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Clonal relationship among Salmonella enterica serovar Enteritidis involved in foodborne outbreaks in Southern Brazil
Food Control 20 (2009) 606–610
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Short Communication
Clonal relationship among Salmonella enterica serovar Enteritidis involved in foodborne outbreaks in Southern Brazil Fernanda A. de Oliveira a,d,*, Mercedes P. Geimba b, Ana P. Pasqualotto a, Adriano Brandelli a, Giancarlo Pasquali c, Wladimir Padilha da Silva d, Eduardo C. Tondo a a
Departamento de Ciências de Alimentos, Instituto de Ciência e Tecnologia de Alimentos – ICTA/UFRGS, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, Prédio 43212, Campus do Vale, Agronomia, CEP 91501-970, Caixa Postal 15090, Porto Alegre, Brazil b Departamento de Microbiologia, Faculdade de Biociências, Pontificia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil c Departamento de Biologia Molecular e Biotecnologia, Instituto de Biociências e Centro de Biotecnologia, UFRGS, Porto Alegre, Brazil d Departamento de Ciência e Tecnologia Agroindustrial, Faculdade de Agronomia, Universidade Federal de Pelotas – UFPel, Pelotas, Brazil
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Article history: Received 26 November 2007 Received in revised form 11 August 2008 Accepted 19 August 2008
Keywords: Salmonella PFGE PCR-ribotyping Foodborne
a b s t r a c t The aim of the present study was to investigate the clonal relationship among Salmonella enterica serovar Enteritidis involved in foodborne outbreaks occurred in Rio Grande do Sul State (RS), Southern Brazil. S. enterica ser. Enteritidis (n = 152) isolated from food involved in salmonellosis were analyzed by PCRribotyping and PFGE. To confirm the clonal relationship, a PCR-ribotyping amplicon was sequenced in 26 randomly chosen S. enterica ser. Enteritidis typed. The results indicate that a specific strain of S. enterica ser. Enteritidis or a very closely related clone of this microorganism was involved in salmonellosis outbreaks occurred in RS. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Human infections by Salmonella Enteritidis have been increased world-wide over the recent years and epidemiological studies have generally implicated the consumption of meat, poultry, eggs and egg products with these foodborne diseases (Boonmar et al., 1998; Rodrigue, Tauxe, & Rowe, 1990). The World Health Organization Global Salm-Surv network reported that in the period of 2000–2002, Salmonella enterica serovar Enteritidis was by far the most common serotype reported from human sources worldwide, accounting for 65% of the isolates (Galanis et al., 2006; Herikstad, Motarjemi, & Tauxe, 2002). In Brazil, an impressive increase in the incidence of this serotype has been reported in the last decade (Fuzihara, Fernandes, & Franco, 2000; Santos, Junior, Fernandes, Tavechio, & Amaral, 2000; Tavechio, Fernándes, Neves, Dias, & Irino, 1996; Tavechio et al., 2002). In RS, a 10 million-people State of the Southern of Brazil, S. enterica ser. Enteritidis was pointed out as the main serotype isolated from foods involved in foodborne outbreaks (Geimba, Tondo, Oliveira, Canal, & Brandelli, 2004). Due to
* Corresponding author. Address: Departamento de Ciências de Alimentos, Instituto de Ciência e Tecnologia de Alimentos – ICTA/UFRGS, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, Prédio 43212, Campus do Vale, Agronomia, CEP 91501-970, Caixa Postal 15090, Porto Alegre, Brazil. E-mail address: [email protected] (F.A. de Oliveira). 0956-7135/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2008.08.014
the increased number of isolations and the public health significance of this serovar in RS, epidemiologic studies have been conducted, aiming to investigate the transmission routes and to characterize the involved strains. Based on these facts, the objective of the present study was to characterize, by PCR-ribotyping and PFGE, S. enterica ser. Enteritidis isolated from foods involved in foodborne outbreaks occurred in the Southern Brazil, during 1999–2002. 2. Materials and methods Strains were isolated from foods involved with foodborne salmonellosis outbreaks that occurred in different localities of RS, in the period of 1999–2002. The outbreaks were investigated by the Division of Sanitary Surveillance Service (DVS/RS) that carried out sample collection and transported suspected food to the Central Laboratory of Rio Grande do Sul (FEPPS/LACEN/RS) or accredited laboratories. Isolates identified as S. enterica ser. Enteritidis were transported to the Instituto de Ciência e Tecnologia de Alimentos (ICTA/UFRGS) for further analysis. 2.1. PCR-ribotyping The primers used have been previously proposed by Jensen, Webster, and Straus (1993) and were specific for the amplification
