Sporogenes Strains Research Articles

Specificity of the AMP-6000 Method for Enumerating Clostridium Endospores in Milk.

Enumeration of endospores of butyric acid-forming clostridia in cheese milk is an essential part of milk quality monitoring for cheese producers to avoid late blowing, severe spoilage caused by clostridia during ripening. However, due to the lack of an internationally standardized method, different methods are used and it is important to consider how the choice of method affects the results. This is particularly relevant when clostridial spore counts in milk are considered for quality payments. The aim of this study was to evaluate the specificity of the AMP-6000 method for the enumeration of endospores of cheese spoiling clostridia in milk. First, to assess the prevalence of Clostridium diversity and to determine potential non-target species, we identified isolates from positive reactions of the AMP-6000 method used to quantify clostridial endospores in raw milk and teat skin samples by MALDI-TOF MS. Based on these results, a strain library was designed to evaluate method inclusivity and exclusivity using pure cultures of target and non-target strains according to ISO 16140-2:2016. Most target Clostridium tyrobutyricum strains, as well as all tested C. butyricum and C. sporogenes strains were inclusive. However, C. beijerinckii may be underestimated as only some strains gave positive results. All non-target strains of bacilli and lysinibacilli, but not all paenibacilli, were confirmed to be exclusive. This study provides performance data to better understand the results of microbiological enumeration of butyric acid-forming clostridia in milk and serves as a basis for future methodological considerations and improvements.

Microbial Characterization of Retail Cocoa Powders and Chocolate Bars of Five Brands Sold in Italian Supermarkets.

A microbial characterization of cocoa powder and chocolate bars of three batches of five different brands sold in Italian markets was performed. The results showed a variable microbial population consisting of mesophilic and thermophilic spore formation in both types of products. The chocolate bars were also contaminated with molds of environmental origin. Bacillus spp. and Geobacillus spp. were found in both products. The chocolate bars were also contaminated by molds belonging to the genera Penicillium and Cladosporium. The sporogenous strains mainly originate from the raw materials, i.e., cocoa beans, as the heat treatments involved (roasting of the beans and conching of the chocolate) are not sufficient to reach commercial sterility. Furthermore, the identified spore-forming species have often been isolated from cocoa beans. The molds isolated from chocolate seem to have an origin strictly linked to the final phases of production (environment and packaging). However, the level of contaminants is limited (<2 log CFU/g); the molds do not develop in both products due to their low Aw (<0.6) and do not affect the safety of the products. However, a case of mold development in chocolate bars was observed. Among the isolated molds, only Penicillium lanosocoeruleum demonstrated a high xero-tolerance and grew under some conditions on chocolate bars. Its growth could be explained by a cocoa butter bloom accompanied by the presence of humidity originating from the bloom or acquired during packaging.

Endogenous CRISPR-Cas Systems in Group I Clostridium botulinum and Clostridium sporogenes Do Not Directly Target the Botulinum Neurotoxin Gene Cluster.

Most strains of proteolytic group I Clostridium botulinum (G1 C. botulinum) and some strains of Clostridium sporogenes possess genes encoding botulinum neurotoxin (BoNT), a potent neuroparalytic agent. Within G1 C. botulinum, conserved bont gene clusters of three major toxin serotypes (bont/A/B/F) can be found on conjugative plasmids and/or within chromosomal pathogenicity islands. CRISPR-Cas systems enable site-specific targeting of previously encountered mobile genetic elements (MGE) such as plasmids and bacteriophage through the creation of a spacer library complementary to protospacers within the MGEs. To examine whether endogenous CRISPR-Cas systems restrict the transfer of bont gene clusters across strains we conducted a bioinformatic analysis profiling endogenous CRISPR-Cas systems from 241 G1 C. botulinum and C. sporogenes strains. Approximately 6,200 CRISPR spacers were identified across the strains and Type I-B, III-A/B/D cas genes and CRISPR array features were identified in 83% of the strains. Mapping the predicted spacers against the masked strain and RefSeq plasmid dataset identified 56,000 spacer–protospacer matches. While spacers mapped heavily to targets within bont(+) plasmids, no protospacers were identified within the bont gene clusters. These results indicate the toxin is not a direct target of CRISPR-Cas but the plasmids predominantly responsible for its mobilization are. Finally, while the presence of a CRISPR-Cas system did not reliably indicate the presence or absence of a bont gene cluster, comparative genomics across strains indicates they often occupy the same hypervariable loci common to both species, potentially suggesting similar mechanisms are involved in the acquisition and curation of both genomic features.

