Inhibition of Bacillus cereus Strains by Antimicrobial Metabolites from Lactobacillus johnsonii CRL1647 and Enterococcus faecium SM21

Bacillus cereus is an endospore-forming, Gram-positive bacterium able to cause foodborne diseases. Lactic acid bacteria (LAB) are known for their ability to synthesize organic acids and bacteriocins, but the potential of these compounds against B. cereus has been scarcely documented in food models. The present study has examined the effect of the metabolites produced by Lactobacillus johnsonii CRL1647 and Enterococcus faecium SM21 on the viability of select B. cereus strains. Furthermore, the effect of E. faecium SM21 metabolites against B. cereus strains has also been investigated on a rice food model. L. johnsonii CRL1647 produced 128 mmol/L of lactic acid, 38 mmol/L of acetic acid and 0.3 mmol/L of phenyl-lactic acid. These organic acids reduced the number of vegetative cells and spores of the B. cereus strains tested. However, the antagonistic effect disappeared at pH 6.5. On the other hand, E. faecium SM21 produced only lactic and acetic acid (24.5 and 12.2 mmol/L, respectively) and was able to inhibit both vegetative cells and spores of the B. cereus strains, at a final fermentation pH of 5.0 and at pH 6.5. This would indicate the action of other metabolites, different from organic acids, present in the cell-free supernatant. On cooked rice grains, the E. faecium SM21 bacteriocin(s) were tested against two B. cereus strains. Both of them were significantly affected within the first 4 h of contact; whereas B. cereus BAC1 cells recovered after 24 h, the effect on B. cereus 1 remained up to the end of the assay. The LAB studied may thus be considered to define future strategies for biological control of B. cereus.     

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

Background

The first step of the bacterial lifecycle is the germination of bacterial spores into their vegetative form, which requires the presence of specific nutrients. In contrast to closely related Bacillus anthracis spores, Bacillus cereus spores germinate in the presence of a single germinant, inosine, yet with a significant lag period.

Methods and Findings

We found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores. The lag period also dissipated when inosine was supplemented with the co-germinator alanine. In fact, HPLC-based analysis revealed the presence of amino acids in the supernatant of germinated B. cereus spores. The released amino acids included alanine in concentrations sufficient to promote rapid germination of inosine-treated spores. The alanine racemase inhibitor D-cycloserine enhanced germination of B. cereus spores, presumably by increasing the L-alanine concentration in the supernatant. Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine. These mutant spores, however, germinated efficiently when inosine was supplemented with alanine. Finally, removal of released amino acids in a washout experiment abrogated inosine-mediated germination of B. cereus spores.

Conclusions

We found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process. Therefore, B. cereus spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination.     

Influence of baking enzymes on antimicrobial activity of five bacteriocin‐like inhibitory substances produced by lactic acid bacteria isolated from Lithuanian sourdoughs

Aim: To evaluate the effect of four different baking enzymes on the inhibitory activity of five bacteriocin‐like inhibitory substances (BLIS) produced by lactic acid bacteria (LAB) isolated from Lithuanian sourdoughs. Methods and Results: The overlay assay and the Bioscreen methods revealed that the five BLIS exhibited an inhibitory effect against spore germination and vegetative outgrowth of Bacillus subtilis, the predominant species causing ropiness in bread. The possibility that the observed antibacterial activity of BLIS might be lost after treatment with enzymes used for baking purposes was also examined. Conclusions: The enzymes tested; hemicellulase, lipase, amyloglucosidase and amylase had little or no effect on the majority of the antimicrobial activities associated with the five BLIS studied. Significance and Impact of the Study: This study suggests a potential application in the sourdough baking industry for these antimicrobial producing LAB strains in the control of B. subtilis spore germination and vegetative outgrowth.     

Inhibitory Effects of Turf Pesticides on Bacillus popilliae and the Prevalence of Milky Disease

