Observation of the effect of gravity on the motion of antimatter.

Einstein's general theory of relativity from 19151 remains the most successful description of gravitation. From the 1919 solar eclipse2 to the observation of gravitational waves3, the theory has passed many crucial experimental tests. However, the evolving concepts of dark matter and dark energy illustrate that there is much to be learned about the gravitating content of the universe. Singularities in the general theory of relativity and the lack of a quantum theory of gravity suggest that our picture is incomplete. It is thus prudent to explore gravity in exotic physical systems. Antimatter was unknown to Einstein in 1915. Dirac's theory4 appeared in 1928; the positron was observed5 in 1932. There has since been much speculation about gravity and antimatter. The theoretical consensus is that any laboratory mass must be attracted6 by the Earth, although some authors have considered the cosmological consequences if antimatter should be repelled by matter7-10. In the general theory of relativity, the weak equivalence principle (WEP) requires that all masses react identically to gravity, independent of their internal structure. Here we show that antihydrogen atoms, released from magnetic confinement in the ALPHA-g apparatus, behave in a way consistent with gravitational attraction to the Earth. Repulsive 'antigravity' is ruled out in this case. This experiment paves the way for precision studies of the magnitude of the gravitational acceleration between anti-atoms and the Earth to test the WEP.

Sympathetic cooling of positrons to cryogenic temperatures for antihydrogen production.

The positron, the antiparticle of the electron, predicted by Dirac in 1931 and discovered by Anderson in 1933, plays a key role in many scientific and everyday endeavours. Notably, the positron is a constituent of antihydrogen, the only long-lived neutral antimatter bound state that can currently be synthesized at low energy, presenting a prominent system for testing fundamental symmetries with high precision. Here, we report on the use of laser cooled Be+ ions to sympathetically cool a large and dense plasma of positrons to directly measured temperatures below 7 K in a Penning trap for antihydrogen synthesis. This will likely herald a significant increase in the amount of antihydrogen available for experimentation, thus facilitating further improvements in studies of fundamental symmetries.

Laser cooling of antihydrogen atoms.

The photon-the quantum excitation of the electromagnetic field-is massless but carries momentum. A photon can therefore exert a force on an object upon collision1. Slowing the translational motion of atoms and ions by application of such a force2,3, known as laser cooling, was first demonstrated 40 years ago4,5. It revolutionized atomic physics over the following decades6-8, and it is now a workhorse in many fields, including studies on quantum degenerate gases, quantum information, atomic clocks and tests of fundamental physics. However, this technique has not yet been applied to antimatter. Here we demonstrate laser cooling of antihydrogen9, the antimatter atom consisting of an antiproton and a positron. By exciting the 1S-2P transition in antihydrogen with pulsed, narrow-linewidth, Lyman-α laser radiation10,11, we Doppler-cool a sample of magnetically trapped antihydrogen. Although we apply laser cooling in only one dimension, the trap couples the longitudinal and transverse motions of the anti-atoms, leading to cooling in all three dimensions. We observe a reduction in the median transverse energy by more than an order of magnitude-with a substantial fraction of the anti-atoms attaining submicroelectronvolt transverse kinetic energies. We also report the observation of the laser-driven 1S-2S transition in samples of laser-cooled antihydrogen atoms. The observed spectral line is approximately four times narrower than that obtained without laser cooling. The demonstration of laser cooling and its immediate application has far-reaching implications for antimatter studies. A more localized, denser and colder sample of antihydrogen will drastically improve spectroscopic11-13 and gravitational14 studies of antihydrogen in ongoing experiments. Furthermore, the demonstrated ability to manipulate the motion of antimatter atoms by laser light will potentially provide ground-breaking opportunities for future experiments, such as anti-atomic fountains, anti-atom interferometry and the creation of antimatter molecules.

Lichenysin is produced by most Bacillus licheniformis strains.

