23S rRNA-targeted Oligonucleotide Probes for the Rapid Identification of Meat Lactobacilli

System. Appl. Microbiol.14, 173-177 (1991) © Gustav Fischer Verlag, StuttgartlNew York

23S rRNA-targeted Oligonucleotide Probes for the Rapid Identification of Meat Lactobacilli CHRISTIAN HERTEL!, WOLFGANG LUDWIG!, MICHAELA OBST2 , Rum F. VOGEL 2 , WALTER P. HAMMES 2 , and KARL HEINZ SCHLEIFER h 1 2

Lehrstuhl fiir Mikrobiologie, Technische Universitat Miinchen, 8000 Miinchen 2, Germany Institut fur Lebensmitteltechnologie, Universitat Hohenheim, 7000 Stuttgart 70, Germany

Received August 31, 1990

Summary A method for the rapid identification of Lactobacillus species that are used as starter organisms for fermented meat products was developed. Sequences of 23S rRNA specific for Lactobacillus curvatus, L. sake, and L.pentosuslL. plantarum were identified and complementary oligonucleotides were synthesized. The specificity of the probes was checked by dot blot and colbny hybridizations. The probes can be used for the analysis of mixed cultures. None of the other Gram-positive bacteria found in meat reacted with the specific oligonucleotide probes.

Key words: Meat lactobacilli - Lactobacillus curvatus - Lactobacillus pentosus - Lactobacillus sake - 23S rRNA Oligonucleotide probe

Introduction Fermented sausages provide an environment which is a habitat for .charateristic bacteria among which Lactobacillus curvatus and L. sake are most competitive (Reuter, 1975). These psychrophilic lactobacilli outnumber other facultatively heterofermentative lactobacilli, e.g., L. plantarum, or L. casei thousandfold (Hammes et al., 1990). On the other hand, dry sausages are produced by application of a variety of lactic acid bacteria (LAB) as starter organisms, either as sole components or in combinations with Micrococcus varians or Staphylococcus carnosus.The composition of the microbial population of fermented sausages is affected by the original microbial contamination of raw materials and factors effective during the fermentation process, e.g., temperature, redox potential, pH and water activity. The study of the microbial population during the fermentation process, especially to follow the growth of starter organisms, requires fast and reliable identification of LAB. This procedure is hampered by the fact, that taxonomic methods resting on physiological or biochemical criteria, are still ambiguous and time consuming (Kandler and Weiss, 1986). " Corresponding author

The development of specific DNA probes for L. curvatus, L. sake and L. pentosuslL. plantarum and their use in a direct hybridisation procedure provides a means for easy quantification of meat lactobacilli. Investigations can be made on changes in microbial populations of meat products and on the distribution of these organisms in other fermented foods or in the meat spoiling population. Hence, the development of DNA probes specific for these organisms can also be seen as a first step towards easy and reliable monitoring of these organisms in the environment and tracing back their hitherto unknown primary habitats. Materials and Methods Organisms and growth conditions. The bacterial strains used to design probes and those used as reference organisms for evaluating the probes are listed in Table 2. Brochothrix thermosphacta,. carnobacteria, lactobacilli, leuconostocs and pediococci were grown anaerobically in MRS (De Man et aI., 1960) broth or on MRS agar plates at 30°C. Enterococci, lactococci and Streptococcus salivarius were grown anaerobically in M17 (Terzaghi and San dine, 1975) broth or on M17 agar plates at 30°C. The remaining strains were cultivated aerobically in yeast extract-

