Bacillus cereus

Journal of Microbiological Methods 34 (1998) 99–106

Journal of Microbiological Methods

Rapid discrimination of psychrotolerant and mesophilic strains of the Bacillus cereus group by PCR targeting of 16S rDNA Felix von Stetten, Kevin P. Francis, Sabine Lechner, Klaus Neuhaus, Siegfried Scherer* ¨ Mikrobiologie, Forschungszentrum f ur ¨ Milch und Lebensmittel, Technische Universitat ¨ Munchen ¨ , Weihenstephaner Berg 3, Institut f ur D-85350 Freising-Weihenstephan, Germany Received 30 March 1998; received in revised form 30 July 1998; accepted 5 August 1998

Abstract The paper describes a novel PCR assay for discriminating psychrotolerant and mesophilic strains of the Bacillus cereus group by targeting of 16S rDNA signatures. Application of the assay circumvents long-term growth tests at low temperature currently used to detect psychrotolerant strains. PCR was performed with pure cultures. A 100% correlation of PCR and growth data at 78C was obtained for the 194 B. cereus group strains tested. Potential applications of the assay for the dairy industry and agriculture are suggested.  1998 Elsevier Science B.V. All rights reserved. Keywords: Bacillus cereus; Bacillus mycoides; Bacillus thuringiensis; PCR; Psychrotolerant; 16S rDNA signature

1. Introduction The closely related Bacillus cereus group strains, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis, are Gram-positive endospore forming rods, originating from soil. As a group these bacteria possess a wide temperature growth range, with individual strains able to grow at temperatures as low as 48C, or as high as 428C. The temperature growth range of a particular strain, however, can usually be defined as either low (e.g. 4–378C) or high (e.g. 10–428C). Strains able to grow at or below 78C (i.e. strains with a low temperature growth range), are termed psychrotolerant (psychrotrophic), whereas strains unable to grow below 78C are termed mesophilic (Meer et al., 1991). Psychrotolerant

*Corresponding author. Tel.: 1 49-8161-713-516; fax: 1 498161-714-512; e-mail: [email protected]

strains of B. cereus have been described as a new species Bacillus weihenstephanensis (Lechner et al., 1998). Although assays such as phage- or biochemical typing allow discrimination of bacterial species within the B. cereus group (i.e. typing a strain as either B. cereus, B. mycoides, or B. thuringiensis), these methods do not allow discrimination of psychrotolerant and mesophilic strains. Present methods for detecting psychrotolerant strains are reliant upon growth at low temperature (78C), which may take up to 14 days to be detected (Meer et al., 1991). Hence, the objective of this study was to accelerate the current protocol for the detection of psychrotolerant strains of the B. cereus group, with the intention that this assay might be used in the food and dairy industry. In order to develop a rapid detection method for such bacteria, at least some molecular aspects of psychrotolerance need to be known. Studies directed at understanding the molecular mechanisms adopted

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F. von Stetten et al. / Journal of Microbiological Methods 34 (1998) 99 – 106

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by bacteria for dealing with growth at low temperature (i.e. cold adaptation) have implicated ribosomes to play a vital role (Condon et al., 1995; Graumann et al., 1996). We therefore focused our attention on one particular component of these molecule complexes, the 16S rRNA subunit. In this study, data are presented showing that PCR targeting of particular psychrotolerant and mesophilic 16S rDNA signatures allows rapid discrimination of these two thermal groups of bacteria. The assay has been developed and evaluated testing 194 B. cereus group strains as well as 50 non-B. cereus group strains.