AIMS: The aim of this study was to elucidate the prevalence of lichenysin production in Bacillus licheniformis and to see whether this feature was restricted to certain genotypes. Secondly, we wanted to see whether cytotoxicity reflected the measured levels of lichenysin. METHODS AND RESULTS: Fifty-three genotyped strains of B. licheniformis, representing a wide variety of sources, were included. lchAA gene fragments were detected in all strains by polymerase chain reaction (PCR). All 53 strains produced lichenysins with four molecular masses as confirmed by LC-MS/MS (liquid chromatography-tandem mass spectrometry) analysis. The amounts of lichenysin varied more than two orders of magnitude between strains and were irrespective of genotype. Finally, there was a strong association between lichenysin concentrations and toxicity towards boar spermatozoa, erythrocytes and Vero cells. CONCLUSIONS: Lichenysin synthesis was universal among the 53 B. licheniformis strains examined. The quantities varied considerably between strains, but were not specifically associated with genotype. Cytotoxicity was evident at lichenysin concentrations above 10 µg ml(-1) , which is in accordance with previous studies. SIGNIFICANCE AND IMPACT OF STUDY: This study might be of interest to those working on B. licheniformis for commercial use as well as for authorities who make risk assessments of B. licheniformis when used as a food and feed additive.

Antibacterial activity of chemically defined chitosans: influence of molecular weight, degree of acetylation and test organism.

Chitosans, polysaccharides obtained from the exoskeleton of crustaceans, have been shown to exert antibacterial activity in vitro and their use as a food preservative is of growing interest. However, beyond a consensus that chitosan appears to disrupt the bacterial cell membrane, published data are inconsistent on the chemical characteristics that confer the antibacterial activity of chitosan. While most authors agree that the net charge density of the polymer (reflected in the fraction of positively charged amino groups at the C-2 position of the glucosamine unit) is an important factor in antibacterial activity, conflicting data have been reported on the effect of molecular weight and on the susceptibility among different bacterial species to chitosan. Therefore, we prepared batches of water-soluble hydrochloride salts of chitosans with weight average molecular weights (M(w)) of 2-224kDa and degree of acetylation of 0.16 and 0.48. Their antibacterial activity was evaluated using tube inhibition assays and membrane integrity assays (N-Phenyl-1-naphthylamine fluorescence and potassium release) against Bacillus cereus, Escherichia coli, Salmonella Typhimurium and three lipopolysaccharide mutants of E. coli and S. Typhimurium. Chitosans with lower degree of acetylation (F(A)=0.16) were more active than the more acetylated chitosans (F(A)=0.48). No trends in antibacterial action related to increasing or decreasing M(w) were observed although one of the chitosans (M(w) 28.4kDa, F(A)=0.16) was more active than the other chitosans, inhibiting growth and permeabilizing the membrane of all the test strains included. The test strains varied in their susceptibility to the different chitosans with wild type S. Typhimurium more resistant than the wild type E. coli. Salmonellae lipopolysaccharide mutants were more susceptible than the matched wild type strain. Our results show that the chitosan preparation details are critically important in identifying the antibacterial features that target different test organisms.

Antibacterial activity of sphagnum acid and other phenolic compounds found in Sphagnum papillosum against food-borne bacteria.

AIMS: To identify the phenolic compounds in the leaves of Sphagnum papillosum and examine their antibacterial activity at pH appropriate for the undissociated forms. METHODS AND RESULTS: Bacterial counts of overnight cultures showed that whilst growth of Staphylococcus aureus 50084 was impaired in the presence of milled leaves, the phenol-free fraction of holocellulose of S. papillosum had no bacteriostatic effect. Liquid chromatography-mass spectrometry analysis of an acetone-methanol extract of the leaves detected eight phenolic compounds. Antibacterial activity of the four dominating phenols specific to Sphagnum leaves, when assessed in vitro as minimal inhibitory concentrations (MICs), were generally >2.5 mg ml(-1). MIC values of the Sphagnum-specific compound 'sphagnum acid' [p-hydroxy-beta-(carboxymethyl)-cinnamic acid] were >5 mg ml(-1). No synergistic or antagonistic effects of the four dominating phenols were detected in plate assays. CONCLUSIONS: Sphagnum-derived phenolics exhibit antibacterial activity in vitro only at concentrations far in excess of those found in the leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: We have both identified the phenolic compounds in S. papillosum and assessed their antibacterial activity. Our data indicate that phenolic compounds in isolation are not potent antibacterial agents and we question their potency against food-borne pathogens.

