67 (2006) 64–69www.elsevier.com/locate/jmicmeth

Journal of Microbiological Methods

AFLP fingerprinting of Colombian Clostridium spp strains,multivariate data analysis and its taxonomical implications

Claudia P. Jaimes a, Fabio A. Aristizábal G. b, Mauricio Bernal M. a,Zulma R. Suárez a, Dolly Montoya a,⁎

a Instituto de Biotecnología, Universidad Nacional de Colombia A.A. 14490, Colombiab Departamento de Farmacia, Universidad Nacional de Colombia A.A. 14490, Colombia

Received 27 July 2005; received in revised form 26 December 2005; accepted 2 March 2006Available online 19 April 2006

Abstract

Amplified Fragment Length Polymorphism (AFLP) analysis was used for characterising 13 native Colombian Clostridium sppstrains. The DNA extraction method was optimised and the use of cetyl trimethyl ammonium bromide (CTAB) and sodiumchloride (NaCl) was incorporated. All strains could be typed in these conditions. The AFLP profiles obtained were submitted tomultivariate analysis and compared with previous pulsed field gel electrophoresis (PFGE) results. The results suggested that the setof native strains could correspond to two new species different to those having been described to date. It is proposed that DNA–DNA hybridisation analysis should be done to produce complementary information for describing the new species.© 2006 Elsevier B.V. All rights reserved.

Keywords: AFLP; PFGE; Bioprospecting; Fingerprinting; Taxonomic classification; Biodiversity

1. Introduction

Knowledge of prokaryote species' biological diver-sity remains incomplete in terms of their richness, overalldistribution and function within Colombian ecosystems.Bioprospecting strategies have thus been implemented

Abbreviations: AFLP, amplified fragment length polymorphism;PCR, polymerase chain reaction; OD, optical density; PFGE, pulsedfield gel electrophoresis; CTAB, cetyl trimethyl ammonium bromide;DNA, deoxyribonucleic acid; NaCl, sodium chloride.⁎ Corresponding author. Tel.: +57 1 3165000 16954; fax: +57 1

3165415.E-mail addresses: cpjaimes@uniboyaca.edu.co (C.P. Jaimes),

faaristizabalg@unal.edu.co (F.A. Aristizábal G.),jmbernalmo@unal.edu.co (M. Bernal M.), zrsuarezm@unal.edu.co(Z.R. Suárez), dmontoyac@unal.edu.co (D. Montoya).

URL: http://www.ibun.unal.edu.co (D. Montoya).

0167-7012/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.mimet.2006.03.002

for exploring bacterial biodiversity, accompanied bymolecular techniques for identifying promising micro-organisms and their taxonomic classification.

Amplified fragment length polymorphism (AFLP)represents one of the molecular characterisation techni-ques used in taxonomic classification, based ondetecting DNA restriction fragments for polymerasechain reaction (PCR) amplification (Zabeau and Vos,1993). AFLP has been applied to studying differentbacterial genera for both taxonomic ends and epidemi-ological typing, showing it to be a useful molecular toolbecause it allows genera, species and even strains to bedifferentiated (Blears et al., 1998; Gaafar et al., 2003;Moreno et al., 2003; Rademaker et al., 2003;Vos et al.,1995; Savelkoul et al., 1999). AFLP fingerprintingstudies have been done recently on Clostridium novyi,Clostridium perfringens, Clostridium botulinum and

65C.P. Jaimes et al. / Journal of Microbiological Methods 67 (2006) 64–69

Clostridium difficile (McLauchlin et al., 2002; Engströmet al., 2003; Van den Berg et al., 2004; Keto-Timonen etal., 2005).

178 strains have been isolated from soil from differentareas of Colombia in the search for solvent-producingmicroorganisms as part of the process of bioprospectingcarried out in the country during the 1990s. Lateranalysis led to it being established that 13 of theseisolated strains exhibited higher total solvent productionthan the Clostridium acetobutylicum ATCC 824 refer-ence strain, using glucose as carbon source (Montoya etal., 2000b). Promising strains were characterised byPFGE fingerprinting (Montoya et al., 2001) andanalysing the 16S rRNA gene sequence (Montoya etal., 2000a). The results suggested that native strains werehomogeneous and that they were closely related to theClostridium butyricum species. TheHindIII enzyme wasselected in preliminary AFLP genotyping assays butonly 7 strains could be typed due to the quality of theDNA and the high endonuclease activity (data noshown). This work presents the results of AFLP fin-gerprinting for the 13 native strains, after DNA ex-traction methods had been optimised and multivariateanalysis for establishing taxonomic relationships.

