Molecular characterization of Escherichia coli from dishwasher rubber seals
Molekularna opredelitev bakterij Escherichia coli z gumijastih tesnil pomivalnih 
strojev
Marjanca Starčič Erjavec, Jerneja Ambrožič Avguštin*
Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
*Correspondence: jerneja.ambrozic@bf.uni-lj.si
Abstract: In this study 35 Escherichia coli isolates obtained from sampled dishwa-
shers rubber seals were analysed with polymerase chain reactions (PCR) using specific 
primers for phylogenetic group, multilocus sequence type (MLST) determination the 
presence of 43 virulence-associated genes (VAGs) linked with intestinal and extrain-
testinal E. coli infections and the presence of some typical E. coli virulence plasmids’ 
sequences in order to assess their virulence potential and/or specific genes, associated 
with the adaption to a specific environment. It was found that all of the 35 E. coli 
isolates belonged to the commensal non-pathogenic phylogenetic group A and that 
the diversity of these E. coli isolates, according to MLST analysis, was relatively low. 
Further, the prevalence of virulence-associated genes among the dishwasher rubber 
seal E. coli isolates was also low. Only the following VAGs were detected: fimH, crl, 
fluA, picU, irp, fyuA, sitA, aslA. Of the five plasmid replication regions tested only 
RepFIA and RepFIIA were detected. The two sequences associated with conjugative 
plasmids namely traJ and traT, were detected in only one isolate. Based on the obtained 
results the studied isolates can be designated as commensal E. coli with low pathogenic 
potential. Due to the low diversity of sequence types, even among isolates obtained 
from dishwashers from different locations, there is a possibility that strains from certain 
clonal groups are more adapted to specific habitats outside warm-blooded hosts than 
strains of other sequence types.
Keywords: dishwasher, E. coli, genes, One Health, plasmids, virulence
Izvleček: V tej raziskavi smo analizirali 35 izolatov bakterije Escherichia coli, 
pridobljenih iz vzorčenih gumijastih tesnil kuhinjskih pomivalnih strojev, da bi oce-
nili njihovo potencialno patogenost in/ali prisotnost genov povezanih z adaptacijo na 
specifično okolje. Z verižno reakcijo s polimerazo (PCR), ob uporabi ustreznih oligo-
nukleotidov, smo vse izolate uvrstili v filogenetske skupine, določili njihov sekvenčni 
tip na podlagi analize multilokusnega zaporedja (MLST), ugotavljali prisotnost 43 
genov, povezanih z virulenco (VAG), ki so pomembni v črevesnih in zunajčrevesnih 
okužbah z E. coli ter ugotavljali prisotnost nekaterih značilnih zaporedij plazmidov, 
povezanih z virulenco E. coli. Ugotovili smo, da je vseh 35 izolatov E. coli pripadalo 
komenzalni nepatogeni filogenetski skupini A in da je bila raznolikost teh izolatov, glede 
na analizo MLST, relativno majhna. Poleg tega je bila razširjenost genov, povezanih 
 ACTA BIOLOGICA SLOVENICA 
 LJUBLJANA 2022              Vol. 65, Št. 1: 18-27
19E. coli from dishwasher rubber seals
z virulenco, med preučevanimi izolati E. coli nizka. Ugotovili smo le naslednje VAG: 
fimH, crl, fluA, picU, irp, fyuA, sitA, aslA. Od petih testiranih plazmidnih replikacij-
skih regij sta bili potrjeni samo RepFIA in RepFIIA. Zaporedji traJ in traT, povezani 
s konjugativnimi plazmidi, smo zasledili samo v enem izolatu. Na podlagi dobljenih 
rezultatov lahko preučevane izolate označimo kot komenzalne E. coli z nizkim pato-
genim potencialom. Glede na majhno diverziteto sekvenčnih tipov, tudi med izolati 
pridobljenimi iz pomivalnih strojev iz različnih krajev, pa obstaja možnost, da so sevi 
iz določenih klonalnih skupin bolj prilagojeni na specifične habitate izven toplokrvnih 
gostiteljev, kot sevi drugih sekvenčnih tipov. 