Fourteen pesticides (fungicides, herbicides, and insecticides) were tested to determine whether they had deleterious effects on the bioinsecticide Bacillus popilliae, the causal agent of milky disease. All of these pesticides reduced levels of spore viability, spore germination, and/or vegetative cell growth when they were tested over a range of concentrations from 0 to 1,000 ppm of active ingredient, and the fungicides had the greatest detrimental effects. As determined by tests in water, the level of spore viability was significantly reduced by chlorothalonil, iprodione, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and 2-[(4-chloro-o-tolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid. In tests performed with iprodione, loss of viability was evident at concentrations less than the concentration calculated to result from recommended use. Tests performed in soil demonstrated that triadimefon, chlorothalonil, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and pendimethalin at concentrations resulting from recommended rates of application reduced spore titers. Spore germination did not occur in the continued presence of 2-[(4-chloro-otolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid, isofenphos, and chlordane, whereas exposure of spores to triadimefon or pendimethalin for 2 days stimulated germination. The tests to determine effects on spore germination were inconclusive for all other pesticides. Triadimefon, chlorothalonil, iprodione, pendimethalin, and chlorpyrifos at concentrations less than the concentrations recommended for use inhibited vegetative cell growth of B. popilliae, and chlordane at a concentration that was twice the concentration expected to result from the recommended rate of application repressed cell growth. My data support the hypothesis that use of synthetic pesticides can contribute to a low incidence of milky disease in white grubs.     

The Bacillus cereus GerN and GerT protein homologs have distinct roles in spore germination and outgrowth, respectively

The GerT protein of Bacillus cereus shares 74% amino acid identity with its homolog GerN. The latter is a Na⁺/H⁺-K⁺ antiporter that is required for normal spore germination in inosine. The germination properties of single and double mutants of B. cereus ATCC 10876 reveal that unlike GerN, which is required for all germination responses that involve the GerI germinant receptor, the GerT protein does not have a significant role in germination, although it is required for the residual GerI-mediated inosine germination response of a gerN mutant. In contrast, GerT has a significant role in outgrowth; gerT mutant spores do not outgrow efficiently under alkaline conditions and outgrow more slowly than the wild type in the presence of high NaCl concentrations. The GerT protein in B. cereus therefore contributes to the success of spore outgrowth from the germinated state during alkaline or Na⁺ stress.     

Diversity of spore germination in response to inosine and L-alanine and its interaction with NaCl and pH in the Bacillus cereus group

Aims: Our aim was to assess the diversity of the nutrient germination response of Bacillus cereus spores. Methods and Results: B. cereus spore germination was monitored by decrease in optical density using a Bioscreen C analyser in response to the major germinant substances inosine and l -alanine. Spores of a set of 12 strains taken to illustrate the diversity of the B. cereus group showed ranging germination capacities. Two strains never germinated in the presence of l -alanine, at any of the germinant concentrations tested. Both the extent and rate of spore germination were affected by low pH and high NaCl concentration, but differently according to the strain. Conclusions: A broad diversity was observed in nutrient-triggered spore germination among the members of the B. cereus group. Spore germination of some strains occurred at low concentrations of inosine or l -alanine, suggesting high receptor sensitivity to germinants. The activity of these receptors was also affected by pH or high NaCl concentration. Significance and Impact of the Study: The greater ability of some strains to germinate in response to l -alanine and inosine is one criterion among others for B. cereus strain selection in food processing or storage studies, before confirmation in complex food or laboratory media. The diversity in response to germinants found among the B. cereus strains suggests a differential expression and (or) absence of some germination genes involved in the response, mainly to l -alanine.     

Live Cell Imaging of Germination and Outgrowth of Individual Bacillus subtilis Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker

Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze Bacillus subtilis spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of B. subtilis vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.     

Production and Characterization of Monoclonal Antibodies against Vegetative Cells of Bacillus cereus

Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus vegetative cells while failing to recognize B. cereus spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.     

Study of Bacillus subtilis Endospores in Soil by Use of a Modified Endospore Stain

The Schaeffer-Fulton endospore stain was modified so that it would stain Bacillus subtilis endospores in soil smears. The modified stain differentiated among dormant spores, spores undergoing activation, and spores which had germinated but had not yet shown outgrowth. These differentiations were seen for spores in soil and for pure spore preparations in the laboratory. This stain was used to show reversible B. subtilis spore activation promoted by an Ensifer adhaerens-like indigenous bacterium in soil and by pure cultures of E. adhaerens added to spores in the laboratory. Under the specific conditions in the laboratory, spore germination did not proceed beyond the activation stage, and relatively little change occurred in the numbers of both E. adhaerens and B. subtilis. This was also true in soil, although some germination with destruction of spores and vegetative cells did occur if the soil had been nutritionally enriched by preincubation with incorporated ground alfalfa.     