2. Materials and methods

2.1. Strains One hundred and ninety-four strains of the B. cereus group were involved in the development and evaluation of this novel PCR assay (Table 1). The majority of these strains (150) were gained from a variety of collections, with an additional 44 B. cereus group strains isolated from food and soil samples prepared in this study. In order to establish the specificity of this

Table 1 Origin and total number of the Bacillus cereus group strains used in this study Country

Source

Total number of Bacillus cereus

Denmark

w

Germany

India i Norway v,w Poland l,w Thailand i

Unknown i Unknown a

Pasteurised milk Soil d Cream w Milk drying plant b,h Milk powder b,h Pasteurised milk m,w UHT-coffee cream w UHT-milk shake w UHT-milk w Soil i Tobacco Whipping cream Butter Soil Kurkuma root Lemon grass Red rice Sweet-potato Chocolate

1 10 2 17 20 28 1 1 1 18 1 3 1

mycoides

thuringiensis

20

5

4

24 8 3 6 1 1 4 a,g,n

3

1g

10 g,f

American Type Culture Collection, Rockville, USA. ¨ Milchforschung, Institut fur ¨ Hygiene, Kiel, FRG. Bundesanstalt fur d Damgaard et al., 1996. f ´ d’Herelle, ´ ´ Felix Reference Centre for Bacterial Viruses, Univ. Laval, Quebec, Canada. g German Collection of Microorganisms and Cell Cultures, Braunschweig, FRG. h Wiebe and Hammer, 1997. i Isolates prepared in this study. l Lonc et al., 1997. m Mayr, 1997. n National Collection of Industrial and Marine Bacteria, Aberdeen, UK. v P. Granum, Norwegian College of Veterinary Medicine, Oslo, Norway. w ¨ Mikrobiologie, Forschungszentrum fur ¨ Milch und Lebensmittel, Tech. Univers. Weihenstephan Bacillus Collection of the Institut fur Munich, Freising-Weihenstephan, FRG. b


assay, 50 non-B. cereus group strains were also tested (Table 2).

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this overnight culture served as inoculum for the growth test, which was performed in 5 ml PCB, shaken at 120 rev. / min and 48C, 78C or 108C.

2.2. Culture conditions and confirmative growth tests

2.3. DNA preparation by osmotic lysis

B. cereus and B. mycoides were isolated on B. cereus selective agar (PEMBA, Unipath, Wesel, Germany), with these plates being incubated for 2 days at room temperature. Growth tests were performed in Plate Count Broth (PCB), composed of 5.0 g casein-peptone (Unipath), 2.5 g yeast-extract (Unipath) and 1.0 g glucose (Sigma-Aldrich, Steinheim, Germany) per litre of deionised water, with the pH adjusted to 7.0. An overnight culture of bacteria was grown in 5 ml PCB, shaken at 150 rev. / min and 308C. Ten ml of

In the majority of cases, transfer of a pinhead sized aliquot of bacteria directly from an agar plate into a tube prior to PCR cycling was sufficient to allow amplification of the target DNA. However, for some strains the results of the PCR could be dramatically improved by previous osmotic lysis. This involved adding 50 ml of an overnight bacterial culture, or a bacterial colony picked from an agar plate, to 100 ml of ice cold Milli-Q purified water (Millipore water purification system, Millipore, Eschborn, Germany). The suspension was either kept

Table 2 Non-Bacillus cereus group strains used in this study. Positive mesophilic (m) or psychrotolerant (p) reactions in the PCR assay are indicated Endospore-forming Gram-positive rods Clostridium perfringens Clostridium sporogenes Bacillus alvei a (p) Bacillus brevis Bacillus circulans a (p) Bacillus coagulans Bacillus firmus Bacillus fusiformis Bacillus laterosporus Bacillus lentus Bacillus licheniformis a (m) Bacillus macerans Bacillus megaterium a (p) Bacillus panthothenticus Bacillus pasteurii Bacillus polymyxa Bacillus psychrophilus Bacillus psychrosaccherolyticus Bacillus pumilus a (m) Bacillus sphaericus Bacillus sporothermodurans (m) Bacillus stearothermophilus Bacillus subtilis (m) Gram-negative aerobic rods Pseudomonas fluorescens Alcaligenes faecalis Flavobacterium flavescens a

Two different strains of this species have been tested.