Ključne besede: E. coli, Eno zdravje, geni, plazmidi, pomivalni stroj, virulenca
Introduction
Recently, the importance of the approach, 
known as “One Health” has been increasingly 
recognized in the world. This approach is based on 
the awareness that microbes, animals and humans 
share ecosystems and that research on the diversity 
and complexity of interactions is needed. Further, 
this approach recognizes that the health of humans, 
domestic and wild animals, plants, and the wider 
environment (including ecosystems) are closely 
linked and interdependent (Miao et al. 2022). 
Escherichia coli (E. coli), which is one of the 
best studied microorganisms in the world and a 
well-known genetic model organism, is a typical 
example of a bacterium that can be found in the 
environment, in water, soil, as well as in animal 
and human hosts (Jang et al. 2017). It usually lives 
in a mutualistic relationship with the host, but can 
also be pathogenic and cause serious, even fatal, 
intestinal and extra-intestinal infections (Allocati at 
al. 2013). E. coli is excreted from the host by faeces 
into the environment, where it can spread and then be 
reintroduced into different animal and human hosts 
by different routes (Jang et al. 2017). It is known for 
its pronounced genetic diversity, reflected in many 
different strains (Chaudhuri and Henderson 2012). 
The E. coli pathogenic strains carry many different 
virulence factors genes including colonization and 
fitness factors, toxins, and other effectors intertwin-
ing with the host’s physiology (Kaper et al. 2004). 
Furthermore, it is known that certain phylogenetic 
groups are associated with pathotypes, for example 
the B2 phylogenetic group is associated with E. coli 
strains causing extraintestinal infections (Čurová 
et al. 2020). On the other hand, commensal strains 
isolated from France and Sweden in the 2000s and 
from North America (USA), Japan, and Australia 
also mainly belong to the B2 group (43%), while 
commensal strains isolated from France and Croatia 
in the 1980s and from Africa (Mali and Benin), Asia 
(Pakistan), and South America (French Guiana, 
Colombia, and Bolivia) belong mainly to the A group 
(55%) (Tenaillon et al. 2010). The genetic diversity 
of E. coli is also reflected in the presence of different 
mobile genetic elements, among which plasmids, 
especially the conjugative ones, are notorious for 
their effect on the genetic diversity and for carriage 
of virulence-associated genes (Kaper et al. 2004).
Although E. coli can exhibit host-associated and 
free-living/environmental state, the data on environ-
mental E. coli strains are particularly scarce as there 
is a huge bias towards data on pathogenic strains 
(Starčič Erjavec and Žgur-Bertok 2015). In order 
to understand the heterogeneity of E. coli strains, 
their ability to adapt and spread within and outside 
hosts and the interactions between these strains, 
including gene exchange with horizontal transfers, 
it is necessary to obtain more data on characteristics 
of strains from specific human-associated environ-
ments such as household dishwashers.  
Zupančič et al. (2019) found that E. coli are 
relatively frequent members of dishwasher rubber 
seal bacterial communities. In their study 35 E. coli 
isolates were obtained from eight out of 30 sampled 
dishwashers rubber seals. The aim of the present 
study was to conduct molecular characterisation 
of these 35 isolates, including the determination of 
phylogenetic group and multilocus sequence type 
(MLST) and screening of virulence-associated 
genes and typical replications regions associated 
with E. coli virulence plasmids. 
Acta Biologica Slovenica, 2022, 65 (1), 18-27 20
Materials and methods
Bacterial isolates and growth conditions
A total of 35 E. coli isolated in six out of 30 
dishwashers randomly selected in kitchens inside 
private dwellings located in seven Slovenian cities 
(i.e., Ljubljana, Velenje, Žalec, Celje, Mislinja, 
Sežana, and Portorož) were obtained from the 
Mycosmo Microbial Culture Collection Ex at 
the Biotechnical Faculty in Ljubljana (more 
informations about the dishwashers are available 
in Zupančič et al. 2019, additional file S1). All 
isolates were grown in liquid LB or on LB plates 
at 37 °C. Liquid cultures were vigorously aerated 
by shaking at 180 rpm. 
