Comparison of Strategies for Isolating Anaerobic Bacteria from the Porcine Intestine.

The isolation of bacteria that represent the diversity of autochthonous taxa in the gastrointestinal tract is necessary to fully ascertain their function, but the majority of bacterial species inhabiting the intestines of mammals are fastidious and thus challenging to isolate. The goal of the current study was to isolate a diverse assemblage of anaerobic bacteria from the intestine of pigs as a model animal and to comparatively examine various novel and traditional isolation strategies. Methods used included long-term enrichments, direct plating, a modified ichip method, as well as ethanol and tyndallization treatments of samples to select for endospore-forming taxa. A total of 234 taxa (91 previously uncultured) comprising 80 genera and 7 phyla were isolated from mucosal and luminal samples from the ileum, cecum, ascending colon, and spiral colon removed from animals under anesthesia. The diversity of bacteria isolated from the large intestine was less than that detected by next-generation sequence analysis. Long-term enrichments yielded the greatest diversity of recovered bacteria (Shannon's index [SI] = 4.7). Methods designed to isolate endospore-forming bacteria produced the lowest diversity (SI ≤ 2.7), with tyndallization yielding lower diversity than the ethanol method. However, the isolation frequency of previously uncultured bacteria was highest for ethanol-treated samples (41.9%) and the ichip method (32.5%). The goal of recovering a diverse collection of enteric bacteria was achieved. Importantly, the study findings demonstrate that it is necessary to use a combination of methods in concert to isolate bacteria that are representative of the diversity within the intestines of mammals.IMPORTANCE This work determined that using a combination of anaerobic isolation methods is necessary to increase the diversity of bacteria recovered from the intestines of monogastric mammals. Direct plating methods have traditionally been used to isolate enteric bacteria, and recent methods (e.g., diffusion methods [i.e., ichip] or differential isolation of endospore-forming bacteria) have been suggested to be superior at increasing diversity, including the recovery of previously uncultured taxa. We showed that long-term enrichment of samples using a variety of media isolated the most diverse and novel bacteria. Application of the ichip method delivered a diversity of bacteria similar to those of enrichment and direct plating methods. Methods that selected for endospore-forming bacteria generated collections that differed in composition from those of other methods with reduced diversity. However, the ethanol treatment frequently isolated novel bacteria. By using a combination of methods in concert, a diverse collection of enteric bacteria was generated for ancillary experimentation.

The Sporobiota of the Human Gut.

The human gut microbiome is a diverse and complex ecosystem that plays a critical role in health and disease. The composition of the gut microbiome has been well studied across all stages of life. In recent years, studies have investigated the production of endospores by specific members of the gut microbiome. An endospore is a tough, dormant structure formed by members of the Firmicutes phylum, which allows for greater resistance to otherwise inhospitable conditions. This innate resistance has consequences for human health and disease, as well as in biotechnology. In particular, the formation of endospores is strongly linked to antibiotic resistance and the spread of antibiotic resistance genes, also known as the resistome. The term sporobiota has been used to define the spore-forming cohort of a microbial community. In this review, we present an overview of the current knowledge of the sporobiota in the human gut. We discuss the development of the sporobiota in the infant gut and the perinatal factors that may have an effect on vertical transmission from mother to infant. Finally, we examine the sporobiota of critically important food sources for the developing infant, breast milk and powdered infant formula.

High counts of thermophilic spore formers in dairy powders originate from persisting strains in processing lines.

During the last decades, thermophilic spore counts became a very important quality parameter for manufacturers with regard to powdered dairy products. Low-spore count powders are highly demanded but challenging to produce when high production volume and long process times are intended. In this study a detailed monitoring of microbial levels in three skim milk powder plants was conducted. Anoxybacillus flavithermus was found to be primarily responsible for increased spore levels with increasing spore numbers being detected after 6-8 h already during initial processing steps. Simultaneously, the species composition shifted from a diverse bulk tank milk microbiota where different Bacillus species represented around 90% of the thermophilic bacteria to a dominance of A. flavithermus in the end product. The analysis of A. flavithermus isolates from different powder batches with RAPD PCR revealed recurring patterns in each of the eight German manufacturers sampled over several months. The high relatedness of isolates exhibiting identical RAPD patterns was exemplified by cgMLST based on whole genome sequences. We assume that A. flavithermus strains persisted in production plants and were not eliminated by cleaning. It is concluded that such persisting strains recurrently recontaminated subsequent powder productions. The data highlight that a targeted optimization of cleaning and disinfection procedures is the most promising measure to effectively reduce thermophilic spore counts in German dairy powders.