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of the spacer region between 16S and 23S rRNA genes. Primer sequences were 50 CAA GGC ATC CAC CGT GT30 and 50 GTG AAG TCG TAA CAA GG30 . Each set of PCRs included a control reaction without template DNA. Every reaction in 25 ll contained 2.5 ll of reaction buffer (100 mmol l1 Tris–HCl pH 8.8, 750 mmol l1 KCl), 2 ll of 10 mmol l1 dNTPs, 1 ll of 50 mmol l1 MgCl2, 2 ll of each primer (20 pmol), 1 U of Taq DNA Polymerase (Biotools, Madrid, Spain), 13.5 ll sterile ultrapure water, and 2 ll of DNA extracted for boiling. The PCR-ribotyping program consisted of a preincubation cycle at 94 °C for 2 min, followed by 25 cycles of 94 °C for 15 s, 55 °C for 4 min and 72 °C for 1 min, and a final extension step at 72 °C for 30 min. All PCRs were performed in a Minicycler (MJ Research, Watertown, MA, USA) and the amplification products were separated by horizontal electrophoresis through 1% agarose gels. The gels were stained with ethidium bromide and visualized under UV light. DNA samples were tested three or more times to evaluate the reproducibility of the method. The discriminatory power of the PCR-ribotyping method was tested by analyzing strains of other Gram negative bacteria as Pseudomonas spp., Escherichia coli, Proteus mirabilis, S. enterica ser. Gallinarum, and S. enterica ser. Typhimurium. Minor differences in band intensity as well as weak bands were not considered to define PCR-ribotypes. Isolates were ascribed to the same PCR-ribotype when their DNA amplified banding patterns were identical. 2.2. PFGE The methods described previously for bacterial human isolates were used (Pang, Chiu, Chiou, Schroeter, & Tsen, 2005; Tsen & Lin, 2001). Briefly, Salmonella cells in 0.1 ml of the cell culture grown in 5 ml of LB broth at 37 °C for 3.5 h were pelleted, washed, resuspended in the same buffer, heated, and mixed with 2% agarose in lysozyme buffer. The mixture was poured into the slots of a plastic mold (Bio-Rad CHEF DRII, Hercules, CA, USA), cooled, and the agarose plugs were transferred to lysozyme solutions containing 2 mg ml1 of lysozyme and cells were lysed for 24 h. The lysis buffer was then replaced by a proteolysis buffer containing 0.1 mg ml1 proteinase K and proteolysis was carried out overnight. Afterwards, the agarose plugs were washed, followed by restriction digestion with XbaI (Invitrogen). After digestion, the plugs were placed into the slots of a 1.2% agarose gel (Bio-Rad, Pulsed field grade) in 0.5 TBE buffer (1 TBE: 89 mM Trisborate, pH 8.3, 2 mM EDTA) and electrophoresis was carried out using CHEF DRII PFGE system (Bio-Rad). The conditions used were 6 V/cm for 18 h at 14 °C with pulsed time ranging from 3 to 63 seg. The XbaI fragments of S. enterica ser. Typhimurium LT2 were used as molecular weight markers (Liu, Hessel, & Sanderson, 1993). The gels were stained with ethidium bromide and photographed. For some strains, PFGE analysis was repeated if the gel patterns were unclear. In addition, for each PFGE pattern, at least one randomly selected isolate was re-typed for the reproducibility of the result in each PFGE batch. PFGE patterns obtained were analyzed by visual assessment. Differences in patterns were judged according to the method described by Thong, Ngeow, Altwegg, Navaratnam, and Pank (1995), Laconcha et al. (1998) and Fernandez et al. (2003). Patterns differing by one band were considered as distinct PFGE patterns.
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2.4. DNA sequencing and analysis Samples were sequenced in the ACTGene Laboratory (Centro de Biotecnologia, UFRGS, Porto Alegre, RS, Brazil) using the automatic sequencer ABI-PRISM 3100 Genetic Analyzer armed with 50 cm capillaries and POP6 polymer (Applied Biosystems, Foster City, USA). Cycle sequencing was carried out using 3.2 pmol of each of the primers described above and 2 ll of BigDye Terminator v3.1 Cycle Sequencing RR-100 (Applied Biosystems) in a final volume of 10 ll. Labeling reactions were performed in a GeneAmp PCR System 9700 (Applied Biosystems) thermocycler with a initial denaturing step of 96 °C for 3 min followed by 25 cycles of 96 °C for 10 seg, 55 °C for 5 seg1 and 60 °C for 4 min. Labeled samples were purified