Gram-positive cocci Micrococcus luteus Staphylococcus aureus Enterococcus faecalis Lactococcus lactis ssp. lactis Regular, nonsporing, Gram-positive rods Lactobacillus casei (m) Lactobacillus delbrueckii ssp. bulgaricus (m) Lactobacillus plantarum a (m) Lactobacillus rhamnosus (m) Listeria monocytogenes Irregular, nonsporing, Gram-positive rods Corynebacterium glutamicum Brevibacterium linens Arthrobacter nicotianae Microbacterium lacticum Gram-negative facultatively anaerobic rods Enterobacter cloacae Proteus vulgaris Salmonella amersfort Klebsiella pneumoniae Escherichia coli

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on ice for at least 1 h or freeze-thaw cycled three times (2108C / 1 608C). The bacterial lysate was stored at 2 208C. Eight ml of lysate were used in each PCR reaction.

GGC and uf: 59CAA GGC TGA AAC TCA AAG GA, respectively.

2.5. Performance of the PCR 2.4. Design of psychrotolerant and mesophilic 16 S rDNA primers Multiple alignments of 16S rDNA sequences (Lechner et al., 1998) from eight mesophilic and 11 psychrotolerant strains of the B. cereus group, revealed the existence of two signatures which specified the temperature growth type (Table 3). These sequence differences were used to design two pairs of oligonucleotide primers, targeting either psychrotolerant or mesophilic 16S rDNA signatures (Fig. 1). Primer pair one (mf-ur) amplifies a 250-bp DNA fragment when the first signature has a mesophilic sequence, whereas primer pair two (uf-pr) amplifies a 130-bp DNA fragment when the second signature has a psychrotolerant sequence. The length of the PCR products can be estimated by agarose gel electrophoresis. Thus, psychrotolerant strains can be recognised by the amplification of a 130-bp fragment, while mesophilic strains are recognised by the amplification of a 250-bp fragment. Sequences of the mesophilic and psychrotolerant specific primers are mf: 59ATA ACA ] TTT TGA ACC ] GCA TG ] and pr: 59GGA GAA GCT CTA TCT CTA GA, respectively ] ] (signature bases underlined). Appropriate universal counter primers are ur: 59CTT CAT CAC TCA CGC

PCR was carried out with a Techne Progene thermocycler in 25-well plates (Advanced Biotechnologies, Hamburg, Germany), sealed with a thermostable polyester tape (neoLab, Heidelberg, Germany). Cycling parameters were: initial denaturation at 948C for 2 min, followed by 27 cycles at 948C for 15 s, 558C for 15 s and 728C for 15 s (Taq polymerase was added at 808C during the initial cycle of 27). A final extension at 728C for 2 min completed the PCR. A two component PCR mastermix was prepared for 1000 reactions. Component ‘cold’ consisted of 16 ml Milli-Q, 3 ml 10 3 buffer (100 mM Tris–HCl, pH 9.0; 15 mM MgCl 2 ; 500 mM KCl, 1.0% gelatine), 5 ml 20% (w / v) Ficoll 400 (Amersham Pharmacia Biotech, Freiburg, Germany) bromophenol blue-stained solution (Kaijalainen et al., 1993), 2 ml 5 mM dNTP-mixture (Eurogentec, Seraing, Belgium), and 25 pmol of each of the four primers (see above). Component ‘hot’ consisted of 14 ml Milli-Q purified water, 1.5 ml 10 3 buffer (see above), 0.4 ml Taq polymerase dilution buffer (Eurogentec), and 100 ml Taq polymerase (5 U ml 21 , Silverstar, Eurogentec). A 25-ml volume of mix ‘cold’ was pipetted into each reaction well and stored

Table 3 Aligned 16S-rDNA sequences of mesophilic (m) and psychrotolerant (p) Bacillus cereus group strains. Sequence numbers are in accordance to the E. coli system (Ehresmann et al., 1972)