Feasibility of using RFLP of PCR-amplified 16S rRNA gene(s) for rapid differentiation of isolates of aerobic spore-forming bacteria from honey.

This study aimed to assess the feasibility of using RFLP of PCR-amplified 16S rRNA gene (s) by using universal primers 27f/1492r and a combination of three restriction enzymes, AluI, CfoI, and TaqI, for a low-cost, rapid screen for a primarily differentiation of isolates of the complex of aerobic spore-forming bacteria commonly found in honey samples. The described method produced unique and distinguishable patterns to differentiate among 80 isolates belonging to 26 different species of Bacillus, Brevibacillus, Lysinibacillus, Rummeliibacillus, and Paenibacillus reported in honey and other apiarian sources.

Exploiting the aerobic endospore-forming bacterial diversity in saline and hypersaline environments for biosurfactant production.

BACKGROUND: Biosurfactants are surface-active biomolecules with great applicability in the food, pharmaceutical and oil industries. Endospore-forming bacteria, which survive for long periods in harsh environments, are described as biosurfactant producers. Although the ubiquity of endospore-forming bacteria in saline and hypersaline environments is well known, studies on the diversity of the endospore-forming and biosurfactant-producing bacterial genera/species in these habitats are underrepresented. METHODS: In this study, the structure of endospore-forming bacterial communities in sediment/mud samples from Vermelha Lagoon, Massambaba, Dois Rios and Abraão Beaches (saline environments), as well as the Praia Seca salterns (hypersaline environments) was determined via denaturing gradient gel electrophoresis. Bacterial strains were isolated from these environmental samples and further identified using 16S rRNA gene sequencing. Strains presenting emulsification values higher than 30 % were grouped via BOX-PCR, and the culture supernatants of representative strains were subjected to high temperatures and to the presence of up to 20 % NaCl to test their emulsifying activities in these extreme conditions. Mass spectrometry analysis was used to demonstrate the presence of surfactin. RESULTS: A diverse endospore-forming bacterial community was observed in all environments. The 110 bacterial strains isolated from these environmental samples were molecularly identified as belonging to the genera Bacillus, Thalassobacillus, Halobacillus, Paenibacillus, Fictibacillus and Paenisporosarcina. Fifty-two strains showed emulsification values of at least 30%, and they were grouped into 18 BOX groups. The stability of the emulsification values varied when the culture supernatants of representative strains were subjected to high temperatures and to the presence of up to 20% NaCl. The presence of surfactin was demonstrated in one of the most promising strains. CONCLUSION: The environments studied can harbor endospore-forming bacteria capable of producing biosurfactants with biotechnological applications. Various endospore-forming bacterial genera/species are presented for the first time as biosurfactant producers.

Diversity and bioactive potential of endospore-forming bacteria cultured from the marine sponge Haliclona simulans.

