Amplified Intergenic Locus Polymorphism as a Basis for Bacterial Typing of Listeria spp. and Escherichia coli

Abstract

DNA-based methods are increasingly important for bacterial typing. The high number of polymorphic sites present among closely related bacterial genomes is the basis for the presented method. The method identifies multilocus genomic polymorphisms in intergenic regions termed AILP (amplified intergenic locus polymorphism). For each locus, a pair of unique PCR primers was designed to amplify an intergenic sequence from one open reading frame (ORF) to the adjacent ORF. Presence, absence, and size variation of the amplification products were identified and used as genetic markers for rapidly differentiating among strains. Polymorphism was evaluated using 18 AILP sites among 28 strains of Listeria monocytogenes and 6 strains of Listeria spp. and 30 AILP markers among 27 strains of Escherichia coli. Up to four alleles per locus were identified among Listeria strains, and up to six were identified among E. coli strains. In both species, more than half of the AILP sites revealed intraspecies polymorphism. The AILP data were applied to phylogenetic analysis among Listeria and E. coli strains. A clear distinction between L. monocytogenes and Listeria spp. was demonstrated. In addition, the method separated L. monocytogenes into the three known lineages and discriminated the most common virulent serotypic group, 4b. In E. coli, AILP analysis separated the known groups as well as the virulent O157:H7 isolates. These findings for both Listeria and E. coli are in agreement with other phylogenetic studies using molecular markers. The AILP method was found to be rapid, simple, reproducible, and a low-cost method for initial bacterial typing that could serve as a basis for epidemiological investigation.Bacterial strain typing has several important applications in microbiology. In clinical practice, strain typing is useful for diagnosis and determining treatment strategy and is essential for rapid identification of disease outbreaks and new virulent strains. In the food industry, strain typing is necessary to ensure food safety and for linking cases of food-borne infections to suspected items in the food chain. Classical bacterial identification is based on selective enrichment, followed by plating on selective media. Species identification is mainly by biochemical characterization, and strain identification is primarily based on serology. These methods do not meet the requirement for rapid identification and typing in clinical, epidemiological, and food industry applications. Recent advances in biotechnology have resulted in the development of numerous methods for detection and typing of microorganisms (11-13, 19, 25) which differ in their sensitivity, rapidity, labor intensiveness, complexity, discriminatory power, reproducibility, and cost (5, 32, 43, 49). In principle, by screening a large number of polymorphic sites, genomic methods should be able to provide very accurate discrimination among closely related strains. The total multilocus output of these methods is often termed "DNA fingerprints" or a "DNA bar code."In the pre...

Vibrio vulnificus Typing Based on Simple Sequence Repeats: Insights into the Biotype 3 Group

“…Two of the loci gave no amplification product (a null allele) for some of the isolates. Null alleles were taken into account in the analysis of relatedness, thus permitting a more accurate assessment than would be possible by ignoring the presence of null alleles (9,24,48,57). An analysis of the two chromosomes revealed a higher number of alleles per locus in chromosome II than in chromosome I (12 and 8 alleles on average, respectively) ( Table 3).…”

Vibrio cholerae Strain Typing and Phylogeny Study Based on Simple Sequence Repeats

“…The NST analysis provides 100% typeability of any strain at any selected locus by combining both sequence variations and product amplification results versus the commonly used sequence based methods that consider only sequence variations (51,79). In addition, NST analysis enables the combination of data from various diagnostics methods, e.g., MNR-MLST, L-SSR and AILP (75), as well as the comparison of results from various typing methods.…”

“…The original MLST method is based on sequence variation at housekeeping genes that are conserved and therefore usually present in all strains but that have limited variation (21,59,69). On the other hand, the MNR loci that present high levels of polymorphism have the disadvantage of missing data from a portion of the strains due to nonamplified ("null") alleles (20,75) as a result of sequence mismatch (SNPs) at the priming sites, as well as insertions or deletions and genome rearrangements (32). We have solved the problem of "no data" from part of the tested strains by using a nonparametric sequence type NST analysis.…”

Rapid Discrimination of Listeria monocytogenes Strains by Microtemperature Gradient Gel Electrophoresis

“…For example, ribotyping requires 8 h (26), PFGE requires 20 to 24 h (34), and microarray typing requires an overnight hybridization (4). RAPD analysis and some of the PCR-electrophoresis-based methods can be performed in a time frame similar to that required for -TGGE (15,21,36), making -TGGE one of the fastest typing methods. In addition, -TGGE showed minimal problems with respect to reproducibility, which can be an issue in RAPD analysis (41).…”

“…Among these, serovars 1/2a, 1/2b, and, especially, 4b are implicated in many cases of human listeriosis (38). Various molecular typing methods beyond serovar typing have been investigated, including ribotyping (26), pulsed-field gel electrophoresis (PFGE) (34,44), randomly amplified polymorphic DNA (RAPD) analysis (21), PCR-electrophoresis-based methods (15,36), microarrays (4), esterase typing (9), amplified fragment length polymorphism analysis (1,17), and multilocus enzyme electrophoresis (11). Among these, ribotyping and PFGE have better discriminatory powers and have been widely used (16).…”