• Bacillus licheniformis;
  • Intraspecies diversity;
  • Pathogen


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Bacillus licheniformis is exploited industrially for the production of enzymes and has been shown to exhibit pathogenic properties. Because of these divergent characteristics, questions arise concerning intraspecies diversity. A comparative study by means of combined repetitive polymerase chain reaction, rpoB and gyrA sequencing, 16S rDNA targeted probe analysis, DNA–DNA hybridizations, gelatinase tests and antibiotic susceptibility tests was performed on a set of strains from diverse sources, including strains with pathogenic potential. B. licheniformis was found to consist of two lineages that are distinguished genotypically.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Bacillus licheniformis is both of interest and concern. On the one hand it is exploited industrially for the large-scale production of enzymes, on the other hand, as reports on the pathogenic effects of B. licheniformis accumulate, more and more questions arise concerning the safety aspects of this organism.

B. licheniformis has a long history in the production of proteases, amylases and lipopeptide surfactants, which are used in detergent production, liquefaction of starch, and oil recovery respectively. Recombinant B. licheniformis strains have been designed and used extensively in large-scale fermentation procedures [1]. B. licheniformis strains produce a variety of peptide antibiotics, such as bacitracin [2]. Recently, a new bacteriocin, produced by B. licheniformis, has been reported [3] and has been examined for its use as a food preservative. These features illustrate the economic importance of the organism. Several attempts have been made to exploit these traits even more by improving production yields [1,4].

B. licheniformis contaminates industrial processes [5–7] and has been involved in food-poisoning incidents [9,10]. Some strains of this taxon were shown to produce a non-proteinaceous toxin, showing physicochemical properties similar to cereulide, the emetic toxin of Bacillus cereus[8]. Although B. licheniformis still has a GRAS (generally regarded as safe) status [8], it is increasingly recognized as a human pathogen. Indeed, various cases of B. licheniformis-associated clinical infections are known [11–15], mainly in immunocompromised hosts. Finally, B. licheniformis strains have also been shown to exhibit an abortifacient potential in cattle [16,17]. Since B. licheniformis has been found as a contaminant in cattle-feeding stocks [16,18], a contamination through foodstuffs has been suggested.

Because of the various economic interests in this species, the abortifacient and toxigenic capacity of some strains, questions arise about intraspecies diversity that could differentiate isolates of potential clinical importance. Duncan et al. [19] described the intraspecific diversity in B. licheniformis based on population studies of soil isolates by means of multilocus enzyme electrophoresis and phenotypic analysis. They observed a high level of diversity among the isolates, which were clustered in two main subgroups. Also Daffonchio et al. [20] found two clusters in the analysis of 10 B. licheniformis strains based on random amplification of polymorphic DNA, internal transcribed spacer polymerase chain reaction (PCR) and tDNA-PCR. Manachini et al. [21] studied 182 isolates attributed to B. licheniformis on the basis of phenotypic tests. Despite the high phenotypic similarities, DNA–DNA reassociation studies showed three very distinct groups that were therefore regarded by Manachini et al. [21] as genomovars of B. licheniformis. Ishihara et al. [22] found two clusters within 22 laboratory stock B. licheniformis strains, based on the distribution and variation of bacitracin synthetase gene sequences and erythromycin resistance.