Microbiota in vitro modulated with polyphenols shows decreased colonization resistance against Clostridioides difficile but can neutralize cytotoxicity

While the knowledge on gut microbiota - C. difficile interactions has improved over the years, the understanding of the underlying mechanisms providing colonization resistance as well as preventative measures against the infection remain incomplete. In this study the antibiotic clindamycin and polyphenol extracts from pomegranate and blueberries were used individually and in combination to modulate fecal microbial communities in minibioreactor arrays (MBRA). Modulated communities were inoculated with C. difficile (ribotype 027). Subsequent 7-day periodical monitoring included evaluation of C. difficile growth and activity of toxins TcdA and TcdB as well as analysis of MBRA bacterial community structure (V3V4 16 S metagenomics). Polyphenols affected multiple commensal bacterial groups and showed different synergistic and antagonistic effects in combination with clindamycin. Exposure to either clindamycin or polyphenols led to the loss of colonization resistance against C. difficile. The successful growth of C. difficile was most significantly correlated with the decrease in Collinsella and Lachnospiraceae. Additionally, we demonstrated that Clostridium sporogenes decreased the activity of both C. difficile toxins TcdA and TcdB. The feature was shown to be common among distinct C. sporogenes strains and could potentially be applicable as a non-antibiotic agent for the alleviation of C. difficile infection.

Proteomic analysis of four Clostridium botulinum strains identifies proteins that link biological responses to proteomic signatures.

Microorganisms alter gene and protein expression in response to environmental conditions to adapt and survive. Whereas the genetic composition of a microbe represents an organism’s biological potential, the proteins expressed provide a functional readout of the organism’s response to the environment. Understanding protein expression patterns in response to specific environmental conditions furthers fundamental knowledge about a microbe, which can be especially useful for understudied organisms such as Clostridium botulinum examined herein. In addition, protein expression patterns that reproducibly occur in certain growth conditions hold potential in fields such as microbial forensics, in which determination of conditions in which an unknown possible biothreat sample had been grown may be important. To investigate the identity and reproducibility of protein profile patterns for varied strains, we defined the proteomic profiles of four Group I strains of Clostridium botulinum, a Category A biothreat agent and the organism responsible for the production of the botulinum neurotoxin (BoNT), in two different culture media grown for five days. The four C. botulinum strains produced one of three neurotoxins (BoNT/A, /B, or /F), and their protein profiles were compared to that of a fifth non-toxigenic strain of C. sporogenes. These strains each had DNA sequences available to assist in accurate protein identification. Differing culture growth phase, bacterial strain, and growth medium resulted in reproducible protein profiles, which were used to calculate relative protein abundance ratios as an internally normalized metric of microbial growth in varying conditions. The resulting protein profiles provide functional information about how four Group I C. botulinum strains and a C. sporogenes strain respond to the culture environment during growth and explores the feasibility of using these proteins to characterize unknown samples.

