Molecular typing and quantification of sporeforming clostridia Grana

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A molecular direct approach have been used to study microbial ecology of spore-forming clostridia present in Grana Padano cheese with late blowing defect. The purpose of this work was to identify the dominant bacteria responsible of this food damage and after this, to determine the average amount of their spoiling cells and spores. A Clostridium-specific denaturing gradient gel electrophoresis (DGGE) performed on total DNA extraction from cheeses, was set to monitor clostridia communities and their synergies in a total of 79 Grana Padano samples from different geographical areas in the north of Italy. Clostridium tyrobutyricum (46) appeared as the dominant strain followed respectively by Clostridium butyricum (31), Clostridium perfringens (13), Clostridium sporogenes (12) and Clostridium septicum (7). These 5 species were often co-present in the same sample showing typical synergic behaviours. SYBR green real-time PCR performed on the prevalent C. tyrobutyricum allowed us to quantify this microrganisms with a detection limit of 60 UFC/gr of cheese. This first study of direct quantification of Clostridium tyrobutyricum in cheese opens the way to further applications also to direct analysis for milk contamination and so as a valid tool to prove milk quality, other than MPN method.

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Keywords: Denaturing gradient gel electtophoresis (DGGE), Grana Padano cheese, Real-time PCR, clostrida

1. Introduction

Grana Padano represents a traditional DOP (Denomination of Protected Origin) Italian cheese worldwide known and largely appreciated for its qualities and taste. It's one of the world's first hard cheeses, created nearly 1,000 years ago by the Cistercian monks of Chiaravalle Abbey, founded in 1135 near Milan, who used ripened cheese as a way of preserving surplus milk. Typical of this hard unpasteurised cheese is the slow cooking and ripening, for at least 9 months, then, if it passes the quality tests, it will be fire-branded with the Grana padano trademark.

Unfortunately during ripening, its crumblier texture is often damaged by spore-forming bacteria contamination, especially clostridia, producing the characteristic late blowing defect due to butyric acid fermentation (Ingham et al., 1998; Klijn et al., 1995; Herman et al., 1995; Le Bourhis et al., 2005; Vissers et al., 2006). This fermentation results from the germination of spores of clostridia due to the anaerobic conditions generated in cheese during its production (Bottazzi V. and Dellaglio F., 1970; Ingham et al., 1998). Late blowing produces the deformation of the cheese loaf, a possible release of a foul-smelling substance (butyric acid) on cutting and, in some cases, an undesirable rancid taste. Damaged cheeses also contain several heterogeneously distributed cavities corresponding to the volume of gas produced and mass digested. This constitutes a great economical loss for cheese makers due also to the high trade value of this cheese.

The main sources of contamination are thought to be silage, water, or unhygienic animal bedding (Lango et al., 1995). In particular, Grana Padano Production Disciplinary states that it must be produced with raw milk from cows which are feed with maize silages and concentrates. Forage preservation by ensilage produces a rapid achievement of a low pH by lactic acid fermentation and the maintenance of anaerobic conditions (McDonald et al., 1991) with a growth advantage for clostridium species. The spores can be transferred to the milk via the faeces, mainly via faecal contamination of the udder. Subsequently, spores present in raw milk can survive during food processing and pasteurisation, and, after, germination and outgrowth to high levels may cause food spoilage.

Clostridium tyrobutyricum is considered to be the primary cause of late blowing (Bottazzi and Dellaglio, 1970). Other clostridia able to produce butyric acid have also been detected in milk and cheeses (Battistotti B., 1985). Molecular methods have been applied to the examination of the late blowing spoilage process in cheese (Cocolin et al., 2004; Klijn et al. 1995; Le Bourhis et al., 2007; Le Bourhis et al., 2005). However, little is known regarding molecular point of view about clostridial concentration and occurrence, population structure and dynamics in a discrete amount of Grana Padano cheeses produced in the northern Italy.

