Determination of resistance and virulence genes in Enterococcus faecalis and E. faecium strains isolated from poultry and their genotypic characterization by ADSRRS-fingerprinting

Abstract

The aim of this study was to determine the antimicrobial resistance of E. faecalis and E. faecium strains isolated from poultry and to carry out genotypic characterization thereof with the ADSRRS-fingerprinting method (amplification of DNA fragments surrounding rare restriction sites) and analysis of the genetic relatedness between the isolates with different resistance and virulence determinants. Samples were collected from 70 4-week-old chickens and tested for Enterococcus. Minimum inhibitory concentrations of 11 antimicrobials were determined using the broth microdilution method. Detection of antibiotic resistance and virulence genes was performed using PCR, and molecular analysis was carried out using the ADSRRS-fingerprinting method. The highest percentage of strains was resistant to tetracycline (60.5%) and erythromycin (54.4%), and a large number exhibited high-level resistance to both kanamycin (42.1%) and streptomycin (34.2%). Among 8 genes encoding AME, the tested strains showed mainly the presence of [aph(3΄)-IIIa], [ant(6)-Ia], [aac(6΄)-Ie-aph(2΄΄)-Ia], and [ant(9)-Ia] genes. Phenotypic resistance to erythromycin was encoded in 98.4% strains by the ermB gene. Genotypic resistance to tetracycline in E. faecium was associated with the presence of tetM and tetL (respectively, in 95.5 and 57.7% of the isolates); in contrast, E. faecalis strains were characterized mainly by the presence of tetO (83.3%). The virulence profile was homogenous for all E. faecium strains and included only efaAfm and ccf genes. All E. faecalis strains exhibited efaAfs, gelE, and genes encoding sex pheromones. The strains tested exhibited 34 genotypic profiles. Comparative analysis of phenotypic and genotypic resistance and virulence profiles and confrontation thereof with the genotypes of the strains tested showed that strains assigned to a particular genotype have an identical phenotypic resistance profile and a panel of resistance and virulence genes. The results of this study confirm that poultry can be a reservoir of resistant E. faecium and E. faecalis strains with multiple combinations of resistance and virulence genes, whose specific panel determines not only phenotypic characteristics but also has a strong correlation with the genotypic profiles of the strains.

Characterization of Multidrug Resistant E. faecalis Strains from Pigs of Local Origin by ADSRRS-Fingerprinting and MALDI -TOF MS; Evaluation of the Compatibility of Methods Employed for Multidrug Resistance Analysis

“…faecalis (ADSRRS-fingerprinting) from the current and previous study (samples for current analysis were collected about one year later than for previous analysis), the full homogeneity of ADSRRS profiles within the same phenotype of resistance has been shown in the previous study, while the strains in the present study were characterized by certain diversity within the same phenotype, both for ADSRRS profiles and resistance genes. This phenomenon may confirm the high discriminatory potential of ADSRRS-fingerprinting method allowing for diversification of strains with the same or similar phenotypic resistance profile, but differing in relation to the genes that determine this resistance [ 12 ]. A similar regularity in the consistency of genetic profiles (obtained with the PFGE method) with resistance profiles was observed by other authors [ 6 , 7 , 32 , 42 , 57 ].…”

“…Identification to the genus Enterococcus was performed as previously described [ 12 ]. Species identification was performed using 16S–23S rRNA intergenic region restriction endonuclease analysis according to previously described protocols [ 25 ].…”

“…DNA was isolated using ready-made kits (Bacterial and Yeast Genomic DNA Purification Kit; Eurx, Gdańsk, Poland) according to protocols described elsewhere [ 12 ]. The isolates were tested for the presence of selected genes encoding resistance to macrolides [ erm A, erm B, erm C, erm F, msr A, mef A], tetracycline [ tet M, tet K, tet O, tet L, tet S], phenicols [ cfr , fex A, cat ], lincosamides [ lnu B, lnu F], copper [ tcr B] as well as aminoglycosides and aminocyclitols [ aac (6’)-Ie- aph (2”)- Ia , aph (3’)- IIIa , aph (2”)- Ib , aph (2”)- Ic , aph (2”)- Id , ant (6)- Ia , ant (4)- Ia , ant (9)- Ia , and aad A], and genes encoding virulence factors: gel E (gelatinase), cyl A, cyl B, cyl M (hemolysin-cytolysin production), efa Afs (cell wall adhesin), agg (aggregation protein), esp (cell wall-associated protein involved in immune evasion), hyl (hyaluronidase) cpd , cob , and ccf (sex pheromones facilitate conjugation).…”

“…Amplification of DNA fragments surrounding rare restriction sites (ADSRRS- fingerprinting) was performed as described by Krawczyk et al [ 8 ] with a few modifications [ 12 ]. The reaction of restriction endonuclease digestion of genomic DNA (150–250ng) was carried out with 25 μl of a reaction mixture composed of 10 U Xba I, 5 U Bgl II, and 5 μl Tango buffer (ThermoScientific ® , Waltham, USA) at 37°C, for 60 min (the reaction was stopped by thermal inactivation at 80°C for 2 min).…”

“…Its discriminatory potential is comparable to the "gold standard" of PFGE [ 8 ] and using this method does not require prior knowledge of the sequences of the analysed strains. Moreover, this method is characterized by repeatability and ease of interpretation of results and has been successfully used for molecular typing of bacteria not only from the genus Enterococcus , [ 8 , 12 ], but also other bacterial species [ 13 – 16 ].…”