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glucose (containing per liter, 10 g of peptone, 8 g of NaCl, 5 g of yeast extract, and 5 g of glucose, pH 7.0) broth or on the corresponding agar plates at 30°C, Designing of probes. DNA of L. curvatus LTH 1432 (previously referred to as L.curvatus Lc2-6), L. pentosus DSM 20314 and L. sake LTH 1644 was extracted and purified as previously described (Knauf et aI., 1989). A variable region of the 5'-terminus of the 23S rRNA genes was amplified from the bulk DNA by the polymerase chain reaction (Saiki et aI., 1988). The sequences of the amplification primers 5'are: 5' AT ATGCGGCCGCCGGGRYKAAGTCGT AAC-3' (all strains), 5'-CACGTGTYCCGCCGTACTC-3' L. curvatus, L. sake) and 5' -CTTTCCCTCRCGGT ACT-3' (L. pentosus) (K = G and T, R = A and G, Y = C and T). The reaction conditions were l~lg template DNA, 1 min at 96 DC, 2 min at 48°C, 3 min at 72 DC; 32 cycles. The amplified fragments were cloned into the vector pBluescript (Stratagene, La Jolla, USA) and sequenced using the dideoxy chain termination method (Sanger et aI., 1977; Chen and Seeburg, 1985). Comparative analysis of the 23S rRNA gene fragment sequences revealed regions that can be used as specific target sites. Oligonucleotides complementary to the 23S rRNA were synthesized on a Cyclone DNA synthesizer (MilliGenJBiosearch, Eschborn). They were 5'-end labeled by using T4 polynucleotide kinase (Boehringer Mannheim, F.R.G.) and ATP (New England [y_ 32 Pl Nuclear, Dreieich, Fed. Rep. Germany) according to the procedure described by Sambrook et a1. (1989). Dot blot hybridization. Nucleic acids were extracted from pure cultures of test strains by a rapid lysis technique as follows: Cells from 10ml overnight cultures were harvested by centrifugation and suspended in 400 [ll of 0.01 M Tris-HCl-O.OOl M EDTA, pH 8.0. The same volume of glass beads (diameter 0.1 mm) was added together with 50 [ll 10% SDS, 400 [ll phenol (equilibrated in 1 M Tris-HCI, pH 8.0). The cells were lysed by sonication (1-2 min) and subsequent incubation at 60°C (10 min). The nucleic acids were precipitated by adding 2.5 volumes of ethanol. One microgram of crude nucleids acids was denatured at 55°C for 5 min in 100 [ll of a buffer containing 50% formamide, 4% formaldehyde, 0.02 M 3-(N-morpholino) propanesulfonic acid, 0.005 M sodium acetate, and 0.001 M EDTA. The denatured nucleic acids were bound to nylon membranes (Zeta Probe, BioRad, Munich,) in a dot blot apparatus (Minifold, Schleicher & Schiill, Dassel). The nucleic acids were immobilized on the membrane by heating at 80°C for 2 h. The membranes were incubated in 0.1 x SSC (standard saline citrate; 1 x SSC is 0.15 NaCI, 0.015 M sodium citrate, pH 7.0) containing 0.5% sodium dodecylsulfate (SDS) at room temperature for 1 hand then prehybridized in a solution containing 6 x SSC, 0.1 % SDS, 0.5% sodium N-lauroyl sarcosine, and 5 x Denhardt's solution (Denhardt, 1966; 1 x Denhardt's solution is 0.02% Ficoll, 0.02% polyvinylpyrrolidone and 0.02% bovine serum albumin)

at 38°C for 2 h. Hybridization was performed in prehybridization solution containing 5 pmol of labeled probe for 4 h. The incubation temperatures used for the particular probes are listed in Table 1. The membranes were washed twice in 2 x SSC containing 0.1 % SDS for 5 min each time at room temperature and subsequently once at the probe-dependent temperature as specified in Table 1. After autoradiography, the hybrids were denatured in 2 x SSC containing 0.1 % SDS at 65 DC for 10 min. The probe was washed off but the nucleic acids are still bound to the membrane and can be used for further hybridizations. Colony hybridization. Colony hybridizations were carried out as described previously (Betzl et aI., 1990). Lactobacilli were grown on Zeta Probe membranes layered on MRS agar medium. Subsequently the membranes were placed on filter paper (Whatman 3) soaked in 2 x SSC containing 5% SDS and heated in a microwave oven (700 W) for 1 to 2 min. The membranes were dried at 80°C for 10 min. Before hybridization the colonies were fixed by spraying with a colorless, fast-drying acrylic lacquer (e.g., Wacolux, Ziirich, Switzerland). Hybridization conditions were the same as described for dot blot hybridization.

