An improved 16S rRNA based PCR method for the specific detection of Salmonella enterica

International Journal of Food Microbiology 80 (2003) 67 – 75 www.elsevier.com/locate/ijfoodmicro

An improved 16S rRNA based PCR method for the specific detection of Salmonella enterica Marija Trkov, Gorazd Avgusˇtin* Zootechnical Department, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domzˇale, Slovenia Received 15 January 2001; received in revised form 27 August 2001; accepted 10 March 2002

Abstract A molecular method for the detection of Salmonella enterica strains based on 16S rRNA sequence analysis was developed by a modification of the previously described PCR primer 16SFI [J. Appl. Bacteriol. 80 (1996) 659], which was combined with a newly developed primer annealing at the position 66 – 82. Only approximately two thirds of now determined Salmonella 16S rRNA sequences contained a region identical to the 16SFI primer sequence and the reverse primer 16SIII was also not specific. Combined, these two primers have been claimed to allow the specific detection of all Salmonella; however, in this study, they did not recognize S. bongori and 3 out of 78 tested S. enterica strains. They also identified some of the tested Enterobacter cloacae strains as Salmonella. On the contrary, the new primer pair, MINf and MINr, made it possible to recognize correctly all of the 78 tested S. enterica strains, representing 31 different Salmonella serovars. None of the 23 non-Salmonella strains from the related g-proteobacterial genera was incorrectly recognized as belonging to S. enterica. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Molecular detection; Salmonella; PCR; 16S rRNA

1. Introduction The need for the development of rapid and accurate detection methods for Salmonella has been intensified in recent years due to increasing incidence of salmonellosis in industrialised countries over the past decades (Baird-Parker, 1990; Tauxe, 1991; Lewis, 1997). Since the traditional microbiological method for the detection of Salmonella requires up to 5 –7 days, and involves several subcultivation steps followed by biochemical and serological tests, quicker approaches have been searched for, mainly at the DNA level. A *

Corresponding author. Tel.: +386-1-721-78-27; fax: +386-1724-10-05. E-mail address: [email protected] (G. Avgusˇtin).

fairly large number of PCR and hybridization methods for Salmonella detection have been developed as reviewed by Olsen et al. (1995) and Scheu et al. (1998). Since no virulence factor that might be common for all Salmonella strains is known, and the Salmonella strains do not have a common plasmid that could form the basis for a genus-specific probe, the detection methods for Salmonella giving the best results often have been based on the utilisation of randomly cloned fragments (Olsen et al., 1991; Aabo et al., 1993). In 1996, 16S rRNA gene-based PCR primers have been described, which should make the specific detection of Salmonella possible (Lin and Tsen, 1996). The detection systems developed from ribosomal gene sequences have certain advantages when

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 2 ) 0 0 1 3 8 - 1

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Table 1 Salmonella isolates used in this study and the results of the PCR assays Salmonella serovar

Strains and sourcesa

Number of strains tested

Number of positive results with the MINf and MINr primer pair

Number of positive results with the 16SFI and 16SIII primer pair (Lin and Tsen, 1996)

Agona Anatum Bareilly bongori 66:z65:Bovis morbificans Brandenburg Bredeney Chingola Coeln Derby Dublin Enteritidis

SVIa 133, IASa MFa MI8, VFa S-205/90, S-193/90 IASa SSIa JEO 4162 IASa VFa 115/93 SVIa 116 IASa MFa MI9 MFa MI5 CODAa 856SA97 VFa 30/93, 13, 66/2, S-200/90, S-22/95, DVKa P31, CODAa 821SA97, 824SA97, 396SA97, 540SA97, CCMa 4420 VFa 85/94, S-99/94, S-100/94 MFa MI2, CODAa 354SA97, 660SA97, 839SA97, 801SA97 VFa 24/93, S-518/90, BFa 134 IASa VFa 35/94, 52, 57, MFa MI4, MI14, CODAa 733SA97, 1301SA97, 936SA97, 1265SA97 IVZa VFa VFa 56/94, MFa MI10 MFa MI1 RIVMa ALM41, CODAa 453SA97, MFa MI3 MFa MI6 VFa S-13/95, S-20/95 VFa 615/98 MFa MI15 MFa MI12 DVKa P32, CODAa 820SA97, 829SA97, 692SA97, 641SA97, 790SA97, MFa MI16, VFa S-210/90, S-224/90, 428, SVIa 135, CCMa 4419, ATCCa 14028, IASa Stm1 MFa MI7 VFa S-160/90, S-14/90, CODAa 494SA97, IASa sw1