Sphagnan--a pectin-like polymer isolated from Sphagnum moss can inhibit the growth of some typical food spoilage and food poisoning bacteria by lowering the pH.

AIMS: Investigate if the antibacterial effect of sphagnan, a pectin-like carbohydrate polymer extracted from Sphagnum moss, can be accounted for by its ability to lower the pH. METHODS AND RESULTS: Antibacterial activity of sphagnan was assessed and compared to that of three other acids. Sphagnan in its acid form was able to inhibit growth of various food poisoning and spoilage bacteria on low-buffering solid growth medium, whereas sphagnan in its sodium form at neutral pH had no antibacterial activity. At similar acidic pH, sphagnan had comparable antibacterial activity to that of hydrochloric acid and a control rhamnogalacturonan pectin in its acid form. CONCLUSIONS: Sphagnan in its acid form is a weak macromolecular acid that can inhibit bacterial growth by lowering the pH of environments with a low buffering capacity. SIGNIFICANCE AND IMPACT OF THE STUDY: It has previously been suggested that sphagnan is an antimicrobial polysaccharide in the leaves of Sphagnum moss with a broad range of potential practical applications. Our results now show that sphagnan in its acid form can indeed inhibit bacterial growth, but only of acid-sensitive species. These findings represent increased knowledge towards our understanding on how sphagnan or Sphagnum moss might be used in practical applications.

Cytotoxicity in Bacillus mojavensis is abolished following loss of surfactin synthesis: implications for assessment of toxicity and food poisoning potential.

Bacillus subtilis and the closely related species Bacillus pumilus and Bacillus licheniformis have periodically been suggested to play a role in the aetiology of food poisoning despite the fact that the organisms do not possess the genes associated with enteropathogenicity in Bacillus cereus. We show here that Bacillus mojavensis, an organism closely related to B. subtilis, is able to produce toxic components which identify as a complex of three different surfactin analogues. These cyclic lipopeptides were soluble in methanol, heat stable after treatment in a boiling water bath for 10 min, resistant to enzymatic degradation by pepsin, trypsin, endoprotease V8 and proteinase K and formed pores in planar lipid bilayers. They were cytotoxic when tested in a series of commonly used laboratory cytotoxicity assays, namely, lactate dehydrogenase release, haemolysis, inhibition of both protein synthesis in Vero cells and motility in boar sperm. We show that such in vitro markers of enterotoxicity are due entirely to production of cyclic lipopeptides since deletion of sfp, a gene essential for surfactin synthesis which abolished the cytotoxicity to Vero cells, boar sperm motility and haemolytic activity. Thus, the relevance of cyclic lipopeptides as food poisoning toxins needs to be evaluated in assays other than the cell cytotoxicity assays in common use.

Cytotoxic Bacillus spp. belonging to the B. cereus and B. subtilis groups in Norwegian surface waters.