Results 1.Design and specificity of probes Comparative partial sequence analysis of 235 rRNA genes of L. curvatus, L. pentosus and L. sake indicated differences in the vicinity of the 5' -termini that can be used for the design of specific oligonucleotide probes. The sequences of the probes and the hybridization and washing temperatures are summarized in Table 1. The oligonucleotide probe for L. pentosus contains six and five base changes in comparison to those of L. curvatus and L. sake, respectively, whereas the latter two probes are distinguished by two base changes.

2. Specificity of oligonucleotide probes The specificity of the oligonucleotide probes was evaluated by dot blot hybridization to membrane- bound crude nucleic acids extracted from pure cultures of various bacteria occurring in meat or used for fermentation of meat. The results are summarized in Table 2 and Fig 1. Oligonucleotide probes specific for L. curvatus and L. sake hybridized only to rRNA targets of strains belonging to the corresponding species. The oligonucleotide probe for L. pentosus also hybridized to rRNA of a strain of the closely related species L. plantarum.

Table 1. Sequences, specificities, and hybridization and washing temperatures of the probes. Base differences within the Lactobacillus species-specific probes are indicated by bold-faced letters Temp (0C) for: Probe Lbc Lbs Lbp 302

Sequence 5' ATGATAATACCCGACT AA3' 5' TTAATGATAATACTCGATT3' 5' ATCTAGTGGTAACAGTTG3' 5'CGGAACTTACCCGAC3'

Specificity

Hybridization

Washing

L. curvatus L. sake L. pentosusfplantarum

38 38 38

45 43 47

Universal

38

44

23S rRNA-targeted Oligonucleotide Probes for the Identification of Meat Lactobacilli Table 2. Strains studied and reaction of blotted nucleic acids with specific probes

Reaction with probe: a

Species

Strain

Lactobacillus curvatus Lactobacillus curvatus Lactobacillus curvatus Lactobacillus sake Lactobacillus sake Lactobacillus sake Lactobacillus sake Lactobacillus sake Lactobacillus pentosus Lactobacillus plantarum Lactobacillus alimentarius Lactobacillus brevis Lactobacillus casei Lactobacillus farciminis Lactobacillus halotolerans Lactobacillus viridescens Leuconostoc mesenteroides Leuconostoc carnosum Leuconostoc gelidum Pediococcus acidilactici Pediococcus pentosaceus Lactococcus cremoris LactococCus lactis Streptococcus salivarius Carnobacterium divergens Carnobacterium piscicola Carnobacterium piscicola Enterococcus avium Enterococcus faecalis Enterococcus faecium Enterococcus malodoratus Brochotrix .thermosphacta Micrococcus varians Micrococcus varians Micrococcus varians Stap./Jylococcus aureus Staphylococcus carnosus

DSM 20010 LTH 1432 LTH 1636 DSM 20017T LTH 1642 LTH 1644 LTH 1648 LTH 1649 DSM 20314 T DSM 20174 T DSM 20249 T DSM 20054T DSM 20011 T LTH 1136 DSM 20190 T DSM 20410 T DSM 20343 T DSM 5576 DSM 5578 DSM 20333 T DSM 20336 T DSM 20069 T DSM 20481 T DSM 20560 T DSM 20623 T DSM 20722 DSM 20730 T DSM 20063 DSM 20478T DSM 20477T DSM 20681 DSM 20171 T DSM 20033 T LTH 1520 LTH 1529 LTH 906 DSM 20501 T

