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Journal ArticleDOI

PCR-RFLP analysis of fliC, fimH and 16S rRNA genes in Salmonella Typhimurium isolates of varied origin

01 Mar 2014-Annals of Microbiology (Springer Berlin Heidelberg)-Vol. 64, Iss: 1, pp 177-183
TL;DR: The results demonstrate that PCR-RFLP based on these genes showed good typeability but low discriminatory power and suggests the importance of these genes as immunization and diagnostic factors in Salmonella Typhimurium.
Abstract: Restricted fragment length polymorphism (RFLP) was used in analyses on the typing and heterogeneity, typeability and polymorphism of the 16S rRNA, fliC and fimH genes in Salmonella Typhimurium isolates of varied origin. The digestion of PCR products with restriction enzymes EcoRV, ClaI, HaeIII and ScaI (fliC genes), HincII, ClaI, EcoRV and MluI (fimH genes) and EcoRI, SmaI and HaeIII (16S rRNA genes) generated two to four bands of ranging in size from 100 to 1,104 bp. Of all the restriction profiles obtained, only the ClaI profile for fimH could be used to classify Salmonella Typhimurium isolates into different groups. According to this profile, pattern A with uncut fimH was observed in eight isolates (36.36 %) and pattern B with 755- and 253-bp bands was observed in 14 isolates (63.63 %). No pattern was allotted for a special region or source. These results demonstrate that PCR-RFLP based on these genes showed good typeability but low discriminatory power. Moreover, the highly conserved nature of fliC, fimH and 16S rRNA illustrated in our study suggests the importance of these genes as immunization and diagnostic factors in Salmonella Typhimurium. Simultaneously, our results also illustrate the potential of ClaI-based fimH analysis as a marker for the sub-serotype level differentiation of Salmonella Typhimurium isolates.
Citations
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Journal ArticleDOI
TL;DR: It is suggested that Salmonella species in cattle are transmitted to beef and, therefore, the consumption of undercooked beef could pose severe health complications on consumers.
Abstract: In this study, three hundred presumptive Salmonella strains isolated from cattle faeces and raw beef samples were subjected to both preliminary and confirmatory tests specific for Salmonella. PCR assays revealed that 100%, 20% and 26.7% of the isolates were positive for 16S rRNA, fliC and fljB gene fragments, respectively. Large proportions (62.4 to 94.3%) of these isolates were multiple antibiotic resistant (MAR) strains that were resistant to three or more antibiotics belonging to different classes. MAR phenotypes Ab1, Ab2, Ab3, Ab7, Ab8, Ab9, Ab26 and Ab27 were dominant among the isolates. Cluster analysis of antibiotic inhibition zone diameter data revealed two major clusters (clusters 1 and 2), and each cluster contained two sub-clusters (1A, 1B, 2A and 2B). PCR data revealed that 27.1% and 30.7% of the isolates possessed the spvC and invA virulent genes, respectively. There was a significant correlation between the possession of MAR phenotypes and virulent gene determinants. Analysis of restriction fragment length polymorphism (RFLP) of 16S rRNA gene fragments using EcoRI and HaeIII showed that large proportions of isolates from beef and cattle faeces produced similar genetic fingerprints. From these results, it is suggested that Salmonella species in cattle are transmitted to beef and, therefore, the consumption of undercooked beef could pose severe health complications on consumers. These findings provide baseline data that could be of great epidemiological importance and, thus, the need to utilise more sensitive typing tools in determining the genetic relatedness of isolates from different sources.

9 citations


Cites background or methods or result from "PCR-RFLP analysis of fliC, fimH and..."

  • ...…16S rRNA gene 572 55 Lin and Tsen 1996 16S RNARa CAC AAATCCATC TCT GGA fliCFb AAGGAATTCATCATGGCACAAG fliC 1488 55 Perera and Murray 2008; Sumithra et al. 2014fliCRb GAAGAATTCAACGCAGTAAA GAGAG fliBFc GGCAACCCGACAGTAACTGG CGATC fljB 135 47 fliBRc ATCAACGGTAACTTCATATTTG INVA-1d…...

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  • ...This is an indication that these isolates could have originated from a common ancestral strain, and similar observations have previously been reported (Jin et al. 2011; Sumithra et al. 2014)....

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  • ...In addition, flagellin gene fragments, fliC (1488 bp) and fljB (135 bp), were also amplified using standard protocols (Perera andMurray 2008; Sumithra et al. 2014....

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  • ...Considering the fact that the 16S rRNA gene segment is highly conserved in bacterial isolates, including Salmonella species, it is routinely used for sequence analysis-based identification (Sumithra et al. 2014)....

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  • ...Restriction enzymes EcoRV and HaeIII supplied by New England Biolabs, UK, were used in the analysis for RFLP (Dauga et al. 1998; Sumithra et al. 2014) according to the manufacturer’s instructions....

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Journal ArticleDOI
TL;DR: The results suggested that the PFGE subtyping is the prominent method for the evaluation and benchmarking of molecular subtyped in food-poisoning outbreaks.
Abstract: Currently, Salmonella enterica is the most common bacterial foodborne pathogen, causing serious extraintestinal disease. Typing methods play an important role on pathogens’ source tracking, knowing...

