Comparison of methods for discrimination between strains of Listeria monocytogenes from epidemiological surveys

Abstract

Total cellular DNA from 28 strains of Listeria monocytogenes isolated from food implicated in food-borne illness and from patients with listeriosis was digested with the restriction endonucleases HindIII, HaeIII, and EcoRI. Following agarose gel electrophoresis, the fragments were subjected to Southern blot hybridization with a digoxigenin-labeled cDNA probe transcribed from Escherichia coli 16S and 23S rRNA. The patterns of bands from genomic (DNA fingerprints) and rDNA fingerprints (ribotypes) were used for classifying L. monocytogenes strains, and the resulting subtypes were compared with serotyping and multilocus enzyme electrophoresis classffication schemes. A total of 15 distinct and identical groups were obtained when genomic DNA was digested with either HindIII or HaeIII. The most discriminating enzyme for ribotyping of strains was EcoRI, which divided the 28 strains of L. monocytogenes into 6 ribotype groups. DNA fingerprinting and ribotyping differentiated L. monocytogenes from other Listeria spp., including L. ivanovii, L. welshimeri, and L. innocua as well as the lactic acid bacteria Lactococcus lactis subsp. lactis and subsp. cremoris. L. monocytogenes strains isolated from four independent food-borne illness incidents were analyzed by all typing methods. Patient and product isolates were not distinguishable by serotyping, ribotyping, or multilocus enzyme electrophoresis. DNA fingerprinting was the only method capable of differentiating these strains, or conversely, of proving relatedness of patient-product pairs of isolates. This method was a relatively simple, sensitive, reproducible, and highly discriminating method for epidemiological tracking of L. monocytogenes implicated in food-borne illness.

Random amplification of polymorphic bacterial DNA: evaluation of 11 oligonucleotides and application to food contaminated with Listeria monocytogenes

“…A schematic presentation which is often applied in connection with restriction fragment length polymorphism analysis (Tveten et al 1991 ;Lew and Desmarchelier 1992) or ribotyping (Baloga and Harlander 1991;Jacquet et al 1992) is, according to our experience, not possible with RAPD profiles, because of the appearance of weak bands in every RAPD system described so far. The interpretation and comparison is possible only if the strains to be compared are investigated together and loaded adjacent on the same gel and compared directly.…”

Restriction fragment length polymorphism analysis of Fusobacterium necrophorum using a novel repeat DNA sequence and a 16S rRNA gene probe

“…A valuable tool to study the epidemiology of bacterial diseases is to use repeated DNA sequences as probes to detect restriction fragment length polymorphisms (RFLPs) between bacterial isolates. Although ribosomal RNA gene probes have been used to great effect to generate RFLPs [5][6][7] we sought a novel repeat DNA sequence to provide a greater degree of sensitivity and flexibility to the analysis. Repeated DNA sequences that have commonly been found on bacterial chromosomes, in addition to ribosomal RNA genes [19][20][21], include short, interspersed, repetitive DNA such as repetitive extragenic palindromic (REP) sequences [22], enterobacterial intergenic consensus sequences (ERICs; [23]) and 209 insertion (IS) elements.…”

“…A common method of typing bacterial isolates is to compare DNA restriction fragment length polymorphism (RFLP) patterns. These can be generated by hybridising total DNA restriction profiles with DNA 206 probes made from repeated DNA sequences such as ribosomal RNA genes (ribotyping [5][6][7][8]) or insertion sequences [9,10].…”

Methods for the isolation and identification ofListeriaspp. andListeria monocytogenes: a review

“…With only minor adjustments this technique can be applied to almost any bacterium or strain. In comparative studies with other typing methods it was found to be the most discriminatory test, in particular for typing of L. monocytogenes serotypes 1/2 and 4 [65,158,203,214,217].…”

“…In general, ribotyping of Listeria isolates involves the restriction enzyme digestion of chromosomal DNA followed by DNA hybridization using an rRNA gene probe. Resulting banding patterns are used to sort Listeria isolates into ribotypes [202] and establish the relatedness of isolates [65,194,204,205]. Ribotyping has been extensively used in epidemiological studies [170,[206][207][208] and automation has allowed the adaptation of this method for routine analysis [209][210][211][212][213][214].…”