The Genetically Remote Pathogenic Strain NVH391-98 of the Bacillus cereus Group Is Representative of a Cluster of Thermophilic Strains

Bacteria of the Bacillus cereus group are known to cause foodpoisoning. A rare phylogenetically remote strain, NVH391-98,was recently characterized to encode a particularly efficientcytotoxin K presumably responsible for food poisoning. Thispathogenic strain and its close relatives can be phenotypicallydistinguished from other strains of the B. cereus group by theinability to grow at temperatures below 17°C and by theability to grow at temperatures from 48 to 53°C. A temperatephage, phBC391A2, residing in the genome of NVH391-98 allowsus to distinguish the three known members of this thermophilicstrain cluster.

INTRODUCTION

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INTRODUCTION

The Bacillus cereus group includes gram-positive aerobic spore-formingbacteria commonly found in soil and sometimes implicated infood poisoning. These opportunistic pathogens cause gastrointestinaldiseases manifested by diarrheic or emetic syndromes (5, 12).The practical importance of B. cereus group studies is growingbecause of the increasing number of related food poisoning cases,especially in developed countries. Since this problem has anobvious relevance to the ability of some B. cereus strains tomultiply in chilled products, psychrotolerant strains have beenthe focus (3, 4, 8, 22). It was shown that psychrotolerant andmesophilic strains have optimal growth temperatures in the rangeof 25 to 35°C, but psychrotolerant strains can be distinguishedby their ability to grow at 4 to 7°C but not at 43°C(14). Based on several distinctive features of psychrotolerantstrains, including the presence of a specific signature in the16S rRNA sequences, a new species, Bacillus weihenstephanensis,was proposed (14, 21). In contrast, the data available in regardto the ability of the B. cereus group bacteria to grow at moderatelyhigh temperatures, that is, close or slightly higher than 50°C,are scarce, nonsystematic, and not sufficiently detailed. Someisolates were reported to grow on plates at 55°C after 5days of incubation (18). A strain, NVH200, was able to growat up to 50°C in a liquid medium after a long lag phaseof 72 h (1). Here we show that only a very few strains of theB. cereus group are able to grow at temperatures higher than48°C. In fact, this ability seems to be restricted to afew strains represented by the genetically remote strain NVH391-98,isolated from a severe food poisoning outbreak, which causedthree fatal cases (15). This strain is able to synthesize inelevated amounts a particularly efficient diarrheic cytotoxinK (2, 7). NVH391-98 and its close relatives are the unique thermophilicisolates of the B. cereus group, presumably representing anothernovel species.

NVH391-98 represents a cluster of thermophilic strains.

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NVH391-98 represents a cluster...

Psychrotolerant B. weihenstephanensis strains KBAB4 (20, 24)and WSBC10206 (14), obtained from V. Sanchis (INRA, La Minière,France) and S. Scherer (IM, Freising, Germany), were isolatedfrom soil in France and Germany, respectively. Mesophilic B.cereus strains ATCC 14579 (9), obtained under the designation6A5 from D. R. Zeigler (BGSC, Columbus, OH), and ATCC 10987(19), obtained from D. Lereclus (INRA, La Minière, France),were of air and dairy origin from the United Kingdom and Canada,respectively. The strain NVH391-98, obtained from D. Lereclus,was isolated from a vegetable purée in France in 1998(15). The strains INRA AF2, obtained under the designation INRA398,and NVH883/00 (10) were from M.-H. Guinebretiere (INRA, Avignon,France). Bacillus subtilis 168 was from the laboratory collection.Standard manipulations with bacteria, phages, and DNA were doneas described previously (16).

In a preliminary experiment, we noted that strain NVH391-98was able to grow rapidly at 48°C, while none of the othertested strains of the B. cereus group grew at this temperature.This is illustrated by a simple plate growth test (Fig. 1).The B. subtilis strain 168, known to be able to grow up to 52°C(11), was used as the high-temperature growth control. Thisexperiment clearly indicated that at least at the high temperature,there is a large difference in the growth abilities of strainNVH391-98 and other representatives of the B. cereus group.To characterize these differences quantitatively, we examinedthe growth of five strains of the B. cereus group, includingtwo psychrotolerant strains, two mesophiles, and NVH391-98.We used three different liquid media, Luria-Bertani (LB), brainheart infusion, and LB medium supplemented with 6 g/liter Bactotryptone and 5 g/liter yeast extract, at temperatures rangingfrom 8 to 55°C.

