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J Clin Microbiol. Nov 2006; 44(11): 4057–4065.
Published online Sep 13, 2006. doi:  10.1128/JCM.01275-06
PMCID: PMC1698353

Identification of Clostridium Species and DNA Fingerprinting of Clostridium perfringens by Amplified Fragment Length Polymorphism Analysis[down-pointing small open triangle]

Abstract

An amplified fragment length polymorphism (AFLP) method was applied to 129 strains representing 24 different Clostridium species, with special emphasis on pathogenic clostridia of medical or veterinary interest, to assess the potential of AFLP for identification of clostridia. In addition, the ability of the same AFLP protocol to type clostridia at the strain level was assessed by focusing on Clostridium perfringens strains. All strains were typeable by AFLP, so the method seemed to overcome the problem of extracellular DNase production. AFLP differentiated all Clostridium species tested, except for Clostridium ramosum and Clostridium limosum, which clustered together with a 45% similarity level. Other Clostridium species were divided into species-specific clusters or occupied separate positions. Wide genetic diversity was observed among Clostridium botulinum strains, which were divided into seven species-specific clusters. The same AFLP protocol was also suitable for typing C. perfringens at the strain level. A total of 29 different AFLP types were identified for 37 strains of C. perfringens; strains initially originating from the same isolate showed identical fingerprinting patterns and were distinguished from unrelated strains. AFLP proved to be a highly reproducible, easy-to-perform, and relatively fast method which enables high throughput of samples and can serve in the generation of identification libraries. These results indicate that the AFLP method provides a promising tool for the identification and characterization of Clostridium species.

The genus Clostridium is a heterogeneous group of bacteria which currently consists of 181 described species. Clostridia are widely distributed in the environment as well as in the intestinal tract of humans and of many animals. Several Clostridium species are pathogenic to humans, domestic animals, or wildlife and are responsible for well-known clostridial diseases such as tetanus, gas gangrene, botulism, pseudomembranous colitis, and food-borne illness (10). In addition, clostridia can be involved in a variety of human infections, such as cholecystitis, pneumonia, bacteremia, empyema, and abscesses, and can thus be isolated from various clinical specimens. However, many of the isolates can be occasional contaminants, or nonpathogenic clostridia, and may not be involved in the disease process. Therefore, the reliable identification of clostridia, isolated from clinical specimens, is important. In addition, a link must be established between isolated clostridia and pathological changes.

Despite the clinical significance of clostridia, reliable, practical, and fast identification methods are few. Although simple tests can serve to identify most commonly isolated Clostridium species, the identification of other clostridia by conventional biochemical testing and gas-liquid chromatography is still laborious, expensive, and time-consuming. Furthermore, several commercial identification systems for anaerobic bacteria have failed to accurately identify Clostridium species (23, 29, 30, 43). Due to these evident drawbacks of conventional methods, there is a growing trend toward molecular diagnostics of bacteria that are difficult to identify by phenotypic characters (28, 42).

Amplified fragment length polymorphism (AFLP) is a DNA fingerprinting method based on the digestion of total DNA with two restriction enzymes, followed by the ligation of restriction site-specific adapters and the amplification of a subset of fragments by PCR (46). The AFLP approach, using a fluorescently labeled primer and detection of the fragments with an automated DNA sequencer, allows the use of internal size markers for accurate band sizing as well as partial automation and straightforward computer-assisted analysis, which make AFLP advantageous for the generation of extensive fingerprinting databases.

AFLP has proven to be a useful tool in epidemiological and outbreak studies (1, 5, 9, 20). In addition to strain typing, AFLP has been utilized in taxonomic studies. AFLP has been used to differentiate Aeromonas (16), Acinetobacter (19), Arcobacter (36), avian mycoplasma (13), Burkholderia (3), Campylobacter (7, 35), and Xanthomonas (18), and the results appear to agree closely with results obtained by DNA-DNA hybridization experiments (2, 3, 16, 38).