Bioethanol and biobutanol production from sugarcorn juice

Corn hybrids with high stalk sugar content or ‘sugarcorn’, are fast-growing energy crops recently developed by Agriculture and Agri-Food Canada. For the first time, this study uses juice extracted from sugarcorn plants for bioethanol and biobutanol production via microbial fermentation. Characterization results for sugarcorn juice from two different crop harvest times are presented. Juice characteristics such as, moisture content, total solids, total dissolved solids, pH, density, elemental analysis, protein, reducing sugars and total carbohydrates were determined for the two juice batches. Sugarcorn juice used in the study contained a maximum of 145 g L−1 carbohydrates, with sucrose, glucose and fructose accounting for 80% of the sugars. Saccharomyces cerevisiae grown in sugarcorn juice supplemented with 3 g L−1 yeast extract produced 45.6 g L−1 ethanol in 72 h of fermentation (yield = 0.41 g ethanol per g carbohydrates). For biobutanol fermentation, a sporogenic strain of Clostridium beijerinckii was cultivated anaerobically in sugarcorn juice-P2 medium, achieving a butanol concentration of 8.3 g L−1 in 257 h (yield = 0.31 g butanol per g total fermentable sugars). Sugarcorn is a new Canadian energy crop and a source of readily fermentable sugars, that has the potential to save on energy and enzyme costs, when compared to corn grain based biofuel production processes.

Genetic Characterization of the Exceptionally High Heat Resistance of the Non-toxic Surrogate Clostridium sporogenes PA 3679

Clostridium sporogenes PA 3679 is a non-toxic endospore former that is widely used as a surrogate for Clostridium botulinum by the food processing industry to validate thermal processing strategies. PA 3679 produces spores of exceptionally high heat resistance without botulinum neurotoxins, permitting the use of PA 3679 in inoculated pack studies while ensuring the safety of food processing facilities. To identify genes associated with this heat resistance, the genomes of C. sporogenes PA 3679 isolates were compared to several other C. sporogenes strains. The most significant difference was the acquisition of a second spoVA operon, spoVA2, which is responsible for transport of dipicolinic acid into the spore core during sporulation. Interestingly, spoVA2 was also found in some C. botulinum species which phylogenetically cluster with PA 3679. Most other C. sporogenes strains examined both lack the spoVA2 locus and are phylogenetically distant within the group I Clostridium, adding to the understanding that C. sporogenes are dispersed C. botulinum strains which lack toxin genes. C. sporogenes strains are thus a very eclectic group, and few strains possess the characteristic heat resistance of PA 3679.

Substrate dependent modulation of butanol to ethanol ratio in non-acetone forming Clostridium sporogenes NCIM 2918

Present study reports a non-acetone producing Clostridium sporogenes strain as a potential producer of liquid biofuels. Alcohol production was positively regulated by sorbitol and instant dry yeast as carbon and nitrogen sources respectively. Media optimization resulted in maximum butanol and ethanol titer (gL−1) of 12.1 and 7.9 respectively. Depending on the combination of carbon sources, the organism was found to manipulate its metabolism towards synthesis of either ethanol or butanol, thereby affecting the total alcohol titer. Among various dual substrate combinations, glucose-glycerol mixture in the ratio of 60:40 resulted in maximum butanol and ethanol titer (gL−1) of 11.9 and 12.1 respectively with total alcohol productivity of 0.59gL−1h−1. In the mixture, when pure glycerol was replaced with crude glycerol, butanol and ethanol titer (gL−1) of 11.2 and 11.7 was achieved. Hence, the strain shows immense potential for biofuels production using crude glycerol as cheap substrate.

Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains

Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75–100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores.

Systemic colonization of clover (Trifolium repens) by Clostridium botulinum strain 2301.

