Limits...
Differentiation of Salmonella strains from the SARA, SARB and SARC reference collections by using three genes PCR-RFLP and the 2100 Agilent Bioanalyzer.

Soler-García AA, De Jesús AJ, Taylor K, Brown EW - Front Microbiol (2014)

Bottom Line: PCR products were individually cut with two different restriction enzymes and the resulting 930 restriction patterns were collected using the Agilent 2100 Bioanalyzer followed by cluster analysis.The combined RFLP results of five sets of restriction patterns allowed us to assign each of the 160 strains to one of 128 restriction types.During inoculation studies we were able to identify S.

View Article: PubMed Central - PubMed

Affiliation: Molecular Methods and Subtyping Branch, Division of Microbiology, Center for Food Safety and Applied Nutrition, US Food and Drug Administration College Park, MD, USA.

ABSTRACT
Rapid molecular typing methods are important tools in surveillance and outbreak investigations of human Salmonella infections. Here we described the development of a three-genes PCR-RFLP typing method for the differentiation of Salmonella species, subspecies and serovars using the Agilent 2100 Bioanalyzer. The fliC, gnd, and mutS genes were PCR-amplified in 160 Salmonella strains representing the two Salmonella species, six subspecies, and 41 different serovars of S. enterica subspecies enterica. PCR products were individually cut with two different restriction enzymes and the resulting 930 restriction patterns were collected using the Agilent 2100 Bioanalyzer followed by cluster analysis. Both species of Salmonella were differentiated by conventional PCR. All of S. bongori tested were gnd PCR negative due to a mismatch at the 3'-end in one the PCR primers. Salmonella subspecies were differentiated into third-teen homogeneous groups representing each of the six subspecies by cluster analysis of restriction patterns generated from the mutS gene cut with AciI. S. enterica subspecies enterica serovars were further differentiated by the combination of the three target genes and five out the six sets of restriction patterns with a discriminatory power of 0.9725 by cluster analysis. The combined RFLP results of five sets of restriction patterns allowed us to assign each of the 160 strains to one of 128 restriction types. During inoculation studies we were able to identify S. Saintpaul and Typhimurium from 24 h pre-enrichment samples using the described method. The use of fliC, gnd, and mutS PCR-RFLP with the Agilent 2100 Bioanalyzer can provide an accessible and automated alternative method for differentiation of Salmonella pathogens.

No MeSH data available.


Related in: MedlinePlus

Concatenated seven housekeeping genes sequences evolutionary relationships of taxa. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. The analysis involved 160 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 3010 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011). Specific, Type I and mix clusters are represented in red, blue, and green, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4127528&req=5

Figure 6: Concatenated seven housekeeping genes sequences evolutionary relationships of taxa. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. The analysis involved 160 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 3010 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011). Specific, Type I and mix clusters are represented in red, blue, and green, respectively.

Mentions: The length of the seven concatenated housekeeping genes was 3,138 bp. The number of variable nucleotides was 59.4% among the 160 Salmonella species, subspecies, and serovars. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011). The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site (Figure 6). Forty-one different clusters and 21 single serovars were identified for a total of 62 different types (Figure 6). This cluster distribution corresponds to a DP of 0.9652. The 41 clusters and the relationship among strains on each cluster are described in Table 4. Twenty-six out of 41 (63.4%) clusters consisted of different homogeneous serovar groups. Twenty out of the 28 (71%) serovars and/or subspecies represented by more than one strain were grouped into homogeneous clusters (Table 4). In eleven out of these 20 (52.4%) homogeneous clusters representing serovars and subspecies containing more than one strain, 100% of the representing strains were grouped together. Fourteen out of 41 (34.1%) clusters were defined as Type I clusters consisting of S. enterica subsp. enterica strains. Five (35.7%), one (7.1%) and eight (57.1%) of the 14 Type I clusters did not share, shared one or two elements in their antigenic formula, respectively. Seven out of eight (87.5%) clusters sharing two elements shared O and the H2 antigen. The remaining cluster sharing two elements of the antigenic formula was composed of strains sharing the O and the H1 (1/8; 12.5%). Only one cluster out of 41 (2.4%) was composed of strains from two different subspecies (Table 4).


