Genetic structure of population of Bacillus cereus and B. thuringiensis isolates associated with periodontitis and other human infections.

Helgason E; Caugant DA; Olsen I; Kolstø AB

doi:10.1128/jcm.38.4.1615-1622.2000

Genetic structure of population of Bacillus cereus and B. thuringiensis isolates associated with periodontitis and other human infections.

Affiliations

1. Biotechnology Centre of Oslo and Institute of Pharmacy, University of Oslo, 0316 Oslo, Norway.
Authors
Helgason E¹
(1 author)

ORCIDs linked to this article

Caugant DA | 0000-0003-2765-9844

Journal of Clinical Microbiology, 01 Apr 2000, 38(4):1615-1622
https://doi.org/10.1128/jcm.38.4.1615-1622.2000 PMID: 10747152 PMCID: PMC86502

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Abstract

The genetic diversity and relationships among 35 Bacillus cereus and Bacillus thuringiensis isolates recovered from marginal and apical periodontitis in humans and from various other human infections were investigated using multilocus enzyme electrophoresis. The strains were isolated in Norway, except for three strains isolated from periodontitis patients in Brazil. The genetic diversity of these strains was compared to that of 30 isolates from dairies in Norway and Finland. Allelic variation in 13 structural gene loci encoding metabolic enzymes was analyzed. Twelve of the 13 loci were polymorphic, and 48 unique electrophoretic types (ETs) were identified, representing multilocus genotypes. The mean genetic diversity among the 48 genotypes was 0.508. The genetic diversity of each source group of isolates varied from 0.241 (periodontal infection) to 0.534 (dairy). Cluster analysis revealed two major groups separated at a genetic distance of greater than 0.6. One cluster, ETs 1 to 13, included solely isolates from dairies, while the other cluster, ETs 14 to 49, included all of the human isolates as well as isolates from dairies in Norway and Finland. The isolates were serotyped using antiflagellar antiserum. A total of 14 distinct serotypes were observed. However, little association between serotyping and genotyping was seen. Most of the strains were also analyzed with pulsed-field gel electrophoresis, showing the presence of extrachromosomal DNA in the size range of 15 to 600 kb. Our results indicate a high degree of heterogeneity among dairy strains. In contrast, strains isolated from humans had their genotypes in one cluster. Most strains from patients with periodontitis belonged to a single lineage, suggesting that specific clones of B. cereus and B. thuringiensis are associated with oral infections.

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J Clin Microbiol. 2000 Apr; 38(4): 1615–1622.

https://doi.org/10.1128/jcm.38.4.1615-1622.2000

PMCID: PMC86502

PMID: 10747152

Genetic Structure of Population of Bacillus cereus and B. thuringiensis Isolates Associated with Periodontitis and Other Human Infections

Erlendur Helgason,^1,² Dominique A. Caugant,² Ingar Olsen,³ and Anne-Brit Kolstø^1,^*

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Abstract

The genetic diversity and relationships among 35 Bacillus cereus and Bacillus thuringiensis isolates recovered from marginal and apical periodontitis in humans and from various other human infections were investigated using multilocus enzyme electrophoresis. The strains were isolated in Norway, except for three strains isolated from periodontitis patients in Brazil. The genetic diversity of these strains was compared to that of 30 isolates from dairies in Norway and Finland. Allelic variation in 13 structural gene loci encoding metabolic enzymes was analyzed. Twelve of the 13 loci were polymorphic, and 48 unique electrophoretic types (ETs) were identified, representing multilocus genotypes. The mean genetic diversity among the 48 genotypes was 0.508. The genetic diversity of each source group of isolates varied from 0.241 (periodontal infection) to 0.534 (dairy). Cluster analysis revealed two major groups separated at a genetic distance of greater than 0.6. One cluster, ETs 1 to 13, included solely isolates from dairies, while the other cluster, ETs 14 to 49, included all of the human isolates as well as isolates from dairies in Norway and Finland. The isolates were serotyped using antiflagellar antiserum. A total of 14 distinct serotypes were observed. However, little association between serotyping and genotyping was seen. Most of the strains were also analyzed with pulsed-field gel electrophoresis, showing the presence of extrachromosomal DNA in the size range of 15 to 600 kb. Our results indicate a high degree of heterogeneity among dairy strains. In contrast, strains isolated from humans had their genotypes in one cluster. Most strains from patients with periodontitis belonged to a single lineage, suggesting that specific clones of B. cereus and B. thuringiensis are associated with oral infections.