Here a denaturing gradient gel electrophoresis approach based on total DNA extraction from cheese allowed us to detect contaminating clostridia species amplifying first by PCR a peculiar region of 270 base pairs of the 16S rRNA gene. Considering Clostridium tyrobutyricum as the favourite spoiling agent, we also set a SYBR green quantitative real-time PCR using as the main target the phosphotransacetylase gene (pta) associated with the acetate formation pathway in this microorganisms.

2. Materials and Methods

2.1. Clostridium type strains culture conditions and genomic DNA isolation

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Reference strains used in this study were Clostridium cochlearium DSM 1285(T), Clostridium tyrobutyricum ATCC 25755(T), Clostridium sporogenes ATCC 7955(T), Clostridium butyricum DSM 10702(T) and isolated Clostridium perfringens UC9018. All strains of Clostridium were harvested in RCM broth (Oxoid) and cultures were incubated at 37°C for 24-48 h in an anaerobic chamber. For DNA extraction, 1 ml of culture was collected and centrifuged at 8,000g for 5 min. Genomic DNA was isolated from the pelleted cells using the Nucleospin Tissue DNA Isolation Kit (Macherey Nagel, Germany) and DNA was verified on a 1% agarose gel containing SYBR® Safe (Invitrogen Corporation Life technologies).

2.2. Cheese sampling procedure

Samples of cheese with anomalous pastry defects and cavities were collected from nine different areas of production of Grana Padano cheese in the North of Italy, all part of the Consorzio per la tutela del formaggio Grana Padano (Bergamo, Brescia, Cremona, Cuneo, Mantova, Padova, Piacenza, Trento, Vicenza). Portions received were 50cm large and 20-30 cm high and were cut directly from the entire cheese shapes; the damaged area of each sample was grounded and suddenly frozen at -20°C in our laboratory until processing.

2.3. DNA extraction from cheese

50 g of grounded cheese was homogenized in 125-µm filter stomacher bags (Biochek, Foster City, Calif.) with 50 ml of distilled water for 2 minutes. The homogenate was collected and spinned for 10 minutes at 4°C. So obtained pellet was the starting material for total DNA extraction using the FastDNA® SPIN kit and the FastPrep® Instrument (Qbiogene, Inc., CA). The isolated DNA was then finally resuspended in 100 µl of DES solution. All samples were extracted in triplicate for DGGE analysis.

2.4. Clostridium-specific PCR

The PCR strategy comprised a first amplification of most of the 16S rDNA gene (1.5 kb) using universal primers P1 (5'- GCGGCGTGCCTAATACATGC-3') and P4 (5'- ATCTACGCATTTCACCGCTAC-3') in order to assure bacterial DNA extraction from samples. PCR amplifications were performed in a final volume of 25 μl which included 12.5 μl of 2X MasterMix PCR (Promega), primers (20 μM), and 3 μl of genomic DNA. Amplifications were performed in a 9700 Thermal Cycler, (Applied Biosystems, CA). Template DNA was generally diluted 10-fold and 100-fold to minimize PCR inhibitors. Reactions were heated to 95°C for 5 min before the addition of the polymerase and cycled at 94°C for 1 min, 56°C for 1 min and 72°C for 1 min. Cycles were repeated 30 times for all samples. Finally, 5 μl of each PCR product was used for visualization on a 2% agarose gel containing SYBR® Safe (Invitrogen Corporation Life technologies). Negative (without DNA) and positive (with DNA from reference strains) controls were performed in every amplification run.

A second PCR was then performed using forward Clostridium-specific primer DGGE2Cl (5'-GGGACGATAATGACGGTACC-3') and reverse P4 (5'- ATCTACGCATTTCACCGCTAC-3') internal to the first primer set producing an amplicon of 250 bp. Templates were 10- to 100-fold diluted products from the purified DNA. Reaction mixture was performed in a final volume of 25 μl and thermal cycles were the following: 5 min at 95°C, 35 cycles of 30 sec at 94°C, 30 sec at 60°C, 30 sec at 72°C, and 7 min at 72°C. Primers were tested before on different clostridia and other species that could frequently be present in dairy products such as Lactobacillus, Streptococcus, Leuconostoc, Bacillus in order to avoid cross-reactions.

