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Journal Article

PCR-Restriction Fragment Length Polymorphism analysis of fljB gene in Salmonella enterica subspecies enterica serovar Typhimurium isolated from avians.

01 Dec 2010-Iranian journal of microbiology (Tehran University of Medical Sciences)-Vol. 2, Iss: 4, pp 178-184
TL;DR: The results of the present study showed that fljB gene is highly conserved among avians in different geographical regions, suggesting not only the importance of flJB gene in survival of organism in different environmental conditions but also the relation between proteins encoded by flj B gene and serotyping scheme.
Abstract: Background and Objectives: Economic constraint of diseases arising from Salmonella Typhimurium causes the study of this zoonotic organism more important. Most studies on identification and characterization of S. Typhimurium are conducted at DNA level. Flagellin genes (fliC and fljB genes encoding phase-1 and phase-2 flagella, respectively) are useful as a model system for studying genetic differentiation. The objectives of the present study were to identify the polymorphism of fljB among avians in different regions by the PCR-RFLP method. Materials and Methods: Fifty-two S. Typhimurium isolates out of 1,870 intestine samples were identified using culture and serotyping as well as multiplex-PCR (broiler (n = 13), layer (n = 12), duck (n = 5), goose (n = 5), sparrow (n = 8), canary (n = 3), pigeon (n = 5) and casco parrot (n = 1) ). Amplification of fljB gene was performed and amplified products subjected to restriction digestion with Hha I enzyme. Results: Two RFLP patterns generated DNA fragments between approximately 50 to 800 bps. Pattern A was observed in 33 (63.46%) and pattern B in 19 (36.54%) of isolates. Salmonella Typhimurium recovered from 13 broilers (ten with pattern A and 3 with pattern B) and 8 sparrow (three with pattern A and 5 with pattern B) showed both A and B patterns. Twelve layers, 5 pigeons and 3 canaries showed pattern A and 5 ducks, 5 geese and one casco parrot showed pattern B. None of these patterns was allotted for a special region. Conclusion: The results of the present study showed that fljB gene is highly conserved among avians in different geographical regions, suggesting not only the importance of fljB gene in survival of organism in different environmental conditions but also the relation between proteins encoded by fljB gene and serotyping scheme.

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Citations
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Journal ArticleDOI
TL;DR: The results confirm the potential presence of multidrug-resistant bacteria in canary facilities, suggesting that measures to educate the public about this risk are necessary.

16 citations

Journal ArticleDOI
TL;DR: The results indicate that MS-H can be used as a sensitive, rapid, and straightforward approach for the typing of Salmonella flagella at the molecular level without antiserum and phase inversion.
Abstract: Forty-three reference strains involving the 24 most common serovars of Salmonella enterica were examined by using a mass spectrometry-based H antigen typing platform (MS-H). The results indicate that MS-H can be used as a sensitive, rapid, and straightforward approach for the typing of Salmonella flagella at the molecular level without antiserum and phase inversion.

7 citations

Journal ArticleDOI
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.

6 citations


Cites background or result from "PCR-Restriction Fragment Length Pol..."

  • ...Paradoxically, Dilmaghani et al. (2010) found that fljB gene in Salmonella Typhimurium is highly conserved....

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  • ...Our observations corroborate earlier observations of Dilmaghani et al. (2010) who showed that the fljB gene is highly conserved among Salmonella Typhimurium isolates from different geographical regions....

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  • ...No pattern was allotted for a special region or source which is similar to the observation of Dilmaghani et al. (2010) in the fljB gene....

    [...]

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 from "PCR-Restriction Fragment Length Pol..."

  • ...Dilmaghani et al (2010) identified the polymorphism of fljB gene among avian in different regions by PCR-RFLP method....

    [...]

Journal Article

2 citations


Cites background from "PCR-Restriction Fragment Length Pol..."

  • ...mutations, deletion and insertions (Dilmaghani et al., 2010)....

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  • ...Antigenic polymorphism of flagella seems to have been generated by the accumulation of ordinary genetic events in flagellin genes, such as point mutations, deletion and insertions (Dilmaghani et al., 2010)....