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Fig. 1. Principle of the 16S-rDNA assay for the discrimination of mesophilic and psychrotolerant B. cereus group strains: the mesophilic-specific forward primer (mf) only anneals when a mesophilic signature 1 is present, the psychrotolerant-specific reverse primer (pr) only anneals if a psychrotolerant signature 2 is present. The universal reverse and forward primers (ur, uf) anneal in either case. The distance of their binding site to the concerned signature determines the length of the amplified DNA. A 250-bp fragment detected on the agarose gel indicates a mesophilic target (lane m), a 130-bp fragment indicates a psychrotolerant target (lane p). The molecular weight marker is a 100-bp ladder (100 bpl).

at 2 208C prior to use, together with a separate 400 ml volume of mix ‘hot’. Before thermocycling, 8 ml of bacterial lysate were added to mix ‘cold’, which was then heated to 948C for 2 min. The temperature was then reduced to 808C and 15-ml volumes of mix ‘hot’ were injected with a stepper before starting PCR cycling.

2.6. Agarose gel electrophoresis Eight-ml volumes of each PCR product were directly transferred into the wells of a 2% agarose gel. This was made possible by the inclusion of Ficoll 400 into the PCR mixture (Kaijalainen et al., 1993). After subsequent electrophoresis the gel was stained with ethidium bromide, and then visualised using a Pharmacia Image Master (Amersham Pharmacia Biotech, Freiburg, Germany).

3. Results and discussion

3.1. Correlation between PCR amplification and bacterial growth at low temperature Of 194 B. cereus group strains tested, 100% correlation was seen between the PCR data (example shown in Fig. 2) and the growth data, with regard to classifying the strains as psychrotolerant or mesophilic. All strains grew at 258C and 308C. Fiftyone strains were psychrotolerant, showing growth at 78C, with 41 of these strains also able to grow at 48C. Forty-five of the remaining 143 mesophilic strains were tested for growth at 108C, all except seven (isolates from Thailand) of these 45 strains grew. Interestingly one strain of B. cereus that was isolated from soil (strain V1R12, this study), and which was shown to grow at 48C, gave both a

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Fig. 2. Example of PCR products amplified from mesophilic and psychrotolerant B. cereus group strains by targeting specific 16S-rDNA signatures: (1) 100-bp ladder; (2) Danish soil, B. mycoides; (3) Thai soil, B. cereus; (4, 5) South German soil, B. cereus; (5) South German soil, B. cereus; (6, 7) North German milk-powder, B. cereus; (8–10) South German pasteurised milk, stored at 78C, B. cereus; (11) Thai rice, B. mycoides (see text for PCR methodology).

mesophilic and a psychrotolerant PCR product. This still occurred when further purified by subculturing several times with single colonies. Provided that the further colony purification was successful, this observation could be explained by the coexistence of mesophilic and psychrotolerant 16S rDNA operon copies within a single organism. Sequence heterogeneities within multiple rRNA operon genes have already been reported for E. coli (Condon et al., 1995). We are currently investigating this hypothesis.

3.2. Specificity of the PCR assay Due to the high degree of conservation found between 16S rDNA sequences in closely related bacteria, 17 of the 50 investigated non-B. cereus group strains gave a PCR product (see Table 2). For this reason, PCR should be performed only on appropriately identified strains. Whether or not it can be done directly following selective enrichment broths remains to be shown.

3.3. Time constraints of conventional microbiology compared to PCR Current protocols for the discrimination of psychrotolerant strains of the B. cereus group from