Differentiation of Salmonella strains from the SARA, SARB and SARC reference collections by using three genes PCR-RFLP and the 2100 Agilent Bioanalyzer.

Soler-García AA, De Jesús AJ, Taylor K, Brown EW - Front Microbiol (2014)

Concatenated seven housekeeping genes sequences evolutionary relationships of taxa. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. The analysis involved 160 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 3010 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011). Specific, Type I and mix clusters are represented in red, blue, and green, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4127528&req=5

Figure 6: Concatenated seven housekeeping genes sequences evolutionary relationships of taxa. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. The analysis involved 160 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 3010 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011). Specific, Type I and mix clusters are represented in red, blue, and green, respectively.
Mentions: The length of the seven concatenated housekeeping genes was 3,138 bp. The number of variable nucleotides was 59.4% among the 160 Salmonella species, subspecies, and serovars. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011). The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site (Figure 6). Forty-one different clusters and 21 single serovars were identified for a total of 62 different types (Figure 6). This cluster distribution corresponds to a DP of 0.9652. The 41 clusters and the relationship among strains on each cluster are described in Table 4. Twenty-six out of 41 (63.4%) clusters consisted of different homogeneous serovar groups. Twenty out of the 28 (71%) serovars and/or subspecies represented by more than one strain were grouped into homogeneous clusters (Table 4). In eleven out of these 20 (52.4%) homogeneous clusters representing serovars and subspecies containing more than one strain, 100% of the representing strains were grouped together. Fourteen out of 41 (34.1%) clusters were defined as Type I clusters consisting of S. enterica subsp. enterica strains. Five (35.7%), one (7.1%) and eight (57.1%) of the 14 Type I clusters did not share, shared one or two elements in their antigenic formula, respectively. Seven out of eight (87.5%) clusters sharing two elements shared O and the H2 antigen. The remaining cluster sharing two elements of the antigenic formula was composed of strains sharing the O and the H1 (1/8; 12.5%). Only one cluster out of 41 (2.4%) was composed of strains from two different subspecies (Table 4).

Bottom Line: PCR products were individually cut with two different restriction enzymes and the resulting 930 restriction patterns were collected using the Agilent 2100 Bioanalyzer followed by cluster analysis.The combined RFLP results of five sets of restriction patterns allowed us to assign each of the 160 strains to one of 128 restriction types.During inoculation studies we were able to identify S.

View Article: PubMed Central - PubMed

Affiliation: Molecular Methods and Subtyping Branch, Division of Microbiology, Center for Food Safety and Applied Nutrition, US Food and Drug Administration College Park, MD, USA.

ABSTRACT
Rapid molecular typing methods are important tools in surveillance and outbreak investigations of human Salmonella infections. Here we described the development of a three-genes PCR-RFLP typing method for the differentiation of Salmonella species, subspecies and serovars using the Agilent 2100 Bioanalyzer. The fliC, gnd, and mutS genes were PCR-amplified in 160 Salmonella strains representing the two Salmonella species, six subspecies, and 41 different serovars of S. enterica subspecies enterica. PCR products were individually cut with two different restriction enzymes and the resulting 930 restriction patterns were collected using the Agilent 2100 Bioanalyzer followed by cluster analysis. Both species of Salmonella were differentiated by conventional PCR. All of S. bongori tested were gnd PCR negative due to a mismatch at the 3'-end in one the PCR primers. Salmonella subspecies were differentiated into third-teen homogeneous groups representing each of the six subspecies by cluster analysis of restriction patterns generated from the mutS gene cut with AciI. S. enterica subspecies enterica serovars were further differentiated by the combination of the three target genes and five out the six sets of restriction patterns with a discriminatory power of 0.9725 by cluster analysis. The combined RFLP results of five sets of restriction patterns allowed us to assign each of the 160 strains to one of 128 restriction types. During inoculation studies we were able to identify S. Saintpaul and Typhimurium from 24 h pre-enrichment samples using the described method. The use of fliC, gnd, and mutS PCR-RFLP with the Agilent 2100 Bioanalyzer can provide an accessible and automated alternative method for differentiation of Salmonella pathogens.

No MeSH data available.


Related in: MedlinePlus