AFLP has been used to genotype Clostridium botulinum (21), Clostridium difficile (24), Clostridium novyi (32), and Clostridium perfringens (31), but the ability of AFLP analysis in differentiating Clostridium species remains unevaluated. Therefore, we applied an AFLP protocol previously used to characterize C. botulinum on 24 different Clostridium species, with special emphasis on pathogenic clostridia of medical or veterinary interest, to examine whether AFLP is a suitable tool for the identification of clostridia. In addition, we assessed the ability of the same AFLP protocol to type clostridia at the strain level by focusing on C. perfringens strains of all five toxin types.

MATERIALS AND METHODS

Bacterial strains.

A total of 129 strains of 24 different Clostridium species from the Culture Collections of the Department of Food and Environmental Hygiene, University of Helsinki, Finland; the Department of Medical Microbiology, University of Turku, Finland; the Institute of Food Research, Norwich, United Kingdom; and the Finnish Food Safety Authority, Kuopio Research Unit, Kuopio, Finland, were studied (Table (Table1).1). Strains consisted of type strains (n = 18) and strains originating from diverse sources and locations, including clinical, environmental, and food samples. Three C. perfringens isolates originated from the same outbreak and three of the C. perfringens NCTC reference strains (NCTC 3180, NCTC 8237, and NCTC 8346) were also obtained from another culture collection with different strain codes (CCUG 2036, ATCC 13124, and CCUG 2037, respectively).

TABLE 1.
List of Clostridium strains used in the study

DNA extraction.

Total DNA was extracted as previously described by Hyytiä et al. (17), with slight modifications. Strains were grown in a tryptose-peptone-glucose-yeast medium (Difco Laboratories, Detroit, MI) under anaerobic conditions at the optimal growth temperature for each strain for 14 to 16 h. The cells were resuspended with 400 μl TE (10 mM Tris-HCl, 1 mM EDTA) and incubated with 7.9 mg/ml lysozyme (Sigma, St. Louis, MO), 159 IU/ml mutanolysin (Sigma), and 476 μg/ml RNase (Sigma) at 37°C with gentle shaking for 15 min (C. botulinum group I), 2 h (C. botulinum group II), or 1 h (other Clostridium species). Lysis was completed by adding 52 μg/ml proteinase K (Finnzymes, Espoo, Finland), 0.23 M NaCl, 9.1 mM EDTA, and 0.8% (vol/vol) sodium dodecyl sulfate. After thorough mixing, the mixture was incubated at 60°C for 1 h with gentle shaking. Phenol-chloroform-isoamyl alcohol (25:24:1 [vol/vol]) and chloroform-2-pentanol (24:1 [vol/vol]) extractions were performed, and the DNA was precipitated with ethanol (95% [vol/vol]), rinsed with 70% ethanol, and resuspended with 100 μl sterile, distilled, deionized water; DNA was stored at −70°C. DNA concentrations were determined using a BioPhotometer (Eppendorf, Hamburg, Germany).

AFLP analysis.

An AFLP protocol previously used to characterize C. botulinum strains (21) was used with some modifications. Total DNA (400 ng) was digested with 15 U HindIII (New England Biolabs, Beverly, MA) and 15 U HpyCH4IV (New England Biolabs) in 1× One-Phor-All buffer plus (Amersham Biosciences, Buckinghamshire, United Kingdom), 5 mM dithiothreitol, and 0.1 mg/ml bovine serum albumin. Restriction site-specific HindIII adapter (0.04 μM; Oligomer, Helsinki, Finland) and HpyCH4IV adapter (0.4 μM; Oligomer) (Table (Table2)2) were ligated with 1.1 U T4 DNA ligase (New England Biolabs) in 1× One-Phor-All buffer plus (Amersham Biosciences), 5 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, and 200 μM ATP. Samples were stored at −20°C prior to PCR amplification.