During a screening program for gelatinase-producing contaminants from crude gelatine extracts, we found B. licheniformis as the dominant contaminant in several European and American production plants (De Clerck et al., submitted). Moreover, it was shown that B. licheniformis strains were able to survive the extreme conditions of temperature, pH, drying and even a UHT treatment applied during gelatine production, ending up in the final product. Since gelatine is used in different food and pharmaceutical applications, a better knowledge about the characteristics of this type of contamination may be of extreme importance for human health. A first genotypic screening of these isolates was performed by means of (GTG)5-PCR. Since these profiles were very homogeneous on the one hand, and because of the various reports that mention consistent groupings within B. licheniformis on the other hand, questions arose concerning the real homogeneity of the gelatine isolates. Are these gelatine isolates highly similar and do they belong to a distinct subgroup within the species, or is the discriminatory power of (GTG)5-PCR insufficient to reveal subgroups? Despite previous studies on the intraspecific diversity of B. licheniformis, it is also unknown whether or not the B. licheniformis strains that contaminate industrial settings and foods and the strains that cause abortion and/or produce toxins belong to different lineages. The present study aims, therefore, to clarify these issues by including a set of 52 strains, from diverse sources such as gelatine, foods, farm environment (including abortion-associated isolates), clinical environment, two strains of each cluster described by Ishihara and co-workers [22] and strains that have been shown to produce a toxin in a comparative study. Combined rep-PCR (REP-PCR, BOX-PCR and (GTG)5-PCR), rpoB and gyrA sequencing, 16S rDNA targeted probe analysis, DNA–DNA hybridizations, gelatinase tests and antibiotic susceptibility tests were performed.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Bacterial strains and growth conditions

Details of the origin and the affiliation of the strains tested are shown in Table 1. Unless stated otherwise, all strains were grown aerobically on Trypticase soy agar (BBL) at 37°C.

Table 1.  List of strains studied
  1. Abbreviations: DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany; DVL, Danish Veterinary Laboratory, Department of Pathology and Epidemiology, Copenhagen, Denmark; IFO, Institute of Fermentation, Osaka, Japan; LMG, BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie Gent, Ghent University, Belgium; NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, UK.

Strain numberOther designationSource (if known)Reference
LMG 6934DSM 394garden soil (Germany) 
LMG 7558ATCC 102, IFO 12195milk[22]
LMG 7559ATCC 9945flour[22]
LMG 7562ATCC 10716septic wound[22]
LMG 7633Bonde 126chinchilla feces 
LMG 12244ATCC 102milk 
LMG 12245Logan B0028  
LMG 12248Logan B0091  
LMG 12363TATCC 14580T [22]
LMG 17334DVL 8400227aborted bovine, placenta 
LMG 17335DVL 9315319aborted bovine fetus, lung 
LMG 17336DVL 9315323aborted bovine, placenta[17]
LMG 17337DVL 9315375aborted bovine fetus, lung 
LMG 17338DVL 9410348aborted bovine fetus, lung 
LMG 17339DVL 9410670-3silage 
LMG 17340DVL 9410670-4potato pulp for cattle feeding 
LMG 17341DVL 9410670-6molasses for cattle feeding 
LMG 17652Logan B0755clinical pathogen 
LMG 17655Logan B1077sheep, feces 
LMG 17657Logan B0451salt mash 
LMG 17658Logan B0698yoghurt 
LMG 17659ATCC 6598horse, blood stream 
LMG 17661Logan B0965cheese 
LMG 19409 cider (France) 
R-977 gelatine production chain (Belgium)[5]
R-1210 gelatine production chain (Belgium)[5]
R-6452B019milking installation 
R-6646B143animal feed 
R-6979B345raw milk 
R-7199B433raw milk 
R-7478B541animal feed 
R-13577 gelatine, final product (Belgium) 
R-13752 gelatine, final product (Belgium) 
R-16197 gelatine, final product (France) 
R-16201 gelatine, final product (France) 
R-16479 gelatine, final product (France) 
R-16510 gelatine, final product (France) 
R-18681 gelatine, final product (Argentina) 
R-18693 gelatine, final product (Argentina) 
R-18837 gelatine, final product (Argentina) 
R-20753F2896/95tandoori king prawn (UK)[8]
R-20756F231/97profiteroles (UK)[8]
R-20759553/2infant feed formula (Finland)[8]
R-20761575E/Pinfant feed formula, unused package (Finland)[8]
R-21381 aborted bovine (Belgium)[16]
R-21382 aborted bovine (Belgium)[16]
R-21383 aborted bovine (Belgium)[16]
R-21384 aborted bovine (Belgium)[16]
R-21385 aborted bovine (Belgium)[16]

