Provedenim istraživanjem je po prvi puta u školjkašima istočne obale Jadrana dokazana
prisutnost verotoksigene E. coli (VTEC), a ujedno i enteropatogene E. coli (EPEC). Cilj
istraživanja je bio dokazati prisutnost i mogućnost detekcije VTEC u morskih školjkaša.
VTEC O157:H7 uz 3-5% sojeva E. coli ne posjeduju enzim ß-glukuronidazu (ß-GLUC(-)) pa
se ne mogu otkriti propisanom MPN metodom za E. coli u školjkašima. Modifikacijom
klasičnih metoda za izdvajanje i brojanje ß- GLUC(+) E. coli i metoda za izdvajanje ß- GLUC
(-) E. coli O157:H7, u periodu od 2007. do 2009. godine sakupljene su kulture iz 900 uzoraka
školjkaša. Istovremeno je 258 uzoraka školjkaša pretraženo ELISA metodom za detekciju Stx
u fecesu, prilagođenoj školjkašima. U jednom uzorku školjkaša utvrđen je Stx, a u izolatu E.
coli PCR postupkom je potvrđena prisutnost stx gena. Pokusom zagađenja školjkaša
prirodnom filtracijom mora onečišćenog s VTEC O157:H7 potvrđena je mogućnost prijenosa
VTEC iz mora u školjkaše uz očuvanu sposobnost stvaranja verotoksina, što je i istovremena
potvrda izabranih metoda. U izolatima E. coli, izdvojenima iz uzoraka školjkaša tijekom
redovite kontrole, PCR pretragom su iz četiri uzorka dagnji i jednog uzorka prnjavica utvrđeni
različiti genotipovi, stx1; stx2, stx1+eae, te stx1+eae+hlyA - genotip visoko patogenih sojeva
EHEC. Prevalencija VTEC iznosi 1% (5/521) u uzorcima s izdvojenom E. coli, odnosno 0,6%
(5/900) u odnosu na ukupni broj pretraženih školjkaša. U 1,9% (17/900) uzoraka školjkaša
utvrđena je i EPEC. Utvrđeni serotipovi VTEC su O7, O78, te O55 povezani sa slučajevima
hemoragijskog uremičnog sindroma - HUS. Prevalencija VTEC u ukupnom broju pretraženih
uzoraka je u prnjavicama iznosila 0,9% dok je u dagnji bila 0,6%. U kamenicama i kunjkama
bakterija nije utvrđena. Opažene razlike u prevalenciji VTEC između različitih vrsta školjkaša
nisu statistički značajne (p=0,8). VTEC su pronađene u 2/12 proizvodnih područja s najvećim
brojem u Malostonskom zaljevu (4/498), ali je prevalencija VTEC od 0,8% nešto manja nego
u području Ušća rijeke Krke (0,9%) sa 1/110 VTEC u pretraženim školjkašima. Razlike u
prevalenciji VTEC između proizvodnih područja nisu statistički značajne (p=0,87 i p=0,9).
Opažene razlike MPN E. coli/100g bez obzira na vrstu školjkaša i proizvodno područje nisu u
korelaciji s nalazom VTEC i EPEC (p=0,39 i p=0,07). VTEC je utvrđena u uzorcima i s
najmanjom brojivom količinom od 20 MPN E. coli/100g. Sezonske varijacije pojave VTEC u
školjkašima između toplijeg i hladnijeg dijela godine nisu statistički značajne (p=0,17) za
razliku od pojavnosti VTEC između različitih godina (od 0% u 2007. godini do 3,9% u 2010.
godini) što je statistički značajno (p=0,04).
E. coli bacteria is a very common bacterium and normal inhabitant of the
intestinal tract in humans and animals. A great majority of strains of E.coli are harmless,
whereas strains of VTEC which produce verotoxin (Stx) cause serious diseases. Due to the
different ways of spreading, it is possible to find strains of VTEC in the sea. Shellfish feed by
filtering the sea water and concentrate substances in their organisms, so the aim of the
research was to prove the presence and possibility of detection of VTEC during the regular
monitoring of shellfish products. The prescribed MPN method of enumeration and detection
of E. Coli in shellfish is based on the fact that the majority of strains of E. Coli rise at 44 oC
and contain the β-glucuronidase enzyme; ß-gluc(+). The strains of VTEC cover around 200
strains of E. Coli of which 3-5% of strains as well as the extremely pathogenic strain of E.
coli O157:H7 do not have the β-glucuronidase enzyme - ß-gluc(-) and cannot be detected
using the standard prescribed method.
