Distribution of virulence genes in Salmonella serovars isolated from man & animals

Transcription

1 Indian J Med Res 117, February 2003, pp Distribution of virulence genes in Salmonella serovars isolated from man & animals H.V. Murugkar*, H. Rahman* & P.K. Dutta Department of Microbiology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India Received January 7, 2003 Background & objectives: Pathogenesis of Salmonellosis depends upon a large number of factors controlled by an array of genes that synergise into the actual virulence of Salmonella. A study was undertaken to observe the distribution of three such genes, namely, Salmonella enterotoxin (stn), Salmonella Enteritidis fimbrial (sef) and plasmid encoded fimbrial (pef) genes, among different serovars of Salmonella enterica isolated from man and animals. Methods: A total of 95 isolates belonging to S. Typhimurium (51), S. Enteritidis (36), S. Bareilly (3), and S. Paratyphi B (5) serovars were subjected to polymerase chain reaction (PCR) assay for the detection of stn, sef and pef genes using their specific primers and the PCR products were analysed by 1 per cent agarose gel electrophoresis for the presence of the respective genes. Results: Varying distribution pattern of these genes was observed amongst the isolates. While, stn was found in all the 95 strains, sef was found only among the S. Enteritidis isolates. The pef gene was found to be absent in 10 isolates including the three S. Bareilly isolates. Interpretation & conclusion: Findings indicated that the stn gene is widely distributed among Salmonella irrespective of the serovars and source of isolation. However, the sef gene appears to be serovar specific. Since the stn gene is found in all the isolates, it can be a viable target gene to explore the possibility of direct detection of Salmonella from samples from biological sources. Key words Enterotoxic gene - fimbrial genes - polymerase chain reaction (PCR) - Salmonella Epidemiology and pathogenic process in salmonellosis are dictated by an array of factors that act in tandem and ultimately manifest in the typical symptoms of salmonellosis. Virulence genes encode products that assist the organisms in expressing its virulence in the host cells. Some genes are known to be involved in adhesion and invasion viz., sef 1, pef 2, spv 3 or inv 4 ; others are associated with the survival in the host system- mgtc 5 or in the actual manifestation of pathogenic processes viz., sop 6, stn 7, pip A, B, D 8. Nucleic acid based diagnostic techniques are being employed for the detection of various gene -encoded virulence factors viz., Salmonella enterotoxin (stn) 9,10, Salmonella Enteritidis fimbriae (sef) and plasmid encoded fimbriae (pef) 11 genes. However, the distribution of these genes among the various isolates obtained from biological source is yet to be elucidated. The present study reports on the distribution of these genes among the Salmonella strains isolated from clinical cases of infections in man and animals. Material & Methods

2 Bacterial strains: The study was carried out in the Department of Microbiology, College of Veterinary Sciences, Assam Agricultural University, Khanapara, Guwahati. A total of 95 strains of Salmonella isolated from various sources, viz., poultry (34), pigs (25), humans (23), and cattle (13) were used for the present study (Table). All the isolates were obtained from various biological sources in northeastern India. A strain of Salmonella enterica serovar Typhimurium DT096 (stn + ) was used as positive control for stn gene and a strain of S. enterica serovar Enteritidis SE7 (sef +, pef + ) was used as positive control for pef and sef genes. A strain each of S. bongori and Escherichia coli (C-600) were used as negative controls. All the positive and negative control strains were provided by Dr H. Tschape, Director, Robert Koch Institute, Germany. The cultures were maintained on nutrient agar slants before use. Detection of stn, sef and pef genes by polymerase chain reaction (PCR): For the detection of stn, sef and pef genes by PCR analysis, bacterial cells from the overnight cultures were suspended in 350 µl distilled water and boiled at 100 o C for 10 min. After boiling, the cell suspensions were cooled on ice and were immediately tested for the presence of stn, sef and pef genes by PCR analysis 10,11. Primers used for stn gene were: Stn P1 5 - TTG TGT CGC TAT CAC TGG CAA CC - 3 (upper primer) and Stn M ATT CGT AAC CCG CTC TCG TCC - 3 (lower primer) 10 (GENSET, Singapore), which flank a 617 bp segment in the stn gene sequence. The PCR mixture (25 µl) included 12.5 µl master mix (QIAGEN, USA) containing 2.5 U Taq DNA polymerase, 20µM each of datp, dctp, dttp and dgtp and PCR buffer, 5 µl (1µM) each of upper and lower primers and 2.5µl of template DNA (bacterial cell suspension). PCR incubation was performed in a thermocycler (Perkin-Elmer, USA) in 25 cycles of denaturation (94 o C for 1 min), primer annealing (59 o C for 1 min) and primer extension (72 o C for 1 min) followed by incubation at 72 o C for 10 min. For sef gene, the primers used were, sef C 5 - GCG AAA ACC AAT GCG ACT GTA - 3 (upper primer) and sef C 5 - CCC ACC AGA AAC ATT CAT CCC - 3 (lower primer) 11 that flank a 1103 bp segment in the sef gene sequence. PCR incubation was performed in 25 cycles of denaturation (94 o C for 1 min), primer annealing (55 o C for 1 min) and primer extension (72 o C for 1 min) followed by incubation at 72 o C for 10 min. For pef gene the primers used were, pef A1 5 TGT TTC CGG GCT TGT GCT - 3 (upper primer) and pef A2 5 - CAG GGC ATT TGC TGA TTC TTC C - 3 (lower primer) 11. These primers flank a 700 bp segment in the pef gene sequence. The PCR was run for 25 cycles of denaturation (94 o C for 55 sec), primer annealing (55 o C for 55 sec) and primer extension (72 o C for 55 sec) followed by incubation at 72 o C for 10 min. A 15 µl aliquot of each PCR product was electrophoretically separated on agarose gel (1%, containing 0.5 µl/ml ethidium bromide, Pharmacia, Sweden), analyzed under UV light (300 nm) and photographed using Gel doc 2000 documentation system (Pharmacia, Sweden). Results & Discussion Salmonella induced diarrhoea is a complex phenomenon involving several pathogenic mechanisms including production of enterotoxin 12. This enterotoxin production is mediated by the stn gene 13. In this study, PCR assay carried out for the detection of the stn gene in 95 Salmonella isolates from five different serovars and four different sources has revealed that the gene was present in all the isolates (Table) that was demonstrated by the presence of a 617 bp PCR product (Fig.1). These findings are in agreement with earlier reports 9,10. Observations from the present study indicated that the stn gene is widely distributed among the Salmonella irrespective of their serovars and source of isolation. This stn gene has been reported to be absent in S. bongori strains 9 and also the other members of Enteriobacteriaceae or Vibrio, which have enterotoxigenic potential 10.

