Bacillus probiotics: an alternative to antibiotics for livestock production

The use of probiotics as feed supplements in animal production has increased considerably over the last decade, particularly since the ban on antibiotic growth promoters in the livestock sector. Several Bacillus sp. are attractive for use as probiotic supplements in animal feed due to their ability to produce spores. Their heat stability and ability to survive the low pH of the gastric barrier represent an advantage over other probiotic micro‐organisms. This review discusses important characteristics required for selection of Bacillus probiotic strains and summarizes the beneficial effect of Bacillus‐based feed additives on animal production. Although the mechanism of action of Bacillus probiotics has not been fully elucidated, they are effective in improving the growth, survival and health status of terrestrial and aquatic livestock. Bacillus strains also have utility in bioremediation and can reduce nitrogenous waste, thereby improving environmental conditions and water quality. Finally, recent innovative approaches for using Bacillus spores in various applications are discussed.     

Assessing the activity of microbicides against bacterial spores: knowledge and pitfalls

Bacterial endospores (spores) have a higher intrinsic resistance to microbicides as compared to other microbial forms, most likely due to their impermeable outer layers and low water content. Though structural differences between the spores of various bacterial species may account for observed variations in their resistance to microbicides, flaws in methods for testing the sporicidal activity of microbicides often exaggerate the differences. This has major implications when considering the selection of one or more surrogates to assess microbicides against clinically relevant spore‐formers such as Clostridium difficile. The mounting significance of Cl. difficile as a pathogen is leading to a corresponding increase in the number of commercially available microbicidal formulations claiming activity against its spores without proper differentiation between the product's sporistatic and sporicidal actions. In this review we critically assess the situation and the implications of product claims on the field use of microbicidal products.     

THE INLAND CHAETOCEROS (BACILLARIOPHYCEAE) SPECIES OF NORTH AMERICA1

Five taxa of Chaetoceros occur in inland waters of North America. These most commonly occur in waters with elevated total dissolved solids in arid regions of the western United States and Canada. Chaetoceros amanita Cleve-Euler is characterized by consistently forming relatively long chains of cells and having very spinose primary resting spore valves. Chaetoceros elmorei Boyer also forms long chains of cells which are connected by evident valvar processes; spores are nearly always smooth. Chaetoceros muelleri Lemm. may form short chains with processes between sibling valves, but also produces solitary cells lacking processes. Chaetoceros muelleri var. subsalsum (Lemm.)Johansen et Rushforth is similar to the nominate but never produces cells with Processes. Both of the C. muelleri varieties produce spores with smooth primary valves. Chaetoceros simplex Ostenfeld is characterized by a noncolonial habit, cells lacking processes and the production of resting spores with warty to some what spinose primary valves.     

Effect of radioprotective agents in sporulation medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and environmentally relevant UV radiation

Aims: To determine the effects of cysteine, cystine, proline and thioproline as sporulation medium supplements on Bacillus subtilis spore resistance to hydrogen peroxide (H₂O₂), wet heat, and germicidal 254 nm and simulated environmental UV radiation. Methods and Results: Bacillus subtilis spores were prepared in a chemically defined liquid medium, with and without supplementation of cysteine, cystine, proline or thioproline. Spores produced with thioproline, cysteine or cystine were more resistant to environmentally relevant UV radiation at 280–400 and 320–400 nm, while proline supplementation had no effect. Spores prepared with cysteine, cystine or thioproline were also more resistant to H₂O₂ but not to wet heat or 254‐nm UV radiation. The increases in spore resistance attributed to the sporulation supplements were eliminated if spores were chemically decoated. Conclusions: Supplementation of sporulation medium with cysteine, cystine or thioproline increases spore resistance to solar UV radiation reaching the Earth’s surface and to H₂O₂. These effects were eliminated if the spores were decoated, indicating that alterations in coat proteins by different sporulation conditions can affect spore resistance to some agents. Significance and Impact of the Study: This study provides further evidence that the composition of the sporulation medium can have significant effects on B. subtilis spore resistance to UV radiation and H₂O₂. This knowledge provides further insight into factors influencing spore resistance and inactivation.     

Implications of paper vs stainless steel biological indicator substrates for formaldehyde gas decontamination

Aims: This study was undertaken to determine the effectiveness of biological indicators currently being employed during formaldehyde decontamination. Data suggest that detectable amounts of formaldehyde are absorbed into the paper strips contained in currently used biological indicators. Absorbed formaldehyde has the potential to inhibit the growth of indicator spores, thus leading to false negative results. Indicators composed of either stainless steel carriers or paper strips were investigated to determine whether stainless steel carriers can be used as an alternative to paper strip indicators. Methods and Results: Biological indicators were exposed to formaldehyde gas and were tested for the presence of formaldehyde and any possible inhibition of spore growth. Absorbed formaldehyde was detected in the paper strip carriers while no formaldehyde was detected from any of the stainless steel carriers. Exposed paper strips were found to inhibit growth of up to 1 × 10⁶ spores while the stainless steel carriers did not inhibit the growth of spores. Conclusions: During decontamination, biological indicators composed of paper spore strips absorb formaldehyde and inhibit growth of any surviving spores. Stainless steel carriers do not absorb formaldehyde and are an ideal alternative substrate for biological indicators. Significance and Impact of the Study: The popular paper strip biological indicator can lead to false negative results during decontamination and is unsuitable for validating formaldehyde decontamination.     