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FIG. 1. Growth of different Bacillus strains at 30°C and 48°C. B. subtilis 168 is used as a positive control for high-temperature growth. The strains B. cereus ATCC 14579, B. weihenstephanensis KBAB4, and NVH391-98 are of the B. cereus group.

For each strain, an aliquot of the overnight culture was diluted100-fold into fresh medium. Growth was monitored by measuringthe increase of the optical density at 600 nm with an automaticcell growth analyzer (Bioscreen C; Labsystems). Microtiter platescontaining 300 µl per well were shaken continuously for96 h.

Extrapolation of the growth profile curves for the psychrotolerantB. weihenstephanensis strains KBAB4 and WSBC10206 to the zerogrowth rates, using a model of Ratkowsky et al. (17), indicateda minimal theoretical growth temperature of 1 to 4°C. Experimentally,growth was detected at the minimal temperature of 8°C (Fig.2) (see the data for all tested strains and the equation parametersin the supplemental material). Above the optimal growth temperatureof 30 to 32°C, the specific growth rates rapidly decreasedwith an increase of temperature. Above 38°C, no growth wasexperimentally detected, while the theoretical maximal growthtemperature was in the range of 40 to 46°C. For the twomesophilic strains, the experimentally detected minimal growthtemperature was 12°C, while 2 to 7°C was the theoreticalestimation, and an optimal growth was observed between 35 and40°C (Fig. 2) (see the supplemental material for all data).The B. cereus strain ATCC 10987 was able to grow at up to 47°C,while the B. cereus strain ATCC 14579 did not grow above 46°C.The theoretical maximal growth temperature for the mesophilicstrains was in the range of 49 to 53°C, again slightly higher(1°C) for the B. cereus strain ATCC 10987. Multiple platetests for about 100 B. cereus group strains from our laboratorycollection (not shown) confirmed the general conclusion thatalmost all strains of the B. cereus group are able to maintainexperimentally detectable growth between 8 and 47°C butnot beyond this range. If the Ratkowsky model adequately describesthe whole range of the temperature dependence of growth, thesetemperatures can be extended to 2 to 53°C. Presumably, longerincubation times are needed to detect the slow growth at theextreme temperatures, but also some growth induction constraintsmay exist in our experimental conditions that do not allow usto detect growth up to the theoretical limits. These constraintscan result in very long lag times, hampering the measurementsof very slow exponential-phase growth. That is why it is necessaryto apply a theoretical model for the estimation of the extremegrowth temperatures. The strain NVH391-98 did not grow at temperaturesbelow 18°C but was able to grow at up to 53°C (Fig.2). The optimal growth temperature was 40°C in LB medium,42°C in LB medium supplemented with 6 g/liter Bacto tryptoneand 5 g/liter yeast extract, and 46°C in brain heart infusionmedium, displaying a relatively high dependence of growth abilityof the strain on the medium used. The theoretical limits ofgrowth, based on Ratkowsky's model, were 8 to 15°C and 58°C.The strain NVH391-98 is therefore able to grow at temperatures6 to 8 degrees higher than the mesophilic strains of the B.cereus group.

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FIG. 2. Growth of Bacillus cereus group strains at different temperatures. Growth rates (r) for representatives of psychrotolerant (B. weihenstephanensis KBAB4), mesophilic (B. cereus ATCC 14579), and thermophilic ("B. cytotoxicus" NVH391-98) strains in different media are plotted against the growth temperature (T). The experimental points are the mean values of three independent experiments. The solid lines show nonlinear least-squares approximations according to the Ratkowsky equation (17). C, degrees Celsius.

Phylogenetic remoteness of NVH391-98 and distinction from its close relatives.

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Phylogenetic remoteness of...

The phylogenetic remoteness of the strain NVH391-98 from otherrepresentatives of the B. cereus group was demonstrated recently(6, 13). Figure 3A illustrates this using the multiple locussequence typing (MLST) schema proposed by Tourasse et al. (23)for the extended set of strains in comparison with that reportedearlier (6). This comparison includes the sequences of the strainsNVH883/00 (GenBank accession no. EF108377 to -383) and INRAAF2, which are closely related to NVH391-98 (6, 10) and havesimilar thermophilic phenotypes. We resequenced the relevantloci for NVH883/00 and confirmed the differences with NVH391-98.MLST and several other independently determined sequences (accessionno. EF108376 and EF108384 to -390) for the strain INRA AF2 donot allow us to distinguish it from NVH391-98.