Material and methods: Combining the classical cultural methods used for isolation
and enumeration of ß-gluc(+) E. coli and method of allocation of ß-gluc(-) E. coli O157:H7,
in the period from 2007 till 2010, a culture from 900 samples of shellfish was collected for
further identification of VTEC. At the same time 258 samples of shellfish (29%) were tested
using ELISA method for detection of verotoxin in feces which was adapted for shellfish by
enrichment. Classical methods of isolation of E. coli and ELISA methods were verified with
three ß-gluc(+); Stx(-) E. coli strains and ß-gluc(-); Stx (+) E. coli O157:H7, and with the
experiment of contaminating the shellfish with natural filtration of sea polluted with VTEC
O157:H7. The experiment proved the spreading of VTEC from the sea to shellfish with a
preserved possibility of producing verotoxin, thus confirming the successful implementation
of methods. The isolates of E. coli obtained by the earlier methods, were tested using
multiplex PCR test of genes on markers virulence stx1, stx2, eae and hlyA. PCR method was
verified with bacteria culture VTEC O157:H7, and the extracted DNA E. coli O26 and O111,
origin of people with symptoms of HUS. The compatibility of ELISA with PCR is 100%.
Results: In isolates of E. coli, with PCR test of genes on VTEC markers, from 4
samples of mussels and one from a warty venus, the presence of stx gene was confirmed.
ELISA method identified verotoxin in one shellfish sample, and using PCR in the stored
isolate E. coli, the presence of stx gene was also confirmed. The compatibility of ELISA with
PCR is 100%. Prevalence is 1% VTEC in samples with isolated E. coli and prevalence of
VTEC of 0,6% in the complete number of 900 tested shellfish samples. In 3,3% isolated E.
coli EPEC was established which is a prevalence of 1,9% of the complete number of shellfish.
In mussels, genotypes were found; two stx2; one stx1, eae; and stx1, eae, hlyA – genotype of
highly pathogenic EHEC strains. In warty venus only one stx1 gene was found. Not one
culture has both genes. Serotyping of VTEC culture established serotypes O7, O78 ,and O55
which is connected with cases of HUS and sporadic epidemics. Prevalence of VTEC in the
complete number of tested samples is the highest in warty venus; 0.9% as compared to
mussels; 0,6%. In oysters and Noah's ark shell prevalence is 0%. The noted differences in
prevalence VTEC and EPEC in the complete number of tested samples of various shellfish is
not statistically significant (both p=0.8). Value of MPN E. coli/100g sample with VTEC
varies from 16000 MPN to the lowest value of 0 MPN E. coli /100g shellfish. The noticed
differences in the frequency of unsatisfactory market test results (>230 MPN) among the
various types of shellfish are statistically significant (p<0.001). VTEC have been found in
2/12 production areas, whilst EPEC positive samples have been found in nearly half of the
areas, 5/12. The greatest number of established VTEC positive samples is in the Malostonski
bay (4/498), but the prevalence of 0,8% VTEC somewhat lower than 0,9% in the area of the
mouth of the river Krka with the established VTEC in 1/110 of tested shellfish. Differences in
prevalence VTEC and EPEC samples in the complete number of tested samples from various
production areas of the Dalmatian part of the Eastern coast of the Adriatic are not statistically
significant (p=0.87 and p=0.9). The highest value of 16000 MPN E. coli/100g shellfish with
VTEC established in the samples in the area of the mouth of the river Krka. With regards to
the level of MPN E. coli /100g shellfish, the noticed differences in the number of
unsatisfactory samples (>230 MPN) among the different locations are statistically significant
(p<0.001). Within the same area the differences among the values of MPN E. coli in the four
different species of shellfish is not statistically significant as compared to the variations
among the species in the whole tested area, so with regards to the level of MPN E. Coli, there
is a more significant influence of the production area rather than species. Regardless of the
species of shellfish and production area, the noticed differences MPN E. coli/100g in shellfish
are not in correlation with the findings of enteropathogenic and verotoxigenic E. coli (p=0.07
and p=0.39). The presence of VTEC is not conditioned by the level of MPN E. coli because
VTEC has been established in samples with the lowest enumeration quantity 20 MPN E.
coli/100g. Seasonal variations of evidence of VTEC in shellfish between the warmer and
colder part of the year is from 1,7% to 12,5%, but overall in the research period from
September 2007 till June 2010, the seasonality are not statistically significant (p=0,17) as
compared to the occurrence of VTEC during the different years (from 0% in 2007 to 3,9% in
2010) which is statistically significant (p=0.04).
Conclusion: The research on the Eastern coast of the Adriatic sea, for the first time,
proved presence of verotoxigenic E. coli – VTEC. It also proved the presence of
enteropathogenic E. coli – EPEC.