Differentiation of Bacillus anthracis and other ‘Bacillus cereus group’ bacteria using...

FEMS Microbiology Letters 128 (1995) 113-118

Differentiation of Bacillus anthracis and other ‘Bacillus cereus group’ bacteria using IS231 -derived sequences

Ian Henderson a,*, Yu Dongzheng b, Peter C.B. Turnbull a

a Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, UK h Institute of Epidemiology and Microbiology, P.O. Box 5, Changping, Beijing 102206, People’s Republic of China

Received 10 February 1995; revised 2 March 1995; accepted 7 March 1995

Abstract

Sequences based on the conserved 20 bp inverted repeat of IS231 variants were used as polymerase chain reaction-based fingerprinting primers of the member species of the Bacillus cereus group (B. anthracis, B. cereus, B. thuringiensis and B. mycoides), because of their close association with transposons, principally Tn4430 in B. thuringiensis. Fingerprints of B. anthracis were simple, and specifically allowed its identification and sub-differentiation from other members of the group. Fingerprints for B. cereus were strain-specific; those for B. thuringensis gave a 1650 bp product, characteristic of IS231 variants A-F. The same reaction conditions gave one or two bands for both f?. anthrucis and B. cereus that differed by

restriction endonuclease mapping from the B. thuringiensis PCR product and established IS231 restriction maps; this does not preclude some kind of relationship between these products and IS231.

Keywords: Bacillus anthracis; Bacillus cereus; Bacillus thuringiensis; IS231; Polymerase chain reaction differentiation

1. Introduction

The ‘Bacillus cereus group’, B. anthracis, B.

cereus, B. thuringiensis and B. mycoides, are no-

table for their phenotypic relatedness. The species

and subspecies of the group can be differentiated only on the basis of highly mutable characteristics such as colony morphology, penicillin and gamma phage susceptibility, motility and lack of haemolysis, and the elaboration of certain virulence factors. Avir- ulent environmental isolates of B. anthracis are only distinguishable from B. cereus if they retain penicillin and gamma sensitivity, are weakly haemolytic

* Corresponding author. Tel: 01980 612361; Fax: 019801

610898.

and are non-motile. This indicates the need for a suitable means of differentiating between members

of the group. The increasingly frequent identification of avirulent B. anthracis in the environment [I], and

the strategic interest in the organism, underscores a need not only for a system of rapid identification, but also for a strain differentiation and epidemiological tracing capability.

Probes of chromosomal organisation seem to be the most likely route to fulfill these requirements. We have recently identified the randomly amplified polymorphic DNA polymerase chain reaction (RAPD PCR) technique to be capable of at least specifically identifying B. anthracis from other B. cereus group members [2]. Slight differences between B. anthracis isolates were detected using this method

0378-1097/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved SSDI 0378.1097(95)00092-5

114 I. Henderson el al. / FEMS Microbiology Letters I28 (I 995) 113-l 18

when pure chromosomal DNA was used, but the reproducibility of the technique is likely to be

severely challenged when complex environmental

samples are presented such as soil, due mostly to the sensitivity of PCR to cation concentrations and the

presence of phenolics. The alternative is to simplify PCR fingerprint patterns, and to aim for targets that are more likely to have some influence on chromoso-

ma1 organisation, such as insertion sequences and transposons.

One of the best characterised of these types of sequence in the B. cereus group is IS231. At least

eight variants (A-F, V and W) of this sequence exist in B. thuringiensis [3-51, and have been shown to be

related to the IS4 family in Escherichia coli [6].

They are delimited by an 11 bp street repeat sequence and a highly conserved 20 bp inverted repeat

sequence, and have been shown to be associated with Tn4430 in B. thuringiensis strain berliner 1715 [7].

Such sequences have yet to be characterised in B.

anthracis and B. cereus although Tn4430 has been

shown to facilitate transfer of the B. anthrucis virulence encoding plasmids pXO1 and pXO2 [8,9] be-

tween members of the B. cereus group. The purpose of this study was to use PCR primers

based on the IS232 20 bp direct repeat as determi-

nants of species-to-species and isolate-to-isolate variation in the members of the B. cereus group, and specifically in B. anthrucis. It was not the purpose to

isolate further IS231 variants from any of the organ- isms examined but clearly some of the PCR products produced may represent such elements.

2. Materials and methods

2.1. Bacterial strains and isolates

.B. anthracis, B. cereus, B. thuringiensis and B.

mycoides isolates and strains are presented in Table 1. Chromosomal DNA was isolated by a method described elsewhere 121.