2. Materials and methods

2.1. Bacterial strains and culture conditions

Strains IBUN 13A, IBUN 18A, IBUN 18Q, IBUN18S, IBUN 22A, IBUN 62B, IBUN 62F, IBUN 64A,IBUN 95B, IBUN 125C, IBUN 137K, IBUN 140B,IBUN 158B were isolated from Colombian soils asdescribed previously (Montoya et al., 2000b). Referencestrains C. acetobutylicum ATCC 824, C. butyricumDSM 2478, C. kainantoi DSM 523, C. beijerinckii werekindly provided by Wolfgang Schwarz from TechnicalUniversity of Munich. Reference strain C. saccharo-perbutylacetonicum N1-4 was kindly provided by D.JJones. (University of Otago, New Zealand).

Conserving the microorganisms, activating thestrains, anaerobic growth conditions and determiningcellular biomass were carried out following the metho-dology described by Montoya et al., 2000b.

2.2. DNA extraction

A preinoculum was prepared by adding 1.5 ml ofpreviously activated culture to 40 ml RCM medium inanaerobic conditions and incubated overnight at 37 °C.7.5 ml of the preinoculum were transferred to flaskscontaining 100 ml RCM medium. It was grown at 37 °C

until 0.3–0.5 OD and placed in an ice bath for 30 to45 min. The cells were harvested by spinning at5000 rpm for 10 min. 500 μl of the pellet were centri-fuged at 12,000 rpm for 5 min and suspended in 567 μlTE buffer (10 mM Tris HCl, 1 mM EDTA). This wasmixed by repeated pipetting using tips which had beencut shorter to widen them. SDS was added at final 0.5%concentration and proteinase K at final 100 μg/ml con-centration. The mixture was incubated in a water bath at37 °C for 1 h; 100 μl NaCl 5 M and 80 μl CTAB/NaClsolution were then added and completely mixed. Afteradding an equal volume of 24:1 chloroform:isoamylalcohol mixture, it was incubated at 65 °C for 10 minand mixed for inversion until the two phases formed ahomogeneous emulsion. It was centrifuged at12,000 rpm for 10 min to remove the complex formedbetween CTAB-proteins and polysaccharides. Chloro-form-isoamyl alcohol extraction was repeated threetimes until the aqueous phase became translucent. DNAwas extracted from the aqueous phase with an equalvolume of phenol/chloroform/isoamyl alcohol solution(25:24:1), mixed and centrifuged at 12,000 rpm for10 min. DNase-free RNAse was added to the superna-tant at final 20 μg/μl concentration and incubated at37 °C for 30 min. The DNA was precipitated with twovolumes of isopropanol and then recovered for centri-fuging at 12,000 rpm for 20 min at 4 °C. The DNA pelletthus obtained was then washed with a volume of pre-chilled absolute ethanol and centrifuged at 12,000 rpmfor 10 min. This step was repeated 3 times with pre-chilled 70% ethanol. The supernatant was skimmed offand the pellet was dried at 37 °C for 1 h. The pellet wassuspended in 100 μl TE buffer and stored at −20 °C.DNA quality was verified by electrophoresis on 0.8%agarose gels.

2.3. AFLP fingerprinting

One μg DNAwas digested with 10 U HindIII enzyme(Promega) in a final volume of 20 μl at 37 °C for 12 h.20 μl of the digested DNAwere transferred to a new tubecontaining 25 pmoles of adapters (5′ CTCGTA-GACTGCGTACC 3′; 3′ CTGACGCATGGTCGA 5′)(Janssen et al., 1996) and 1 U DNAT4 Ligase, (Promega)in a final volume of 30 μl at 4 °C for 12 h. Enzymes wereinactivated at 65 °C for 20min after the digestion–ligationreaction had been finalised (Clerc et al., 1998). 50 ngdigested–ligated DNA was amplified by PCR in 25 μlreaction volume with HindIII primer (5′ GACTGCG-TACCAGCTT 3′ with an additional C at the 3′ extremeacting as the basis for selection) at 0.2 μM concentration,with 0.1 mM dNTPs, 1.25 U Taq polymerase (Promega).

