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Applied and Environmental Microbiology, May 2006, p. 3793-3796, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3793-3796.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Combination of Multiplex PCR and PCR-Denaturing Gradient Gel Electrophoresis for Monitoring Common Sourdough-Associated Lactobacillus Species

Luca Settanni,1 Sara Valmorri,1 Douwe van Sinderen,2 Giovanna Suzzi,1 Antonello Paparella,1 and Aldo Corsetti1*

Dipartimento di Scienze degli Alimenti, Sezione di Microbiologia Agro-Alimentare ed Ambientale, Università degli Studi di Teramo, Teramo, Italy,1 Alimentary Pharmabiotic Centre and Department of Microbiology, University College Cork, Cork, Ireland2

Received 30 November 2005/ Accepted 16 March 2006


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ABSTRACT
 
A combination of denaturing gradient gel electrophoresis (DGGE) and a previously described multiplex PCR approach was employed to detect sourdough lactobacilli. Primers specific for certain groups of Lactobacillus spp. were used to amplify fragments, which were analyzed by DGGE. DGGE profiles obtained from Lactobacillus type strains acted as standards to analyze lactobacilli from four regional Abruzzo (central Italy) sourdoughs.


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INTRODUCTION
 
PCR-based methods are frequently applied for the purpose of bacterial typing (13) or monitoring changes in microbial populations (2). Although a polyphasic methodology, based on both culture-dependent and culture-independent methods, is the preferred approach to determine the presence of metabolically active microflora of fermented foods (1), culture-independent methods are useful tools to rapidly follow microbial communities that are significant for the development of the specific characteristics of the final product, e.g., the population dynamics of various lactobacilli during sourdough fermentation (15).

Among culture-independent methods, developed to circumvent the limitations of conventional microbial community analysis (17), denaturing gradient gel electrophoresis (DGGE) analysis is one of the most widely applied genetic methods to quickly and economically study microbial diversity (10, 12). DGGE represents a method to obtain species information through analysis of PCR products of the same length but with different sequences and is based on differential mobility in an acrylamide gel matrix of increasing denaturant (urea and formamide) concentration. Generally, total bacterial DNA from the habitat of interest is extracted and a region with a hypervariable nucleotide base sequence is amplified by PCR and subjected to DGGE.

This work aimed to develop a single-step identification methodology for the differential detection of 16 Lactobacillus species that are generally associated with the sourdough ecosystem by combining DGGE with a previously described multiplex PCR method (15). The applicability of the system was verified with four sourdoughs from artisan bakeries of the Abruzzo region of central Italy.

Strains used in this study are listed in Table 1. Sourdough lactic acid bacteria were propagated in sourdough bacterial broth (8) at 30°C for 24 h, while all other strains were propagated as indicated by the respective culture collections for 24 h.


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  		TABLE 1. Lactic acid bacterial strains used in this study

Genomic DNAs were extracted as reported by De Los Reyes-Gavilán et al. (6) from 2-ml samples of overnight cultures.

The combination of multiplex PCR and PCR-DGGE developed in this study was performed as outlined in Table 2. Briefly, group-specific DNA fragments obtained following multiplex PCR were directly amplified (group 1) or amplified after band excision and purification (groups 2 to 4) (GFX PCR DNA and gel band purification kit [Amersham Biosciences]) using primer combinations in which one of the primers contained a GC clamp for DGGE purposes (18). For groups 3 and 4, the latter primer combinations were based on sequences within the multiplex-generated DNA fragment (nested set of primers) so as to generate a shorter PCR fragment in order to suit the DGGE, which allows separation of DNA fragments with a maximum length of 500 bp (16). All PCRs were carried out using the same conditions as those for Grouping-multiplex PCR (15). The high annealing temperature (65°C) of the PCR replaced the touchdown PCR approach normally associated with DGGE (11). The optimal range of band separation was determined by a perpendicular gel prepared as indicated by the Bio-Rad DCode apparatus (Bio-Rad) supplier. Parallel electrophoresis was run at 60°C for 10 min at 20 V, 14 h at 40 V, and 2 h at 140 V. Gels were stained with ethidium bromide for 15 min and rinsed for 30 min in distilled H2O.


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  		TABLE 2. Groups of Lactobacillus species, oligonucleotide primer sequences and locations, and DGGE conditions

Four standard ladders, one for each group, were developed, representing 15 Lactobacillus species in total (Fig. 1). Although methods for qualitative analysis of specific populations of complex ecosystems based on genus-specific PCR and DGGE have been developed (7, 14), the commonly used universal 16S rRNA gene-derived primers can readily amplify yeast, fungal, or plant DNAs copurified with bacterial DNA (9). In order to avoid misleading DGGE profiles, we approached this problem by using primers specific for lactobacilli based on parts of the hsp60 gene for group 1 species and on parts of the 23S rRNA gene for species belonging to groups 2 to 4.