2.2. PCR

PCR primers (Table 2) were synthesized by the automated phosphoramidite method using an Ap- plied Biosystems model 380B DNA synthesizer. PCR

Table I

Sources of ‘Bacillus cereus group’ isolates

Organism a.’ Other designation Host/Source ’ Reference

B. anthracis: NCTC 8234 Sterne cow, 1937 11

South Africa ’

ASC 182 Pasteur France, pre-1880’s 12

ASC 328 Vellum Cow, pre-1939 13

United Kingdom

ASC 184 Vellum e pxo1+ pxo2-

ASC 185 Vollum e pxo1- pxo2+

ASC 68 Ames Cow, 1980

USA

ASC 69 New Hampshire Human, 1957 14

USA

ASC 52 Human, 1982 15

Zimbabwe

ASC 183 pxo1- pxo2+,

penicillin-resistant

isolate ’

ASC 58 Elephant, 1983 16

Namibia

ASC 60 As ASC 58

a Other B. cereus group strains used in this study: B. thuringiensis HD37, HD102 and HD225, obtained from H.T. Dulmage, US

Department of Agriculture, Brownsville, Texas, USA; B.

thuringiensis F2113/78 (ssp. entomocidus); B. cereus NCTC 2599, F4810/72, F2532/74, F3484/77, F4433/73, ASC 109,

ASC 112 and ASC 113; E. mycoides NCTC 09680. All strains

with an F prefix were obtained from the Food Hygiene Labora-

tory. Central Public Health Laboratory, Cohndale, London, UK.

h ASC: Anthrax Section Culture, Research Division, CAMR, Por-

ton Down, Salisbury, UK.

’ Unless otherwise stated, B. anthracis isolates are

pxo1+ pxo2+.

d Livestock and UK human vaccine strain.

’ Kindly supplied by Defence Microbiology Division, Chemical

and Biological Defence Establishment, Porton Down, Salisbury,

UK.

f ASC 32 cured by Dr. C.P. Quinn, Molecular Microbiology

Group, CAMR, Porton Down, Salisbury, UK.

mixtures of 100 ~1 consisted of 1.25 mM each of dATP, dCTP, dGTP and dTTP, 3.0 mM MgCl,, 2 PM of single PCR primer, and 10 ~1 of 10 X

reaction buffer (160 mM (NH,),SO,, 670 mM Tris- HCl (pH 8.81, 0.1% Tween-20). PCR cycling conditions consisted of 95°C for 5 min and 30 cycles of 94°C for 2 min, 40°C for 1 l/2 min, 72°C for 2 min (primer 1, Table 2) or 3 min (primer 2, Table 21, and finally 72°C for 5 min, in an MJ Research thermocy- cler. PCR products were analyzed by agarose gel electrophoresis [ 101.

I. Henderson et al. / FEMS Microbiology Letters 128 (1995) 113-I 18 11s

2.3. PCR product characterisation

To analyze the PCR products by restriction en-

donuclease mapping, samples were separated in low melting point agarose (Sigma) and cut from the gel; the DNA was subsequently purified using Wizard

minicolumns (Promega). A restriction map of each band was determined using several restriction en- donucleases. These enzymes were used according to manufacturer’s instructions.

Results and discussion

Primers 1 and 2 (Table 2) provide the means to

assess (i) the possibility of IS231-like sequences being present in the genomic DNA of B. anthracis

and (ii) variability in the spacing between these

IS23I-like sequences in the genome, respectively. Using the 20 bp inverted repeat sequence as a PCR target means that only a single primer needs to be used in each PCR reaction allowing easy optimisa- tion of the reaction.

Fig. 1 shows PCR profiles for primer 1. Reactions were optimised by varying primer annealing temper-

ature and the magnesium ion concentration. All the B. thuringiensis strains analyzed produced a single band of approximately 1650 bp, the characteristic size of IS231 A-F variants (Fig. 1A). PCR products from B. anthracis gave characteristic one or two

band patterns (lanes 2-13, Fig. 1B). The lower band has a,molecular mass of approximately 1.9 kb, within the correct size range of IS231 variants V and W

Table 2

Terminal inverted repeats of IS231 variants and primers used for PCR

from B. thuringiensis var israeliensis. The upper

band had a molecular mass in excess of 3.1 kb and was produced only from certain isolates. The pres-

ence of this extra band is independent of virulence. B. cereus has a much greater pattern variety compared to both B. thuringiensis and B. anthracis.

Among these varieties are two B. cereus strains

which appear identical to B. thuringiensis (B. cereus

F4810/72 Fig. lA, lane 7, and F564/49 Fig. lA, lane 6), and one identical to the B. anthracis double

band pattern (B. cereus F3484/77 Fig. lB, lane 9).