by isopropanol precipitation followed by 70% ethanol rinsing. Precipitated products were suspended in 10 ll formamide, denatured at 95 °C for 5 min, ice-cooled for 5 min and eletroinjected in the automatic sequencer. Sequencing data were collected using the software Data Collection v1.0.1 (Applied Biosystems) programmed with the following parameters: Dye Set ‘‘z”; mobility File ‘‘DT3100POP6 {BDv3}v1.mob”; BioLIMS Project ‘‘3100_Project1”; Run Module 1 ‘‘StdSeq50 POP6 50 cm cfv 100”; and Analysis Module 1 ‘‘BC-3100SR Seq FASTA.saz”. A BLAST search of the nucleotide sequences of S. enterica ser. Enteritidis 16S–23S intergenic spacer regions containing the tRNA-Ile and tRNA-Ala genes (accession no. Z96979) and the tRNA-Glu gene (accession no. Z96978) was conducted through the National Center for Biotechnology Information homepage (http://www.ncbi.nlm.nih.gov/). Phylogenetic and molecular evolutionary analyses were conducted using the Molecular Evolutionary Genetics Analysis (MEGA) software version 3.0. A dendrogram was constructed using the unweighted pair group method with arithmetic mean (UPGMA) method (Sneath & Sokal’s, 1973), and its reliability was tested by bootstrap with 1000 replications (Hedges, Parker, Sibley, & Kumar, 1996). 3. Results 3.1. PCR-ribotyping PCR-ribotyping analysis was able to discriminate other bacterial genera from Salmonella serovars since samples containing DNA of Pseudomonas spp., E. coli, P. mirabilis, S. enterica ser. Gallinarum, and S. enterica ser. Typhimurium yielded distinct banding patterns. The PCR-ribotyping technique produced very reproducible DNA banding patterns for S. enterica ser. Enteritidis strains. Minor differences in the intensity of bands were observed when the same strain was submitted to repeated amplifications, but it did not compromise the accuracy of discrimination. Among the 152 S. enterica ser. Enteritidis strains examined, two banding patterns were identified, being designated R1 and R2 (Fig. 1). Profile R1 grouped 94.7% (n = 144) of the isolates and presented a banding pattern composed by four bands of approximately 300, 400, 600, and 800 bp. Profile R2 grouped 5.3% (n = 8) of the strains, presenting a banding pattern of only two bands (400 and 600 bp). The bands of about 400 and 600 bp were detected in all strains tested. 3.2. PFGE
2.3. Purification of PCR products A fragment of approximately 600 bp present in all PCR-ribotyping profiles was removed from the agarose gels and purified with the ConcertTM Matrix Gel Extraction System (Gibco.BRL) according to manufacturer’s protocol.
During PFGE analysis, XbaI digestion revealed five DNA banding profiles, which comprising 11–13 bands with sizes varying between 50 and 800 kb (Fig. 2). The predominant profile (P1), presenting 12 bands, and grouped 92.8% of the strains (n = 141). Two patterns were represented by only one strain, which showed
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Fig. 1. PCR-ribotyping profiles of Salmonella Enteritidis isolated from foods involved in foodborne outbreaks occurred in Rio Grande do Sul State, South of Brazil, during the years 1999 and 2002. Lane M: molecular size marker (100 bp DNA ladder, Invitrogen).
able region was positioned in 363–463 bp. Sequence variations were very restricted and they seemed to be caused by minor deletions/insertions and individual base mutations. Strains 2048 and 2055, which were classified as R2, showed 100% similarity. When these strains were compared to the other S. enterica ser. Enteritidis sequenced, the similarities varied from 98% to 100%, suggesting high similarity among the microorganisms. To improve the understanding of the molecular structure of the spacer regions and to evaluate the presence of tRNA genes, samples were aligned with sequences of the tRNA-Ile and tRNA-Ala genes (present in the rrnH operon), and the tRNA-Glu gene (present in the rrnE operon) of S. enterica ser. Enteritidis described in the GenBank. All 16S–23S intergenic regions sequences presented the tRNA-Ile and the tRNA-Ala genes. The S. enterica ser. Enteritidis rrnH operon sequence described in GenBank demonstrated homology to the sequences studied. The presence of tRNA-Glu gene was not found among the strains analyzed. Fig. 2. Pulsed field gel electrophoresis (PFGE) patterns for XbaI-digested genomic DNA of Salmonella Enteritidis strains from foods. Lane numbers correspond to the different patterns. Lane 4 contains a re-typed strain of lane 1. Lane M contains XbaIdigested DNA of Salmonella Typhimurium LT2.