2 3 1 1 1 1 1 1 1 1 1 11

2 3 1 0 1 1 1 1 1 1 1 11

2 3 1 0 1 1 1 1 1 1 1 11

3 5

3 5

3 5

3 1 9

3 1 9

2 0 9

1 1 2 1 2 1 1 2 1 1 1 14

1 1 2 1 2 1 1 2 1 1 1 14

1 1 2 1 2 1 1 2 1 1 1 14

1 3 1

1 3 1

1 3 0

Give Hadar Heidelberg Houten Infantis Liverpool Livingstone Luidelberg Ohio Panama Paratyphi B Reading Saint-paul Senftenberg Takoradi Thompson Typhimurium

Uganda Virchow Weltevreden

a Bacterial isolates were obtained from: The American Type Culture Collection (ATCC), Rockville, MD; University of Ljubljana, Biotechnical Faculty (BF), Food Technology Department, 1000 Ljubljana, Slovenia; Czech Collection of Microorganisms (CCM), Brno, Czech Republic; The Spanish Type Culture Collection (CETC), University of Valencia, Burjasot, Spain; Veterinary and Agrochemical Research Centre (CODA), Brussels, Belgium; Department for Animal Product Quality (DVK), Centre of Agricultural Research, Melle, Belgium; University of Malaya, Institute of Advanced Studies (IAS), Kuala Lumpur, Malaysia; Department of Sanitary Microbiology (IVZ), Institute of Public Health of the Republic of Slovenia, 1000 Ljubljana, Slovenia; Japan Collection of Microorganisms (JCM), The Institute of Physical and Chemical Research, Japan; Laboratorium voor microbiologie universiteit Gent (LMG), Gent, Belgium; University of Ljubljana, Medical Faculty (MF), Institute of Microbiology and Immunology, 1000 Ljubljana, Slovenia; Poultry farm Pivka (PKP), 6257 Pivka, Slovenia; National Institute of Public Health and Environmental Protection (RIVM), Netherlands; Statens Serum Institut (SSI), Division of Microbiology, Copenhagen, Denmark; State Veterinary Institute (SVI), Bratislava, Slovakia; University of Ljubljana, Veterinary Faculty (VF), Institute for Microbiology and Parasitology, 1000 Ljubljana, Slovenia.

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compared to the use of other DNA or RNA targets (Amann et al., 1995). Utilising the fast-growing ribosomal database (Maidak et al., 2000), screening can be performed through a larger number of known sequences in comparison to databases compiling other nucleotide sequences and a search can be carried out for sequence regions which could be used as narrow or broad specific probes or primers. Further, the number of targets may be higher if ribosomal gene sequences are exploited, since most bacteria have multiple ribosomal operons in the genome (Fogel et al., 1999) and therefore the detection could be facilitated. However, the analysis of currently available ribosomal gene sequences from various proteobacteria showed that previously designed PCR primers do not share homology with target regions in all Salmonella. In this study, we present our work on improving the previously designed Salmonella specific 16S rRNA PCR primers.

2. Materials and methods 2.1. Bacterial strains and culture conditions Various Salmonella and non-Salmonella isolates, listed in Tables 1 and 2, were used in the study. The

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cultures were grown overnight in nutrient broth (Oxoid, Basingstoke, UK) with constant shaking (150 rpm), at 37 jC. 2.2. Cell lysis and PCR amplification The method described by Trkov et al. (1999) was used for genomic DNA preparation for PCR amplification. One milliliter of the cell culture was centrifuged at 13,000  g for 5 min and the pellet was washed twice with distilled water. The washed pellet was resuspended in 100 Al of 0.125% sodium dodecylsulfate and 0.05 M NaOH (1:1). The suspension was incubated at 95 jC for 15 min and kept at 20 jC until used. One microliter of the cell lysate was used for PCR amplification. The 20-Al reaction mixture consisted of 15 pmol of each primer (MINf: 5V-ACGGTAACAGGAAGMAG-3V, M = A/C and MINr: 5V-TATTAACCACAACACCT-3V), 1.9 mM MgCl2, 0.2 mM each of dNTP (Pharmacia, Uppsala, Sweden), 0.5 U of GoldStar DNA polymerase (Eurogentec, Seraing, Belgium), 1  PCR buffer, 0.05% Tween 20 and a DNA template. Amplification was carried out in a thermo-cycler (GeneAmp PCR System 2400, Perkin Elmer, USA) using a temperature programme consisting of initial denaturation (90 s at 94 jC), 30 cycles (denaturation for 15