Evidence for specificity of cultivable bacteria associated with arbuscular mycorrhizal fungal spores

Bacteria associated with arbuscular mycorrhizal (AM) fungal spores may play functional roles in interactions between AM fungi, plant hosts and defence against plant pathogens. To study AM fungal spore-associated bacteria (AMB) with regard to diversity, source effects (AM fungal species, plant host) and antagonistic properties, we isolated AMB from surface-decontaminated spores of Glomus intraradices and Glomus mosseae extracted from field rhizospheres of Festuca ovina and Leucanthemum vulgare. Analysis of 385 AMB was carried out by fatty acid methyl ester (FAME) profile analysis, and some also identified using 16S rRNA gene sequence analysis. The AMB were tested for capacity to inhibit growth in vitro of Rhizoctonia solani and production of fluorescent siderophores. Half of the AMB isolates could be identified to species (similarity index 0.6) within 16 genera and 36 species. AMB were most abundant in the genera Arthrobacter and Pseudomonas and in a cluster of unidentified isolates related to Stenotrophomonas. The AMB composition was affected by AM fungal species and to some extent by plant species. The occurrence of antagonistic isolates depended on AM fungal species, but not plant host, and originated from G. intraradices spores. AM fungal spores appear to host certain sets of AMB, of which some can contribute to resistance by AM fungi against plant pathogens.     

Sensitization of Bacillus subtilis spores to dry heat and desiccation by pretreatment with oxidizing agents

Aims: To determine if pretreatment with oxidizing agents sensitizes Bacillus subtilis spores to dry heat or desiccation. Methods: Bacillus subtilis spores were killed approx. 90% by oxidizing agents, and the sensitivity of treated and untreated spores to dry heat and desiccation was determined. The effects of pyruvate on spore recovery after oxidizing agent pretreatment and then dry heat or desiccation were also determined. Conclusions: Spores pretreated with Oxone? or hypochlorite were not sensitized to dry heat or freeze‐drying. However, hydrogen peroxide or t‐butylhydroperoxide pretreatment sensitized spores to dry heat or desiccation, and the desiccation caused mutagenesis in the survivors. Pyruvate increased recovery of spores treated with hydrogen peroxide alone or plus dry heat or desiccation, and with t‐butylhydroperoxide and desiccation, but not with t‐butylhydroperoxide alone or plus dry heat. Significance and Impact of the Study: Pretreatment with peroxides sensitizes bacterial spores to subsequent stress. This finding may suggest improved regimens for spore inactivation.     

The effect of peroxyacetic acid-based sanitizer, heat and ultrasonic waves on the survival of Clostridium estertheticum spores in vitro

AIM: To determine the effect of selected physical and chemical treatments on the survival of 'blown pack'-causing Clostridium estertheticum. METHODS AND RESULTS: The study investigated the survival of the spores of 'blown pack'-causing C. estertheticum following the four treatments, which include: heat alone, ultrasound followed by heat treatment, peroxyacetic acid (POAA)-based sanitizer followed by heat treatment and POAA sanitizer followed by heat treatment in the presence of 20% animal fat. No C. estertheticum survivors were recovered in spore preparations that underwent either of the two treatments with the sanitizer, resulting in the inactivation of 4 to 5 log CFU ml(-1) of spores. Similarly, no survivors were detected in spore preparations that were treated with the sanitizer for 5 min at room temperature without further heat treatment. When using heat alone and ultrasound followed by heat treatment, complete spore inactivation did not occur for spores heated at times and temperature combinations other than 240 s at 100 degrees C. CONCLUSIONS: POAA sanitizer used with or without heat is capable of in vitro inactivation of at least 4 log CFU ml(-1)C. estertheticum spores. SIGNIFICANCE AND IMPACT OF THE STUDY: The data generated in the study provide background information for controlling 'blown pack'-causing clostridia on dressed carcasses and in meat plant environment.     

Ultrastructure of Buddenbrockia allmani n. sp. (Myxozoa, Malacosporea), a parasite of Lophopus crystallinus (Bryozoa, Phylactolaemata)

Development of a new species of malacosporean myxozoan (Buddenbrockia allmani n. sp.) in the bryozoan Lophopus crystallinus is described. Early stages, represented by isolated cells or small groups, were observed in the host's body wall or body cavity. Multiplication and rearrangement of cells gave an outer cell layer around a central mass. The outer cells made contact by filopodia and established adherens junctions. Sporoplasmosomes were a notable feature of early stages, but these were lost in subsequent development. Typical malacosporean sacs were formed from these groups by attachment of the inner (luminal) cells by a basal lamina to the outer layer (mural cells). Division of luminal cells gave rise to a population of cells that was liberated into the lumen of the sac. Mitotic spindles in open mitosis and prophase stages of meiosis were observed in luminal cells. Centrioles were absent. Detached luminal cells assembled to form spores with four polar capsules and several valve cells surrounding two sporoplasms with secondary cells. Restoration of sporoplasmosomes occurred in primary sporoplasms. A second type of sac was observed with highly irregular mural cells and stellate luminal cells. A radially striated layer and dense granules in the polar capsule wall, and previous data on 18 rDNA sequences enabled assignment of the species to the genus Buddenbrockia, while specific diagnosis relied on the rDNA data and on sac shape and size.