Diversity of plasmids and Tn1546-type transposons among VanA Enterococcus faecium in Poland

Abstract

The objective of this study was to investigate the antimicrobial resistance, Tn1546 transposon variability and plasmid diversity among Polish vancomycin-resistant Enterococcus faecium (VREfm) isolates of VanA phenotype in the context of their clonal structure. Two hundred sixteen clinical VREfm isolates collected between 1997 and 2010 were studied by antimicrobial susceptibility testing, MLST, MLVA and detection of IS16, esp _Efm, pilA, intA and plasmid-specific genes by PCR. Tn1546 structure was revealed by overlapping PCR and sequencing. Selected isolates were subjected to PFGE-S1 and Southern hybridization analyses. The vast majority of the isolates (95.8 %) belonged to lineages 17/18 (during the whole study period 1997-2010) and 78 (mostly in 2006-2010) of hospital-adapted meroclone of E. faecium. All isolates displayed a multi-drug resistance phenotype. Twenty-eight Tn1546 types (including 26 novel ones) were associated with eight different ISs (IS1216, IS1251, ISEfa4, ISEfa5, ISEfm2, ISEf1, IS3-like, ISEfm1-like). The vanA-determinant was typically located on plasmids, which most commonly carried rep2_pRE25, rep17_pRUM, rep18_pEF418, rep1_pIP501, ω-ε-ζ and axe-txe genes. VanA isolates from 1997-2005 to 2006-2010 differed in clonal composition, prevalence of gentamicin- and tetracycline-resistance and plasmidome. Our analysis revealed high complexity of Tn1546-type transposons and vanA-plasmids, and suggested that diverse genetic events, such as conjugation transfer, recombination, chromosomal integration and DNA mutations shaped the structure of these elements among Polish VREfm.

Fig. 1

Diversity of Tn1546 transposon types among E. faecium VanA isolates. Position of primers used in PCR mapping and sequencing indicated by arrows with primer names; open rectangles, transposon genes; stars, positions of point mutations; analyzed areas of the transposon shadowed; dashed lines, deletions in the left arm of the transposon; filled rectangles, deletions within the transposon; vertical arrow, triangles with arrows, the IS positions; single-nucleotide insertion in vanY

Fig. 1

Diversity of Tn1546 transposon types among E. faecium VanA isolates. Position of primers used in PCR mapping and sequencing indicated by arrows with primer names; open rectangles, transposon genes; stars, positions of point mutations; analyzed areas of the transposon shadowed; dashed lines, deletions in the left arm of the transposon; filled rectangles, deletions within the transposon; vertical arrow, triangles with arrows, the IS positions; single-nucleotide insertion in vanY

Fig. 1

Diversity of Tn1546 transposon types among E. faecium VanA isolates. Position of primers used in PCR mapping and sequencing indicated by arrows with primer names; open rectangles, transposon genes; stars, positions of point mutations; analyzed areas of the transposon shadowed; dashed lines, deletions in the left arm of the transposon; filled rectangles, deletions within the transposon; vertical arrow, triangles with arrows, the IS positions; single-nucleotide insertion in vanY

Fig. 1

Diversity of Tn1546 transposon types among E. faecium VanA isolates. Position of primers used in PCR mapping and sequencing indicated by arrows with primer names; open rectangles, transposon genes; stars, positions of point mutations; analyzed areas of the transposon shadowed; dashed lines, deletions in the left arm of the transposon; filled rectangles, deletions within the transposon; vertical arrow, triangles with arrows, the IS positions; single-nucleotide insertion in vanY

Fig. 2

Plasmid-associated gene distribution among Polish VREfm VanA. Number of isolates with a particular gene given above the graph bars

Fig. 3

Hypothetical evolution of Tn1546 structures among Polish VREfm VanA. Type A1, found in different cities, is a presumable ancestor variant with remaining types being its direct or indirect derivatives. Types A2, A3 and A5 developed by point mutations in wt type. A4 developed from A3 through single deletion events in the vanS-vanH intergenic region. A6 is an A3 derivative that lacks ca. 1900 bps in the 5′ end. The E type transposon, a third potential derivative of A3, arose through acquisition of ISEfa4 between vanX and vanY. A3 and its derivatives were typical for Poznań (Po) medical centres. B3 and G variants presumably developed from A5 after insertion events of IS1216 and ISEf within vanX-vanY intergenic region in Kraków (Kr) and Warsaw (Wa), respectively. The ubiquitous B2 type, typical for Warsaw, probably emerged from a single insertion event of IS1216 within the A1 type with a concomitant deletion of the 5′ end of the transposon. The B4 (additional single nucleotide insertion within vanY) and BH (ISEfm1-like insertion between vanX and vanY) types represent possible derivatives of B2. The B1, D and F transposon types are potential derivatives of A1 formed by IS1216, ISEfa5 and ISEfm2 insertions, respectively. Another group of transposon variants, encompassing types BI, BBI, BBBI1, BBBI2, BB1 and BB2 emerged through complex insertion and deletion events in different regions of wt transposon promoted mostly by IS1216 elements. This group was detected mainly in Gdańsk (Gd). The activity of another insertion sequence, IS1251, followed by IS1216 insertions and several point mutations resulted in the formation of C- and BC-types in Kraków and Warsaw, respectively