AIMS: Despite the frequent isolation of endospore-formers from marine sponges, little is known about the diversity and characterization of individual isolates. The main aims of this study were to isolate and characterize the spore-forming bacteria from the marine sponge Haliclona simulans and to examine their potential as a source for bioactive compounds. METHODS AND RESULTS: A bank of presumptive aerobic spore-forming bacteria was isolated from the marine sponge H. simulans. These represented c. 1% of the total culturable bacterial population. A subgroup of thirty isolates was characterized using morphological, phenotypical and phylogenetic analysis. A large diversity of endospore-forming bacteria was present, with the thirty isolates being distributed through a variety of Bacillus and Paenibacillus species. These included ubiquitous species, such as B. subtilis, B. pumilus, B. licheniformis and B. cereus group, as well as species that are typically associated with marine habitats, such as B. aquimaris, B. algicola and B. hwajinpoensis. Two strains carried the aiiA gene that encodes a lactonase known to be able to disrupt quorum-sensing mechanisms, and various isolates demonstrated protease activity and antimicrobial activity against different pathogenic indicator strains, including Clostridium perfringens, Bacillus cereus and Listeria monocytogenes. CONCLUSIONS: The marine sponge H. simulans harbours a diverse collection of endospore-forming bacteria, which produce proteases and antibiotics. This diversity appears to be overlooked by culture-dependent and culture-independent methods that do not specifically target sporeformers. SIGNIFICANCE AND IMPACT OF STUDY: Marine sponges are an as yet largely untapped and poorly understood source of endospore-forming bacterial diversity with potential biotechnological, biopharmaceutical and probiotic applications. These results also indicate the importance of combining different methodologies for the comprehensive characterization of complex microbial populations such as those found in marine sponges.

Spore-forming bacteria in soil cultivated with GM white poplars: isolation and characterization.

The impact of transgenic white poplars (Populus alba L. cv. 'Villafranca') was assessed on the soil aerobic spore-forming bacteria (SFB). The genetically modified poplars, expressing either the StSy gene for resveratrol production or the bar gene for herbicide tolerance, were cultivated in greenhouse. The occurrence of SFB was monitored in soil samples collected at eight different timepoints over a two-year period. The total culturable bacterial population of the StSy and bar trials underwent significant seasonal fluctuations in the range of 10(6)-2.5 x 10(8) CFU/g dry soil and of 10(4)-5 x 10(8) CFU/g dry soil, respectively. Changes occurred also within the culturable SFB population with size varying at 10(3)-5 x 10(4) CFU/g dry soil and 10(2)-2 x 10(5) CFU/g dry soil in the StSy and bar trials, respectively. No significant differences in the size of the total and SFB culturable populations were observed when comparing each transgenic line with the nontransformed control line while seasonal shifts of soil bacterial populations were evident in both trials. The culturable SFB fraction included three isolates (SFB-1, SFB-2 and SFB-3) classified by 16S rDNA sequence analysis as members of the Bacillus genus. According to the reported data, cultivation of both herbicide-resistant and resveratrol-producing GM white poplars did not affect the culturable SFB population at the soil level.

[Phylogenetic diversity of endophytic endospore-forming bacteria isolated from Cinnamomum longepaniculatum N. Chao ex H. W. Li].

OBJECTIVE: In order to study the diversity of endophytic endospore-forming bacteria in Cinnamomum longepaniculatum. METHODS: We took modified nutrient agar medium for isolation and cultivation and analyzed the 16S rRNA gene sequences of the isolates. RESULTS: Forty non-redundant endospore-forming bacterial isolates were ascertained, which accounted for 38.1% of all the endophytic bacterial isolates. Of them, 24 isolates were from roots, 7 from stems and 9 from leaves. Phylogenetic analysis based on 16S rRNA gene sequences showed that 35 of them belonged to 16 species of the genera Bacillus, Lysinibacillus and Paenibacillus, and 5 isolates with < 97% sequence similarities to their closely related members were presumed to be potential novel species. CONCLUSION: The results showed that the cultivable endospore-forming bacteria diversity was abundant and there were some potential novel strains in Cinnamomum longepaniculatum. The microflora of endophytic endospore-forming bacteria in individuals of C. longepaniculatum showed that some bacteria distributed in different organs, but the others were organ-specific bacteria.

Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria.

Minimal standards for describing new taxa within the aerobic endospore-forming bacteria are proposed, following Recommendation 30b of the Bacteriological Code (1990 Revision). These minimal standards are recommended as guidelines to assist authors in the preparation of descriptions for novel taxa. They encourage broad polyphasic characterization and the construction of descriptions that are practically useful in routine diagnostic laboratories. The proposals have been endorsed by the Subcommittee on the Taxonomy of the Genus Bacillus and Related Organisms of the International Committee on Systematics of Prokaryotes.