2.5. DGGE analysis

Clostridium cluster I-specific amplicons obtained by PCR amplification (see above) were re-amplified using primers DGGE2Cl Clamp and P4 targeting the V3-V4 region of the 16S rDNA and producing a 270-bp PCR product suitable for DGGE analysis (Edwards et al., 1989). A GC-clamp (5'-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCC-3') was incorporated in the 5' region of the DGGE2Cl primer to improve the sensitivity in the detection of DNA sequence differences by DGGE analysis as previously described (Sheffield et al., 1989). The PCR reaction mixture was performed in a final volume of 50 μl containing 25 μl of 2X MasterMix PCR (Promega) and 0.1 μM of each primer. After DNA addition (1 μl from the previous PCR reaction), the samples were subjected to the amplification in a 9700 Thermal Cycler (Applied Biosystems, CA) using an amplification cycle in which the annealing temperature was 63°C for 35 cycles. A denaturation of 95°C for 5 min was used and extension was performed at 72 °C for 1 min, with a final extension of 72 °C for 7 min. Five microliters of the product were analyzed by standard agarose gel electrophoresis before DGGE analysis. The IngenyPhor U-2 (Ingeny International BV Netherlands) apparatus was used for the sequence specific separation of the PCR products. Electrophoresis was performed in a 1-mm acrylamide gel (8% [wt/v] acrylamide:bis-acrylamide 37.5: 1) containing a 45-65% denaturant gradient (100% denaturant corresponds to 7 M urea and 40% [v/v] deionised formamide) increasing in the direction of the electrophoretic run with a stacking gel on top. PCR products (20 μl) were loaded and migrated at 90 V for 16 h in Tris-acetate buffer at 60°C (0.04 M Tris-acetate and 0.001 M EDTA, pH 8.0). Together with samples, amplicons of the V3-V4 region of five Clostridium reference strains (see above) were loaded on the gel as a ladder.

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After electrophoresis, they were stained for 20 min with 1X SYBR Green I Gel Staining solution (Roche), and analyzed under UV illumination. Pictures of the gels were digitized by using a CDC camera (Olympus).

Small pieces of selected DGGE bands were punched from the gel using a cutter. The blocks were, then, transferred in 50 μl of sterile water and let to diffuse overnight at 4°C.

5 μl of the eluted DNA were used for the re-amplification and the PCR products, generated with the GC clamped primer, was checked by DGGE. Only products migrating as a single band and at the same position in respect of the control, were amplified with the primer without the GC clamp, purified and sent to a commercial sequencing facility (BMR Cribi, Padova, Italy) for sequencing.

2.6. Sequence analysis

16S rRNA gene sequences of isolated were compared with the annotated Bacterial and Archaeal small-subunit 16S rRNA sequences present in the Ribosomal Database Project (http://rdp.cme.msu.edu/). In addition, a further check with the BLAST program (Altschul et al., 1997) was done to define the closest relatives of the PCR products separated in DGGE gel.

