Vancomycin Resistance in Enterococcus faecium from the Dallas, Texas, Area Is Conferred Predominantly on pRUM-Like Plasmids

Abstract

Vancomycin-resistant E. faecium (VREfm) is a significant public health concern because of limited treatment options. Genomic surveillance can be used to monitor VREfm transmission and evolution. Genomic analysis of VREfm has not been reported for the Dallas/Fort Worth/Arlington, TX, area, which is currently the 4th largest metropolitan area in the United States. Our study aimed to address this gap in knowledge by analyzing the genomes of 46 VREfm strains and 1 vancomycin-sensitive comparator collected during routine fecal surveillance of high-risk patients upon admission to a Dallas, TX, hospital system (August to October 2015). Thirty-one complete and 16 draft genome sequences were generated. The closed VREfm genomes possessed up to 12 extrachromosomal elements each. Overall, 251 closed putative plasmid sequences assigned to previously described and newly defined rep family types were obtained. Phylogenetic analysis identified 10 different sequence types (STs) among the isolates, with the most prevalent being ST17 and ST18. Strikingly, all but three of the VREfm isolates encoded vanA-type vancomycin resistance within Tn1546-like elements on a pRUM-like (rep17) plasmid backbone. Relative to a previously reported typing scheme for the vanA-carrying Tn1546, new variants of the Tn1546 were identified that harbored a combination of 7 insertion sequences (IS), including 3 novel IS elements reported here (ISEfa16, ISEfa17, and ISEfa18). We conclude that pRUM-like plasmids are important vectors for vancomycin resistance in the Dallas, TX, area and should be a focus of plasmid surveillance efforts. IMPORTANCE Vancomycin is an antibiotic used to treat infections caused by multidrug-resistant Gram-positive bacteria. Vancomycin resistance is common in clinical isolates of the Gram-positive pathogen Enterococcus faecium. Among E. faecium strains, vancomycin resistance genes can be disseminated by plasmids with different host ranges and transfer efficiencies. Surveillance of resistance plasmids is critical to understanding antibiotic resistance transmission. This study analyzed the genome sequences of VREfm isolates collected from the Dallas, TX, area, with particular focus on the mobile elements associated with vancomycin resistance genes. We found that a single plasmid family, the pRUM-like family, was associated with vancomycin resistance in the majority of isolates sampled. Our work suggests that the pRUM-like plasmids should continue to be studied to understand their mechanisms of maintenance, transmission, and evolution in VREfm.

Keywords: Enterococcus; mobile genetic element; mobile genetic elements; plasmid; plasmids; vancomycin resistance.

FIG 1

Phylogeny and plasmid content of Dallas E. faecium isolates. The phylogenetic tree of 49 E. faecium strains is based on an alignment of SNPs in 1,706 core genes. The branches colored green, gold, and red are supported by bootstrap values of >70, 40 to 60, and <30, respectively. The reference genomes E. faecium 1,231,501 and ATCC 700221 are labeled in red. Data for MLST, number of presumptive plasmids (shown as “##” for incomplete assemblies), and plasmid rep types are shown. Plasmid replicon conserved domains RepA_N (i), Rep_3 (ii), Rep_trans (iii), Rep1 (iv), Inc18 (v), Rep_2 (vi), and Rol_Rep_N (vii) (A), rep family (B), plasmid size ranges (C), and reference plasmid names (D) present in each strain are shown. A dark purple box indicates a chromosomally integrated plasmid whose size range was not determined. The rep17 and mosaic of rep17/rep18 are indicated with the gray and light green boxes, respectively. Plasmid groups defined in our study (pMI1 to pMI9; see main text) are highlighted in light blue. N/A, not applicable.

FIG 2

Sequence type (ST) distribution of Dallas E. faecium isolates. A total of 10 STs were observed across 47 isolates. The frequency of STs (percentage of total isolates) is shown.

FIG 3

Distribution of predicted antibiotic resistance genes among Dallas isolate genomes. Shown are 12 resistance genes {6′-N-aminoglycoside acetyltransferase [aac(6′)I-i], bifunctional enzyme 6′-aminoglycoside acetyltransferase-2″-aminoglycoside phosphotransferase [aac(6′)-aph(2″)], aminoglycoside nucleotidyltransferase [ant(6)-Ia], aph(3′)III, vanA, erm(B), erm(T), lnu, acquired macrolide resistance-like protein [msr], tet(M), tet(L), and dihydrofolate reductase [dfrG]}, and the antibiotics they provide resistance to. A colored box indicates the presence of a certain resistance gene in the isolate. The boxes are color-coded to indicate the location of the gene (chromosome or plasmid).

FIG 4

Genetic map of a representative group 2a pRUM-like plasmid. The 39-kb plasmid p97_1_39kb (GenBank accession number CP066571) from the Dallas VREfm isolate 97-1 is shown. Arrows represent genes. The genes are color-coded based on their predicted activity. Maintenance genes are predicted to be responsible for general maintenance of the plasmid within the bacterial host. Resistance genes provide antibiotic resistance.

FIG 5

Classification and distribution of pRUM-like plasmid groups. (A) pRUM-like plasmids were classified based on the presence or absence (indicated by a plus or minus, respectively) of 2 replication modules (rep17 and rep18) and 2 stability modules (toxin-antitoxin system; TA_axe-txe and TA_relE). (B) Frequency of the pRUM-like plasmid groups among 43 Dallas isolates.

FIG 6

Cluster of pRUM-like plasmids identified from PLSDB with 99.9% ANI. (A) BLASTn alignment of plasmid sequences with Easyfig. (B) Data for E. faecium strains and plasmids, arranged in the same order as in the alignment.

FIG 7

Tn1546 structural variations in the Dallas VREfm isolates. A reference Tn1546 structure that includes transposase, resolvase, and van gene cluster flanked by IR_L and IR_R (inverted repeats, left and right, respectively) is delineated at the top. The Tn1546 groups are represented below the reference Tn1546, and the name of the group (C3, BC2, BC6, BC7, BC8, BC9, BC10, BC11, BCB, BBC, BCK, BCL, BCM, BBCD, BCCL, D1, or J) is shown on the left. Red boxes represent deleted regions relative to the reference Tn1546. Vertical lines with numbers at the bottom represent the nucleotide position within the reference Tn1546. IS elements are represented by downward arrows. Black stars indicate point mutations. The figure is not drawn to scale.