Diversity of aerobic and facultative alkalitolerant and halotolerant endospore formers in soil from the Alvord Basin, Oregon.

Many proteins produced by Bacillus species isolated from extreme environments have been utilized for industrial purposes, as these extreme environments often promote evolution of unique protein properties. The Borax Lake area is unusual due to its geothermal activity, elevated pH, and high arsenic and salt concentrations in its soils. Soils from this region are likely to harbor alkalitolerant, halotolerant, endospore-forming strains that may be of potential ecological and/or commercial interest. The objectives of this study were to develop new PCR primers that could target Bacillus or closely related 16S rRNA genes, to characterize the diversity of alkalitolerant, halotolerant, endospore-forming organisms in the soils surrounding Borax Lake, and to identify novel organisms that may ultimately provide new enzymes for applied use. A three-pronged approach was used to identify such bacteria in soil samples. Organisms were isolated using two different techniques. Finally, metagenomic DNA from soil samples was subjected to 16S rRNA gene amplification using the newly designed primers. Assays were performed to characterize the halotolerance and alkalitolerance of isolates. Four different endospore-forming genera and 22 different species were identified by sequencing their 16S rRNA genes. Twenty-five organisms had 96% or less identity to known organisms. Thus, the newly designed Bacillus-related PCR primer sets proved useful for the detection of new species of endospore-forming bacteria in these unique soils. Results indicate that the collection of strains obtained from the Borax Lake region represents a rich source of alkalitolerant, halotolerant, endospore formers.

Dominance of endospore-forming bacteria on a Rotating Activated Bacillus Contactor biofilm for advanced wastewater treatment.

The bacterial diversity inherent to the biofilm community structure of a modified rotating biological contactor wastewater treatment process, referred to as the Rotating Activated Bacillus Contactor (RABC) process, was characterized in this study, via both culture-dependent and culture-independent methods. On the basis of culture-dependent methods, Bacillus sp. were found to exist in large numbers on the biofilm (6.5% of the heterotrophic bacteria) and the microbial composition of the biofilms was quite simple. Only three phyla were identified-namely, the Proteobacteria, the Actinobacteria (High G+C Gram-positive bacteria), and the Firmicutes (Low G+C Gram-positive bacteria). The culture-independent partial 16S rDNA sequence analysis revealed a considerably more diverse microbial composition within the biofilms. A total of eight phyla were recovered in this case, three of which were major groups: the Firmicutes (43.9%), the Proteobacteria (28.6%), and the Bacteroidetes (17.6%). The remaining five phyla were minor groups: the Planctomycetes (4.4%), the Chlorobi (2.2%), the Actinobacteria (1.1%), the Nitrospirae (1.1%), and the Verrucomicrobia (1.1%). The two most abundant genera detected were the endospore-forming bacteria (31.8%), Clostridium and Bacillus, both of which are members of the Firmicutes phylum. This finding indicates that these endospore-forming bacteria successfully colonized and dominated the RABC process biofilms. Many of the colonies or clones recovered from the biofilms evidenced significantly high homology in the 16S rDNA sequences of bacteria stored in databases associated with advanced wastewater treatment capabilities, including nitrification and denitrification, phosphorus accumulation, the removal of volatile odors, and the removal of chlorohydrocarbons or heavy metals. The microbial community structures observed in the biofilms were found to correlate nicely with the enhanced performance of advanced wastewater treatment protocols.

Paenibacillus sepulcri sp. nov., isolated from biodeteriorated mural paintings in the Servilia tomb.