13 bands. Profile P1 grouped strains isolated from different regions of RS, not allowing the discrimination among these isolates. Among 144 strains grouped in R1 profile, 97.9% (n = 141) were also classified as P1, suggesting a clonal relationship among the majority of the S. enterica ser. Enteritidis analyzed. No R2 strains were grouped in P1, however, three strains classified as R1 were sub-classified as P2, P4, and P5, demonstrating the higher discrimination power of the PFGE method. 3.3. DNA sequencing The 600 bp DNA fragment generated by PCR-ribotyping of 26 strains of S. enterica ser. Enteritidis were sequenced. The strains analyzed were randomly chosen among the 152 S. enterica ser. Enteritidis. According to the sequencing results, the strains studied in this work were clearly grouped into a single cluster. All samples showed high similarity, varying from 98% to 100%. Sequenced nucleotides were numbered from 1 to 528 bp. Regions from 82 to 194 bp, 196 to 285 bp, 287 to 362 bp, 372 to 402 bp, 318 to 425 bp, 430 to 461 bp and 463 to 528 bp were identical in all S. enterica ser. Enteritidis strains tested. The most vari-
4. Discussion Prior to the analysis of S. enterica ser. Enteritidis strains, the discrimination power of the PCR-ribotyping method was evaluated analyzing non-S. enterica ser. Enteritidis bacteria. PCR-ribotyping was able to generate different banding profiles for the other bacteria tested, suggesting an appropriate discrimination power to type S. enterica ser. Enteritidis strains at the serotype level. The similarity of the results obtained by PCR-ribotyping and PFGE analysis supports the adequacy of PCR-ribotyping to study the epidemiological relationship among S. enterica ser. Enteritidis strains. The PCR-ribotyping method also presented very reproducible results, in a short time, even when performed by different persons or in diverse equipments (results not shown). In this study, the primers developed by Jensen et al. (1993) and after used by Jensen and Hubner (1996) were able to type strains of S. enterica ser. Enteritidis involved in foodborne outbreaks and presented similar results described by other studies (Baudart, Lemarchand, Brisabois, & Lebaron, 2000; Lagatolla et al., 1996). The amplification of the intergenic spacer sequences between the 16S and 23S rRNA genes has proved to be relevant for typing bacteria (De Cesare, Manfreda, Dambaugh, guerzoni, & Franchini, 2001), because these regions show extensive sequence and length variation that can be used to characterize bacteria at the genus, species and subspecies level (Dolzani, Tonin, Lagatolla, Prandin, & Monti-Bragadin, 1995; Kostman, Edlind, LiPuma, & Stull, 1992).
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Generally, the use of diverse typing methods is recommended to confirm the clonal relationship among different microorganisms. PFGE and DNA sequencing could be very useful especially when the target microorganism presents a low genetic variability, as S. enterica ser. Enteritidis. Our results demonstrated that strains with a predominant PFGE profile were also classified in a PCR-ribotyping banding pattern, suggesting that the same strain of S. enterica ser. Enteritidis was involved with different salmonellosis outbreaks. Aiming to confirm the clonal similarity of these microorganisms, DNA sequencing was carried out in this study. Christensen, Moller, Vogensen, and Olsen (2000) have indicated that typing of bacteria by DNA sequence comparison is beneficial because of its high precision. The same researchers reported that the investigation by cloning all of the rnn operons would be too laborious to employ such technique for routine application, suggesting that one alternative strategy would consist in sequencing individual operons using PCR techniques, as was carried out in the present work. The sequencing of a 600 bp PCR-ribotyping fragment generated in our study revealed DNA sequences with high degrees of similarity. This result corroborates the finding of a low number of PCRribotyping and PFGE profiles identified, suggesting a high genetic similarity among the S. enterica ser. Enteritidis strains studied. The low polymorphism observed among the sequences suggests that they are probably the result of point insertions and/or deletions affecting blocks of nucleotides, mainly in the region between 363 and 463 bp. Because rRNA intergenic spacer regions are more subject to genetic drift than the 16S rRNA and the 23S rRNA genes (Hain, Ward-Rainey, Kroppensted, Stackebrandt, & Rainey, 1997), and since such variations have been used as identification and typing tools (Gurtler & Stanisich, 1996), the low variation found in the sequences studied by us suggests that the strains analyzed may originate from a unique ancestor. Other studies also have demonstrated low levels of genetic diversity of S. enterica ser. Enteritidis isolated in different parts of the world (Liebisch & Schwarz, 1996; Maré, Dicks, & van der Walt, 2001). Confirming these findings, Pang et