Periodontal diseases, whether marginal or apical, are initiated and sustained by factors produced by the microbiota of dental plaque. Plaque microorganisms may damage cellular and structural components of the periodontium via release of proteolytic and noxious waste products. Among the enzymes released by these bacteria are proteases capable of digesting collagen, elastin, fibronectin, fibrin, and various other components of the intercellular matrix of epithelial and connective tissue (22). These organisms also release numerous metabolic products such as ammonia, indole, hydrogen sulfide, and butyric acid.

A growing literature has suggested some reasonable candidates for destructive periodontal disease, among which are Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Bacteroides forsythus, Prevotella intermedia, Fusobacterium nucleatum, Campylobacter rectus, Eikenella corrodens, Peptostreptococcus micros, Selenomonas spp., Eubacterium spp., spirochetes, and Streptococcus intermedius (34). However, all periodontal pathogens have probably not yet been identified. The presence of unusual species in periodontal lesions suggests the possibility that they may play a role in the etiology of periodontal diseases.

Bacillus cereus is frequently isolated as a contaminant of milk, cereals, and various other foods and has been the cause of isolated cases and outbreaks of food poisoning. Occasionally it can be responsible for wound and eye infections, as well as systemic infections (10, 38). This organism may produce an emetic or diarrheal syndrome induced by an emetic toxin and enterotoxins, respectively. Other toxins produced during growth include phospholipases, proteases, and hemolysins (10), which may contribute to the pathogenicity of B. cereus in nongastrointestinal disease. B. cereus isolated from clinical material other than feces and vomitus was commonly dismissed as a contaminant, but it is likely to contribute to disease when it is part of the predominant microflora of the site. The possible role of B. cereus in periodontitis has not previously been assessed. Bacillus thuringiensis is primarily a biopesticide for insects due to its production of insecticidal toxins, which is the only established difference from B. cereus (reviewed by Crickmore et al. [7]). It is also isolated from human wounds (8) and gastroenteritis infection (19).

We have previously analyzed the genetic diversity of B. cereus and B. thuringiensis isolates from soil (18). In the present study we have extended our analyses of the genetic diversity of B. cereus and B. thuringiensis by focusing mainly on the genetic diversity of strains isolated from human infections, including periodontal infections.

To analyze the genetic diversity of B. cereus and B. thuringiensis, multilocus enzyme electrophoresis was used. The strains were also serotyped using antiserum raised against flagellar antigen of B. thuringiensis. Since it is well known that most B. cereus and B. thuringiensis strains contain large plasmids (1, 3, 4, 15), several strains were also analyzed by pulsed-field gel electrophoresis (PFGE) for the detection of large extrachromosomal DNA.

In this study we wanted to determine whether strains of B. cereus isolated from patients differed from those in the environment that we have previously examined (18). Since strains isolated from dairies were available, we included those in this study to get an even broader view of the genetic diversity and population structure of B. cereus and B. thuringiensis.

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MATERIALS AND METHODS

Strain isolation.

The 35 human strains were isolated from diverse sources, i.e., oral cavity, urine, blood, and skin (Table (Table1).1). Of the 20 strains from the oral cavity, 11 were isolated from marginal periodontitis, 8 were isolated from apical periodontitis, and 1 was isolated from lichen planus. Bacillus isolates were among the most predominant in all of the clinical samples. Sixteen of the patients lived in different counties or cities of Norway, and three were from Brazil. Two of the patients in Norway were immigrants. All of the oral strains had been isolated from consecutive samples submitted to the Microbiological Diagnostic Service, Department of Oral Biology, University of Oslo, during a 26-month period. Bacillus identification had been based on cultivation under aerobic and anaerobic atmospheres, colony morphology, hemolysis, Gram stain, spore formation, and biochemical reactions using the API 50 CHB Medium and CH kit (bioMérieux Sa, Marcy l'Etoile, France). The biochemical tests were read by an automatic reader (ATB 1525 Reader; bioMérieux), and the results were transformed into numerical codes which were submitted to a database (API Plus; bioMérieux) for identification.