In recent years, cases of botulism in cattle and other farm animals and also in farmers increased dramatically. It was proposed, that these cases could be affiliated with the spreading of compost or other organic manures contaminated with Clostridium botulinum spores on farm land. Thus, soils and fodder plants and finally farm animals could be contaminated. Therefore, the colonization behavior and interaction of the botulinum neurotoxin (BoNT D) producing C. botulinum strain 2301 and the non-toxin producing Clostridium sporogenes strain 1739 were investigated on clover (Trifolium repens) in a field experiment as well as in phytochamber experiments applying axenic and additionally soil based systems under controlled conditions. Plants were harvested and divided into root and shoot parts for further DNA isolation and polymerase chain reaction (PCR) assays; subsamples were fixed for fluorescence in situ hybridization analysis in combination with confocal laser scanning microscopy. In addition, we observed significant differences in the growth behavior of clover plants when inoculated with clostridial spores, indicating a plant growth promoting effect. Inoculated plants showed an increased growth index (shoot size, wet and dry weight) and an enlarged root system induced by the systemic colonization of clover by C. botulinum strain 2301. To target C. botulinum and C. sporogenes, 16S rDNA directed primers were used and to specifically detect C. botulinum, BoNT D toxin genes targeted primers, using a multiplex PCR approach, were applied. Our results demonstrate an effective colonization of roots and shoots of clover by C. botulinum strain 2301 and C. sporogenes strain 1739. Detailed analysis of colonization behavior showed that C. botulinum can occur as individual cells, in cell clusters and in microcolonies within the rhizosphere, lateral roots and within the roots tissue of clover.

Implications of Genome-Based Discrimination between Clostridium botulinum Group I and Clostridium sporogenes Strains for Bacterial Taxonomy.

Taxonomic classification of Clostridium botulinum is based on the production of botulinum neurotoxin (BoNT), while closely related, nontoxic organisms are classified as Clostridium sporogenes. However, this taxonomic organization does not accurately mirror phylogenetic relationships between these species. A phylogenetic reconstruction using 2,016 orthologous genes shared among strains of C. botulinum group I and C. sporogenes clearly separated these two species into discrete clades which showed ∼93% average nucleotide identity (ANI) between them. Clustering of strains based on the presence of variable orthologs revealed 143 C. sporogenes clade-specific genetic signatures, a subset of which were further evaluated for their ability to correctly classify a panel of presumptive C. sporogenes strains by PCR. Genome sequencing of several C. sporogenes strains lacking these signatures confirmed that they clustered with C. botulinum strains in a core genome phylogenetic tree. Our analysis also identified C. botulinum strains that contained C. sporogenes clade-specific signatures and phylogenetically clustered with C. sporogenes strains. The genome sequences of two bont/B2-containing strains belonging to the C. sporogenes clade contained regions with similarity to a bont-bearing plasmid (pCLD), while two different strains belonging to the C. botulinum clade carried bont/B2 on the chromosome. These results indicate that bont/B2 was likely acquired by C. sporogenes strains through horizontal gene transfer. The genome-based classification of these species used to identify candidate genes for the development of rapid assays for molecular identification may be applicable to additional bacterial species that are challenging with respect to their classification.

Spoilage of oat bran by sporogenic microorganisms revived from soil buried 4000 years ago in Iranian archaeological site

The Bronze Age archaeological site of Shahr-i Sokhta (30° 39′ N; 61° 24’ E), located today in southeastern Iran, Sistan region, is a special archaeological deposit in which the exceptional preservation of human, plant and animal remains, due to the dry climate of the region, can provide detailed information on one of the first complex proto-urban societies. In recent years, there has been growing interest in changes in local climate and environment as major reasons why the settlement was abandoned about 4000 years ago. Food shortage has been regarded as a direct effect of these changes. No attention has been paid to the potential health hazards associated with ancient urban/domestic pollution, although large garbage deposits have been found in several parts of the site.During excavations in 2007, four soil samples were taken under aseptic conditions at a depth of 1.5–2 m in a stratified deposit sealed by the floor of a small house, dated to the second half of the third millennium BC. Microbiological, palynological, carpological and microanalytical studies were performed on the four soil samples. Site C was identified as the most affected by human activity. Failure of conventional methods of detecting culturable and unculturable microbes in site C indicated the need for specific culture conditions suggested by palynological observations. Since oat seed residues were identified among the archaeobotanical material, oat bran was used as carbon and energy source to make a new medium to revive microbes. Coarsely ground oat bran was sterilized twice and soaked with minimal medium as sole carbon source. About 50 mg of buried soil from site C was added to the medium in cell culture flasks with aerobic and anaerobic stoppers and incubated at 28–30 °C and at 4 °C.After incubation under aerobic and anaerobic conditions, five sporogenic microbes were identified by sequencing 16S rDNA and ITS rRNA regions: a sporogenic strain IRC3 identified as Bacillus sp. was the only isolate under anaerobic conditions, whereas under aerobic conditions four moulds were isolated: Aspergillus flavus IRC1, Penicillium crysogenum IRC2, Cladosporium sp. IRC4, and the psychrotroph Aspergillus restrictus IRC5. Bacillus sp., with 99.7% similarity to Bacillus subtilis, broke down oat bran, producing a gel, while Cladosporium sp., with 99.8% similarity to Cladosporium sphaerospermium, grew on oat bran by synthesizing intracellular lipids. All these microbes are known to spoil food and they are common where there is intense human activity.

