VanA-Enterococcus faecalis in Poland: hospital population clonal structure and vanA mobilome

Abstract

The aim of our study was to characterize the epidemiological situation concerning nosocomial vancomycin-resistant Enterococcus faecalis of VanA-phenotype (VREfs-VanA) in Poland by investigating their clonal relationships and the vanA-associated mobilome. One-hundred twenty-five clinical isolates of VREfs-VanA collected between 2004 and 2016 were studied by phenotypic assays, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR detection of plasmid-specific genes, and Tn1546 structure and localization mapping. Selected isolates were subjected to PFGE-S1, Southern hybridization, genomic sequencing and conjugation experiments. The majority of isolates (97.6%) belonged to clonal complexes CC2 and CC87 of E. faecalis. All isolates were resistant to vancomycin and teicoplanin, and resistance to ciprofloxacin and aminoglycosides (high level) was very prevalent in this group. VanA phenotype was associated with 16 types of Tn1546, carrying insertion sequences IS1216, ISEfa4, IS1251 and IS1542, located on repUS1_pVEF1, rep1_pIP501, rep2_pRE25, rep9_{pAD1/pTEF2/pCF10} and rep6_pS86 replicons. The most common Tn1546 B- and BB-type transposons, harbouring one or two copies of IS1216, were inserted between rep18a_p200B and repUS1_pVEF1 genes and located on ~ 20 kb and 150-200 kb plasmids. VREfs-VanA in Poland represent a polyclonal group, indicating a number of acquisitions of the vanA determinant. The repUS1_pVEF1-vanA plasmids, unique for Poland, were the main factor beyond the acquisition of vancomycin resistance by E. faecalis, circulating in Polish hospitals.

Keywords: Enterococcus faecalis; HAI; Plasmid; Tn1546; VRE.

Fig. 1

Diversity of Tn1546 transposon types among E. faecalis VanA isolates. Black arrows, transposon genes; stars, positions of point mutations; areas of the transposon analysed by PCR mapping and sequencing shadowed; the A5912G and A10086G point mutations detected on Illumina reads; dashed lines, deletions in the left arm of the transposon; grey arrows, ISs; DR, direct repeats; ins, insertion; del, deletion; n.d.,– not determined; ^a number of isolates, if larger than one, are given in brackets; ^b reference Tn1546 (M97297). Nucleotide positions correspond to the reference Tn1546 transposon

Fig. 1

Diversity of Tn1546 transposon types among E. faecalis VanA isolates. Black arrows, transposon genes; stars, positions of point mutations; areas of the transposon analysed by PCR mapping and sequencing shadowed; the A5912G and A10086G point mutations detected on Illumina reads; dashed lines, deletions in the left arm of the transposon; grey arrows, ISs; DR, direct repeats; ins, insertion; del, deletion; n.d.,– not determined; ^a number of isolates, if larger than one, are given in brackets; ^b reference Tn1546 (M97297). Nucleotide positions correspond to the reference Tn1546 transposon

Fig. 2

PT-based dendrogram of VREfs-VanA in Poland (n = 125) collected during 2004–2016. Bp, Biała Podlaska; El, Elbląg; Gd Gdańsk,; Gr, Grajewo; Gro, Grodzisk Mazowiecki; Gry, Gryfice; Je, Jelenia Góra; Ka, Katowice; Lo, Łódź; Lu, Lublin; My, Myślenice; Op, Opole; Ost, Ostrów Mazowiecki; Ot, Otwock; Pl, Płock; Ple, Pleszew; Po, Poznań; Rz, Rzeszów; Si, Siedlce; So, Sosnowiec; Sw, Świdnica; Sz, Szczecin; To, Toruń; Wa, Warszawa; Wr, Wrocław; Zi, Zielona Góra; the city abbreviation is followed by the centre number; presumable outbreaks designated with letters A-K; TgR, isolate resistant to tigecycline; ChR, isolate resistant to chloramphenicol; CpS, isolate susceptible to ciprofloxacin; not HLSR, isolate susceptible to high concentration of streptomycin; not HLGR, isolate susceptible to high concentration of gentamicin; only phenotypes that differentiate the isolates are shown. Filled and empty circles indicate strong and weak haemolysis activity, respectively. The first Polish VREFs-VanA isolate (7946/1998) included for comparative purposes

Fig. 2

PT-based dendrogram of VREfs-VanA in Poland (n = 125) collected during 2004–2016. Bp, Biała Podlaska; El, Elbląg; Gd Gdańsk,; Gr, Grajewo; Gro, Grodzisk Mazowiecki; Gry, Gryfice; Je, Jelenia Góra; Ka, Katowice; Lo, Łódź; Lu, Lublin; My, Myślenice; Op, Opole; Ost, Ostrów Mazowiecki; Ot, Otwock; Pl, Płock; Ple, Pleszew; Po, Poznań; Rz, Rzeszów; Si, Siedlce; So, Sosnowiec; Sw, Świdnica; Sz, Szczecin; To, Toruń; Wa, Warszawa; Wr, Wrocław; Zi, Zielona Góra; the city abbreviation is followed by the centre number; presumable outbreaks designated with letters A-K; TgR, isolate resistant to tigecycline; ChR, isolate resistant to chloramphenicol; CpS, isolate susceptible to ciprofloxacin; not HLSR, isolate susceptible to high concentration of streptomycin; not HLGR, isolate susceptible to high concentration of gentamicin; only phenotypes that differentiate the isolates are shown. Filled and empty circles indicate strong and weak haemolysis activity, respectively. The first Polish VREFs-VanA isolate (7946/1998) included for comparative purposes

Fig. 3

PFGE-S1 Southern blotting results for selected 50 VREfs-VanA and the first Polish VREFs-VanA isolate (7946/1998) (A) and comparative PFGE-S1 Southern blotting with the vanA probe for conjugation-positive isolates (donors D1-D15) and their E. faecalis and/or E. faecium transconjugants (B). Eight isolates analysed by WGS underlined; ^a city abbreviations and outbreak designation as in Fig. 2; isolates positive for conjugation in bold

Fig. 3

PFGE-S1 Southern blotting results for selected 50 VREfs-VanA and the first Polish VREFs-VanA isolate (7946/1998) (A) and comparative PFGE-S1 Southern blotting with the vanA probe for conjugation-positive isolates (donors D1-D15) and their E. faecalis and/or E. faecium transconjugants (B). Eight isolates analysed by WGS underlined; ^a city abbreviations and outbreak designation as in Fig. 2; isolates positive for conjugation in bold

Fig. 4

Map of the p1207_4 vanA-plasmid (GenBank accession no. CP075608); positions and directions of predicted coding sequences are indicated by arrows, with antimicrobial resistance genes in red, plasmid replication genes in blue, genes associated with conjugative transfer, mobilization and transposition in green and other CDSs in black