66 C.P. Jaimes et al. / Journal of Microbiological Methods 67 (2006) 64–69

The PCR programme consisted of: 94 °C 150 s; 94 °C30 s, 60 °C 60 s, 72 °C 150 s (30 cycles); 72 °C 4 min.AFLP products were separated on 2% agarose gels at 3 V/cmwith 0.5× TBE and visualised by staining with 5 ng/μlethidium bromide. The gels were digitalised in Gel Doctransilluminator (BioRad®) and the bands and their sizeswere calculated by using Quantity One software. Thetechnique's reproducibility was evaluated with assays onDNA extracted during different sessions, on differentdays.

2.4. Statistical analysis

Once amplified DNA fragment sizes had beenestablished, they were ordered in terms of decreasingsize. Each band consisted of a categorical variablewhich might have been present or absent for construct-ing a matrix of zeros and ones. Bands which did notpresent variability between strains were excluded.SPAD MN 5.6.1 software (Decisia, France) was usedfor analysing this matrix's by means of multiplecorrespondence analysis (MCA). Strains' factorial coor-dinates were calculated by using this software andvisualised on a factorial plane. Hierarchical cluster ana-lysis was done by means of RECIP/SEMIS application(i.e. trees were drawn). Ward's aggregation criterion wasused for minimising intra-classes variance and maxi-mise variance between classes.

The information provided by AFLP fingerprinting wascomplemented by PFGE results already published

Fig. 1. Banding profiles obtained by standardised AFLP technique for Colowith HaeIII mixed with pUC18 digested with HindIII (sizes indicated on theDSM 523 (Lane 6), C. acetobutylicumATCC 824 (Lanes 7,11), Escherichia cstrains IBUN 22A (Lane 1), IBUN 18S (Lane 2), IBUN 62B (Lane 3), IBUN 6(Lane 9), IBUN 158B (Lane 10), IBUN 95B (Lane 12), IBUN 62F (Lanes 1317). Note that banding profiles obtained for reference strain Clostridium acetColombian strains.

(Montoya et al., 2001). Data from both types of analysiswere combined in a single matrix and the previousanalysis repeated. This was done because it was consi-dered that strains would be better characterised if thegreatest amount of variables describing them wereincluded in such analysis (Diaz, 2002; Franco et al., 2001).

TheDiscrimination index (DI), the probability that twounrelated strains would be placed into different typinggroups, was calculated by Simpson index of diversity(Hunter and Gaston, 1988).

3. Results

3.1. Importance of DNA purification

The DNA extraction's proceedure described bySambroock et al. (1989) was initially used. However,the DNA obtained for the strains being studied were notreproducibly digested by enzymes. CTAB/NaCl wasthus assayed for cellular lysis for obtaining better DNApurification. This procedure has been described for ge-nomic DNA extraction from plants (Ausubel et al.,1994) and previously reported for Pseudomonas sy-ringae (Clerc et al., 1998). DNA from native and patternstrains showed total restriction when CTAB was used.This fact is probably associated with polysaccharide andproteins forming complexes with CTAB which becomedetached during extraction with chloroform in DNAextraction allowing best purification (Ausubel et al.,1994). The modified CTAB method presented

mbian native strains. Lanes M are Molecular Marker: ϕX174 digestedleft). Reference strains are located as follows: C. butyricum (kainantoi)oli pUC18 (Lane 18). The remaining lanes show AFLP of the following4 A (Lane 4), IBUN 140B (Lane 5), IBUN 18 A (Lane 8), IBUN 140B,14), IBUN 125C (Lane 15), IBUN 18Q (Lane 16), IBUN 18 A (Laneobutylicum (Lanes 7,11) were clearly different from those obtained for

67C.P. Jaimes et al. / Journal of Microbiological Methods 67 (2006) 64–69

advantages over other methods tested in our work. Itreduced final proteinase K concentration from 1 mg/mlto 100 μg/ml and its incubation time from 2 h to 45 min.The standardised protocol did not require lysozyme,thus reducing costs and shortening extraction time.