TABLE 1

Characteristics of 65 B. cereus and B. thuringiensis isolates from dairies, periodontal infections, and other human infections and of three culture collectionstrains

ET(s)	Isolate^a	Mo isolated	Sex of patient^b	Age (yr) of patient	Location^c	Source^d	Disease^e	Serotype^f
1	AH 610 (80)				N, Porsgrunn	Dairy		H−
2, 3	AH 605 (17)				N, Kongsberg	Dairy		H3a3b3c
	AH 613				N, Sortland	Dairy		ND^g
4	AH 614				N, Porsgrunn	Dairy		H−
5	AH 612				N, Orkdal	Dairy		H34
6	AH 407				F	Dairy		H34
7	AH 406				F	Dairy		H−
8	AH 403				F	Dairy		H3a3b3c
9	AH 400				F	Dairy		AA
10	AH 402				F	Dairy		H29
11	AH 401				F	Dairy		H15
12	AH 404				F	Dairy		H29
13	AH 408				F	Dairy		AA
14	AH 727				N, Skien	Pus		H37
15, 16	AH 595 (1)				N, Gjøvik	Dairy		ND
	AH 598 (4)				N, Gjøvik	Dairy		H−
	AH 599 (5)				N, Hamar	Dairy		H−
17	AH 606 (23)				N, Tynset	Dairy		H−
18	AH 830	October 1995	F	32	N, Oslo	GR	AP	H54
19	AH 833				F	Dairy		AA
20	AH 730				N, Skien	Urine		H−
21	AH 728				N, Skien	Urine		H−
	AH 810	February 1994	F	49	N, Telemark	Gingiva	MP	H−
	ATCC 10987				Reference strain			AA
22, 23	AH 820	October 1995	F	76	N, Akershus	PP	MP	H22
	AH 813	September 1995	F	30	B, Curitiba	RC	AP	H22
	AH 812 [OH 599]	September 1995	M	48	N, Oslo	RC	AP	H−
24, 25	AH 816	November 1995	M	35	N, Akershus	GR	AP	H22
	AH 818	September 1995	F	38	B, Curitiba	RC	AP	H22
	AH 819 [OH 600]	September 1995	M	48	N, Oslo	PP	MP	H18
	AH 826	April 1996	M	55	N, Rogaland	RC	AP	AA
	AH 817	November 1995	F	56	N, Akershus	CH	LP	H−
	AH 823	January 1996	F	78	N, Oslo	PP	MP	H−
	AH 827	March 1996	M	82	N, Hedmark	PP	MP	H−
	AH 828	March 1996	F	60	N, Oslo	PP	MP	H−
	AH 829	March 1996	M	30	N, Akershus	DA	AP	H−
	AH 824	February 1996	F	50	N, Akershus	PP	MP	H18
	AH 825	March 1996	F	69	N, Akershus	PP	MP	H18
	AH 831	April 1996	F	45	N, Oslo	PP	MP	H18
	ATCC 4342				Reference strain	H19
26	AH 723				N, Skien	Eye		H−
27	AH 717				N, Skien	Skin		H−
28	AH 611 (87)				N, Oslo	Dairy		H−
29	AH 604 (11)				N, Odalen	Dairy		H−
30	AH 607 (59)				N, Tynset	Dairy		H−
31	AH 608 (61)				N, Tynset	Dairy		H−
32, 33	AH 724, 726				N, Skien	Urine		H13 (2)
34	AH 722				N, Skien	Urine		H27
35	AH 719				N, Skien	Cervix		AA
36	AH 720				N, Skien	Blood		AA
37	AH 729				N, Skien	Wound		H−
38	AH 721				N, Skien	Urine		H18
39	AH 815	October 1995	M	47	N, Møre og Romsdal	PP	MP	H15
40	AH 811	August 1995	M	25	B, Sao José dos Pinhais	RC	AP	H23
41	AH 718				N, Skien	Nose		H27
	AH 725				N, Skien	Eye		H−
	AH 814	October 1995	M	63	N, Akershus	OC	MP	H27
42	ATCC 14579^T				Type strain			H6
43	AH 609 (68)				N, Tynset	Dairy		H−
44	AH 601 (8)				N, Trysil	Dairy		H6
45	AH 600 (7)				N, Gjøvik	Dairy		H6
46	AH 405				F	Dairy		H5a5b
47	AH 596 (2)				N, Gjøvik	Dairy		H6
48	AH 597 (3)				N, Otta	Dairy		H13
	AH 602 (9)				N, Lillehammer	Dairy		H13
	AH 603 (10)				N, Gjøvik	Dairy		H13
49	AH 716				N, Skien	Pus		H27

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^aNumbers in parentheses are those used by Granum et al. (16); names in brackets are those used by Kotiranta et al. (24).