2.2Repetitive element primed genomic fingerprinting

Template DNA was prepared using a slight modification of the method of Pitcher et al. [23], as previously described by Heyndrickx et al. [24]. The REP2I (5′-ICGICTTATCIGGCCTAC-3′), REP1R (5′-IIIICGICGICATCIGGC-3′), BOX A1R (5′-CTACGGCAAGGCGACGCTGACG-3′) and (GTG)5 (5′-GTGGTGGTGGTGGTG-3′) primers were used as previously described by Versalovic et al. [25]. PCR amplifications were performed as described before [25], using a DNA thermocycler (Perkin Elmer 9600) and Goldstar DNA polymerase (Eurogentec, Belgium). The PCR products were electrophoresed in a 1.5% agarose gel (15×20) for 16 h at 1.9 V cm−1 in 1×TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) at 4°C. The rep-PCR profiles were visualized after staining with ethidium bromide under ultraviolet light, followed by digital image capturing using a CCD camera. The resulting fingerprints were analyzed by the BioNumerics V3.0 software package (Applied Maths, Belgium). The digitized REP-, BOX- and (GTG)5-PCR patterns were linearly combined, assigning equal weights to each analysis. Similarities were calculated using Pearson correlation and an average linkage (UPGMA) dendrogram was obtained.

2.3Partial sequencing of rpoB and gyrA genes

An rpoB fragment corresponding to Bacillus subtilis rpoB numbering positions 6–585 was PCR-amplified using primers rpoB-f (5′-AGGTCAACTAGTTCAGTATGGAC-3′) and rpoB-r (5′-AAGAACCGTAACCGGCAACTT-3′). A gyrA fragment corresponding to B. subtilis gyrA numbering 43–1070 was PCR-amplified using primers gyrA-f (5′-CAGTCAGGAAATGCGTACGTCCTT-3′) and gyrA-r (5′-CAAGGTAATGCTCCAGGCATTGCT-3′). The reactions were carried out in a 50 μl reaction mixture, containing 20 pmol of each primer, 10 nmol of each dNTP, 5 μl 10×PCR buffer (Applied BioSystems), 1 U Taq polymerase (Applied Biosystems) and 50 ng template DNA. The PCR profile consisted of denaturation at 94°C for 2 min; 40 cycles of denaturation at 94°C for 30 s, annealing at 51°C for 45 s and extension at 68°C for 50 s (or 60 s for the gyrA gene) with a final extension at 68°C for 90 s (or 10 min for the gyrA gene). The resultant amplicons were purified using the NucleoFast® 96 PCR system (Millipore) and sequenced in both directions using the same primers. Sequencing was performed as described previously [26] using an ABI 3100 automated DNA sequencer. Phylogenetic analysis was performed using the BioNumerics V3.0 software package.

2.416S rDNA targeted probe analysis

A 5′ hypervariable region of the 16S rDNA, corresponding to B. subtilis 16S rDNA numbering positions 41–307, was amplified with primers RT-f (5′-GCGGCGTGCCTAATACATGC-3′) and RT-r (5′-CTCAGGTCGGCTACGCATCG-3′). A B. licheniformis-specific Taqman probe (5′-FAM-GAGCTTGCTCCCTTAGGTCAG-DabSyl-3′) was designed targeting a section of this region corresponding to B. subtilis 16S rDNA numbering positions 79–99. The Smartcycler (Cepheid) real-time PCR setting was used for a combined PCR probe analysis. The reaction mixture was prepared using the ‘Lightcycler-FastStart DNA Master Hybridization Probes’ kit, as described by Woo et al. [27]. The PCR profile consisted of an initial denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 15 s with optical devices off and annealing at 60°C for 1 min with optical devices on.

2.5DNA–DNA hybridizations

Large-scale DNA preparation was performed as described previously [28]. DNA–DNA hybridizations were performed with photobiotin-labelled probes in microplate wells as described by Ezaki et al. [29], using a HTS7000 Bio Assay Reader (Perkin Elmer) for the fluorescence measurements. The optimal renaturation temperature was determined according to the equation of De Ley et al. [30] as 37°C.

2.6Gelatinase tests

Gelatinase activity of the isolates was investigated by two different tests as described previously [5].