In 2001, a Gram-variable, facultatively anaerobic, endospore-forming bacterium isolated from biodeteriorated mural paintings in the Servilia tomb of the Roman necropolis of Carmona was deposited as Paenibacillus strain LMG 19508. Subsequently, the strain was characterized in detail using phenotypic and molecular methods. The 16S rRNA gene sequence confirmed that the strain belongs to the genus Paenibacillus and indicated its relationship to Paenibacillus mendelii CCM 4839(T) (96.7 % sequence similarity). The predominant menaquinone was MK-7. The cell wall contained meso-diaminopimelic acid of the A1gamma type. The DNA G+C content (50 mol%) and the major fatty acid (anteiso-C(15 : 0)) of strain LMG 19508(T) were also consistent with its affiliation to the genus Paenibacillus. DNA-DNA hybridization distinguished strain LMG 19508(T) from other phylogenetically related Paenibacillus species. Therefore, the isolate represents a novel species, for which the name Paenibacillus sepulcri sp. nov. is proposed. The type strain is CCM 7311(T) (=LMG 19508(T)).

Screening of Bacillus strains as potential probiotics and subsequent confirmation of the in vivo effectiveness of Bacillus subtilis MA139 in pigs.

A total of 124 samples were collected from the intestine of broiler chickens, piglet faeces, fermented foods, soils and Chinese herbs. More than 750 strains of aerobic, spore-forming bacteria were isolated from these samples. The inhibitory activity of these spore-forming strains against Escherichia coli K88, E. coli K99, Salmonella typhimurium and Staphylococcus aureus was assessed using a disc plate diffusion assay. The six bacilli with the largest inhibition zones against the four indicator bacteria were chosen and assessed for their resistance to unfavorable conditions within simulated gut environments. The strain Bacillus subtilis MA139 showed full resistance to pH 2, 0.3% bile salts and exhibited the highest antimicrobial activity. Based on these results, B. subtilis MA139 was selected as a potential probiotic and fed to piglets at concentrations of 2.2 x 10(5), 2.2 x 10(6) or 2.2 x 10(7) CFU/g of feed during a 28-day feeding trial. A negative control consisting of the basal diet with no additives and a positive control consisting of the basal diet supplemented with 16 g/ton flavomycin were also included. Ninety piglets between 35 and 40 days old were used in the in vivo animal trials. B. subtilis MA139 enhanced daily gain (P = 0.10) and feed conversion (P = 0.03) compared with the negative control. The performance of pigs fed B. subtilis MA139 supplemented diets did not differ from that of pigs fed the antibiotic diet. There was a significant increase in Lactobacilli cell counts (P = 0.02) and a numerical decrease in E. coli counts (P = 0.05) in the faecal samples of pigs fed B. subtilis MA139 with 2.2 x 10(5) CFU/g of feed. The overall results of this study show that the use of initial co-culture with indicator pathogens, a disc plate diffusion assay and simulated gut environment tolerance tests is one of effective methods of screening Bacillus for probiotic use and that B. subtilis MA139 is a promising alternative to antibiotics for use as a feed additive in piglet diets.

Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov., Paenibacillus anaericanus sp. nov. and Paenibacillus terrae strain MH72, N2O-producing bacteria isolated from the gut of the earthworm Aporrectodea caliginosa.

Earthworms emit nitrous oxide (N(2)O) via the activity of bacteria in their gut. Four N(2)O-producing facultative aerobes, ED1(T), ED5(T), MH21(T) and MH72, were isolated from the gut of the earthworm Aporrectodea caliginosa. The isolates produced N(2)O under conditions that simulated the microenvironment of the earthworm gut. ED1(T) and ED5(T) were Gram-negative, motile rods that carried out complete denitrification (i.e. the reduction of nitrate to N(2)) and contained membranous c-type cytochromes. ED1(T) grew optimally at 30 degrees C and pH 7. ED1(T) oxidized organic acids and reduced (per)chlorate, sulfate, nitrate and nitrite. The closest phylogenetic relative of ED1(T) was Dechloromonas agitata. ED5(T) grew optimally at 25 degrees C and pH 7. ED5(T) grew mainly on sugars, and nitrate and nitrite were used as alternative electron acceptors. The closest phylogenetic relatives of ED5(T) were Flavobacterium johnsoniae and Flavobacterium flevense. MH21(T) and MH72 were motile, spore-forming, rod-shaped bacteria with a three-layered cell wall. Sugars supported the growth of MH21(T) and MH72. Cells of MH21(T) grew in chains, were linked by connecting filaments and contained membranous b-type cytochromes. MH21(T) grew optimally at 30-35 degrees C and pH 7.7, grew by fermentation and reduced low amounts of nitrite to N(2)O. The closest phylogenetic relatives of MH21(T) were Paenibacillus borealis and Paenibacillus chibensis. Based on morphological, physiological and phylogenetic characteristics, ED1(T) (= DSM 15892(T) = ATCC BAA-841(T)), ED5(T) (= DSM 15936(T) = ATCC BAA-842(T)) and MH21(T) (=DSM 15890(T) = ATCC BAA-844(T)) are proposed as type strains of the novel species Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov. and Paenibacillus anaericanus sp. nov., respectively. MH72 is considered a new strain of Paenibacillus terrae.