^bF, female; M, male.

^cN, Norway; F, Finland; B, Brazil.

^dGR, granuloma; PP, periodontal pocket; RC, root canal; CH, cheek; DA, detoalveolar abscess; OC, oral cavity.

^eAP, apical periodontitis; MP, marginal periodontitis; LP, lichen planus.

^fH−, nontypeable; AA, autoagglutinable

^gND, not determined.

The strains isolated from patients with diseases other than periodontitis were provided by B. E. Kristiansen. The strains isolated from dairies were gifts from P. E. Granum and M. Salkinoja-Salonen.

Three culture collection strains, B. cereus ATCC 4342, ATCC 10987, and ATCC 14579^T, were also included (Table (Table11).

Serotyping.

H serotyping based on flagellar antigens was performed by the agglutination method, as described by de Barjac (9).

Electrophoresis of enzymes.

Protein extracts of the isolates were electrophoresed on starch-gel, and selective enzyme staining was performed as described by Selander et al. (32). Thirteen enzymes (see Table Table3)3) were assayed as previously described (18).

TABLE 3

Allele frequencies and genetic diversity at 13 enzyme loci in 48 ETs of 65 B. cereus and B. thuringiensis isolates from dairies andpatients

Locus^a	No. of alleles	Diversity of isolates from:
Locus^a	No. of alleles	Dairies (n = 30)	Nonperiodontal human infections (n = 15)	Periodontal infections (n = 20)
NSP	1	0.000	0.000	0.000
P3T	7	0.772	0.714	0.639
P3M	2	0.433	0.527	0.500
P3B	4	0.593	0.264	0.000
ALD	4	0.630	0.143	0.222
PGI	5	0.618	0.538	0.000
ADK	5	0.721	0.396	0.000
MDH	5	0.276	0.484	0.000
ME	4	0.729	0.264	0.000
EST	7	0.729	0.648	0.389
ACO	4	0.675	0.648	0.667
IPO	2	0.000	0.264	0.000
CAT	8	0.766	0.670	0.722
Mean	4.5	0.534	0.428	0.241

^aThe 13 enzymes assayed were nucleoside phosphorylase (NSP), three leucyl-leucyl-glycine peptidases (P3T, P3M, and P3B), alanine dehydrogenase (ALD), phosphoglucose isomerase (PGI), adenylate kinase (ADK), malic enzyme (ME), malate dehydrogenase (MDH), α-naphthyl-propionate esterase (EST), aconitase (ACO), indophenol oxidase (IPO), and catalase (CAT).

Statistical analysis.

Genetic diversity at an enzyme locus among the electrophoretic types (ETs) or isolates was calculated as h = 1 − Σx_i² [n/(n − 1)], where x_i is the frequency of the ith allele and n is the number of ETs or isolates. The mean genetic diversity (H) is the arithmetic average of h values over all loci (32). The genetic distance between pairs of ETs was expressed as the proportion of enzyme loci at which dissimilar alleles occurred (mismatches). Clustering was performed from a matrix of genetic distances by the average-linkage method (33).

The index of association (I_A) was calculated as described by Maynard-Smith et al. (28). The I_A value is a measure of the degree of association between alleles at different loci and has an expected value of zero for large randomly mating bacterial populations undergoing frequent recombination or a panmictic population structure. A population with an I_A value that is significantly different from zero has infrequent genetic recombination and is considered clonal. For the I_A calculation, a computer program, kindly provided by Brian G. Spratt, Department of Zoology, University of Oxford, was used.

Digestion of DNA.

The restriction enzymes AscI and NotI were obtained from New England BioLabs, Beverly, Mass. Agarose blocks were treated as plugs as described previously (5, 23).

PFGE.

