A study was undertaken to observe the distribution of three such genes, namely, Salmonella enterotoxin ( stn ), Salmonella Enteritidis fimbrial (sef) and plasmid encoded fimbrial [r]
(1)Indian J Med Res 117, February 2003, pp 66-70
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), SalmonellaEnteritidis 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, sefandpef genes using their specific primers and the PCR products were analysed by 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., sef1, pef2, spv3 or inv4; others are
associated with the survival in the host system- mgtC5 or in the actual manifestation of pathogenic
processes viz., sop6, stn7, pip A, B, D8 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
(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 100oC for 10 After boiling, the cell suspensions were
cooled on ice and were immediately tested for the presence of stn, sef and pef genes by PCR analysis10,11 Primers used for stn gene were: Stn P1 - TTG TGT CGC TAT CAC TGG CAA
CC - 3 (upper primer) and Stn M13 - ATT CGT AAC CCG CTC TCG TCC - (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, 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 (94oC for
min), primer annealing (59oC for min) and primer extension (72oC for min) followed by
incubation at 72oC for 10 For sef gene, the primers used were, sef C - GCG AAA ACC
AAT GCG ACT GTA - (upper primer) and sef C - 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 (94oC for min), primer annealing (55oC for min) and
primer extension (72oC for min) followed by incubation at 72oC for 10 For pef gene the
primers used were, pef A1 TGT TTC CGG GCT TGT GCT - (upper primer) and pef A2 - 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 (94oC for 55
sec), primer annealing (55oC for 55 sec) and primer extension (72oC for 55 sec) followed by
incubation at 72oC for 10 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 enterotoxin12 This enterotoxin production is mediated by
the stn gene13 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 reports9,10 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 strains9 and also the other members of Enteriobacteriaceae or Vibrio, which have
(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 24 24 22
S. Enteritidis 8
S. Paratyphi B 2
Pigs
S. Typhimurium 12 12 12
S. Enteritidis 11 11 11
S. Paratyphi B 1
S. Bareilly 1 0
Humans
S Typhimurium 10 10 10
S. Enteritidis 11 11 11 11
S. Paratyphi B 2
Cattle
S. Typhimurium 5
S. Enteritidis 6 6
S. Bareilly 2 0
Total 95 95 36 85
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 reported14
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 cell15 and in mediation of excessive fluid accumulation2 in the murine small
intestine Salmonella Enteritidis fimbriae 14 (SEF 14), one of the major fimbriae of Salmonella,
consists of 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, sefBand sefC1 and sefD16, 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 sefCgene, 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 al10 who showed that except for strains of S Enteritidis
and S Gallinarum, none of the other serotypes (S Typhimurium, S Newport, S. Kentucky, S
(4)Fig Detection of stn gene by polymerase chain reaction (PCR) Lane to and 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 Detection of sef gene by polymerase chain reaction (PCR) Lane - Negative control (S bongori); Lane - Negative control (Esch coli C-600); Lane - 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
Fig Detection of pef gene by polymerase chain reaction (PCR) Lane 1- Negative control (S bongori); Lane 2- Negative control (Esch coli C-600); Lane - 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
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 earlier11
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
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