Inhibitory activity of reuterin, nisin, lysozyme and nitrite against vegetative cells and spores of dairy-related Clostridium species

The butyric acid fermentation, responsible for late blowing of cheese, is caused by the outgrowth in cheese of some species of Clostridium, resulting in texture and flavor defects and economical losses. The aim of this study was to evaluate the effectiveness of different antimicrobial compounds against vegetative cells and spores of C. tyrobutyricum, C. butyricum, C. beijerinckii and C. sporogenes strains isolated from cheeses with late blowing defect. Minimal inhibitory concentration (MIC) for reuterin, nisin, lysozyme and sodium nitrite were determined against Clostridium strains in milk and modified RCM (mRCM) after 7d exposure. Although the sensitivity of Clostridium to the tested antimicrobials was strain-dependent, C. sporogenes and C. beijerinckii generally had higher MIC values than the rest of Clostridium species. The majority of Clostridium strains were more resistant to antimicrobials in milk than in mRCM, and vegetative cells exhibited higher sensitivity than spores. Reuterin (MIC values 0.51–32.5mM) and nisin (MIC values 0.05–12.5μg/ml) were able to inhibit the growth of vegetative cells and spores of all assayed Clostridium strains in milk and mRCM. Strains of C. tyrobutyricum exhibited the highest sensitivity to lysozyme (MIC values<0.20–400μg/ml) and sodium nitrite (MIC values 18.75–150μg/ml). These results suggest that reuterin and nisin, with a broad inhibitory activity spectrum against Clostridium spp. spores and vegetative cells, may be the best options to control Clostridium growth in dairy products and to prevent associated spoilage, such as late blowing defect of cheese. However, further studies in cheese would be necessary to validate this hypothesis.

Diversity of Cultivable Fungi Isolated from the Chenghai Lake in Yunnan Province

The aims of this study were to investigate the fungal diversity and the ecological distribution of these fungi in the plateau lake and surrounding soil, to analyze the relations between fungi quality and the physical and chemical factors of lake water. Fungi were identif ied with classical morphological method. In Addition, fungal diversity, evenness and similarity in different habitats were analyzed. Software IBM SPSS Statistics 19 was used to carry out Pearson correlation analysis and signif icance test on the physical and chemical factors as well as fungi quantity from 8 winter water samples. Among the total 1 760 strains isolated, 1 504 sporogenous strains were identified as 98 genera. The dominant genera were Aspergillus and Penicillium, with isolation frequency 14.89% and 12.39%, respectively. Both fungal species(65 genera) and isolation frequency(60.13%) were the highest in the spring soil. The analysis of diversity and evenness index showed that fungi in the spring soil and the winter sediment had the highest diversity, with Shannon-Wiener index and Simpson index as 2.917 7, 0.907 4 and 2.910 7, 0.918 5, respectively; the evenness index of winter lake water was the largest(0.793 1). Similarity coeff icient revealed that fungal similarity between winter lake water and winter sediment was the highest, with Jaccard index and Sorenson index 0.365 4 and 0.535 2, separately. Pearson correlation analysis showed that temperature, total nitrogen, total hardness were positively correlated with fungi quantity, whereas pH, conductivity, turbidity, total phosphorus, and total organic carbon were negatively correlated with fungi quantity. Significance test showed that eight physical and chemical factors were not significantly related to fungi quantity. The investigation suggested that a large number of fungi exist in great diversity in the Chenghai Lake and surrounding soil. The results laid the foundation for subsequent activity screening and metabolic product research. Tab 6, Ref 23

