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)