Paenibacillus hodogayensis sp. nov., capable of degrading the polysaccharide produced by Sphaerotilus natans.

Sphaerotilus natans is a sheathed bacterium often found in activated sludge that has a bulking problem. A bacterial strain that is able to degrade the extracellular polysaccharide produced by S. natans was isolated. The isolate was a spore-forming, aerobic, rod-shaped bacterium. The Gram reaction was variable or negative. The optimum growth temperature was 30 degrees C and the optimum pH was 8. The G+C content of the DNA was 55 mol%. The major cellular fatty acid and respiratory quinone were anteiso-C(15 : 0) and MK-7, respectively. Phylogenetic analysis based on the 16S rRNA gene indicated that the isolate was a member of the genus Paenibacillus. The nearest relative, with a similarity of 94.2 %, was Paenibacillus koleovorans, a bacterium capable of degrading the sheath of S. natans. The phenotypic characteristics of the isolate were apparently different from those of related species in the genus Paenibacillus. It is proposed that the isolate be designated Paenibacillus hodogayensis sp. nov. The type strain is SG(T) (=JCM 12520(T)=KCTC 3919(T)).

Paenibacillus phyllosphaerae sp. nov., a xylanolytic bacterium isolated from the phyllosphere of Phoenix dactylifera.

A bacterial strain, designated PALXIL04(T), was isolated from the phyllosphere of Phoenix dactylifera. Phylogenetic analysis placed the isolate within the genus Paenibacillus with the closest relatives being Paenibacillus curdlanolyticus and Paenibacillus kobensis. DNA-DNA hybridization measurements showed low DNA relatedness (15-20 %) between the isolate and its closest relatives. Cells were Gram-variable, facultatively anaerobic, motile, sporulating rods. Catalase and oxidase were produced by the organism. Cellulose, starch, aesculin and xylan were hydrolysed. Growth was supported by many carbohydrates as the carbon source. MK-7 was the predominant menaquinone and anteiso-C(15 : 0) the major fatty acid. The G+C content of the DNA was 50.7 mol%. Phylogenetic, DNA-DNA relatedness and phenotypic analyses indicated that strain PALXIL04(T) represents a novel species of the genus Paenibacillus, for which the name Paenibacillus phyllosphaerae sp. nov. is proposed. The type strain is PALXIL04(T) (=LMG 22192(T)=CECT 5862(T)).

Desulfosporomusa polytropa gen. nov., sp. nov., a novel sulfate-reducing bacterium from sediments of an oligotrophic lake.