AIMS: To investigate the presence and numbers of Bacillus spp. spores in surface waters and examine isolates belonging to the B. cereus and B. subtilis groups for cytotoxicity, and to discuss the presence of cytotoxic Bacillus spp. in surface water as hazard identification in a risk assessment approach in the food industry. METHODS AND RESULTS: Samples from eight different rivers with variable degree of faecal pollution, and two drinking water sources, were heat shocked and examined for the presence of Bacillus spp. spores using membrane filtration followed by cultivation on bovine blood agar plates. Bacillus spp. was present in all samples. The numbers varied from 15 to 1400 CFU 100 ml(-1). Pure cultures of 86 Bacillus spp. isolates representing all sampling sites were characterized using colony morphology, atmospheric requirements, spore and sporangium morphology, and API 50 CHB and API 20E. Bacillus spp. representing the B. cereus and B. subtilis groups were isolated from all samples. Twenty-one isolates belonging to the B. cereus and B. subtilis groups, representing eight samples, were screened for cytotoxicity. Nine strains of B. cereus and five strains belonging to the B. subtilis group were cytotoxic. CONCLUSIONS: The presence of cytotoxic Bacillus spp. in surface water represents a possible source for food contamination. Filtration and chlorination of surface water, the most common drinking water treatment in Norway, do not remove Bacillus spores efficiently. This was confirmed by isolation of spores from tap water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of food with water containing low numbers of Bacillus spores implies a risk for bacterial growth in foods. Consequently, high numbers of Bacillus spp. may occur after growth in some products. High numbers of cytotoxic Bacillus spp. in foods may represent a risk for food poisoning.

Characterization of spore appendages from Bacillus cereus strains.

AIMS: Further characterization and comparison of spore appendages from Bacillus cereus strains. METHODS AND RESULTS: Appendages were isolated from 10 B. cereus strains from the food industry and food-borne outbreaks. The appendage proteins were dissolved in sample buffer containing 2% SDS and 5% mercaptoethanol at 100 degrees C, and subjected to SDS-PAGE. None of the appendages showed identical protein patterns. Western blots, using antibodies raised against a 3.5 kDa appendage protein, showed that the majority of the appendage proteins reacted with the antibody. Removal of the appendages by sonic treatment of the spores did not alter their heat resistance. The appendages were digested by proteinase K, pepsin, and the enzymes in the detergent Paradigm 10, but not by trypsin or chymotrypsin. Spore adhesion to stainless steel was scarcely affected by removal of the appendages. Digestion of adhered intact spores (with appendages) with Paradigm 10 showed a high degree of variation. CONCLUSIONS: Spore appendages from B. cereus are complex proteinaceous structures that differ among strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Information about spore appendages and their involvement in spore adhesion is crucial for improving cleaning methods used for control of bacterial spores in the food industry.

Growth and toxin profiles of Bacillus cereus isolated from different food sources.

Eleven strains of Bacillus cereus isolated from milk and meat products have been used to study growth and sporulation profiles in detail. Polymerase chain reaction (PCR) using primers detecting cold shock protein A gene signatures (cspA), showed that none of the strains were the newly suggested species in the B. cereus group, B. weihenstephanensis, comprising psychrotolerant cereus strains, although one of the strains grew at 4 degrees C, two at 6 degrees C and seven grew at 7 degrees C. One of the two strains that grew at 6 degrees C had a maximum growth temperature of 42 degrees C, while the remaining 10 strains all grew at temperature of 43 degrees C or higher. Only three strains grew at 48 degrees C. At 42 degrees C, the generation time varied between 11 and 34 min. Spore germination was much faster for the two strains that grew at 6 degrees C than for the other nine strains in milk at 7 degrees C and 10 degrees C. All strains were cytotoxic and contained the non-haemolytic enterotoxin gene (nhe), 10 strains contained the enterotoxin T gene (bceT), and only six had the gene (hbl) encoding haemolytic enterotoxin. Two strains showed some microheterogeneity in the nhe operon. but contained all three genes. We can conclude that true B. cereus strains can have growth profiles as expected for B. weihenstephanensis, and that nhe and bceT were not correlated with growth profiles. However, the two psychrotolerant strains with minimal growth temperature of 4 degrees C and 6 degrees C did not contain hbl, as judged from our PCR results.

Clostridium perfringens type A enterotoxin forms mepacrine-sensitive pores in pure phospholipid bilayers in the absence of putative receptor proteins.

