Molecular characterization of resistance and virulence
genes
We analysed the presence of virulence genes and determinants of
resistance to the selected antimicrobials. The str A gene was
detected in 75% of all streptomycin-resistant isolates. Genes
conferring kanamycin resistance were only detected in eight out of the
19 resistant strains. The aph(3’)Ia gene was identified in seven
isolates, while the aph(3’)IIa gene was present in only one
strain. We detected one strain with resistance to gentamicin encoded by
the aac(3)-II gene. The distribution of genes encoding resistance
to phenicols was highly diverse. In most chloramphenicol-resistant
strains, the flo R gene (16/27) was present alone (7/27) or in
combination with cml A and/or cat . However, 15 strains
showed the presence of the cml A gene, which was found as single
(6/27) or with the other analysed genes (Table 2). The tet A gene
dominated in the tetracycline-resistant strains and was present as
single in 29/45 isolates and together with tet B in four strains.
In ten strains, we found a single tetB gene. In two cases, we did
not detect any of these two genes. Sulfamethoxazole resistance was
mainly related to the sul2 gene (26/32) detected as single or
together with the other genes tested (Table 2). A single E. coliESBL producer was a carrier of the bla CTX M-27gene.
The pathotypes were determined based on the presence of selected
virulence-associated genes (VAGs). We found eight strains with specific
pathotypes, and the ExPEC pathotype dominated. We identified theE. coli isolates as ExPEC based on the presence of at least two
VAGs including P fimbrial genes papC , S fimbrial genessfa/foc , the iron acquisition gene iut A , afimbrial
adhesion genes afa/dra , and the group 2 polysaccharide capsule
gene kpsMTII , as proposed by Johnson et al. (2003). We found four
strains with the ExPEC pathotype. E. coli ExPEC obtained from the
squirrels contained the same gene profile: papC, kpsMTII, iutA ,
and additionally the papAH gene coding for the
pyelonephritis-associated pilus. The R67/KAN strain contained only three
genes: papC, papAH , and kpsMTII , while the H80/CHL strain
had additionally the sfa/foc and iutA genes and the
α-hemolysin hlyA- coding gene. We also found two isolates from the
squirrels that had the ETEC pathotype. The S140/KAN strain had both
genes encoding heat-stable enterotoxins, whereas S42/TET had only theestI gene. Two strains, one from the rats (R59/TET) and one from
the red deer (J165/TET), were defined as EHEC since they contained the
Shigatoxin 2-encoding gene. We did not obtain any EPEC isolate (Table
1).
The comprehensive analysis also showed that 16 animals tested (29% out
of the strain-positive samples) were carriers of more than one E.
coli isolate. Moreover, the strains isolated from the same individuals
differed in both resistance (phenotypic and gene profiles) and virulence
profiles and were characterized by completely separate
ADSRRS-fingerprinting profiles (Fig.S1)