A PFGE instrument from Beckman was used. Electrophoresis was done in 25 mM Tris-borate buffer–0.05 mM EDTA (pH 8.3) at 14°C with a pulse time of 4 s for the first 10 min at 170 mA, after which the pulse time was 30 or 60 s for 20 h at 150 mA. The bacterial DNA was isolated in agarose plugs as described previously (5, 23) and electrophoresed in 1% agarose. Size markers used were Saccharomyces cerevisiae chromosomes (New England BioLabs) and a mixture of phage lambda DNA concatemers and HindIII fragments of lambda (New England BioLabs). After electrophoresis, gels were stained for 10 min in ethidium bromide (100 μg/ml) and destained for about 1 h in water. After treatment for 10 min in 0.25 M HCl, twice for 30 min each in 0.5 M NaOH–1.5 M NaCl, and twice for 30 min each in 0.5 M Tris (pH 8)–1.5 M NaCl, the gels were blotted overnight in 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) onto nitrocellulose membrane filters (Micron Separations Inc.). The filters were baked for 2 h at 80°C after blotting.

Probes.

An extrachromosomal DNA of about 300 kb from strain AH 818 was used as a probe. This strain is from the cluster ET 24-25 and was randomly chosen. The probe DNA was isolated from a low-melting-point agarose gel by cutting out the band of interest and digesting it with β-agarase as described by the manufacturers (New England BioLabs). The probe was labeled using the Klenow enzyme (New England BioLabs) and ³²P-labeled dCTP and dATP (13, 14).

Hybridization.

The filters were prehybridized in 3× SSC–0.1% sodium dodecyl sulfate (SDS)–10× Denhardt solution (0.1% bovine serum albumin, 0.1% FIcoll, 0.1% polyvinylpyrrolidone) for 2 h at 68°C and hybridized in 25 ml of 3× SSC–0.1% SDS–10× Denhardt solution–10% dextran sulfate (Pharmacia, Uppsala, Sweden) with a denatured labeled probe overnight at 68°C. The filters were washed twice for 30 min each in 3× SSC–0.1% SDS at 68°C, twice for 30 min each in 1× SSC–0.1% SDS at 68°C, and once for 30 min in 0.3× SSC–0.1% SDS at 68°C.

Parasporal crystal observation.

The bacteria were grown aerobically on Schaeffer sporulation medium (31) plates for 5 to 7 days at 28°C. The bacterial cells were stained with carbolfuchsin and observed under a microscope (Labophot-2; Nikon).

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RESULTS

Overall genetic diversity.

In the collection of 65 strains, 12 of the 13 enzyme loci were polymorphic. The number of alleles per locus ranged from 2 to 8 with an average of 4.5 alleles per locus (Tables (Tables22 and and3).3). Among the 65 strains, a total of 48 ETs were distinguished. Among all ETs, the mean genetic diversity over all loci (H) was 0.508 (Table (Table2).2).

TABLE 2

Mean genetic diversity per locus in B. cereus and B. thuringiensis isolates from humans anddairies

Source	No. of isolates	No. of ETs	Mean no. of alleles	Diversity^a
Source	No. of isolates	No. of ETs	Mean no. of alleles	Isolates	ETs
Periodontitis	20	9	1.7	0.150	0.241
Other human infection	15	14	2.8	0.411	0.428
Dairies	30	27	4.0	0.526	0.534
Total	65	48	4.5	0.457	0.508

^aGenetic diversity for ETs and isolates was calculated as described in Materials and Methods.

In the collection of the strains we have analyzed until now, i.e., soil samples (18) and human and dairy isolates (this study) (a total of 219 isolates), all of the enzyme loci were polymorphic. Among all 131 ETs found within the 219 isolates, the mean genetic diversity over all loci (H) was 0.545.

The constructed dendrogram shows the genetic relationships among all of the analyzed strains from a previous study (18) and this study (Fig. (Fig.1).1). To simplify the dendrogram, isolates on the same branch in the dendrogram have a genetic distance of less than 0.1. At a genetic distance of 0.6, there were two clusters of ETs, designated I and II. Cluster I included isolates from soil samples and from dairies, while cluster II included all isolates from patients and from soil samples from the Moss area in Norway (18), in addition to strains from dairies.

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FIG. 1

Genetic relationships among 222 strains of B. cereus and B. thuringiensis from this study and a previous study of soil isolates (18). The dendrogram was generated by the average-linkage method of clustering from a matrix of coefficients of genetic distance based on 13 enzyme loci. Isolates are placed on the same branch when the genetic distance is less than 0.1. Numbers in the dendrogram indicate ETs in this study as listed in Table Table1.1. Symbols indicate strains isolated from patients ( [filled triangle] ), moss (soil sample) (●), other soil samples (○), and dairies (■). Arrows indicate reference strains: a, B. cereus ATCC 10987; b, B. cereus ATCC 4342; c, B. cereus type strain ATCC 14579.