Table 2 Non-Salmonella isolates used in this study and the results of the PCR assays Species

Strains and sources

Number of strains tested

Number of positive results with the MINf and MINr primer pair

Number of positive results with the 16SFI and 16SIII primer pair (Lin and Tsen, 1996)

Citrobacter brakii Citrobacter freundii Enterobacter cloacae Enterobacter sakazakii Escherichia coli Hafnia alvei Klebsiella ornithinolytica Klebsiella oxytoca Klebsiella planticola Klebsiella pneumoniae Pantoea agglomerans Proteus mirabilis Proteus sp. Shigella sp. Yersinia enterocolitica

ATCC 6750 VF 198/97, SVI 142 LMG 2783, PKP 167/1, 168, VF 1126/98 JCM 1233 ATCC 11775, 11229, CECT 515, MF MI20 ATCC 13337 JCM 6096 JCM 1665 JCM 7251 ATCC 13883 JCM 1236 ATCC 14153 ATCC 14159 IVZ IVZ, VF 265/98

1 2 4 1 4 1 1 1 1 1 1 1 1 1 2

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 3 0 0 0 0 0 0 0 0 0 0 0 0

For explanation of origin of strains, see Table 1.

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s at 94 jC, annealing for 10 s at 60 jC, extension for 30 s at 72 jC) and final extension for 4 min at 72 jC. PCR amplifications with primers 16SF and 16SIII were performed as described by Lin and Tsen (1996). For PCR amplification of the almost complete 16S rRNA genes, the universal PCR primers published by Weisburg et al. (1991) (fD1: 5V-AGAGTTTGATCCTGGCTCAG-3V) and Olsen et al. (1986) (1392: 5V-ACGGGCGGTGTGTRC-3V, R = A/G) were used. The 20-Al reaction mixture consisted of 6 pmol of each primer, 1.6 mM MgCl2, 0.2 mM each of dNTP, 0.75 U of GoldStar DNA polymerase, 1  PCR buffer, 0.05% Tween 20 and 1 Al of the cell lysate. Amplification was carried out using a temperature programme consisting of initial denaturation (90 s at 94 jC), 30 cycles (denaturation for 15 s at 94 jC, annealing for 1 min at 60 or 58 jC, extension for 90 s at 72 jC) and final extension for 7 min at 72 jC. The amplification products were examined by electrophoresis in a 1% agarose (Seakem ME, BioWhittaker Molecular Applications, Rockland, USA) gel and 1  TBE buffer at 5 – 7 V cm 1 and documented with the Bio-Rad Gel Doc 1000 Documentation System (BioRad, Hercules, California). 2.3. Sequence analysis The isolated PCR products (genes for 16S rRNA) were purified with QIAquick Gel Extraction Kit (Qiagen, Germany) and sequenced at the Department of Biochemistry, Colorado State University, USA (http://mmr.bmb.colostate.edu). Standard protocol for cycle sequencing using ABI Prism 377 DNA Sequencer was used with following sequencing primers: fD1 (Weisburg et al., 1991), 519f (Lane, 1991) and 907f (Lane, 1991). All reference sequences were obtained from the GenBank database. The sequences were automatically aligned (multiple sequence alignment) by the Clustal W program (Higgins et al., 1992; Thomson et al., 1994), using the default settings.

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2.4. Nucleotide sequence accession numbers The nucleotide sequences of the 16S rRNA genes from the strains Enterobacter cloacae 2783 and S. Heidelberg 134 were submitted to the GenBank nucleotide sequence database, under accession numbers AF276988 and AF276989, respectively.