Conjugal transfer between Bacillus thuringiensis and Bacillus cereus strains is not directly correlated with growth of recipient strains

Bacillus thuringiensis and Bacillus cereus belong to the B. cereus species group. The two species share substantial chromosomal similarity and differ mostly in their plasmid content. The phylogenetic relationship between these species remains a matter of debate. There is genetic exchange both within and between these species, and current evidence indicates that insects are a particularly suitable environment for the growth of and genetic exchange between these species. We investigated the conjugation efficiency of B. thuringiensis var. kurstaki KT0 (pHT73-Em^R) as a donor and a B. thuringiensis and several B. cereus strains as recipients; we used one-recipient and two-recipient conjugal transfer systems in vitro (broth and filter) and in Bombyx mori larvae, and assessed multiplication following conjugation between Bacillus strains. The B. thuringiensis KT0 strain did not show preference for genetic exchange with the B. thuringiensis recipient strain over that with the B. cereus recipient strains. However, B. thuringiensis strains germinated and multiplied more efficiently than B. cereus strains in insect larvae and only B. thuringiensis maintained complete spore germination for at least 24 h in B. mori larvae. These findings show that there is no positive association between bacterial multiplication efficiency and conjugation ability in infected insects for the used strains.     

Involvement of alanine racemase in germination of Bacillus cereus spores lacking an intact exosporium

The l-alanine mediated germination of food isolated Bacillus cereus DSA 1 spores, which lacked an intact exosporium, increased in the presence of d-cycloserine (DCS), which is an alanine racemase (Alr) inhibitor, reflecting the activity of the Alr enzyme, capable of converting l-alanine to the germination inhibitor d-alanine. Proteomic analysis of the alkaline extracts of the spore proteins, which include exosporium and coat proteins, confirmed that Alr was present in the B. cereus DSA 1 spores and matched to that encoded by B. cereus ATCC 14579, whose spore germination was strongly affected by the block of conversion of l- to d-alanine. Unlike ATCC 14579 spores, l-alanine germination of B. cereus DSA 1 spores was not affected by the preincubation with DCS, suggesting a lack of restriction in the reactant accessibility.     

Chemical and Morphological Studies of Bacterial Spore Formation : III. The Effect of 8-Azaguanine on Spore and Parasporal Protein Formation in Bacillus cereus var. Alesti

The purine analogue, 8-azaguanine, was added to cultures of the parasporal crystal-forming organism Bacillus cereus var. alesti at different times during growth and synchronous sporulation. The effect of its incorporation has been studied with particular reference to cell growth, nucleic acid composition, cytology, and the synthesis of the spore and crystal protein. Additions of the analogue during any stage of growth prevented further cell proliferation and all spore and crystal formation. Since both nucleic acids continued to be formed, cells of an increased size developed, containing large masses of chromatin in the form of condensed balls or axial cords. Lipid-containing inclusions also appeared following these additions and were usually aggregated at the centre or poles of the cells. The analogue could be isolated as the ribonucleotide from both the acid soluble and RNA fractions of these inhibited cells. Additions of the analogue following commencement of sporulation did not prevent either spore or crystal formation or affect the nucleic acid content of the sporulating cells. However, as before, the 8-azaguanine was incorporated into both the acid soluble and RNA of the cells, but not into these fractions of the spores ultimately formed. The implications of these findings are discussed in relation to crystal protein synthesis.     

Chemical and Morphological Studies of Bacterial Spore Formation : II. Spore and Parasporal Protein Formation in Bacillus cereus var. Alesti

The development of both the spore and parasporal protein crystal of Bacillus cereus var. alesti was followed using chemical and cytological techniques. The changes which led to the formation of the fore-spore were similar to those already described for Bacillus cereus. However, adjacent to the developing fore-spore a small inclusion became discernible in phase contrast. This protein inclusion during its growth was differentiated from the chromatin and lipid-containing inclusions by sequential staining techniques. During spore and crystal formation no net synthesis of either nucleic acid was detected. Tracer studies with radioactive phosphorus confirmed that the spore chromatin was derived from that in the vegetative cell. These same studies also indicated that a turnover of ribonucleic acid occurred during the sporulation process. During their formation both the spore and crystal incorporated methionine-³⁵S from the medium and from cellular material into a bound form. Sequential extractions with alkali and with alkaline-thioglycollate reagent revealed that the solubility characteristics of the mature crystal were possibly related to the presence of intermolecular disulphide bonds which developed after the major synthesis of the crystal was complete. The synthetic nature of sporogenesis and crystal formation is discussed with reference to the concept of "endotrophic" sporulation.     

Sporulation Temperature Reveals a Requirement for CotE in the Assembly of both the Coat and Exosporium Layers of Bacillus cereus Spores

The Bacillus cereus spore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers of B. cereus ATCC 14579 spores and on the resulting spore properties. The cotE deletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ΔcotE spores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ΔcotE spores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ΔcotE spores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H₂O₂ and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly of B. cereus spore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation.     