Genetic diversity within and between source groups.

The genetic diversity of each source group of ETs varied from 0.241 (periodontal infection) to 0.534 (dairy isolates) (Tables (Tables22 and and3).3). Only two ETs contained isolates from different sources, periodontal infection and another human infection (ET 21 and ET 41). No isolate from dairies shared ETs with isolates from humans, and no dairy isolates from Norway and Finland shared ETs.

The genetic diversity for ETs of the periodontal infection strains was 0.241, with an isolate diversity of 0.150 (Tables (Tables22 and and3).3). This low isolate diversity compared to ET diversity indicated that many isolates shared the same ET. Only 6 out of 13 loci were polymorphic for the periodontal infection isolates (Table (Table33).

Of the 20 strains isolated from periodontal infections, 12 were identical except for the lack of activity (null allele) at ACO (ETs 24 and 25), and three differed from these by expressing an allele at one locus. The B. cereus reference strain ATCC 4342, isolated from milk in the 1950s, had the same genotype as most of the periodontal isolates (Fig. (Fig.1;1; Table Table1).1). All of the 15 isolates of ETs 22 to 25 had been isolated from different patients except AH 812 and AH 819, which were from the same patient but from two different types of periodontitis (Table (Table11).

Index of association.

The degree of association between alleles at different loci was measured by the index of association. When a given population has an I_A value significantly different from zero, the population exhibits linkage disequilibrium; i.e., it is clonal (28).

The I_A was calculated for the two clusters I and II. For cluster I, the I_A for the ETs was 0.48 ± 0.29, and for cluster II, it was 0.17 ± 0.12 (Table (Table4).4). The I_A in both clusters was significantly different from zero and therefore indicated a clonal structure. However, considering human isolates as a specific source or environmental niche, clonality disappears for the ETs, with an I_A of 0.26 ± 1.00. This population, however, shows a clonal structure when the calculation is done for the isolates, with an I_A of 0.92 ± 0.68. According to Maynard-Smith et al. (28), this indicates an epidemic structure for all of the human isolates.

TABLE 4

Measures of association between loci in isolates and ETs of strains in clusters I and II and from human sources, as presented in Fig.1

Group	No. of isolates	No. of ETs	I_A
Group	No. of isolates	No. of ETs	Isolates	ETs
Cluster I	97	75	0.65±0.24	0.48±0.29
Cluster II	124	86	0.34±0.12	0.17±0.12
Human sources	35	21	0.92±0.68	0.26±1.00

Genetic variation in relation to serotype.

The serotype classification was previously developed exclusively for B. thuringiensis strains. Of the 58 serovars (45 H serotypes and their subgroups) described worldwide among B. thuringiensis strains (25), 14 were represented among the 65 sample isolates in this study (Table (Table1).1). The number of isolates assigned to each serotype varied from one to five, and the most common serotypes were H13 and H18. Except for a few strains that were autoagglutinable (and thus nontypeable), the remaining isolates (48%) did not cross-react with any of the known B. thuringiensis antisera. Of the 13 isolates of ETs 24 and 25, three different serotypes were found, while six strains were nontypeable. There was no close association between serotypes and genotypes, which is in agreement with previous studies (3, 18, 40).

PFGE of digested DNA.

PFGE of strains of ETs 24 and 25 showed at least two different profiles, with the third profile being that of the reference strain ATCC 4342 (Fig. (Fig.2).2).

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FIG. 2

PFGE of DNAs from three B. cereus and B. thuringiensis strains of ETs 24 and 25 digested with AscI (lanes 2 to 4) and NotI (lanes 7 to 9). Lanes: 1 and 6, lambda concatemers; 2 and 7, AH 817; 3 and 8, AH 831; 4 and 7, B. cereus ATCC 4342; 5 and 8, S. cerevisiae chromosomes. The electrophoresis was run as described for Fig. Fig.33.

PFGE of undigested DNA and hybridization.

PFGE analysis of 62 strains showed that most of them (51 strains) contain extrachromosomal DNA with an apparent size of 15 to 600 kb. Examples of the results are given in Fig. Fig.3.3. Sixteen strains had extrachromosomal DNA of about 300 kb, including 11 of the 12 strains in ETs 24 and 25 isolated from patients with periodontitis (Fig. (Fig.3;3; Table Table1).1). The reference strain ATCC 4342 of the same ET cluster did not contain the extrachromosomal element of 300 kb. Three of the strains containing and 300-kb extrachromosomal DNA were from periodontal infection and belong to ET 18 and ETs 22 and 23, while two were isolated from dairies and belonged to ET 48 and ET 29. The extrachromosomal DNAs in the strains of ETs 24 and 25 appeared to be linear, since they always migrated in a pulse time-dependent manner (data not shown).