Determination of phylogenetic groups of E. coli 
isolates
The phylogenetic groups for all of the E. coli 
isolates were determined by PCR, as described by 
Clermont et al. (2000) and improved by Clermont 
et al. (2013). The phylogenetic groups obtained by 
the method of Clermont et al. (2000) were assigned 
to their phylogenetic (sub)groups, according to the 
interpretation of Escobar-Páramo et al. (2006).
Multilocus sequence typing of E. coli isolates
Multilocus sequence typing was carried out 
according to Wirth et al. (2006) using PCR primers 
and protocols specified on the E. coli MLST web-
site (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli) to 
amplify housekeeping genes adk, fumC, gyrB, icd, 
mdh, purA and recA. The purified PCR products 
were sent to Microsynth AG (Switzerland) for 
DNA sequencing. The sequences were analysed 
for allelic profiles and sequence types through the 
E. coli MLST website. 
Detection of virulence-associated genes (VAGs) 
and plasmid replication regions of the E. coli 
isolates
The presence of 43 VAGs, including adhesins 
(fimH, crl, eae, bmaE, gaf, aaf, papGI, papGII, 
papGIII, matA, sfa/foc, iha, afa/dra, and hra), 
autotransporters (sat, vat, hbp (tsh), picU, and fluA 
(Ag43)), iron acquisition systems (fyuA, irp, sitA, 
iutA, iucD, and iroN), genes involved in increased 
serum survival and protectins (iss, ompTAPEC, 
kpsMTII, neuCS, cvi), toxins (stx1, stx2, astA, eltA, 
cnf2, hlyA), invasins (aslA, ibeA, gimB, tia), and 
uropathogenic specific protein usp, was tested in 
all isolates. This was achieved using amplification 
procedures as described previously (Dozois et 
al. 1992, Yamamoto et al. 1996, Yamamoto and 
Echeverria 1996, Maurer et al. 1998, Paton and 
Paton 1998, Schubert et al. 1998, Hoffman et al. 
2000, Johnson and Stell 2000, Vila et al. 2000, 
Janβen et al. 2001, Ruiz et al. 2002, Ewers et al. 
2007, Runyen-Janecky et al. 2003, Starčič et al. 
2003, Tóth et al. 2003, Watt et al. 2003, Parham 
et al. 2005, Vidal et al. 2005) and the instructions 
for the ‘DEC Primer Mix’[Statens Serum Institut, 
Denmark]. FluA was amplified using the primer 
pair FluAF 5’-GCGGTGTACTGCTGGCCG-3’ 
and FluAR 5’-CGTTGTGGCTGCCCAGAC-3’, 
under the following cycling conditions: initial 
denaturation for 5 min at 95 °C, 30 cycles of 30 s 
at 94 °C and 30 s at 60 °C, followed by extension 
for 90 s at 72 °C. Likewise for usp, using UspDe-F 
5’-ATGCTACTGTTTCCGGGTAGTGTGT-3’ /
UspDe-R 5’-CRTGTAGTCKGGGSGTAACAAT-3’: 
initial denaturation for 5 min at 95 °C, 30 cycles 
of 30 s at 94 °C and 30 s at 55 °C, followed by 
extension for 2 min at 72 °C. Both final extensions 
were performed at 72 °C for 10 min. The plasmid 
replication regions were determined by PCR as 
described previously for RepFIA, RepFIIA (Starčič 
Erjavec et al. 2003), RepFIC (Carattoli et al. 2005) 
and IncP (Sobecky et al. 1997). For RepFIB, the 
following protocol was used: initial denaturation 
at 94 °C for 4.5 min, followed by 30 cycles, each 
consisting of 30 s of denaturation at 94 °C, 30 s of 
annealing at 63 °C, and 1.5 min of elongation at 72 
°C. Additionally, the E. coli isolates were screened 
for F-like plasmid conjugative transfer genes traJ 
(the main positive regulator of conjugal transfer) 
and traT (surface exclusion and serum resistance), 
as described previously (Starčič Erjavec et al. 2003, 
Johnson and Stell 2000).
