Ribotyping of Salmonella Enteritidis strains reveals the spread of a single genotype in the Brazilian city of Ribeirão Preto

1. Professor at Faculdade de Biomedicina, Universidade de Franca. 2. Biologist at the milk bank of the university hospital of Faculdade de Medicina de Ribeirão Preto of Universidade de São Paulo (FMRP/USP). 3. Professor at Departamento de Análises Clínicas Toxicológicas e Bromatológicas of Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP. 4. Researcher at Instituto Adolfo Lutz, São Paulo. 5. Associate professor at Departamento de Clínica Médica of FMRP/USP. y


Introduction
Non-typhoid salmonellosis is one of the commonest foodborne diseases and most frequently reported bacterial infections.The usual causative agent of this disease is Salmonella typhimurium or the emerging pathogen S. Enteritidis (1) .In the last decade the incidence of gastrointestinal infections caused by S. Enteritidis has increased (16,21) ; outbreaks have been reported in the United States and Europe since the 1970s (3) .Also, in many developing countries, such as Brazil, foodborne outbreaks and nosocomial infections caused by Salmonella enterica subsp.enterica serotype Enteritidis (S.Enteritidis) remain an important public health problem.In the emergency unit at the university hospital of Faculdade de Medicina de Ribeirão Preto of Universidade de São Paulo (FMRP/USP), during the years 1985-1999, S. Enteritidis exhibited a steady and significant rise, becoming the most frequently isolated Salmonella serotype in 1998 (6) .This increase could be linked to a rapid growth of the international trade of food products, changes in the type of food consumed, food contamination and salmonellosis in acquired immunodeficiency syndrome patients.
The worldwide rise of S. Enteritidis nowadays constitutes a public health concern; it is a challenge to epidemiologist and clinical staff alike (22,24) .The main approach taken to control the dissemination of salmonellosis is to employ molecular techniques.Methods based on DNA restriction analysis, such as ribotyping, can discriminate Salmonella strains involved in human infection and give information on the epidemiological and genetic relationship among serotypes.Hence, to differentiate serotypes such as S. Enteritidis, ribotyping has been employed in epidemiological investigation all over the world (2,9,10) .
The aim of this study was to analyze the rRNA gene restriction pattern of the S. Enteritidis strains isolated from patients at the university hospital of FMRP/USP, between 1996 and 1998.

Material and methods
A total of 83 strains of Salmonella (9.5%) were isolated from 872 patients admitted to the university hospital of FMRP/USP, State of São Paulo, Brazil, between March 1996 and April 1998.Salmonella strains were identified by standard bacteriological methods (biochemical and agglutination methods) (7) and serotyped, as detailed in Popoff and Le Minor (17) .S. Enteritidis was the most frequently cultivated serotype from clinical samples, totalizing 38 strains (45.8%), listed in Table 1.Salmonella chromosomal DNA was extracted, purified, as described by Brenner et al. (4) , and digested with SphI enzyme in accordance to manufacturer's instructions (Pharmacia Biotech, Herts, United Kingdom).Previous findings (14) had shown that this enzyme was the best to discriminate strains of S. Enteritidis.DNA fragments were separated by horizontal electrophoresis in 0.8% agarose gel (Sigma) with Tris-acetate EDTA as running buffer (23) , transferred to nylon membranes probe obtained by reverse transcription from E. coli rRNA (Boerhinger Mannheim, Germany), and labeled with digoxigenin (DIG), as in Popovic et al. (18) .In this method, genes are arranged in seven to eight bands (Figure 1) and a single difference in the number or position of these bands is considered as a different ribotype.DNA fragment sizes were estimated using DNA STAR software (DNA STAR Computer System for Molecular Biology and Genetics, London, UK), and Haemophilus aegyptius 3031 EcoRI DNA digest as molecular marker.

r results
The commonest occurrence of infection was in children in their first ten years.At the time of hospital admission, most patients presented acute gastroenteritis and fever.Some of the characteristics of the isolated strains, one from each patient, are displayed in Table 1.
In the ribotyping analysis following SphI digestion, two ribotypes (A and B) were detected among the 38 S. Enteritidis strains (Figure 1).Thirty-six strains of S. Enteritidis (94.7%) had ribotype A and the other two (5.3%) had ribotype B, differing from the absence of one band at a position corresponding to the 17.6 Kbp marker.discussion Salmonellosis continues to be the major problem for food industries and the public health system (6,13,21) .Recent data report the increasing incidence of S. Enteritidis strains worldwide (13,16,21) .In the state of São Paulo, a remarkable rise in the number of patients infected by S. Enteritidis was observed in 1993.Since 1994, it has become the most frequent serotype responsible for foodborne outbreaks and sporadic cases of human disease.There has also been an increase in the frequency with which S. Enteritidis has been isolated from blood cultures, mainly in children (9,24) .Laboratory characterization of this pathogen is epidemiologically important because it helps establish the connection between clinical cases and possible sources of infection.Investigators have shown that ribotyping can differentiate Salmonella serotypes according to source, regardless of host or geographic origin (8) .
Ridley et al. (20) and Laconha et al (10) investigated the genotypic differences between strains of Salmonella by plasmid analysis, ribotyping and pulsed-field gel electrophoresis (PFGE).The results obtained by those researchers indicated that PFGE may offer a better level of discrimination of S. Enteritidis types than other genotypic methods.Conversely, other epidemiological studies of S. Enteritidis have demonstrated that PFGE methodology has a lower discriminatory capacity than ribotyping (11,12,15,19,25,26) .
Fernandes et al. (9) used rRNA gene restriction patterns to investigate the relatedness of S. Enteritidis strains isolated in São Paulo, from 1975 to 1995, and showed that ribotyping is a genomic profiling method that is reproducible and suitable for tracing the spread of S. Enteritidis.They found that the restriction endonuclease SphI discriminated best between subtypes of this serotype.Thus, to discriminate S. Enteritidis strains in the present study, ribotyping was performed with the enzyme SphI.This approach separated these Salmonella isolates into two ribotypes (A and B).Ribotype A was much the most prevalent and is probably endemic in the community of Ribeirão Preto.Otherwise, the infected patients were exposed to the same source of infection.This ribotype was the same as that most frequently identified among S. Enteritidis strains isolated in different geographic locations in São Paulo (and designated R11) (9) .We conclude that rRNA gene-restriction patterns were used effectively to identify subtypes among Salmonella Enteritidis strains, confirming the value of ribotyping as an epidemiological tool, as well as revealing that in Riberão Preto these strains display great clonal homogeneity.

Characteristics and results of the ribotyping test of the 38 S. Enteritidis isolates Date of isolate Strain number Clinical material Age Ribotype
MARTINS, C. H. G. et al.Ribotyping of Salmonella Enteritidis strains reveals the spread of a single genotype in the Brazilian city of Ribeirão Preto • J Bras Patol Med Lab • v. 42 • n. 1 • p. 19-23 • fevereiro 2006 y: years; m: months; N: Newborn.

Table 1
MARTINS, C. H. G. et al.Ribotyping of Salmonella Enteritidis strains reveals the spread of a single genotype in the Brazilian city of Ribeirão Preto • J Bras Patol Med Lab • v. 42 • n. 1 • p. 19-23 • fevereiro 2006