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FIG. 3

PFGE of undigested DNAs from B. cereus and B. thuringiensis. (A) The electrophoresis was run on a Beckman apparatus with 4-s pulses for 10 min at 170 mA and 30-s pulses for 20 h at 150 mA. (B) Hybridization of extrachromosomal DNA of 300 kb from AH 818. Lanes 1 to 7 and 10 to 13 are of ETs 24 and 25. Lanes: 1, AH 825; 2, AH 827; 3, AH 828; 4, AH 831; 5, AH 829; 6 AH 826; 7, AH 818; 8, S. cerevisiae chromosomes; 9, lambda concatemers; 10, AH 817; 11, AH 819; 12, AH 823; 13, AH 818; 14, AH 812 (ETs 22 and 23); 15, AH 810 (ET 21); 16, AH 814 (ET 41); 17, S. cerevisiae chromosomes; 18, lambda concatemers; 19, AH 725 (ET 41); 20, AH 814 (ET 41); 21, AH 718 (ET 41); 22, AH 811 (ET 40); 23, AH 815 (ET 39); 24, AH 601 (ET 44); 25, AH 818; 26, S. cerevisiae chromosomes; 27, lambda concatemers; 28, AH 407 (ET 6); 29, AH 406 (ET 7); 30, AH 610 (ET 1); 31, AH 612 (ET 5); 32, AH 608 (ET 31); 33, AH 818; 34, AH 722 (ET 34). Isolate AH 818 is used as a control in lanes 13, 25, and 33. Symbols indicate strains isolated from patients ( [filled triangle] ) or dairies (■).

A probe made of a 300-kb extrachromosomal DNA from strain AH 818 hybridized to all of the extrachromosomal DNAs (an example is shown in Fig. Fig.3B)3B) except for strains of ET 18 and ET 29. A weak hybridization to extrachromosomal DNA of different sizes was observed in some strains. However, no hybridization was detected in 26 strains, indicating that these plasmids were entirely different, although some were of similar sizes (Fig. (Fig.33).

Parasporal crystal observation.

Only one strain, AH 400, isolated from a dairy, produced an obvious parasporal inclusion.

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DISCUSSION

The genetic diversity of B. cereus and B. thuringiensis isolates was analyzed using multilocus enzyme electrophoresis. The advantage of this method is that the characters tested are minimally subject to evolutionary convergence, because the electrophoretic variants (allozymes) are essentially selectively neutral or nearly so (11, 12, 17, 39).

The total genetic diversity of the strains in this study (0.508) was somewhat lower than the genetic diversity found among B. cereus and B. thuringiensis from reference strains and culture collections (0.556) (3) and from soil samples (0.526) (18). The high genetic diversity observed in these and other strains is not surprising, since the genetic diversity of rRNA gene restriction patterns displayed by B. thuringiensis was greater than those for all other species examined by that technique (30). The genetic diversity of the ETs from dairies was also high (0.526) (Table (Table2),2), although 13 out of 30 strains formed a separate cluster that differed from all other isolates by more than 0.6 (Fig. (Fig.1).1). Several of the dairy strains, found in both clusters I and II of the dendrogram, are psychrotrophic (16). A cold shock protein has been described for B. cereus (29), and expression of this gene may be common in B. cereus strains that live in the cold environment of dairies. Some or all of those strains probably belong to a new psychrotolerant B. cereus-like group of isolates that recently has been described as a new species, Bacillus weihenstephanensis (26). All psychrotrophic strains in that study were found to belong to this new species. B. weihenstephanensis was found to be more related to Bacillus mycoides than to B. cereus and B. thuringiensis (26). This is thus possibly the reason why some of the psychrotrophic strains in ETs 6 to 12 cluster together with a distance of 0.45 with respect to other isolates, but whether those strains belong to this new species remains to be elucidated.