their mesophilic counterparts are extremely slow, involving monitoring of growth at 78C for up to 14 days (Meer et al., 1991). Since the majority of pasteurised food and dairy products in which this bacterium causes spoilage are usually consumed within this period however (Notermans et al., 1997), little benefit is gained by using such low temperature growth protocols. The PCR procedure developed in this study for discriminating these two thermal groups of B. cereus is both rapid and cost effective. The most laborious step in this procedure is the initial isolation of pure cultures of B. cereus. Isolation of these bacteria on selective agar took 2 days, due to the incubation at ambient temperature to ensure the recovery of all B. cereus strains. Refinement of the PCR to allow quantification of both psychrotolerant and mesophilic templates simultaneously (e.g. using a 59 nuclease assay; Witham et al., 1996), would eliminate the time necessary for the initial isolation of bacteria. Such a refinement could allow separation of the bacteria directly from a food or soil matrix by using techniques such as immunomagnetic capture (Koo et al., 1996; Quinlan and Foegeding, 1997; Blake and Weimer, 1997), magnetic capture-hybridization (Jacobsen, 1995), centrifugation (Herman et al., 1995), or two-phase partitioning (Sacks and Alderton, 1961). Such approaches are currently under investigation.


A further advantage of the PCR assay as opposed to growth experiments, is that such a molecular approach does not depend on the experimental conditions, such as different media, but exclusively reflects a genetic determinant.

3.4. Potential applications of the PCR assay Due to the heat resistance of their spores, psychrotolerant B. cereus and B. mycoides are known to limit the shelf life of pasteurised refrigerated food, such as fresh milk (Meer et al., 1991; Griffiths, 1992; te Giffel et al., 1995). The high frequency of this bacterium in food and dairy products is of particular concern due to the majority of B. cereus isolates producing enterotoxins, leading to such strains being commonly associated with food poisoning (Becker et al., 1994; van Netten et al., 1997). As part of the food industry’s hazard analysis of critical control points (HACCP), this novel PCR assay could be applied to monitor foods for psychrotolerant B. cereus group strains prior to pasteurisation. Strains of the Bacillus cereus group play an important role in agricultural applications (Salama et al., 1986; Maplestone and Campbell, 1989; Halverson and Handelsman, 1991; Blagoeva et al., 1995; Paterno et al., 1995). Their beneficial abilities, however, strongly depend on the bacterium’s temperature growth type. Therefore a preliminary screening, using the novel PCR assay could support the selection of suitable strains.

Acknowledgements We would like to thank Ralf Mayr (this lab), Per Damgaard (The Royal Veterinary and Agricultural University Frederiksberg, Denmark), Per Granum (Norwegian College of Veterinary Medicine, Oslo, Norway), E. Lonc (Uniwersytetu Wroclawskiego, ¨ Poland) and Christian Wiebe (Bundesanstalt fur Milchforschung, Kiel, Germany) for supplying us with strains from their collections. Part of this work was supported by the Deutsche Forschungsgemeinschaft MA 1702 / 3-1.

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ELSEVIER

Journal

of Microbiological

Methods 35 (1999)

185

Journal ofMicrobiological Methods

Erratum

Erratum to “Rapid discrimination of psychrotolerant and mesophilic strains of the Bacillus cereus group by PCR targeting of 16s rDNA” [J. Microbial. Methods 34 (1998) 99-1061” Felix von Stetten, Kevin P. Francis, Sabine Lechner, Imtitutfir Mikrobiologie.

Forschungszentnrm

Received

The Publisher

30 March

Siegfried

Scherer”

fiir Milch wui Lebensmittel, Technischr Univer.sitiit Miinchen. Weihenstephaner D-85.150 FreisinRWeihenstephull. Germane

1998; received

regrets the following

Klaus Neuhaus,

in revised form 30 July 1998; accepted

5 August

Berg 3.

1998

error in the above paper.

The primer-sequence provided on page 102 (pr: S’GGA GAA GCT CTA TCT CTA GA) is not correct. The correct sequence is pr: 5’ GAG AAG CTC TAT CTC TAG A. The results are not reproducible with the incorrect sequence of pr. The authors would like to apologize for this error to all who tried the novel assay without success.

“Corresponding author. ‘PII of the original article: SO167-7012(98)00077-3

0167.7012/99/$ - see front matter PII: SO167-7012(99)00036-6

0

1999 Published

by Elsevier Science B.V. All rights reserved.