Five strains of sulfate-reducing bacteria were isolated from the highest positive dilutions of a most probable number (MPN) series supplemented with lactate and inoculated with sediments from the oligotrophic Lake Stechlin. The isolates were endospore-forming and were motile by means of laterally inserted flagella. They stained Gram-negative and contained b-type cytochromes. CO difference spectra indicated the presence of P582 as a sulfite reductase. Phylogenetic analyses of the 16S rDNA sequences revealed that the isolates were very closely affiliated with the genus Sporomusa. However, sulfate and amorphous Fe(OH)(3), but not sulfite, elemental sulfur, MnO(2), or nitrate were used as terminal electron acceptors. Homoacetogenic growth was found with H(2)/CO(2) gas mixture, formate, methanol, ethanol, and methoxylated aromatic compounds. The strains grew autotrophically with H(2) plus CO(2) in the presence or absence of sulfate. Formate, butyrate, several alcohols, organic acids, carbohydrates, some amino acids, choline, and betaine were also utilized as substrates. The growth yield with lactate and sulfate as substrate was 7.0 g dry mass/mol lactate and thus two times higher than in sulfate-free fermenting cultures. All isolates were able to grow in a temperature range of 4-37 degrees C. Physiologically and by the presence of a Gram-negative cell wall, the new isolates resemble known Desulfosporosinus species. However, phylogenetically they are affiliated with the Gram-negative genus Sporomusa belonging to the Selenomonas subgroup of the Firmicutes. Therefore, the new isolates reveal a new phylogenetic lineage of sulfate-reducing bacteria. A new genus and species, Desulfosporomusa polytropa gen. nov., sp. nov. is proposed.

The microbial content of non-sterile pharmaceuticals distributed in Norway.

Seventy-seven registered trademark pharmaceuticals and allied products, not required by the relevant monographs to comply with the test for sterility, were investigated for their microbial content. All the products examined complied with current regulations with respect to the numbers and types of microbes isolated, indicating the effectiveness of existing production practices in meeting existing standards. Gram-positive endospore-forming rods accounted for the majority of the bacteria isolated. Gram-negative rods were present for the most part in incidental numbers. However, some of these were of species that have been previously indicated as opportunistic pathogens and which should be considered as objectionable in pharmaceuticals. A number of the isolates showed antibiotic resistances unusual for the species in question.

Isolation, characterization, and identification of bacterial contaminants in semifinal gelatin extracts.

Bacterial contamination of gelatin is of great concern. Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products. In a previous study (E. De Clerck and P. De Vos, Syst. Appl. Microbiol. 25:611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated. In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined. Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced. In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants. Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated. For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups. Representative strains were identified by means of 16S rRNA gene sequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity. The majority of isolates belonged to members of Bacillus or related endospore-forming genera. Representative strains were identified as Bacillus cereus, Bacillus coagulans, Bacillus fumarioli, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus sonorensis, Bacillus subtilis, Bacillus gelatini, Bacillus thermoamylovorans, Anoxybacillus contaminans, Anoxybacillus flavithermus, Brevibacillus agri, Brevibacillus borstelensis, and Geobacillus stearothermophilus. The majority of these species include strains exhibiting gelatinase activity. Moreover, some of these species have known pathogenic properties. These findings are of great concern with regard to the safety and quality of gelatin and its applications.

Screening of bacterial contamination during gelatine production by means of denaturing gradient gel electrophoresis, focussed on Bacillus and related endospore-forming genera.

AIMS: To screen for bacterial contamination during gelatine production by means of denaturing gradient gel electrophoresis (DGGE). As members of Bacillus and related genera were found to persist in the final product, this study focussed on these taxa. METHODS AND RESULTS: Template DNA was extracted from gelatine samples at five crucial points of a gelatine production process. A primer specific for Bacillus and related genera was designed and used in a selective PCR, followed by a nested DGGE-PCR targeting the V9 region of the 16S rDNA. DGGE analysis of the resulting amplicons, and sequence analysis of selected bands, showed high sequence similarities of these bands with Bacillus fumarioli, B. licheniformis, B. coagulans and Clostridium perfringens. When the selective PCR was omitted, primarily Lactobacillus bands were retrieved. CONCLUSIONS: PCR-DGGE analysis of gelatine extracts can be used for tracing and screening of bacterial contamination during gelatine production. A selective PCR, nested with DGGE-PCR, gave much more accurate information about endospore-forming contaminants than did the direct DGGE procedure alone. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of this nested DGGE-PCR protocol may provide important information about possible hazards to the final microbiological quality and/or safety of gelatine, so allowing production parameters and/or remediation procedures may be adjusted on-line.