In the results presented here we find the I_As for ETs in clusters I and II to be 0.481 ± 0.29 and 0.171 ± 0.12, respectively, which indicates a clonal population structure (28), although some DNA uptake is likely to occur. Calculation of the I_A value for isolates from patients (0.92 ± 0.68) indicates linkage disequilibrium, i.e., restriction in recombination, and thus a clonal population. On the other hand, the calculation of I_A values for the patient ETs (0.26 ± 1.00) did not indicate linkage disequilibrium, hence suggesting a recombinant population. The isolates from the patients are thus consistent with the epidemic model of population structure described by Maynard-Smith et al. (28).

We find the high clonality observed for the strains isolated from patients with periodontal infections very interesting. This probably indicates a more stable and homogenous virulent clone of B. cereus as is found for its close relative Bacillus anthracis (21). A high clonality has also been reported for Porphyromonas gingivalis and Prevotella intermedia recovered from persons with periodontal disease, where most subjects appeared to be colonized by the same genotypes of those species (36).

Reduced genetic diversity is often observed in disease-causing bacteria. For example, in Neisseria meningitidis, most of the disease-causing strains belong to a few clones or complexes of related clones in comparison to the genotypic diversity observed in strains from healthy carriers (6, 27).

PFGE analysis of the NotI and AscI DNA fragment profiles of the strains from ETs 24 and 25 isolated from patients divides this lineage further into at least two subclasses (Fig. (Fig.2).2). This heterogeneity indicates that these strains may not have originated from a single source during the sampling period. Still, the strains in this cluster are very similar and possibly have kept the same virulence factor(s). Although the reference strain ATCC 4342 belongs to this cluster, the DNA fragment profile was clearly divergent compared to the other strains of the cluster (Fig. (Fig.2).2). In addition it lacks the extrachromosomal DNA fragment of 300 kb. This may not be so surprising, taking into account that the strain was originally isolated from milk almost 50 years ago (35).

Many survival traits are located on plasmids, such as genes coding for virulence factors and antibiotic resistance (20). B. anthracis, belonging to the B. cereus group and thus a close relative of B. cereus and B. thuringiensis (and the causative agent of anthrax) (2), has its crucial virulence factors located on two plasmids, and when one or both are lost the B. anthracis is not virulent (37). One may speculate that the extrachromosomal DNA of 300 kb observed in the strains from the patients with periodontitis has a virulence factor(s) that makes the strains capable of adhering to type I collagen, laminin, and fibronectin, as observed for strains OH 599 (AH 812) and OH 600 (AH 819) (24). It would be interesting to test whether the patient strains lacking the 300-kb plasmid have reduced adherence to type I collagen, laminin, and fibronectin.

Although it is interesting that all strains isolated from patients were in cluster II, together with the soil isolates from southern Norway (Moss) (18), none of the patients from whom strains were isolated were living in the central or northern part of Norway, where the soil samples in cluster I were from. Yet, the strains from periodontitis infections belonging to the ET cluster 24-25 were from patients living more than 400 km apart in Norway, or in Brazil, indicating that these strains most likely have important virulence factors in common.

In conclusion, this study indicated low genetic diversity among B. cereus and B. thuringiensis isolates from patients but high genetic diversity among dairy isolates, as observed previously for soil isolates. The B. cereus strains isolated from patients show an epidemic population structure, where 60% of the isolates share the same ET.

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ACKNOWLEDGMENTS

We acknowledge M.-M. Lecadet for serotyping strains and B. E. Kristiansen and P. E. Granum for gifts of strains. We thank B. G. Spratt for letting us use his computer program.

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Genetic structure of population of Bacillus cereus and B. thuringiensis isolates associated with periodontitis and other human infections.

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Genetic Structure of Population of Bacillus cereus and B. thuringiensis Isolates Associated with Periodontitis and Other Human Infections

Erlendur Helgason

Dominique A. Caugant

Ingar Olsen

Anne-Brit Kolstø

Abstract

MATERIALS AND METHODS

Strain isolation.

TABLE 1

Serotyping.

Electrophoresis of enzymes.

TABLE 3

Statistical analysis.

Digestion of DNA.

PFGE.

Probes.

Hybridization.

Parasporal crystal observation.

RESULTS

Overall genetic diversity.

TABLE 2

Genetic diversity within and between source groups.

Index of association.

TABLE 4

Genetic variation in relation to serotype.

PFGE of digested DNA.

PFGE of undigested DNA and hybridization.

Parasporal crystal observation.

DISCUSSION

ACKNOWLEDGMENTS

REFERENCES

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