TABLE 2.
Adapter and primer oligonucleotides used in AFLP

The digested and ligated DNA samples were diluted with sterile, distilled, deionized water (1:2) and amplified by preselective PCR (72°C for 2 min; 20 cycles of 94°C for 20 s, 56°C for 2 min, and 72°C for 2 min) in a 20-μl reaction mixture containing 4 μl of diluted template DNA, 15 μl amplification core mix (Applied Biosystems, Foster City, CA), 25 nM Hind-0 primer (Oligomer), and 125 nM Hpy-0 primer (Oligomer) (Table (Table2).2). After preselective amplification, the templates were diluted with sterile, distilled, deionized water 1:20. Selective amplification was performed in a 10-μl reaction mixture containing 1.5 μl of diluted template, 50 nM 6-carboxyfluorescein (FAM)-labeled Hind-C primer (Oligomer), 250 nM Hpy-A primer (Oligomer) (Table (Table2),2), and 7.5 μl amplification core mix (Applied Biosystems) (94°C for 2 min; 1 cycle of 94°C for 20 s, 66°C for 30 s, 72°C for 2 min; then the annealing temperature was lowered by 1°C each cycle to 56°C [10 cycles], followed by an additional 19 cycles at a 56°C annealing temperature and a final 30-min extension at 60°C). All PCR amplifications were performed with a PTC-200 Peltier thermal cycler (MJ Research, Inc., Waltham, MA).

Selective amplification products (1 μl) were mixed with 0.5 μl of internal size standard (GS-500 LIZ; Applied Biosystems) to enable accurate band sizing and 11.5 μl Hi-Di formamide (Applied Biosystems) and denatured at 95°C for 2 min. Denatured fragments were electrophoresed on POP-4 polymer (Applied Biosystems) on an ABI PRISM 310 genetic analyzer (Applied Biosystems) in 1× genetic analyzer buffer with EDTA (Applied Biosystems). The electrophoresis conditions were 15 kV at 60°C for 28 min. Data preprocessing was performed using GeneScan 3.7 fragment analysis software (Applied Biosystems).

Reproducibility testing.

Reproducibility of the method was determined by performing duplicate (n = 17), triplicate (n = 5), fivefold (n = 2), or sixfold (n = 1) experiments, including DNA extraction, AFLP analysis, electrophoresis, and numerical data analysis, with a total of 25 strains representing different Clostridium species (Table (Table1).1). In addition, reproducibility among different data sets was assessed using C. botulinum type E strain K-51 as an internal reference, which underwent each step of the DNA extraction and AFLP analysis a total of 11 times, thereby providing a standard for comparison among different data sets.

AFLP pattern analysis.

AFLP patterns were analyzed using BioNumerics software, version 4.5 (Applied Maths, Kortrijk, Belgium), and similarity between normalized AFLP patterns (range 75 to 450 bp) was calculated with the Pearson product-moment correlation coefficient. Clustering and construction of dendrograms were performed by using the unweighted pair-group method with arithmetic averages.

RESULTS

All clostridial strains studied were typeable by AFLP. The enzyme and primer combination used in AFLP analysis generated evenly distributed banding patterns in the range of 75 to 450 bp. Based on the cluster analysis, the 45% similarity level served to differentiate Clostridium strains at the species level (Fig. (Fig.1).1). By this criterion, AFLP analysis divided Clostridium strains into 21 clusters (I to XXI); 20 of the clusters consisted of strains belonging to a single species. In addition, eight strains, which were the only representatives of the particular species studied, occupied separate positions. AFLP failed, however, to discriminate between Clostridium ramosum and Clostridium limosum, which clustered together with a 45% similarity level (cluster XIV).

FIG. 1.
Reconstructed AFLP banding patterns and a dendrogram of 24 Clostridium species based on AFLP analysis. The cluster containing 37 C. perfringens strains is shaded. A similarity analysis was performed using the Pearson product-moment correlation coefficient, ...

In reproducibility testing among different data sets, the internal reference C. botulinum strain K-51 showed 93% or higher similarity due to small variations in peak heights. However, the resulting AFLP banding patterns, measured based on fragment sizes, remained constant during each separate run. In addition, after independent repeated experiments in the 25 different Clostridium strains tested, none of the AFLP banding profiles changed. The 93% cutoff value served to define the AFLP type of C. perfringens strains.