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FIG. 3. Phylogenetic and phenotypic distinctions of the "Bacillus cytotoxicus" strain cluster. (A) Neighbor-joining phylogenetic tree for a representative set of the B. cereus group strains. Strain names and concatenated sequences are taken from the MLST database described in reference 23, GenBank, or genomic sequences. Vertical bars labeled C, T, and W indicate the major strain clusters according to reference 20. The cluster of the three strains closest to NVH391-98 is labeled Y. The sequences of the four bacillus strains closest to the B. cereus clade, B. subtilis 168, Bacillus amyloliquefaciens FZB42, Bacillus licheniformis ATCC 14580, and Bacillus pumilis SAFR-032, were extracted from GenBank entries (accession no. NC_000964, NC_009725, NC_006270, and NC_009848) and used to represent an out-group. Completely sequenced strains are labeled seq. (B) Turbid (left) and clear (right) plaques formed by phage phBC391A2 and by its mutant, phBC391B3vir, respectively, on the INRA AF2 strain used as an indicator strain. (C) Partial nucleotide and corresponding amino acid sequences of the Bcer982969 gene in the phages phBC391A2 (top) and phBC391B3vir (bottom). The location of this mutation in the putative repressor gene of phBC391A2 proves the identity of phBC391B3vir as a clear-plaque mutant of the former phage.

We noted that after overnight propagation on solid media at50°C and subsequent longtime storage at room temperature,two strains, NVH391-98 and INRA AF2 (but not NVH883/00), showtypical autolytic morphology, characterized by plaque-like clearingsin the areas of dense growth on agar. Using double-layer agarassays, we found that the NVH391-98 strain produces bacteriophagethat formed turbid plaques on the INRA AF2, but not the NVH883/00,cell lawn. This phenotype allows us to distinguish the NVH391-98,INRA AF2, and NVH883/00 strains. We detected clear-plaque-formingphage mutants among turbid plaques on INRA AF2 with a frequencyof approximately 2 x 10^–4 (Fig. 3). Analysis of the genomicsequence of the strain NVH391-98 (accession no. NC009674) revealedtwo regions, nearly 2,690 and 3,010 kb, designated phBC391A1and phBC391A2, respectively, containing clusters of phage-relatedgenes and thus potentially encoding inducible prophages.

To identify the phage infecting the strain INRA AF2, we isolatedDNA from a clear-plaque-forming phage mutant, designated phBC391B3vir(Fig. 3B), and analyzed it by EcoRV digestion. The resultingprofile corresponded to the theoretical profile of phage phBC391A2DNA, rather than to that of phBC391A1 (not shown). Direct sequencingof this DNA by use of primers specific to NVH391-98 chromosomalDNA produced readable chromatograms, with the average signalstrength 7- to 10-fold higher, only with primers correspondingto the phBC391A2 (not shown). Moreover, we determined that themutation causing the clear-plaque phenotype of phBC391B3virwas due to the insertion of an additional A into the stretchof five A's, resulting in a reading frame shift in the Bcer982969gene encoding a potential phage repressor (Fig. 3C). Therefore,the three strains can be phenotypically distinguished by usingtheir different levels of susceptibility to the phage phBC391A2.Only the strain NVH391-98 produces this phage, and only thestrain INRA AF2 can be used as an indicator strain to detectPFU.

In conclusion, the formal sequence-based comparisons allow usto consider the strain NVH391-98 and its close relatives asa genetically rather remote species of the B. cereus group.At present, NVH391-98 and its two close relatives describedhere are the only known strains of this group for which thermophilicgrowth is confirmed. We proposed earlier that strain NVH391-98be considered a representative of a new species, for which thename "Bacillus cytotoxicus" was suggested (13). The existenceof two closely related but different strains, INRA AF2 and NVH883/00,validates the novel species status of NVH391-98.

ACKNOWLEDGMENTS

We thank S. Scherer and M.-H. Guinebretiere, D. R. Zeigler,D. Lereclus, and V. Sanchis for the gifts of strains.

This work was partially supported by the French National ResearchAgency (project ANR-05-PNRA-013).

FOOTNOTES

* Corresponding author. Mailing address: Génétique Microbienne, INRA Domaine de Vilvert, 78352 Jouy-en-Josas cedex, France. Phone: 33 1 34 65 27 24. Fax: 33 1 34 65 25 21. E-mail: alexei.sorokine{at}jouy.inra.fr

Published ahead of print on 21 December 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

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