21E. coli from dishwasher rubber seals
Results
All tested dishwasher E. coli isolates belong to 
the non-pathogenic phylogenetic group A
Phylotyping revealed, that all of the 35 E. coli 
isolates from the dishwasher rubber seals belonged 
to the commensal non-pathogenic phylogenetic 
subgroup A0 according to Clermont et al. (2000) 
and Escobar-Páramo et al. (2006), and to phyloge-
netic group A according to Clermont et al. (2013). 
Multilocus sequence typing analysis of E. coli 
dishwasher isolates reveals low strain diversity 
The diversity of the E. coli isolates according 
to MLST analysis was relatively low. Twenty-seven 
(77%) of the 35 E. coli isolates were assigned to four 
known sequence types: ST189 (12 isolates, 44%), 
ST216 (7 isolates, 26%), ST399 (7 isolates, 26%), 
and ST1316 (1 isolate, 4%). The remaining eight 
isolates belonged to a new sequence type (Tab. 1).
Prevalence of virulence-associated genes 
among the E. coli isolates from dishwasher 
rubber seals is low 
The prevalence of virulence-associated genes 
among the dishwasher rubber seal E. coli isolates 
was low (Tab. 1, Fig. 1). All isolates carried the 
curli fimbriae regulator gene crl and, except for 
strain L-436, the type 1 fimbrial adhesion gene 
fimH. Isolates L-687 and L-748 were positive for 
the autotransporter gene picU. PicU has mucinolytic 
activity through which colonisation of the host 
intestine is facilitated, by helping the pathogenic 
enteric bacteria to penetrate the mucus layer that 
coats the intestinal epithelium (Bhullar et al. 2015). 
Among the genes associated with iron acquisition, 
sitA was detected in isolate L-436, and irp and 
fyuA in isolates L-595 and L-1071. Further, 14 
isolates (L-439, L-437, L-591, L-1101, L-778, 
L-781, L-785, L-786, L-790, L-808, L-814, L-815, 
L-821, L-836) were positive for aslA, a gene that 
is associated with invasion of the blood–brain 
barrier (Hoffman et al. 2000). 
Figure 1: Prevalence of virulence-associated genes and plasmid replication regions in all E. coli, isolated from 
dishwasher rubber seals.
Slika 1: Prevalenca genov, povezanih z virulenco in plazmidnimi regijami za podvojevanje, v vseh izolatih E. coli 
z gumijastih tesnil pomivalnih strojev.
Acta Biologica Slovenica, 2022, 65 (1), 18-27 22
Prevalence of virulence plasmid associated 
sequences among the E. coli isolates from 
dishwasher rubber seals is low 
In E. coli, plasmids can encode virulence fac-
tors and antibiotic resistance genes. Therefore, all 
of the isolates were analysed for plasmid-related 
characteristics; namely, the replication region and 
the tra region associated with conjugation. Of the 
five plasmid replication regions tested, only two 
were detected: RepFIA in eight isolates (L-396, 
L-434, L-431, L-433, L-594, L-736, L-983, L-982), 
and RepFIIA in one isolate (L-436) (Tab. 1). One 
isolate (L-429) was shown to have F-like plasmid 
tra region genes, traJ and traT (Tab. 1).
Table 1: MLST sequence type, virulence-associated genes and virulence plasmid associated sequences among the 
E. coli isolates from the dishwasher rubber seals. The following genes were not detected and are not 
included in the table: Adhesins: eae, bmaE, gaf, aaf, papGI, papGII, papGIII, matA, sfa/foc, iha, afa/
dra and hra,  Protectins: ompT, kpsMTII, neuCS, cvi and iss,  Autotransporters: sat, vat and hpb/tsh, 
Iron acquisition genes: iutA, iucD and iroN,  Toxins: stx1, stx2, astA, , cnf2, hlyA and elt,  Invasins: ibeA, 
gimB and tia,  Uropathogenic specific protein: usp. The RepFIC, RepFIB and IncP plasmid replication 
regions were not present, and are not included in the table.