al. (2007) suggested a major world-wide clone of S. enterica ser. Enteritidis based on PFGE analysis. The presence of the tRNA-Ala and the tRNA-Ile genes were verified in all the strains sequenced in the present work. However, none of them presented the tRNA-Glu gene. Similar results were reported by Chiu, Chen, Hwang, and Tsen (2005). Christensen et al. (2000) found the genes enconding for tRNA-Ile, tRNA-Ala and tRNA-Glu in the intergenic spacer regions of 25 S. enterica strains belonging to 18 different serovars. In the last decades, S. enterica ser. Enteritidis has emerged as the main causative agent of gastroenteritis in humans in diverse countries (Chmielewski, Wieliczko, Kuczkowski, Mazurkiewicz, & Ugorski, 2002; Muresu et al., 2001), including Brazil (Tavechio et al., 1996; Peresi et al., 1998). In previous works, Salmonella spp. was reported as the main cause of foodborne diseases in RS (Costalunga & Tondo, 2002; Silveira & Tondo, 2006). In addition, S. enterica ser. Enteritidis was the serovar mostly isolated from foods involved with salmonellosis in this Brazilian State (Geimba et al., 2004). Considering that Brazil is currently one of the world’s most important poultry and bovine meat producers, the knowledge about what kind of strains are causing salmonellosis assume great importance. The results of the present study suggested the spread of a specific strain, or closely related strains of S. enterica ser. Enteritidis among several cities of the RS State. Acknowledgements We thank to FAPERGS – Fundação de Amparo à Pesquisa do Rio Grande do Sul for the financing support of this Project. We would like to thank to Dr. Suzana Costalunga, Dr. Josete Baialardi Silveira,
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Short Communication
Clonal relationship among Salmonella enterica serovar Enteritidis involved in foodborne outbreaks in Southern Brazil Fernanda A. de Oliveira a,d,*, Mercedes P. Geimba b, Ana P. Pasqualotto a, Adriano Brandelli a, Giancarlo Pasquali c, Wladimir Padilha da Silva d, Eduardo C. Tondo a a
Departamento de Ciências de Alimentos, Instituto de Ciência e Tecnologia de Alimentos – ICTA/UFRGS, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, Prédio 43212, Campus do Vale, Agronomia, CEP 91501-970, Caixa Postal 15090, Porto Alegre, Brazil b Departamento de Microbiologia, Faculdade de Biociências, Pontificia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil c Departamento de Biologia Molecular e Biotecnologia, Instituto de Biociências e Centro de Biotecnologia, UFRGS, Porto Alegre, Brazil d Departamento de Ciência e Tecnologia Agroindustrial, Faculdade de Agronomia, Universidade Federal de Pelotas – UFPel, Pelotas, Brazil
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Article history: Received 26 November 2007 Received in revised form 11 August 2008 Accepted 19 August 2008
Keywords: Salmonella PFGE PCR-ribotyping Foodborne
a b s t r a c t The aim of the present study was to investigate the clonal relationship among Salmonella enterica serovar Enteritidis involved in foodborne outbreaks occurred in Rio Grande do Sul State (RS), Southern Brazil. S. enterica ser. Enteritidis (n = 152) isolated from food involved in salmonellosis were analyzed by PCRribotyping and PFGE. To confirm the clonal relationship, a PCR-ribotyping amplicon was sequenced in 26 randomly chosen S. enterica ser. Enteritidis typed. The results indicate that a specific strain of S. enterica ser. Enteritidis or a very closely related clone of this microorganism was involved in salmonellosis outbreaks occurred in RS. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Human infections by Salmonella Enteritidis have been increased world-wide over the recent years and epidemiological studies have generally implicated the consumption of meat, poultry, eggs and egg products with these foodborne diseases (Boonmar et al., 1998; Rodrigue, Tauxe, & Rowe, 1990). The World Health Organization Global Salm-Surv network reported that in the period of 2000–2002, Salmonella enterica serovar Enteritidis was by far the most common serotype reported from human sources worldwide, accounting for 65% of the isolates (Galanis et al., 2006; Herikstad, Motarjemi, & Tauxe, 2002). In Brazil, an impressive increase in the incidence of this serotype has been reported in the last decade (Fuzihara, Fernandes, & Franco, 2000; Santos, Junior, Fernandes, Tavechio, & Amaral, 2000; Tavechio, Fernándes, Neves, Dias, & Irino, 1996; Tavechio et al., 2002). In RS, a 10 million-people State of the Southern of Brazil, S. enterica ser. Enteritidis was pointed out as the main serotype isolated from foods involved in foodborne outbreaks (Geimba, Tondo, Oliveira, Canal, & Brandelli, 2004). Due to
* Corresponding author. Address: Departamento de Ciências de Alimentos, Instituto de Ciência e Tecnologia de Alimentos – ICTA/UFRGS, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, Prédio 43212, Campus do Vale, Agronomia, CEP 91501-970, Caixa Postal 15090, Porto Alegre, Brazil. E-mail address: [email protected] (F.A. de Oliveira). 0956-7135/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2008.08.014