Pichia insulana sp. nov., a novel cactophilic yeast from the Caribbean

A novel species of ascomycetous yeast, Pichia insulana sp. nov., is described from necrotic tissue of columnar cacti on Caribbean islands. P. insulana is closely related to and phenotypically very similar to Pichia cactophila and Pichia pseudocactophila. There are few distinctions between these taxa besides spore type, host preference and locality. Sporogenous strains of P. insulana that produce asci with four hat-shaped spores have been found only on Curaçao, whereas there was no evidence of sporogenous P. cactophila from that island. In addition, sequences of the D1/D2 fragment of the large-subunit rDNA from 12 Curaçao strains showed consistent differences from the sequences of the type strains of P. cactophila and P. pseudocactophila. The type strain of P. insulana is TSU00-106.5(T) (=CBS 11169(T) =UCD-FST 09-160(T)).

Characterization of Clostridium spp. Isolated from Spoiled Processed Cheese Products

Of 42 spoiled cheese spread products, 35 were found to harbor Clostridium spp. Typical signs of spoilage were gas production and off-odor. The identity was determined for about half of the isolates (n = 124) by Analytab Products (API), Biolog, the RiboPrinter System, 16S rDNA sequencing, cellular fatty acid analysis, or some combination of these. The majority of isolates were identified as Clostridium sporogenes (in 33% of products), but Clostridium cochlearium (in 12% of products) and Clostridium tyrobutyricum (in 2% of products) were also retrieved. Similarity analysis of the riboprint patterns for 21 isolates resulted in the identification of 10 ribogroups. A high degree of relatedness was observed between isolates of C. sporogenes originating from products produced 3 years apart, indicating a common and, over time, persistent source of infection. The spoilage potential of 11 well-characterized isolates and two culture collection strains was analyzed by inoculating shrimp cheese spread with single cultures and then storing them at 37°C. Tubes inoculated with C. tyrobutyricum did not show any visible signs of growth (e.g., coagulation, discoloration, gas formation) in the cheese spread. After 2 weeks of incubation, tubes inoculated with C. cochlearium or C. sporogenes showed gas-holes, syneresis with separation of coagulated casein and liquid, and a change in color of the cheese. The amount of CO2 produced by C. cochlearium strains was approximately one-third that produced by the majority of C. sporogenes strains. To our knowledge, this is the first study to isolate and identify C. cochlearium as a spoilage organism in cheese spread.

Isolation, In vitro Antibacterial Activity, Bacterial Sensitivity and Plasmid Profile of Lactobacilli

The present research work was conducted to evaluate the beneficial effects as well as the safety aspects of lactobacilli as probiotic. Lactobacilli were isolated from poultry faecal samples, feed samples and from some known preparations procured from poultry feed manufacturers. L. acidophilus and L. sporogenes were tested for the antibacterial activity against four poultry pathogens viz. Escherichia coli, Salmonella spp., Proteus spp. and Pseudomonas aeruginosa. Cell free supernatant (CFS) of L. acidophilus exhibited significantly higher antibacterial activity against Salmonella spp. at original pH (4.50±0.02). At the adjusted pH (6.50±0.02) significantly higher antibacterial activity was recorded against indicator organism except for P. aeruginosa. Likewise, L. sporogenes exhibited similar antibacterial activity at original as well as adjusted pH except for E. coli. Antibacterial activity against E. coli was significantly higher at adjusted pH than at original pH of CFS. The competitive exclusion of E. coli by lactobacilli over the intestinal epithelial cells (IEC) was checked. L. acidophilus strain I, which was of poultry origin, exhibited maximum attachment over IEC as compared to other three strains of non-poultry origin viz. L. acidophilus strain II, L. sporogenes strain I and II. Overall, L. acidophilus exhibited higher competitive exclusion as compared to L. sporogenes. All the lactobacilli of poultry origin were most sensitive to penicillin G, amoxycillin, ampicillin and chloramphenicol, least sensitive to sulphamethizole, ciprofloxacin, neomycin, norfloxacin and pefloxacin and resistant to metronidazole and nalidixic acid. The isolates from probiotic preparations were most sensitive to ampicillin, amoxycillin and tetracycline, least sensitive to sulphamethizole, norfloxacin, neomycin and ceftriazone and resistant to nalidixic acid and metronidazole. Eight of the multiple drug resistant lactobacilli isolates were studied for the presence of plasmids. Plasmids could be extracted from six isolates of lactobacilli. These plasmids could be responsible for bacteriocin production or for antibiotic resistance of the strains. The lactobacilli need further studies regarding their safety for use in the probiotic preparations. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 9 : 1336-1342)