Clostridium perfringens enterotoxin (CPE) is an important cause of food poisoning with no significant homology to other enterotoxins and its mechanism of action remains uncertain. Although CPE has recently been shown to complex with tight junction proteins, we have previously demonstrated that CPE increases ionic permeability in single Caco-2 cells using the whole-cell patch-clamp technique, thereby excluding any paracellular permeability. In this paper we demonstrate that CPE forms pores in synthetic phospholipid membranes in the absence of receptor proteins. The properties of the pores are consistent with CPE-induced permeability changes in Caco-2 cells suggesting that CPE has innate pore-forming ability.

Enterotoxin plasmid from Clostridium perfringens is conjugative.

Clostridium perfringens enterotoxin is the major virulence factor involved in the pathogenesis of C. perfringens type A food poisoning and several non-food-borne human gastrointestinal illnesses. The enterotoxin gene, cpe, is located on the chromosome of food-poisoning isolates but is found on a large plasmid in non-food-borne gastrointestinal disease isolates and in veterinary isolates. To evaluate whether the cpe plasmid encodes its own conjugative transfer, a C. perfringens strain carrying pMRS4969, a plasmid in which a 0.4-kb segment internal to the cpe gene had been replaced by the chloramphenicol resistance gene catP, was used as a donor in matings with several cpe-negative C. perfringens isolates. Chloramphenicol resistance was transferred at frequencies ranging from 2.0 x 10(-2) to 4.6 x 10(-4) transconjugants per donor cell. The transconjugants were characterized by PCR, pulsed-field gel electrophoresis, and Southern hybridization analyses. The results demonstrated that the entire pMRS4969 plasmid had been transferred to the recipient strain. Plasmid transfer required cell-to-cell contact and was DNase resistant, indicating that transfer occurred by a conjugation mechanism. In addition, several fragments of the prototype C. perfringens tetracycline resistance plasmid, pCW3, hybridized with pMRS4969, suggesting that pCW3 shares some similarity to pMRS4969. The clinical significance of these findings is that if conjugative transfer of the cpe plasmid occurred in vivo, it would have the potential to convert cpe-negative C. perfringens strains in normal intestinal flora into strains capable of causing gastrointestinal disease.

CytK toxin of Bacillus cereus forms pores in planar lipid bilayers and is cytotoxic to intestinal epithelia.

CytK is a cytotoxin isolated from a strain of Bacillus cereus cultured from cases of necrotic enteritis and the amino acid sequence of the protein suggests that it may belong to the family of beta-barrel pore-forming toxins. We show here in planar lipid bilayers the toxin is able to form pores which are weakly anion selective and exhibit an open channel probability close to one. The predicted minimum pore diameter is approximately 7 A. We also show that cytK is a potent cytotoxin against human intestinal Caco-2 epithelia. CytK, like other beta-barrel pore-forming toxins, spontaneously forms oligomers which are resistant to sodium dodecyl sulphate (SDS), but not to boiling. CytK represents a pore-forming toxin linked with human cases of necrotic enteritis.

Psychrotolerant species from the Bacillus cereus group are not necessarily Bacillus weihenstephanensis.

Twenty-six strains of Bacillus cereus from different sources were determined to be either mesophilic or psychrotrophic by growth at 6 and 42 degrees C. The strains were also screened by two polymerase chain reaction (PCR) methods designed to discriminate between mesophilic and psychrotrophic types. Seventeen of the 26 strains were able to grow at 6 degrees C, but only four conformed to the new psychrotolerant species Bacillus weihenstephanensis. Among the 26 strains were two which caused outbreaks of food poisoning in Norway, and three others that were isolated from food suspected of causing illness. The presence of the gene components encoding production of enterotoxins Nhe, Hbl, EntT and a recently described cytotoxin K was determined by PCR. All the strains possessed genes for at least one of these toxins, and 19 of the 26 strains were cytotoxic in a Vero cell assay. We conclude that there are psychrotrophic B. cereus strains which cannot be classified as B. weihenstephanensis, and that intermediate forms between the two species exist. No correlation between cytotoxicity and the growth temperature of the strains was found.