the increased number of isolations and the public health significance of this serovar in RS, epidemiologic studies have been conducted, aiming to investigate the transmission routes and to characterize the involved strains. Based on these facts, the objective of the present study was to characterize, by PCR-ribotyping and PFGE, S. enterica ser. Enteritidis isolated from foods involved in foodborne outbreaks occurred in the Southern Brazil, during 1999–2002. 2. Materials and methods Strains were isolated from foods involved with foodborne salmonellosis outbreaks that occurred in different localities of RS, in the period of 1999–2002. The outbreaks were investigated by the Division of Sanitary Surveillance Service (DVS/RS) that carried out sample collection and transported suspected food to the Central Laboratory of Rio Grande do Sul (FEPPS/LACEN/RS) or accredited laboratories. Isolates identified as S. enterica ser. Enteritidis were transported to the Instituto de Ciência e Tecnologia de Alimentos (ICTA/UFRGS) for further analysis. 2.1. PCR-ribotyping The primers used have been previously proposed by Jensen, Webster, and Straus (1993) and were specific for the amplification
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of the spacer region between 16S and 23S rRNA genes. Primer sequences were 50 CAA GGC ATC CAC CGT GT30 and 50 GTG AAG TCG TAA CAA GG30 . Each set of PCRs included a control reaction without template DNA. Every reaction in 25 ll contained 2.5 ll of reaction buffer (100 mmol l1 Tris–HCl pH 8.8, 750 mmol l1 KCl), 2 ll of 10 mmol l1 dNTPs, 1 ll of 50 mmol l1 MgCl2, 2 ll of each primer (20 pmol), 1 U of Taq DNA Polymerase (Biotools, Madrid, Spain), 13.5 ll sterile ultrapure water, and 2 ll of DNA extracted for boiling. The PCR-ribotyping program consisted of a preincubation cycle at 94 °C for 2 min, followed by 25 cycles of 94 °C for 15 s, 55 °C for 4 min and 72 °C for 1 min, and a final extension step at 72 °C for 30 min. All PCRs were performed in a Minicycler (MJ Research, Watertown, MA, USA) and the amplification products were separated by horizontal electrophoresis through 1% agarose gels. The gels were stained with ethidium bromide and visualized under UV light. DNA samples were tested three or more times to evaluate the reproducibility of the method. The discriminatory power of the PCR-ribotyping method was tested by analyzing strains of other Gram negative bacteria as Pseudomonas spp., Escherichia coli, Proteus mirabilis, S. enterica ser. Gallinarum, and S. enterica ser. Typhimurium. Minor differences in band intensity as well as weak bands were not considered to define PCR-ribotypes. Isolates were ascribed to the same PCR-ribotype when their DNA amplified banding patterns were identical. 2.2. PFGE The methods described previously for bacterial human isolates were used (Pang, Chiu, Chiou, Schroeter, & Tsen, 2005; Tsen & Lin, 2001). Briefly, Salmonella cells in 0.1 ml of the cell culture grown in 5 ml of LB broth at 37 °C for 3.5 h were pelleted, washed, resuspended in the same buffer, heated, and mixed with 2% agarose in lysozyme buffer. The mixture was poured into the slots of a plastic mold (Bio-Rad CHEF DRII, Hercules, CA, USA), cooled, and the agarose plugs were transferred to lysozyme solutions containing 2 mg ml1 of lysozyme and cells were lysed for 24 h. The lysis buffer was then replaced by a proteolysis buffer containing 0.1 mg ml1 proteinase K and proteolysis was carried out overnight. Afterwards, the agarose plugs were washed, followed by restriction digestion with XbaI (Invitrogen). After digestion, the plugs were placed into the slots of a 1.2% agarose gel (Bio-Rad, Pulsed field grade) in 0.5 TBE buffer (1 TBE: 89 mM Trisborate, pH 8.3, 2 mM EDTA) and electrophoresis was carried out using CHEF DRII PFGE system (Bio-Rad). The conditions used were 6 V/cm for 18 h at 14 °C with pulsed time ranging from 3 to 63 seg. The XbaI fragments of S. enterica ser. Typhimurium LT2 were used as molecular weight markers (Liu, Hessel, & Sanderson, 1993). The gels were stained with ethidium bromide and photographed. For some strains, PFGE analysis was repeated if the gel patterns were unclear. In addition, for each PFGE pattern, at least one randomly selected isolate was re-typed for the reproducibility of the result in each PFGE batch. PFGE patterns obtained were analyzed by visual assessment. Differences in patterns were judged according to the method described by Thong, Ngeow, Altwegg, Navaratnam, and Pank (1995), Laconcha et al. (1998) and Fernandez et al. (2003). Patterns differing by one band were considered as distinct PFGE patterns.