Lbc

+ + +

Lbs

+ + + + +

Lbp

+ +

175

Location

302

in Fig.1 b

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

a5 a12, elO a6 b3 a1 a4, ell a2 a3 bIO, el2 bl b9 b5 b8 b4 b6 a7 a8 ns b12 a9 a10 c8 c6 c9 b7 c2 el c4 all, c5 c7 c3 b2 ns ns ns ns ns

DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, F.R.G.; LTH, Institut fur Lebensmitteltechnologie, Universitat Hohenheim, F.R.G. b Designation of the location refers to Fig. lA, B, C and D. ns, not shown in Fig. 1.

a

3. Colony hybridization Pure and mixed cultures were analyzed by colony hybridization. Mixtures of L. curvatus with L. sake or L. pentosus, and L. sake with L. pentosus were grown on Zeta Probe membranes layered on MRS agar as described in Materials and Methods. The colony hybridization experiments are documented in Fig. 2. The accessibility of the 235 rRNAs of the three lactobacilli to the oligonucleotide probes in colony hybridization was proven by hybridization to a universal probe (a, d and g in Fig. 2). After autoradiography the hybrids were denatured, the probe washed off and the membranes were rehybridized to a second, specific probe an~by repeating the procedures to another or universal probes. These studies demonstrate that the probes can be used for the specific and rapid

enumeration of L. curvatus, L. pentosuslL. plantarum and L. sake as colony forming units in a mixed population. Discussion Nucleic acid probes are especially useful for the rapid detection and identification of clinically or economically important organisms. They have been used successfully for the rapid detection of pathogenic and spoilage bacteria in food products (Schleifer, 1990). There are different approaches to design nucleic acid probes. They can be derived from randomly cloned DNA fragments or from DNA or RNA with known functions. At present all DNA probes for the species specific detection of lactobacilli are based upon randomly cloned DNA fragments (Petrick et

Ch. Hertel et al.

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Fig. 2. Colony hybridization of mixed cultures of lactobacilli (a, b, c: L. curvatus and L. pentosus; d, e, f: L.sake and L.pentosus; g, h, i: L.sake and L. curvatus) to a universal (a, d, and g) and specific (b and i: Lbc; e and h: Lbs; c and f: Lbp) probes. The specificities of the probes are defined in Table 2.

ai., 1988; Delley et ai., 1990). The disadvantage of these probes is that the biological function of a random DNA is not known. It may be part of a gene of vital importance or of a DNA sequence without any essential function. Therefore, nothing is known about its stability or genetic variability. In comparison, the use of nucleic acid probes targeted against 16S or 23S rRNA or the corresponding genes has several advantages. Firstly, these rRNAs are ubiquitously distributed and show differences in the extent of conservation within the molecule. Thus, probes can be designed for various ranges of specificity. It is even possible to design a probe that reacts with any cellular life form (see Table 1, universal probe). Such a universal probe is an important control tooi. Secondly, rRNAs are present in very high copy numbers (about 10 4 in exponentially growing cells of E. coli). Thus, a considerable increase of sensitivity can be achieved by targeting rRNA instead of DNA. Thus, rRNA-targeted oligonucleotide probes can be used for in situ identification of whole cells (DeLong et ai., 1989; Amann et ai., 1990) and colony hybridization of gram-positive bacteria (Betzl et ai., 1990). Thirdly, the single strandedness of rRNA provides better accessibility to the probe and DNA-rRNA hybrids are more stable than DNA-DNA hybrids. Lactobacillus curvatus and L. sake are genetically closely related and phenotypically not easy to differentiate (Kandler and Weiss, 1986; Hastings and HolzaPfel, 1987). On the other hand, there is a growing demand for a rapid and reliable differentiation of the two species, since they are of predominant importance in fermented meat production (Hammes et ai., 1990) and as spoilage organisms of vacuum-packed meat (Reuter, 1981). A 1.2 kilobase random DNA probe specific for L. curvatus has been described (Petrick et ai., 1988). However, nothing is known about the stability of the target region of this probe and because of its size it can probably not be used for colony hybridization in contrast to the oligonucleotide probes described in the present paper. They are ideal for analyzing mixed cultures as usually present in fermented meat products. The oligonucleotide probes specific for L. curvatus and L. sake do not hybridize to 23S rRNA of the other gram-positive bacteria found in meat. The oligonucleotide probe directed against the 23S rRNA of L. pentosus also reacted with that of L. plantarum. This reflects the close phylogenetic relatedness of these two species (Kandler and Weiss, 1986). Acknowledgements. This work was supported by Bundesministerium fiir Forschung und Technologie grants 0319274A and 0319280A.