3 Table. Prevalence of stn, sef and pef genes among Salmonella isolated from various sources Source/serotype No. of isolates No. positive for virulence genes Stn sef pef Poultry S. Typhimurium S. Enteritidis S. Paratyphi B Pigs S. Typhimurium S. Enteritidis S. Paratyphi B S. Bareilly Humans S. Typhimurium S. Enteritidis S. Paratyphi B Cattle S. Typhimurium S. Enteritidis S. Bareilly Total The findings from the present study show that the gene is present in all the Salmonella serotypes and reports that the stn gene contained sequence unique to Salmonella strains, makes this gene a suitable PCR target for detection of Salmonella strains in field samples. Development of a PCR system for diagnosis of Salmonella on the basis of stn detection has been reported 14 wherein a single organism could be detected per gram of faecal or meat sample by the application of this method. The role of both sef and pef genes in the pathogenesis of Salmonella is still undefined. Preliminary studies on mice have shown that these play an important role in bacterial adhesion to the epithelial cell 15 and in mediation of excessive fluid accumulation 2 in the murine small intestine. Salmonella Enteritidis fimbriae 14 (SEF 14), one of the major fimbriae of Salmonella, consists of 4 major protein subunits SefA (14 kd), SefB (28 kd), SefC (90kd), and SefD (10 kd), which are arranged sequentially in the fimbrial structure and are encoded by fimbrial operons sefa, sefb and sefc 1 and sefd 16, respectively. SefC forms the largest component of the fimbriae and its presence indicates the presence of the SEF adhesin. The primer for this gene therefore, was considered for detection of sef gene among the isolates in the present study. The sefc gene, detected by the presence of a 1103 bp product (Fig. 2), was found to be present in only 36 of the 95 isolates, all of which belonged to S. Enteritidis serovar (Table). The present findings corroborate the findings of Rahman et al 10 who showed that except for strains of S. Enteritidis and S. Gallinarum, none of the other serotypes (S. Typhimurium, S. Newport, S. Kentucky, S. Weltevreden and S. Indiana) tested were found to contain these genes. In the present study, presence of pef gene was evaluated with the help of primer for pef gene, which encodes for the major portion of the pef operon.

4 Fig. 1. Detection of stn gene by polymerase chain reaction (PCR). Lane 1 to 5 and 9 to 12- Test isolates (S. Enteritidis); Lane 13- S. Bareilly; Lane 14- S. Paratyphi B; Lane 7- Negative control (S. bongori); Lane 15- Negative control (Esch. Coli C-600); Lane 6- Positive control (S. Typhimurium DT096); Lane 8,16- DNA Ladder. Fig. 2. Detection of sef gene by polymerase chain reaction (PCR). Lane 1 - Negative control (S. bongori); Lane 2 - Negative control (Esch. coli C-600); Lane 3 - Positive control (S. Enteritidis SE 7); Lanes 4,5,8,9,12,13 - S. Enteritidis; Lanes 6,11 - S. Typhimurium; Lane 10 - S. Bareilly; Lanes 7,14 - DNA Ladder. The pef gene, detected by the presence of a 700 bp PCR product (Fig. 3), was found to be present in 85 of the 95 isolates). There was no serotype specific presence or absence of this gene. Forty nine of 51 S. Typhimurium strains showed the presence of this gene (Table). Thirty one of the 36 S. Enteritidis strains and all the five S. Paratyphi B isolates were also positive for pef gene. All the S. Bareilly isolates were negative for the gene. This inter-serotype variation in the presence of pef gene has also been recorded earlier 11. Fig. 3. Detection of pef gene by polymerase chain reaction (PCR). Lane 1- Negative control (S. bongori); Lane 2- Negative control (Esch. coli C-600); Lane 3 - Positive control (S. Enteritidis SE7); Lanes 5, 10- S. Typhimurium; Lane 4, 6, 11, 12,13- S. Enteritidis; Lane 9- S. Bareilly; Lane 14- S. Paratyphi B; Lane 7, 15- blank; Lane 8, 16- DNA Ladder. A detailed study involving all the major serotypes needs to be carried out to obtain a definite status regarding the presence of this gene among various serotypes of Salmonella and the role of the products phenotypically expressed by these genes on the pathogenesis of salmonellosis in man and animals.

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