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2.4. DNA sequencing and analysis Samples were sequenced in the ACTGene Laboratory (Centro de Biotecnologia, UFRGS, Porto Alegre, RS, Brazil) using the automatic sequencer ABI-PRISM 3100 Genetic Analyzer armed with 50 cm capillaries and POP6 polymer (Applied Biosystems, Foster City, USA). Cycle sequencing was carried out using 3.2 pmol of each of the primers described above and 2 ll of BigDye Terminator v3.1 Cycle Sequencing RR-100 (Applied Biosystems) in a final volume of 10 ll. Labeling reactions were performed in a GeneAmp PCR System 9700 (Applied Biosystems) thermocycler with a initial denaturing step of 96 °C for 3 min followed by 25 cycles of 96 °C for 10 seg, 55 °C for 5 seg1 and 60 °C for 4 min. Labeled samples were purified by isopropanol precipitation followed by 70% ethanol rinsing. Precipitated products were suspended in 10 ll formamide, denatured at 95 °C for 5 min, ice-cooled for 5 min and eletroinjected in the automatic sequencer. Sequencing data were collected using the software Data Collection v1.0.1 (Applied Biosystems) programmed with the following parameters: Dye Set ‘‘z”; mobility File ‘‘DT3100POP6 {BDv3}v1.mob”; BioLIMS Project ‘‘3100_Project1”; Run Module 1 ‘‘StdSeq50 POP6 50 cm cfv 100”; and Analysis Module 1 ‘‘BC-3100SR Seq FASTA.saz”. A BLAST search of the nucleotide sequences of S. enterica ser. Enteritidis 16S–23S intergenic spacer regions containing the tRNA-Ile and tRNA-Ala genes (accession no. Z96979) and the tRNA-Glu gene (accession no. Z96978) was conducted through the National Center for Biotechnology Information homepage (http://www.ncbi.nlm.nih.gov/). Phylogenetic and molecular evolutionary analyses were conducted using the Molecular Evolutionary Genetics Analysis (MEGA) software version 3.0. A dendrogram was constructed using the unweighted pair group method with arithmetic mean (UPGMA) method (Sneath & Sokal’s, 1973), and its reliability was tested by bootstrap with 1000 replications (Hedges, Parker, Sibley, & Kumar, 1996). 3. Results 3.1. PCR-ribotyping PCR-ribotyping analysis was able to discriminate other bacterial genera from Salmonella serovars since samples containing DNA of Pseudomonas spp., E. coli, P. mirabilis, S. enterica ser. Gallinarum, and S. enterica ser. Typhimurium yielded distinct banding patterns. The PCR-ribotyping technique produced very reproducible DNA banding patterns for S. enterica ser. Enteritidis strains. Minor differences in the intensity of bands were observed when the same strain was submitted to repeated amplifications, but it did not compromise the accuracy of discrimination. Among the 152 S. enterica ser. Enteritidis strains examined, two banding patterns were identified, being designated R1 and R2 (Fig. 1). Profile R1 grouped 94.7% (n = 144) of the isolates and presented a banding pattern composed by four bands of approximately 300, 400, 600, and 800 bp. Profile R2 grouped 5.3% (n = 8) of the strains, presenting a banding pattern of only two bands (400 and 600 bp). The bands of about 400 and 600 bp were detected in all strains tested. 3.2. PFGE
2.3. Purification of PCR products A fragment of approximately 600 bp present in all PCR-ribotyping profiles was removed from the agarose gels and purified with the ConcertTM Matrix Gel Extraction System (Gibco.BRL) according to manufacturer’s protocol.
During PFGE analysis, XbaI digestion revealed five DNA banding profiles, which comprising 11–13 bands with sizes varying between 50 and 800 kb (Fig. 2). The predominant profile (P1), presenting 12 bands, and grouped 92.8% of the strains (n = 141). Two patterns were represented by only one strain, which showed
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Fig. 1. PCR-ribotyping profiles of Salmonella Enteritidis isolated from foods involved in foodborne outbreaks occurred in Rio Grande do Sul State, South of Brazil, during the years 1999 and 2002. Lane M: molecular size marker (100 bp DNA ladder, Invitrogen).
able region was positioned in 363–463 bp. Sequence variations were very restricted and they seemed to be caused by minor deletions/insertions and individual base mutations. Strains 2048 and 2055, which were classified as R2, showed 100% similarity. When these strains were compared to the other S. enterica ser. Enteritidis sequenced, the similarities varied from 98% to 100%, suggesting high similarity among the microorganisms. To improve the understanding of the molecular structure of the spacer regions and to evaluate the presence of tRNA genes, samples were aligned with sequences of the tRNA-Ile and tRNA-Ala genes (present in the rrnH operon), and the tRNA-Glu gene (present in the rrnE operon) of S. enterica ser. Enteritidis described in the GenBank. All 16S–23S intergenic regions sequences presented the tRNA-Ile and the tRNA-Ala genes. The S. enterica ser. Enteritidis rrnH operon sequence described in GenBank demonstrated homology to the sequences studied. The presence of tRNA-Glu gene was not found among the strains analyzed. Fig. 2. Pulsed field gel electrophoresis (PFGE) patterns for XbaI-digested genomic DNA of Salmonella Enteritidis strains from foods. Lane numbers correspond to the different patterns. Lane 4 contains a re-typed strain of lane 1. Lane M contains XbaIdigested DNA of Salmonella Typhimurium LT2.