These findings suggest the possibility that all three species and sub-species may harbour variants

of IS231-like sequences. To examine this, limited

restriction mapping analysis was performed on the 1.6 kb band of B. cereus and B. thuringiensis and the 1.9 kb band of B. anthracis. These comparisons

are presented in Fig. 2. Although direct comparisons between known and newly determined restriction maps are difficult without sequence data, one can see

from these maps that known IS231 variants have highly similar site distributions (and hence conserved DNA sequences). This is borne out by alignments of

the DNA sequences of IS231 variants [3-51. There appears to bo good correlation B: thuringiensis

HD102 PCR product and B. cereus ASC 109 with established restriction maps of IS231 variants. There was no correlation between the B. anthracis 1.9 kb band map and these variants (Fig. 2).

Of particular interest to this study are the fingerprints generated with the PCR primers. Fingerprints with primer 1 were not species-specific; certain patterns were common to B. thuringiensis and B. cereus

IS.231

Variant

A

B

C

D

E” F

V

W

Primer 1

Primer 2

Sequence

S-CAT GCC CAT CAA Cl-T AAG AA-3’

5’.CAT GCC CAT CAA CTT AAG AA-3’

S-CAT GCC CAT CAA CIT AAG AA-3’

5’.CAT GCC CAT CAA ATT AAA GA-3’

5’-CAT ACC CAT CAA CIT AAG GG-3’

5’-CAT GCC CAT CAA Cl-f AAG AA-3’

5’-CAT CGC CAT CAA GCT AAG GA-3’

S-CAT CGC CAT CAA GCT AAG GA-3’

5’-CAT GCC CAT CAA CTI AAG AA-3’

3’-GTA CGG GTA G’IT FAA TK ‘IT-5’

Source/Reference

5 This study

This study

’ This sequence is only found at the 3’ end of 1.9231.

116 I. Henderson et al. /FEMS Microbiology Letters 128 (1995) 113-118

4072bp

2036bp

1635bp

in the laboratory, where it appears to be reluctant to grow on B. anthracis selective media and in horse

blood (for capsule production tests). This difference is seen further with primer 2 in the next section, and

confirmed using rRNA fingerprinting approaches (Henderson, I. and Duggleby, C.J., manuscript in preparation).

1018bp

0 123

Primer 2 fingerprint patterns were different for each strain of B. cereus, B. thuringiensis and B.

mycoides tested and therefore could not be used for their differentiation. In contrast, patterns for B. an-

thracis were highly specific. Only three pattern variants could be detected for all of the B. anthracis

isolates tested (Fig. 3); these did not overlap with patterns for B. cereus, B. mycoides and B.

thuringiensis. Variation was found principally in the presence or absence of the PCR product of approximately 1.6 kb. Those isolates that do not have this band also lack the 3.1 kb band with primer 1. ASC

182 also lacks this band and has a different fingerprint with primer 2 compared to other B. anthracis

isolates, confirming the suspected difference of this

isolate [3]. This difference may be as a result of numerous laboratory manipulations in the > 100 years since it was first isolated. As with primer 1, the reasons for the differences observed with primer 2 are unclear, showing no relationship with documented histories. We must conclude, therefore, that

possibly with the exception of ASC 182, these differences at the level of chromosomal DNA appear to be at random in the absence of other data.

Consistency of DNA fingerprints derived by any technique does not mean that isolates are identical.

Although a high degree of band-sharing does occur

=bp

1635bp

1018bp

Fig. 1. PCR fingerprints generated using primer 1. Isolates and

strains are (A) 1 kb ladder (l), E. fhuringiensis HD37 (2), HD102

(3), HD225 (4), F2113/78 (S), 8. cereus 564/49 (6) F4810/72

(7) F2532/74 (8), F3484/77 (9), NCTC 2599 (IO), B. mycoides NCTC 09680 (11) and 1 kb ladder (12). (B) B. anthracis NC’K

8234 (1) AX 11 (2), ASC 58 (3), ASC 68 (4), ASC 69 (5), ASC

328 (6) ASC 184 (7) ASC 185 (8), ASC 81 (9), ASC 182 (lo),

ASC 183 (ll), ASC 55 (12), ASC 60 (13) 8. cereus ASC 109

(14) ASC 112 (15), ASC 113 (16) 1 kb ladder (GibcoBRL) (17).

Electrophoresis was carried out in 0.8% (w/v) agarose for 16 h at

55 v.