3. Results and discussion 3.1. Design of the PCR primers Lin and Tsen (1996) have published PCR primers for the specific detection of Salmonella strains based on the 16S rRNA sequence analysis. None of these primers was specific for Salmonella. The primers were previously used as labelled probes and they hybridized to the Citrobacter, Klebsiella and Serratia strains (Lin and Tsen, 1995). However, when used in combination, the primers allowed specific detection of Salmonella. Since then, several new 16S rRNA sequences from various Salmonella strains as well as strains from related genera have been deposited in the DNA databanks. The analysis of sequences available in databanks showed that in approximately one third of the Salmonella sequences deposited mismatches can be found when targeted regions are compared with the sequence of the primer 16SFI, which anneals, according to Escherichia coli numbering, at positions 454 –473 of the 16S rRNA gene (Fig. 1). Seven mismatches were found when analysing 16S rRNA sequences from two S. bongori strains, five mismatches were found when analysing sequences from S. Houten, S. Bareilly, S. Weltewreden and S. Typhymurium, three mismatches were found when analysing sequence from S. Agona, and one mismatch was found when analysing S. Chingola and S. Bovis morbificans sequences. On the other hand, the sequence of the 16SFI primer was identical to two sequences from related bacteria belonging to the genera Pantoea and Enterobacter, whereas only one mismatch could be identified in the sequence from K. planticola.

Fig. 1. Multiple sequence alignment of 16S rRNA sequences for Salmonella and non-Salmonella isolates. Sequence regions from positions 451 – 489 according to E. coli numbering are shown. The target sites for the MINr and 16SF1 primers are shaded. The second column contains database accession numbers of the analysed sequences.

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When analysing the reverse primer 16SIII of Lin and Tsen (1996), which anneals at positions 1008 – 1025 of the 16S rRNA gene according to E. coli numbering, only two of the deposited Salmonella sequences in the GenBank database were found to contain no mismatches with the primer sequence. Other 32 Salmonella sequences had between one and eight mismatches and only 6 of the 32 sequences were found to contain one mismatch only (results not shown). The sequence analysis also showed that by modification of the forward 16SFI primer, the specificity

can be increased, so that the sequence would be identical to all known Salmonella sequences with the exception of the two sequences from the S. bongori strains. This was achieved by using the upstream sequence at positions 451 – 467 of the 16S rRNA gene, starting three nucleotides further upstream than the 16SF1 primer, and being three nucleotides shorter (Fig. 1). Only sequences with a maximum of three mismatches are shown in Fig. 1. Since this sequence was not absolutely specific for Salmonella strains, being identical to sequences from K. planticola, P. agglomerans and E. sakazakii strains (see above), and

Fig. 2. Multiple sequence alignment of 16S rRNA sequences for Salmonella and non-Salmonella isolates. Sequence regions from positions 49 – 88 according to E. coli numbering are shown. The target site for the MINf primer is shaded. The second column contains database accession numbers of the analysed sequences.

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the primer 16SIII was shown to be non-specific, we searched for another region within the 16S rRNA gene that would increase the specificity. The appropriate region was found at position 66 –82 according to E. coli numbering (Fig. 2). Since some Salmonella sequences had adenine and others cytosine at position 80, a forward primer containing both nucleotides at this position in an equimolar ratio was constructed and named MINf. The downstream region described above, annealing at positions 451 – 467, was used for constructing the reverse PCR primer, and was named MINr. Some E. coli strains were found to have the same sequence in the region where the MINf primer anneals, but the reverse complements of sequences of these strains had more than three mismatches when compared to the MINr primer, and should therefore pose no problem for specific amplification. 3.2. The specificity of MINf and MINr The specificity of MINf and MINr was compared with the specificity of the primer pair 16SF1/16SIII by amplification of targeted regions from template DNAs in the cell lysates from the Salmonella and non-Salmonella isolates shown in Tables 1 and 2. Seventy-nine Salmonella strains were analysed, representing 32 different Salmonella serovars, and 23 non-Salmonella strains were analysed as negative controls (these strains were from nine different genera of the family Enter-

Fig. 3. Agarose gel electrophoresis of PCR products amplified with the primers 16SFI and 16SIII. Line M: molecular weight marker (yX 174/HaeIII, Eurogentec, Searing); line 1: S. Heidelberg 134; line 2: S. Weltevreden sw1; line 3: S. Houten; line 4: S. bareilly; line 5: E. cloacae 167/1; line 6: E. cloacae 168; line 7: E. cloacae 2783.