3.2. AFLP fingerprinting of Colombian native strains

The standardised methodology was applied to thestrains being studied and AFLP patterns obtained for12–15 DNA bands, ranging in size from 300 to 2000 bp(Fig. 1). A value of 1.0 was calculated for the power ofdiscrimination when applying Hunter and Gaston'sformula (1988). The banding profiles were reproduciblewhen the assays were repeated with DNA extracted bythe method on different days.

3.3. Clustering analysis

AFLP and PFGE data were analysed apart and thenput taken together in a single matrix. 100 bands corre-sponding to restriction fragments produced by ApaI andSmaI enzymes by means of PFGE and 53 electropho-retic bands obtained by AFLP were analysed.

Hierarchical Cluster Analysis for AFLP fingerprint-ing showed that native strains were separately clustered

Fig. 2. Hierarchical Cluster Analysis tree based on multiple correspondence abetween Colombian (IBUN strains) and reference strains. SPAD 5.6.1 softwfrom both techniques by means of multiple correspondence analysis (MCAapplication. The cut-off line produces 4 groups; Note that eleven Colombianincluded in this study.

from that of solventogenic clostridia pattern strains in-cluded in the analysis. When analysing the AFLP–PFGE matrix set of data 147 bands (variables) wereincluded and those which did not represent variabilityfor the analysis were automatically eliminated. 4 patternstrains shared by the two techniques were taken. Theresults from the jointed analysis (Fig. 2) showed thatwhen a greater number of variables were used, it gave acomprehensive strains description. The groupingobtained showed the robustness of the individual analy-sis and confirmed that the native strains constitute agroup which is separated from the pattern strains.

4. Discussion

4.1. DNA purification

DNA extraction in species from theClostridium genuspresents difficulties due to the anaerobic requirements asnuclease activity becomes increased on coming into con-tact with oxygen in the atmosphere, leading to rapidautolysis. Purification becomes more difficult during thelate growth phase as there ismoreDNAse activity becausethere are a greater proportion of proteins respecting DNA.Itmay be true that the quality ofDNA isolatedwhen usinga 0.3 OD becomes improved, but there is a low yield of

nalysis for AFLP–PFGE profiles showing the taxonomic relationshipsare was used for analysing a jointed matrix's containing data obtained). Hierarchical cluster analysis was done by means of RECIP/SEMISNative strains (IBUN) are separately clustered from reference strains

68 C.P. Jaimes et al. / Journal of Microbiological Methods 67 (2006) 64–69

extracted DNA. The process becomes less efficient, ac-companied by greater consumption of reagents (unpub-lished data) and thus a 0.5 optical density was selected. Ithas been suggested that ProteinaseK resolves the problemof nucleases by degrading proteins; however, the successof isolating DNA from Clostridium in this work couldhave been associated with two factors. The simultaneoususe of SDS and Proteinase K has been described as beinga combinationwhich reinforces proteolysis (Ebeling et al.,1974).Also, whenCTAB is used in concentrations greaterthan 0.5 M NaCl, it forms protein and polysaccharideprecipitation complexes, thereby improving nucleic acidpurification (Ausubel et al., 1994).

4.2. Obtaining AFLP

An original protocol (Janssen et al., 1996), based onusing a single enzyme, a single adaptor, just one primerand electrophoretic analysis on agarose gels, wasmodified for use on this study. Suitable banding profilesfor the taxonomic analysis of strains were obtained inthe conditions used here. A good banding pattern wasobtained when using the original method and digestionwith HindIII (Fig. 1).

4.3. Analysing AFLP–PFGE grouping

The cut-off line on the trees based on the level index(for establishing the statistically most representativegroups) formed 5 groups for strains analysed by AFLPand 4 groups for strains analysed by PFGE, when thesame strains were included in each analysis (Data notshown).Similar trees were obtained for the twotechniques, and a native strains group was clearlyseparated from reference strains included in both cases.However an additional branch including strains IBUN62F and IBUN18Q was observed in the AFLP tree. Thissuggests the consistency of the groupings obtained.