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23S rRNA-targeted Oligonucleotide Probes for the Identification of Meat Lactobacilli rRNA-targeted oligonucleotide probes. Environ. Appl. MicrobioI. 56, 2927-2929 (1990) Chen, E. Y., Seeburg, P. H.: Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4, 165-170 (1985) Delley, M., Mollet, B., Hottinger, H.: DNA probe for Lactobacillus delbrueckii. Appl. Environ. Microbio!. 56, 1967-1970 (1990) Delong, E. F., Wickham, G. S., Pace, N. R.: Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science 243, 1360-1363 (1989) De Man, J. C, Rogosa, M., Sharpe, M. E.: A medium for the cultivation of lactobacilli. J. Appl. Bact. 23, 130-135 (1960) Denhardt, D. T.: A membrane filter technique for the detection , of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-646 (1966) Hammes, W. P., Banthleon, A., Min, S.: Lactic acid bacteria in meat fermentation. FEMS Microbiol. Rev. (1990) Hastings, j. W., Holzapfel, W. H.: Conventional taxonomy of lactobacilli surviving radurization of meat. J. Appl. Bact. 62, 209-216 (1987) Kandler, 0., Weiss, N.: Genus Lactobacillus, pp. 1209-1234. In: Bergey's Manual of Systematic Bacteriology, Vol. 2 (P. H. A. Sneath, ed.). Baltimore, Williams and Wilkins 1986 Knauf, H. j., Vogel, R. F., Hammes, W. P.: Introduction of the transposon Tn 919 into Lactobacillus curvatus Lc2-c. FEMS Microbiol. Lett. 65, 101-104 (1989)

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Petrick, H. A. R., Ambrosio, R. E., Holzapfel, W. H.: Isolation of a DNA probe for Lactobacillus curvatus. Appl. Environ. Microbiol. 54, 405--408 (1988) Reuter, G.: Classification problems, ecology and some biochemical activities, pp. 221-229. In: Lactic acid bacteria in beverages and food U. G. Carr, C V. Cutting, and G. C Whiting, eds.). London, Academic Press 1975 Reuter, G.: Psychrotrophic lactobacilli in meat production, pp. 253-258. In: Psychrotrophic microorganisms in spoilage and pathogenicity U. H. B. Christian, G. Hobbs, T. A. Roberts, and N. Skovgaard, eds.). New York, Academic Press Inc. 1981 Saiki, R. K., Gelfaud, D. H. Stoffel, S., Scharf, S. j., Higuchi, R., Horn G. T., Mullis, K. B., Ehrlich, H. A.: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 293, 487--491 (1988) Sam brook, j., Fritsch, E., Maniatis, T.: Molecular cloning, a laboratory manual, 2nd ed. Cold Spring HarborlNY, Cold Spring Harbor Laboratory 1989 Sanger, F., Nicklen, S., Coulson, A.: DNA sequencing with chainterminating inhibitors. Proc. Nat. Acad. Sci. USA 74, 5463-5467 (1977) Schleifer, K. H.: DNA probes in food microbiology. Food Biotechno!' 4, 585-598 (1990). Terzaghi, B. E., Sandine, W. E.: Improved medium for lactic streptococci and their bacteriophages. App!. Environ. MicrobioI. 29, 807-813 (1975)

Professor Dr. Karl Heinz Schleifer, Lehrstuhl fiir Mikrobiologie, Technische Universtat Miinchen, Arcisstr. 21, D-8000 Miinchen 2