Figure 1
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  		FIG. 1. DGGE ladders. The Lactobacillus organisms and strains are as follows: lane 1, L. plantarum ATCC 14917T; lane 2, L. pentosus ATCC 8041T; lane 3, L. paraplantarum DSM 10667T; lane 4, L. alimentarius LMG 9187T; lane 5, L. paralimentarius DSM 13238T; lane 6, L. farciminis DSM 20184T; lane 7, L. mindensis DSM 14500T; lane 8, L. sanfranciscensis DSM 20451T; lane 9, L. fructivorans DSM 20203T; lane 10, L. hilgardii DSM 20176T; lane 11, L. brevis ATCC 14869T; lane 12, L. fermentum ATCC 14931T; lane 13, L. pontis DSM 8475T; lane 14, L. panis DSM 6035T; and lane 15, L. frumenti DSM 13145T.

In order to validate the above-described system and verify the standard ladders, DGGE fragments from lactobacilli, found at the same position of the type strains, were excised from gel and eluted in 20 µl sterile double-distilled H2O overnight at 4°C. One microliter of the eluted DNA of each DGGE band was amplified as reported above. This operation was repeated once or twice until a single band was obtained on gel, and then the single bands were amplified with primers that did not contain the GC clamp for their subsequent insertion in plasmid vectors for direct sequencing (MWG Biotech AG).

Since a 180-bp band and a 280-bp band, characteristic of groups 2 and 3, respectively, were obtained with Weissella confusa DSM 20196T and Lactococcus lactis subsp. lactis DSM 20481T, respectively, when the strains were subjected to Grouping-multiplex PCR (15), these two strains were subjected to DNA amplification with primers containing the GC clamp and loaded on DGGE gel together with the respective group ladder. Neither strain generated an amplification product, thus excluding false-positive results.

Under the DGGE conditions used in this study, all Lactobacillus strains tested could be distinguished on the basis of their unique band migration distances. The system was subsequently tested on total DNA samples isolated from dough environments.

Four doughs were inoculated with a mixture of Lactobacillus strains that belonged to the same multiplex group, thus representing the most complex situation in terms of species discrimination, since all strains are identified by an identically sized PCR product. In addition, two reference doughs, named R (without starter, not fermented) and FR (without starter, fermented at 30°C for 24 h), were prepared and treated as previously described by Settanni et al. (15) for analysis by the multiplex PCR/PCR-DGGE combined system. Total DNA from doughs was extracted by means of two different protocols following the methods reported by Settanni et al. (15) and Ampe et al. (1). Isolated DNAs were first subjected to Grouping-multiplex PCR, and then the bands characteristic of doughs 1 to 4 were excised from the agarose gel and, following amplification with primers specific for DGGE analysis (Table 2), separated by DGGE. Each species showed the band produced when the pure culture was processed to develop the DGGE ladders. As expected, no amplification products from doughs R and FR were obtained with Grouping-multiplex PCR.

The system was also applied to analyze four artisan wheat sourdoughs (A to D) collected in the Abruzzo region (central Italy) at the end of fermentation (6 h at about 25°C). Analysis showed a prevalence of Lactobacillus plantarum and Lactobacillus sanfranciscensis (results not shown), common species in Italian sourdoughs (5), after DNA extraction using both methods. Furthermore, doughs A and C also contained Lactobacillus rossiae (formerly Lactobacillus rossii [3]) and L. paralimentarius (results not shown), respectively, but these species were detected only by use of DNA extracted with the protocol reported by Ampe et al. (1), probably since the detection level of this method is more sensitive than that of the method reported by Settanni et al. (15). These results were in agreement with those obtained with the cultivation method performed as previously described by Corsetti et al. (4), and they showed isolation of L. plantarum and L. sanfranciscensis from the 10–8 plate count. In contrast, L. rossiae and L. paralimentarius were not found; they were isolated from 10–7 and 10–6 plate counts, respectively (data not shown).

The multiplex PCR/PCR-DGGE combined system developed in this study is highly specific for the sourdough Lactobacillus species considered; it is based on differently sized PCR products generated from the various groups of Lactobacillus spp., combined with different nucleotide sequences among species of each group. Hence, it allows the reduction of species to follow to just those belonging to each group.


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ACKNOWLEDGMENTS
 
This study was financially supported by the Italian Ministry for University and Research (Nuovopane Research Project, S637-P).


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FOOTNOTES
 
* Corresponding author. Mailing address: Dipartimento di Scienze degli Alimenti, Sezione di Microbiologia Agro-Alimentare ed Ambientale, Università degli Studi di Teramo, V. C. R. Lerici 1, 64023 Mosciano Sant'Angelo, Teramo, Italy. Phone: 39 (0)861 266896. Fax: 39 (0)85 8071509. E-mail: acorsetti{at}unite.it. Back


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Applied and Environmental Microbiology, May 2006, p. 3793-3796, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3793-3796.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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