2.7. Cluster analysis

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2.8. SYBR green real-time PCR

PCR real-time reactions were set in 20 µl final reaction volume using LightCycler® instrument capable of two-colour detection and LightCycler-FastStart DNA Master SYBR Green I kit from Roche Diagnostics (Penzberg, Germany). PCR mixtures contained 1Ã- Fast Start reaction mix (FastStart Taq DNA polymerase with buffer, dNTPs, SYBR Green I dye) and 0.3 μM PTACtyrRT F (5' -GGAAACACAAGGGTATGACTCC- 3') and PTACtyrRT R (5' -GCACCTGGTAGTCCTGTAGC- 3') primers. 5 μl of 100-fold diluted DNA extracted from cheese was used in each reaction. The PCR programme consisted of an initial denaturation for 10 min at 95 °C followed by 45 cycles (10 sec at 95 °C, 15 sec at 63 °C, 25 sec at 72 °C). Tm analysis cycle consisted of an initial heating to 95 °C, incubation for 15 sec at 65 °C and heating to 95 °C at a rate of 0.1 °C s−1 while continuously measuring the fluorescence in the channel FL1. At the end, cooling was reached at 40 °C. PCR results were recorded as crossing point (Cp) values using the 2nd derivative maximum method of the instrument software (version 3.0). The melting temperatures were estimated from the inflection point of the melting curve by the software. Samples having a Cp value of ≤41 and a characteristic Tm were considered positive.

2.9. Quantification standard curve

A standard curve was constructed using 10-fold dilutions of Clostridium tyrobutyricum ATCC 25755(T) DNA, extracted from pure culture with Nucleospin Tissue DNA Isolation Kit (Macherey Nagel, Germany) as above described and quantified with Qubit Quantitation Platform (Invitrogen, UK). The copy number of the pta gene was established considering that, based on the molecular weight of the 3.76-mbp-sized genome of Clostridium botulinum A str. Hall (NC009698) (REF…), 1 ng of DNA is 2.46 x 105 times the entire genome and that pta gene is a single-copy gene. The 7 points composing the standard curve were expressed as CFU/ml calculated by multiplying the number of pta gene copy in the extracted type DNA with the ratio of the final suspension volume of the DNA isolated from 1 ml of pure culture of C. tyrobutyricum ATCC 25755 and 5, as 5µl of the final DNA suspension were object of PCR.

The pta gene copy number per 50 g of cheese was calculated in the same way, only considering the 100-fold dilution of the starting extracted DNA, since the extraction method was exactly the same as the standard sample.

2.10. Statistical analysis

…

3. Results

3.1. Efficiency of the PCR protocol and DGGE normalization

The first aim of this work was to develop a reliable molecular method to identify and typify clostridia strains in cheese samples. To reach this goal different primers sets and DGGE conditions were tested and, among all, the best results were achieved using DGGECl2-P4 primers. The choice of the Clostridium specific primers was based on the analysis of the 16S rDNA sequence of Clostridiales. This analysis revealed that members of the Clostridium genera, differently from related taxonomical units of Firmicutes, present a deletion of about 17bp (positions 582-600) + 15 bp (positions 604-618) in the central region of 16S rRNA gene amplified. This allowed to design a couple of primers based on the sequences upstream and downstream the gap that has been demonstrated to amplify almost uniquely 16S rDNA from Clostridium species (data not shown). The PCR protocol allowed amplification of DNA from reference clostridial strains (Clostridium cochlearium DSM 1285 (T), Clostridium tyrobutyricum ATCC 25755 (T), Clostridium sporogenes ATCC 7955 (T), Clostridium butyricum DSM 10702 (T) and Clostridium acetobutylicum LMG 5710 (T)) but not from non-clostridial reference strains (Lactobacillus plantarum, Lactobacillus sakei, Lactobacillus helveticus, Bacillus coagulans, Enterococcus faecium and Streptococcus thermophilus). Upon DNA amplification with DGGE2Cl and P4 primers, each reference strain produced a single DGGE band and these bands constituted a normalization ladder.