13 bands. Profile P1 grouped strains isolated from different regions of RS, not allowing the discrimination among these isolates. Among 144 strains grouped in R1 profile, 97.9% (n = 141) were also classified as P1, suggesting a clonal relationship among the majority of the S. enterica ser. Enteritidis analyzed. No R2 strains were grouped in P1, however, three strains classified as R1 were sub-classified as P2, P4, and P5, demonstrating the higher discrimination power of the PFGE method. 3.3. DNA sequencing The 600 bp DNA fragment generated by PCR-ribotyping of 26 strains of S. enterica ser. Enteritidis were sequenced. The strains analyzed were randomly chosen among the 152 S. enterica ser. Enteritidis. According to the sequencing results, the strains studied in this work were clearly grouped into a single cluster. All samples showed high similarity, varying from 98% to 100%. Sequenced nucleotides were numbered from 1 to 528 bp. Regions from 82 to 194 bp, 196 to 285 bp, 287 to 362 bp, 372 to 402 bp, 318 to 425 bp, 430 to 461 bp and 463 to 528 bp were identical in all S. enterica ser. Enteritidis strains tested. The most vari-
4. Discussion Prior to the analysis of S. enterica ser. Enteritidis strains, the discrimination power of the PCR-ribotyping method was evaluated analyzing non-S. enterica ser. Enteritidis bacteria. PCR-ribotyping was able to generate different banding profiles for the other bacteria tested, suggesting an appropriate discrimination power to type S. enterica ser. Enteritidis strains at the serotype level. The similarity of the results obtained by PCR-ribotyping and PFGE analysis supports the adequacy of PCR-ribotyping to study the epidemiological relationship among S. enterica ser. Enteritidis strains. The PCR-ribotyping method also presented very reproducible results, in a short time, even when performed by different persons or in diverse equipments (results not shown). In this study, the primers developed by Jensen et al. (1993) and after used by Jensen and Hubner (1996) were able to type strains of S. enterica ser. Enteritidis involved in foodborne outbreaks and presented similar results described by other studies (Baudart, Lemarchand, Brisabois, & Lebaron, 2000; Lagatolla et al., 1996). The amplification of the intergenic spacer sequences between the 16S and 23S rRNA genes has proved to be relevant for typing bacteria (De Cesare, Manfreda, Dambaugh, guerzoni, & Franchini, 2001), because these regions show extensive sequence and length variation that can be used to characterize bacteria at the genus, species and subspecies level (Dolzani, Tonin, Lagatolla, Prandin, & Monti-Bragadin, 1995; Kostman, Edlind, LiPuma, & Stull, 1992).
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Generally, the use of diverse typing methods is recommended to confirm the clonal relationship among different microorganisms. PFGE and DNA sequencing could be very useful especially when the target microorganism presents a low genetic variability, as S. enterica ser. Enteritidis. Our results demonstrated that strains with a predominant PFGE profile were also classified in a PCR-ribotyping banding pattern, suggesting that the same strain of S. enterica ser. Enteritidis was involved with different salmonellosis outbreaks. Aiming to confirm the clonal similarity of these microorganisms, DNA sequencing was carried out in this study. Christensen, Moller, Vogensen, and Olsen (2000) have indicated that typing of bacteria by DNA sequence comparison is beneficial because of its high precision. The same researchers reported that the investigation by cloning all of the rnn operons would be too laborious to employ such technique for routine application, suggesting that one alternative strategy would consist in sequencing individual operons using PCR techniques, as was carried out in the present work. The sequencing of a 600 bp PCR-ribotyping fragment generated in our study revealed DNA sequences with high degrees of similarity. This result corroborates the finding of a low number of PCRribotyping and PFGE profiles identified, suggesting a high genetic similarity among the S. enterica ser. Enteritidis strains studied. The low polymorphism observed among the sequences suggests that they are probably the result of point insertions and/or deletions affecting blocks of nucleotides, mainly in the region between 363 and 463 bp. Because rRNA intergenic spacer regions are more subject to genetic drift than the 16S rRNA and the 23S rRNA genes (Hain, Ward-Rainey, Kroppensted, Stackebrandt, & Rainey, 1997), and since such variations have been used as identification and typing tools (Gurtler & Stanisich, 1996), the low variation found in the sequences studied by us suggests that the strains analyzed may originate from a unique ancestor. Other studies also have demonstrated low levels of genetic diversity of S. enterica ser. Enteritidis isolated in different parts of the world (Liebisch & Schwarz, 1996; Maré, Dicks, & van der Walt, 2001). Confirming these findings, Pang et al. (2007) suggested a major world-wide clone of S. enterica ser. Enteritidis based on PFGE analysis. The presence of the tRNA-Ala and the tRNA-Ile genes were verified in all the strains sequenced in the present work. However, none of them presented the tRNA-Glu gene. Similar results were reported by Chiu, Chen, Hwang, and Tsen (2005). Christensen et al. (2000) found the genes enconding for tRNA-Ile, tRNA-Ala and tRNA-Glu in the intergenic spacer regions of 25 S. enterica strains belonging to 18 different serovars. In the last decades, S. enterica ser. Enteritidis has emerged as the main causative agent of gastroenteritis in humans in diverse countries (Chmielewski, Wieliczko, Kuczkowski, Mazurkiewicz, & Ugorski, 2002; Muresu et al., 2001), including Brazil (Tavechio et al., 1996; Peresi et al., 1998). In previous works, Salmonella spp. was reported as the main cause of foodborne diseases in RS (Costalunga & Tondo, 2002; Silveira & Tondo, 2006). In addition, S. enterica ser. Enteritidis was the serovar mostly isolated from foods involved with salmonellosis in this Brazilian State (Geimba et al., 2004). Considering that Brazil is currently one of the world’s most important poultry and bovine meat producers, the knowledge about what kind of strains are causing salmonellosis assume great importance. The results of the present study suggested the spread of a specific strain, or closely related strains of S. enterica ser. Enteritidis among several cities of the RS State. Acknowledgements We thank to FAPERGS – Fundação de Amparo à Pesquisa do Rio Grande do Sul for the financing support of this Project. We would like to thank to Dr. Suzana Costalunga, Dr. Josete Baialardi Silveira,
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