Changes in Colonic Bile Acid Composition following Fecal Microbiota Transplantation Are Sufficient to Control Clostridium difficile Germination and Growth

Fecal microbiota transplantation (FMT) is a highly effective therapy for recurrent Clostridium difficile infection (R-CDI), but its mechanisms remain poorly understood. Emerging evidence suggests that gut bile acids have significant influence on the physiology of C. difficile, and therefore on patient susceptibility to recurrent infection. We analyzed spore germination of 10 clinical C. difficile isolates exposed to combinations of bile acids present in patient feces before and after FMT. Bile acids at concentrations found in patients’ feces prior to FMT induced germination of C. difficile, although with variable potency across different strains. However, bile acids at concentrations found in patients after FMT did not induce germination and inhibited vegetative growth of all C. difficile strains. Sequencing of the newly identified germinant receptor in C. difficile, CspC, revealed a possible correspondence of variation in germination responses across isolates with mutations in this receptor. This may be related to interstrain variability in spore germination and vegetative growth in response to bile acids seen in this and other studies. These results support the idea that intra-colonic bile acids play a key mechanistic role in the success of FMT, and suggests that novel therapeutic alternatives for treatment of R-CDI may be developed by targeted manipulation of bile acid composition in the colon.     

Germination of Bacillus thuringiensis var. israelensis spores in the gut of Aedes larvae (Diptera: Culicidae)

In laboratory experiments, germination and growth of Bacillus thuringiensis israelensis in the gut of Aedes aegypti and A. vexans larvae (Culicidae: Diptera) was observed. The number of spores and vegetative cells in the gut of living larvae and in cadavers was estimated by plaing homogenized larvae on selective agar plates. The number of spores per gut increased in the first 40–140 min of exposure to a maximum, and decreased in the subsequent time, demonstrating spore germination in living larvae, moribunds, and in cadavers. Twenty-four hours after the death of the larvae, a minimal amount of spores, but an increased number of vegetative cells, was found in cadavers. In A. aegypti larvae, germination and growth of B. thuringiensis israelensis in the larval gut was photographically documented.     

Characterization of Reutericyclin Produced by Lactobacillus reuteri LTH2584

Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains of Escherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C₈ chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. Höltzel, M. G. Gänzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766–2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of L. reuteri LTH2584 corresponded to that of purified as well as synthetic reutericyclin.     

Detection of<Emphasis Type="Italic"> Bacillus cereus</Emphasis> group bacteria from cardboard and paper with real-time PCR

The aim of this study was to develop a PCR-based rapid method to detect Bacillus cereus group cells from paper and cardboard. Primers targeting the 16S rDNA and real-time PCR with SYBR green I detection were used in order to be able to also quantify the target. Both autoclaved cardboard samples spiked with B. cereus vegetative cells or spores and naturally contaminated paper and cardboard samples were studied. Results were compared with culturing verified by commercial (API) tests. Several different methods were tested for DNA isolation from the paper and cardboard samples. Two commercial kits intended for soils, the UltraClean soil DNA kit and the FastDNA spin kit for soil, gave the most reproducible results. In spiked samples, the average yield was 50% of added vegetative cells, but spore yield was only about 10%. PCR results from adding vegetative cells correlated with added colony-forming unit (cfu) values (r=0.93, P <0.001) in the range 100–10,000 cfu g^–1. Three out of nine studied paper and cardboard samples contained B. cereus group bacteria, based both on culturing and real-time PCR. The numbers were 10²–10³ bacteria g^–1; and PCR gave somewhat higher results than culturing. Thus, real-time PCR can be used as a rapid semi-quantitative method to screen paper and cardboard samples for contamination with B. cereus group bacteria.     

Isolation and characterization of bacteriocin-like inhibitory substances from lactic acid bacteria isolated from Azerbaijan cheeses

The search for lactic acid bacteria (LAB) producing bacteriocin-like inhibitory substances (BLISs) was performed in traditional Azerbaijan cheeses. Eight strains have been isolated and identified, four of which (S1a, S2, Q2b, and M1b) were identified as Lactobacillus buchneri. The remaining four strains (M3a, A4b, Q4a, and A3) were identified as L. pentosus, L. brevis, L. fermentum, and L. collinoides, respectively. Antimicrobial agents of all isolated LAB strains suppressed the growth of Lactobacillus bulgaricus 340, Escherichia coli, Enterococcus durans, and Listeria innocua. Saccharomyces cerevisiae and Candida pseudotropicalis were resistant to these BLISs. Proteinase K and trypsin completely inactivated BLIS whereas lipase and amylase partially inactivated BLIS.