Suitability of PCR fingerprinting, infrequent-restriction-site PCR, and pulsed-field gel electrophoresis, combined with computerized gel analysis, in library typing of Salmonella enterica serovar enteritidis

Abstract

Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D = 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.

FIG. 1

Similarity dendrograms of band patterns of Salmonella serovar Enteritidis strains produced by PCR fingerprinting and M13 primer. (A) Amplification of DNA was performed with a Linus Autocycler 32 thermocycler in several batches. (B) Amplification of DNA was performed with a PE Applied Biosystems 9600 thermocycler in one batch.

FIG. 2

IRS-PCR of different serovars of Salmonella obtained with the enzyme combination XbaI-HhaI. (A) Lane M, molecular mass marker (pGEM). Lane 1, serovar Arizonae; lane 2, serovar Litchfield; lane 3, serovar Virchow; lane 4, serovar Miami; lane 5, Salmonella serogroup 11; lane 6, serovar Abony; lane 7, serovar Virchow; lane 8, serovar Dublin; lane 9, serovar Enteritidis; lane 10, serovar Typhimurium. Numbers on the left indicate the size of the marker in base pairs. (B) Similarity dendrogram of the IRS-PCR patterns.

FIG. 3

Similarity dendrogram of the IRS-PCR patterns of 36 epidemiologically unrelated Salmonella serovar Enteritidis strains obtained with enzyme combination XbaI-HhaI. En, England; Dk, Denmark; Sp, Spain.

FIG. 4

Agarose gels of the PFGE profiles of Salmonella serovar Enteritidis obtained in the intercenter reproducibility study of PFGE. Fifteen duplicated strains were processed in two laboratories. Pictures on the left were obtained in center C (Denmark) and those on the right in center B (Spain). (A) DNA digested with restriction enzyme XbaI. (B) DNA digested with restriction enzyme SpeI. (C) DNA digested with restriction enzyme BlnI. Lanes M, lambda ladder used as a molecular marker. Numbers on the right indicate the size of the marker in kilobases.