 16S rDNA sequences were obtained (including the HV region, corresponding to positions 70–344 of the E. coli numbering). For R-10919, R-11590 andR-11600 a more complete sequence was generated.2Available at the EMBL database.3 Best matches, obtained by FASTA analysis at the EMBLdatabase.(see review [15]). B. thuringiensis is used as a biological insecticide. Difference between B. cereus and B. t hurin-giensis is merely based on the presence of genes coding for insecticidal toxins in B. thuringiensis, usually present on plasmids. If strains are cured from their plasmids, the two species are difficult to distinguish [8]. Recently, a food poisoning potential of enterotoxic B. t huringiensis strains is described for vegetables treated with a biological insecticide [2].B. licheniformis and B. cereus are well known contam-inants of industrial processes [19], foods [4] and clinical environments [5, 14]. More surprising was the presence of isolates identified as B. fumarioli in gelatine samples originating from pig skin in 2000, as this species was de-scribed for isolates from soils present near fumaroles on Antarctica [13] and, to our knowledge, has not been re-ported elsewhere. However, growth conditions in these Antarctic habitats (50 °C, pH 5.5) are not in contradic-tion with conditions during the production of gelatine. So far, no pathogenic properties have been reported for this species.Isolates identified as B. badius and Brevibacillus agri were found in 1995 in samples originating from the bovine bone production chain. Strains identified as B. co-agulans and Paenibacillus sp. were isolated in the present study from samples originating from the same process. Isolates identified as Bacillus subtilis and Alicyclobacillus acidocaldarius were found in the present study in samples originating from the pig skin process. Brevibacillus agri was found in water samples. B. coagulans, B. subtilis and Brevibacillus species have been associated with food-borne illness in a few cases [16, 18]. The genus Alicy-clobacillus consists of thermoacidophilic bacteria, some of them responsible for the spoilage of acidic beverages, such as fruit juices [6, 28]. Paenibacillus species have been reported as contaminants in industrial processes and in foods [18, 20]. No pathogenicity for humans has been reported for members of this genus.In conclusion, aerobic sporeforming bacteria may con-taminate gelatine production processes. Bacillus, Bre-vibacillus, Paenibacillus and Alicyclobacillus isolates were shown to be able to liquefy gelatine, herewith de-stroying its gelling properties. Finally, some of the isolates were assigned to species, known to exhibit pathogenic properties for humans, which is of great concern since gelatine is applied in foods and pharmaceutical products. Further investigation is needed to reveal whether the en-dosporeforming species present along the gelatine pro-duction process dominate the bacterial flora in the final product of pharmaceutical and food applications causing the actual problems of viscosity loss and form a threat for human or animal health.AcknowledgementsElke De Clerck was supported by a fellowship of the IWT (Institution for the Promotion of Innovation by Science and Technology in Flanders). Paul De Vos is indebted to the FWO for financial support (FWO grant 36015602). We thank Rous-selot NV for their collaboration.References1.Ash, C., Farrow, J. A. E., Dorsch, M., Stackebrandt, E.,Collins, M. D.: Comparative analysis of Bacillus anthracis, Bacillus cereus and related species on the basis of reverse transcriptase sequencing of 16S ribosomal RNA. Int. J.Syst. Bacteriol. 41, 343–346 (1991).2.Bishop, A. 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