Combining the two sets of data (AFLP and PFGEmatrices) clearly showed that 11 of the native strainsformed a separate group from that of the pattern strainsincluded in this study in both the MCA (multiplecorrespondence analysis) and the tree obtained (Fig. 2)and that the remaining strains seemed to be very remotefrom other native strains. IBUN 62F and IBUN 18Qwere similar to pattern strain C. butyricum, a species towhich native strains have been shown to be more closelyrelated, according to previous 16S rRNA results(Montoya et al., 2001; Klaassen et al., 2002).

These results suggest that the set of native strainscould correspond to two new species: group 1 (IBUN18Q and IBUN 62F strains) and group 2 (IBUN 13A,

IBUN 18A, IBUN 18S, IBUN 62B, IBUN 64A,IBUN 95B, IBUN 125C, IBUN 22 A, IBUN 158B,IBUN 137K and IBUN 140B). A DNA–DNAhybridisation analysis is needed for complementingthe results presented in this work as it has beendefined as being an essential test when describing newspecies (Stackerbrandt and Goebel, 1994).

Acknowledgements

This work was carried out within the framework ofthe project entitled, “Anaerobic solvent producingbacteria: biomass fermentation and isolation of cellulo-lytic strains,” financed by COLCIENCIAS (the InstitutoColombiano Francisco José de Caldas) and theUniversidad Nacional de Colombia. We would like tothank Bernardo Chávez Córdoba for his help inanalysing the statistical data, and Jason Garry forpatiently reading the manuscript.

References

Ausubel, F., Brent, R., Kingston, R., Seidman, J., Smith, J., Struhl, K.,1994. Current Protocols in Molecular Biology. Current ProtocolsEd John Wiley and sons inc., Suplement 27, Vol I Cap IIpag. 2.4.1–2.4.2.

Blears, M.J., De Grandis, S.A., Lee, H., Trevors, J.T., 1998. Amplifiedfragment length polymorphism (AFLP): a review of the procedureand its applications. J. Ind. Microbiol. Biotech. 21, 99–114.

Clerc, A., Manceau, C., Nesme, X., 1998. Comparison of randomlyamplified polymorphic DNA with amplified fragment lengthpolymorphism to assess genetic diversity and genetic relatednesswithin genospecies III of Pseudomonas syringae. Appl. Env.Microbiol. 64, 1180–1187.

Diaz, L.G., 2002. Estadística multivariada: inferencia y metodos,Editorial Panamericana Formas e Impresos. 1st edition. EditorialPanamericana Formas e Impresos S.A. Bogotá, Colombia.

Ebeling,W., Hennrich, N., Klockow,M., Metz, H., Orth, H.D., Lang, H.,1974. Proteinase K from Tritirachium album Limber. Proteinase K.Eur. J. Biochem. 15, 91–97.

Engström, B.E., Fermèr, C., Lindberg, A., Saarinen, E., Bâverud, V.,Gunnarsson, A., 2003. Molecular typing of isolates of Clostridiumperfringens from healthy and diseased poultry. Vet. Microbiol. 94,225–235.

Franco, J., Crossa, F., Ribaut, J.M., Beltrán, J., Warburton, M.L.,Khairallah, M., 2001. A method for combining molecular markersand phenotypic attributes for classifying plant genotypes. Theor.Appl. Genet. 103, 944–952.

Gaafar, A., Unzaga, M.J., Cisterna, R., Clavo, F.E., Urra, E., Ayarza,R., Martín, G., 2003. Evaluation of a modified single-enzymeamplified-fragment length polymorphism technique for finger-printing and differentiating of Mycobacterium kansasii Type Iisolates. J. Clin. Microbiol. 41, 3846–3850.

Hunter, P., Gaston, M., 1988. Numerical index of discriminatoryability of typing systems: an application of Simpson index ofdiversity. J. Clin. Microbiol. 26, 2465–2466.