2.7Antibiotic susceptibility tests

All isolates were screened for their resistance against a selection of six antibiotics, i.e. tetracycline (30 μg), ampicillin (10 μg), bacitracin (10 μg), penicillin G (10 μg), erythromycin (10 μg) and streptomycin (25 μg), using a modification of the agar disk diffusion method [31]. A cell suspension was plated on Iso-sensitest agar (Oxoid). Antibiotic disks (Oxoid) were placed on the inoculated plates using an Oxoid Disc Dispenser. Following 24 h incubation at 37°C, inhibition zones around the disks were measured using digital calipers. Inhibition zones were compared with interpretation tables recommended for Gram-positive bacteria.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Repetitive element primed genomic fingerprinting

Based on the combined BOX-, (GTG)5 and REP-PCR profiles, two subgroups can be distinguished (Fig. 1). Subgroup A encompasses seven strains and subgroup B comprises 45 strains including the type strain LMG 12363T. The (GTG)5 profiles do not contribute to the group structure. Indeed, with some exceptions, (GTG)5 profiles consist of only a few bands (often five or fewer) and are homogeneous throughout the strain set. However, BOX- and REP-PCR profiles are more complex and clearly illustrate the grouping, each showing group-specific bands. Subgroup A shows more diversity, mainly in weaker bands, than subgroup B.


Figure 1. Clustering based on linearly combined BOX-, (GTG)5- and REP-PCR profiles of all B. licheniformis strains tested in this study. The grouping based on rpoB and gyrA sequences is also given.

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3.2Partial sequencing of rpoB and gyrA genes

Comparative studies of partial rpoB and gyrA sequences both demonstrate the same subgroups, A and B, found with BOX- and REP-PCR (Fig. 1).

In the rpoB sequence, subgroup A (among which LMG 7559, accession number AJ579781) and B (among which LMG 12363T, accession number AJ579782) differ from each other at 10 positions. Subgroup B shows a further subdivision into B1 (among which LMG 12363T) and B2 (among which LMG 17659, accession number AJ579783), differing in one position only. All nucleotide substitutions occur on the third codon position and are silent.

In the gyrA sequence, subgroup A (among which LMG 7559, accession number AJ579784) and B (among which LMG 12363T, accession number AJ579786) differ from each other at 28 positions. Subgroup A shows a subdivision into A1 (among which LMG 7559) and A2 (among which LMG 12248, accession number AJ579785), differing at 17 positions. Again, all nucleotide substitutions occur on the third codon position. All substitutions are silent, with the exception of one. At position 609, a substitution of T (A1 and B) by A (A2) results in a glutamate group (A2) instead of an aspartate group (A1 and B).

3.316S rDNA targeted probe analysis

When DNA of strains LMG 12363T, LMG 7558 and LMG 17334, all clustering within subgroup B based on BOX- and REP-PCR, rpoB and gyrA sequencing, was used as a template, a clear fluorescence signal was measured (Fig. 2). Strains LMG 6934 and LMG 7562, clustering within subgroup A based on the above-mentioned techniques, do not reveal such a strong signal. This lower signal may be due to one or more mismatches. These data suggest that also at the 16S rDNA level a distinction can be made between both subgroups.


Figure 2. Real-time PCR signal with 16S rDNA targeted probe of five B. licheniformis strains.

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3.4DNA–DNA hybridizations

A DNA–DNA reassociation value of 79% was found between LMG 6934, clustered within subgroup A, and LMG 12363T, clustered within subgroup B. According to the recommendations for species delineation [32,33], these strains belong to the same species.

3.5Gelatinase tests

All strains were shown to exhibit a gelatinase activity.

3.6Antibiotic susceptibility tests

All strains were found to be susceptible to tetracycline. A susceptibility range was found for the other antibiotics, but no link could be made with the subgroups found in BOX- and REP-PCR, rpoB and gyrA sequencing.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Rep-PCR has been shown to be a useful technique in the subtyping of Bacillus species [34,35]. Although in many cases a sufficiently detailed profile can be obtained by applying a single primer or primer pair, the use of combined profiles incorporates more fragments and thus a larger portion of the genome in the rep-PCR analysis. As a result, a more reliable grouping is revealed. In this study BOX-, (GTG)5- and REP-PCR profiles were combined to demonstrate intraspecies diversity.