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Fig. 4. Agarose gel electrophoresis of PCR products amplified with the primers MINf and MINr. Line M: molecular weight marker (yX 174/HaeIII, Eurogentec, Searing); line 1: S. Heidelberg 134; line 2: S. Weltevreden sw1; line 3: S. Houten; line 4: S. bareilly; line 5: E. cloacae 167/1; line 6: E. cloacae 168; line 7: E. cloacae 2783; line 8: K. ornithinolytica 6096; line 9: K. planticola 7251; line 10: E. sakazakii 1233.

obacteriaceae, all closely related to the genus Salmonella). MINf and MINr PCR primers generated an approximately 402 bp long PCR product, which was observed with all 78 Salmonella strains belonging to the species S. enterica, as defined by Le Minor and Popoff (1987), but not when the strain S. bongori JEO 4162 and other non-Salmonella strains were analysed (Tables 1 and 2). 16SFI and 16SIII primers, however, did not enable the generation of the expected 575 bp long PCR product with three Salmonella strains belonging to the serovars S. Heidelberg, S. Weltevreden and S. Houten or with the S. bongori JEO 4162 strain. On the other hand, a 575-bp long product was observed when DNA from three different strains out of four tested strains of the species E. cloacae was amplified (Tables 1 and 2, Fig. 3). As stated above, MINf and MINr enabled correct amplification of the targeted regions from DNAs from all S. enterica strains, and did not permit incorrect amplification from non-S. enterica DNAs like the 16SFI and 16SIII primer pair (Fig. 4). In order to find out whether the sequence variation was actually the cause of the incorrect amplification when the 16SFI and 16SIII primer pair was used, we set out to determine the 16S rRNA sequences from the strains S. Heidelberg 134 and E. cloacae 2783. The analysis showed that the sequences of MINf and MINr primers are absolutely complementary to the targeted regions in S. Heidelberg 134, whereas six mismatches were found between the primer 16SIII and the appropriate region in the 16S rRNA gene of S.

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Heidelberg 134. Five mismatches were found when comparing the MINf primer sequence to the E. cloacae 2783 sequence, whereas only one mismatch was found when comparing the 16SFI and 16SIII primer sequences with the sequence of the E. cloacae strain 2783 (not shown). It is therefore obvious that sequence variation is the cause of false results obtained with the primers developed by Lin and Tsen (1996). We can conclude that by modifying one of the PCR primers developed by Lin and Tsen (1996), and by constructing a new PCR primer, a specific PCR detection system for S. enterica strains based on the 16S ribosomal RNA sequences was successfully developed. The fact that the targets are ribosomal genes permits further development of the detection system by modifying the PCR primers into oligonucleotide probes. The latter could then be labelled and used for in situ hybridization techniques, which may in combination with epifluorescent microscopy or flow cytometry (Amann et al., 1995; Davey and Kell, 1996) allow further physiological and ecological studies of this organism. The method described here may be used for the detection of Salmonella in food samples despite being specific for S. enterica only, since S. bongori strains are rare and represent only a minute portion of all Salmonella isolates.

Acknowledgements This work was financially supported by the Slovenian Ministry of Science and Technology. For providing the bacterial strains, we would like to thank Dr. Lieve Herman from the Department of Animal Product Quality, Centre of Agricultural Research, Melle, Belgium, Dr. Hein Imberechts from Veterinary and Agrochemical Research Centre, Brussel, Belgium, Dr. Tomas Kuchta from the Department of Sanitary Microbiology, Food Research Institute, Bratislava, Slovakia, Mrs. Natasˇa Klun from the Institute of Public Health of the Republic of Slovenia, Professor Dr. Janez Mahle from the Institute for Microbiology and Parasitology of the Veterinary Faculty, University of Ljubljana, Slovenia, and Professor Dr. Peter Raspor from Food Technology Department of Biotechnical Faculty, University of Ljubljana, Slovenia.

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