3.2. Detection of Clostridia in cheese samples by DGGE

For the purpose of this study, 79 samples have been processed. Results of Clostridium cluster I-specific PCR analyses suggest that clostridia are nearly ubiquitous members of the Grana Padano ecosystem. Using the PCR protocol, putative Clostridium cluster I-specific amplicons have been obtained for all 79 farm samples (100 % of the total). The results obtained by DGGE analysis on control strains from international collections are shown in Fig. 1. (vedere se mettere una foto con solo I type o con type+campioni). Species-specific migration patterns were obtained, making possible the identification of C. butyricum (Fig. 1, lane …), C. sporogenes (Fig. 1, lane …), C. tyrobutyricum (Fig. 1, lanes …), and C. cochlearium (Fig. 1, lane…). Some strains showed patterns formed by different bands; among type strains, C. butyricum is the one which presented two DGGE bands due to the amplification of multicopies of the 16S rRNA gene that contained differences detectable by DGGE as previously described by Cocolin et al. (2001).

The results obtained are summarized in table … and the DGGE profiles for farm samples processed are shown in figure ….

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Fig. 1. DGGE profiles of the PCR products originated from the control strains (numbers in red) and from the farm samples (numbers in black). Lane 1, C.butyricum DSM#10702; lane 2, 28 C.sporogenes UC9000; lane 3, C.sporogenes ATCC#7955; lane 24-25, C.tyrobutyricum ATCC#25755; lane 26, C.acetobutylicum LMG#5710; lane 27 C.cochlearium DSM#1285.

Clostridia bands were obtained without pasteurization and enrichment. In the positive cheese samples the clostridia population was mixed. We considered the most indicative bands for each sample. The results of the direct identification of the Clostridium spp. in cheese samples were confirmed by sequencing of the DGGE bands migrating in the spreading region of the clostridia species.

A total of … sequences, obtained from the bands indicated in Fig. 1, showed an high homology( 85-100%) with 16S rRNA genes from Clostridium species considered in the study and already present in GeneBank. The other strain identification have been obtained by comparison of bands with the fingerprint of the sequenced bands.

For the 6 samples of cow faeces analysed from farm A, 5 of them showed presence of C. disporicum and 1 of Clostridium sp., and 3 both C. disporicum and Clostridium sp. The fingerprints also demonstrated that isolates from faeces had identical patterns, indicating and confirming that they have a similar microbiota.

For what concerns feed, different types were detected. In the 2 grass samples, C. sporogenes/botulinum and C. disporicum were present; in the 7 hay samples, C. butyricum (1/7) and C. disporicum (5/7) were frequent but also C. botulinum, C. beijerinckii and C. tunisiense were found; in maize silage we detected mostly C. intestinale, C. botulinum/C. sporogenes and C. disporicum. Finally, in the concentrate mix the principal types were Clostridium sp., C. disporicum, C. intestinale and C. butyricum.

Table 2. Results obtained by DGGE analysis and strain identification of the farm samples processed in the study

ID

Lane

Farm

Sample

Band Number

Strain Identification

Similarity (%)

1

4

A

Grass

1

C. botulinum/sporogenes

98

2

5

A

Hay

1-2

3

C.butyricum

C. disporicum

99

3

6

A

MaizeSilage

1

2

3

C.botulinum or C. beijerinckii

C.intestinale or C.sp UsIt102-1

C.botulinum/C.sporogenes

93

99

100

4

7

A

Concentrate

1

2

C. sp. (T)

C.sp. (T)

92

99

5

8

A

Cow Faeces

1

C.disporicum

6

9

A

Cow Faeces

1

C.disporicum

7

10

A

Cow Faeces

1

2

C. disporicum

C.sp.

92

8

11

A

Cow Faeces

1

C.sp.

9

12

A

Cow Faeces

1

2

C.disporicum

C. sp.

10

13

A

Cow Faeces

1

2

C.disporicum

C.sp.

