Presence of cytotoxicAeromonas and Plesiomonas shigelloides in

Presence of cytotoxicAeromonas and Plesiomonas shigelloides in

176
Rev Biomed 1998; 9:176-180.
Brief Communication
Presence of cytotoxic Aeromonas
and Plesiomonas shigelloides in
fresh vegetables.
Rafael Monge1, María L. Arias-Echandi2, Dagmar Utzinger2.
1
Instituto Costarricense de Investigación y Enseñanza en Nutrición y Salud. Aptd 4, Tres Ríos. 2Facultad de Microbiología, Universidad de Costa Rica. Costa Rica.
SUMMARY.
Introduction. Fresh vegetables are an essential
part of the human diet, but they also represent
an important vehicle for the dissemination of
different pathogenic microorganisms. The
objective of this study was to evaluate the
potential impact of Aeromonas species and
Plesiomonas shigelloides isolated from fresh
produce, in Costa Rica.
Material and Methods. 150 samples of fresh
vegetables, including lettuce, cilantrum leaves
and celery were evaluated for the presence of
Aeromonas species and Plesiomonas
shigelloides according to the methodology
described by von Graevenitz. Also, a
cytotoxicity determination was carried out
according to the methodology described by
Cumberbatch et al.
Results. Aeromonas sp. were found in 30% (15
samples) of cilantrum leaves, 52% (26 samples)
of lettuce and 46% (23 samples) of celery.
Plesiomonas shigelloides were found in 6% (5
samples) of lettuce and 3% (3 samples) of
celery. Twenty nine (45%) of the Aeromonas
isolates were cytotoxin positive.
Discussion. Even though the specific public
health significance of cytotoxic Aeromonas and
Plesiomonas shigelloides in fresh produce is
unknown, its presence in vegetables suggests
that the sanitary quality of these represents a
risk for public health. Hazard Analisis and
Critical Control Point (HACCP) programs must
be developed in order to minimize the risk of
illness which the consumption of these products
represent.
(Rev Biomed 1998; 9:176-180)
Key words: Aeromonas, Plesiomonas shigeloides, human diet.
RESUMEN.
Presencia de Aeromonas citotóxicas y Plesiomonas shigelloides en vegetales frescos.
Corresponding address: María Laura Arias E. Facultad de Microbiología, Universidad de Costa Rica, Ciudad Universitaria "Rodrigo Facio", Costa
Rica, América Central.
Received December 3, 1997; Accepted January 21, 1998.
This paper is also available at http://www.uady.mx/~biomedic/rb98935.html
Revista Biomédica
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Aeromonas y Plesiomonas shigelloides en vegetales.
Introducción. Los vegetales frescos forman una
parte esencial de la dieta humana, pero también
representan un vehículo importante para la transmisión de diferentes microorganismos patógenos. El objetivo de este estudio fue evaluar el
impacto potencial de las especies de Aeromonas y Plesiomonas shigelloides, aisladas a partir de productos frescos en Costa Rica.
Material y métodos. 150 muestras de vegetales frescos, incluyendo lechuga, hojas de culantro y apio fueron evualadas para la presencia de
Aeromonas sp. y Plesiomonas shigelloides según la metodología descrita por von Graevenitz. También se determinó la citotoxicidad de las
especies de Aeromonas según la metodología
descrita por Cumberbatch et al.
Resultados. Aeromonas sp. fue aislada en el
30% (15 muestras) de las hojas de culantro, 52%
(26 muestras) de las lechugas y 46% (23 muestras) del apio. Plesiomonas shigelloides fue aislada en el 6% (5 muestras) de la lechuga y 3%
(3 muestras) del apio. Veintinueve (45%) de las
Aeromonas aisladas eran citotoxina positivas.
Discusión. Aún cuando el significado específico de la presencia de Aeromonas citotóxica y
Plesiomonas shigelloides en productos frescos
es desconocido, su presencia en vegetales sugiere que la calidad sanitaria de estos representa un riesgo para la salud pública. Se debe desarrollar programas de Análisis de Riesgos y Puntos Críticos de Control (ARPCC) para minimizar el riesgo de enfermedad que el consumo de
estos productos representa.
(Rev Biomed 1998; 9:176-180)
Palabras clave: Aeromonas, Plesiomonas shigelloides, dieta humana.
INTRODUCTION.
Fresh vegetables are an essential part of
the diet of people worldwide, and during the last
years their consumption has significantly increased due to their promotion by health authorities
in order to prevent the development of some
chronic diseases such as atherosclerosis and cancer (1). Nevertheless, the presence of some pathogenic microorganisms in fresh produce has
been recognized for many years. Several outbreaks of human gastroenteritis have been linked
to the consumption of fresh vegetables contaminated with pathogenic microorganisms such
as enterotoxigenic Escherichia coli, E. coli
0157; H7, Shigella flexneri, Salmonella enteritidis, Vibrio cholerae and Bacillus cereus (2).
Vegetables may become contaminated with
pathogenic microorganisms during irrigation.
Aeromonas species and Plesiomonas are especially important, since both bacteria are of acquatic origin, also, they have been linked with
gastroenteritis and the consumption of raw vegetables (2-4). Callister and Agger (5) isolated
cytotoxic Aeromonas from parsley, spinach, celery, alfalfa, sprouts, brocoli, lettuce, endive,
escarole and kale, confirming the role of fresh
vegetables as vehicles of pathogenic microorganisms.
In Costa Rica, vegetables can play an
important role in the spread of these bacteria,
since an important percentage of superficial
water used for their irrigation does not fulfill the
sanitary normatives established by international
organisms (6). The aim of this study is to
evaluate the potential impact of Aeromonas
species and Plesiomonas shigelloides, isolated
from fresh produce, in Costa Rican inhabitants.
