Print version ISSN 1517-8382
Braz. J. Microbiol. vol.34 suppl.1 São Paulo Nov. 2003
Detecção de genes de virulência in Salmonella Enteritidis isoladas de diferentes fontes
Sílvia Dias de OliveiraI,II,III; Carla Rosane RodenbuschIII; Geovana B. MichaelIII; Marisa I.R. CardosoIII; Cláudio Wageck CanalIII; Adriano BrandelliII
IDepartamento de Ciências Microbiológicas, Faculdade de Biociências, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brasil
IIInstituto de Ciências e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil
IIIFaculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil
The presence of three virulence genes, invA, spvR, and spvC, was determined in Salmonella Enteritidis isolated from poultry, pigs, humans and food. All isolates were positive for the invA gene, with 91.2% being positive for spvR and 90.2% for spvC. There was no significant difference in the prevalence of the virulence genes between isolates from different sources. The results indicate that there is a putative high virulence potential for the S. Enteritidis isolates characterized.
Key words: Salmonella Enteritidis, virulence, spv, PCR.
A presença de três genes de virulência (invA, spvR e spvC) foi determinada em Salmonella Enteritidis isoladas de aves, suínos, humanos e alimentos. Todos os isolados foram positivos para o gene invA, 91,2% também foram positivos para o spvR e 90,2% para o spvC. Não existiu diferença significativa na prevalência dos genes de virulência entre isolados de diferentes origens. Os resultados indicaram que, provavelmente, exista um alto potencial de virulência nos isolados de S. Enteritidis caracterizados.
Palavras-chave: Salmonella Enteritidis, virulência, spv, PCR.
Salmonellosis is one of the most common infectious diseases of both humans and animals and since the 1980's there has been a dramatic world-wide increase in the number of reported isolations of S. Enteritidis (6).
The virulence of Salmonella is linked to a combination of chromosomal and plasmid factors, the chromosomally located invasion gene invA being thought to trigger the invasion of salmonellae into cultured epithelial cells (1), while an operon (spvRABCD), containing five genes, is present on plasmids commonly associated with some serotypes, the spv genes possibly having the ability to increase the severity of enteritis and allow infection and persistence at extra-intestinal sites (3).
The purpose of our study was to asses the potential virulence of S. Enteritidis isolates from poultry, pigs, humans and food by detecting the presence of the invA, spvR and spvC virulence genes using the Polymerase Chain Reaction (PCR).
MATERIALS AND METHODS
Our study used 102 S. Enteritidis strains isolated in Southern Brazil, 31 from food involved in foodborne outbreaks, 22 from broiler carcasses, 21 from poultry (viscera and environmental samples), 17 from humans and 11 from pigs (lymph nodes, faeces and fresh pork sausage).
DNA was extracted as previously described (4). PCR was performed with three sets of primer pairs: 139-141, specific for the invasion gene invA (4); PG 48-PG49, specific for the spvR gene (7); and VIR113-VIR561 specific for the spvC gene (5), amplifying 284 bp, 890 bp and 472 bp DNA fragments, respectively (Fig. 1). PCR amplifications were performed in a final volume of 25 µL containing DNA template, 1.5 mM MgCl2, 10 mM Tris HCl (pH 8.0), 50 mM KCl, 0.2 mM of each nucleotide, 0.8 rmol/µL of each primer and 1 U of Taq DNA polymerase. The amplification of the invA gene fragment was carried out as previously described (4), the amplification conditions for the spvC gene fragment being similar except that the annealing temperature was 58°C. Amplification of the spvR gene fragment was carried out as previously described (7). Amplification products were separated by electrophoreses on 1.2% agarose gel stained with 5 µg/mL of ethidium bromide with a 100 bp DNA ladder as molecular weight marker.
RESULTS AND DISCUSSION
All 102 S. Enteritidis isolates contained the invasion gene invA, other studies having reported similar results (4,8), which was expected since the invA is an invasion gene conserved among Salmonella serotypes.