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References
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Journal ArticleDOI
TL;DR: The presently described multiplex PCR can serve as an alternative to the tedious time-consuming procedure of Salmonella culture and identification in food safety laboratories and to detect S. typhimurium from water and food samples.
Abstract: The aim of the work was to specifically differentiate S. typhimurium from other closely related Salmonella serovars by monoplex or multiplex PCR and to detect it from water and food samples. Genes targeted were invA, iroB, STM4497, STM2755, fliC, fljB and rfbJ and evaluated on 58 Salmonella standard serovars/strains including 9 S. typhimurium strains, 7 suspected Salmonella isolates and 8 other organisms as negative controls. Both invA and iroB showed a uniform amplification with all serovars of S. enterica group. STM2755 and STM4497 gene based PCR’s specifically exhibited amplification in all the nine confirmed S. typhimurium strains. The rfbJ PCR produced amplification with confirmed S. typhimurium strains, in addition showed reaction with S. abony. Both STM4497, STM2755 PCR’s and rfbJ could identify two of the seven biochemically suspected Salmonella isolates that were later confirmed to be S. typhimurium on the basis of sequence data. PCR for fliC genes had amplification exhibited by a large number of serovars of the S. enterica group, including S. typhimurium strains but not to S. brunei,S. newporti, S. abony and S. weltevreden. fljB was detected in all strains of S. enterica and E. coli with the exception of S. typhi. fljB and fliC were amplified in 6/7 and 5/7 presumptive Salmonella isolates. The same PCR’s were converted into two multiplex formats for simultaneous identification of the Salmonella genus, S. enterica group and S. typhimurium as a species. The first multiplex set comprised on invA, iroB, STM4497, STM2755 and the IAC. The second multiplex set comprised of invA, iroB, fljB, fliC, rfbJ along with IAC. The detection limit for S. typhimurium in the two multiplex PCR sets was in the range of 350–400 cfu/PCR reaction and that of DNA around 2 pg. The multiplex PCR (format 1) was first evaluated on spiked water, chicken and mutton samples and the detection limit for S. typhimurium was in the range of 100 cfu/100 ml, <60 and <50 cfu/gm, respectively. Further evaluation of multiplex PCR (format 1) was undertaken on 50 natural samples of chicken, eggs, litter, soil etc. and the comparison done with conventional culture isolation and identification procedure. The multiplex PCR could identify the presence of Salmonellla in three samples and the same three samples also yielded Salmonella by the conventional method. Therefore, the presently described multiplex PCR can serve as an alternative to the tedious time-consuming procedure of Salmonella culture and identification in food safety laboratories.

46 citations

Journal ArticleDOI
TL;DR: DNA sequence analysis of the flk gene has revealed it to be identical to a gene of Escherichia coli of unknown function which has an overlapping, divergent promoter with the pdxB gene promoter.
Abstract: The flagellum of Salmonella typhimurium is assembled in stages, and the negative regulatory protein, FlgM, is able to sense the completion of an intermediate stage of assembly, the basal body-hook (BBH) structure. Mutations in steps leading to the formation of the BBH structure do not express the flagellar filament structural genes, fliC and fljB, due to negative regulation by FlgM (K. L. Gillen and K. T. Hughes, J. Bacteriol. 173:6453-6459, 1991). We have discovered another novel regulatory gene, flk, which appears to sense the completion of another assembly stage in the flagellar morphogenic pathway just prior to BBH formation: the completion of the P- and L-rings. Cells that are unable to assemble the L- or P-rings do not express the flagellin structural genes. Mutations by insertional inactivation in either the flk or flgM locus allow expression of the fljB flagellin structural gene in strains defective in flagellar P- and L-ring assembly. Mutations in the flgM gene, but not mutations in the flk gene, allow expression of the fljB gene in strains defective in all of the steps leading to BBH formation. The flk gene was mapped to min 52 of the S. typhimurium linkage map between the pdxB and fabB loci. A null allele of flk was complemented in trans by a flk+ allele present in a multicopy pBR-based plasmid. DNA sequence analysis of the flk gene has revealed it to be identical to a gene of Escherichia coli of unknown function which has an overlapping, divergent promoter with the pdxB gene promoter (P. A. Schoenlein, B. B. Roa, and M. E. Winkler, J. Bacteriol. 174:6256-6263, 1992). An open reading frame of 333 amino acids corresponding to the flk gene product of S. typhimurium and 331 amino acids from the E. coli sequence was identified. The transcriptional start site of the S. typhimurium flk gene was determined and transcription of the flk gene was independent of the FlhDC and sigma28 flagellar transcription factors. The Flk protein observed in a T7 RNA polymerase-mediated expression system showed an apparent molecular mass of 35 kDa, slightly smaller than the predicted size of 37 kDa. The predicted structure of Flk is a mostly hydrophilic protein with a very C-terminal membrane-spanning segment preceded by positively charged amino acids. This finding predicts Flk to be inserted into the cytoplasmic membrane facing inside the cytoplasm.