In general, 16S rDNA sequences are used in Bacillus classification as a framework for species delineation [36]. On the other hand, protein-coding genes such as gyrA and rpoB exhibit much higher genetic variation and have been used for the classification of closely related taxa within the B. subtilis group [37,38]. We investigated the high discriminatory power of these genes to assess intraspecies diversity of B. licheniformis strains. Based on sequence analysis of both genes, the same grouping of strains as demonstrated by means of BOX- and REP-PCR was obtained. All rpoB substitutions are silent, while one gyrA substitution leads to a change from aspartate to glutamate. As these amino acids are homologues, a minor effect on the overall protein structure is expected.

We used the BioNumerics V3.0 software package (Applied Maths, Belgium) to illustrate the correspondence of the grouping between all techniques mentioned above by comparison of the individual similarity matrices (Table 2). The combined BOX-/(GTG)5-/REP-PCR clustering and a clustering only based on a combination of BOX- and REP-PCR are included. Table 2 also illustrates (GTG)5-PCR does not contribute (low congruence) to the group structure found with other techniques. The grouping was confirmed by a combined PCR probe analysis targeting the 16S rDNA, suggesting that group-specific signature sequences are also present in this gene. Notwithstanding the clear-cut subgroups, DNA–DNA reassociation values support the maintenance of these strains in a single species.

Table 2.  Congruence between the results of different techniques applied on all B. licheniformis strains tested in this study, expressed by percentage Pearson correlation between similarity matrices formulated for each technique

The subgroups found in this study are in agreement with the grouping reported by Ishihara et al. [22] based on bacitracin synthetase gene sequences and erythromycin resistance. However, we did not find that erythromycin resistance complies with this grouping. This could be due to differences in the methods used by the two research groups to test the antibiotic resistance or to a different strain selection. This observation demonstrates once again the importance of standardization of the methodologies testing phenotypic characteristics that are used in comparative studies. The DNA reassociation study presented here supports the classification of the strains in a single species, hence indicating that representatives of genomovars as suggested by Manachini et al. [21] were not included.

Subgroup B was found to be more than six times bigger than subgroup A. Remarkably, although all isolates were shown to exhibit a gelatinase activity, gelatine isolates all grouped in the largest cluster together with the type strain of the species. Food and environmental isolates, clinical pathogens, strains with abortifacient potential and toxin-producing strains are encountered in both subgroups. This is again a strong indication that the spread of important clinical characteristics is not necessarily linked to internal genotypic groupings of taxa. Acquisition of pathogenic characteristics may be the result of coincidental horizontal transfer that occurs independently of the genetic characteristics that are currently used in bacterial taxonomy.

In conclusion, we demonstrate the potential of BOX-PCR, REP-PCR, gyrA and rpoB sequencing and 16S rDNA targeted probe analysis to distinguish between two subgroups within B. licheniformis, reported earlier on the basis of bacitracin synthetase gene sequences. In contrast to previous studies, the large strain set we used was obtained from a variety of sources and contains strains known to exhibit pathogenic properties. Although B. licheniformis is of economic interest and clinically important representatives may form a threat for public health, we show that the subdivision of B. licheniformis does not comply with known expressed pathogenic capacities of the strains included. Therefore, a division into subspecies at this moment is of no practical use. B. licheniformis consists of at least two lineages that can be clearly distinguished genotypically, both containing strains with a pathogenic potential.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

E.D.C. was supported by a fellowship of the IWT (Institution for the Promotion of Innovation by Science and Technology in Flanders). P.D.V. is indebted to the FWO Vlaanderen for Research Grant G.0156.02. We thank Kurt Van Mol and Stefanie Hubeau for excellent technical assistance and Jef Hommez, Patsy Scheldeman and Mirja Salkinoja-Salonen for providing strains. We thank Rousselot NV for their collaboration.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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