| Abstract | U RH školjkaši se uzgajaju cijelom obalom Jadranskoga mora. Uzgoj, izlov i promet
školjkašima reguliran je brojnim zakonskim aktima kojima je cilj stavljanje na tržište
sigurne hrane, no zakonodavstvo ne predviđa kontrolu Vibrio spp., pa postoji mogućnost
da se na tržištu nalaze školjkaši kontaminirani bakterijama ovoga roda koje mogu
uzrokovati pojavu bolesti u ljudi. Kako su potencijalno patogeni vibriji vrlo prošireni u
prirodi, osobito u morskoj i slatkoj vodi, obalama mora i koritima rijeka, često se dokazuju
u različitim vrstama akvatičnih životinja. U našem istraživanju pretražena su ukupno 632
uzorka školjkaša iz istarskoga akvatorija kako bi se dokazala prisutnost najznačajnijih,
potencijalno patogenih vibrija, V. cholerae, V. parahaemolyticus i V. vulnificus. Korištena
je standardna mikrobiološka metoda HRN EN ISO 21872-1:2017, te molekularne PCR
metode koje su dokazale veću prevalenciju (23,89 %) u odnosu na 2,37 %, koliko je
postignuto mikrobiološkom metodom. PCR metodama za dokazivanje genoma bakterija
dokazane su sljedeće prevalencije: V. cholerae 0,95 %; V. parahaemolyticus 17,72 % (uz
temperaturu prednamnažanja 41,5 °C), odnosno 15,36 % (uz temperaturu prednamnažanja
37 °C); V. vulnificus 3,96 % (uz temperaturu prednamnažanja 41,5 °C), odnosno 7,87 %
(uz temperaturu prednamnažanja 37 °C). Po prvi put u RH dokazana je prisutnost bakterije
V. cholerae u školjkašima, te geni bakterije V. parahaemolyticus tdh i trh uz prevalenciju
od 1,6 % odnosno 6,4 %, koji kodiraju enterotoksine odgovorne za simptome oboljenja
ljudi. Utvrđeni izgledi za dokazivanje vrste V. parahaemolyticus bili su 5,18 puta veći ako
je temperatura mora iznad 15 °C (P<0,001). S javnozdravstvenog gledišta od izrazite je
važnosti nalaz potencijalno patogenih bakterija roda Vibrio. Uz povišenu temperaturu mora
i zraka, u vrhuncu turističke sezone kada je najveća potražnja za dagnjama, kamenicama,
kapicama, kunjkama i rumenkama, ti morski plodovi mogu biti izvor infekcije, te je
potrebno pratiti njihovu pojavnost s ciljem zaštite zdravlja ljudi. |
| Abstract (english) | INTRODUCTION
The cultivation of bivalve molluscs is an activity performed by numerous
individuals and organizations in the Republic of Croatia, as shellfish are a traditional food
of the country’s whole coastal area. Their peculiarity lies in the consumption without
thermal processing, or eventually, with only partial thermal processing. Shellfish
cultivation, harvesting, and trade are all regulated by numerous legislations, which aim to
put healthy foods on the market. However, the European Legislation does not call for the
Vibrio spp. examination – this could result in placing the shellfish contaminated by the
bacteria of this genus on the market, their consumption, and consequently, human illness.
The potentially pathogenic vibrios are widely spread in nature, especially within salt and
fresh water, at the seacoasts and riverbeds, and are often detected in various species of
aquatic animals.
METHODS
This paper presents the most important potentially pathogenic halophilic vibrios, V.
cholerae, V. parahaemolyticus and V. vulnificus, detected in bivalve molluscs harvested in
the Istrian aquatory. A total of 632 samples from 18 locations were analysed. The bivalve
molluscs samples collected in the period of time from March 2017 to March 2020 included
mussels, oysters, scallops, Noah's Ark shell and Smooth Venus clam. The following
methods were used: the microbiological method for the detection of potentially
enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus (HRN
EN ISO 21872-1:2017), and the polymerase chain reactions (PCR) for the detection of V.
parahaemolyticus, V. cholerae and V. vulnificus. The polymerase chain reaction (PCR) was
also used for the detection of tdh and trh genes of the positive Vibrio parahaemolyticus.
The PCR methods were used for confirmation of the bacterial isolates, after biochemical
identification. They were also used for the detection of presence of all potentially
pathogenic Vibrio spp. in the secondary enrichment broths.
RESULTS
The classical microbiological method, used on a total of 632 shellfish samples,
detected the prevalence of 2.37 % for the potentially pathogenic Vibrio spp., while
molecular PCR techniques detected a total prevalence of 23.89 % of potentially pathogenic
Vibrio spp., when applied in secondary enrichment broth of all 632 samples. Among 15
confirmed isolates, one was V. cholerae detected in Noah's Ark shell which presents the
first detection of V. cholerae in shellfish sample in Croatia; two isolates were V. vulnificus
detected in mussels and oysters and 12 isolates of V. parahaemolyticus detected in mussels,
Noah's Ark shell and oysters in 8, 3 and 1 samples respectively. The PCR method for the
detection of V. cholerae genes detected a prevalence of 0.95 %. The use of the PCR
method for the detection of V. parahaemolyticus genes detected a prevalence of 17.72 %
with the enrichment temperature of 41.5 °C, while the prevalence of 15.36 % was detected
with the enrichment temperature of 37 °C. The use of the PCR method for the detection of
V. vulnificus genes detected a prevalence of 3.96 % with the enrichment temperature of
41.5 °C, while the prevalence of 7.87 % was detected with the enrichment temperature of
37 °C. The PCR method for the detection of tdh and trh genes of the V. parahaemolyticus
revealed no positive among 12 isolates, while the same method detected a prevalence of
1.6 % and 6.4 % respectively within the total number of positive V. parahaemolyticus
samples when applied on secondary enrichment broth. Detection of tdh and trh genes of
the V. parahaemolyticus presents the first finding in Croatia indicating possible risk for
consumers.
CONCLUSIONS
The use of the ISO method within the classical microbiological method show lower
prevalence compared to the results obtained with the molecular PCR technique, which
implies that the quality of selective culture media and incubation conditions needs further
improvement. The used PCR methods are reliable, but time-consuming, since an individual
PCR test has to be conducted for each bacterial species. Hence, it is necessary to try to
implement multiplex PCR methods, according to the references, which would reduce the
time required to obtain results. A larger percentage of positive samples within the Noah's
Ark shell and the Smooth Venus clam population were noticed at the harvesting areas of
multiple shellfish species, which could be used for the selection of indicator types for the occurrence monitoring of potentially pathogenic Vibrio spp. The determined odds for the
detection of V. parahaemolyticus species are 5.18 times higher with the sea temperature
above 15 °C (P<0.001). Obtained results in all indicate the presence of potentially
pathogenic Vibrio spp. in shellfish harvested in the Istrian aquatory and the need for the
introduction of regular monitoring of their presence. Using the results achieved, the time of
greatest risk for consumers can be selected, when it is necessary to monitor the presence of
potentially pathogenic Vibrio spp. with the aim of protecting human health. |