Genotyping Clostridium botulinum toxinotype A isolates from patients using amplified rDNA restriction analysis

In this study, the application of amplified rDNA restriction analysis (ARDRA) for characterizing Clostridium botulinum toxinotype A strains isolated from individuals with botulism was evaluated. Ten restriction enzymes were tested for their suitability in ARDRA as a typing method and HhaI was selected for the best outcome. Analysis of HhaI restriction profiles of the amplified products divided C. botulinum isolates into three clusters. Non-toxigenic Clostridium sporogenes strains showed an ARDRA restriction pattern that was distinct from those observed for C. botulinum. The successful use of ARDRA for subdivision of C. botulinum in this study confirmed that this technique is a powerful method for typing of C. botulinum toxinotype A clonal diversity. In addition, it is rapid, sensitive and simple.

Investigation of action of peroxoacetic acid on lipid component of bacterial spores and contribution to the standardization of efficiency evaluation test

The aim of this research was to contribute to a more detailed insight in the action of peroxygen disinfectants based on peroxoacetic acid (PAA), on lipid components of sporogenic forms of microorganisms. Sporogenic forms are recognized to be significantly more resistant in comparison to vegetative forms. Bacillus cereus ATCC 11778 and Bacillus subtilis NCTC 10480 where chosen as referent bacterial sporogenic strains used to investigate the acting mechanism of PAA. After treatment of the above mentioned bacterial strains with PAA, fatty acids have been isolated from untreated and treated spores and consequent changes in the lipid component were analyzed. The obtained methyl-esters of fatty acids (MEFA) were analyzed by gas chromatography/mass spectrometry (GC/MS), using a standard of bacterial MEFA. Results confirmed that after treatment, in Bacillus cereus certain quantitative changes occurred, which included the decrease in quantity of fatty acids with 16 and 17 carbon atoms, i.e. 16:0, 16:1, /so 17:0 and anteiso 17:0. At the same time, in B. Subtilis the significant decrease in quantity of fatty acids with 15 and 17 carbon atoms occurred, i.e. anteiso 15:0 and anteiso 17:0.

Survival and conjugation of Bacillus thuringiensis in a soil microcosm.

The survival and conjugation ability of sporogenic and asporogenic Bacillus thuringiensis strains were investigated in broth, in non-amended sterile clay soil monoculture and in mixed soil culture. The 75 kb pHT73 plasmid carrying an erythromycin resistance determinant and a cry1Ac gene was transferred in mating broth and soil microcosm. Survival of strains was assessed in soil monoculture and in mixed soil culture for up to 20 days. Sporogenic strains rapidly formed viable spores which were maintained until the end of the experiment. The asporogenic strains were no longer recovered after 8 days of incubation. This study shows that the environmental impact of asporogenic B. thuringiensis strains is lower than that of sporogenic B. thuringiensis strains. Thus, the use of asporogenic strains may significantly reduce any potential risk (gene transfer, soil and plant contamination) due to the dissemination of B. thuringiensis-based biopesticides in the environment.