43 citations

Journal ArticleDOI
TL;DR: The profiles of the insertion element IS200, which has been shown to provide phylogenetic markers for serogroup D1 salmonellae, were analyzed and provide unequivocal evidence that Salmonella 9,12:l,v:- arose from a strain of S. goettingen.
Abstract: The emergence in several countries of the monophasic serogroup D1 serovar Salmonella 9,12:l,v:- provided the opportunity to study its evolutionary origin. According to current models, such a variant serovar could have arisen by horizontal transfer of a new flagellar gene to a preexisting monophasic Salmonella strain or, alternatively, by the loss of the phase 2 flagellar gene of an originally biphasic Salmonella strain. Five known serovars of Salmonella, S. panama, S. kapemba, S. goettingen, S. zaiman, and S. mendoza, could have been possible ancestors of the new variant. The profiles of the insertion element IS200, which has been shown to provide phylogenetic markers for serogroup D1 salmonellae, were analyzed in relation to the restriction fragment length polymorphisms of the phase 2 flagellar gene. Together they provide unequivocal evidence that Salmonella 9,12:l,v:- arose from a strain of S. goettingen. Analysis of the flj operon of the variant indicated that loss of phase 2 flagellar antigen expression occurred through deletion of the hin gene and adjacent DNA, thereby blocking the phase 2 flagellar gene in the off position.

37 citations

Journal ArticleDOI
Noriko Okazaki1, S Matsuo1, K Saito1, Akira Tominaga1, Masatoshi Enomoto1 
TL;DR: The mechanisms for gene conversion between the two genes are discussed and also some intriguing aspects of flagellar antigenic specificities in various Salmonella serovars from the viewpoint of gene conversion.
Abstract: The Escherichia coli-Salmonella typhimurium-Salmonella abortus-equi hybrid strain EJ1420 has the two Salmonella flagellin genes fliC (antigenic determinant i) and fljB (determinant e,n,x) at the same loci as in the Salmonella strains and constitutively expresses the fliC gene because of mutations in the genes mediating phase variation. Selection for motility in semisolid medium containing anti-i flagellum serum yielded 11 motile mutants, which had the active fliC(e,n,x) and silent fljB(e,n,x) genes. Genetic analysis and Southern hybridization indicated that they had mutations only in the fliC gene, not in the fljB gene or the control elements for phase variation. Nucleotide sequence analysis of the fliC(e,n,x) genes from four representative mutants showed that the minimum 38% (565 bp) and maximum 68% (1,013 bp) sequences of the fliC(i) gene are replaced with the corresponding sequences of the fljB(e,n,x) gene. One of the conversion endpoints between the two genes lies somewhere in the 204-bp homologous sequence in the 5' constant region, and the other lies in the short homologous sequence of 6, 8, or 38 bp in the 3' constant region. The conversions include the whole central variable region of the fljB gene, resulting in fliC(e,n,x) genes with the same number of nucleotides (1,503 bp) as the fljB gene. We discuss the mechanisms for gene conversion between the two genes and also some intriguing aspects of flagellar antigenic specificities in various Salmonella serovars from the viewpoint of gene conversion.

25 citations

Journal ArticleDOI
TL;DR: The results indicated that the invasive ability of the pathogen is significantly increased after 2 h of hyperosmotic stress, and regulator PhoP and sigma factors RpoE, RpoD appear to participate in the network regulatory mechanisms that benefit the pathogenic to adapt hyperosMotic environmental conditions.
Abstract: Salmonella enterica serovar Typhi is a human enteroinvasive pathogen that can overcome the stress caused by the high osmolarity of the human small intestine and cause systemic infection. To investigate the global transcriptional regulations of S. enterica serovar Typhi exposed to a hyperosmotic environment, a genomic oligo-DNA microarray containing 4474 Salmonella genes was prepared. A wild strain of S. enterica serovar Typhi GIFU10007 was grown in LB medium containing 50 mM NaCl to simulate a low osmotic environment. The hyperosmotic stress was simulated by an osmotic up-shift, which increased the concentration of NaCl in the LB from 50 mM to 300 mM. Genome-wide gene expressions of S. enterica serovar Typhi at 15 min, 30 min, 60 min, and 120 min after the osmotic up-shift were investigated by the microarray analysis. Gene expression profiles in somewhat later stage (60 ~120 min) of the stress were quite different from those in the early stage (0 ~ 30 min) of the stress. At 120 min after the osmotic stress, the expression levels of 889 genes were obviously changed. However, expression levels of only 382 genes were significantly changed at 15 min after the osmotic stress. The expression levels of most SPI-1 genes associated with invasion of the pathogen were increased at 120 min after the osmotic up-shift, but were not obviously changed at 15 min or 30 min after the osmotic stress. Expressions of a central regulatory gene, phoP, and sigma factor genes rpoE, rpoD, and rpoS were also changed with different profiles during the osmotic stress. These results indicated that the invasive ability of the pathogen is significantly increased after 2 h of hyperosmotic stress, and regulator PhoP and sigma factors RpoE, RpoD appear to participate in the network regulatory mechanisms that benefit the pathogen to adapt hyperosmotic environmental conditions. The later increased invasive ability of S. enterica serovar Typhi after hyperosmotic stress may be one reason why the pathogen performs invading in the distal ileum of human and not in areas of the upper small intestine.

25 citations

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