Janssen, P., Coopman, R., Huys, G., Swings, M., Bleeker, M., Vos, P.,Zabeau, M., Kersters, K., 1996. Evaluation of the DNA fingerprinting

69C.P. Jaimes et al. / Journal of Microbiological Methods 67 (2006) 64–69

method AFLP as a new tool in bacterial taxonomy. Microbiology 142,1881–1893.

Keto-Timonen, R., Nevas, M., Korkeala, H., 2005. Efficient DNAfingerprinting of Clostridium botulinum Types A, B, E, and F byamplified fragment length polymorphism analysis. Appl. Environ.Microbiol. 71, 1148–1154.

Klaassen, C.H., Van Haren, H.A., Horrevorts, A.M., 2002. Molecularfingerprinting of Clostridium difficile isolates: pulsed-field gelelectrophoresis versus amplified fragment length polymorphism.J. Clin. Microbiol. 40, 101–104.

McLauchlin, J., Salmon, J.E., Ahmed, S., Brazier, J.S., Brett, M.M.,George, R.C., Hoodk, J., 2002. Amplified fragment lengthpolymorphism (AFLP) analysis of Clostridium novyi, C. perfrin-gens and Bacillus cereus isolated from injecting drug users during2000. J. Med. Microbiol. 51, 990–1000.

Montoya, D., Perdomo, L., Arévalo, C., Aristizábal, F., Schwarz, W.,2000a. Caracterización de cepas nativas de Clostridium spp porsequenceción parcial del gen Ribosomal 16SrRNA. Rev. Colombi-ana Biotecnol. 2, 35–39.

Montoya, D., Spitia, S., Silva, E., Schwarz, W.H., 2000b. Isolation ofmesophilic solvent-producing clostridia from Colombian sources:physiological characterisation, solvent production and polysac-charide hydrolysis. J. Biotechnol. 79, 117–126.

Montoya, D., Arévalo, C., Aristizábal, F., Schwarz, W., 2001. Newsolvent-producing Clostridium spp. strains, hydrolysing a widerange of polysaccharides, are closely related to Clostridiumbutyricum. J. Ind. Microbiol. Biotech. 27, 329–335.

Moreno, A.M., Baccaro, M.R., Ferreira, A.J.P., Pestana de Castro,A.F., 2003. Use of single-enzyme amplified fragment lengthpolymorphism for typing Pasteurella multocida subsp. multocidaisolates from pigs. J. Clin. Microbiol. 41, 1743–1746.

Rademaker, J.L., Hoste, B., Kersters, K., Louws, F.J., Swings, J.,Vauterin, L., Vauterin, P., de Bruijn, F.J., 2003. Comparison ofAFLP and rep-PCR genomic fingerprinting with DNA–DNAhomology studies: Xanthomonas as a model system. Int. J. Syst.Bacteriol. 50, 665–677.

Sambroock, J., Frisch, E.F., Maniatis, T., 1989. Molecular Cloning: aLaboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press,Cold Harbor NY.

Savelkoul, P.H., Aarts, H.J., De Haas, J., Dijkshoorn, L., Duim, B.,Otsen, M., Rademaker, J.L., Schouls, L., Lenstra, J.A., 1999. Ampli-fied-fragment length polymorphism analysis: the state of an art.J. Clin. Microbiol. 37, 3083–3091.

Stackerbrandt, E., Goebel, B.M., 1994. Taxonomic note: a place forDNA–DNA reassociation and 16S rRNA sequence analysis in thepresent species definition in bacteriology. Int. J. Syst. Bacteriol. 44,846–849.

Van den Berg, R.J., Claas, E.C., Oyib, D.H., Klaassen, C.H.,Dijkshoorn, L., Brazier, J.S., Kuijper, E.J., 2004. Characterisationof toxin A-negative, toxin B-positive Clostridium difficile isolatesfrom outbreaks in different countries by amplified fragment lengthpolymorphism and PCR ribotyping. J. Clin. Microbiol. 42,1035–1041.

Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van der Lee, T., Hornes,M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., Zabeau, M., 1995.AFLP: a new technique for DNA fingerprinting. Nucleic AcidsRes. 23, 4407–4424.

Zabeau, M. and Vos, P., 1993. Selective restriction fragment ampli-fication: a general method for DNA fingerprinting. EuropeanPatent Application Number: 9240269.7, Publication Number053485.