Tabela 1: Sekvenčni tip MLST, z virulenco povezani geni in zaporedja, značilna za plazmide, povezanih z virulenco 
pri izolatih E. coli z gumijastih tesnil kuhinjskih pomivalnih strojev. Naslednjih genov nismo zasledili 
pri preučevanih izolatih in zato niso vključeni v tabelo: Adhezini: eae, bmaE, gaf, aaf, papGI, papGII, 
papGIII, matA, sfa/foc, iha, afa/dra in hra,  Protektini: ompT, kpsMTII, neuCS, cvi in iss,  Avtotransporterji: 
sat, vat in hpb/tsh,  Sistemi za privzem železa: iutA, iucD in iroN,  Toksini: stx1, stx2, astA, , cnf2, hlyA 
in elt,  Invazini: ibeA, gimB in tia,  Uropatogeni specifični protein: usp. Plazmidnih replikacijskih regij 
RepFIC, RepFIB in IncP prav tako nismo zasledili in niso vključene v tabelo.
Dish-
washer
E. coli 
isolate
Sequence 
type
Adhesins Autotrans-
porters
Iron acquisition Invasin Plasmid 
replication 
regions
Plasmid 
conjugation 
regions
fimH crl fluA picU irp fyuA sitA aslA RepFIA RepFIIA traT traJ
1 L-429 ST 399 + + + – – – – – – – + +
L-595 ST 399 + + + – + + – – – – – –
L-1071 ST 399 + + + – + + – – – – – –
6 L-1101 ST 189 + + – + – – – + – – – –
L-790 ST 189 + + – – – – – + – – – –
L-396 ST 399 + + – – – – – – + – – –
10 L-431 UN + + – – – – – – + – – –
L-433 UN + + – – – – – – + – – –
L-594 UN + + – – – – – – + – – –
L-736 UN + + – – – – – – + – – –
L-983 UN + + – – – – – – + – – –
L-982 UN + + – – – – – – + – – –
13 L-434 ST 216 + + – – – – – – + – – –
23E. coli from dishwasher rubber seals
L-589 ST 216 + + – – – – – – – – – –
L-590 ST 216 + + – – – – – – – – – –
L-592 ST 216 + + – – – – – – – – – –
L-745 ST 216 + + – – – – – – – – – –
L-747 ST 216 + + – – – – – – – – – –
L-748 ST 216 + + – + – – – – – – – –
22 L-436 ST 1316 – + – – – – + – – + – –
25 L-591 ST 189 + + – – – – – + – – – –
L-778 ST 189 + + – – – – – + – – – –
L-781 ST 189 + + – – – – – + – – – –
L-785 ST 189 + + – – – – – + – – – –
L-786 ST 189 + + – – – – – + – – – –
L-808 ST 189 + + – – – – – + – – – –
L-814 ST 189 + + – – – – – + – – – –
L-815 ST 189 + + – – – – – + – – – –
L-821 ST 189 + + – – – – – + – – – –
L-593 ST 399 + + – – – – – – – – – –
L-940 ST 399 + + – – – – – – – – – –
L-687 ST 399 + + – + – – – – – – – –
L-439 ST189 + + – + – – – + – – – –
L-437 UN + + – – – – – + – – – –
L-836 UN + + – – – – – + – – – –
UN, unknown.
Discussion
The primary source of the studied dishwasher as-
sociated E. coli isolates might be both the household 
water supply system connected to the dishwasher 
and the contaminated vessels (Zupančič et al. 2019). 
In either case, it can be expected that studied E. 
coli strains are mainly commensal. Indeed, all of 
the E. coli isolates were assigned to phylogenetic 
group A0, which is associated primarily with non-
pathogenic commensal strains (Stoppe et al. 2017). 
This phylogenetic placement agrees with their low 
virulence potential and environmental adaptability. 
All of these isolates carried genes involved in 
adhesion; crl, and except one, fimH, the key fac-
tors in adhesion and biofilm formation on abiotic 
surfaces (Pratt and Kolter 1999). Furthermore, 14 
isolates carried genes for AslA, a member of the 
arylsulphatase family of enzymes. In bacteria, 
the asl genes are expressed under conditions of 
sulphur starvation, in order to scavenge sulphate 
from exogenous substrates. In human meningitis 
Acta Biologica Slovenica, 2022, 65 (1), 18-27 24
strains of E. coli K1, AslA contributes to invasion 
of the blood–brain barrier (Hoffman et al. 2000). 