MATERIAL AND METHODS.
A total of 150 samples of fresh vegetables,
including 50 samples of lettuce (Lattuca sativa),
50 samples of cilantrum leaves (Coriandrum
sativcim) and 50 samples of celery (Apium
graveolens) were tested for the presence of both
bacteria. Samples of these vegetables were
collected from five open markets (agricultural
fairs) during the first trimester of 1997. Each
sample was analyzed according to the
Vol. 9/No. 3/Julio-Septiembre, 1998
178
R Monge, ML Arias-Echandi, D Utzinger.
methodology described by von Graevenitz (7).
Briefly, 25 g of each sample was enriched in
alcaline peptonated water 0.1% (pH 8.4) for 24
hours at 35ºC. After this incubation period, two
plates of inositol-bile-brillant green agar were
streaked, and incubated for 18 h at 35ºC. Typical
colonies were purified using blood agar for
further biochemical identification, which
included the following tests: Gram stain, TSI
(triple-sugar-iron), oxidase, catalase, motility,
lysine, arginine and ornitine descarboxilase,
esculine hydrolisis, Voges Proskaues, and
glucose, inositol, manitol, salicine, arabinose,
xilose fermentation.
The determination of citotoxicity of the
Aeromonas species was done according to the
methodology described by Cumberbatch et
al.(8). Aeromonas strains were propagated in 8
ml of brain heart infusion broth incubated at 35ºC
with agitation at 100 rpm for 18 to 24 h. The
broth cultures were cleared by centrifugation at
10000 x g for 30 min at 4ºC, followed by
filtration through a 0.45mm filter (Millipore
Corp). Cell free supernatants were stored at 4ºC
for use within one day. Tests for cytotoxin were
performed with culture supernatants added to
Hela cell monolayers maintained in minimal
essential medium containing 5% fetal calf serum.
The supernatants were added at a final dilution
of 1:5 to monolayers in 96 well microtiter plates.
Following an 18 to 24 h incubation at 35ºC under
5% CO2 and 90% relative humidity, the cells
were examined for cell death. Brain heart
infusion broth diluted with minimal essential
medium served as a negative control.
Only supernatnats which caused 100% detachment were considered as cytotoxin positive. Vital dye exclusion was not needed to confirm cell death because of the easily recognizable morphological cell changes, including cell
shrinkage, loss of adherence, and pyknotic nuclei.
Revista Biomédica
RESULTS.
Aeromonas sp. were found in 30% (15
samples) of cilantrum leaves, 52% (26 samples)
of lettuce and 46% (23 samples) of celery.
Plesiomonas shigelloides were found in 6% (5
samples) of lettuce and 3% (3 samples) of celery.
Twenty nine (45%) of the Aeromonas isolates
were cytotoxin positive. Citotoxic Aeromonas
sp. was recovered in ca 50% of the three kinds
of produce sampled.
Even though the climate changed from cold
and windy (January) to hot and sunny (FebruaryMarch), no difference was observed on the
isolation of both bacteria.
DISCUSSION.
The specific public health significance of
cytotoxic Aeromonas and Plesiomonas shigelloides in fresh produce is unknown. However, its
presence in fresh vegetables represents a potential risk for the development of gastroenteritis
Aeromonas species are considered to be ubiquous in nature (2-3); therefore it is not surprising
that they have also been isolated from fresh produce, nevertheless, the identification of cytotoxic
Aeromonas represents a risk for public health,
even more if one considers that other researchers
have correlated Aeromonas toxigenicity with
hemolysin production (9-10). Burke et al. (9),
Callister and Agger (5) reported that 90-97%
of cytotoxic Aeromonas strains could be correctly classified by the hemolysin assay.
This is important since, althouth Aeromonas role as a foodborne illness agent has not been
fully confirmed (2-3,5), its potential as infectious agent exists through several mechanisms (enterotoxin similar to Cholera toxin, cytotoxin and
hemolysin) as occurs in Escherichia coli (2-3,5).
Aeromonas species are psychrotrophic organisms (5,9), therefore its growth at 5ºC is significant. Refrigeration will not prevent Aeromonas sp. growth, and only a small temperature
increase can lead to rapid propagation of this
179
Aeromonas y Plesiomonas shigelloides en vegetales.
bacteria in produce as has been shown in other
works (2,5). P. shigelloides presents a different
behavior, since it can not grow under 8 to 10
ºC, but it can survive under these conditions, representing a risk for the consumer (3,11).
Recent studies confirm the role of Plesiomonas shigelloides as a primary pathogenic
agent in diarrheal episodes (3). Different studies
have shown an increase in the reported cases of
diarrhea, as well as high P. shigelloides isolation from environmental sources during warm
season (3,4) and no recovery occurred during
the colder months. Anyway, in Costa Rica this
bacteria may be isolated from vegetables any
time during the year, as has been reported in
Zaire (12), since the temperature remains relatively warm year round and never drops to
freezing.
The presence of different pathogenic microorganisms, such as cytotoxic Aeromonas,
Plesiomonas shigelloides, Listeria monocytogenes, Cryptosporidium, Giardia intestinalis, Entamoeba histolytica HAV and Rotavirus in fresh
vegetables consumed in Costa Rica (13-16), suggests that the sanitary quality of these represents a risk for public health.
The methods of culture, harvest, handling,
processing, packaging and distribution of fresh
produce on a regional or local scale within the
country, must receive attention in terms of identifying and controlling microbiological hazards.
Hazard analysis and critical control point (HACCP) programs must be developed in an effort to
minimize the risk of illness that the consumption
of fresh produce represent.
ACKNOWLEDGEMENT.
We thank the cooperation of the Virus Section,
Faculty of Microbiology, for the preparation of cellular
cultures.
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