Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus Group occasionally associated with food poisoning.

An aerobic endospore-forming bacillus (NVH 391-98(T)) was isolated during a severe food poisoning outbreak in France in 1998, and four other similar strains have since been isolated, also mostly from food poisoning cases. Based on 16S rRNA gene sequence similarity, these strains were shown to belong to the Bacillus cereus Group (over 97% similarity with the current Group species) and phylogenetic distance from other validly described species of the genus Bacillus was less than 95%. Based on 16S rRNA gene sequence similarity and MLST data, these novel strains were shown to form a robust and well-separated cluster in the B. cereus Group, and constituted the most distant cluster from species of this Group. Major fatty acids (iso-C(15:0), C(16:0), iso-C(17:0), anteiso-C(15 : 0), iso-C(16:0), iso-C(13:0)) supported the affiliation of these strains to the genus Bacillus, and more specifically to the B. cereus Group. NVH 391-98(T) taxon was more specifically characterized by an abundance of iso-C(15:0) and low amounts of iso-C(13:0) compared with other members of the B. cereus Group. Genome similarity together with DNA-DNA hybridization values and physiological and biochemical tests made it possible to genotypically and phenotypically differentiate NVH 391-98(T) taxon from the six current B. cereus Group species. NVH 391-98(T) therefore represents a novel species, for which the name Bacillus cytotoxicus sp. nov. is proposed, with the type strain NVH 391-98(T) (= DSM 22905(T) = CIP 110041(T)).

Low temperatures and fermentative metabolism limit peptidoglycan digestion of Bacillus cereus. Impact on colony forming unit counts.

The impact of fermentative metabolism at low temperature on cell division of Bacillus cereus was studied. Fermentation at 37 °C had no influence on the division of bacteria. Aerobic cultures at 15 °C produced larger cells than at 37 °C, but cell division was normal. In fermentative cultures at 15 °C, no increase in CFU ml(-1) was observed. However, A(600) increased, due to formation of long filaments. Transmission electronic microscopy and light microscopy with fluorescent staining showed several nucleic acid entities separated by a hydrophobic membrane, indicating that each filament contained several individual cells attached by peptidoglycan. When left in air at room temperature, one filament gave several daughter cells, this means that one CFU formed by one filament may represent a greater contamination potential than one CFU formed by a single cell. Division was observed in cultures at 15 °C with anaerobic respiration in the presence of nitrates. Possible filamentous growth must thus be taken into account to avoid underestimating B. cereus growth in vacuum or modified atmosphere packaged foods stored at low temperature.

Unsaturated fatty acids from food and in the growth medium improve growth of Bacillus cereus under cold and anaerobic conditions.

In a chemically defined medium and in Luria broth, cold strongly reduced maximal population density of Bacillus cereus ATCC 14579 in anaerobiosis and caused formation of filaments. In cooked spinach, maximal population density of B. cereus in anaerobiosis was the same at cold and optimal temperatures, with normal cell divisions. The lipid containing fraction of spinach, but not the hydrophilic fraction, restored growth of B. cereus under cold and anaerobiosis when added to the chemically defined medium. This fraction was rich in unsaturated, low melting point fatty acids. Addition of phosphatidylcholine containing unsaturated, low melting point, fatty acids similarly improved B. cereus anaerobic growth at cold temperature. Addition of hydrogenated phosphatidylcholine containing saturated, high melting point, fatty acids did not modify growth. Fatty acids from phospholipids, from spinach and from hydrogenated phosphatidylcholine, although normally very rare in B. cereus, were inserted in the bacterium membrane. Addition of phospholipids rich in unsaturated fatty acids to cold and anaerobic cultures, increased fluidity of B. cereus membrane lipids, to the same level as those from B. cereus normally cold adapted, i.e. grown aerobically at 15 °C. B. cereus is therefore able to use external fatty acids from foods or from the growth medium to adapt its membrane to cold temperature under anaerobiosis, and to recover the maximal population density achieved at optimal temperature.

Influence of anaerobiosis and low temperature on Bacillus cereus growth, metabolism, and membrane properties.

The impact of simultaneous anaerobiosis and low temperature on growth parameters, metabolism, and membrane properties of Bacillus cereus ATCC 14579 was studied. No growth was observed under anaerobiosis at 12°C. In bioreactors, growth rates and biomass production were drastically reduced by simultaneous anaerobiosis and low temperature (15°C). The two conditions had a synergistic effect on biomass reduction. In anaerobic cultures, fermentative metabolism was modified by low temperature, with a marked reduction in ethanol production leading to a lower ability to produce NAD(+). Anaerobiosis reduced unsaturated fatty acids at both low optimal temperatures. In addition, simultaneous anaerobiosis and low temperatures markedly reduced levels of branched-chain fatty acids compared to all other conditions (accounting for 33% of total fatty acids against more 71% for low-temperature aerobiosis, optimal-temperature aerobiosis, and optimal-temperature anaerobiosis). This corresponded to high-melting-temperature lipids and to low-fluidity membranes, as indicated by differential scanning calorimetry, 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy, and infrared spectroscopy. This is in contrast to requirements for cold adaptation. A link between modification in the synthesis of metabolites of fermentative metabolism and the reduction of branched-chain fatty acids at low temperature under anaerobiosis, through a modification of the oxidizing capacity, is assumed. This link may partly explain the impact of low temperature and anaerobiosis on membrane properties and growth performance.

Fatty acid profiles and desaturase-encoding genes are different in thermo- and psychrotolerant strains of the Bacillus cereus Group.

BACKGROUND: The Bacillus cereus Group consists of closely-related bacteria, including pathogenic or harmless strains, and whose species can be positioned along the seven phylogenetic groups of Guinebretière et al. (I-VII). They exhibit different growth-temperature ranges, through thermotolerant to psychrotolerant thermotypes. Among these, B. cytotoxicus is an atypical thermotolerant and food-poisoning agent affiliated to group VII whose thermotolerance contrasts with the mesophilic and psychrotolerant thermotypes associated to the remaining groups I-VI. To understand the role of fatty acid (FA) composition in these variable thermotypes (i.e. growth behavior vs temperatures), we report specific features differentiating the FA pattern of B. cytotoxicus (group VII) from its counterparts (groups I-VI). FINDINGS: The FA pattern of thermotolerant group VII (B. cytotoxicus) displayed several specific features. Most notably, we identified a high ratio of the branched-chain FAs iso-C15/iso-C13 (i15/i13) and the absence of the unsaturated FA (UFA) C16:1(5) consistent with the absence of ∆5 desaturase DesA. Conversely, phylogenetic groups II-VI were characterized by lower i15/i13 ratios and variable proportions of C16:1(5) depending on thermotype, and presence of the DesA desaturase. In mesophilic group I, thermotype seemed to be related to an atypically high amount of C16:1(10) that may involve ∆10 desaturase DesB. CONCLUSION: The levels of i15/i13 ratio, C16:1(5) and C16:1(10) UFAs were related to growth temperature variations recorded between thermotypes and/or phylogenetic groups. These FA are likely to play a role in membrane fluidity and may account for the differences in temperature tolerance observed in B. cereus Group strains.