Gene Scanning of an Internalin B Gene Fragment Using High-Resolution Melting Curve Analysis as a Tool for Rapid Typing of Listeria monocytogenes

Abstract

The ability to accurately track Listeria monocytogenes strains involved in outbreaks isListeria monocytogenes, the causative agent of listeriosis, is a facultative intracellular pathogen of humans and animals. It is widespread in the environment and has the ability to survive and grow under extreme conditions. Patients with listeriosis show symptoms such as gastroenteritis, encephalitis, meningitis, and septicemia. The high case-fatality proportion of approximately 20% to 30% makes L. monocytogenes an important human pathogen. [1][2][3] In listeriosis outbreaks and for epidemiological investigations, a fast and accurate protocol for subtyping L. monocytogenes is essential. 4 In outbreak situations the L. monocytogenes serotyping scheme based on somatic (O) and flagellar (H) antigens 5 has limited value for tracking isolates. Of the 13 serotypes of L. monocytogenes, only a small fraction (serotypes 4b, 1/2a, and 1/2b) account for more than 96% of reported human listeriosis cases in Austria. 2 Insufficient reproducibility of serotyping, 6 relatively low discriminatory power, and antigen sharing among serotypes all impede the value of serotyping in outbreak investigations and so demand more accurate molecular-based typing solutions. 7 Research toward development of molecular typing protocols based on virulence gene analysis, ribotyping, and microarray analysis revealed that L. monocytogenes comprises five phylogenetic groups (PG). 8 -11 Recently developed multiplex PCR serotyping methods allow a differentiation of isolates on the PG level. 12,13 Phylogenetic groups have been correlated with serotypes: PG I.1 with serotype 1/2a, 3a; I.2 with 1/2c, 3c; II.1 with 4b, 4d, 4e; II.2 with 1/2b, 3b, 7; and III with 4a, 4c. 10 In cases of outbreak, the discriminatory power of multiplex PCR serotyping methods is insufficient, and it is necessary to type isolates by pulsed-field gel electrophoresis (PFGE), which is the current gold standard for L. monocytogenes typing. The PFGE method is time-consuming, however, and is difficult to standardize, which hampers interlaboratory exchange and comparison of typing results. 14 Typing methods by DNA sequence analysis and single nucleotide polymorphism (SNP) detection appear more promising for fast and accurate strain typing. Recently developed multilocus sequence typing and multivirulence locus sequence typing protocols are accurate and highly discriminative procedures for subtyping of L. monocytogenes strains. 14 -17 The improved discriminatory power of multivirulence locus sequence typing, compared with multilocus sequence typing and PFGE, was demonstrated by subtyping of genetically diverse L. monocytogenes isolates. 18,19 For rapid identification and typing of isolates in routine diagnostics, a PCR-based typing method targeting a sin-

Listeriosis: The Dark Side of Refrigeration and Ensiling

“…High resolution melting (HRM) curve-PCR analysis for L. monocytogenes was developed by Pietzka et al (2011). Genomic bacterial DNA (gDNA) is extracted from bacterial cells grown overnight at 37 ° on blood agar plates and a 500-bp fragment located in the virulence gene internalin B ( inlB) is amplified for subsequent HRM analysis.…”

Technique d’analyse des courbes de fusion haute résolution (FHR) ou High Resolution Melting analysis (HRM) en infectiologie

“…• soit elles permettent d'identifier des espèces microbiennes ; • soit, en épidémiologie, elles permettent de typer des souches virales ou bactériennes [7][8][9][10][11] ou de différencier les souches vaccinales des souches sauvages [12,13] ; • soit elles permettent de détecter des mutations notamment sur les gènes de résistance et, ainsi, de différencier les souches sauvages des souches mutées [14].…”

High resolution melting analysis for the characterization of lineage II Listeria monocytogenes serovars 1/2a and 1/2c based on single nucleotide polymorphisms identification within the Listeria Pathogenicity Island-1 and inlAB operon: a novel approach for

“…); genotyping of L. monocytogenes isolates from clinical cases targeting inl B (Pietzka et al . ), and from poultry carcasses and slaughterhouse environments by characterizing the inl B and ssr A genes (Sakaridis et al . ).…”