(Fig. lA), and B. cereus and B. anthrucis (Fig. 1B). This primer could not be used to differentiate between this group of closely related species. The differences observed between B. anthracis isolates do not correspond to the known documented histories of these isolates. For example ASC 184 (Fig. lB, lane 7) and ASC 185 (Fig. lB, lane 8) are from a common ancestor yet give different fingerprint patterns. However, ASC 182, which only gives a single band (Fig. lB, lane lo), is seen to be different

000 D lsssbp IS231A

D 0 0 0 165=Q IS2376

0 D 0 lassbp I I IS231C

D OD lssmp IS237D

D D D lassbp IS237E

00 0 DD lassbp IS237F 0 D D -SW I

D 0 IS23N 0 lssobp D D HD102 lewbp 1 B. cweus D 0 lombp 8. anthrads

Fig. 2. Alignment of Dral (D) restriction enzyme sites for known

IS231 variants and PCR products of B. thuringiensis HD102, B.

cereus ASC 109 and all B. anthracis.

I. Henderson et al. / FEMS Microbiology Letters 128 (1995) 113-I 18

with the products from the two specific primers in this study for B. anthmcis, the individual bands themselves may differ at the DNA sequence level.

To this end, bands of equal electrophoretic mobility generated from both primer 1 and 2 were isolated and purified from low melting point agarose for

several of the B. anthracis isolates analysed. Cloning

and sequencing was beyond the scope of this study, but isolated DNA’s were restriction-mapped. Typical results are presented in Fig. 4. Bands of comparable electrophoretic mobility were identical at this level of analysis. Isolate-to-isolate variation was not ob-

served, reinforcing the notion of the highly conserved nature of B. anthracis.

In summary, sequence-specific or sequence-di- rected oligonucleotide primers are useful fingerprinting tools for the B. cereus group. This is particularly the case for B. anthracis where the high degree of conservancy between isolates and strains makes fin-

gerprinting the best approach for identification and sub-differentiation. A similar conclusion was drawn

from Ml3 repeat sequence fingerprinting using PCR [2] but those fingerprints were much more complex and difficult to reproduce. Fingerprints in this in-

stance are simple and reproducible, with differences

1 2 3 4 5 6 7 8 910111213141516

3054bp

2036bp

1635bp

1016bp

5OObp

Fig 3. PCR fingerprints generated using primer 2. 1 kb ladder (11,

B. anthracis NCTC 8234 (2), ASC 11 (31, ASC 58 (4), ASC 68

(51, ASC 69 (6), ASC 328 (71, ASC 184 (81, ASC 185 (91, ASC

327 (101, ASC 91 (111, ASC 182 (121, ASC 183 (131, ASC 55

(14), ASC 60 (151, and 1 kb ladder (16). Electrophoresis was

carried out in 0.8% (w/v) agarose for 16 h at 55 V.

1 2 3 4 5 6 7 8 910111213141516

117

4072bp

2036bp

1635bp

1018bp

500bp

Fig. 4. Restriction mapping of the 1.9 kb PCR product of B.

anthracis ASC 91 and ASC 182 generated with primer 1. Lanes

are: 1 kb ladder (l), ASC 91 DraI (21, DraI/XbaI (3). XbaI (4), XbaI/SspI (51, SspI (61, DraI/SspI (71, ASC 182 DraI (81,

DraI/XbaI (9), XbaI(lO), XbaI/SspI (111, SspI (121, DraI/SspI (131, and 1 kb ladder (14). Enzymes that did not cut the PCR

products were BamHI, BscI, CfoI, EcoRI, EcoRV, HaeII,

HindIII, Mlul, NraI, PstI, Scul, StuI and XhoI. Digested DNA

was analysed by electrophoresis in 0.8% (w/v) agarose for I6 h

at 55 V.

between individual B. anthracis isolates being much

more obvious. Fingerprint variation between B. anthracis iso-

lates is limited to gross band differences. Differences

between bands of comparable electrophoretic mobility do not seem to be apparent although extensive DNA sequencing of such PCR products needs to be

performed to confirm this. The life cycle of B.

anthracis depends on its finding a suitable host in which to multiply; in the environment it exists al- most invariably in the spore form. The opportunity for influences of DNA variation, such as exposure to phages and other DNA transforming events, is therefore limited to the relatively infrequent times when the organism is in the vegetative stage. Fingerprint- ing methods using specific targets show promise for studies of the variation of the chromosome for B. anthracis as opposed to those which probe the chromosome in a random manner.

118 I. Henderson et al. /FEMS Microbiology Letters I28 (1995) 113-I 18

Acknowledgements

The authors thank the Royal Society for the Study Visit award to Dr. Yu which made his contribution to this research possible. The technical assistance of Ms. Caroline Redmond is also gratefully acknowl- edged.

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