Almeida, Gonçalo N.

Carneiro, L. M.

Hogg, T. A.

Magalhães, R. S.

Oliveira, B. D.

Santos, I. R.

Teixeira, Paula

English

Repositório Institucional da Universidade Católica Portuguesa

COMPARISON OF TWO SEROTYPING METHODS OF L. MONOCYTOGENES ISOLATED FROM HUMAN CASES OF LISTERIOSIS, FOOD AND FOOD PLANTS ENVIRONMENTAlmeida GN*, Magalhães RS, Santos IR, Carneiro LM, Oliveira BD, Teixeira PC and Hogg TAIntroductionListeria monocytogenes is the causative agent of serious epidemic and sporadic food-borne listeriosis. Although rare when compared to other food-borne diseases, a significant feature of listeriosis is the high lethality rate (about 30%), which makes L. monocytogenes a pathogen of considerable concern. Apparently, not all strains of L. monocytogenes are capable of causing disease in humans. Of the 13 serovars only 4 (1/2a, 1/2c, 1/2b, and 4b) are associated with more than 98% of known reported human cases of listeriosis. Despite it’s poor discriminatory power, with only four serotypes being found in foods or involved in cases of human listeriosis, serotyping is usually the first step in a typing scheme. When an outbreak or contamination route, are under investigation, serotype information allows investigators to exclude strains from further study.Serotyping by conventional methods requires several days (3-5) before final results are available, whilst using multiplex PCR, results can be obtained in 6 hours.The aim of this study was to compare the performance of these two methods using strains from clinical, food or from food plant environments deposited at the Listeriastrain collection at Escola Superior de Biotecnologia. Materials and MethodsA total of 185 strains - 39 of food origin, 55 from food plant environments and 91 from human clinical cases - were used in this study.Classical agglutination serotyping was performed using anti O and anti H antisera according to the manufacturers’ instructions (Denka Seiken Co., Ltd, Tokyo, Japan) and the modifications described by Lukinmaa (2003). Multiplex PCR “serotyping” was performed according to Doumith et al (2004). ResultsTable 1 shows the results obtained by both methods tested. Three different serotypes were identified by the agglutination methodology: 19 strains were serotype 1/2a, 97 were serotype 1/2b and 69 were serotype 4b. By multiplex-PCR strains were grouped in three groups: 14 strains were 1/2a-3a, 103 were 1/2b-3b and 68 were 4b-4d-4e. Universidade Católica Portuguesa · Escola Superior de BiotecnologiaGonçalo Nieto AlmeidaRua Dr. António Bernardino de Almeida  · 4200-072 PORTOTel.: + 351 225 580 00 81  · Fax: + 351 225 580 111E-mail: gnalmeida@esb.ucp.ptConclusionA linear relationship was found between results obtained using classical agglutination serotyping (Denka Seiken antisera) and multiplex-PCR: Classical agglutination = 0,95 multiplex-PCR+ 6,75; R2= 1,00. The overall concordance between the two methods was 94%. For serotypes 1/2a, 1/2b and 4b it was 68%, 98% and 94%, respectively.It was demonstrated that molecular serotyping can be a useful rapid method although it does not distinguish strains 1/2b from 3b and 7, 1/2c from 3c and 4b from 4e and 4d. PCR patterns are, however,  less subjective to interpret  than the agglutination method.References(1) Lukinmaa S, Aarnisalo K, Suihko M, Siitonen A. Diversity of Listeria monocytogenesisolates of human and food origin studied by serotyping, automated ribotyping and pulsed-field gel electrophoresis. Clin Microbiol Infect. 2004; 10:562-8.(2) Doumith M., Buchrieser C., Glaser P., Jacquet C. and Martin P., 2004. J. Clin. Microbiol. 42 : 3819-3822AcknowledgmentsThis work was funded by Fundação para a Ciência e Tecnologia project POCTI/AGR/44889/2002.AgglutinationPCR1/2a – 3a 1/2b – 3b 4b – 4d – 4e1/2a 13 5 11/2b 0 95 24b 1 3 65Table 1 – Typing results obtained by multiplex PCR and agglutination Denka commercial antiseraIn Figure 1 the linear regression  obtained for both methodologies is shown. Figure 2 show an example of the multiplex assay profilesFigure 1 – Linear regression obtained for typing by multiplex PCR vs classical agglutination.Figure 2 –Lane 1 DNA ladder (Biorad, EZ Load™ Molecular Rulers), lanes 2 – 5 control strain profiles for multiplex groups: 4b-4d-4e, 1/2c-3c, 1/2b-3b-7, 1/2a-3a, respectively, lanes 8 -12 profiles obtained from isolates.y = 0,9524x + 6,7471R2 = 1,0001020304050607080901000 20 40 60 80 100Multiplex PCRDenka antisera1     2    3     4    5    6    7    8     9   10   11   12

Comparison of two serotyping methods of L. monocytogenes isolated from human cases of listeriosis, food and food plants environment

https://repositorio.ucp.pt/bitstream/10400.14/5659/1/com-inter_2007_esb_1062_Almeida_Gon%c3%a7alo_22.pdf

Comparison of two serotyping methods of L. monocytogenes isolated from human cases of listeriosis, food and food plants environment

Abstract

Similar works

Full text

Available Versions

Repositório Institucional da Universidade Católica Portuguesa