11

14

B

MaizeSilage

1

2

Cl.intestinale or Cl.sp.UsIt102-1

C.disporicum

100

12

15

B

Grass

1

2

C.disporicum

Uncultured Firmicutes bacterium

97

13

16

B

Hay

1

2

3

C.tunisiense or C.sp.PPf35E4

C.disporicum

Uncultured Firmicutes bacterium

86

14

17

B

Grass Hay

1

2

C.disporicum

Uncultured Firmicutes bacterium

21

22

B

Concentrate Mix

1

2

C.intestinale

C.disporicum

85

28

23

C

Concentrate Mix

1-2

3

C.butyricum

Uncultured Firmicutes bacterium

29

18

C

Grass Hay

1-2

3

C.disporicum

Uncultured Firmicutes bacterium

30

19

C

Comm Grass Hay

1

2

3

C.botulinum or C. beijerinckii

C.disporicum

Uncultured Firmicutes bacterium

31

20

C

"Cooked"Loietto Hay

1

C.botulinum or C. beijerinckii

32

21

C

Loietto Hay

1

2

C.botulinum or C. beijerinckii

Uncultured Firmicutes bacterium

4. Discussion

This study was conducted to develop a PCR system for amplification of Clostridium species suitable for subsequent analysis via DGGE and to understand the diffusion of such species along the Italian farms production chain. The approach used is exclusively based on molecular methods without the help of standard microbiological techniques.

Primers DGGE2Cl-P4, used in this study, proved to be selective for Clostridium; when combined with the reverse primer P4, the forward primer DGGE2Cl, designed on the region V3-V4 of the 16S rDNA gene sequence, produces a 250-bp PCR fragment which is highly specific for clostridia. Encouragingly, primer DGGE2Cl didn't show high numbers of mismatches at the 3'-end with a range of gram-positive non-Clostridium species.

The applicability of the PCR-DGGE method for assessing the diversity of Clostridium spp. obviously depended also on the proper lysis of Clostridium cells and, most of all, of spores by the DNA extraction method used, making the 16S rDNA genes accessible for amplification. The lysis and extraction protocol used in this study was based on a mechanical and enzymatic treatment; this provided sufficient lysis of Clostridium cells and spores from silage and faeces yielding sufficient specific target DNA for successful amplification.

We know that field operations (e.g. mowing, tedding, raking, chopping) may contaminate the crop with soil and therefore with spores (McDonald et al., 1991; Rammer et al., 1994). Proliferation of clostridia may occur during the later stages of aerobic spoilage of silage (Lindgren et al., 1985). When animals consume feed contaminated with spore-forming bacteria, large quantities of spores are excreted in their faeces. Significant correlations between the levels of anaerobic spores in silage and faeces could be demonstrated, confirming that silage is an important source of spores (Dasgupta and Hull, 1989; Te Giffel, 1997; Cook and Sandeman, 2000).

Among the samples analysed, we have observed that in one closed farm environment it's possible to have a transmission from feed to faeces; in the case of farm A (Grana Padano producing), Cl. disporicum and Clostridium sp. have been found both in feed samples than in faeces, demonstrating that this two kind of bacteria transmit from feed to animals being resistant and multiplying into the digestive gastrointestinal tract.

In all feed samples examined, different Clostridium species have been detected (Cl. botulinum/sporogenes, Cl. butyricum, Cl. intestinale, Cl. beijerinckii, Cl. tunisiense), both in grass and hay forages than in maize and concentrate silage. This phenomenon is probably due to bad hygienic agricultural practises, during crop manipulations and stocking. Moreover, it has been ascertained that on grass fertilized with cow faeces the levels of spores were higher than those on grass inorganically fertilized (Rammer et al. 1994), validating also the opposite passage from faeces to soil. For this, the introduction of improved agricultural practices to avoid deterioration and health hazards is suggested.

A more wide study has to be enlarged also to milk and cheese in order to find correlations between feed and milk clostridia contamination in the same farm environment.

Generally, the use of molecular techniques proposed for the analysis of complex microbial community in silage require a pre-definition of the species to search for; here the PCR-DGGE system developed in this work is quite fully specific for Clostridium and it is extremely useful for studying the diversity of this species and closely related groups in farm samples.