The prevalence of other tested virulence-associated 
genes was very low. Only three isolates carried one 
or two genes involved in iron acquisition. Three 
isolates carried fluA for autotransporter Ag43a, 
which mediates diffuse adherence and bacterial 
intercellular aggregation, thus promoting biofilm 
formation and persistence (Zude et al. 2013). flu 
genes are predominantly found in clinical isolates, 
although Restieri et al. (2007) reported that 56% 
of commensal isolates screened in their study were 
positive for Ag43 coding genes. Furthermore, four 
isolates carried the autotransporter gene picU, which 
has been associated with phylogenetic group B2 
isolates. Pic has mucinolytic activity, promotes 
species-specific haemagglutination, host colonisa-
tion, and immune evasion, by direct cleavage of 
complement proteins. The potential role of Pic in 
commensal strains is unknown (Abreu et al. 2015, 
Bhullar et al. 2015).
Contrary to our initial expectations related to 
tap water as the environmental inoculum, MLST 
analysis indicated low diversity of E. coli isolates 
on dishwasher rubber seals. This might be partly 
due to isolation of the same strain from individual 
dishwashers, although isolates with the same se-
quence type isolated from the same dishwasher 
often carried different tested genes, e.g.: dishwasher 
1 isolate L-429 (traJ, traT) and isolates L-595 
and L-1071 (irp, fyuA) all belong to ST399. From 
dishwasher 25, isolates of three different sequence 
types were detected, namely ST189, ST399 and 
an as yet unknown sequence type. Again, isolates 
from the same ST also differed; e.g., ST189 strain 
L-439 was positive for picU and aslA, and isolate 
L-778 was positive only for aslA. The same applied 
to dishwasher 6, with isolates L-1101 (picU, aslA) 
and L-790 (aslA); both ST189. The same sequence 
type of isolated bacterial strain was found in different 
dishwashers (different time in use) with different 
frequency of use, which were even geographically 
located in different cities (e.g. Ljubljana and Celje), 
thus indicating the strong selective pressure for 
certain sequence type genetic background in such 
specific extreme environments. 
Analysis of the traits characteristic for horizontal 
gene transfer (traT, traJ and IncP-replication region) 
showed that vast majority of the studied isolates does 
not possess conjugative F-like and IncP plasmids. 
In the light of the knowledge that virulence factor 
genes can be spread in bacterial populations via 
conjugative plasmids, the studied isolates are po-
tential recipients which could have high acceptance 
for conjugative IncF or IncP group plasmids and 
hence might obtain additional virulence-associated 
genes via conjugation. Therefore, further stud-
ies revealing the ability of acquiring conjugative 
plasmids are needed.
Conclusion
To the best of our knowledge, this is the first 
molecular characterization of E. coli isolates from 
Slovenian dishwasher rubber seals. Based on the 
obtained results the studied isolates can be desig-
nated as commensal E. coli with low pathogenic 
potential. However, further studies should shed 
light on the genome traits enabling specific adapta-
tion of some clonal linages to harsh environments 
such as the studied dishwasher rubber seals and 
the potential of horizontal gene exchange in such 
bacterial communities.
Povzetek
Koncept Enega zdravja vse bolj pridobiva 
na pomenu, saj smo spoznali, da je zdravje ljudi, 
domačih in divjih živali, rastlin in širšega okolja 
(vključno z ekosistemi) tesno povezano in soodvisno. 
Escherichia coli (E. coli), ki je eden najbolje razis-
kanih mikroorganizmov na svetu in dobro poznan 
genetski modelni organizem, je tipičen primer 
bakterije, ki jo najdemo v okolju, v vodi, zemlji, 
pa tudi v živalskih in človeških gostiteljih. E. coli 
sicer velja za komenzalno, nepatogeno bakterijo, ki 
s svojim gostiteljem živi v mutualističnem odnosu, a 
obstajajo sevi, ki zaradi nabora virulentnih dejavni-
kov lahko povzročajo okužbe. Za patogene seve E. 