2 citations


Cites methods from "PCR-RFLP analysis of fliC, fimH and..."

  • ...[45] used RFLP to analyse the typing and according to their results, PCR-RFLP was used with four endonucleases which digested 16S rRNA, fliC and fimH genes....

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Journal ArticleDOI
TL;DR: It is demonstrated that using HhaI restriction endonuclease could differentiate Salmonella enteritidis andSalmonella colindale but there is similarity between pattern of SalmoneLLA typhimurium and Salmoneella infantis.
Abstract: The genus of Salmonella is very polymorphic and comprised of a number of genetically closely related serotypes. It is one of the emerging pathogen in food-borne disease which is often found in contaminated chicken eggs. Salmonella enterica is considered one of the major pathogens in public health worldwide. A total of 31 Salmonella isolates identified by specific antisera, which included Salmonella enteritidis (51.6%), Salmonella typhimurium (25.8%), Salmonella infantis (19.4%) and Salmonella colindale (3.2%). DNA was extracted using phenol- chloroform- isoamylalchol method. All the isolates showed fliC gene (1500bp) by using specific primers. PCR products were subjected to digestion using HhaI restriction endonuclease. PCR- RFLP results showed 3 patterns between all isolates. Our research gained in this study demonstrated that using HhaI restriction endonuclease could differentiate Salmonella enteritidis and Salmonella colindale but there is similarity between pattern of Salmonella typhimurium and Salmonella infantis .

2 citations


Cites methods or result from "PCR-RFLP analysis of fliC, fimH and..."

  • ...According to the results based on Matsui et al (2001) and Sumithra et al (2013) research showed that PCR-RFLP with more than one endonuclease and genes give good typeability and increase the differentiating power....

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  • ...Sumithra et al (2013) used RFLP to analysis of typing, heterogeneity, typeability and polymorphism of the 16S rRNA, fliC and fimH genes in Salmonella typhimurium isolates from different origin....

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References
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Journal ArticleDOI
TL;DR: An index of discrimination for typing methods is described, based on the probability of two unrelated strains being characterized as the same type, which may be used to compare typing methods and select the most discriminatory system.
Abstract: An index of discrimination for typing methods is described, based on the probability of two unrelated strains being characterized as the same type. This index may be used to compare typing methods and select the most discriminatory system.

2,982 citations


"PCR-RFLP analysis of fliC, fimH and..." refers methods in this paper

  • ...The numerical index of discrimination (D) of restriction enzymes which can result in discrimination among Salmonella Typhimurium isolates was calculated using Simpson’s index of diversity (Hunter and Gaston 1988)....

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Journal ArticleDOI
TL;DR: This procedure is effective in producing digestible chromosomal DNA from a variety of gram-negative bacteria, all of which normally produce large amounts of polysaccharides.
Abstract: Most protocols for the preparation of bacterial genomic DNA consist of lysis, followed by incubation with a nonspecific protease and a series of extractions prior to precipitation of the nucleic acids. Such procedures effectively remove contaminating proteins, but are not effective in removing exopolysaccharides which can interfere with the activity of enzymes such as restriction endonucleases and ligases. In this unit, however, the protease incubation is followed by a CTAB extraction whereby CTAB complexes both with polysaccharides and with residual protein, effectively removing both in the subsequent emulsification and extraction. This procedure is effective in producing digestible chromosomal DNA from a variety of gram-negative bacteria, all of which normally produce large amounts of polysaccharides. If large amounts of exceptionally clean DNA are required, the procedure can be scaled up and the DNA purified on a CsCl gradient, as described in the alternate protocol.

1,992 citations

Book ChapterDOI
01 Jan 2000
TL;DR: This chapter describes two procedures for the isolation of chromosomal DNA from E. coli that can be used for most gram-negative and gram-positive bacteria or modified to isolate DNA from organisms other than bacteria.
Abstract: This chapter describes two procedures for the isolation of chromosomal DNA from E. coli. These procedures can be used for most gram-negative and gram-positive bacteria or modified to isolate DNA from organisms other than bacteria.

546 citations


"PCR-RFLP analysis of fliC, fimH and..." refers methods in this paper

  • ...Isolation of genomic DNA Genomic DNA of all isolates was isolated using the CTAB method (Wilson 1987)....

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  • ...After characterization, a glycerol stock was made of each culture which was kept at −20 °C. Isolation of genomic DNA Genomic DNA of all isolates was isolated using the CTAB method (Wilson 1987)....

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Book
15 May 2000
TL;DR: The genetic basis of Salmonella virulence fimbriae structure ofSalmonella antibiotic resistance host susceptibility, resistance and immunity, and Epidemiology and control: vaccines competitive exclusion environmental aspects Salmoneella in animal feed epidemiology public health aspects are studied.
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377 citations

Journal ArticleDOI
TL;DR: This supplement reports the characterization of 18 new Salmonella serovars recognized in 2002 by the WHO Collaborating Centre for Reference and Research onSalmonella.

271 citations


"PCR-RFLP analysis of fliC, fimH and..." refers background in this paper

  • ...The most common of the total of 2,668 Salmonella enterica serovars (Popoff et al. 2004) is Salmonella Typhimurium (Rahman 2002), and the number of Typhimurium infections in human and animals are dramatically increasing (Mikasova et al. 2005)....

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