C. botulinum strains were divided into seven distinct clusters. Group I (proteolytic) C. botulinum strains were linked together at a similarity value of 56% (cluster III). Group II (nonproteolytic) C. botulinum strains formed three clusters; cluster XV consisted of strains of C. botulinum types B and F, whereas C. botulinum type E strains were divided into clusters XVI and XVII. C. novyi and group III C. botulinum types C and D clustered together with a similarity value of 22%. C. novyi and C. botulinum type D formed single clusters XI and XIII, respectively, whereas C. botulinum type C strains were divided into two separate clusters (X and XII). The AFLP analysis clearly differentiated between C. botulinum group I and Clostridium sporogenes. However, C. sporogenes and C. botulinum group I strains were linked together at a similarity value of 22%.

C. perfringens strains (n = 37) clustered together with a similarity value of 58% (Fig. (Fig.11 and and2).2). Two subclusters of C. perfringens strains were observed. Strains belonging to subclusters 1 and 2 clustered together with a similarity value of 62% and 85%, respectively. In subcluster 1, toxin types A, B, C, D, and E were detected, whereas subcluster 2 consisted only of strains of toxin type A (Fig. (Fig.2).2). With a 93% cutoff value, C. perfringens strains were divided into 29 different AFLP types. However, visual examination of AFLP patterns of C. perfringens revealed minor fragment differences in strains of AFLP types aflp3 and aflp5. In three events, the same C. perfringens strain was obtained from two different commercial culture collections. The AFLP analysis of these pairs that initially originate from the same isolate resulted in identical fingerprinting patterns. Identical AFLP profiles were also observed for isolates originating from the same outbreak.

FIG. 2.
Reconstructed AFLP banding patterns and a dendrogram of 37 Clostridium perfringens strains based on AFLP analysis. A similarity analysis was performed using the Pearson product-moment correlation coefficient, and clustering was performed by using the ...

DISCUSSION

In this present study, we applied AFLP to 24 different Clostridium species and a total of 129 strains. AFLP differentiated all species tested, except for C. ramosum and C. limosum. Thirteen species were separated into single species-specific clusters, and eight strains, which were the only representatives of those particular species, also occupied separate positions. Furthermore, C. botulinum strains were divided into seven species-specific clusters. Although AFLP failed to discriminate between C. ramosum and C. limosum at the 45% similarity level, these species were linked together at a relatively low similarity level of 49%, and the differences between AFLP patterns were evident in visual examination. Since it is necessary to extend the identification library with several AFLP patterns of well-defined strains for each species to obtain reliable species identification (7), further AFLP analysis on larger numbers of strains of C. ramosum and C. limosum may facilitate differentiation between these species as well.

Some Clostridium strains are known to produce extracellular DNase, which may limit the use of DNA fingerprinting methods such as pulsed-field gel electrophoresis (PFGE) (11, 25, 41, 44, 45). Since all strains studied were typeable by AFLP, the AFLP method seemed to overcome the problem of extracellular DNase production. Furthermore, the AFLP method proved to be highly reproducible and the similarity level of ≥93% for internal reference strains is in accordance with earlier studies (3, 4, 6, 22, 36). The slight variation seen in peak heights of AFLP patterns during reproducibility testing may result from differences in the effectiveness of digestion-ligation or PCR amplification steps (27). The 93% cutoff value used for defining the AFLP type of C. perfringens and the 45% similarity level, which served to differentiate Clostridium strains at the species level, are in agreement with previous AFLP studies of different bacteria (6, 22, 39). AFLP also proved to be a relatively fast, easy-to-use method. The AFLP analysis, including numerical data analysis, can be completed within two working days when initiated with pure DNA.

The results of the AFLP analysis confirmed the phylogenetic finding based on 16S rRNA sequencing of three distinct lineages of C. botulinum groups I, II, and III (15). The distribution of C. botulinum strains in more than one cluster may also stem from the wide genetic diversity observed among group II C. botulinum strains. This is in accordance with previous studies based on PFGE (12) and AFLP (21).

C. sporogenes, which displays high 16S rRNA sequence homology (15) and DNA relatedness (26) with group I C. botulinum types A, B, and F, is considered a nontoxigenic counterpart of group I C. botulinum. Ghanem et al. (8) also reported that 17% of C. sporogenes strains were incorrectly identified as C. botulinum type A or B by cellular fatty acid analysis. The AFLP results are in agreement with those of previous studies on the close relationships of C. botulinum group I and C. sporogenes, since these species were linked together with AFLP, albeit with a relatively low similarity value. With AFLP, however, a clear distinction emerged between C. botulinum group I and C. sporogenes species-specific clusters, indicating that AFLP analysis is capable of discriminating between these Clostridium species.

