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Tipagem molecular de Listeria monocytogenes
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In this work, a set of 59 isolates and six reference strains of Listeria monocytogenes
collected, in the period of March to October of 1992, from nine cheese dairies of the
Ribatejo and West region and Algarve were characterized. The isolates were from
pasteurized cow's milk, ewe’s milk, mixed (ewe, cow and goat) milk, fresh cheeses and
the environments of the cheese dairies where the milk was processed. In three out of
the nine cheese dairies, L. monocytogenes was re-isolated after an intensive cleaning
and disinfection program.
In order to determine the prevalence of the serogroups and the phylogenetic
relatedness among the isolates, the techniques of serotyping by multiplex-PCR
(MPCR) and the analysis of profiles by pulsed-field gel electrophoresis (PFGE) were
used. Multiplex-PCR and PFGE are currently used by several public and private
laboratories for the characterization of L. monocytogenes. Multiplex-PCR constitutes a
practical alternative to slide agglutination serotyping and separates L. monocytogenes
isolates belonging to the twelve serovars into five distinct serogroups. PFGE is
considered the gold standard method for subtyping foodborne pathogens, because of
its high discriminatory power and reproducibility.
The percentage of isolates belonging to serogroup (4b-4d-4e), usually associated with
human cases of listeriosis, was 86% (51/59), confirming recent findings by other
authors and by our team, with isolates from cheese and related environments.
From the 65 isolates analysed, 48 pulsotypes (combined ApaI and AscI profiles) were
identified. The relationships among L. monocytogenes strains based on their combined
ApaI and AscI PFGE profiles are supported by a cophenetic correlation coefficient (ρ)
equal to 0.97. This value explained the goodness of fit of the clustering to the original
data and values higher than 0.80 are considered reasonable. A higher SID value was
achieved with ApaI (0.80) compared to AscI (0.64). The combination of patterns
produced by both enzymes increased the discriminatory power up to 0.99.
In accordance with the visual analysis of the dendrogram, 45 dairy isolates with
indistinguishable ApaI and AscI profiles were identified, suggesting that these might be
isolates of a persistent strain.
The comparison of these isolates with 24 isolates of dairy products in the database of
CBISA (Colecção de Bactérias do Instituto Superior de Agronomia) showed, at a
similarity of 0.71, a subgroup that gathered 10 from these isolates with the 45
mentioned above. The low genetic variability of L. monocytogenes isolates from milk,cheeses and related environments, suggests that some strains of L. monocytogenes
can be better adapted to this specific ecological niche.
Relatively to the 24 isolates of dairy products in the database of CBISA, the higher
prevalence of serogroup (4b-4d-4e) (58%) was also verified.
The contamination of cheese with L. monocytogenes can be explained by a decrease
in the effectiveness of sanitization procedures in use, or by poor hygienic practices in
milking and in cheesemaking.
The application of molecular typing techniques to determine the origin of the
contamination of the cheeses can lead to a better understanding of the routes of
propagation of L. monocytogenes, allowing the planning of more effective prevention
measures.
Computer-assisted data analysis of PFGE data simplifies the processing of a large
number of samples while allowing data sharing if a stringent compliance to standard
protocols is achieved. This standardization includes not only a rigorous
accomplishment of the Pulse-Net standardized protocol but also standardization of
procedures in computer assisted data analysis for pattern normalization and transfers
of information intra and inter laboratories. PulseNet Europe recommends settings of
both optimization and position tolerance at 1.5% for band comparison. Although
complying with these settings, strains visually indistinguishable may still be considered
different according to the clustering analysis. Nevertheless, to improve correct
interpretation of subtyping data, the availability of a large and diverse PFGE type
database is needed.
The PFGE analysis of the data presented here suggests the existence of a persistent
strain in one or more cheese dairies. The high prevalence of serogroup (4b-4d-4e)
among isolates from milk, cheese and dairy environment reported here is particularly
concerning, since this group includes isolates usually associated with both sporadic
and epidemic cases of human listeriosis. These results stress the risk of listeriosis
associated with the consumption of cheese contaminated with L. monocytogenes. The
implementation and maintenance of good manufacturing practices (GMPs) together
with sanitation standard operating procedures (SSOPs) all along the food chain will
contribute to the effective erradication of persistent strains of this pathogenic
bacterium.----------------------------------------Neste trabalho, as técnicas de serotipagem por multiplex-PCR (MPCR) e a análise de
perfis de DNA por electroforese em campo pulsado (PFGE) foram aplicadas a um
conjunto de 65 isolados de Listeria monocytogenes, 59 dos quais com origem em
leites pasteurizados de vaca, de ovelha e de mistura, queijos frescos e ambientes de
nove fábricas onde estes leites foram processados, de modo a determinar
proximidades filogenéticas entre os isolados e a prevalência dos serogrupos.
A percentagem de isolados pertencentes ao serogrupo (4b-4d-4e), habitualmente
associado a casos de listeriose humana, foi de 86% (51/59), confirmando resultados
recentes de outros autores em isolados de queijos e ambientes relacionados.
A partir dos 65 isolados produziram-se 48 pulsotipos (perfis ApaI e AscI combinados).
No entanto, a análise visual identificou 45 isolados com perfis ApaI e AscI
indistinguíveis, sugerindo tratar-se de uma estirpe persistente.
A comparação destes isolados com 24 isolados de produtos lácteos existentes na
base de dados da CBISA mostrou, a 0,71 de similaridade, um subgrupo que reunia 10
destes isolados com os 45 isolados referidos anteriormente, sugerindo uma adaptação
a este tipo de nicho ecológico.
Os resultados apresentados enfatizam o risco associado ao consumo de queijo
contaminado com L. monocytogenes.
Descrição
Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia
Palavras-chave
Listeria monocytogenes multiplex-PCR PFGE cheese queijo prevalência do serogrupo