A new cytotoxin from Bacillus cereus that may cause necrotic enteritis.

A cytotoxin (CytK) has been isolated from a Bacillus cereus strain that caused a severe food poisoning outbreak killing three people. A protein of 34 kDa was highly cytotoxic, and the addition of other secreted proteins gave no synergistic effect. CytK was also necrotic and haemolytic. No known B. cereus enterotoxins were produced by this strain. A DNA sequence from 1.8 kb upstream to 0.2 kb downstream of the toxin gene was sequenced. The deduced amino acid sequence of the toxin showed similarity to Staphylococcus aureus leucocidins, gamma-haemolysin and alpha-haemolysin, Clostridium perfringens beta-toxin and B. cereus haemolysin II, all belonging to a family of beta-barrel channel-forming toxins. There was no sequence similarity between CytK and enterotoxins of B. cereus. The upstream sequence contained a partial sequence of a putative histidine kinase gene. A recognition site for PlcR, which regulates the transcription of enterotoxins HBL and Nhe of B. cereus, was found in the promoter region of the toxin. This new cytotoxin may be responsible for a disease that is similar to, although not as severe as, the necrotic enteritis caused by the beta-toxin of C. perfringens type C.

Common occurrence of enterotoxin genes and enterotoxicity in Bacillus thuringiensis.

Seventy-four strains of Bacillus thuringiensis thuringiensis representing 24 serovars were examined for the presence of three enterotoxin genes/operons; the non-haemolytic enterotoxin Nhe, the haemolytic enterotoxin hbl and the Bacillus cereus toxin bceT using polymerase chain reaction. The nheBC genes were found in all strains examined, the hblCD genes in 65 of the 74 strains and bceT in 63 strains. There was little consistency of the distribution of enterotoxin loci among strains of the same serovar in serovars that were well represented in our collection. Culture supernatants from all but one strain inhibited protein synthesis in Vero cells, generally with a toxicity equivalent to that seen in strains of B. cereus isolated from incidents of food poisoning. Microbiological Societies.

Prevalence, characterization and growth of Bacillus cereus in commercial cooked chilled foods containing vegetables.

In cooked-chilled and pasteurized vegetable products, initial numbers of Bacillus cereus were below 10 cfu g-1. Before the appearance of spoilage, numbers reached 6-8 log cfu g-1 at 20 degrees C and 4-6 log cfu g-1 at 10 degrees C. Bacillus cereus was not detected in samples stored at 4 degrees C. Ten percent of strains isolated from the products were able to grow at 5 degrees C and 63% at 10 degrees C. Bacillus cereus strains unable to degrade starch, a feature linked to the production of emetic toxin, did not grow at 10 degrees C and had a higher heat resistance at 90 degrees C. Using immunochemical assays, enterotoxin was detected in the culture supernatant fluid of 97.5% of the strains. All culture supernatant fluids were cytotoxic but important variations in the level of activity were found. Psychrotrophic isolates of B. cereus were unable to grow in courgette broth at 7 degrees C whereas they grew in a rich laboratory medium. At 10 degrees C, these isolates grew in both media but lag time in courgette broth was 20-fold longer than in the rich laboratory medium.

The 105-kDa protein component of Bacillus cereus non-haemolytic enterotoxin (Nhe) is a metalloprotease with gelatinolytic and collagenolytic activity.

A sequence of 91 amino acids residues, probably starting from the N-terminal of the mature protein, was determined for the 105-kDa protein of the non-haemolytic enterotoxin of Bacillus cereus. The last part of this sequence was similar to parts of the N-terminal portions of two collagenases of Clostridium histolyticum and Clostridium perfringens. Zymography, with intact collagen fibril and gelatin as substrates, showed that the 105-kDa protein had collagenolytic and gelatinolytic activity. The 105-kDa protein also showed activity against a typical collagenase substrate, azocoll, and was inhibited by EDTA and 1,10-phenanthroline. We conclude that the 105-kDa protein is a collagenase.