The spvR virulence gene was detected in 91.2% of the S. Enteritidis strains and the spvC gene in 90.2%, there being no significant difference in the prevalence of virulence genes among the isolates from different sources. Only two strains contained spvR but not spvC; and one strain contained spvC but not spvR. The analysis of the spv operon has shown that spvR is an essential virulence gene while the spvC gene plays an accessory role but is probably needed for full virulence (2) and, therefore, it may be concluded that the isolates without the spvR gene are probably avirulent while absence of the spvC gene may indicate partial virulence.
High prevalence of these virulence genes has also been found by other authors (5,9), although in our isolates from humans and poultry the prevalence of virulence genes was even higher than that already reported. It appears that there may be some variation in the prevalence of some virulence genes because a study involving poultry products, wastewater and human sources found that only 15% of isolates contained the spvC gene (8).
A further conclusion of our study is that because they are not present in all isolates, spv genes are not appropriate targets for the specific detection of the Enteritidis serovar, although they have been used as such in several studies. However, our results regarding the presence of the invA gene do agree with those of other authors who have found that this gene is a good target for detecting salmonellae.
1. Galan, J.E.; Curtiss III, R. Cloning and molecular characterization of genes whose products allow S. typhimurium to penetrate tissue culture cells. Proc. Natl. Acad. Sci. USA, 86:6383-6387,1989. [ Links ]
2. Guiney, D.G.; Fang, F.C.; Krause, M.; Libby, S.; Buchmeier, N.A.; Fierer, J. Biology and clinical significance of virulence plasmids in Salmonella serovars. Clin. Infect. Dis., 21:S146-S151, 1995. [ Links ]
3. Libby, S.J.; Lesnick, M.; Hasegawa, P.; Weidenhammer, E.; Guiney, D.G. The Salmonella virulence plasmid spv genes are required for cytopathology in human monocyte-derived macrophages. Cell. Microbiol., 2:49-58, 2000. [ Links ]
4. Oliveira, S.D.; Santos, L.R.; Schuch, D.M.T.; Silva, A.B.; Salle, C.T.P.; Canal, C.W. Detection and identification of salmonellas from poultry by PCR. Vet. Microbiol., 87:25-35, 2002. [ Links ]
5. Rexach, L.; Dilasser, F.; Fach, P. Polymerase chain reaction for salmonella virulence-associated plasmid genes detection: a new tool in salmonella epidemiology. Epidemiol. Infect., 112:33-43, 1994. [ Links ]
6. Rodrigue, D.C.; Tauxe, R.V.; Rowe, B. International increase in Salmonella Enteritidis: a new pandemic? Epidemiol. Infect., 105:21-27, 1990. [ Links ]
7. Rubino, S.; Muresu, E.; Solinas, M.; Santona, M.; Paglietti, B.; Azara, A.; Schiaffino, A.; Santona, A.; Maida, A.; Cappuccinelli, P. IS200 fingerprint of Salmonella enterica serotype Typhimurium human strains isolated in Sardinia. Epidemiol. Infect., 120:215-222, 1998. [ Links ]
8. Swamy, S.C.; Barnhart, H.M.; Lee, M.D.; Dreesen, D.W. Virulence determinants invA and spvC in salmonellae isolated from poultry products, wastewater, and human sources. Appl. Environ. Microbiol., 62:3768-3771, 1996. [ Links ]
9. Threlfall, E.J.; Hampton, M.D.; Chart, H.; Rowe, B. Use of plasmid profile typing for survillance of Salmonella enteritidis phage type 4 from humans, poultry and eggs. Epidemiol. Infect., 112:25-31, 1994. [ Links ]
Sílvia Dias de Oliveira
Departamento de Ciências Microbiológicas
Faculdade de Biociências, Pontifícia Universidade Católica do Rio Grande do Sul
Av. Ipiranga 6681, 90619-900
Porto Alegre, RS, Brasil
This paper corresponds to an "extended abstract" selected for oral presentation in the 22nd Brazilian Congress of Microbiology, held in Florianópolis, SC, Brazil, in November 17-20, 2003