coli so značilni številni različni dejavniki virulence, 
vključno z dejavniki, ki omogočajo kolonizacijo, 
sintezo toksinov in efektorskih molekul, ki se vpletajo 
v fiziologijo gostitelja. Nadalje je znano, da so dolo-
čene filogenetske skupine povezane s patogenostjo 
sevov. Podatkov o okoljskih sevih E. coli in njihovih 
lastnostih je v literaturi malo. Namen te raziskave je 
25E. coli from dishwasher rubber seals
tako bil opredeliti molekularne značilnosti izolatov 
E. coli z gumijastih tesnil pomivalnih strojev in 
oceniti njihov patogeni potencial. V tej raziskavi smo 
35 izolatov bakterije E. coli, osamljenih iz osmih 
vzorčenih gumijastih tesnil kuhinjskih pomivalnih 
strojev, analizirali z verižno reakcijo s polimerazo 
(PCR) z uporabo specifičnih začetnih oligonukleo-
tidov za njihovo filogenetsko skupino in sekvenčni 
tip na osnovi multilokusnega zaporedja (MLST), za 
prisotnost 43 genov, povezanih z virulenco (VAG) 
E. coli pri črevesnih in zunajčrevesnih okužbah 
(geni adhezinov fimH, crl, eae, bmaE, gaf, aaf, 
papGI, papGII, papGIII, matA, sfa/foc, iha, afa/
dra, in hra; avtotransporterjev sat, vat, hbp (tsh), 
picU, in fluA (Ag43); sistemov za privzem železa 
fyuA, irp, sitA, iutA, iucD, in iroN; protektinov iss, 
ompTAPEC, kpsMTII, neuCS, cvi; toksinov stx1, 
stx2, astA, eltA, cnf2, hlyA; invazinov aslA, ibeA, 
gimB, tia, in uropatogenega specifičnega proteina 
usp) ter za prisotnost nekaterih značilnih zaporedij 
plazmidov, tudi povezanih z virulenco E. coli (zapo-
redja plazmidnih replikacijih regij RepFIA, RepFIB, 
RepFIIA, RepFIC, IncP ter zaporedji, povezani s 
konjugacijo, traJ in traT). Ugotovili smo, da je vseh 
35 izolatov E. coli pripadalo komenzalni, nepatogeni 
filogenetski skupini A po Clermontu in sod. (2013) 
in da je bila raznolikost teh izolatov glede na analizo 
MLST relativno majhna. Sedemindvajset izolatov 
smo uvrstili v enega izmed štirih sekvenčnih tipov: 
ST189 (12 izolatov), ST216 (7 izolatov), ST399 (7 
izolatov), in ST1316 (1 izolat). Tudi razširjenost 
VAG med preučevanimi izolati E. coli je bila nizka. 
Potrdili smo le naslednje VAG: fimH, crl, fluA, picU, 
irp, fyuA, sitA, aslA. Vsi izolati so imeli genski 
zapis crl, fimH pa smo našli pri vseh, razen pri 
enem izolatu. Pri 14 izolatih smo potrdili prisotnost 
aslA, pri štirih izolatih picU, pri treh izolatih fluA 
ter pri dveh izolatih irp in fyuA (Tab. 1, Sl. 1). Od 
petih testiranih plazmidnih replikacijskih regij sta 
bili odkriti samo RepFIA in RepFIIA. Zaporedji 
traJ in traT, povezani s konjugativnimi plazmidi, 
sta bili odkriti samo pri enem izolatu (Tab. 1, Sl. 
1). Majhna raznolikost sekvenčnih skupin (ST) 
ter podatka, da so bili iz istega pomivalnega stroja 
izolirani sevi iste ST z nekoliko drugačim naborom 
genov za dejavnike virulence, ter prisotnost sevov 
iz enake ST iz strojev na različnih lokacijah, bi 
lahko bila posledica na specifično okolje prilago-
jenih klonalnih skupin. Kolikor vemo, je to prva 
študija molekularne opredelitve izolatov E. coli z 
gumijastih tesnil pomivalnih strojev v Sloveniji. 
Na podlagi dobljenih rezultatov lahko preučevane 
izolate označimo kot komenzalne E. coli z nizkim 
patogenim potencialom.
Acknowledgements
The study was financially supported by 
Slovenian Research Agency (grant P1-0198). Our 
acknowledgements also go to Katja Molan for her 
technical assistance. 
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