C. novyi type A and C. botulinum types C and D cannot be differentiated from each other by their phenotypic properties, and C. novyi type A is considered a nontoxigenic variant of group III C. botulinum (14). That group III C. botulinum and C. novyi clustered together with AFLP suggests that they are closely related. This is in accordance with previous studies based on 16S rRNA gene sequence analysis, which revealed that C. novyi type A and C. botulinum types C and D are grouped as a separate phylogenetic lineage (15). With AFLP, single species-specific subclusters were observed for C. novyi and C. botulinum type D, while C. botulinum type C strains were divided in two separate subclusters. Wide diversity among C. botulinum type C has also been reported by Nakamura et al. (34), who found that based on DNA-DNA homology studies, one strain of C. botulinum type C (Stockholm strain) was more closely related to a group consisting of C. novyi type B and Clostridium haemolyticum strains than to the other strains of C. botulinum type C examined.

Phenotypically, C. bifermentans closely resembles C. sordellii. The main criterion for the differentiation of these species is the lack of urease production by C. bifermentans, although researchers have reported urease-negative strains of C. sordellii (33). In this present study, one of the C. sordellii strains (LV1765/1988) was urease negative, which could hamper identification based on phenotypic testing. However, AFLP clearly differentiated between C. sordellii and C. bifermentans strains.

The AFLP method previously described for the characterization of C. botulinum (21) also proved suitable for the characterization of C. perfringens. Although some toxinotype-related subclustering was observed, in general, AFLP was deemed unsuitable for differentiation between various toxinotypes of C. perfringens. This finding was expected, since genes encoding three major toxins of C. perfringens (β, epsilon, and ι toxins) are located on plasmids, thus enabling even toxinotype change of a strain by loss or acquisition of plasmids (37). The existence of strains of different toxin types in the same cluster has also been revealed by multiple-locus variable-number tandem-repeat analysis, suggesting that the acquisition of plasmid-borne major toxin genes is a rather recent event and that strains of different toxin types may not have distinct evolutionary histories (40). Unrelated C. perfringens strains resulted in divergent AFLP banding patterns, whereas C. perfringens strains initially originating from the same isolate or from the same outbreak showed identical fingerprinting patterns, thus indicating that AFLP is a highly discriminative genotyping method. With AFLP, we observed excellent typeability; thus, AFLP is an attractive alternative to PFGE in outbreak situations. In addition to the high diversity of AFLP profiles of C. perfringens and C. botulinum, the AFLP patterns of strains of C. bifermentans, C. septicum, C. sordellii, and C. sporogenes showed substantial diversity, suggesting that AFLP may be able to subtype these species at the strain level. Further research is warranted to evaluate the usefulness of AFLP in epidemiological studies of these Clostridium species.

We conclude that AFLP is a highly reproducible, easy-to-use, and relatively fast method which can be applied to different clostridia and used for the generation of identification libraries. Therefore, libraries of AFLP profiles of well-defined Clostridium strains provide a valuable additional tool in the identification of Clostridium species. Due to partial automation, which enables high throughput of samples, AFLP seems particularly well suited for screening large numbers of isolates. Moreover, the same protocol can be used in typing at the strain level. This present study demonstrated the value of AFLP for distinguishing between strains of C. perfringens, and AFLP can thus be utilized in epidemiological and outbreak studies of C. perfringens.

Acknowledgments

This study was supported by the Finnish Graduate School on Applied Bioscience (ABS), the Finnish Veterinary Foundation, and the Walter Ehrström Foundation.

We gratefully acknowledge Mike Peck, Sinikka Pelkonen, and Seppo Sivelä for providing the various Clostridium strains and Hanna Korpunen for her excellent technical assistance.

Footnotes

[down-pointing small open triangle]Published ahead of print on 13 September 2006.

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